Chapter – 2

10

Chapter-2

Methods for the Analysis of Some Chemotherapeutic

Agents.

Introduction

The chemical substances, which interact at molecular level altering the body

functions therapeutically, are generally referred to as drugs or chemotherapeutics.

Drugs vary in molecular size and chemical nature. Based on the effect a compound

has on the body, drugs have been variously classified as analgesics, antibiotics,

narcotic analgesic agonists/antagonists, central nervous system (CNS), stimulants/

depressants, tranquilizers, etc. The pharmacological properties of such drugs are

commonly misused for euphonic, homicidal, suicidal and doping purposes. Fatalities

due to the accidental use of excessive dose of drugs are also of very common

occurrence. In this regard, constant updating of the analytical methods for the drug

analysis is very important. The author has given a brief account of the drugs selected

for investigation in the present chapter in the following paragraphs.

Metoclopramide hydrochloride

Metoclopramide hydrochloride (MCP) chemically, 4-amino-5-chloro-N-[2-

(diethylamino) ethyl]-2-methoxy benzamide is an important derivative of benzamide.

It is a potent dopamine receptor antagonