Research Collection

Doctoral Thesis

Stability studies of the parental solutions of sympatol andnoradrenaline

Author(s): Agrawal, Devendra Kumar

Publication Date: 1960

Permanent Link: https://doi.org/10.3929/ethz-a-000131813

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STABILITY STUDIES

OF THE PARENTAL SOLUTIONS

of

SYMPATOL AND NORADRENALINE

THESIS

PRESENTED TO

THE SWISS FEDERAL INSTITUTE

of

TECHNOLOGY, ZURICH

for

THE DEGREE OF

DOCTOR OF NATURAL SCIENCES

by

DEVENDRA KUMAR AGRAWAL

Citizen of India

Accepted on the recommendation of

Prof. Dr. K. Steiger—Trippi

and

Prof. Dr. J. BUchi

Job Printers, Allahabad

1960

Printed by Job Printers, 99, HewettRoad; Allahabad

1959—60

PREFACE

Physiological action may be achieved by the administration of drugsin various forms. One of the most rapid means of obtaining such an

action is by administering the drug in the form of an injection. The

inclusion of various monographs in different Pharmacopoeias relating to in¬

jections points to the significant advantages of such a form of medica¬

ment.

However one of the most intriguing problems before the pharmacisthas been that of the stabilization of the galenicals in general and injec¬tions in particular. In the words ofSchou (1); "How is it that a questionlong known to be of great importance is still characterised by beinginsufficiently considered if not at all touched."

The present work has been carried out to determine the con¬

ditions necessary for the stability of the parental solutions of two of the

sympathomimetic amines : Sympatol and Noradrenaline.

ACKNOWLEDGEMENTS

The experimental work in connection with these investigationshas been carried out at the School of Pharmacy of the Swiss FederalInstitute of Technology, Zurich, under the supervision and guidance of

Prof. Dr. K. Steiger. I have feelings ofdeep gratitude towards my teacher,Prof. Dr. K. Steiger for taking a keen inrerest in me, and giving his valu¬

able advice and guidance throughout the course of this work.

My sincerest thanks are due to Messrs Volkart Foundation, Winter-

thur, for their scholarship to assist me to complete my research,which was a material financial help to me throughout the course of

my work.

I am also thankful to Dr. Hippenmeier of the Kantons Apotheke,Zurich, for allowing me to work in his laboratories with the Zeiss

Spectrophotometer, to Messrs Boehringer Sohn, Ingelheim am Rhein for

supplying their original product of Sympatol and to Messrs Hoechst Farb-

werke, Frankfurt for supplying Noradrenaline.

DEDICATED TO MY DEAR PARENTS

ERRATA

Page Errata Correct

2 Aquoeus Aqueous

18 Stndarad Standard

33

+

NH31

+

NH21

39 apporximate approximate

55 ehl the

77 SB as

119 fig. 15 fig 16

120 fig. 16 fig. 15

fig. 16 (in Table) fig. 15

124 ZUSAMVIEMFASSUNG ZUSAMMEMFA&

124 siehe page siehe seite

125 abbau Abbau

127 Sprtzein Spritzen

Hohlandeln Hohlnadeln

CONTENTS

Some considerations of Injections and Sympathomimetic amine s

CHAPTER I Page

Injections

1. Requirements .. .. .. . . 1

2. Manufacture of injections .. .. 2

3. Stability of injections . . 13

CHAPTER II

Sympathomimetic amines

1. General considerations .. ....16

2. Therapeutic uses of sympathomimetic amines..

18

3. Relation between chemical structure and

pharmacological activity of sympathomimeticamines 19

4. Classification of sympathomimetic amines .. 20

CHAPTER III

Stability of Sympatol solutions

1. Problem

2. Working procedure

CHAPTER IV

Sympatol

1. Synonyms

2. Synthesis3. Tests

4. Methods of quantitative analysis described

in the literature

5. Pharmacological actions

6. Clinical uses

29

29

31

31

31

33

..34

..35

7. Dosage ..••

••

..36

CHAPTER V

Testing of the materials and apparatus

1. Sympatol •• ••••

..37

2. Distilled water39

V1U

Page

3. Ampules .. • • .. ..39

4. Other materials .. .. 41

5. Apparatus .. .. .. ..41

CHAPTER VI

Methods of analysis for Sympatol1. Quantitative methods

.. .. .. 42

2. Colour standards for comparison of the colour..

57

3. pH determination .. .. 61

CHAPTER VII

Determination of the stability of Sympatol solutions

1. Principles ..62

2. Preparation of the solution.. 62

3. Testing of the ampules .. 64

4. Discussion and conclusions of the results

of colour and pH determination.. 70

5. Injection of Sympatol .. 71

CHAPTER VIII

Stability studies of Noradrenaline (NA) solutions with respect to

racemization

1. Introduction.. .. .. ..72

2. Problem.. .. .. ..72

CHAPTER IX

Noradrenaline

1. Synonyms and proprietary names.. 73

2. Synthesis .. 73

3. Physical properties .. 74

4. Qualitative tests.. 74

5. Methods of quantitative analysis described..

75in the literature

6. Pharmacological actions.. 78

7. Clinical uses.. 80

8. Dosage

CHAPTER X

Testing of the material

.. 80

1. Noradrenaline hydrochloride ..81

IX

PageCHAPTER XI

Determination of fresh and deteriorated Noradrenalinein solution

1. Introduction.. .. .. ...

83

2. UV. Spectrophotometric method.. ..

84

3. Colorimetric methods.. .. ..

87

4. Summary '

.. .. ..94

CHAPTER XII

Racemizations studies of the dilute solution of Noradrenaline

1. Method for concentrating the solution and for deter¬

mining the optical rotation

2. Preparation of the solution

3. Results

4. Summary of the results of racemization studies

5. Discussion and conclusions

CHAPTER XIII

96

97

98

102

105

Prediction of the stability of the solutions of Noradrenaline with

respect to racemization

1. Introduction 106

2. Order of reaction...

106

3. Temperature coefficient 108

4. Experimental 109

5. Racemization studies at 50°, 60° and 70° 117

6. Summary ..

121

CHAPTER XIV

Summary 122

References 127

The following abbreviations have been used in the text

°= Temperature has been given in centigrade scale

g = Gramme

ml = Millilitre

AR = Analytical reagent

NA = Noradrenaline

Some Considerations of Injections and

Sympathomimetic amines

CHEATER I

INJECTIONS .

Injections are sterile aqueous or nonaqueous solutions or suspen¬sions intended for administration under, or through one or more layersof skin or mucous membrane. This form of medicament has several

advantages (2) :—

(a) Instantaneous physiological action is achieved especiallyif the drug is given intravenously.

(b) Drugs which may be useless or may lose their potency if

given orally can be easily administered.

(c) Drugs can be administered even when the patient is unable

to take anything orally.

(d) The physician controls the amount of medicament given,and so there is no possibility of over dosage.

1. Reqnirenjents

General requirements for the injections may be summarised as

follows :-—

(a) They should be clear, free from dust particles and

homogeneous if the substance is insoluble.

(b) They should be sterile.

(c) They should be isotonic with blood serum.

(d) They should have the same pH value as that of the blood

serum, if the conditions permit.

(e) They should not give rise to any undesirable effects when

injected.

(f) They should be stable.

In order to comply with the above requirements it is importantthat every care is being taken during the manufacture of injections.

2

2. Manufacture of Injections

Butikofer (3) has discussed the different aspects of the manu¬

facture of injections. The following scheme summarises the steps invol¬

ved for their manufacture :

Solvent

Itest

(physical &chemical)

(Pyrogen test

Active ingredient

test

(physical &

chemical)

Solution

Filtration

IFilling

Closing

Sterilization

ITests

Additive

I '

test

.(physical &

chemical)I

Pyrogen test

Container

Itest

(alkali)

Chromic-acid-bath

Icleaning

Isterilization

Sterility Pyrogen pH checking Visual (dustparticles, colouration)

LabellingI

Packing

Expedition

In the following pages we shall discuss the important points con¬

cerning the requirements of injections.

2.1. Solvent

2. 1. 1. Aquoeus :ol\ents

Water for injection is used for the preparation of parentalsolutions for which most of the pharmacopoeias give a separatemonograph. It is prepared by distilling potable or distilled water

from a neutral glass still, fitted with an efficient device to preventthe entrainment of droplets.

It is important that water for injection is free:from dissolved gaseslike carbon dioxide as it could precipitate dissolved 'substances like

phenobarbitone sodium, sulphadiazine sodium, theophylline with

ethylenediamine, pentobarbitone sodium, sodium dihydrocholic etc., while

the presence of oxygen could oxidize such substances as adrenaline,noradrenaline, ascorbic acid, para-amino-salicylic-acid, apomorphine,mersalyl etc. Further it should be free from metallic ions especially iron

and copper as they considerably accelerate the processes of oxidation.

Schou and Gredsted (4) have investigated the oxygen content of differ¬ent types of water which is reproduced in table I.

Table I

Oxygen content of water at 20°.

1. Collected directly from the apparatus 1-2 to 2-34 ml/litre

2. Distilled water stored in ordinary containers 6-35 ml/litre

3. Boiled and rapidly cooled 4-0 ml/litre

4. Sterilized by autoclaving in Erlenmeyer flasks, 2-46 to

plugged with nonabsorbent cotton wool. (120*20 min). 3-58 ml/litre

5. Water freed of oxygen through liberation of COa 0-45 ml/litre

by added bicarbonate.

From the above table' it is clear that the oxygen content of water

varies considerably under different conditions of storage. In the case

of those substances which are easily oxidized it is essential that the

water which is used for their solution should contain the minimum amount

of dissolved oxygen, and therefore it is recommended to give in the

monographs ofinjections of such substances a limit for the oxygen content

of the solvent.

2.1. 2. Non aqueous solvents

Fixed oils or isolated esters of the higher fatty acids are generallyused as non-aqueous solvents.' They should have no odour or taste

suggesting rancidity and should comply with the following tests :

(a) Cooling test showing the absence of solid paraffin.

(b) Iodine value.

(c) Saponification value.

(d) Test for the presence of peroxides.

. (e) Acid value.

(f) Test for mineral oils.

(g) Viscosity test.

Beside the above solvents ether, ethyl alcohol, propylene glycol,glycerin, benzyl benzoate and different polyethylene glycols are alsoused .as non-aqueous vehicles. Recently glycofurol (tetrahydrofurfurylalcohol-polyethyleneglycolether, containing an average of two ethyleneglycol groups) has been reported by Spiegelberg et al. (5) as a verysuitable non-aqueous solvent.

2. 2. Isotonicity ofthe solution

A solution is said to be isotonic with respect to blood serum,when the two show the same osmotic pressure against water. In the

following we consider isotonicity only with respect to blood serum. In

4

order to prevent haemolysis it is important that the injections are isotonic,

especially when great quantities are to be injected directly into the

blood stream.

A o.9% solution of sodium chloride is isotonic with blood serum

since it has the same osmotic pressure as that of the blood serum cor¬

responding to a freezing point depession of 0*56° (against water),' thoughLund el al. (6) found this value to be 0-52°. The solutions can be made

isotonic in one of the following ways :

2. 2. 1. By calculation of the freezing point depression.

2. 2. 2. By sodium chloride equivalent method.

2. 2. 3. By tables of the freezing point depression.

2. 2. 4. By graphical method.

While making calculations by any of the above methods it should

be noted that in the case of a solution containing more than one

substance, the calculations are valid only when there is no chemical reac¬

tion. Szekely and Goyan (7) have given a critical reveiw of the com¬

monly used methods for making solutions isotonic.

2.2. 1. By calculation of the freezing point depression

Depression of the freezing point follows Raoult's law which can

be expressed as :

k. W. 1000. n

t =- where

M. L

t = freezing point depression of the solution in relation to the

solvent (°C)

k = molar freezing point depression.

W = weight of the dissolved substance (g).

n = number of ions in which the molecule is dissociated.

M = molecular weight of the dissolved substance.

L = weight of the dissolved substance and solvent (g).

Now if it is required to prepare solutions which are to be isotonic withblood serum by means of the above equation, the freezing point depres¬sion of the blood serum is introduced instead of t, and the molar freezingpoint depression of a substance which does not dissociate when dissolvedin water instead of k, this value being —1-86° when one mole of thesubstance is dissolved in lOOg of water and then by introducing thevalues of M and L in the above equation one will get the value of W.The above law is however valid only in case of dilute solutions.

2.2. 2- Sodium chloride equivalent method

Wells (8) and Goyan et al. (9) have described a method based on thefact that the molal* lowering of the freezing point for substances of the

*moles of a dissolved substance per lOOOg of water.

same ionic type is fairly constant. They divided the substances in different

groups having approximately the same value of molal lowering of the

freezing point.

Molal lowering of the freezing point is defined by the equation :

A t

L = where

C"

L - molal lowering of the freezing point (°C).

A t = depression of the freezing point produced by the solution

in question (°C).

C = concentration of the solution (moles per 1000 g of water).

The calculations are made by finding out the value of sodium chlo¬

ride equivalent E for a substance by the following equation :

L X 58-45

E = or

M X 3-44

E = 17. (L/M) where

M = molecular weight of the active substance.

L = molal freezing point depression ofthe active substance (°C)

58-54 = molecular weight of sodium chloride.

3-44 = molal freezing point depression for sodium

chloride (CC).

The sodium chloride equivalent E for a substance means the amount

ofsodium chloride which shall give the same osmotic pressure as 1 g of

the substance in question when dissolved in an equal volume of water.

The practical application of the above method may be seen

from the following example :

Ephedrine sulphate 0*60 g

Chlorobutanol 0*12 g

Aqua 30 ml

to make the above solution isotonic.

Amount of sodium chloride required to make the above solutionisotonic = 0-9 x 30/100 = 0-27 g.

Sodium chloride equivalent for ephedrine sulphate = 0*60 X E

for ephedrine sulphate = 0-60 x 0-17 = 0*10 g.

Sodium chloride equivalent for chlorobutanol = 0*12 X E for

chlorobutanol = 0-12 X 0*18 = 0-02 g.

6

Amount of sodum chloride required to make the solution isotonic

= 0-27 — (0.10 + 0-02) = 0-15 g.

2.2.3. By tables of freezing point depression

Ph. Helv. V. Suppl. II (10) has given a table of the depressionof the freezing point of a 1 % w/v solution of the adjusting substances

commonly used in practice. The weight of the substance required to make

the solution isotonic is then calculated from the follwoing formula :—

0-56 — a

W = where

b

W * weight of the adjusting substance (g).

0-56 = the depression offreezing point of blood serum (°C).

a = depression of the freezing point of the unadjustedsolution (°C).

b = depression of the freezing point of a solution of the ad¬

justing substance (1 g +water to make 100 ml)

The values of a and b can be read directly from the table. In case

of solutions having more than one substance, the value of a is obtained byadding the depression of the freezing point of different substances.

The utility of the above method is limited for substances having a

linear.relationship between the depression of the freezing point with in¬

creasing concentration of the substance in solution.

2.2.4. Graphical method

Lund et al. (6) have worked out a graphical method for makingthe solutions isotonic. The method has been adopted in the Ph. Dan.

IX. They plotted the experimental values of the depression of the

freezing point first in a system where the ordinate is the log of the

concentration and the abscissa is the log of the freezing point depression.The straight line thus formed is then moved into a corresponding arithme¬

tical system, where the ordinate is the concentration and the abscissathe freezing point depression.

Now by drawing a "mirrored" sodium chloride curve, starting fromthe point on abscissa which corresponds to the freezing point depressionat which the prepared solution is to be fixed, it is possible to read directlythe amount of sodium chloride to be added (see fig. I).

In the following figure the amount of sodium chloride required to

make a 2 % w/v solution of resorcinol isotonic with blood serum is

0-325 g, which is directly obtained on the ordinate at point d.,

Cone, in % .

30 —

20a i

^^Resorcinol

10 '

|i-^_. "~

~"

1

c

Sodium Chloride

'oi 02 '0-3 0-4'

05 0-6

Fig. I.

2.2.5. Test for the isotonicity of the solution

This may be simply done by determining the depression of the

freezing point of the solution by means of Beckmann's thermometer, or

indirectly by the thermoelectric method oi Hill (11), in which the de¬

pression of the freezing point is indirectly measured by the difference

of vapour pressure of a 0'9 % solution of sodium chloride and that of

the solution in question. Lund et al. (6) have used the apparatusoi Hill with slight modifications to determine the depression of the freez¬

ing point of several substances.

2.3. Clarity of the solution

A perfectly clear solution may be obtained by the use of sintered

glass funnels, of which several makes are available in the market, (Jenaerglass filter, Pyrex glass filter). In table 2, a summary is given of the

different tvpes of filters available, as well as their practical applications(12)-

Table 2

Summary of the different types of Pyrex and Jenaer glass filters with

their porosity no., diameter of the pores and applications.

Porosity number Jenaer or French English pyrex

pyrex filters filters

Practical application!

Calcuculated diameter of the pores in /i

1

2

90 — 150 100 — 120

40—90 40—50

for heavy precipitates,for preparative work with

medium sized crystals or

precipitates.

15 — 40 20 — 30 for preparative work withfine ppt. or analytical workwith medium fine ppt.

5 — 15 '5 — 10 for preparative and ana¬

lytical work with veryfine precipitates.

1 — 1-5 1-2 — 1-3 for bacteria-proof filtration.

8

Beside the above filters the filters mentioned below are also often

used when large quantities of the solution have to be filtered :

(a) Asbestos filters : In this category different types of Seitz fil¬

ters, Filtrox filters and many others are used.

(b) Candles of kieselguhr and porcelain, of which different

makes are available, are also extensively used.

(c) There are also many synthetic filters available of which

the following may be mentioned : polyvinyl chloride

filters, polytetrafluorethylene filters etc.

(d) Metal filters : some types of metal filters are also availa¬

ble which can be used when the solution does not react

with the metal.

Ordinary filter paper is not suitable as it usually gives solutions

with fine fibres. Further for clarity of the solution it is essential that the

container is properly cleaned and freed from dust and glass particleswhich we shall discuss under the next heading.

2.4. pH of the solution

Solutions for injection should maintain their optimum pH value

during heat sterilization and on storage as it plays a very important role

with respect to the following :

(a) precipitation of the free base from the alkaloidal solutions,for example : solutions of morphine, strychnine, etc.

(b) hydrolysis of ester alkaloids and glycosides, e.g. atropine,scopolamine etc.

(c) oxidation of such important drugs like adrenaline, apomor-

phine, eserine etc.

(d) change in. optical rotation.

i

Beside the above changes, pH can also effect the stability of collo¬

idal substances and emulsions.

Since most of the parental solutions are dispensed in glass containers

-it is important that they should not impart any alkalinity to their contents.

Pharmaceutical preparations of varying composition are stored in glasscontainers under different conditions of storage, so the containers are

required to fulfil several specifications which we shall discuss below :

2.4.1. Tiie glass containers should be chemically resistant

Chemical resistance is the most important requirement for glass con¬

tainers. To fulfil this condition glass is required to have certain

specific compositions. In table 3 compositions of typical glass containers,glass tubing and some chemically resistant glasses is given.

Table 3 after Dimbleby (13) and Fahrig (14)

Percentage composition of typical container glasses, tubing and some

chemically resistant glasses.

Constituent Colourless containers Soft soda Colourless Pyrcx Jenaeroxides British U.S.A. tubing neutral glass apparat¬

ampule us glass-20

SiOa 73-4 73-3 7012 670 80-7 74-5

Ba03 ~ —— 0-78 7.5 120

to 12-6

4-6

Ti03 004 1 ] U.S. n.s. 005 _

A120,Fe2o;r

0-75 > 0-48 }• 2-58 8-5 2-2-30 8-5

0045 J J n.s. 008 —

MnO — — n.s. —

CaO 8-9 5-3 5-4 40 0-20 0-8

MgO 0-1 39 3-6 0-3 — 01

Na20 15-9

116-31 16-82 8-7 3-9-41 7-6

K20 0-4 0-35 40 — —

As2Or 001 — — — Z. 001 —

S<V n.d. n-d. 0-20 n.s. —

Coefficient of — _ 9-6 7-3 3-3

linear expansionXlO^

n.s. ;= not stated, n.d. = not determined

2.4.1.1. Test for chemical resistance of glass continers

All glass containers used for injections are required to comply with

the tests for the limit of alkalinity of glass as given in different pharmaco¬

poeias. There are generally two types of tests, one being the crushed

glass test, U. S. P. XV (15), and the other being the surface test, Brit.

Ph. 1958 (16), Ph. Helv. V. (17).

In the crushed glass test a certain amount of powdered glass is au-

toclaved for a definite period together with a specified amount of re¬

distilled water of a defined pH. Then the water is titrated aginst stan¬

dard acid for any given alkali. The amount of acid used is a measure of

the alkalinity of the glass. Thus the quality of the whole galss is tested.

The principle of the surface test as given in Brit. Ph. 1958 and in Ph.

Helv. V. is the same, i.e. the neutralization of a limited amount of acid.

However Ph. Helv. V. directs that the interior surface of the container

should be calculated and a quantity of the standard acid + indicator

per unit area of the surface added, thus ensuring that the quality of the

glass is tested and the size eliminated as a factor. But by this method largecontainers are subjected to a much more rigorous test than small con¬

tainers.

On the other hand Brit. Ph. 1958 directs that the container should

be filled to its prescribed capacity with the standard acid + indicator..

In this case the volume of the container is the deciding factor of the result.'

For if one compares the ratio of the surface area with the volume of the

10

standard acid, it is obvious that the smaller containers will be subjectedto a much more rigorous test than the larger contianers of the same

quality of glass. In table 4 the approximate surface area of different

sizes of containers is given together with the quantity of 0.1 N acid

per 100 ml of the test solution for different pharmacopoeias.

Table 4

Surface area and the amount of acid per ioo ml of the test solution

according to different pharmacopoeias

Capacity Inner

surface

Ph. Helv NachtragV. bottles

;. DAB6

ampulesPh. Dan

IX.

. U. S.P.

XV

Codex.Gall. 7.

Brit.

Ph. 1958.

cm2 ml of 0-01 N acid„

1 ml 5-9 1-18 (2-1) (0-42) 0-9 1-4 1-5 1-66

10 ml 28-8 0-57 2-1 0-42 0-42 1-4 1-5 1-66

100 ml 98-2 0-196 0-95 019 019 1-4 1-5 1-66

500 ml 306-2 0-122 0-55 0-11 011 1-4 1-5 1-66

1000 ml 511-1 0-102 0-43 0086 0086 1-4 1-5 1-66

By looking at the above table it is interesting to note that the amount

of acid per 100 ml of the test solution for 1 litre containers is about 15

times less compairing the Ph. Helv. V. or the Ph. Dan. IX. methods

with those of U. S. P. XV, Brit. Ph. 1958 or Codex Gall. 7.

Some work has been done in the laboratories of this institute on this

problem and on the basis of these results the following quantities of acid

to be used for different containers is being recommended :

Table 5.

Recommended acid for alkali test of different size of containers.

Sterilizationtime at 120°

Capacity of

container

ml of 0.01 N

H2S04ml of water neutralized

with methyl red

20 min. up to 2 ml 1-0 99 0

20 min. 2 to 10 ml 0-9 991

30 min. 10 to 100 ml 0-8 99-2

40 min. 100 to 500 ml 0-5 99-5

50 min- 500 to 1000 ml 0.4 99-6

Beside the above official methods, the method of Mylius (18) is worth

mentioning in which the quality of the glass is tested by determining the

amount of [Na201 per 100 cm'• of the surface area of the container.

Berry (19) and Fahrig (14) have discussed the methods which are used

for testing the quality of glass containers.

11

2.4.2. The glass containers should be mechanically strong

Containers for parental solutions should be strong enough so as not

to give way on sterilization or during handling. Christensen (20) has worked

out a formula by which the pressure exerted on the container during ster¬

ilization can be calculated. Miinzel (21) has used his method to test the

infusion bottles which are used in Switzerland.

2.4.3. The glass containers should resist thermal shocks

The thermal coefficient of expansion of glass containers should be

such that on heating for sterilization and on cooling the container does not

break. In table 3 on page 9 the coefficient of linear expansion of

a few types of glass is given.

2.4.4. The glass should be easy to melt and seal and should not

splinter on opening'

The glass for containers should be such that it can be easily melted

and sealed. This property mainly depends on the silica content of

the glass. From table 3 on page 9, one may see that the silica content

of soft soda tubing or ampules is much less than that of Pyrex or Jenaerglass. In order to avoid glass splinters in the container care should be

taken while opening and cleaning. In an investigation by Dreweny(22) in which he examined the finished ampules from different firms,he found that in some cases glass splinters were present. Further he

showed that the amount of splinters was reduced to a minimum when the

ampules "were opened by downward pressure instead of horizontal

or vertical pressure. He also proposes a method in which the ampules are

opened after heating at 250°. By this method he showed that the amount

of splinters in the containers become negligible.

2 4.4.1. Test for splinters and otl.er foreign particles

This is done by viewing the contents in incident light against a

white and a black background. The details of the method are givenin U. S. P. XII (23).

2. 5. Sterility of the solutions

Solutions for injection may be sterilized by one of the followingmethods. The choice of the method however depends on the propertiesof the medicament.

(a) Sterilization by dry heat in the hot air oven. This method

is used for the sterilization of non-aqueous solutions, since

the sterilization in an autoclave has the same effect as that

of the dry heat which is not adequate to kill the spore-

bearing bacteria due to the inability of the steam to

penetrate below the non-aqueous solutions.

(b) Sterilization by moist heat, i.e,. by heating in an auto¬

clave in saturated steam under pressure. This method is

commonly used for aqueous solutions of those substances

which do not get decomposed by heating at high tempera¬tures.

12

(c) Sterilization in presence of a bactericide. This method isapplied for those substance which can not be heated above100' without decomposition.

(d) Sterilization by the use of bacteria-proof filters or by the use

of aseptic methods. These two methods are used forthose substances which can not be heated sufficiently to kill

micro-organisms without undergoing decomposition.

In table 6 the time is given for different methods of sterilizationas mentioned in different pharmacopoeias .

Table 6

Table showing the different methods cf sterilization together withthe temperature and duration as given in different pharmacopoeias.

Pharmacopoeia Method of sterilization

Dry

temp

heat

•C time

Saturated steam

under pressure

tempt "C time

In presence of a

bactericide

tempt"C time

Brit. Ph. 1958 150 1 h 115-116 30 min 98-100 30 min

Ph. Int. I 150 2h 115-116 30 mm 98-100 30 min

Ph. Dan. IX 140

160

3h2h

120 20 min

Ph. Helv. V 160

120

1-5 h

2h

110-120 15-20 min 100 30 min

Codex Gall. 7 120 1 h no 20 min 100 30 min

Ph. U.S.S.R. 8 120 12-15 min

Nachtr. DAD 6 130

140

180

200

1 h

45 min

20 min

10 min

120 15 min

For a detailed study of the subject we refer to the works of Baumann

(24), Stick (25), Cazzani (26) and that of Lesure and Lavagne (27).

2.5.1. Tests for Sterility

Injections sterilized by method number c, b or c are generallynot required to be tested for sterility, however, when the injection hasbeen prepared by filtration or by means of aseptic techniques it must be

tested for sterility. Most of the pharmacopoeias give the details of the

sterility test. The principle of testing the sterility is the creation of

optimum conditions for bacterial growth. The sample passes the test if

the growth of microorganisms does not occur in any of the incubatedtubes.

13

3 Stability of injections

One of the important obligations of the pharmacist is to market phar¬maceutical preparations that will maintain their label therapeutic value

and initial appearance for the duration of their shelf life.

Different destructive processes which affect the stability of injections

may be divided into three broad categories :

3.1. Biological (due to the development of micro-organisms)

3. 2. Chemical, which may be due to any one of the following

processes

3.2.1. Hydrolysis.

3.2.2. Oxidation.

3.2.2. Racemization.

The above processes are mostly dependent on light, temperature,pH, oxygen and enzymes.

3.3. Physicl factors :

3.1. Biological

The development of micro-organisms can be checked very easily

by sterilization or by the addition of a suitable bacteriostatic agent.

3.2. Chemical

3.2.1. Hydrolysis : It may be explained by the following general

equation :

R-C-O-R+H 0-> R—COOH+R-OHu

"

O

It is an important destructive factor in the case of all esters, manyalkaloids like atropine, scopolamine etc. and local anesthetics like

procaine etc. The process is dependent on pH and temperature.

3.2.2. Oxidation

Oxidation plays an important role in the case of all oxidizablesubstances like adrenaline, noradrenaline, ascobic acid, ergot alkaloids,apomorphine, physostigmine, eserine etc. The process is dependenton oxygen pressure or on the presence of 02 donors, temperature, pH and

is easily catalysed by different ions like Cu+ ,Fe+^ . It may be

checked by the replacement of oxygen with an inert gas, the use of anti¬oxidants in the form of reducing agents and by inactivating the above

mentioned ions.

3.2.2.1. Inert gases. In many instances replacement of air with an

inert gas like nitrogen or carbon dioxide helps to check the oxidation.For example: Whittel (28) reported that the injections of sulphonamidescan be protected from colouration if air is replaced by nitrogen. Foster

and Stewart (29) showed that injections of ergometrine when filled with

14

nitrogen retained 60 % of the activity after a period of about five

years while those filled with air were completely inactive. Brunnhofer and

Steiger (30) showed that indigocarmine solutions in ampules filled with

oxygen lose 63 % of their chemical activity while those filled with nitrogenlost only 6 %.

3.2.2.2. Antioxidants. So far the most commonly used antioxidants

are sodium and potassium metabisulphite. They act by being more

readily oxidized than the material, having the following reaction :

NaaS.,04 + 0, + H30 = Na3S04 + HaS04

Berry and West (31), West (32) showed that in the pesence of

0.1 % of potassium metabisulphite and at pH 4.2 the solutions of

adrenaline are least oxidized. West (33) further found that for solutions of

noradrenaline 0.1 °/„ of sodium metabisulphite and a pH of. 3.5 fo 3'9

are essential to check the oxidation. In table 7 a comparative list is

given of the antioxidants used as mentioned in different pharmacopoeias.

Table 7

Injection of Antioxidant Ph. Int. I Brit. Ph.

1958

U.S.P.

XV

Ph. Dan.

IX

Adrenaline Na metabisulphite 0.1% 0.1 % none 0 05 •>/„

Adrenaline &

Procaine

Na metabisulphite — 0.1 % none 0.1 %

Hydromorphine Na metabisulphite — — — 0.05 %

Morphine Na metabisulphite none 0.1 % none —

Metaoxedrine Na metabisulphite — — — 0.05 %

Menadioni Na metabisulphite — — — ;0.l %

Noradrenaline Na metabisulphite — — none 0.05 %

Oxedrine Na metabisulphite — — — 0.6 %

Physostigrainc Na metabisulphite 0.05% — — —

Procainamide Na metabisulphite — 0.1 % — —

Stibophen Na metabisulphite 0.1 %*

none — —

Vitamin A & D Nordihydroguajaretic acid DAK 7th ed.

3.2.2.3. 'Effect of different ions. Schou (34) reported that Cu + +

acts as a catalyst on autooxidative processes and thus can affect thestability of parental solutions to a large extent. Girard and Kerny (35)reported that the injections of adrenaline deteriorated in the presence ofmetallic ions. Pfeiffer and Offermann (36) reported that ethylenediamine

15

tetraacetate retards the oxidation by forming a complex with metallic ions,

e.g. Cu "*"+, Fe+ + +

etc, which in turn does not catalyse the reaction.

Solutions of ascorbic acid (37) and of para-amino-salicylic-acid (38) havebeen reported to be stable in presence of ethylene-diamine-tetra acetate.

3.2.3. Racemization

The process of converting an optically active compound into its

optically inactive modification, containing an equal amount of the op¬tically active components, is called racemization.

500S*

Optically inactive mixture

The mechanism by which racemization takes place is not always clear,but where tautomerism is possible it may be explained on the basis (39):

G — c - 0

The laevorotatory form of such important natural occuring alkaloidslike hyoscyamine, ergot alkaloids, ephedrine etc. and of adrenaline, no¬radrenaline etc. is physiologically much more active than the racemicform. Racemization takes place as a function of pH and temperature.Kisbye and,Schou (40) found that the solutions of adrenaline are moststable at pH 4.2 with regard to racemization. Kisbye (41), Kisbye andBoh (42) have investigated the relation between pH and racemizationof many sympathomimetic amines. Their results show that the specificrotation is highly dependent on hydrogen ion concentration, as therotation shows a marked change in alkaline solutions.

3.3. Physical factors

Separation of suspensions, emulsions, colloidal solutions, changein size of the suspended crystals, e. g. of hormone preparations, changein viscosity of nonaqeous solutions etc. are a few of the physicl factorswhich can appreciably affect the stability of some parental soulutionsand make them unfit for dispensing.

CHAPTER II

SYMPATHOMIMETIC AMINES

1. General considerations

Sympathomimetic amines are a class of basic compounds the generalcharacteristics ofwhich are to mimic or act like the effects of stimulatingthe sympathetic nerves to the organs of the body.

The human body is controlled by two types of nerves : sensory and

motor. Sensoiy nerves carry impulses away from the surface or organwhile motor nerves carry impluses towards the surface or organ. Motor

nerves are of two types: voluntary and autonomic (vegetative or involun¬

tary). Voluntary nerves control the limbs which are directly operated by the

will, while autonomic nerves control the organs of the body, e.g. heart, liver

etc. Autonomic nerves have been further divided into two types of nerves:

sympathetic and parasympathetic. Most organs which are innervated

by autonomic nerves possess both sympathetic and parasympathetic nerves

which are almost antagonistic to one another in function. The division

is as follows :

NERVES

SENSORY MOTOR Nerves carrying impulsesNerves carrying impulsesaway from the surface or

organ

towards the surface or

organ

I |VOLUNTARY . AUTONOMIC

Nerves controlling the limbs Nerves controlling the

organs of the body

SYMPATHETIC PARASYM-

PATHIC

Sympathetic nerves have their origin in the thoracolumbar segmentsof the spinal cord. The effects of stimulation of the sympathetic and

parasympathetic nerves on the-chieforgans of the body are given intable 8

17

Table 8

The effects of stlmnlation of the Sympathetic and Parasympatheticnerves on the chief organs of the body

Organ Sympathetic Parasympathetic

Blood-vessels Constriction, except coronary Nil (except in certain specialvessels which arc dilated. cases where dilation occurs,

and iti the coronary vessels

which are constricteJ.

Heart-rate Acceleration and augmenta- Inhibition,

tion.

Eye:Iris Contraction of radial musck Contraction of circular muscle

(Mydriasis). (Miosis).

Ciliary muscle Nil. Contraction.

Skin :

Sweat secretion Augmentation Nil.

Erection of hairs Increased Nil.

Salivary glands

Stomach :

Contractions

Slight viscid secretion

Inhibition

Free secretion and vasodila¬

tion.

Augmentation.

Secretions ••Increase.

Sphincters Contraction'or relaxation Relaxation or contraction.

Intestinal movements Inhibition Augmentation.

Gall bladder Relaxation Contraction.

Liver Glycogenosis Nil.

Spleen Contraction Nil.

Pancreatic secretion •• Increase.

Bronchial muscles Relaxation Contraction.

Bronchial secretion Nil increase.

Suprarenal glands Secretion Nil.

Ureter Relaxation Contraction.

Bladder:

Fundus Relaxation Contraction.

Sphincter Contraction Relaxation.

Uterus Contraction and relaxation

Wihon and Schild (43).

18

Stimulation of sympathetic or parasympathetic nerves results in theliberation of acetylcholine at the synapse and the transmission of the

nerve impulse from pre to post ganglionic fibres is achieved by the inter¬vention of acetylcholine. However in case of parasympathetic division

acetylcholine is liberated at the post-ganglionic endings, while in case

of sympathetic division sympathin is liberated at the post-ganglionicendings.

The liberation of acetylcholine and sympathin in the autonomic

nervous system is illustrated below (44).

Sympathetic

Q^Zj^VonSympathin

1st neuroni

Acetylcholine

1st neuron

ParasympatheticV-"' Acetylcholine-&

f£3T reacting cell

The concept of chemical mediation of nerve impulses to various or¬

gans was developed by Loewi (45). Cannon and Rosenblueth (46) suggestedthat Sympathin was more than one substance, or a mixture of substances.

(Excitatory effect was attributed to Sympathin E, and Inhibitory effect to

Sympathin I). Later the experiments of Euler (47), (48), Bulbring and

Burn (49), Gaddum and Goodwin (50), West (51) and others confirmed that

Sympathin was a mixture of Adrenaline and Noradrenaline. SympathinE being pure 1-Nor-adrenaline and Sympathin I being pure 1-Adrenaline.

2. Therapeutic uses of Sympathomimetic amines

Sympathomimetic amines have got a wide use in therapy. Below

we shall mention only their mam clinical uses (52), (53) based on the

following effects :,

2.1. Vascular effects -:'

In control of haemorrhage of skin and mucous membranes for

congestion ofnasal mucosa in cases of chronic and vasomotor rhinitis, si¬

nusitis, acute coryza, etc. and in conjugation with local anesthetics, etc.

2.2. Cardiac effects

In cases of cardiac arrest, acute cardiac failure, spinal anesthesia

and in post operative shocks, etc.

2 3. Allergic disorders

In bronchial asthma, miscellaneous, allergic disorders like serum

reactions, serum sickness, hayfever, etc.

2.4. Miscellaneous uses

In severe hypoglycemia, chronic urticaria, milder forms of whoopingcough, mydriatic uses, in cases of narcolepsy, depressions, etc.

Besides the above mentioned uses, sympathomimetic amines havegot a wide field of clinical application for which the stndarad works on

Pharmacology and Therapeutics may be referred to.

19

3. Relation between chemical structure and pharmacological acti¬

vity of sympathomimetic amines

No other known group of chemically related and pharmacologicallysimilar compounds reveals such a high degree'of positive corelation

between structure and activity as is revealed in the case of sympathomi¬metic amines. Barger and Dale (54) were the pioneers to investigatethe relationship between chemical structure and activity ofsympathomi¬metic amines.

In the following pages only the main points will be metioned . The

literature on the subjct is very large, however, the following authors have

given excellent reviews on the subject : (55) (56), (57), (58).

18 «

3.1. Alkyl amines(

CHS— (CH2)n—CH-CH31

' 1N

A

(a) In alkyl amines a minimum of a four carbon atom structure

is required for sympathomimetic activity ; maximum

activity is achieved with a carbon chain of six, while on

further increasing the carbon chain, activity decreases with

increasing toxicity.

(b) The optimum position of the amino group seems to be in

the o< or fi position, though this is not always the case.

(c) Substitution in the aminogroup results in the loss of activity.

/3 '«

3.2. Phenylalkylamines ^—G—G-N<

(a) An alcoholic group on the ft position of the aliphatic side

chain generally increases the pressor activity, e.g. propa-

drine is seven times more active than benzidrine.

(b) The presence of an << methyl group reduces the pressor

activity, however, it confers great stability with an increase

in duration of action and oral activity as in the case oi

ephedrine. By increasing the carbon chain to more than

three carbon atoms pressor activity is adversely affected

with increased toxicity.

(c) By saturating the phenyl ring pressor activity is some¬

what diminished, though the duration of action is increased.

20

3.3. Monohydrox> alky1 amines HO—/==\_ci c N<^

I I

(a) By introduction of a phenolic group pressor activity is

very much enhanced. Meta and para compounds beingequally active, though ortho compounds are feebler.

(b) Substitution on the N atom results in the loss of pressor

activity.

3. 4. Dihydroxyphenylalkylamines

OHI jB «

HO—<\ A-O-C—N<

I I

(a) By the introduction of two OH groups in the benzene ringin meta and para positions in relation to the aliphatic side

chain, the compound becomes truely sympathomimetic and

the activity is very much increased, as in the case of adrena¬

line. But these compounds are very unstable due to the

presence of catechol nucleus.

(b) By methylating the amino group pressor activity is reduced.For example : noradrenaline is 5/3 times as potent as ad¬

renaline when tested on the blood pressure of the cat.

However the methyl group seems to confer bronchodilator

property which is well marked in adrenaline.

(c) If the methyl group attached to the amino group is replacedby higher aliphatic groups, the activity is changed from

pressor to depressor, though the compounds retain the peri¬pheral properties Of adrenaline, e.g. isoprenaline, aleudrine,neo-epinipe, etc. art powerful bronchodilators.

4. Classification of Sympathomimetic amines

In the following table (no. 9) those sympathomimetic amines which

are commonly used (59) are listed together with their chemical name,formula and poprietary names in the following order of classification :

4.1. Alkylamines4.2. Cycloalkylamines

4.3. Arylalkylamines4.3.1. Phenylalkylamines4.3.2. Monohydroxy or methoxyphenylalkylamines

.

4.3.3. Dihydroxyphenylalkylamines

4.4. Miscellaneous

F.)

K.

(S.

Benzedrex

CH,

ICH2-CH-NH-CH,

Propylhexedrin

e

—\

/-C

yclo

hexy

l-2-

meth

ylam

ino-

prop

ane

1

Pari

s)Hue

(Lab.

Pacamine

Bell

on)

(Roger

Heptedrine

(Lilly)

sulfate

Tuamine

Knol

l).

(Bil

hube

rOenethyl

—

.so.

.CI

/nh-ch,3

I

CH„

—CH

—CH,2

•

CH,

CH3-(CHa)4-CH-NH3

4-

o:

formula

GeneralCycl

oalk

ylam

ines

.

4.2.

,

(Lilly)

Forthane

.C03

CH,

CH-NH2-CH8

CH3-(CH,)4-

+r

CH,

CH,

II

NH3

-CH3—CH2—CH—CH2—CH

sulfate

Tuamineheptane

2-Am

ino-

hept

ane-sulfate

2-Me

thyl

amin

o-he

ptan

e-hy

droc

hlor

ide

2—Amino-4-methyl-hexane-carbonate

names

Prop

rietar

yFormula

names

used

commonly

other

and

name

Chemical

NH„

CH3-(CH2)n-CH-CH3

:formula

General

atoms

4-8

from

of

chain

Carbon

ahaving

Alky

laim

nes

4.1.

9TABLE

(Grimault)

Phenedrine(Delagrange)

Maxiton

(S.K

.F.)

Dexedrine

Haen)

(Riedelde

Apophcdrin

(Spe

cia)

Ortedrine

Werke,

(Nordmark

tonon

Elas-

F.).,

K.

(S.

Benzedrine

(Minden)

Eventin

CI

CH,

OH

II

Q—CH—CH—NH3—CH

+

2J

3CH

so4

.

so4

Q-CHa-CH-NH3

OH

1

NH,

1.

+

Q-CH-CH,-]

so4

CH3

^J-CH2-CH-NH3

.CI

.CI

-CH.-CH,/NH-CH,

o+

0-CH9-CH2

CH,

Q-CH9-CH-NH9-

4-

1-Ephedrine

rochloride

-Phenyl-2-me

thyl

amin

o-pr

opan

ol-h

yd-

-1

1d-Benzedrine

d-Amphetamine

d-l-Phenyl-2-amino-propyl-sulfate

Phenylethanolamine

-Phenyl-2-am

inoethanol-sulfate

dl-1

Amphetamine

-Phe

nyl-

2-am

inopropanesulfate

dl-1

alkylamines

Phenyl

4.3.1.

mines

laalky

Aryl

4.3.

hydr

ochloride

Gyverine

chloride

methylamine-hydro-

(cyc

lohe

xyle

thyl)

Di-chloride.

Gycl

ohex

yl-i

sopropyl-methylamine-hydro-

names

Proprietary

Formula

names

used

commonly

other

and

name

Chemical

K5

Engl

.)(Wyeth

Mephine

"

(Wyeth)

Wyamine

S04

(Mer

rell

)Vonedrine

CI

Dohme)

&Sharp

(Merck,

Propadrine

CI

Wellcome)

(Burroughs

Methedrin

(Kno

ll)

Isophen

(Abb

ott)

,

'

'(Temmler),''Desoxyne

Pervitin

CI

(Mer

ell)

Nethamine

CI

(Hoechst)

Racedrin

(Merck)

Ephetonin

CI

CH„

Q_CH2-C-NH.-CH3

CH3

CH,

^-CH-CH^NH^CHg.

*

CH,

OH

11-CH—CH—NH3

4-

'

CH3

y)~CH2—CH—NH2—CH3

^

Mephentermine

'"

prop

ane-

sulf

ate

-Phenyl-2-

meth

yl-2

-met

hyla

mino

-1

hydr

ochl

oride

dl-l-Pheny

l-2-

meth

ylam

ino-

prop

ane-

Phen

ylpr

opanolamine

dl-N

or-E

phed

rine

chloride

dl-l

-Phe

nyl-

2-amino-propanol-hydro-

d-De

soxy

-eph

edri

nerochloride

l-Ph

enyl

-2-m

ethy

lami

no-p

ropa

ne-h

yd-

CH3

CH.

OH

I.

I1

I*—^

|1-

N-Et

hyle

phedrine-hydrochloride

6^)—CH—CH—NH—C3HB

panol-hydrochloride.

+P-—

l-l-

Phen

yl-2

-met

hyl-

ethy

l-am

ino-

pro-

',

CH,

OH

1CH—CH—NH2—CH,

o-?

hydrochloride

Racephedrine

hydr

ochl

oride

dl-l

-Phe

nyl-2-methylamino-propanol-

names

Proprietary

Formula-

names

used

commonly

other

and

name

Chemical

4»

(Diwag)

Novadral

CI

.

(Boehringer)

Effortil

CI

OH

£>>—

CH-C

H2—N

H3

OH

OH

)—CH—CH9—NH_—CUS

(x

CH

+CH,

OH

IOH

werke)(Tropon-

Dilatol

CI

Dohme).

~

CHOH-COO

&Sharp

(Merck

Aramine

GHOH-COOH

HO—Q—CH—CH—NH2—CH,—CH2—CH2—^

CH,

OH

!I

I—CH—CH—NH,

1OH

(Boo

ts)

Phenylephrine

Stea

rns)

.Winthrop-

Neo-synephrine)

(Boe

hrin

ger)

Adrianol

CI

OH

^_GH—CHa—NHa—GH3

OH

etha

nolh

ydrochloride

dl-l-(3-hydr

oxyp

heny

l)-2

-ami

no-

amino-etha

nol-

hydr

ochl

orid

edl

-l-(

3-Hy

droxyphenyl)-2-ethyl

hydr

ochl

oridepropyl-aminopropanol

(3-methyl)

-2-[

phen

yI-

-(4-

Hydr

oxyp

heny

l)dl-1

Metaraminol

panol-bita

rtra

tel-l-(3-Hyd

roxy

phen

yl)-

2-am

inop

ro-

no-ethanol

-hydrochloride

-2-methylami-

-(3-

Hydrox

yphe

nyI)

1-1

names

Propri

etar

yFormula

names

used

commonly

other

and

Chemical

phenylalkylamines

methoxy

or

Monohydroxy

4.3.

2.

ento

Wellcome)

(Burroughs

Vasoxine

Wellcome)

(Burroughs

Vasoxyl

CI

Upjohn)

Orthoxin(

CI

(Boo

ts)

Pholetone(K

noll

)Veritol

S04

.

CHJ

OH

OCH3

^_J>

—CH—

CH—N

H3AOCH3CH3

Q>-CH,—CH—NH2—CH3

+\

rocH3

CH,

Q—CH,—CH—NH2—CH3

HO—

teams)

(Win

throp-Startrate

Synephrine

CHOH-COO-

(Boe

hrin

ger)

.Sympatol

CHOH-COO

CH,

1

OH

1{J—CH—CH2—NH2

HO—

jCHS

(S.K

.F.)

Paredrin

Br

.O—CH2—CH—NH

—HO

J+

CH

OH

(Hoechst)

Suprifen

CI

.

IQ—CH—CH—NHa—CH3

HO—

hydrochloride

Methoxyamine

prop

anol

-hydrochloride

dl-l

-(2,

5-Dimethoxyphenyl)-2-amino-

hydrochloride

Methoxyphenamine

prop

ane-

hydr

ochl

orid

edl

-l-(

2-Me

thox

yphe

nyl)

-2-m

ethy

lami

no-

Paredrinol

propane-sulfate

-2-m

ethy

lami

no-

(4-H

ydroxy

phen

yl)

-dl-1

Oxedrinetartrate

aminoethanol-tartrate-2

-methyl-

(4-H

ydro

xyph

enyl

)-

dl-1

2-Aminopropylphenol

propane-hydrobromide

dl-l

-(4-

Hydr

oxyp

heny

l)-2

-ami

no-

propanol-hyd

roch

lori

de-2-m

ethy

lami

no-

(4-h

ydro

xyphenyl)

-dl-1

names

Prop

riet

ary

Formula

names

used

commonly

other

and

Chemical

N3

(Hoechst)

Corbasil

Stea

rns)

(Winthrop

Buta

neph

rine

(Hoechst)

1-Arterenol

Stea

rns)

,(Winthrop

CH,

OH

i}—CH—CH—NH2

HO-

1OH

CI

C2HS

OH

HO"W~CH—CH—NH3

Levophed

(DGI),

Levarterenolum

(Sch

erin

g)Aktamin

CHOH-COOH

1CHOH-COO

.

(Abbott)

Norisodrine

Wellcome)

(Burroughs-

Neo-Epinine

3

Stea

rns)

,(Winthrop

Isup

rel

(Lil

ly),

Aludrin(B

oehr

inge

r).

Aludrin

S04

OH

OH

HO'\J—CH_CH3—NH3

OH

OH

propanol

dl-l

-(3,

4-Di

hydroxyphenyl)-2-amino-

Ethy

l-no

radr

enaline

buta

nol-

hydrochloride

dl--

l-(3

,4-D

ihydroxyphenyl)-2-amino-

Noradrenaline

Arterenol,

ethanol-bita

rtra

tel-

l-(3

,4-D

ihydroxyphenyl)-2-amino-

1HO—^JJ

CH—CHa—NH,—GH(GH3)2

HO—t^s—

pylamino-ethanol-sulfate

-2-isopro-

-(3,

4-Di

hydroxyphenyl)

dl-1

names

Proprietary

Formula

names

used

commonly

other

and

name

Chemical

Dihydroxyphenylalkylamines

4.3.3.

(Chemosan)

Stryphnon

Stea

rns)

(Winthrop

Kephrine

CI.

(Hoechst)

Suprarenin

CI.

(Lak

esdi

e)Methadren

CHOH-COOH

(Cil

ag)

Isolevin

CHOH-COO

OIIv—'

CH3

C—CH,-^NH2—

C\-

HO—

+l_OH

OH

\_J—CH—CH,—NH2—CH3

HO—

+kOH

OH

Q-CH-CH2-N(CH3)9

HO-

1OH

OH

^—CH-CHa—NHa—CH(CH3),

HO—

+"

_

f

IOH

Wellcome)

(Bur

roughs

Epinin

CI.

}—CH,—CH3—NHa—CH,

6HO—

1OH

Adrenalon

thylamino-ethane-hydrochloride

-oxo-2-me-

-1-(3,4-Dihydr

oxyphenyl)

1

Epin

ephr

ine

Adre

nali

ne,

tartrate)

(or

aminoethanol-hydrochloride

-2-methyl-

(3,4

-Dih

ydroxyphenyl)

--1

1N-Me

thyl

epinephrine

thylamino-ethanol

-2-dimer

-(3,

4-Di

hydroxyphenyl)

1dl

pylamino-ethanol-bitartrate

l-l-(3"4-Dih

ydroxyphenyl)-2-isopro-

methylamine-hydrochloride

ethyl-

2-(3,4-Dihydro

xyph

enyl

)

names

Proprietary

Formula

names

used

commonly

other

and

Chemical

CO

N3

CI

(Pfize

r)Tyzine

(Ciba)

Privine

NOs

(Boe

hringer)

Preludin

(Lil

ly)

Clopane

.CI

(Raschig)

Ascensil

CH,

CH,

"NH2.

~

;N

I

y

o

CH,

"CHo

NH9

.N"

Q_CHa-C^

+NH9

k^-CH3

CI.

CH,

+1

f'S—©

2-Ph

enyl

-3-m

ethy

l-mo

rpho

line

-hyd

rc-

O

\-CH2—CH—NHa—CH3

I

N%j>—NH,

CH,

hydrochloride

Tetr

ahyd

razo

linehydrochloride

imidazoline

3-4-Tetr

ahydro-l-napthyl)-

2-(l-2

nitrate

Naphazoline

nitrate

2—(1—NapthyImethyl)-imidazoline-

chloride

hydrochloride

Cyclopentamine

hydr

ochl

orid

el-

Cycl

open

tyl-

2-me

thyl

amin

opro

pane

-

4-Me

thyl

-2-a

mino

-pyr

idin

e

names

Proprietary

Formula

names

used

commonly

other

and

Chemical

Miscellaneous

4.4.

STABILITY OF SYMPATOL SOLUTIONS

Chapter III

PROBLEM AND WORKING PROCEDURE

1. Problem

Aqueous solutions of sympatol are used in therapy by parental and

oral administration. On heat sterilization and after storage, the above

solutions become coloured and may loose their potency. Stockton et al.

(60) observed that the solutions of sympatol turned pink in about three

weeks time after sterilization by boiling, however, without any loss

of biological activity.

The purpose of this investigation was to evaluate the conditions under whichthe solutions ofsympatol used parentally, could be heat sterilized and stored without

developing any unpleasant colour or loss in sympatol content. However due to the

costly and time consuming biological methods of analysis it was not possibleto evaluate the biological activity under different conditions of storage.

2. Working Procedure

In order to ascertain the stability ofsympatol solutions, the followinginvestigations were carried out :

2.1. Quantitative estimation of sympatol content

2.1.1. Colorimetric method.

2.1.2. Ultra-violet spectrophotometric method.

2.1.3. Chromatographic method.

2.2. pH value : Determination by potentiometric method.

2.3. Colour estimation : By comparison with standard colour solu¬

tions.

2.4. Material used in the following experiments were of the

following standards :

2.4.1. Sympatol (Boehringer) Nachtr. DAB 6 (61) standard.

2.4.2. Ampules conforming with the "Powdered glass test" of U.S.

P. XV (15).

2.4.3. Water conforming with the requirements of "water for injec¬

tion", Ph. Int. I (62).

2.4.4. Other chemicals were of A. R. standard.

2.5. Preparation of the solution

A 6 % w/v solution of sympatol was prepared in distilled water

and the following conditions were made to obtain :

30

(a) pH values : 3, 4, 5, 6 and 7

(b) each containing :

rongalit 0.2 %sodium metabisulphite 0.1 %filled with nitrogenfilled with air.

2.6. Storage i The ampules were stored at 4°,. 20° (room temp.)and 37°.

2.7. Testing : The above prepared solutions were- analysed: after

20, 40, 80, 160 and 350 days.

CHAPTER IV

SYMPATOL: SYNONYMS, SYNTHESIS, TESTS (PHYSICAL AND

CHEMICAL), METHODS OF QUANTITATIVE ANALYSIS,

PHARMACOLOGY, CLINICAL USES AND DOSAGE.

1. Synonyms

SympatolOxedrine tartrate

Synephrine tartrate

Aethaphenum

Vasocordin

(C9H1303N)2.C4HaOe

BoehringerPh. Dan. IX (63)NNR. 1934 (64)

(65)

Leo (Copenhagen)

Mol. Wt. 484. 49

HO-f~^—CH—CHa—NH,-CH3

OH

CHOH

1CHOH

0

11 -

—C-O

I —

—C-O

11

0

Chemical name: Sympatol is dl-l-(4-Hydroxyphenyl)-2-mcthylamino-ethanol-d-tartrate.

2. Synthesis

Sympatol was first synthesised in the year 1927. Several methods

are available for its synthesis under the patents of different countries ;

(66), (67), (68), (69), (70), (71),. One of the best methods for the

preparation ofsympatol is achieved by condensation of w-Chloro-p-ben-zoyloxy-acetophenone with methylbenzyl amine, followed by hydrolysisand subsequent hydrogenation, (66).

Sympatol can also be prepared by treating w-Methylamino-p-hydroxy-acetophenone hydrochloride with hydrogen under ordinaryor raised pressure in the presence of a hydrogen carrying catalyst such as

Pd, Pt or their oxides. After addition of ammonia p-Oxyphenyl-ethanol-methylamine crystallizes, (67).

Lately some new methods of synthesis have also been described

by Bergmann and Sulzbacher (72), Fodor and Kovacs (73) and Asscher (74).

3. Tests

The following tests are described in Nachtr. DAB 6 -(61) and in

Ph. Dan. IX (63).

32

3. 1. Physical

3.1.1. Description. Sympatol is a white crystalline powder ;

odourless ; tastes bitter.

3.1.2. Melting 'range, for tartrate 188° to 192° Ph. Dan. IX

for base 185° to 189" Ph. Dan. IX

for base 176° to 180° Nachtr. DAB 6.

3.1.3 Solubility. Easily soluble in water ; sparingly soluble in

alcohol; insoluble in solvent ether and in chloroform."

3.1.4. Specific rotation. The specific rotation is 4-10° 'to+15'!,corresponding to a rotation of+0*40° to +0*60" when 0'50 g of sympatolis dissolved in 25 ml of water and a 20 cm long polarimeter tube at 20J

is used, Ph. Dan. IX.

3. 2. Chemical

3.2.1. Identification tests

3.2.1.1. To 2 ml of aqueous solution of sympatol (14-49 ) add

1 ml of copper sulphate solution (10 %) and 1 ml of sodium hydroxidesolution (15%); a deep ultramarine blue colour is formed ; shake with

2 ml of solvent ether; the ether layer must remain colourless.

3.2.1.2. Add a drop of ferric chloride solution (14-9) to 1ml of the

aqueous solution of sympatol (1+49); an intense yellow coulor is formed

which changes to greenish brown on further addition of two drops of ferric

chloride solution. *

3.2.1.3. To 2 ml of the aqueous solution of sympatol (1+49)add 2 drops of glacial acetic acid, 1 drop of ferrous sulphate solution

(1+9) followed by 3 drops of hydorgen peroxide (3%); a green colour

is fomed which turns brown by adding 4 ml of 2 N sodium hydroxidesolution.

3.2.1.4. Moisten a few milligrams of sympatol with a drop of water,add to 2 ml of sulphuric acid, followed by a crystal of rcsorcinol; on care¬

fully warming a red violet colour* is formed.

3.2.2. Parity tests

3.2.2.1. On dissolving 0.01 g of the sympatol in 1 ml of sulphuricacid; the colour of the solution should not be more than very slightlyyellowish (foreign organic matter).

3.2.2.2. The aqueous solution of sympatol (1+9) must be clear

and colourless ; it should not change the colour of litmus paper to

more than slight red (pH 5.8-6.6).

3.2.2.3. The sympatol should be free from chloride and sulphate-ions.

33

3.2.2.4. To the aqueous solution of sympatol (1+49) add 3 dropsof sodium sulphide solution (10%); there should be no change in the

solution (salts of heavy metals).

3.2.2.5. To the aqueous solution of sympatol (1 +49) add 1 drop of

ammonia solution (10%) and 1 ml of dimethylglyoxime solution (1% in

C,HgOH),; after keeping for several hours no red colour or turbidityshould develop (nickel compounds).

3.2.2.6. To 2 ml of the aqueous solution of sympatol (1 +49) add

1 drop of phenyl hydrazine ; there must not be any change after keepingthe solution for 5 minutes (test for methylaminomethyl-(4-oxyphenyl)-ketone).

3.2.2.7. 0.20 g of the sympatol on drying at 105° must not

loose more than 0*0010 g in weight.

3.2.2.8. 0"50 g of the sympatol on ignition must not give a residue

of more than 0"0005 g.

4. Methods of quantitative analysis described in the literature

Methods available in the literature for the quantitative estimation of

sympatol may be divided into two broad categories :

4.1. Volumetirc methods

4.2. Colorimetric methods

We shall discuss them in detail on the following pages.

4.1. Volumetric methods

4.1.1. Bromometric methd. This method is based on the quan¬titative formation ofdibromosympatolwith bromine. The excess ofbromine

is then determined iodometrically. Thus, from the amount of bromine

used up in the reaction, the sympatol content is calculated. KSllstram

(75) found that the accuracy of the method depends on the bromine con¬

centration and bromination time. Awe and Stohlmann (76) further showed

that the acid concentration and sympatol content used for the bromina¬

tion are also equally important.

The reaction may be formulated as follows :

H0~0~CH—ch»~nh»OH CH„

CHOH.COO

CHOH.COO+ 4Br, =

J 2

Br

oBr OH

HO-Q-Cf-^-1*CH,

CHOH.COO

CHOH.COO +4HBr

2

34

4.1.2. By estimation of nitrogen content. Here the nitrogen content

is determined by the Kjeldahl method bv which the sympatol content

is calculated. The method is official in Ph.' Dan. IX (63) and NNR. 1934

(64).

4.1.3. By precipitation as tetraphfenyl borate. Flaschka et al. (77)and Worell and Ebert (78) have used the reaction of tetraphenyl borates with

organic bases for the estimation of sympathomimetic amines. Aklin (79)has applied the method to assay the opium alkaloids. Sodium-tetra-

phenyl-borate gives an insoluble precipitate with organic bases which

reacts with mercuric chloride with the liberation of free hydrochloric acid

according to the following equation :

+ — + —

RNH3C1 +Na (BPh4) -> RNH3 (BPh4) + NaCl

RNH3 (BPh4) + 4 HgCl2 + 3 HaO -» RNH3C1 + 4 PhHgCl+ 3 HC1+ HaBOa

The acid content is determined by titration against standard alkali.

The method can be applied for the quantitative determination of sympatol.

4..2. Colorimetric methods

4.2.1. Sinodinos and VuillUume (80) have described a colour reaction

which can be used for the estimation of sympatol content. Diazotized

p-nitro-aniline couples with sympatol and gives a rose colour in an alkalinemedium. The colour can be concentrated by extracting with a mixture of

ethanol and butanol; then the intensity can be measured in a suitable

photometer. Jindra et al. (81) reports that the colour formed after

diazotization gives a peak at 425 mju and that it does not exactlyconfirm with Lambert-Beer's law and therefore it is necessary to draw

calibration curves.

None of the methods described above permit an unambiguousdetermination of the undecomposed sympatol content in a solution

because they are all based on the presence of either an amino group or a

hydroxy phenyl nucleus, whereas the decomposed product of sympatol can

give the same results by the above methods like the pure sympatol, as

the amino group or the hydroxy phenyl nucleus may remain intact in

the decomposed product.

So we had to find out some other method which could be used for

determining the undecomposed as well as the decomposed sympatolin a solution.

'

5. Pharmacological actions

The action of sympatol is,dn general, qualitatively similar to that of

adrenaline, exhibiting more prolonged but less intense action. It raises

the blood pressure, constricts the peripheral vessels, dilates the pupil, re¬

laxes the smooth muscles of the intestines and increases the muscular tone

ofthe uterus as reported by Ehrismann (82), Lasch (83), Ehrismann and Malqff(84) and Kusehinsky (85).

35

Ehrismann (82), Kuschinsky (85) and Schuntermann (86) reported that

sympatol does not cause cardiac irregularities, or arrhythymia as re¬

ported by Oberdisse (87), which are observed in the.case of adrenaline injec¬tions. Ergotamine diminished or abolished but did not reverse the pres¬sor action of sympatol (84), (85), (88), but it reverses the vasoconstrictor

action in perfused vessels as reported by Schretzenmayr (89), Barkan et al.

(90), and Behrens and Taeger (91). Tainter and Seidenfeld (88) showed that

the cause of the pressor action ofsympatol was due to the direct stimulationof the vascular muscles as indicated by the results of analyses in experi¬ments on ergotaminised and cocanised cats. Pressor action is unaffected

(88) or possibly slightly increased by cocaine (85), though Gurd (92)reported that pressor action is reduced by cocaine.

The median pressor dose of 1-sympatol is 0.5 mg/k.g. intravenouslyas reported by Tainter and Seidenfeld (88).. On comparing the pressor acti¬

vity of optical isomers they found that racemic form was \ and d-isomer

only 1//60 as active as the 1-sympatol.

5.1. Potency ratio of sympatol to adrenaline

There is a wide variation in the results of various workers for the

potency of sympatol when compared to adrenaline. The following figureswill illustrate the results which have been obtained for 1-sympatol andadrenaline by different workers.

Table 10

Poteney ratio of 1-sympatol to adrenaline

Activity

Pressor Constrictor Intestines Uterus

1/25 — —

1/25 — —

1/25 1/100 —

1/58 — —

1/60 to 1/100 1/100

1/375 1/700 —

Reference

tcrus

Ehrismann (82)

— Lasch (83)

— Ehrismann and

Maloff(84)

— Tainter and

Seidenfeld(88)

1/70 to

1/100Kuschinsky (85)

— Gurd (92)

5.2. Toxicity

Kuschinsky (85) reported that the toxicity of 1-sympatol comparedwith 1-adrenaline is 150 times less as against the findings of Ehrismann (82)who reported it to be 200 to 400 times less toxic.

6. Clinical uses

Sympatol is used as a vasoconstrictor in mild collapse due to failing

perepheral blood flow and in conjugation with procaine in local anesthesia.

36

It is used to shrink the mucous membrance in hay fever and in asthma

(64), (93). It is superior to ephedrine for topical application in the nose

as reported by Stockton et al. (60). It has been reported to be useful

in chronic urticaria (94), in combination with quinine for the treatment

of arrhythymia perpetua (95), in cases of circulatory disorders in dip-theria (96), etc.

Besides the above mentioned uses syihpatol has many other clinical

applications which have been summarised by Boehringer and Sohn (97),

7. Dosage

Orally : 0.1 to 0.3 g daily.

Intravenously : 0.03 to 0.06 g.

CHAPTER V

TESTING OF THE MATERIALS AND APPARATUS

1. Syrnpatol

The sample of sympatol used gave the following results when tested

as decribed previously on page 32.

1.1. Physical tests

1.1.1. Melting range of sympatol : between 189°-I91°.

Melting range of the base : To 2.5 ml of the (1 +9 ) solution

of sympatol 1*5 ml of ammonia solution (10 %) was added. On

rubbing the sides of the test tube free base was precipitated. It was

washed with distilled water and dried at 105°. The melting range of the

base was between 176°-178°. Heating was so maintained that the rise

in temperature of the bath was 5° per minute.

1.1.2. Specific rotation : (test as described on page32) + 13'5° at20°.

1.2. Chemical tests

1.2.1. Identification tests : the sample of sympatol was tested for

identity tests numbers 3.2.1.1., 3.2.1.2. and 3.2.1.4. as described on page

32, to which it complied.

1.2.2, Parity test*

Sympatol complied with the following purity tests which have been

described on page 32.

1.2.2.1. Foreign organic matter : 0.01 g of sympatol were dissolved in

1 ml of sulphuric acid. The solution was practically colourless.

1.2.2.2. Acidity : pH of (1+9) solution of sympatol was 6.2.

1.2.2.3. Chloride and sulphate ions : the sample of sympatol did not

give any positive tests for chloride or sulphate ions.

1.2.2.4. Heavy metals : the sample of sympatol did not give anypositive tests for heavy metals.

(a) Copper : For testing the presence of Cu"*" "Hons a 5 mg %solution of dithizone (diphenylthiocarbazone) in carbon tetrachloride

was used. To 5 ml of the 6% solution of sympatol 0*1 ml of the above

solution of dithizone were added. The colour of the carbon tetrachloride

layer turned pink after shaking for 60 seconds, while on the additionof more than 0*5 ml of the dithizone reagent the colour of the carbon

tetrachloride layer remains unchanged showing that the amount of

Cu + 'present is less than 10 /igper 100 mlof the sympatol solution. (1 ml

of dithizone reagent =l'6/*g of Cu '"•").

38

(b) Nickel : the sample of sympatol did not give any positivetests for nickel.

1.2.2.5. Methylaminomethyl-{4'oxyphenyl)-ketone: the sample of

sympatol did not give any positive tests for methylaminomethyl-(4-oxy-phenyl)-ketone.

1.2.2.6. Residue on ignition : residue of the sample of sympatol on

ignition was within limits.

1,3, Quantitative estimation of sympatol

For quantitative determination of sympatol the following procedure,as given in Analysmetoder (98), was used.

1.3.1 Method : weigh accurately 50 to 80 mg of sympatol in a 200

ml glass stoppered flask. Add 30 ml of water, 20 ml of 0.1 N bromide-

bromate reagent and 10 ml of2N hydrochloric acid. Close the stopper and

keep it in the dark for 15 minutes. Add lgof potassium iodide and keepin the dark for five minutes. Titrate the liberated iodine with

0.1 N sodium thiosulphate solution, using mucilage of starch as indicator.

Each ml of 0*1N bromide-bromate solution = 0*006053 g of sympatol.

1.3.2. Results

The results of the quantitative estimation of sympatol are givenin table U.

Table 11

Table showing the volume of 0.1 N bromide-bromate solution

used for different amounts of the sample of sympatol.

Weight of Volume of 0.1 N bromide- Volume of 0.1 N Volume of 0.1 N

sympatol bromate solution added NaaSOj sol. used bromide-.

bromate sol.

0.0880 g 20 ml 5.55 ml 14.45 ml

0.0656 g 20 ml 9.2 ml 10.8 ml

blank 20 ml 20.0 ml 20.0 ml

14.45 .0.6053

(a) Sympatol content = = 99*4

0.0880

10.8.

0.6053

(b) Sympatol content = ' = 99-7

0.0656

Sympatol content as found by bromometric method = 99*5%.

In the following experiment the above sample ofsympatol was alwaysused.

39

2. Distilled water

Double distilled water was prepard as follows, and it complied with

the requirements of Ph. Int. I (62) for "water for injection". Fresh

distilled water was distilled from a neutral glass still (pyrex) which was

fitted with an efficient device for preventing entrainment. The first

portion of the distillate was rejected and the rest was collected in a neutral

galss container leaving about 1/10 of the original volume in the still.

+ +Distilled water was free from Cu ions when tested with dithizon reagentas described on page 37.

The above prepared distilled water was used immediately.

3. Ampules

The ampules complied with the following test (powdered glass test

of U.S.P. XV (15).

3 1. Method of U.S P. XV for powdered glass test

3.1.1. Apparatus:—

3.1.1.1. Autoclave—For these tests use an autoclave capable of

maintaining a temperature of 121 i 0.5°, equipped with the thermometer,a pressure gauge and a vent cock.

3.1.1.2. Mortar—Use a steel mortar made according to the

specifications of U.S.P. XV.

3.1.1.3. Other equipment—Also required are 8" sieves includingthe No. 20, No. 40. and No. 50 sieves, 250 ml Erlenmeycr flasks made of

resistant glass aged as specified, a 2-lb hammer, a permanent magnet,

a desiccator, tin foil, and adequate volumetric apparatus.

3.1.2. Reagents :

3.1.2.1. Special distilled water. The water to be used in these

tests is distilled water, re-distilled from an all-glass still constructed

of chemically resistant glass and equipped with ground glass joints. In

operating the still, add 1 drop of phosphoric acid to each liter of water

contained in the still. Reject the first 10 or 15% of the distillate and

retain the next 75#%.

3.1.2.2. Methyl red solution—Dissolve 200 mg. of methyl red in

60 ml of alcohol, then add sufficient purified water, to make 100 ml.

If necessary, neutralise the solution with 0.02 N sodium hydroxide so

that the titration of 100 ml of special distilled water, containing 5 dropsof indicator, does not require more than 0.20 ml of 0.20 N sodium

hydroxide to effect the colour change of the indicator.

3.1.3. Powdered glass test.

Rinse thoroughly with purified water six or more containers selected

at random and dry them in a stream of clear dry air. Crush the

containers into fragments about 25 mm. in size, divide about 100 g of

the coarsely crushed glass into 3 apporximately equal portions, and

40

place one of the portions in the special raqrtar. With the pestle in

the place, crush the glass further by striking 3 or 4 blows with the

hammer. Nest the sieves and empty the mortar into the No. 20 sieve.

Repeat the operation on each of the two remaining portions of glass,emptying the mortar each time into the No. 20 sieve. Shake the

assembled nest of sieves for 5 minutes, preferably with a mechanical

shaker and return the glass remaining on the No. 20 and No. 40 sieves

to the mortar and repeat twice the crushing and sieving operations.Discard the contents of the receiving pan, reassemble the sieves, and

continue the shaking for 5 minutes. Transfer the portion retained on

the No. 50 sieve, which should weigh in excess of" 10 g, to a closed

container and store in a desiccator until used for the test.

Spread the sample on a piece of glazed paper and pass a magnetthrough it to remove particles of iron that may be introduced duringcrushing. Transfer the sample to a 250 ml Erlenmeyer flask of

resistant glass and wash it with six successive, 30 ml portions of acetone,

swirling each time for about 50 seconds, and carefully decanting the

acetone. After washing, the sample should be free from agglomerationsof glass powder, and the surface of the grains should be practically free

from adhering fine particles. Dry the flask and the contents for 20

minutes at 140°, transfer the grains to a weighing bottle and cool in

a desiccator. Use the test sample within 48 hours after drying.

3.1.4. Procedure

Transfer exactly 10 g of the prepared sample to a 250 ml

Erlenmeyer flask that has been digested (aged previously) with specialdistilled water for at least 24 hours in a bath at 90°or for 1 hour at 121°.

Add exactly 50 ml of special distilled water. Prepare a blank by adding50 ml of special distilled water to a similarly prepared flask and cap

all flasks with crimped new tin foil that has been rinced twice with

acetone. Place the containers on the rack in the autoclave and close

it securely, leaving the vent cock open. Heat until steam issues

vigourously from the vent cock and continue heating for 10 minutes.

Close the vent cock and adjust the temperature so that it rises 1° per

minute until it reaches 121°, taking 19 to 21 minutes to reach the

desired temperature. Hold the temperature at 121±0.5° for 30 minutes,

counting from the time when this temperature is reached. Then

release the steam and cool at the rate of 0.5° per minute, ventingto prevent the formation of a vacuum and allowing 38 to 46 minutes

to drop to atmospheric pressure. Cool the flask at once in runningwater, add 5 drops of methyl red solution, and titrate immediatelywith 0.02 N sulphuric acid. If the volume of the titrating solutions

is expected to be less than 10 ml; use^a microburet. Record the

volume of 0.02 N sulphuric acid used to neutralize the extract from

10 g of the prepared sample of glass, corrected for a blank on the flask.

Powdered glass test requires no more than 1.0 ml. of 0.02 N acid

per 10 g of glass.

3.1.5. Results

Two sets of observations were taken for each type of ampule. The

results are tabulated in table 12.

41

Table 12

Table showing the amount of 0'02 N acid used for glass.

Size of ampule Weight of the Initial reading Final reading

powder ml ml

Amount of 0.02N

H2S04 used ml

Blank — 0.0

Blank — 0.5

5 ml 10.300 g 0.0

5 ml 10.310 g 0.0

25 ml 12.486 g 0.0

25 ml 10.21 g 0.0

0.05

0.55

0.35

0.35

0.60

0.47

0.05

0.05

0.35

0.35

0.60

0.47

Amount of acid used for :

(0.35(a) 5 ml ampules

(0.35(b) 5 ml ampules

(0.60

— 0.05) .10

10.30— 0.05) .

10

10.31

— 0.05) .10

= 0.29 ml

= 0.29 ml

= 0.44 ml

= 0.40 ml(0.47

(d) 25 ml ampules

12.486— 0.05) .

10

10.21

Both typs of ampules passed the powdered glass test as the amount

of acid used was well within the prescribed limits.

It is to be noted that we have made the above tests with sieves

No. Ill, No. IV and No. IV a (Ph. Helv. V) with a mesh diameter of

1.5 mm, 0.47 mm. and 0.32 mm. respectively instead of sieves No. 20,

No. 40 and No. 50. The sieves correspond very closely to the sieves of

U.S.P. XV. Further the mortar and pestle were also not of the same

dimensions as described in U.S.P.XV.

4. Other materials

All the other materials used in the following experiments were of

A. R. standard.

5. Apparatus

5.1. UV-Spectrophotometer

For spectrophotometric measurements the Zeiss Spectrophotometermodel PMQ;II, with 1 cm quartz cuvettes to hold the solution and the

solvent bank, was used throughout our work.

CHAPTER VI

METHODS OF ANALYSIS FOR SYMPATOL

1. Quantitative methods

None of the methods which we have discussed on page 33 permitan estimation of the deteriorated product in a solution of sympatol. So

we have investigated the following methods which may be useful to estimate

the deteriorated product in a solution.

1 1. Colorimetric method

The use of Millon's reagent for the quantitative determination of

phenols and phenolic derivatives was first described by Folin and Ciocalteau

(99) Lugg (100) and Arnow (101). The formation of colour is based on the

reaction ofnitrous acid with phenols or phenolic derivatives in the pre¬

sence of mercury. The nitrous acid may react in a position orthotoOH

group. Mercury compunds accelerate the colour formation due to their

great reactivity towards organic compounds, However the actual mechani¬

sm of the reaction is not very clear.

Ellin and Kondritzer (102) have made use of the above reaction for

determination of the phenylephrine content by measuring the absorbancyat495 m/*. Wehave used theirmethodwith thefollowing modifications:

(a) In the original method the solutions were diluted immediatelyafter addition of sodium nitrite solution. However, we found that in order

to obtain the full development of the colour, the solutions should be diluted

15 minutes after the addition of sodium nitrite solution. t

1.1.1. Method

1.1 1.1. Extinction wavelength curve

Initial experiments showed that a maximum peak is obtained at

500 m/t corresponding to filter no. 10 of Pulfrich's photometer. The

readings are given in table 13 and the relationship between extinction and

wavelength is graphically shown in fig. 2.

Table 13

l

Relation between extinction and wavelength by using4mg% solution of sympatolfor development of the colour.

Filter no. Corresponding wavelength Extinction values 4 rng.%E

1 cm.

1 570 m/t 0-280

2 590 m/* 0-22

3 610 ml* 0.10

43

Filter no. Corresponding wavelength Extinction values 4 mg%E

1 cm.

4

5

6

7

8

9

10

666 m/*

720 m/»

750 m/A

430 m/»

450 m/i

470 m/*

500 m M

006

006

006

0-395

0-48

0-57

0-62

The above readings are mean af 3 different sets of observations

E1cm

0/

/I

0.2

^n,.

„ .

500 BOO'

700'

8fxmg

ABSORPTION-CURVE of SYMPAT0L

after color reaction with HgSO^

Fig. 2

1.1.1.2. Stability of the colour. The colour is very stable and there is

no change for at least 90 minutes after development of the colour.

1.1.1.3. Time of dilution.

It is very important that after the addition ofsodim nitrite solution,flasks are kept at room temperature for 15 minutes and then diluted. If the

solution is diluted immediately after the addition of sodium nitrite

44

solution the colour does not develop fully, as is clear from the followingreadings. :

Table 14

Effect of time of dilution, on extinction values

Concentration of Extinction values at 500 m/n

sympatol Immediately after dilution After 15 minutes dilution

After the addition of sodium nitrite solution

40 j".g / ml 0-57 0-63

20 f»g / ml'

0-295 0-32

1.1.2. Final method

To a 50 ml volumetric flask add sympatol solution representing about

1.5 mg of sympatol. Add 3 ml of mercuric sulphate solution (15% w/v).Heat on a boiling water bath for 10 minutes ; cool the flasks to room

temperature ; add 3 ml of freshly prepared solution of sodium nitrite

(0.1 %). Keep the flasks at room temperature for 15 minutes then make

the volume to 50 ml by the addition of" distilled water; mix. Then read

the extinction at 500 imi in the Pulfrich's photometer using 1 cm cuvettes

against a blank prepared in the same way as the coloured solution but

without sympatol.

1.1.2.1. Reagents used

1.1.2.1.1. Standard solution of sympatol. Sympatol which

complied with the tests described on page 38 was used to prepare the

standard solution. A 1 mg per ml solution in water was prepared.

1.1.2.1.2. Mercuric sulphate solution. A 15 % w/v solution of

HgS04 in 5 N H2 S04.

1.1.2.1.3. Sodium nitrite solution. 0.1% solution of sodium

nitrite in cold water (freshly prepared).

1.1.2.2 Apparatus

Pulfrichs photometer with 1 cm cuvettes was used.

1.1.2.3. Standard curve of sympatol at 500 m/(,

Sympatol solutions representing 400, 700, 1000, 1500 and 2000 ^gof sympatol were accurately measured in 50 nil volumetric flasks. Then

the colour was developed as described above. At least 3 sets ofobser¬

vations were taken for each solution. The results are given in table 15.

The maxium deviation of readings was^ 1.5 to 2%.

45

Table 15

Extinction values at 500 mji, for different cone, of sympatol

Concentration of

sympatolMean extinction values of 3 different sets of

readings, at 500 m/l.

40 fg / ml

30 /*g / ml

20 /*g / ml

14 fig / ml

8 fig / ml

0-625

0-470

0-320

0-220

0-125

A graph showing the relationship between the extinction and theconcentration of sympatol was drawn (See fig. 3).

06

0.4 -

0.2 -

10» .

sympatol

20. .

content

30 40vg/ml.

STANDARD CURVE of SYMPATOL at 500 mji.

Fig. 3

The relationship between the extinction and the concentration of

sympatol is linear as is clear from the above Fig. Thus it is possible to

use the method for quantitative estimation of sympatol.

1.1.3. Application of colori metric method for estimation ofdestroyedsympatol in solution

For this investigation a 6% w/v solution of sympatol in water

was heated at 120° for 10 hours. After heating the solution turned yellowwith an orange precipitate, showing that some of the sympatol hadd eteriorated.

46

The precipitate was filtered and the extinction readings for the fil¬

tered solution were taken at 500 imi after developing the colour as des¬

cribed on page 44. The results are shown in table 16.

Table 16

Extinction readings of the freshly prepared and of the heated solution of sympatol.

(a) freshly prepared solution 3 mg%E

1 cm . = 0.470

(b) heated solution (120°-10 h.) 3 nig %E

1 cm . = 0.455

1.1.4. Results

The results show that the colour formed after development with

sodium nitrite in the presence of mercuric sulphate is vey stable and obeysLambert-Beer's law for the range of concentration of sympatol studied.

Thus it is possible to use the above method for the quantitative estimation

of sympatol.

However, a solution of sympatol which was heated at 120° for

10 hours, did not give any appreciable difference in the extinction values

(the maxium loss was found to be 3%), though the solution, after heating,turned yellow with an orange precipitate, showing that the product was

deteriorated. As the reults of the fresh solution and that of the deteri¬

orated solution practically remain the same, we conclude that the method

is not good to measure the amount of destroyed sympatol in solution.

1.2. U. V. Spectropliotomctric method

In the literature we did not find any ultraviolet absorption studies

of sympatol, though similar studies were available for phenylephrine bySchou and Rhodes (103) and Ellin and Kondritzer (102). Since sympatoldifferes from phenylephrine only in respect to the position of the hydroxylgroup in the benzene ring where it occupies a para position instead of

meta position, it was expected that sympatol might also give absorptionspectra which could be used as a basis for the estimation of oxidized and

unoxidized sympatol content in aqueous solutions.

1.2.1. Experimental

1.2.1.1. Apparatus

U.V. Spectrophotometer which has been described on page 41

was used.

1.2.1.2. Method

Standard aqueous solutions containing 1.2 mg% of sympatol were

prepared by diluting 0.01 ml of the 6% w/v solution of sympatol to

50 ml. The solution was measured by means of an Agla micro

pipette. Spectral extinction curves between the wavelengths of 200

and 290 tap. were drawn. The results are tabulated in table 20. Theyare the mean values ofat least 3 different sets of observations. The maxi¬

mum difference of readings wasi 1%.

47

Sympatol shows absorption maxim at 223 nut and at 273 mp and a

minimum at 247 rap.. The ratio ofthe absorption values at 223 m^ and at

273 m/* is 5-86 : 1. The graphical relatloship between the extinction and

wavelength is shown in fig. 5.

. 1.2.1.3. Standard curve of sympatol at 223 m/x

: Solutions representing 2.4, 4.8, 7.2, 9.6, 12.0, 14.4, 16.8, 19.2,

21.6 and 24.0/tg/ml of sympatol, were prepared by accurately measuringdifferent volumes of a 6% w/v solution of sympatol by means of an Agla

pipette and then diluted to 50 ml. The extinction readings were taken at

223 m/i by using 1 cm cuvettes to hold the solution and the solvent blank.

The results are given in table 17.

Table 17

Relationship between the extinction values and the sympato 1 content at 223mfi

Sympatol content p.g / ml 'Extinction values at 223 m/i using 1 cm cuvettes

2-4 0.091

,4-8 0.178

7-2 0.2725

9-6 0.3625

120 0.4450

14-4.

0.538

16-8 0.608

19-2 0.750

21-6 0-805

240 0.890

* The above results are the mean values of 3 different sets of observationsFor ench 3 readings were taken.

48

A graph showing the relationship between the extinction values andthe sympatol content at 223 mM is shown in fig. 4, which is a straight line.

*/

0.8_

o.s-/•

y

0A-

0.2-

-

s 0 5 20 l»a/ml.

STANDARD CURVE of SYMPATOL at 223 m>i.

Fig. 4

1.2.1.4. Effect of pH on the extinction wavelength curve of a 6

w/v solution of sympatol

For this study a 6% w/v solution of sympatol of pH values 3, 4, 5,6 and 7 was prepared. The extinction wavelength curve between the

wavelengths of 200 and 290 rmt was drawn, using a concentration of 1.2

mg% of sympatol, as described on page 44.

Results

The results of the extinction values for solutions of different pH values

are given in table 18. They are the mean values of at least 3 different

sets of observations. From the following extinction readings at different

pH values it is clear that by varying the pH of the sympatol soultions from

3 to 7, the extinction wavelength curve remains unchanged.

49

Table 18

Effect ofpH on the extinction wavelength curve of sympatol.

Wave -length in m/i Extinction values for 1.2 mg% solution 1.2 mg%E

of sympatol with 1 cm cuvettes. 1 pm

pH 7.

pH 6 pH 5 pH 4 pH 3

200 0-67 0-67 0-665 0-67 0-665

205 0-27 0-265 0-27 0-27 0-26

210 0-285 0-28 0-235 0-28 0-285

215 0-34 0-3375 0-3375 0-335 0-335

220 0-425 0-425 0-425 0-42 0-415

221 0-44 0-4375 0-435 0-435 0-435

222 0-4425 0-445 0-445 0-44 0-445

223 0-445 0-445 0-445 0-4425 0-445

224 0-44 0-44I

0-44 0-435 0-435

225 0-43 0-425 0-425 0-425 0-42

230 0-26 0-255 0-255 0-25 0-245

235 0-072 0072 0074 0072 0.074

240 0-0195 0018 0-0195 0-018 .0018

245 0-016 0015 0016 0-015 0015

250 0-018 002 0-02 0-02 0018

255 0-028 0026 0-026 0024 0-026

260 0-038 0038 0040 0-038 0-038

265 0-054 0054 0-053 0053 0054

270 0068 0068 0068 0064 0-066

272 0072 0072 0072 0-072 0-072

273 0074 0074 0074 0073 0 074

274 0072 0-072 0072 0072 0072

275 007 007 007 0-07 0068

280 0-06 006 0058 0059 006

285 0-024 0-026 0026 0024 0024

290 0008 0-012 002 001 0-009

50

1.2 1.5. Effect of heating on absorption spectra andpH of sympatolsolution

A 6 % w/v solution of sympatol ofpH 6.2 was filled to 2/3 capacityin 5 ml ampules. The ampules were sealed and heated in an autoclave

at 120° for a period of 1 to 10 hours. The pH and extinction values at

223 mp were measured at different time intervals after filtering the

solution. The readings are given in table 19.

Table 19

Effect of heat sterilization on extinction values at 223 rn/t, and pH of a 6% w/vsolution of symxpatol.

Time of sterilization at Extinction values at 223 m/i 1.2 mg %E pH

120° with a cone, of 1.2 mg% l cm

Unsteril solution 0-445 6-2

30 min. 0-445 60

1 h. 0-445 5-7

2 h. , 0-4425 5-5

3 h. 0-44 5-4

4 h. 0-44 5-2

7 h. 0-1375 515

10 h. 0-435 50

Further the results of the extinction wavelength for a 6% w/v solution

ofsympatol which was heated at 120° for 10 hours are j-iven in table

20. The readings were taken after filtering the solution.

Table 20

Extinction wavelength curves of:

(1) freshly prepared solution of sympatol.

(2) heated solution (120°-10 h.). The ppt. was filtered off.

(3) The product which formed on heating a 6% w/v solution of

sympatol. The product was recrystallized and then dissolved in 20%

alcohol.

51

Concentration of each solution = 1.2 mg%

Extinction values 1.2 mg%E

Wavelength in mf* 1 cm

solution solution solution

(1) (2) (3)

200 0-700 0-627 0-720

205 0-390 0366 0-505

210 031 0295 0.315

215 035 0335 0-357

220 0-42 041 0-338

221 0-43 0-42 0-338

222 0-44 0-43 0-338

823 0-445 0-435 0338

224 0-44 0425,

0-338

225 0-43 0-42 0-338

230 0-285 0-27 0-298

240 0045 0037 0-159

245 0033 0029 0123

247 0025 0020

250 0034 0027 0114

260 005 0045 0121

270 007 007 0153

272 0075 0081 0-162

273 0076 00825 0-165

274 0075 0-082 0-165

275 0074 0080 0-174

280 0-065 0071 0192

290 0019 0-024 0-216

293 0-216

294 0-216

295 0015 0019 0-216

296 0-2145

300 0013 0-017 0-211

320 0180 •

340 0087

360 0-026

52

Results:

(a) After heating at 120° for 10 hours the pH of the 6 % w/vsolution of sympatol drops down from 6.2 to 5.0.

(b) The maximum loss of sympatol content found by measuringthe extinction at 223 mp, is about 2%.

(c) There is practically no change in the extinction wavelengthcurve of the freshly prepared solution and that of the heated solution (120°-10 h.) of sympatol.

1.2.1.6. Absorption studies of the product formed on heating the

sympatol solutions

On heating at high temperatures the sympatol solutions turn yellowwith an orange precipitate, showing the deterioration of the product.However the extinction wavelength curve of the heated solution (120°-10 h.) remains unchanged as is clear from fig. 5. So we have also studiedthe nature of the extinction wavelength curve of this orange product.

1.2.1.6,1. Experimental

20 ml of a 6% w/v solution of sympatol was heated at 120°for 10 hours. The solution was filtered. The orange precipitatewas washed with cold water and recrystallized from 45% alchohol

(3 times). The product was dissolved in 20 % alcohol so as to givea concentration of 1.2 mg % Extinction readings were taken betweenthe wavelengths of 200 to 360 m^ as described on page 46. The results

are given in table 20. A graph showing the relation between the extinctionand the wavelength is given in fig. 5.

Results

(a) Melting range of the product = 202°—204°.

(b) Extinction wavelength curve. The curve of this product is

completely different from that of the sympatol. There is no maximumeither at 223 tap, or at 273 mp, instead another maximum at 290-295

ntyi is obtained. However, by referring to fig. 5, it is seen that the extinc¬tion value of this product at 223 rap. is about 3/4 and at 273 mp. about 2times that of the pure sympatol.

Thus it is not possible to estimate the amount of destroyed sympatolby measuring the extinction at the two. maxima i.e. either at 223 mpor at 273 imw

53

1.SYMPATOL-fr«sh solution , ertaxat x.223&273mu.2.SYMPATOLSOLUTIONJhtatedat12u° lOh.E^,, at » = 223»273mu

3.THE PRODUCT formed on heating.Solution made m20°oalcohot

£„,„ at X. 290-295 mp.

Fig. 5

1.2 1.7. Summary of the results ofU. V. Spectrophotomc'ric method

1 2.1 7.1. ^Extinction wavelength curve

Sympatol gives absorption maxima at 223 m/* and 273 mju. The

ratio of the absorption values at 223 m/iand273m/tis5,86: 1. Further

the extinction obeys Lambert Beer's law within the concentration

range of sympatol studied. So the method can be used to assay the

sample of sympatol.

1.2 1 7.2 Effect of pH oa extinction wavelength curve

There is no effect by varying the pH of the sympatol solution from

3 to 7 on the extinction wavelength curve.

1.2.1.7.3. Effect ofheating on absorption curve and pH

(a) after 10 hours heating at 120° sympatol solutions turned yellowwith an orange precipitate and the pH dropped from 6.2 to 5.0.

(b) The extinction wavelength curve of the heated solution (120°-10 h.) remains practically unaffected.

(c) The maximum loss by measuring the extinction at 223 mjuof a heated solution of sympatol (120°-10 h.) was 2.3%.

54

1.2.1.7.4. Absorption curve of the product formed on beating

The product does not show any maximum either at 223 nty or at

273 m/t further the melting range of the product is different from that of

the pure sympatol, showing that it is some different product. However it

gives another maximum at 290-295 rmi.

So we conclude that this method can not be used for quantitativeestimation of the deteriorated product in solution.

13. Quantitative chromatographicmethod

For quantitative estimation of substances by paper chromatography,the spot area of an unknown substance must be compared with spotsobtained from the same volume of a known concentration of the same

substance developed on the same sheet of paper. Fisher and Holmes (104),Fisher et al. (105) showed that the spot area of round and ovoid spotsincreases proportionaly with the logarithm of the amount of substance

forming the spot.

For evaluation of the spots one of the following techniques can be

employed :

(a) Planimetric measurement. Fisher et al. (105) obtained an

accuracy of db 2 % by this method.

(b) Counting of squares on a graph paper. The spot area can be

traced out with a sharp pencil and copied on a graph paper and then the

number of squares counted. Reid and Lederer (106) obtained an accuracy

of i 5 % by this method.

(c) By elution of the substance. In this method the piece of filter

paper containing the substance is pinched between two pieces of glass res¬

ting in a petric dish. The liquid rises between the glass plates due to

capillary attraction and after eluting the substance from the filter paper

drops in a beaker. Then it is analysed by a suitable method.

We have used methods (a) and (b) in our studies due to their

relative simplicity and very small percentage of errors.

1.31. Experimental1.3.1.1. Apparatus1.3.1.1.1. A chromatographic chamber of 30x25x50 cm (L.

B. H.) was used.

1.3.1.1.2. Paper. Whatman No. 1 paper cut into 46x20 cm

strips was used.

1.3.1.1.3. Planimeter made by Amsler & Co., Schaffhausesn.

1.3.1.2. Reagents1.3.1.2.1. Solvent system. The following solvent system described

by Wickstrom and Salveson (107) has been used :

Ethyl acetate : acetic acid (95%) : water: : 3:1:3.

The solvents were shaken and kept over night before use.

1.3.1.2.2. Developing reagent. As developing reagent diazotised

p-nitro-aniline was used. 0.25 g of p-nitroaniline is dissolved by gentleheating in 25 ml ofN HCl and diluted with ethanol to 50 ml. 0.01 g ofsodium nitrite is added to every 10 ml of the above solution before

spraying (cooling under running water).

55

Sprayed chromatograms were allowed to dry for 3-5 minutes andthen passed through a 0.5 N ethanolic solution of sodium hydroxide. The

excess ofsodium hydroxide was removed with a clean filter paper.

1.3.13. Tectnique

The same technique for descending chromatograms as described

by Consden et al. (108) has been used.

1.3.1.3.1. Application of eht substance. 0.01 ml of solutions of

sympatol ofdifferent concentrations were applied on the starting line .bymeans of an Agla pipette so as to give spots of 200, 150, 100, 50 and 25

p.g of sympatol content.

1.3.1.3.2. 'Measurement of the spot area.. The spots were outlined

by means of a sharp pencil. Then the area was measured by a planimeterand by counting the squares on the graph paper after tracing the spot area.

The results are given in table 21.

Table 21

Relationship between the sympatol content and the spot area

No. of chromato¬ 1 2 3 4 5 6 Averagegrams

Quantity of sympatol Area in square centimeters

200 MS a

b

9.0

9.07

8.8

8.78

8.4

7.88

8.0

7.99

9.1

9.31

8.0

8.01

8.53

150/*3 a

b

7.0

7.1

6.6

6.56

7.0

6.89 •

6.8

6.98

6.7

6.75

7.0

6.91

6.86

100>g a

b

5.6

5.65

5.2

5.24

6.1

6.05

5.9

5.91

5.7

5.71

5.5

5.51

5.67

50 /»3 a

b

3.7

3.84

4.0

3.96

4.2

4.13

4.0

4.14

3.8

3.91

4.2

4.23

4.18

25 f-5 a

b

3.1

3.14

3.0

2.89

2.9

2.93

2.3

2.41

2.5

2.7

2.7

2.68

2.77

a — Measurement on the chromatogram by planimeter.b — Measurement on graph by counting the number ofsquares.

1.3 2. Seperation of the oxidized product

A solution of sympatol was heated at 120° for 10 hours. The

precipitate was filtered and the solution was chromatographed with the

following solvent systems :

(a) Ethyl acetate : acetic acid 95%: water : : 3:1:3.

56

(b) N-butanol : acetic acid 95% : water :: 4 : 1 ; 5.

(c) N-butanol : toluene : water : acetic acid 95%: : 2:2:1:1:

However in no case two spots were obtained. Only one spot cor¬

responding to the pure product was obtained.

1.3.3. Results

1.3.3.1. On plotting the log of concentration against the spot

area, we got a linear relationship for spot contents of 25 to 150 /tg of sy¬

mpatol. However, when the sympatol content was more than 150 jugthere was no more a linear relationship, as would be clear from fig. 6.

_

/ •

/ / •

8-/ s

/ /

I •

/ •

/ •

/ /

w _

' I /f

iw

a y

V tf

6 //

//

S

-

in tf

Jd jf

I4"-

/2_

•

1.0 1'5X xu

i » i i

2.0 2,5log of tho cone.

RELATION BETWEEN THE LOG OF THE CONC

OF SYMPATOL & AREA.

Fig. 6

Thus the method can be used for quantitative estimation of sympatolcontent.

1.3.3.2. On chromatographing the heated • solution of sympatol120°-10h.), there was neither a difference in the spot area when comparedwith that of the freshly prepared solution nor did we get two different

spots, one for the pure product and another for the deteriorated

product.

Thus by this method too, it is not possible to estimate the amount

of deteriorated product in a solution.

57

1.4. Sommary of the analytical methods

1.4.1. Colorimetric method. From the foregoing a colorimetric

method for the estimation of sympatol may be stated as follows : To a 50

ml volumetric flask add sympatol solution representing 1.5 mg of sympatolfollowed by 3 ml of HgS04 solution (15%). Heat in a water bath for 10

minutes, cool the flasks to room tepmerature, then add 3 ml of NaNO,solution (0.1%). Keep the flasks for 15 minutes and then make the

volume to 50 ml, mix and read the extinction at 500 m^ in a sutiable pho¬

tometer.

The colour is stable for at least 60 minutes and obeys Lambert-Beer's

law within the range of cone, of sympatol studied.

1.4.2. Spectrophotometric method. Sympatol gives maxima at 223

and 273 m/x. The ratio of the absorption values at the two maxima is

5.86 : 1. Both maxima can be used to assay the sympatol solutions.

Change in pH from 3 to 7 does not have any eifect on the spectral

transmission curve.

On heating the sympatol solutions for a long time, an orange productis formed which gives absorption maximum at 290-295 nip.

1.4.3. Paper chromatographic method. A linear relationship between

spot area and the log of the cone, of sympatol between 25 and 150 pg is

obtained when the solutions are chromatographed on the same sheet of

paper by the descending technique. Thus the method may be used to

estimate the sympatol. content quantitatively.

However all the 3 methods failed to give any difference in the results

between fresh and deteriorated solution of sympatol. Since none of the

foregoing methods could be used to estimate the stability of sympatol solu¬

tions, we have based the results of the stability experiments of sympatol on

comparison with the standard colour solutions described in'the following

pages.

2. Colour standards for comparisonof the colour

Solutions.of sympatol become coloured after heat sterilization and

storage as mentioned before. The intensity of the colour increases with

time. The coloured solutions do not give any maximum absorption in

the visible range of light as would be clear from the extinction readings

given in table 22. So it was not possible to express the intensity of the

colour in terms of figures.Table 22

Relation between the extinction and wavelength for solutions ofsympatol (6% w/v)which were coloured after heating

Wave length in mf* Extinction values for 6 % w/v solution of sympatol, using1 on cuvettes.

Solution of pH 6 Solution of pH 5

375 2.00 1.90

400 1.05 0.95

58

Table 22

Wave lenghtmft Extinction values for 6 % w/vsolution of sympatol, using1 cm cuvettes.

Solution of pH6 Solution of pH 5

425 070 062

450 045 042

475 0-30 6-27

500 0-215 018

525.

0-15 013

550 0105 009

575 007 006

600 005 004

625 004 003

650 004 003

700 0025 0015

750 0015 00075

800 0015 00075

900 0005 00025

However in order to express the development of colour quantitativelyand to impose a certain standard limit for the colouration of the injectionsof sympatol, the colour of the sympatol solutions was compared with the

standard colours proposed by Buchi (109), in the following way.

For comparison of the colour 2.0 ml of the solution are taken in a

test tube with an inner diameter of 13 mm. The comparison is madewith a standard in diffused daylight against a white background and viewinghorizontally.

2.1. Stock coloured solutions

The following reagents are required to prepare the stock colouredsolutions.

2.1.1. Ferric chloride standard solution. Dissolve 55g of FeCl3.6HaO in a portion of a mixture of 25 ml of concentrated sulphuric acidRS and 975 ml of water and make the volume to 1000 ml with the abovemixture. Mix 10 ml of the above solution with 15 ml of water, 5ml of conesulphuric acid RS and 4g of potassium iodide in a glass stopperedErlenmeyer flask and keep in the dark for 15 minutes. Dilute with 100 mlof water and titrate the liberated iodine with 0"1 N sodium thiosulphatesolution using starch solution as indicator.

1 ml of 0.1 N NaaS2Os = 0.02703g FeCl3. 6H20.After the titration, dilute the solution with suphruic acid water

mixture so that each ml contains 0.0450g of FeCl8. 6H20.

59

2.1.2. Cobalt chloride standard solution. Dissolve 65g of CoCl^. 6

H40 in a portion of a mixture of 25 ml of cone, sulphuric acid RS and750 ml of water and make the volume to 1000 ml with the above

mixture. Mix 5 ml of this solution in a 250 ml Erle'nmeyer glass stopperedflask with 5 ml of hydrogen peroxide RS (30% w/v) and 10 ml of cone,

sodium hydroxide solution RS (10 N). Boil the mixture for 10 minutes,cool and then mix with 2 g of potassium iodide and 40 ml of dilute sul¬

phuric acid RS (10% w/v). As soon as the precipitate dissolves titrate the

liberated iodine with 0.1 N sodium thiosulphate solution.

1 ml of 0.1 N NaaS203 = 0.0238 g CoCl2.6 HaO.

After the titration dilute the solution with sulphuric acid water mix¬

ture so that each ml contains 0.0595 g of CoCl^.OHgO.

2.1.3. Copper sulphate standard solution. Dissolve 65 g of CuS04.5H20 in a portion of a mixture of 25 ml of cone, sulphuric acid and

750 ml of water and make the volume to 1000 ml with the above mixture.

Dilute 10 ml of this solution with 50 ml of water and add 12 ml ofdilute

acetic acid RS (30 % w/v) and 3 g of potassium iodide. Titrate the

liberated iodine with 0.1 N sodium-thiosulphate solution using starch as

indicator.

1 ml of 0.1 N Na2SaOa = 0.02497 g of CuS04.5H20.

After the titration dilute the solution with sulphuric acid water mix¬

ture so that each ml contains 0.0624 g of GuS04.5 H sO.

Then prepare the stock coloured solutions by mixing the followingquantities of standard solutions.

Table 23

Stock coloured solutions.

Stock coloured Standard solutions 1% HC1

FeCl,

ml

RS C0CI2 RS

mlCuS04

ml

RS ml

B Brown 30 3-0 2-4 1-6

BY Brownish-yellow 24 10 0-4 62

Y Yellow 24 06 00 70

R Red 06 1-2 00 82

2.2. Standard colour solutions

Standard colour solutions were prepared by mixing the following

quantities of stock solutions and 1 % hydrochloric acid.

60

2.2.1 Browrish-yellow solution BY

Solution ml of stock

solution BY

ml of 1 '/,i HC1

BY 1 20 00

BY 2 1-5 05

BY 3 10 1-0

BY 4 05 1-5

BY 5 025 1-75

BY 6 01 1-9

2.2.2. YcUow solution Y

solution ml of stock

solution Y

ml of 1 % HC1

Y 1 20 00

Y 2 1-5 05

Y 3 10 1-0

Y 4 05 1-5

Y 5 025 175

Y 6 01 1-9

2.2.3. Red solution R

Solution ml of stock

solution R

ml of 1% HC1

R 1 20 00

R 2 1-5 0-5

R 3 10 10

R 4 05 1-5

R 5 025 1-75

R 6 01 1-9

2.2.4. Brown solution B

Solution ml of stock

solution B

ml of 1% HC1

B 1

B 2

B 3

B 4

B 5

B 6

1-5

10

075

0-50

0-25

010

05

10

1-25

1-5

1-75

1-9

61

One more stock solution BY2 was prepared in the same way as

that of stock solution BY (p. 59), however, twice more concentrated

solutions of ferric chloride, cobalt chloride and copper sulphate were

used.

Stock solution BY2

FeCl3 solution (each ml contains 0.09 g of FeCl3. 6H20) .. 2*4 ml

CoCla solution (each ml contains 0.119 g ofCoCl2. 6Ha0) ..1-0 ml

CuS04 solution (each ml contains 0.1248 g of CuS04.5H20). 0-4 ml

Hydrochloric acid 2% .. .. .. ..6-2 ml

From this stock solution the following standard colour solutions

were prepared.

2.2 5. Standard colour solutions (Brownish-yellow solutionBV2)

Solution ml of stock ml of 2% HC1

solution BY2

BY2 1 2-0 00

BY2 2 15 0-5

BY2 3 10 10

BY2 4 05 1-5-

BY2 5 0-25 1-75

BY2 6 01 1-9

3. pH determination

All the pH determinations were done with Philips pH meter modelGM 4491, with a glass electrode.

Before making measurements the apparatus was always checkedby means of a standard buffer solution of pH 4-632.

CHAPTER VII

DETERMINATION OF STABILITY OF SYMPATOL SOLUTIONS

1. Principle

In order to examine the stability of autoclaved sympatol soslutionthe follwoing variables were used :

'

(a) storage time :

(b) storge temperature :

(c) pH values :

(d) antioxidants :

(e) gas

2. Preparation of the solution

Sympatol was accurately weighed and dissolved in freshly prepareddouble distilled water in a volumetric flask, so as to give a concentration

of6% w/v ofsympatol. The solution was divided into 5 parts and the pHwas adjusted to 3, 4, 5, 6-25 and 7 by the addition of the followingquantities of either tartaric acid or sodum bicarbonate :

Table 24

PH Substance added

3-0 3-06% of tartaric acid

4-0 0'565% of tartaric acid

5-0 0-072% of trartaric acid

6'25 none

7*0 0-l % of sodium bicarbonate

In the case of solutions of pH 3 and 4, there was a slight increase involume of the solution due to the addition of tartaric acid. The sympatolcontent was adjusted exactly to 6 % w/v by the addition of sympatoland the concentration rechecked by measuring the extinction at 223 me,

The solutions were then filtered through a sintered glass funnel (JenaerG 4).

Each of the solutions of different pH values was then divided into 4

equal parts. To one part 0.1 % ofsodium metabisulphite, to the 2 nd part0.2 % of rongalit and to the other two parts no antioxidant was added.

2.1. Filling

Filling was done with a graduated burette with a large glass capillaryso thai the volume in each ampule could be controlled. The solution was

20, 40, 80, 160 and 350 days.4°, 20° and 37°.

3, 4, 5, 6 and 7.

sodium metabisulphiterongalit.

nitrogenair

.

63

exposed to air throughout the filling operation. Each of the four partsof each soultion was filled into 5 ml ampules approximately to 2/3 of their

capacity (3.8 ml of the solution was filled in each ampule).

2.2. Sealing

Ampules containing the 1st part (namely 0-l% sodium metabisul-

phite) of each solution of different pH values were sealed in the presence of

air. Similarly, ampules containing the 2nd and 3rd parts (namely0.2 % rongalit and no antioxidant respectively) were sealed in the presenceof air. The 4th part of each solution containing no antioxidant was,

however, sealed in the presence of nitrogen. For this, the ampules were

placed under a bell-jar from which air was then removed by means of a

vacuum pump. Nitrogen was then allowed to enter the bell-jar and the

ampules were sealed immediately one by one.

2.3. Sterilization

The ampules were sterilized at 120° for 30 minutes in an Egroautoclave.

2 4. Storage

Sterilized ampules were stored at 4°, 20° (room temp.) and 37°

in cardboard boxes, protected from light, for a period of 20, 40, 80, 160

and 350 days.

In all, the following sympatol solutions were prepared :

Table 25

Sympatol solution (6 % w/v)

pH Antioxidant present Gas Lot number

None

Sodium metabisulphite

None

None

Rongalit

Sodium metabisulphiteNone

None

Rongalit

Sodium metabisulphiteNone

None

Rongalit

Sodium metabisulphite

None

None

Rongalit

Sodium metabisulphite

Air 1 A

0.1 % Air 2 SA

Air 3 A

Nitrogen 4 N2

0.2 % Air 5 RoA

0.1 % Air 6 SA

Air 7 A

Nitrogen 8 N2

0.2 % Air 9 RoA

0.1 % Air 10 SA

Air 11 A

Nitrogen, 12 Na

0.2 % Air 13 RoA

0.1 % Air 14 SA

Air 15 A

Nitrogen 16 Na

0.2% Air 17 RoA

0.1 % Air 18 SA

64

3. Testing of the ampules

Sterilized ampules were then analysed after different storage timingsfor development of colour, change in pH and sympatol content.

3.1. Colour test

For testing the development of colour the method as described on

page 58 was applied.3.1.1. Results

The results of the colour test for sterilized sympatol solutions which

were performed immediately after sterilization, and after storage for 20,40, 80, 160 and 350 days at 4°. 20° 37° are given in tables 26, 27 and 28

respectively.

Table 26

Effect of storage time on the COLOUR of the sterilized solution

of sympatol (6 % w/v) at 4°.

Solution Time in days after which the colour was compared

pH Lot number

160 350

DY3 BY2-BY3

BY2 BY2

BY2 BY1

BY4 BY4

BY2 BY2

BY2 BY1

Y3 Y3

Y4 Y4

CL CL

CL CL

BY4 BY3

Y5 Y5

Y6 Y6

CL CL

Y6 Y5

CL CL

CL CL

CL CL

20 40 80

1 A BY3 BY3 BY3 BY3

2 SA R4 BY4 BY3 BY3

3 A B3 B3-BY3 BY2 BY2

4Na R6 BY6 BY5 BY5

5 RoA BY3 Y3 Y3 BY3

6 SA B5 BY3-BY4 BY3 BY2

7 A BY6 BY5 Y4 Y3

8N2 CL BY6 BY6 Y6

9 RoA CL CL CL CL

10 SA CL CL CL CL

11 A BY5 BY5 BY4 BY4

12 Na CL CL CL Y6

13 RoA CL Y6 Y6 Y6

14 SA CL CL CL CL

15 A CL Y6 Y6 Y6

16 N2 CL CL CL CL

17 RoA CL CL CL CL

18 SA CL CL CL CL

65

Tabic 27

Effect of storage time on the COLOUR of the sterilized solutions ofSympatol (6%w/v) at 20°.

PH

Solution Time in days after which the colour was compared

lot number

20 40 80 160 350

7 1 A BY3 BY2 BY2 BY2 BY1 C

2 SA B3-B4 BY3 BY3 BY2 BY2 BY1

3 A 133 BY2 B1-BY1 BY1 BY1 C

6 4 N2 R6 BY5 BY4 BY3-BY4 BY3 BY3

5 RoA BY3 Y3 Y3 Y3 Y3 Y3

6 SA B5 BY2-BY3 BY1 BY2 — C

7 A BY6 BY3 BY1 BY3Z BY2 C

5 8 N2 CL BY5 Y5 Y4 Y4 BY3

9 RoA CL CL Y6 Y6 Y5 Y4

10 SA CL CL Y6 Y5 Y5 C

11 A BY5 BY4 BY3 BY3 BY2 BY1

4 12 N2 CL CL Y6 Y5 Y4 Y4

13 RoA CL Y6 Y6 Y6 Y6 Y6

14 SA CL CL CL CL CL CL

15 A CL Y6 Y5 Y5 Y4 Y4

16 N2 CL CL CL CL Y5 Y5

17 RoA CL Y6 Y6 Y5 Y5 Y4

18 SA CL CL CL CL CL CL

18 SA after storage for 24 months was colourless.

66

Table 28

Effect of storage time on the COLOUR of the sterilized solutions

of Sympatol (6% w/v) at 37°

pH Solution

lot number

Time: in days after which the colour was compared

0 20 40 80 160 350

7 1 A DY3 BY1 BY32 BY22 BY1» C

2 SA R4 BY1 BY1 .BY1 BY1 C

3 A B3 BY1 BY22 BY22 BY22 C

6 4 N2 R6 BY4 BY3 BY3 BY3 BY3

5 RoA BY3 — Y3 Y3 Y3 Y2

6 SA B5 B BY22 BY22 — C

7 A BY6 BY1 BY12 BY12 BY12 C

5 8 N2 CL Y4 Y4 Y4 BY3 BY3

9 RoA CL Y6 — Y6 Y4 Y3

10 SA CL CL BY4 Y4 Y4 C

11 A BY5 Yl BY1 BY1 BY1 C

4 12 N2 CL Y5 Y5 Y4 Y3 Y3

13 RoA CL Y6 Y6 Y5 Y5 Y5

14 SA CL CL CL CL BY3 BY3

15 A CL Y5 Y4 Y3 BY4 BY1

3 16 N2 CL CL Y5 Y5 Y4 Y4

17 RoA CL Y5 Y4 Y4 Y4 BY3

18 SA CL CL CL CL CL CL

18 SA aftet storage for 15 months, CL; after 18 months Y6

CL •

A \

N*

RoA

SA

= colourless for colour standards

= air BY1

«s nitrogen Yl

= rongalit 0'2%+air BY2 etc see 'page 60.

= sodium-metabisuphite 0'1 % +air.

= deeply coloured beyond range of standards used. ' 1

67

3.12. Summary of the results of colour determinations

3.1 2 1 Sterilization at 120° for 30 minutes

Heating at 120° for 30 minutes is sufficient to colour the sympatolsolutions of initial pH 6 and 7, irrespective of the antioxidant or gascontent. Solutions of initial pH 5 and 4 containing neither antioxidant

nor gas showed colouration on autoclaving, while those containing either

of the two remained colourless. Further all the solutions of pH 3

remained colourless on autoclaving.3.1.2 2. Storage at 4°

For storage periods between 20 and 350 days, time .factor in

general had no qualitative and indeed little quantitative influence

as a factor in the colouration. Without exception all solutions of

initial pH 7 and 6, and of pH 5, 4 and 3 not containing anyantioxidant, became more coloured. The colour variation were

between CL to Y5 or from B5 to BY1. Further solutions of pH 5

and 4 with nitrogen also developed a colour to the extent of Y4.

Solutions of pH5 with rongalit or sodium metabisulphite, solutions of

pH 4 with sodium metabisulphite and all the solutions of pH 3 with

antioxidant or nitrogen remained colourless even after 350 days.

3 1.2.3. Storage at 20° between 20 and 350 days.

On storage at 20° the development of colour is more prominent than

at 4°. All solutions of initial pH 7, 6 and 5 show colouration to the

extent of colour standard BY3 or brown. The Solution of pH 5 with

rongalit however shows colouration only to the extent of Y4. Solutions of

pH 4 and 3 with sodium metabisulphite remain fully colourless after 350

days while solutions of pH 3 containing nitrogen remain colourless till 80

days. The rest of the solutions of pH 4 and 3 develop colour to the ex¬

tent of Y4 to Y6.

3.1.2.4. Storage at 370 between 20 and 350 days

On storage at 37° the development of colour reacnes tne maximum.Solutions of initial pH 7, 6, 5, 4 and 3 become coloured to the extent ofY5 to brown, with the exception of the solution of pH 4 with sodium

metabisulphite which remained colourless up to 80 days and the solutionof pH 3 with sodium metabisulphite which remained colourless for theentire period.

3.1.2.5. Solutions of pH 3 with sodium metabisulphite remain

completely colourless at least for 24 months when stored at 4° or 20°,while at 37° a yellow colour to the extent of Y6 developed after storage for18 months.

32. Determination of pH

The pH value of the sterilized sympatol solutions was determined as

described on page. 61.

32.1. Results

The results of the pH determinations on the various solutions of

sympatol are given in table 29.

68

Table 29

Effect ofstor age time at 4°,20° and 37° on the pH of the sterilized solutions of

sympatol

pH of Sympatol solutions

before immedi- autoclaved solutions after storage of

ately

pH Solution Storage steril- after Auto-20 days 40 days 80 days 160 days 350 days

temp.in "C ization claving

1 A

2 SA

3 A

4 N2

6 RoA

6 SA

7 A

8 N3

4 7-2 71 69 69 69 69 6-9

20 7-2 7-1 68 68 67 67 6-6

37 72 7-1 6-8 6-7 67 65 66

4 71 69 62 6-2 62 6-2 6,2

20 7-1 6-9 6-2 6-2 62 62 62

37 7-1 6-9 61 6-2 62 62 60

4 62 5-9 5-8 '5-8 58 58 58

20 62 5-9 58 5-7 5-7 5-7 56

37 62 5-9 5-7 5-7 55 56 56

4 62 60 5-9 60 60 60 60

20 62 60 60 60 60 60 60

37 62 60 60 60 60 60 5-9

4 57 55 5-5 5-5 5-5 5-5 55

20 57 55 55 55 5-5 55 55

37 57 55 5-5 5-5 55 55 55

4 5-3 53 5-3 5-3 5-3 5-3 53

53 53 53 52 5-2 5-2 5-2

53 53 52 52 5-3 .5-2 5-1

4 50 50 49 4-9 50 50 50

20 50 50 49 4-9 49 50 4-9

37 50 50 49 4-9 4-9 49 4-9

4 50 50 49 49 50 50 50

20 50 50 49 4-9 50 50 50

37 50 50 50 50 50 50 5-0

69

Storageemp. in '

pH of Sympatol solutions

pH Solution

t

before

'C steril¬

ization

immediately/after auto- —

claving 20 days

autodaved solutions after storage of

40 days 80 days 160 days 350 days

5 4 49 4-9 48 48 49 4-9 49

9 RoAl 20 • 49 4-9 4-8 48 4-9 4-9 4-9

37 4-9 49 48 48 48 4-9 49

4 49 4-9 49 4-9 49 49 49

10 SA 20 4-9 4-9 4-9 4-9 4-9 4-9 4-9

37 4-9 49 49 4-9 49 4-9 4-9

11 A 4 4-0 40 40 40 40 40 40

20 40'

40 40 40 40 40 40

4 37 40 4-0 40 40 40 40 40

12 N2 4 40 40 40 40 40 40 40

20 40 4-0 40 40 40 40 40

37 40 40 40 40 40 40 40

4 40 40 40 40 40 4-0 40

13 Ro A 20 4-0 40 40 40 40 4-0 . . 4-0

37 40 40 4-0 40 40 40 40

4 40 40 40 40 40 40 4-0

4 14 SA 20 40 40 40 40 40 40 40

37 40 40 40 40 40 40 4-0

4 30 30 2-9 30 30 30 3-0

3 15 A 20 30 30 30 30 30 30 30

37 3-0 30 30 29 30 30 30

16 N2 4 30 3-0 29 3-0 30 30 3-0

20 30 30 2-9 30 30 30 3-0

37 30 30 2-9 30 30 30 3-0

70

pH of Sympatol solutions

before immediately autoclaved solution after storage of

pH Solution Storage '

temp, in °C steril- after auto

ization claving 20 days 40 days 80 days 160 days 350 days

4 30 3-0 30 30 30 30 3 0

1 RoA 20 30 30 30 30 30 30 30"

37 3-0 30 30 30 30 30 30

4 30 3-0 30 30 30 30 30

B SA 20 30 30 30 30 30 3-1 30

37 3 0 30 30 30 30 30 30

A = Air, N2 = Nitrogen

RoA = Rongalit 0.2 %-fair ,SA = Sodium metabisulphite 0.1%+air.

3.2.2. Summary of the results of pH determination of sympatolsolutions

3.2.2.1. Immediately after sterilization at 120° for 30 minutes

in an autoclave, the pH of sympatol solutions no. 1 to 5 slightly droppeddown towards the acidic side, while the pH of all other solutions remained

unchanged.

3.2.2.2. After storage at 4°, the pH of autoclaved sympatol solu¬

tions no. 1, 2 and 3 dropped down after 20 days (change of pH 0.1 to

0.7 pH units.) after which there was no change, while the pH of all of

the other solutions remained unchanged after 350 days.

3.2.2.3. After storage at 20° the pH of the autoclaved sympatolsolutions no. I, 2 and 3 tends towards the acidic side even after

350 days (change of pH 0.6 to 0.9 pH units) while the pH of all

other solutions remained unaffected.

3.2.2.4. After storage at 37°, the effect on the autoclaved sym¬

patol solutions no. 1, 2 and 3 is the same as at 20° (change of pH, 0.6

of 1.1 pH units). The pH of all other solutions remained unaffected.

3.2.2.5. There is practically no effect of heat sterilization or storagetime on the pH of those solutions with a pH value less than 6 after

storage at 4°, 20° and 37° for a period of 350 days.

4 Discussion and conclusions of the results of colour and pHdetermination

4.1. The development of colour is dependent on pH of the

solution and storage temperature.

71

4.2. Though nitrogen checks the oxidation of the phenolic OH

group, it is not sufficient to stabilize the sympatol solutions, as we found

that the sympatol solutions containing nitrogen become coloured after

one year.

4.3. Of the two antioxidants used, sodium metabisulphite was

more useful to check the development of colour than rongalit.

4.4. In order to obtain the optimum stability of the autoclaved

sympatol solutions the following conditions must be adhered to :

(a) — pH value should be as low as possible (pH 3) .

(b) — air must be repalced by nitrogen.

(c) — as antioxidant 0-1% of sodium metabisulphite should be

added.

(d) — storage temperature must be as low as possible.

On the grounds of the above findings, the following instructions

are proposed for the preparation and storage of the parental solutions

of sympatol.

5. Injection of sympatol

The following formula is recommended for the injection of sympatol:

Sympatol 6.0 g

Sodium metabisulphite 0.1 g

Acid tartaric 3.1 g

Aqua bidistillata to make 100 ml

Dissolve sodium metabisulphite and tartaric acid in freshly boiled andcooled double distilled water in an atmosphere of nitrogen. Then add

sympatol, filter and make up the required volume. Fill and seal the

ampules in an atmosphere of nitrogen.

Sterilization. Sterilize by autoclaving at 120° for 15 minutes.

pH. Between 2.8 to 3.2.

Storage. The ampules should be stored at low temperatures and

can be used till the development of colour is not more than the standard

colour Y6, which does not take place till after at least 18 months at

storage temperature of37° and 24 months or more at a storage temperature of 20°.

STABILITY STUDIES OF NORADRENALINE (NA) SOLUTIONSWITH RESPECT TO RACEMIZATION

CHAPTER VIII

INTRODUCTION AND PROBLEM

Introduction.

Recently NA has gained a great importance as a therapeuticagent, as would be clear from the fact that it has been recognized byseveral pharmacopoeias. The conditions for the stability of NA solu¬

tions were described by West (33). He recommended a pH of about

3.6 and the presence of 0*1% of sodium metabisulphite. March (110)has assayed solutions ofNA prepared according to Ph. Dan. IX. Add. 1954

(111). However none of the papers deal with the problem of-racemiz¬

ation of NA though similar studies for adrenaline were available byKisbye and Schou (40), Kisbye (41) and Rosenblum el al. (112).

As it is well known that the laevo form of NA is about 40 times

more active than the dextro form, it is important that solutions of NA

do not undergo racemization during sterilization and storage. No test

for the specific rotation of NA solutions is given by any of the pharmaco¬poeias, obviously due to the fact that it is not possible to determine the

rotation of such dilute solutions. Hellberg (113) has described a method

for concentrating the dilute solutions of adrenaline and NA and then

determining the rotation.

Problem. In the following study

(a) the conditions for maintenance of optimum optical activityof the dilute solutions of 1-NA during sterilization and storage have

been investigated.

(b) by carrying out the stability experiments at higher temperatureswe have determined the velocity constant of the racemization of NA

solutions and the temperature coefficient for a difference of 10° and then

have predicted the stability with respect to racemization of NA solutions

at storage temperature on the basis of these results.

CHAPTER IX

NORADRENALINE : SYNONYMS, SYNTHESIS, PHYSICAL PRO-

PERTIES, QUALITATIVE TESTS, METHODS OF QUANTITA¬TIVE ANALYSIS, PHARMACOLOGICAL ACTIONS, CLINICAL

USES AND DOSAGE.

1. Synonyms and proprietary names

Levarterenol bitartrate

Nor-adrenaline acid tartrate

Nor-adrenaline bitartrate

Nor-adrenaline bitartrate

1 -Norepinephrine

Levophed bitartrate

1-Arterenol

Aktanin

Noradrec

Noradrenaline

C,Hu08N.C4Ht0,.Hi0 . .

OH

HO—6}—CH—CH3—NH3AH

U. S. P. XV (114)Brit. Ph. 1958 (115)Ph. Int. I (116)Ph. Dan. IX. Add. 1954 (111)

Winthrop-StearnsHoechst

Schering (Germany)Recordati

Boehringer, Byk

Mol. Wt. 337. 3

0

II —

CHOH-C-O1

CHOH-C-OH

II

0J

Ha0

Chemical name. Noradrenaline bitartrate is (—)— o<—3:4—dihydroxyphenyl -/}- aminoethanol d—bitartrate monohydrate, or the monohydrateof (—)—2— amino —1—3' —4' dihydroxyphenyl ethanol acid tartrate.

2. SynthesisNA was first synthesised by Stolz (117) in the year 1904. According

to his method NA may be prepared by treating pyrocatechol with chloro-

acetylchloride and phosphorous oxychloride. The reaction product is then

treated with ammonia and, finally, moderate reduction of the ketone givesdl—NA.

OH OH

H0*0 + G1COCH»C1 -» CICH/XX)-^OH

POG1a > -\J

OH

HO-6 >COCHaCl

OH

NHg HO-^>-COCHaNH., > HO-^>—CH-CH3-NHaOH

74

Several other methods are also available for the synthesis of NA,for which the following literature is cited : (118), (119), (120), (121),(122), (123).

However the resolution of di—NA was reported only recently byTullar (124) and Tainter et al.' (125) in 1948. The separation was based

on the fact that only 1—NA forms a hydrated salt with d-tartaric acid.

This 1-noradrenaline d-bitartrate monohydrate possesses greater'solubilityin aqueous methanol and considerably lower water solubility than does

the nonhydrated d-noradrenaline d-bitartrate, permitting an easy separationof the diasteromers.

Noradrenaline acid tartrate is required to comply with the

following tests as given in Brit. Ph. 1958 (115)

3. Physical properties9

3. 1. Description

A white or nearly white, crystalline powder; odourless; tastes bitter;gradually darkens on exposure to air and light.

3. 2. Solubility

Freely soluble at 20° in 2.5 parts of water, slightly soluble

in ethanol 95%and practically insoluble in ether and chloroform.

3. 3. Specific rotation

_ 10° to — 12° U.S.P. XV

_ 9.4° to — 12°"

; Ph. Dan. IX. Add. 1954

— 9.8° to — 11.2° Ph. Int. I

3. 4. Melting range

102° Brit. Ph. 1958

100° to 106° U. S. P. XV, Ph. Dan.

IX. Add. 1954, Ph. Int.l

4. Qualitative tests

4. 1. Identification tests

»

4.1.1. Dissolve 10 mg of NA-acid tartrate in 1 ml of water, add

1 drop of ferric chloride solution (15% w/v), an intense green colour

is produced which changes to blue and then to red on the gradual addi¬

tion of sodium bicarbonate solution (5°/0 w/v).

4. 1. 2. Acidity

pH of a l°/0 w/y solution : 3.5 to 5.0.

4. 1. 3. Extinction

Extinction of a 1-cmla.yer of a0.005o/o w/v solution in N/100 HG1at 279 m^: about 0.40. i

75

4. 1. 4. Tartrates

Moisten a few milligramsNA—acid tartrate with a drop of water,add to 2 ml of sulphuric acid followed by a crystal of resorcinol : on

carefully warming a red violent colour is formed.

4. 2. Distinction from similar substances (adrenaline & isoprenaline)Mix 1 ml of 0.1 % NA-acid tartrate solution with 10 ml of a

standard solution of pH 3.6, add 1 ml of N/10 iodine, allow to stand

for 5 minutes, and add 2 ml ofN/10 Na2Sa03 solution : not more than

a very faint red colour is produced. Repeat the operation usingsolution of standard pH 6.6: a strong reddish violet colour is produced.

4. 3. Purity tests

4. 3. 1. Aminomethyl—(3,4—dihydroxyphenyl)—ketone, (Norad-renalone)

Prepare a 0.1% solution of NA—acid tartrate in 0-01 N HC1,measure the extinction at 310 m/i in 1 cm cuvettes : the extinction

must not be more than 0.2 (i. e. E,°

must not be more than 2).1 cm

4. 3. 2. Water

5.0 to 6.0 % w/w when determined by Karl Fischer method.

4. 3. 3. Loss on ignitionOn ignition NA-acid tartrate must not give a residue of more than

0.1 % w/w.

5. Methods of quantitative analysis described in the literature

For the quantitative estimation of NA many methods are available.

They may be divided into two categories.

5.1. Biological5.2. Chemical

5. 1. Biological methods

Biological methods are in some cases more specific and useful whenit is required to estimate the undecomposed product in a solution.However the great disadvantage of the biological methods is that they are

very time consuming, costly and there is always a great deviation inthe results. Consequently we have based our results on chemical methodsof analysis.

5. 2. Chemical methods

The chemical methods for the quantitative determination ofNA maybe classified into four categories.

5. 2. 1. Volumetric

5. 2. 2. Colorimetric

5. 2. 3. Fluorometrici

5. 2. 4. Spectrophotometry

76

5. 2. 1. Volumetric methods

5. 2. 1. 1. By determination of nitrogen content

The method is based on the determination of the nitrogen content

of the cation (+) portion of the molecule. The method is official for the

assay of NA-acid tartrate in U. S. P. XV (114).

5. 2. 1. 2. By titration in a non-aqueous medium

In this method the NA content is determined by titration in a non¬

aqueous medium like glacial acetic acid with perchloric acid. The

method is official for the assay of NA-acid tartrate in Brit. Ph. 1958 and in

Ph. Dan. IX. Add. 1954.

5. 2. 2. Colorimetric methods

Most of the colorimetric methods are based on the formation

of noradrenochrome or iodonoradrenochrome by such oxidizing agentsas iodine, permanganate, ferricyanide etc. The reaction may be for¬

mulated as follows :

HO—ff^, CHOH 0=(^% CHOH 0=^ CHOH

HO-ll^ | -» 0=kJ I -> 0=1^ |/CHs /CH2 \/CHaN N N

H2 Ha H

Further, some diazonium compounds have also been used for the

formation of a coloured product with NA. It is to be noted that

the results obtained by colorimetric methods do not differentiate between

the racemic and the I-form which latter is physiologically more active.

5. 2. 2. 1. Iodine method

Euler aud Hamberg (126) have worked out a method for the deter¬

mination of adrenaline and NA in a mixture. When a solution of NA

buffered at pH 6 is treated with iodine, the maximum formation of

noradrenochrome takes place after 3 minutes iodine treatment. The

colour is measured at 529 m/j, after neutralizing the excess of iodine

with sodium thiosulphate solution. Oesterling (127) showed that the

noradrenochrome is more stable if the concentration of the buffer is

N/10 rather than N/2. The above method has been adopted in Ph.

•Int. 1 (128) for the assay of the injection of NA-acid tartrate.

5. 2. 2. 2. Permanganate method

In this method Suzuki and Ozaki (129) used a solution of potassiumpermanganate in lactic acid at pH 6.5 and 3 minutes oxidizing time.

They showed that their method gives a uniform tint of colour in

comparison to the iodine method of Euler and Hamberg.5 2 2.3 Method of Folin, Cannon and Denis

The phosphomolybdic acid reagent (130) and the phenol reagent(99) which give colour reactions with polyphenols can be applied for theestimation of NA. But the colour formed is very unstable and further,metabisulphites interfere with the reaction as reported by Somer andWest (131).

77

5.2.2.4 Ferrocitrate method

This method is based on the colour reaction of the phenolic OH

groups by Fe* *ions. The intensity of the coloured product is measured

in a suitable photometer and the NA content is found by means of a

standard curve. Richter (132) has given the details of the method for the

estimation of adrenaline and NA. The method is used in U.S.P. IV

(133) for the estimation of adrenaline content.

5.2.25. Other coloritnerric methods

The method of Auerbach and Angell (134), where the colour is deve¬

loped by the action of NA with sodium-napthoquinone-4-sulfonate and

benzalammonium chloride, is very lengthy and too cumbersome.

Schuler and Hdnrich (135) have described a method in which theyused the napthalene sulfonates of 2 or 3 halogen-4-nitro-l-aminobenzeneas the reagent. The formation of the colour is assumed to be due to

transformation or decomposition of the diazonium salt caused by phenoliccompounds in an acid medium.

5 2.3. Fluorometric methods

Fluorometric methods are based on the formation of a fluorescent

product, when NA is oxidized in an alkaline medium. Lund (136),(137) has established the constitution of the fluorescent product formed

by the oxidation of adrenaline which is adrenolutine. It is assumed that

NA also follows the same course with the formation of noradrenolutina

sb follows :

HO—r**S CHOH 0=^ CHOH

:Cr J,HO—%J J -^O

-»HOl1

H H

keto Noradrenolutine enol

Most of the fluorometric methods have been designed to estimate

adrenaline and NA in mixture in body fluids ; however all the methods

can be profitably employed to estimate NA in pure solutions.

5.2.3.1. Method of Lund

The method is based on the formation of 3:5:6-trihydroxyindolewhich fluoresces in ultraviolet light (138). NA is oxidized to noradreno-chrome with manganese dioxide, and the noradrenolutine is formed bythe addition of a strong alkali. Ascorbic acid is added along with the

alkali so that the oxidation does not proceed further than the norad¬

renolutine stage. The NA content is determined by measuring the

fluorescence of the noradrenolutine and subsequent comparison with a

standard curve.

78

5.2.3.2. By condensation with ethylene diamine

The observation of Natelson el al. (139) that the catechol amines

form strongly fluorescent condensation products with ethylene-diaminein the presence of oxygen has been used by Weil-Malherbe and Bones

(140) for the estimation of adrenaline and NA. The samples of adrenaline

and NA are heated with ethylenediamine and the fluorescent productis quantitatively extracted with isobutanol. The fluorescence is then

measured in a suitable spectrophotometer. The reaction probably takes

the following course :

HO- /^—CHOH O = r^s CH.OH

HO—^ !| §:Q-GHa+02 > \/CH2

I N

NH, I

H

CHaNHa N

- + I r^r5^—CHOH

N \/CHaN

AThe method has the advantage that the fluorescent products are

very stable as compared with the method of Lund; however, the method

cannot be applied in the presence of metabisulphites.

Erne and Cant ack (141) have modified the original method of Weil-

Malherbe and Bones for the estimation of adrenaline and NA and have

further proposed a combination of ion-exchange and subsequentfluorimetry to remove metabisulphites.

5.2.3.3 Fluorescent method of Euler and Floding (142), (143)

In this method potassium ferricyanide is used as the oxidizingagent for NA at pH 6. After treating the oxidation product with strongalkali and stabilization of the fluorescent product with ascorbic acid, the

fluorescent intensities are measured.

5.2.4 Spectrophotometric method

NA gives an absorption maxima at 221 and 279 mp-. The absor-

1%bancy index, E at 279 mju for NA-acid tartrate monohydrate being

1 cm

80. The above property can be utilised to estimate the NA content in solu¬

tions. In U.S.P. XV (144) this spectrophotometric method is used to deter¬

mine the NA content only in the injections of NA-aicd tartrate, while in

Ph. Int. I (116) the method is official for the assay of NA-acid tartrate.

However, it is to be noted that for the etimation of NA in a solution,

part of which might have been oxidized, it is essential that the oxidized

product does not give any appreciable absorption at the wavelength at

which the absorption readings for NA are to be taken.

79

6. Pharmacological actions

Comparative pharmacology of NA was first described by Bargerand Dale (54). It is a more general systemic vasoconstrictor agent than

adrenaline. The pharmacological actions of NA have been discussed

in .a comprehensive review by' Euler (145), Lands (146) and Burn and

Hutch'e'on (147). We shall describe them below.

6.1. Action on heart and coronary vessels

The action of NA on the heart is in many respects similar to that of

stimulation of the sympathetic heart nerves.' When given intravenouslyNA causes bradycardi a and a reflex slowing of the heart. NA producesonly a slight and transient increase in ventricular rate, while adrenaline

shows a considerable rise in heart frequency.NA dilates the coronary arteries and its action is about two and one-

half times than that of adrenaline.

6.2. Systemic circulation

There is an almost parallel rise in systolic and diastolic blood pres¬sures after intravenous infusion of NA, with practically no increase in

cardiac output, showing general vasoconstriction where as, a similar dose

of adrenaline, though raising the ventricular pressure lowers the diastolic

pressure with little or no change in mean pressure. Since the cardiac

output is simultaneously increased the action implies vasodilation.

6.3. Muscle and skin circulation

Both adrenaline and NA cause constriction of the skin vessels thoughthe action of adrenaline is more pronounced. The results of different

authors show that whereas adrenaline causes dilation of the vessels of

the muscle, NA causes vasoconstriction.

6.4. Kidneys

NA has a constrictor action on the vessels of the kidneys, the effect,however, being smaller than that of adrenaline. The effect is attri¬

buted to the indirect action of NA on systemic circulation.

6.5. Smooth muscles

Usually NA has a weaker effect on smooth muscles than adrenaline,and is not as potent a bronchodilator as adrenaline.

6.6. Glandular secretion

In general NA increases the glandular secretion, for example of sali¬

vary glands. Though the liberation of adrenaline and NA inhibits gast¬ric secretion.

6.7. Relative potency of Isomers

Luduena et al. (148) found the 1-form of NA to be 12 to 60 times as

active as the d-form, irrespective of the fact whether inhibition or excitation

was caused. Swanson and Chen (149) found the 1-form to be 45 times

more active than the d-form when tested on the blood pressure response of

pithed cats and dogs.

6.8. Toxicity

NA is considerably less toxic than adrenaline. In the followingtable given by Euler (145) the comparative lethal doses of adrenaline and

NA is given as found by different workers.

80

Table 30.

Acute toxicity of intravenously injected adrenaline and noradrenaline in

mice after 24 hours.

Substance LD -. in group cages50

mg/kgLD

,- in single cages50

mg/kg

1—Adrenaline 2.4 ± 0.2 2.5 ± 0.2

1—Noradrenaline 6.1 =fc 2.0 20.5

d—Noradrenaline 70.7 ± 428 66.0

7. Clinical uses

Since the findings of Euler (150), (47), Holtz et al. (151) and others

show that NA is naturally present in the animal body, it has gained impor¬tance as a therapeutic agent.

NA is used as a general vasoconstrictor. Its usefulness as a pressor

drug was first reported by Goldenberg et al. (152). We shall describe below

its main uses taken from the review on NA by Euler (145) :—

7. 1. In acute hypotension due to loss of vasomotor tone and

also after central vasomotor depression of various origins.

7. 2. In shocks of various kinds like haemorrhage, post operativeblood pressure depression etc. It has been reported to be the best vaso¬

constrictor agent against the circulatory shock in acute myocardialinfarct.

7.3. To prolong the duration of spinal anesthesia NA is more

advantageous than adrenaline in conjugation with local anesthetics due

to its greater resistance to oxidation and the absence of tachycardia-shortcomings which are comnlonly associated with adrenaline.

Churchill Davidson (153) has in detail discussed the uses of NA.

8. Dosage

By intravenous infusion 5 to 25 micro grams per minute, accordingto the blood pressure of the patient.

CHAPTER X

TESTING OF THE MATERIAL

1. Noradrenaline hydrochloride

In our expriments we have made the racemization studies of dilutesolutions of NA. It was necessary to obtain maximum optical rotation withinthe limited concentrations of NA. So we have used 1-NA hydrochloridein our experiments because the specific rotation of 1-NA hydrochlorideis about 4 times that of 1-NA acid tartrate.

1-NA hydrochloride must confirm to all the tests as described for

NA acid tartrate on page 74, as all of these tests are based on the cation

(+) portion of the molecule, except tests of melting range, specific rotation

and tartrates which are for NA hydrochloride as follows :

Melting range. 145.2° to 146.4° Tullar (124)

Specific rotation. — 40°

Throughout our experiments we have used the sample of

(1-Noradrenaline hydrochloride (Hoechst) which complied with the tests

described below.

1.1. Physical tests

1.1.1. Melting range

The melting range of NA hydrochloride was between 144°—147°.

1.1.2. Specific rotation

A 2 % w/v solution of NA hydrochloride was prepared in water, and

the rotation was measured at 20°, using a 200 mm long polarimetertube. The specific rotation was found to be —40°.

1.2. Chemical tests

1.2.1. Identification tests

1.2.1.1. Test withferric chloride. NA hydrochloride gave a positivetest with ferric chloride solution as described on page 74.

1.2.1.2. Acidity. pH of a 1 % w/v solution of NA hydrochloride was

4.35.

1.2.1.3. Chloride. NA hydrochloride gave a positive test for

chloride ions.

1.2.2. Distinction from similar substances. (Adrenaline and isopre-naline) NA hydrochloride complied with the test as given on page. 75.

1.2.3. Purity tests

1.2.3.1. Test for aminomethyU'3, 4-dihydroxyphenyl)-ketonenor-adrenalone)). On measuring the extinction of a 5 mg % solutionof NA hydrochloride at 310 m^ using 1 cm cuvettes, the value of

1%E was 0.1., showing that the sample complied with the test described on1 cm.

page 75.

82

1.2.3.2- Test for copper ions. A 5 mg% solution of dithizone

(diphenylthiocarbazone) was prepared in carbon tetrachloride. 5 ml ofthe 1% solution of NA hydrochloride were shaken with 0.1 ml of

the dithizone reagent for 60 seconds. There was no changein the colour of the carbon tetrachloride layer showing the

absence of Cu "*"ions.

1.2.3.3. Loss on ignition. NA hydrochloride complies with thetest of loss on ignition as described on page 75.

1.3. Quantitative estimation of NA

The sample of NA hydrochloride was assayed according to the

Method of Ph. Int. I (116).

1.3.1. Method

A 5 mg % solution of NA hydrochloride was prepared in 0.01 N

HC1. Extinction readings at 279 m^ were taken in the Zeiss Spectropho¬tometer, using 1 cm cuvettes to hold the solution and the solvent blank.

1.3.2. Results

The results are given in table 31. below.

Table 31

Extinction values at 279 ir/l for a 5 mg % solution of NA Hcl. -

Extinction value NA hydrochloride content

0.640 calculated from the value

0.645 eJ c% =80 for CsHnOgN.C^Os-t^O

0.650 98-5 %

CHAPTER XI

DETERMINATION OF FRESH AND DETERIORATED NA IN

SOLUTIONS

1. Introduction

1.1. Oxidation of NA

Solutions of NA are liable to undergo spontaneous oxidation in

the presence of oxygen. The oxidation proceeds quicker in an alkalinemedium than in an acid medium. The oxidation is accelerated by the

presence of metallic ions, especially Cu"*" ions as reported by Schou

(34). In recent years the chemistry of the oxidation of NA has been

thoroughly investigated.

1.1.1. Noradrenochrome

On oxidation NA gives a red colour which has been attributed to theformation of noradrenochrome. Noradrenochrome has been identifiedby Beaudet (154). He prepared it by treatment of NA with silver oxide.Noradrenochrome gives absorption maxima in aqueous solutions at 490,310 and 205 m/*. The same results were obtained by Marquardt and Carl

(155). According to Green and Richter (156), when adrenaline is treatedwith iodine the first stage is the formation of adrenaline quinone, whichis then transformed into adrenochrome. It is assumed that NA followsthe same course. BulockandHarley-Mason (157) have discussed the mechanism-of oxidation of adrenaline and NA.

1.1.2. Iodonoradrenochrome

When NA is treated wit.li potassium iodate iodonoradrenochromeis formed. It was prepared by Barer et al. (158), and has been giventhe following structure :

0=,^ CHOH

\/CH.IN

H

However, we did not find any absorption study of iodonoradreno¬chrome in the literature.

All the colorimetric and fluorometric methods which we havedescribed on page 76, are based on the formation of a colour product byoxidation with iodine, permanganate etc., or on the coupling reaction with

a suitable diazo reagent, or on the formation ofa fluorescent product.

However by looking into the literature it was not clear if these methods

of analysis of NA could be used to determine the deteriorated NA in a so¬

lution. Therefore we have investigated the following methods to find

out if they can differentiate quantitatively between NA and the deteriora¬

ted NA in a solution.

84

2. Spectrophotometry method (UV.)3. Colorimetric methods

3.1. Iodine method

3.2. Persulphate method

3.3. Ferrocitrate method

2. UV spectrophotometric method

This method is official in Ph. Int. I. (116) to assay the sampleof NA-acid-tartrate while in U. S. P. XV (144) only for the injectionof NA-acid-tartrate. However, it is not clear if the method coulddifferentiate between NA and its oxidation products quantitatively Soin this investigation we have tried to use the method for the estimationof deteriorated NA in solution.

2.1. Method

A 5 mg % solution of NA base (1) was prepared in 0.01 N HC1,and that of NA hydrochloride (II) and NA-acid-tartrate monohydrate(III) in water. The extinction readings between the wavelengths of 210to 300 rmi were taken for all the 3 solutions (I, II, III) which are givenin table 33.

2.2. Results

NA gives absorption maxima at 220-221 m/t and at 279 m/*. The

ratio of the extinction values at the 2 maxima is 2.26:1. The graphicalrelationship between extinction and wavelength has been shown in fig. 7.

' In table 32 the extinction values at 279 m^ for the different salts

of NA, together with their corresponding molecular weights are given.

Table 32

Extinction values for NA base, NA hydrochloride and NA acid tartrate

monohydrate at 279 m/^along with their molecular weights

Data NA base NA hydro- NA acid tartrate

chloride monohydrate(I)

.

(II) (III)

Molecular weight 169-1 £05G 3373

Ratio of mol. weights 1:11 = 0.82 I:III a 050

Extinction values for a concen¬

tration of 65 mg .0-775 0-640 0-395

Ratio of extinction values II: I =s 0.82 III: I = 0'50

From the above table it is clear that the extinction value is dependentonly on the cation (+) portion of the molecule of NA.

85

Table 33

Relation between extinction and wavelength for

(I) Noradrenaline base (solution in 0.01 N HC1)

(II) Noradrenaline hydrochloride (aqueous solution)

(III) Noradrenaline acid tartrate monohydrate (aqueous solution)

(IV) A 2 % w/v solution of NA base in 0.01 N HGl, which was

heated at 210° for 2 hours in the presence of oxygen. The precipitatewas filtered after heating.

Concentration of each of the solution was 5 mg %,

Extinction values at a cone, of 5 mg% (4%*)I II III

\ f

IV

Wavelength NA base NA hydrochloride NAacid Heated solution

in mC tart

rate

of NA base

210 1-90 1-80 1-35 1-90

215 1-65 1-375 0-88

217 1-675 1-40 0-895

218 1-725 1-425 0-8975 1-725219 1-725 1-45 0-90 1-725

220 1-75 1-45 0-9025 1-75

221 1-75 1-45 0-9025 1-75

222 1-725 1-425 0-90 1-725

223 1-70 1-425 0-8875

225 1-60 1-38 0-88 1-65

230 1-40 115 0-72 1-40

235 0-83 0-69 0-42

240 0-31 0-26 016 0-36

245 012 008 005

250 009 005 0035 0105

255 0092 0078 0052

260 0175 015 0094 0-22

265 0-32 0-265 0165 0-34

270 0-51 0-4225 0-265 0-535

275 0-70 0-58 0-3575 0-705

276 0-73 0-60 0-375 0-7375

277 0-755 0-62 0-385

278 0-77 0-635 0-3925 0-7725

279 0-775 064 0-395 0-775

280 0-77 0-635 0-3925 0-77

281 0-765 0-63 0-3S0

285 0-65 0-5375 0-33 0-66

290 0-285 0-245 015 0-3125

295 0038 0-036 0024 016

300 000 0002 0002 0-035

310C-029

320 0-026

86

2.3- Effect of heating on the U.V. absorption carve of NA

For this investigation a 0.1 % solution of NA base in hydrochloricacid of pH 3 was filled in 5 ml ampules to half their capacity in the pre¬sence of oxygen. The ampules were heated at 120° for 2 hours in an

autoclave. The solution turned brown with a black precipitate after

heat treatment showing some destruction of NA. The solution was fil¬

tered and the extinction readings between the wavelengths of 210 to 320

tnn were taken by using a concentration of 5 mg%.

Thejreadings of the extinction values are given in table 33 and a

graph showing the relationship between extinction and wavelength is shownin fig. 7.

,.5mgtf 3 2

"1cm

0,8

ABSORPTION CURVES OF NORADRENALINE

0.6-

0,2.

300 mu

1.NA BASE,solutionin 0-01 NHCl .

2.NA Hydrochloride,aqueous solution.

3.NA Acid tartrate .aqueous solution.

4 NABASE.heated solution (120°-2 h. in presence of02)

Fig. 7

87

2.3.1. Discussion

The extinction wavelength curve of NA remains practicallyunchanged after heating the solution at 120° for 2 hours, except that

the extinction values between the wavelengths of 280-320 m/* are slightly

higher than that of the freshly prepared solution of NA, as is clear from

fig. 7.

So we conclude that the method can not be used to estimate the

deteriorated content of NA in a solution.

3. Colorimetric methods.

3.1. Iodine method.

For the iodine oxidation of NA the method of Euler and Hamberg(126), (159) has been applied. They presumed that the developmentof colour was due to the formation of adrenochrome from adrenaline and

noradrenochrome from NA. However, in Ehrlens (160) opinion,adrenaline on oxidation with iodine gives a mixture of adrenochromeand iodoadrenochrome. The formation of iodoadrenochrome is maximum

at lower pH values by the method of Euler and Hamberg. It is assumed

that NA follows the same course.

3.11. Method of oxidation

To 1 ml of the NA solution in a 20 ml volumetric flask 10 mlof the citrate buffer was added followed by 1 ml of iodine solution.

Exactly after 3 minutes the excess ofiodine ws neutralized by the additionof 1 ml of sodium thiosulphate solution. The volume was made to 20

ml by the addition of distilled water. Within a period of 5 minutes the

extinction readings were taken between the wavelengths of 230-600 m,using a concentration of2.5 mg % of NA base, in 1 cm cuvettes againsta blank which was prepared in the same way but without NA in

the Zeiss-spectrophotometer described on page 41.

3.1.2. Reagents3.1.2.1. Citrate buffer of pH 6 which was prepared as follows :

Na2HPCv 2H,0 0.2 M (35.6 g/litre) 63.1 ml

Citric acid 0.1 M (21.01 g/litre) 36.9 ml

3.1.2.2. 0.1 N iodine solution

3.1.2.3. 0.1 N sodium thiosulphate solution.

3.1.2.4. Solution of NA base. A 0.5 mg per ml solution in hydro¬chloric acid of pH 3

.

3.1.3. Results

The extinction readings between the wavelengths of 230-600 m/*

are given in table 34. The coloured product gave two maxima ; one inthe ultra violet range i. e. at 295-296 me and other in the visible rangei

. e. at 520-525 m/*. The extinction values at the two maxima were :

295.296 m, ^ mg%=1-12

520-525 m/xl cm

= 0.37

88

3.2. Persulphate method

The method for the persulphate oxidation is that of Barker et al.

(161), which has been used by Stock and Hinson (162) for the oxidation of

adrenaline and NA.

3.2.1. Method of oxidation

10 ml of the NA solution were mixed with 10 ml of the buffer

solution in a 20 ml volumetric flask, so that the final concentration

ofNA base in the solution was 2.5 mg %. The flasks were kept at room

temperature for 35 minutes and the extinction readings were taken between

the wavelengths of 230-600 mfi in 1 cm cuvettes against a blank which

was prepared in the same way but without NA in the Zeiss-spectrophoto¬meter described on page 41.

3.2.2. Reagents

3.2.2.1. Buffer solution. A buffer solution of pH 5.5 containingthe following quantities of the reagent was prepared.

Na^HP04.12H20 0.329%

NaHiP04.2 H20 0.937%

NaCl 1.0%

Potassium persulphate 0.2%

3.2.2.2. A 5 mg % solution of NA base in the minimum amount

of hydrochloric acid of pH 3.

35.2. Results

The extinction readings between the wavelengths of 230-600 m/* are

given in table 35. The coloured product gave 2 maxima. One at

288-290 m/j and another at 490-495 m/j. The extinction values at the

2 maxima were :

288-290 mM^JO1^ '

= °-98

490-495 mjul cm

= 0.34

3.3. Determination of deteriorated NA in solution by Iodine or

Persulphate method

For this investigation a 0.1% solution of NA base ofpH 3 was heated

at 120° for 2 hours in presence ofoxygen. After heating the solution turned

light brown with a black precipitate. The precipitate was filtered and

the solution oxidized with iodine and with persulphate respectively as

described before. Then the extinction was measured at different

maxima.

3.3.1. Results

The results of the extinction measurements are given in table 34.

-89

Table 34

Extinction values of NA solutions after iodine and persulpliate oxidation.

Wave lengthin m/*

Extinction values at a cone, of 2.5 mg% of NA base

Iodine method Persulphate method

NA solution NA solution

Frseh Deteriotrated % loss Fresh Deteriorated % loss

295-296 1.125 1.080 4.0 %

520-525 0.3675 0.350 4.8 %

288-290 0.9825 0.915 6.8 %

490495 0.3375 0.3185 5.6 %

The above readings are the mean of 3 sets of observations.

Table 35

Extinction reading) of NA after Jodine and after Persulphate oxidation.

Extinction values for a cone, of 2.5 mg% of NA

Wave length in

m/iIodine method Persulphate method

230 0.476 1.100

240 0.560 0.740

250 0.650 0.575

260 0.690 0.550

270 0.750 0.695

280 0.950 0.900

284 0.96

285

286

1.1000.97

288 0.9825

290 1..100 0.9825

295 1.125-

.

'

0.960

296 1.125

300 1.100 0.900...

310•

0.933 0.655'

320 . 0.710 ,0.3825

90

Extinction values for a conc.of2-5 mg % ofNA

Wave length in m/»

Iodine method Persulphate method

330 0.430 0.180

340 0.290 0,092

360 0.170 0.062

380 0.147 0.084

400 0.120 0.130

420 0.123 0.195

440 0.173 0-260

49Q 0.240 Q.313

460 • 0,302 >0.333

«90 0.3375

4950.3375

"&0 0.343 0.326

520 0.3675 0295

625 0.3675

530 0.360

540 0.3425 0.220

560 0.300 0.145

580 0.225 0.086

600 0.160 0.050

3.4. Discussion of the results of iodine and persulphate methods

3.4.1. Iodine method

The absorption intensity of the curve after iodine oxidation is greatlyincreased and the maximum is shifted from 279 m/t to 295 m,ti in the

ultra-violet region. Further, another maximum is obtained between

520-525 m/i in the visible spectrum. By looking into fig. 8 one finds that

91

the general characteristic of the absorption curve in the V. V. region re¬

mains practically unchanged. The differences are only quantitative.

ncrrt.

ABSORPTION-CURVES

OF OX i DIZED AND NONOXIDIZEO

I-NORADRENALINE

Fig. 8

1. Noradrenaline-Base, nonoxidized Emax. at X- 279 m/t.

2. Noradrenaline-Base, oxidized with iodine 3 min.

Emax. at A.=295—96 m^520—25 mjx

3. Noradrenaline—Base oxidized with persulphates 35 min.

Emax. at A=288—90 m^ & 490—95 m/*

3.4.2. Persulphate method

After persulphate oxidation the maximum in the ultra violet regionshifts from 279 to 288-290 ni/i, with a general increase in the absorption

92

intensity, as may be seen from fig. 8." Further, another maximum in

the visible region of the spectrum is obained between 490-495 mj*.

3.4.3. The maximum loss shown by the iodine method for a solution

of NA which was heated at 120° for 2 hours in the presence of oxygen

is 4.8 % while the persulphate method showed a loss of 6.8 %. If

we compare the results with those of West (33) who found that solutions

of NA in half filled ampules lose 25% of the activity after heat sterili¬

zation at 115° for 30 minutes when tested biologically, it would be clear

that the iodine or persulphate method does not give results which show

full deterioration of NA.

So neither of the methods can be used to estimate the deteriorated

NA content in solution.

3.5. Ferro-citrate method,

Rickter (132) described the details for the estimation of adrenaline

and NA by the well known reaction of phenols with Fe+ +ions. We

have investigated the possibility of using this method for the estimation of

deteriorated NA in solution.

3.5.1. Procedure

Pipette a solution representing about 1 mg of NA hydrochloride in a

20 ml volumetric flask. Add 15 ml of sodium bisulphite solution. Then

add 0.2 ml of ferro-citrate solution followed by 2 ml of the buffer solution.

Make the volume to 20 ml by the addition of sodium bisulphite solution.

After 25 minutes, when the development of the colour reached the maxi¬

mum, read the extinction at 350 mju, using 1 cm cuvettes against a blankwithout NA.

3.5.2. Reagents

3.5.2.1. Ferro-citrate solution. 1.5 g of ferrous sulphate (FeS04.7 H^O) were dissolved in 200 ml of water to which 1 g of sodium

bisulphite and 1 ml of N hydrochloric acid were added.

0.60 g of sodium citrate (Ph. Helv. V) were dissolved in the above

solution. This solution was freshly prepared each day.

3.5.2.2. Buffer solution. 42 g of sodium bicarbonate and 50 gof potassium bicarbonate were dissolved in about 180 ml of water. To

another 180 ml of water 37*5 g of amino-acetic acid and 17 ml ofammonia

solution (30% ) were added. The two solutions were mixed and the

volume was made up to 500 ml. '(pH 7.5 approximately) .

3.5.2.3. Sodium bisulphite solution. A 0.2% solution of NaHSOain water.

3.5.3. Standard curve

NA hydrochloride solution representing 400, 600, 800, 1200 and 1600

Ug of NA hydrochloride was taken in a 20 ml volumetric flask and the

colour was developed as described before and the extinction was measured

at 530 m^ in 1 cm cuvettes. The readings are given in table 36.

'93

Tabic 36

Relation between cone, ofNA hydrochloride and extinction at 530 m/*

Final cone, of

NA 4iydrochlorideExtinction at 530 m/t using1 cm cuvettes

20 M3/nil 01825

30 P£/ml 0.2725

40 /ig/ml 0 3650

60 Ms/ml

80 /ig/ml

0.5425

0.7275

(The above values are the mean of 3 different sets of readings).

The relationship between the concentration of NA hydrochlorideand extinction values is shown in fig. 9.

GS

0,0-

*

-

v7"O

y a*-

PX

m

<U

/

aa/

y3'/m\ *20 «° '' '«'

60

'STANDAiiD CURVE F0F! NORADRENALINE HCL

AT530mp

Fig 9.

3.5.4. Application of the ferro-citrate method for the estimationof deteriorated NA in solution

For this investigation a 2% w/v solution of NA hydrochloride ofdifferent pH values was prepared by the addition of either sodiumbicarbonate or IN hydrochloric acid. To half of the solutions of each

pH value, 0.1% of NatS208 was added. The solutions were filled in

5 ml ampules to half of their capacity and sealed in the presence of air.The ampule's were then heated at 120° for 10 hours.

94

All the solutions turned brown with a black precipitate. The

precipitate was filtered and the NA hydrochloride content was determined

by ferro-citrate method as described on page 92.

3.5.5. Results

The results are given in table 37. They are the mean of 3 different

sets of observations.

Table 37

The effect of heating at 120° for 10 hours on the NA hydrochloride content, of

solutions of NA hydrochloride of different pH values, with and without sodium

metabisulphite

Initial pH of the solution

without NajSaOj%loss Initial pH of the solution

with 0.1% NaaSa05% loss

3.1 2.8 3.4 0.5

4.3 6.5 4.2 2.8

5.5 7.1 5.1 3.3

The maximum loss shown by this method is 7 % for a 2 % w/vsolution of NA hydrochloride of pH 5.5, which seems to be much less than

the values of West (33) who obtained a loss of 30% after 6 hours heatingat 115°, the estimations being done biologically. The possible expla¬nation for the above high values of intact NA may be that the oxidized

product itself reacts with the ferro-citrate reagent and gives the colour.

So we conclude that this method is not good to estimate the oxidized

NA in solution.

4. Summary

4.1. U.V. Spectrophotometric method

The extinction values of NA are independent of the anion (—) por¬tion of the molecule. The extinction wavelength curve of a fresh solution

and that ofa heated solution (120°-2 hours, in presence of oxygen) remains

unchanged.

4.2. Iodine method

After iodine oxidation NA gives maxima at 295-296 m/t and at 520-

525 m/». The ratio of the extinction values at the two maxima is 3:1.

The maximum loss shown by this method for a solution of NA which

was heated at 120° for 2 hours in presence of oxygen is only 4.8 %.

4.3. Persulphate method

After persulphate oxidation NA gives maxima at 288-290 m^ and

at 490-495 m/*. The ratio of the extinction values at the two maxima

is 2.9:1.

95

The maximum loss shown by this method for a solution of NAwhich was heated at 120° for 2 hours in presence of oxygen is 6.8%.

4.4. Ferro-citrate method

NA gives maximum at 530 m/» and the colour obeys the Lambert-

Beer's law within the range of concentration of NA studied. The

maximum loss shown by this method for a solution ofNA which was hea¬

ted at 120° for 10 hours is 7%.

CHAPTER XII

RACEMIZATION STUDIES OF THE DILUTE SOLUTIONS OF

NORADRENALINE

In the following study we have determined the effect of storagetime at room temperature (18°-22°) on racemization of 0.2% solutions

of NA hydrochloride under the following variables :

(a) after 30, 60, 160 and 290 days.

(b) at pH values of 2, 3, 4 and 5.

(c) with and without 0.1% of sodium metabisulphite.

(d) in presence of nitrogen and air.

1. Method for concentrating the solution and for determining the

optical rotation

The following method described by Hellberg (113) for concentratingthe dilute solutions of adrenaline and NA has been used.

1.1. Column

About 0.9 g of the resion formed a column 3 X140 mm (0.99 Cm3)in chromatographic tube,

1.2. Absorption20 ml of the dilute solution corresponding to 40 mg of NA hydro¬

chloride was passed through the column with a velocity of 1 ml perminute. Then the column was washed with 20 ml of distilledjvater.

1.3. Elution of noradrenaline

For elution, 2N hydrochloric acid was used, which was

allowed to pass through the column with a velocity of 0.5 ml per minute.

The first 0.8 ml was discarded because this portion of the elute is very

poor in NA content and the follwoing 1.2 to 1*5 ml was collected.

1.4. Optical rotation of the elate

The optical rotation of the elute was measured immediately after

elution. At least 5 different readings were taken for each observation. The

results of the optical rotation measurements given in the following tables

are the mean values'of these readings.

1.5. Noradrenaline content of the elute

0.05 ml of the elute was diluted to 20 ml with distilled water, and

the extinction was read at 279 rmi, using 1 cm cuvettes in the spectropho¬tometer. Three sets of observations were taken for each solution. The

amount of NA content was then calculated by means of a standard,prepared from the same batch samples of NA hydrochloride.

The specific rotation was then calculated from the readings of the

optical rotation and the concentration of the elute.

1.6. Material

1.6.1. 1-noradrenaline hydrochloride confirming with the tests

as described on page 81 was used.

97

1.6.2. Double distilled water was prepared and tested as-describedon page 39.

1.6.3. 25 ml ampules which confirmed to the powdered glass test

of U.S.P. XV (15) were used. The results of the test are given on page40.

1.6.4. Amberlite IRG 50, a cation exchange resin with carboxylgroups as active centres, in its neutralized, ammonium saturated form has

a certain exchange capacity tpwards alkaloidal cations ; Winter and Knnin

(163).-The ion exchanger amberlite IRG 50, IV mesh Ph. Helv. V

(164) was transformed form its proton form to ammonium form bytreating with an excess of2 N ammonia. The resin was then washed with

/ distilled water and air dried.

1.7. Apparatus

1.7.1. A chromatographic column with an inner diameter of 3mm

and 140 mm length, with a capillary fitted with a stop cock was used for

concentrating the solution.

1.7.2. The optical rotation was measured with F. Schmid-Haensch-

Polarimeter, using 10 and 20 cm long polarimeter tubes with an inner

diameter of 2 mm and 1.5 mm respectively. A Zeiss sodium vapour lampwas employed as light source.

.

1.7.3. All the pH measurements were made with Methrom Poten¬

tiometer with a glass electrode.

1.7.4. For spectrophotometric measurements the apparatus des¬

cribed on page 41 was used.

1.8. Discussion of the method

1.8.1 The interfering anions as sulphate or tartrate are removed

during the exchange process. »

1.8.2. The maximum error of the method was 3 to 5 %.1.8.3. There is no risk of racemization during the elutiori step

as would be clear from the results given in table 38.

.1.8.4.. In our experiments we obtained with this method a maxi¬

mum concentration of 8 times of the original which was sufficient for

our work.

2. Preparation ofthe solution

NA hydrochloride was accurately weighed and dissolved in freshlyprepared distilled water containing 0.8 % of sodium chloride, so as t6

give a concentration of 2 in 1000. The solution was then divided intofour equal parts and the pH was adjusted to approximately 2,3,4 and 5

by the addition of either sodium bicarbonate or hydrochloric acid. Thento half of each of the above solutions 0.1% of sodium metabisulphite was

added.v

2.1. Filling• 25 ml portions of the above solutions were filled in 25 ml glass am¬

pules. Ampules containing the solutions with sodium metabisulphite

98

were sealed after replacing the air with nitrogen, while'the other ampuleswere sealed in presence of air.

2.2. Sterilization .,,-..

The above ampules were sterilized at 120° for 30 minutes.,

-

2.3. Storage , , „ .

'

The sterilized ampules were stored at room temperature (I8°-22°),protected from light, for a period of 30, 60, 160 and 290 days, after

which they were analysed for racemization according to the method des¬

cribed in previous page*. .

3. Results

Table 38

Effect of elation on racemization of NA hydrochloride solutions,

pH - Specific rotation of" '

2% w/v solution Same solution 10 times diluted and

the rotation measured after cone, by elution

2 0 40* 40°

30 40» 40 5'

4-2 40° 40°

51 40° 39-5°

The results of the optical rotation measurements of the sterilized

solutions of NA hydrochloride after keeping at room temperature for 4,30, 60, 160 and 290 days are given in the follwoing tables.

Table 39

Specific rotation- of the sterilized solutions of noradrenaline hydro¬chloride after keeping for 4 days at room temp. (18°—22°).

Solutions without sodium mctabisulphite in air filled ampules

Initial pH of the

solutionOpticalrotation

20 cm tube

Extinction at

279 mm in 1

cm cuvettes

NA hydrochlo¬ride content

Specificrotation

20 — 0-265° 0 35 1-125 % — 11-0"

30 — 0-915' 0-42 1-31 % — 34-9''

4-3 — 1-0C° 0-44 1-375 % — 39-3°

5-4 — l-06» 0-415 1-30 % — 40-8°

Solutions with 0;1 % of sodium metabisulphite inNa filled ampules

Initial pH of

the solutionOptical rotation

20 cm tube

Extinction

279 mp. in

at

1 eta

NA hydrochloridecontent

Specificrotation

cuvettes

2.1 — 0-41° 0-335 1-05 % — 19-5°

3.3 — 067° 0-265 0-83 % — 40-4"

4.1 — 0-97" 0-385 1-20 % — 40-4°

5.0' — 1-14° 0-447 1-40 %'

— 41-0"

Table 40

Specific rotation of the sterilized solutions of noradrenaline hydroch¬loride after keeping for 30 days at room temp. {l8°-22°).

Solutions without sodium metabisulphite in air filled ampules

Initial pH of Optica] .Extinction at NA hydrochloride Specificthe solution rotation 279 mp in 1 cm content rotation

20 cm tube cuvettes

2-1 — 0-225° 0-445 1-39 % — 81°

30 — 0-705° 0-37 1-17 % -

— 31-4°

4-3 — 1-06° 0-51 1-59 % — 33-3°

5-4 — 0-965° 0-428 1-34 % — 360°

Solutions with 0-1 % of sodium metabisulphite in N2 filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specificthe solution rotation 279 tnfi in 1 cm content rotation

20 cm tube cuvettes

JU —0-50° 0-4625 1-44% — 17-3°

3.3 —100° 0-4425 1-38 % — 36-2°

4.1 — 0-80° 0-345 1-08 % — 370°

5.0 ~ 0-93° 0-3825 1-19 % • — 390°

Table 41

Specific rotation of the sterilized solutions of noradrenaline hydroch¬loride after keeping for 60 days at room temperature (18°-22t>).

Solutions without sodium metebisulphite in airfilled ampules

Initial pH of

the solutionOpticalrotation

20 cm tube

Extinction at

279 m(t in 1 cm

cuvettes

NA hydrochloridecontent

Specificrotation

20

30

4-3

5-4

— 016°

— 0.43°

N.D,

— 0-77°

0-430

0-3175

N.D.

0-360

1-34-%

1-0 %

N.D.

1-12%

— 60°

— 21-5°

N.D.

— 34-2°

N.D. =Not determined.

100

Solutions with 0~1 % of sodium metabisulphite in N2 filled ampules^-

Initial pH of - Optical Extinction at NA hydrochloride Specificthe solution • rotation 279 m/t in 1 cm content rotation

20 cm tube cuvettes

2-1 — 0-37" 0-3575 1-12 % — 16-5°

33;. — 0-76°' 0-34 1-06 % — 35-8°

4-1 <,

— 0-87° 0-3725 1-16 % — 37-0*

50 — 0-89" 0-3725 M6 % — 33-2°

Table 42

Specific rotation of the sterilized solutions of noradrenaline hydro¬chloride after keeping for 160 days at room temperature. (18°-22°)

Solutions without sodium metabisulphite in air filled ampules

Initial pH of

the solutionOpticalrotation

20 cm tube

Extinction at

279 m/* in 1 cm

cuvettes

NA hydrochloridecontent

Specificrotation

'

20 — 0055° 0-52 1-62 % — 3-4°

3-0 — 0-43° • 0-485 1-51% — 28-5° -

4-3 — 0-39° 0-335 1-30 % — 300O.

5-4 — 0-42° 0-45 1-40 % — 300°

Solutions with 0-1% ofsodium metabisulphite in N2 filled ampules

Initial pH of

the solutionOpticalrotation

10 cm tube

Extinction at

279 m/x in 1 cm

cuvettes

NA hydrochloridecontent

Specificrotation

21 — 0-16" 0-4325 1-35 %' — 11'8°

3-3 - — 0-50", 0-52 1-62 %c — 30'8°

41 — 0-59° 0-515 1-61 % — 36-6°

, 5-0 — 0-57° - 0-51 1-59 % .— 35-8°

• '>,

Table 43 -

Specific rotation of the sterilized solutions of noradrenaline hydro¬chloride after keeping for 290 days at room temp. (18°-22°).

Solutions without sodium metabisulphite in air filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specificthe solution "rotation 279 m/i in 1 cm content rotation

10 cm tube cuvettes

20' 0-00 — 00 000

3-0 — 0-41° • 0-413 1-50 % — 27-3°I

4-3 — 0-46" 0-42 1-53 % — 30'0°

5-6 — 0-37° 0-345 1-26 %. , ..,

— 300°

101

Solutions with 01% of sodium metabisulphite in Na filled ampules

Initial pH of Optical Extinction at NA hydrochloride Specificthe solution rotation .. 279 in/* in 1 cm content rotation

10 cm tube -cuvettes

21 — 0-23° 0-40 1-45 % — 80".f

3-3 — 0-3?" 0-38 1-38 % — 280°

41 — 0-47° 0-385 •1-38 % — 340°

50 — 0-44" 0-42 1-53 % , _ 28-7°

Table 44

Specific rotation of the sterilized solutions of noradrenaline hydro¬chloride without sodium metabisulphite in air filled ampules.

Initial pH, of Specific rotation

the solution'

"

Time in days after sterilization at which the optical rotation was measured

4 30 60 160 290'

;'

I t

2.0 — 11-8° — 81° — 6-C — 3-4° - 0.0 -

3.0 — 349° — 31-4° — 21-5° — 28-5° — 27-3°

4.3 — 39-3° — 33-3"» - — 30-0" — 300°

5.4 — 40-8° — 36-0"" — 34£° — 300° — 300°

Specific rotation of the sterilized solution of noradrenaline hydro-ide containing 0.1 % of sodium metabisulphite in Nj filled ampules.

Initial pH of Specific rotationthe solution

Time in days after sterilization at which the optical rotation was measured

4 30 60<

160 2C0

21 —19-5° — 17-3° —16-5°' "

—11-8° — 80° -

3-3'

— 40-4°' — 36-2° — 35-8° — 30-8° —280°

4-1 — 40-4° -

— 37-0° — 37-3° —36-6° — 340"

. 6>0 _ 41-0- — 390° — 3-82° — 358 — 28-7"

102

Tabic 45

Table showing the relationship between storage time and the

percentage of noradrenaline hydrochloride racemized at different pH values, for

solutions of NA hydrochloride without j^faQS:i0B in air filled ampules.

Initial pH of Time in days

the solution 4 30 60 160 290

Percentage of noradrenaline hydrochloride racemized'-

2-1 70-5 80-0 850 .91-5 100

30 12-5 21-5 460 290 320

4-3 20 170 250 250

-5-4 00 10-0 14-5 25-0 . 250 • -

^

Table 46

Table showing the relationship between storage time and the Percentageofnoradrenaline hydrochloride racemized at different pH values, for solutions of

NA .hydrochloride with 0.1% of Na^O,, in N, filled ampules.

Initial pH of Time in days

the solution 4 30 60 160 290

Percentage of noradrenaline hydrochloride racemized

2-1 510 570 690 70-5 800

3-3 00 9-5 11-0 230 300

41 00 70 70 86 150

50 00 2.5 4-5 10-5 280

4. Summary of the results of racemization studies

4.1. Effect of pH on racemizationofNA solutions

4.1.1. The solution of pH 2 got more or less completely racemized

after about 10 months, irrespective of the fact whether they contained

sodium metabisulphite or not. While the solutions ofpH 3 showed about

30% racemization after this time.

- 4.1.2. The minimum racemization was with'the-solutions of pH4.1 containing 0.1% of sodium metabisulphite in ampules filled with nit¬

rogen instead of air, which showed a racemization of 15% after 290 days,while the solutions without sodium metabisulphite showed a stabilization

of the racemization at 25% after about 5 months.

103

;"

The effect of storage time x>n racemization is graphically shown in

fig. 10.

-«—^0.2%solution of NA Hydrochloride with 0.l7oNa2S205 Fn

N2 filled ampules.

— j0.2°/o solution of NA Hydrochloride without Na2S20ginair filled ampules.

Fig. 10

4.1.3. For the solutions of pH 5 containing 0.1% of sodium meta-

bisulphite the racemization is only 5% after 60 days, but the racemization

curve goes still constantly upwards after 290 days when the racemization

is 28%. While the solutions of pH 4 and 5 containing no sodium meta-

bisulphite show nearly the same loss of optical activity, namely 25% after

160 and 290 days.

' 4.2. Effect of sodium metabisulphite on racemization

Sodium metabisulphite helps "at least to some extent in' checkingthe racemization, which is most prominent at pH value of 4. The effect

104

has been graphically shown in fig. 11 for solutions of different pH values

after a period of 290 days. .

^SJ sterilized solution of NA-hydrochloride containing0.1 e)P of N^S2Os> in Ngfilled ampules.

sterilized solutions of NA hydrochloride, i n air filled ampules.

Fig. 11

The racemization curves in fig. 10 for the solutions without metabi¬

sulphite are flat after about 60 days, showing that the rate of racemi¬

zation is very slow afterwards, while for the solutions with metabisulphite'the curves show a slight slope indicating that the racemization equilibriumis not yet reached even after 290 days. . .

.,

105

5. Discussion and conclusions

5.1. Storage time at room temperature (18°-22°) has very littleinfluence on the racemization. During the first 160 days the influence oftime is pronounced, after which there is very little change.

5.2. Racemization is highly dependent on pH of the solution.The best pH value was in the neighbourhood of 4

.

5.3. Sodium metabisulphite has some influence on checking theracemization.

On the basis of these findings the following instructions are proposedfor the injection of NA, with respect to optical stability.

5.4. Injection of noradrenaline

Noradrenaline 0.1%

Sodium chloride 0,9%

Sodium metabisulphite 0.1%

Aqua bidistillata to make 100 ml.

Dissolve sodium chloride and sodium metabisulphite in freshlyprepared double distilled water. Add noradrenaline, filter and make

the required volume. The above operations should be carried out in

an atmosphere of nitrogen.

Fill and seal the ampules in an atmosphere of nitrogen.

Sterilization. Sterilize by autoclaving at 120° for 15 minutes.

pH between 3.7 to 4.5

It is to be noted that the pH values recommended by different phar¬macopoeias vary to a great extent for the injections of noradrenaline acid

tartrate, as may be seen from table 47.

Table 47

Recommeded pH values for the injections of NA acid tartrate

Pharmacopoeia Recommended pH value

Ph. Int. I (128) 2-5 — 4-5

Ph. Dan. K Add. 1954 (164) 2-5

U. S. P. XV (144) 3 —4-5

On the basis of our findings we recommend that in order to

maintain the maximum optical activity the pH value of the dilute solutions

ofNA must be between 3.7 to 4.5j which also agrees very closely with the

findings of West (33) who recommended a pH value of 3,5 to 3.9 for the

injecions of NA, with respect to biological activity.

CHAPTER XII

PREDICTION OF THE STABILITY OF THE SOLUTIONS OF

NORADRENALINE WITH RESPECT TO RACEMIZATION.

1. Introduction

In recent years one finds a continuous stream of new pharmaceuticalpreparations being introduced to the market. For the pharmacist as

well as for the doctor it is essential to know for how long the preparationwill keep its labelled amount of the drug on storage at room temperature.

One way of knowing the stability of the new preparation is by storingit at the storage temperature, say for a period of 1 to 2 years, and then

finding out the activity by suitable chemical or biological methods from

time to time, and in case the preparation is not stable, then carryingout the lengthy time consuming process once again. But in these

days of competition pharmaceutical firms can ill-afford to wait for such

a long time before they can put their preparation on the market.

However, if the reaction, which inactivates the product, is of the

first order and the temperature coefficient known, it should be possible to

predict the stability of the preparation at the storage temperature bycarrying out the experiments at elevated temperatures within a compera-tively short period. In the following pages we shall discuss the basisof the above method (40), (165).

2. O'der of reaction

The rate of a chemical reaction is proportional to the concentrations

of the reacting substances according to the law of mass action. As thereaction proceeds the concentration of the reacting substances goes on

decreasing and so does the reaction rate.

The reactions can be classified according to their molecularity, i; eithe number of molecules or atoms taking part in the chemical reaction.If only one molecule is involved the reaction is said to be unimolecular,if two molecules are involved bimolecular, and so on. The example of

a unimolecular reaction is the decomposition of nitrogen pentoxide as shownbelow :

N308 > N.,04 + 1/2 O,.

For a bimolecular reaction hydrolysis of an ester may be cited as an

example :

• CH3.COO.CaHs + Hp -» CH3.COOH + G2H8OH'

From a quantitative standpoint reactions may be classified accordingto the order of the reaction 1. e., the number of atoms or molecules whoseconcentrations (or for gases pressure) determine the rate of the reaction.The molecularity and the order of the reaction are often identical, but not

always ; for example, though the hydrolysis of an ester is a bimolecular.,reaction, it is a first order reaction because the concentration of water may.be assumed to be constant since it is present' in large excess. The rate'

of the reaction is therefore solely determined by the concentration of the

ester.

•

'

"107

2.1. First ordet reaction

In a first order reaction the rate is directly proportional to theconcentration of the reacting substances. If c is the concentration of the

reacting substance, t the time and k the velocity constant the above con¬

dition may be expressed as :

dc-— =.kc (1)

dt

According to the equation (1), the rate of decrease of the concehtra-

dc

tion with time, z. e.,at any instant represented by the time t, is pro-

dt

tional to the concentration c at that instant.

The equation (1) can be put in another form. If a is the initial

concentration of the reacting substance and x the decrease during the

time t, then (a-x) would be the amount remaining which is equal to concen¬

tration c at any instant. Substituting this in equation (1), we get.

d (a-x)= k. (a-x) (2)

dt

and since the initial concentration a is constant

d (a-x) dx

dt dt

and hence equation (2) may be written as

dx

= k. (a-x) (3)dt

equation (3) can be rearranged so as to carry out the integrationdx

k . dt = (4)a-x

now when t is zero, the value of x is also zero, while after time t the

change is x. By integrating equation (4) we get

1 a 2.303 a

k = In = . log (5)t a-x t a-x

This equation (5) is known as the kinetic equation for a reaction of

the first order.- By studying this equation, the following conclusions can

be drawn.

a

(a) The quantity is the ratio of the concentrations and

a-x

so it is independent of the units used, provided that the same units are

used for both a and x.

108 •

(b) The time to complete any definite fraction tif the reaction is'

independent of the initial concentration.

2.1.1. Velocity constant

For a first order reaction the value of k should be a constant one

which can be easily determined by carrying out the stability experimentsat a definite temperature for different values oft, and then by calculatingthe value ofk by introducing the experimental values ofa, x and t in equ¬

ation (5), or a graphical method may be used. By rearrangement equa¬

tion (5) becomes

2.303 2.303t = .log a . log {a-x) (6)

k k

Now ift is plotted as ordinate and log (a-x) as abscissa, the plot should

be a straight line if the reaction is of first order.

2.1.2. Examples of first order reaction

Hydrolysis ofan ester, inversion ofsucrose, decomposition of nitrogenpentoxide in liquid or gaseous state are a few examples of the first order

reaction.

3. Temperature coefficient

The velocity of a chemical reaction is invariably increased to a mar¬

ked extent by raising the temperature. In general, by raising the tem¬

perature by 10°, the reaction velocity is doubled, though it is not alwaysthe case.

The relation between time and temperature was first given byArrhenius, as the relation between reaction velocity and temperature with

the equationEa 1

log k = . + q where (7)

T = absolute temperatureR = gas constant

Ea = critical increment

q = a constant

Now the temperature coefficient is the coefficient by which to multi¬

ply the velocity constant in order to obtain the velocity constant at any

temperature. The above statement will be clear from the followingexample :

We shall consider the hydrolysis of procaine where we know the

relation between time and destruction at 20° and wish to know the

stability at 10°. For the hydrolysis at the two temperatures we have :

2.303 a

kI0° = . log (8)tio°t,°

a-x

109

2.303 a

kaoo = —. log (9)

tj0 a-x

If we now specify the time t to olrtain the same amount of hydro-a

lysis, using the same initial -concentration log would have the same

a-x

value in the two equations. Now by division we have

» <10)Tc °

t°

If the temperature coefficient for this process is 0.2 we get

fll)Klo Vjo

k10°. (20-10). 0.2 t10

Experimentally the temperature coefficient is determined by carryingout the stability experiments at definite temperatures at intervals of 10°,and if the time t is plotted as abscissa and the destruction as ordinate'

one will obtain at each specific value ofthe ordinate the relation between

the two times tx and t2, thus giving the temperature coefficient of the

process for 10° as would be clear from fig. 15.

'4. Experimental

In the following experiments we have made the racemization studies

of 2 % w/v solutions of NA hydrochloride of

(a) <pH values : 2, 3, 4 and 5

(b) with and without sodium metabisulphite

(c) for 1, 3, 6 and 10 hours

(d) rat 60°-, 100° and 120°.

4.1. Material an3 apparatus

4.1.1. Double distilled water, 5 ml ampules and 1-Noradrenaline

hydrochloride confirming to the tests as described an .pages ,39, 41 and

81 repectively were used.

4.1.2. The polarimeter which has l>een described on page 97

using a lOcmlongpolarimeteriubewithan inner diameter of2 iron was

used.

4.1.3. pH measurements were made with Methrom-potentiometerwith a glass electrode. The accuracy of the apparatus was 0.05 pH units.

4.1.4. Heating at 60" was done in an electric oven, -while for

heating at 100° and 120°, the Egro autoclave was used. The ampuleswere placed in the autoclave only when a temperature of about 100° was

attained. After the duration of heating the ampules were taken out oftheautoclave as soon as possible and stored in a refrigerator at a temperatureof4° till the optical rotation was determined. The heating ti me was noted

from the moment the autoclave registered a temperature of 100° or 120°

110

as the volume of the solution in each ampule was only 2.5 ml and not

more than 20 ampules were heated at one time.

4.2. Determination of the optical rotation

All the measurements were made between the temperatures of

20° and 22°, except for the solutions of pH 5.5/100° and of pH 5.5/120°whose rotation was measured at 23°-24°. The deviation in the opticalrotation for 15° is about 0.01° therefore the error of the measurements

between the temperatures of 20° and 24° is negligible.

Solutions which were turbid due to the formation of precipitatewere filtered through a small filter paper before the determination of

the optical rotation. Further those solutions which were dark coloured

were diluted so that readings could be taken.

The following results are the mean of 5 to 10 readings fQr each solu¬

tion, the mximum deviation .from the .mean. being± 2.5 %.

4.3. Working procedure

4.3.1. Preparation of the solutions

NA hydrochloride was accurately weighed and dissolved in freshlyprepared double distilled water in a volumetric flask. The solution

was divided into 4 equal parts and the pH value of different portions of

the solution was adjusted to approximately 2, 3, 4 and 5 by the addition

of either sodium bicarbonate or 1 N hydrochloric acid. The solutions

of different pH values were then divided into two equal parts, and to

one portion of the solution of each pH value 0.1% of sodium metabis-

ulphite was added. The final concentration of the different solutions was

adjusted to 2 g of NA hydrochloride per 100 ml of the solution. The

NA content was checked by measuring the extinction at 279 mju.

4.3.2. Filling and sealing ,

2.5 ml quantities of the above solutions were filled in freshly cleanedand dried 5 ml ampules. The ampules containing the solutions without

metabisulphite were sealed in the usual way in the presence of air, while

those ampules which contained the solution with metabisulphite were

sealed after replacing the air by nitrogen as described on pages 62 and 63

The ampules were then stored in a refrigerator at a temperature of 4° for a

maximum period of fifteen weeks.

Throughout the operation, care was taken that the solutions did not

get in contact with any metal.

4.3.3. Heating of the ampules• The above ampules were then heated for 1, 3, 6 and 10 hours

at 60°, 100° and 120°.

4.4. Results

.

4.4.1. pH of 1-noradrenaline hydrochloride solutions

f' 2%w/v( solutions of NA hydrochloride with and without sodium

metabisulphite were prepared, the pH values of which were measured

immediately after preparation. The results are shown in table 48.

Ill

Table 48

pH values of I-noradrenalinc hydrochloride solutions

pH Values

NA hydrochloride solutions without

Na2SaOc, in air filled ampulesNA hydrochloride solutions

•with 0.1% of Na2S3Os, in

nitrogen filled ampules

=. 2-2 2-4

3-1 34"

4-3 4-2

5-5 5-1

4.4.2. Results of the optical rotation determinations* The following observations have beem made between '20°, and

24° and are without temperature correction.

^

In tables 49 and 50 the following signs stand for :

Solution A = freshly prepared solution, whose rotation was

measured immediately after preparation.* solution .for the determination two times diluted.

+ solution for the determination three times diluted.

x solution for the determination four times diluted.

Table 49

Specific rotation of l-noradrcnalinc hydrochloride solutions without sodium

metabisulphitc in air filled ampules

Specific rotation of 2% w/v NA hydrochloridesolutions without Na3S20s, heated to a temp, of

treating time

in hours pH 60» IOC 120°

Solution A

1

3

6

10

Solution A

1

3

6

10

Solution A

1

3

6

10

Solution A

1• 3

6'

10

2.2

3.1

4.3

5.5

39-5* — 40 0° — 400°

390° — 270° • — JD-B*380° — 17-2° 00°370° — 50° 0 0°

34-5° — 1-0* 0 0°

395° — 40 0° — 400°390° — 38-5° — 26-5°380# — 32-5° — 16-5' +

370° — 24-0' + — 40°X35 0° — 12-0'X 0 0°X

39-5° — 400° — 40 0'

390° — 37-5° — 27-5"38 0' — 32-5° — 150°*

370' — 310°* — 80°X31-5" — 270** — 40°X

39-5" — 40 0" — 40 0°390" — 360°* — 26-0«*38-0° — 330°* — 140°X37-0° — 26 0°* — 00°X350° — 24-0° + 00°X

112

Table SO

Specific rotation of I-noradrenaline hydrochloride solutions containing 0.1 %of sodium tnetabisuIpMte in N2 filled ampules

Specific rotation of 2% w/r NA hydro¬chloride solutions with 0.1 % Na3S20e,heated to a temp, of

Heating time in hours pH 60° 100" 120°

Solution A.

1

a

6

10

2.4

39S* — 40-0* — 40-0'

39-5" — 320tt — 260'

380° — 255" — 42"

380" — 110" 00

36-5* — 30" 00

Solution A

I

3

6

10

3.4

39-5° — 400° — 40-0°

39-5* — 40-0° — 24-0"

390" — 33-5* — 10.0"

380" — 25-5° — 2-7"

370" — 13-5° — 1-Q" •

' Solution A

1

3

6

10

4.2

39-5" — 400" — 400"

39-5° — 37-5" — 32-5°

390° — 330" — 18-5"

380" — 30-5° — 100" *

37-5* — 26-5° — 60° +

Solution A

1

3

C

10

5.1

39-5° — 400° — 40 0°

39-5° — 36-5" — 28-5"

390° — 32-2" — 160? *

380" — 260° — 4-0° X

37.0° — 25.0° o.o x

113

Table 51

Racemization of 1-noradrenaline hydrochloride solutions

% of 1-NA hydrochloride % of 1-NA hydrochlorideracemized without pH racemized with 0.1 % of

Na2S2Os, in air filled ampules Na2S2Os, in N2 filled ampules

Heating time

in hourspH

60° 100° 120° 60° 100° 120°

1 1-27 32-5 73-7 • Nil - 200 350

3 22 3-8 570 1000 2-4 1-27 38-0 ' '

900

6 6-35 87-5 1000 3-8 72-5 1000

10 127 97-5 1000 6-3 92-5 1000.

1 1-27 375 33-75 Nil Nil 40 0

3 31 3-8 1875 53-75. 34 .1-27 16-25 750

6 6-35 400 900 3-8 36-25 93-25

10 11-4 700 1000 6-3 66-25 97-5

•1 1-27 6-25 31-2 Nil 6-25 190

4.3 3-8 18-5 62-5 4.2 1-27 17-5 53-7

6 6'35 22-5 800 3-8 23-7 750

10 12-7 32-5 900 51 33-75 .850

1 1-27 100 350 Nil 8-8 23-7

3 55 3-8 17-5 650 51 1-27 19-5 600

6 6-35 350 1000 3-8 350 90 0

10 11-4 400 1000 6-35 37-5 1000

4.5. Discussion of the results

4.5.1. Influence of sodium metabisulphite

(In general the racemization for solutions containing sodium metabi¬

sulphite in nitrogen filled ampules is less when compared with thesolutions containing no metabisulphite in air filled ampules.

4.5.1.1. At 60° the racemization for the solutions containing no

metabisulphite in air filled ampules was practically double than that ofthe solutions with metabisulphite in nitrogen filled ampules.

4.5.1.2. At 100° the difference is much smaller and at pH values

of 4 and 5 there is no difference.

114

4.5.1.3. At 120° there is some difference only after 1 hour heatingtime for the solutions of all pH values, while after heating for more than

1 hour there is very little difference in racemization for the solutions conta¬

ining metabisulphite in nitrogen filled ampules and those without metabi¬

sulphite in air filled ampules, as may be seen from the results given in table

51.

4.5.2. Influence of pH

The results of table 51 show that the pH plays an important role on

the rate of racemization.

4.5.2.1. At 60° there was an equal amount of racemization between

pH values of 2 and 5.

4.5.2.2. At 100° the effect of pH is very clear. On comparingthe racemization results of pH 2, 3, 4 and 5 after 10 hours heatingtime we find that the racemization at pH 3 is 25% less while at pH 4 and

5 about 60% less than the racemization at pH 2. i

4.5.2.3. At 120° the pH has the same influence as at 100°, exceptthat the amount of racemization is greater. The minimum racemiza-

tion is in the case of solutions of pH 4, 2.

4.5.3. Influence of time and temperature

The influence of heating time and temperature for solutions of 1-

noradrenaline hydrochloride, with and without sodium metabisulphite,at different pH values is graphically shown in figures 12 and 13.

3'

6

TIME IN HOURS

RELATION BETWEEN THE PERCENTAGE OF

NORADRENALINE HCL RACEMISED AND

TIME AT DIFFERNT TEMPERATURESAND

pH-VALUES

(2%wfv totutlonof Noradrenalint HCL with

0,1% Sodium mttabtaulfite.1

RELATION BETWEEN THE PERCENTAGE OF

NORADRENALINE HCL RACEMISED AND TIME

AT DIFFERENT TEMPERATURES ANDpH-VAlUES

(2°/oWv solution of Noradranaltna HCl withoutSodium matabtfiAfite)

Fig. 12. Fig. 13.

The results show that by heating the solutions for longer periodsand by increasing the temperature, the rate of racemization goes on

increasing.

115

4.5.4. Calculation of the stability of noradrenaline solutions

In order to determine the order of the reaction for the racemizationofNA hydrochloride solutions, the velocity constant k has been calculatedfrom the formula :

2.303 a

k =. log where

t a-x

a = initial cone, of the unracemized NA = 100

x «=%of racemized NA hydrochloride during t hours.

The k values for different tempratures are given in table 52.

Table 52

Velocity constants for the racemization of 1-noradrenaline hydrochloridesolutions at different temperatures.

PH

Velocity constant k

2 % w/v solution of NA

hydrochloride withoutNa 2S2O5, in air filled

ampules.

pH 2% w v solution ofNA

hydrochloride with 0.1%NaaS205, in nitrogen

filled ampules.

Heating time

in hours. 60° 100° 120° 60° 100° 120°

1 00129 0-3915 0-3357 000 0-2232 0-4309

3 22 00127 0-2809 24 00043 01488 0-7676

6 00109 0-3456 00064 0-2152

10 00136 0-3689 00072 0-2590

1 00129 0-0283 0-4118 000 000 0-5108

3 31 00127 00691 0-2852 34 00043 00591 0-4621

6 00109 00851 0-3838 00064 00751 0-4487

10 00121 0-1204 00051 01090 0-3689

1 00129 0-0645 0-3740 000 00645 0-2107

3 43 00127 00683 0-3270 42 0-0043 00681 0-2567

6 00109 00425 0-2683 00064 0-0451 0-2311

10 00139 00393 0-2303 00065 00412 0-1897

1 00129 01055 0-4320 000 0-0921 0-3385

3 5-5 00127 00641 0-3499 5-1 00043 00722 0-3054

6 0-0109 00718 00064 00718 0-3838

10 00139 0-0511 00065 00470

116

The figures of the above table show that at 120° and at 100°

there is an appreciable difference in the k values for different time

intervals at all the pH values, showing that the reaction is not

of the first order at 120° and at 100°. While at 60° the k values are

more or less constant between the pH values of 2 to 5, proving that the

reaction is of the first order at lower temperatures.

The above is also clear by the graphical relationship of the log of

the % of unracemized NA hydrochloride and time, which is shown in fig.14 for solutions of NA hydrochloride containing 0.1% of metabisulpliitein nitrogen filled ampules.

RELATIONBETWEENTHELOeoF THE%0F

THE UNRACEMISEO NORADRENALINE HCIANO

THE TIME AT DIFFERENT TEMPERATURES AND

pH VALUES.

Fig 14

So we conclude that it is not possible to predict the stability of NA

solutions with respect to racemization at storage temperature from the

racemization studies at 100° or,120°. Further, in order to predict the

stability at storage temperature, the stability experiments must be carried

out in the neighbourhood of 60°.

It is to be noted that the rate of racemization at 60° is very slowand an error of 0-10° in the polarimeter reading will give a difference of

racemization of 1*25% when the optical rotation readings are taken for a

2% w/v solution of NA hydrochloride, using 10 cm long polarimetertube, i. e. there will be an error of as much as 20 % for a solution which

has been heated for 10 hours. So it is necessary that the racemizationstudies at lower temperatures should be carried out over a much longerperiod so as to obtain a racemization ofabout 50%, and thus minimize the

error.

117

5. Racemization studies at 5(P, 60°, 70°

In our previous experiments we found that the best pH for the solu¬

tions of NA hydrochloride was about 4, and that the reaction velocitywas constant in the neighbourhood of 60° with respect to racemization.

So in the following studies we have further carried out the stability

experiments at 50°, 60° and 70°, for a much longer period (about 200-400

h.) for a 2% w'v solution of NA hydrochloride containing 0.1% of sodium

metabisulphite in nitrogen filled ampules of approximately pH 4.

5.1. Experimental

The method for preparation of the solution and determination of the

optical rotation was exactly the same as described on page 110 exceptfor the following differences :

(a) The ampules were heated in a liquid paraffin bath of which the

maximum variation of temperature was ± 1°.

(b) All the optical rotation readings were taken between 20° and 22°

5.2. Results

In tables 53, 54 and 55 results of the optical rotation determinations

at 50°, 60°, and 70°, are given together with the calculated k values.

Table S3

Specific rotation and the velocity constantkof I-noradrenaline hydrochloridesolutions containing 0-1 % of Na2S205 in nitrogen filled ampules after heating at 50°

Heating time pHtSpecific rotation

in hours% of unracemized

NA hydrochlorideVelocity constant k

Solution A — 41-0° 100

48 — 37-5° 91-46 000186

96 — 35-0° 85-37 000164

144

4.25

— 330° 80-5 • 000150

192 — 30-5° 74-4 000154

240 — 27-25°•

66-46 000170

290 — 26-0° 63-4 000157

400 — 23-5° 50-73 000170

Mean k value 0.00164

118

Table 54

Specific rotation and the velocity constant fc of l-noradrenaline hydrochloridesolutions containing 01 % of Na2S2Os in nitrogen filled ampules after heating at 60°

Heating time

in hoursPH Specific rotation % of unracemizcd

NA hydrochlorideVelocity constant k

Solution A — 41-0° 100

8^

— 39-5° 96-34' 000460

16 — 38-5° 93-9 000390

48 — 34-5° 84-15 000360

96

4.25

— 27-5° 67-07 000416

144 — 24-0° 58-54 000370

192 — 190° 46.34 000400

240* — 18-0° 40-4 0-00380

Mean k value.. .. .. 000397

Table 55

Specific rotation and the velocity constant k of l-noradrenaline hydrochloridesolution containing 0-1 % of Na2S2Os in nitrogen filled ampules after heating at 70°

Heating time

in hourspH Specific rotation % of unracemized

NA hydrochlorideVelocity constant k

Solution A — 41-0° 10000 000

8 — 38-25° 93-30 000866

16 — 35-7° 87-10 000860

24

4.25

— 33-5° 81-70 0 00840

48 — 27-0° 65-85 000870

72 — 22-25° 54-30 000847

96 — 18-0° 4390 000856

144* — 6-5° 15-9 001270

200* — 4-5° 110 001120

Velocity constant k, mean value .. •• 000856

Solution A = freshly prepared solution, the rotation of which was

measured immediately after preparation.*= solution which turned slightly yellowish after heating.

5.3. Discussion of the results

5.3.1. Velocity constant

The reaction velocity is practically constant for all the 3 temperatures

i. e. 50°, 60° and 70°, which has been calculated from the formula

119

2.303 a

k =. log

t a-x

for example k value at 60° after 16 hours, when the value of a-x

= 93.9 is

2.303 100

k =. log = 0.00390

16 93.9

The relation between the log of the percentage of the unracemized

NA hydrochloride and time is graphically shown in fig. 15, which islinear showing that the reaction is of the first order.

/o racemized

0.0

20

40

60

80

100

hours

300

RACEMISATION OF NORADRENALINE AT 50°,60°&70ofora2%w/v solution of NA Hydrochloride of phU.2

Fig. 15

120

The solutions, when heated at 50° for 400 hours, remain colourless,but at 60° start developing a yellowish colour after about 240 hours

at which the racemization is 60%, further, at 70° solutions start turningyellowish after 96 hours ofheating time, with a slight brownish precipitateand increase in reaction velocity after 144 hours when the racemization is

about 85%.

5.3.2. Temperature coefficient

The temperature coefficient for a difference of 10° is found from

fig. 16 and from the reaction velocity constants given in table 56.

The temperature coefficient for the above process for a difference

of 10° is 2-3.

togot2.0-

V\^^^^^**^_

1.8 -

--^5{fc

1.6_

x^ fc

U- A^S-

-

Hours 100 200 300 too

RELATION'BETWEENTHELOG 0FTHE%OF

THE UNRACEMJSED NORADRENALINE HCl &

THETIME AT DIFFERENT TEMPERATURES .

Fig. 16.

Table 56

Velocity constants for the racemization ofNA hydrochloride at 50°, 60°, and 70°;and the temperature coefficient for 10°

PH Temperature Velocity constant

kTemperature cofficient

calculated from fig. 16

50° 0.00164

4.25 6G° 0.00397 2.4 2.3

70° 0.00856 2.15 2.3

121

5.3.3. Calculation of the stability at room temperature (20d)

Now if we know the time after which a certain % of NA is racemized

at a fixed temperature, it should be possible to keep the solutions 2.3

times longer at 10° lower temperature. In the following table the

storage time for different percentage of racemization of NA has been cal¬

culated.

Table 57

Calculated storage time for solutions of NA hydrochloride ofpH 425; containing0-1 % of NajSaOg, in nitrogen filled ampules

% racemization Temp. Time in h. Calculated storage time at 20°

10 50° 60 60 X 233 h. = 30 days

20 50° 136 136 X 2-33 h. = 78 days

30 60° 96 96 X 2-34 h. sa 112 days

40 60° 137 137 X 2-34: h. = 160 days

50 70° 82 82 X 235 h. a 220 days

60 70° 105 105 X 2-35 h. s 282 days

5.3.4. Conclusions

5.3.4.1. It is not possible to predict the stability of NA solutions

with respect to racemization by carrying out the stability experimentsat higher temperatures because the results of the experiments of the dilute

solutions of NA hydrochloride at room temperature (18°-22°) show that

for solutions of pH 2,3, 4 and 5 an equilibrium is attained with respect to

racemization after about 60 days, as may be seen from fig. 10, page 103,and so there is a great difference in the predicted storage time and that

found experimentally for the dilute solutions of NA hydrochloride.

5.3.4..2. In order to maintain maximum activity, NA injectionsmust first be stored for about 2 months after manufacture, so that an

equilibrium is attained with respect to racemization and only then theyshould be dispensed after determining their potency.

6. Summary

6.1. For the racemization of NA solutions the reaction is not ofthe first order at 120° and 100°, however at 70°, 60° and 50° the reactionis of the first order.

6.2. The temperature coefficient for a difference of 10° for a

"2% w/v solution of NA hydrochloride of pH 4.25 containing 0.1% ofsodium metabisulphite in nitrogen filled ampules is 2.3.

6.3. The minimum racemization was found for solutions witha pH value of about 4.

6.4. Solutions containing 0.1% of sodium metabisulphite in nit¬

rogen filled ampules showed less racemization when compared with the

solutions containing no sodium metabisulphite in air filled ampules.

CHAPTER XIV

SUMMARY

1. Sympatol1.1. Methods of analysis

The following methods of analysis for the quantitative estimation of

sympatol in solutions have been established :

1.1.1. Colorimetric method

Sympatol gives a colour reaction with sodium nitrite in the

presence of mercuric sulphate. The coloured product gives a peak at

500 m/i and is stable at least for 50 minutes. Thus the method can be

applied for the quantitative estimation ofsympatol content.

1.1 2. UV-Spectrophotometric method

.Sympatol gives absorption maxima at 223 and 273 mp. Theratio of the extinction values at the two maxima is 5.86 :1. Thus

any of the maxima can be used to assay the sympatol solutions.

1.1.3. Paper chromatographic method

Solutions of sympatol gave a linear reltionship between the spotarea and the log of the amount of sympatol between 25 to 150 p.g. Thusthe method may be used for the quantitative estimation of sympatolin solutions.

On heating the sympatol solutions for a long time an orange productis formed, which gives absorption maximum at 290-295 mp.

However none of the above 3 methods can be used to estimate

quantitatively the undeteriorated and deteriorated sympatol content in

a mixture.

1.2. Stability of sympatol solutions

Since none of the previously examined methods can be used to

determine the stability of" sympatol solutions, but as the decomposedproduct of sympatol is coloured, the stability of sympatol solutions has

been determined by means of colour standards.

Through the systematic variation of different influencing factors the

following results have been obtained :

(a) The development of colour and thus the destruction of sympatolsolutions is proportional to the increasing pH and storage temperature.

(b) Though nitrogen checks the oxidation in the phenolic OH

group it is not sufficient to stabilize the sympatol solutions.

(c) Sodium metabisulphite helps to check the development of the

colour.

In order to obtain the optimum stability of sympatol solutions the

following conditions must be adhered to '

123

— pH value should be 3 or lower,— the ampules must be filled in an atmosphere of nitrogen,— as antioxidant 0.1% of sodium metabisulphite must be added,

— storage temperature must be as low as possible..

On the basis of the above, suggestions for the preparation of a stable

solution of sympatol have been given, (see page 71)

2. Noradrenaline

2.1. Methods of analysis

The following methods ofanalysis for the estimation of undeteriorated

and deteriorated noradrenaline content in solutions have been investigated.2.1.1. UV Spectrophotometric method.

The extinction wavelength curve of the deteriorated solution of

NA does* not show any change when compared with that of the fresh

solution.*

2 1.2 Iodine method

After iodine oxidation NA gives absorption maxima at 295-296 mfand at 520-525 m/*. The maximum loss shown by this method for a

solution of NA which was heated at 120° for 2 hours in the presenceof oxygen is 4.8%.

2.13. Persulphate method

After persulphate oxidation NA gives absorption maxima at 288-290 mjxand at 490-495 rmt. The maximum loss shown by this method for a

solution of NA which was heated at 120° for 2 hours in the presenceof oxygen is 6.8 %.

2.1.4. Ferro-citrate method

NA gives an absorption maximum at 530 imt after reaction

with ferro-citrate solution. The maximum loss shown by this method

for a solution of NA which was heated at 120° for 10 hours in the pre¬sence" of air is 7%.

None of these methods can be used for the estimation of the deteri¬

orated NA content, as there is a great deviation in the above results and the

results of the biological assays carried out by other workers.

2.2. Racemization studies of the dilate solutions of NA (0.2%)

As the 1-form ofNA is much more potent than the racemic form,the best conditions for the stability of the optical activity of NA solutions

have been evaluated.

2.2.1. The racemization is highly dependent on pH. Solutions

with a pH value in the neighbourhood of 4, show the maximum opticalactivity.

2.2.2. Storage time at room temperature (18°-22°) has verylittle influence on racemization. During the first 160 days, the influence

of time is pronounced after which there is very little change.

2.2.3. Sodium metabisulphite has some influence on checkingthe racemization.

J24

2.3. Prediction of the stability of NA solutions with respect to

i acemization.

2.3.1. At 120° and 100°, the reaction is not of the first order with

respect to racemization.

2.3.2. At 50°, 60° and 70° the reaction is of the first order with

respect to racemization. The temperature coefficient of the process for a

difference of 10° for a solution of NA of pH 4.25 containing 0.1% of

sodium metabisulphite is 2.3

2.3.3. On the basis of the racemization experiments of dilute

solutions of NA at 20°, the different storage timings obtained for varying

degree of racemization of NA, do not agree with the calculated storage

time. For calculation of the stability, the racemization experiments have

been carried out for a concentrated solution of NA (2%) at 50°, 60° and

70°. The dilute solutions ofNA between the pH values of 3 to 5, show an

equilibrium of racemization after storage for 2 months at 20, against

the calculated storage timings under the same conditions.

ZUSAMMEMFASSUNG

1. Sympatol

1.1. Bestimmungsmethoden

Die folgenden Methoden fiir die quantitative Gehaltsbestimmungvon Sympatollosungen wurden ausgearbeitet :

1.11. Kolorimetrische Methode.

Sympatol gibt bei Anwesenheit von Mercurisulfat mit Natrium-

nitrit eine Farbreaktion. Die Absorptionskurve des gefarbtenReaktionsproduktes, welches mindestens 50 Minuten lang stabil ist,

zeigt ein Maximum bei 500 m/i. Diese Reaktion kann daher fur die

Gehaltsbestimmung von Sympatol in Lbsungen benutzt werden.

11.2. UV-Spektrophotomelrische Methode.

Sympatol selbst zeigt im UV-Gebiet zwei Maxima : eines bei

223 m/t, ein zweites bei 273 imi. Das Verhaltnis der Extinktionswerte

bei diesen beiden Wellenlangen betragt 5.86:1. Beide Maxima sind

daher fiir die Gehaltsbestimmung von Sympatollosungen brauchbar.

11.3. Papierchromatographiche Methode.

Bei der Papierchromatographie von Sympatollosungen nimmt die

Grosse der Sympatolflecken linearmitdem Logarithmus der Sympatol-menge zwischen 25 und 150 ^g zu. Diese Methode scheint daher auch

geeignet zu sein, um den Sympatolgehalt einer L'dsung zu bestimmen.

Wenn Sympatoll'dsungen langere Zeit erhitzt werden, bildet sich

ein orange gefa*rbtes Produkt, welches ein Absorptionsmaximum bei 290-

295 m/t aufweist.

Es zeigte sich jedoch, dass keine dieser 3 Methoden im Stande

ist,,zersetztes und nicht zersetztes Sympatol quantitative nebeneinander

zu erfassen.

125

1.2. Haltbarkeit von SympatoUosungen

Da keine der vorhergeprliften Methoden zur Haltbarkeitsbestimmungvon SympatoUosungen tauglich ist, und da ferner festgestellt wurde,dass die Zersetzungsprodukte von Sympatol gefarbt vvaren, wurde die

Haltbarkeit der SympatoUosungen auf Grund der FarbveranderungenbeurteUt.

Durch systematische Variation der einzelnen Einflussfaktoren ergabsich, dass

(a) die Farbentwicklung und damit der Abbau des Sympatols mit

steigendem pH und steigender Lagertemperatur zunimmt.

(b) die Begasung mit Stickstoff die Oxydation der phenolischenOH-Gruppe wohl hemmt, aber fur eine normale Lagerzeit nicht geniigendverhindert,

(c) Natriummetabisulfit die Verfa"rbung von SympatoUosungenwcitgehend hintanhUlt.

Um moglichst stabile SympatoUosungen zu erhalten, sind folgendeBedingungen einzuhalten:

— der pH-Wert soUte 3 oder weniger sein,

— die Ampullen miissen mit Stickstoff begast werden,

— als Antioxydans ist 0.1% Natriummetabisulfit zuzufiigen,

— die SympatoUosungen miissen bei moglichst niedriger Tempera¬ture gelagert werden.

Auf Grund der erhaltenen Resultate wurde eine Vorschrift fur einehaltbare SympatoUosungen vorgeschlagen (siehe page 71).

2. Noradrenalin

2.1. Bestimmnngsmcthodcn

Es wurde versucht, mit ciner der nachfolgcnden Methoden denGehalt von oxydiertem und nichtoxydiertem Noradrenalin zu

bestimmen.

2.1.1. UV-Spcktrophotometrische Methode. Die Extinktions-

Wellenlangenkurve von weitgehend oxydiertem Noradrenalin zeigtkeine wesentlichen Unterschiede gegeniiber frischzubereiteten Noradrc-

nalinlosungen.

2.1.2. Jod-Methode. Mit Jod oxydiertes Noradrenalin zeigtAbsorptionsmaxima bei 295-296 m/i und bei 520-525 m/*. Der mitdieser Methode nachweisbare abbau des Noradrenalins nach 2 StundenErhitzen auf 120° in sauerstoffgefiillten Ampullen betrug hochstens

4.8%.

2.1.3. Persulfat-Methode. Mit Natriumpersulfat oxydiertesNoradrenalin zeigt Absorptionsmaxima bei 288-290 tnp. und bei 490-

126

495 m/i. Der mit dieser Methode nachweisbare Abbau betrug nach2 Stunden Erhitzen auf 120° in sauerstoffgefiillten Ampullen hochstens

6.8%.

2.1.4. Ferrozitrat-Methode. Das Reaktions produkt von Nora-

drenalin mit Firrozitrat zeigt ein Absorptionsmaximum bei 530 tn/i. Der

mit dieser Methode nachweisbare Abbau von Noradrenalin nach 10

Stunden Erhitzen auf 120° in luftgefullten Ampullen betrug hochas-

Jtens 7%

Da die mit diesen Methoden erhaltenen Resultate unter sich

nicht ubereinstimmen und vor allem grosse Abweichungen gegeniiberden im biologischen Versuch erhaltenen Resultaten anderer Autoren

aufweisen, eignen sie sich fiir die Bestimmung des zersetzten Norad-renalins nicht.

2.2. Untersuchung iiber die Razemisicrung von Noradrenalin in

verdiinnten Losungen

Da die Wirkung des 1-Noradrenalins viel starker ist als diejenigedes Razemates warden die Einfliisse untersucht, die die Erhaltung der

optischen Aktivitat begiinstigen.

2.2.1. Die Razemisierung, von 1-Noradrenalin ist sehr pH abh'angigDie optische Aktivitat wird bei ca. pH 4 am langsten erhalten.

2.2.2. Bei Zimmertemperatur (18°-22°) geht die Razemisicrungim Durchschnitt langsam vor sich. In den ersten 160 Tagen strebt

sie ziemlich rasch einem Gleichgewichtszustand entgegen, welcher

sich nach 160 Tagen kaum weiter verandert.

2.2.3. Natriummetabisulfit besitzt die Fahigkeit, den Razemisier-

ungsvorgang zu hemmen., .

2.3. Berechnung der Haltbarkcit von 1-Noradrenalinlosungen in

Bezug auf die Razemisicrung

2.3.1. Im Bereich von 100° bis 120° folgt die Razemisierungnicht dem Gesetze der Reaktionen erster Ordnung.

2.3.2. Bei 50°, 60° und 70° folgt die Razemisierung dem Gesetze

fiir Reaktionen erster Ordnung. Der Tempcraturkoeffizient fiir eine

Temperaturdifferenz von 10° betragt fiir eine Noradrenalinlosung vom

pH 4.25 mit einem Zusatz von 0.1% Natriummetabisulfit 2.3.

2.3.3. Bei der experimentellen Bestimmung der Razemisierungvon verdiinnten Noradrenalinlosungen bei 20° resultierten fiir die

verschiedenen Razemisierungsgrade Lagerfristen, welche gar nicht

mit den berechneten iibereinstimmten. Als Grundlage fiir diese Berech-

nungen dienten Razemisierungsversuche mit konzentrierten Noradrenalin¬

losungen (2%) bei 50°, 60°, und 70°. Die Razemisierung erreicht in den

verdiinnten Noradrenalinlosungen vom pH 3-5 bei 20° nach 2 Monatenim Gegensatz zu den Berechnungen einen Gleichgewichtszustand.

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CURICULUM VITAE

of

Devendra Kumar AgrawalI

I was born at Bilgram (Uttar Pradesh), India, on the 26th of

January 1932.

After attending School at Hardoi, I passed the High School exami¬

nation of the U. P. Board of High School and Intermediate Educationin the year 1947. After" further studying for 2 years I passed the Inter¬

mediate (science) examination from the Government College, Allahabad,in the year 1949.

I studied at the Pharmaceutical department of the College of Tech¬

nology of Banaras Hindu University, and passed the final degreeexamination of the Bachelor of Pharmacy in June 1954.

After taking apprenticeship for 1 year at various manufacturinglaboratories I joined the School of Pharmacy of the Swiss Federal Insti¬tute of Technology, Zurich, in October 1955. After passing the admissionexamination in August, 1956, I started work on research, the results ofwhich are herein submitted,

\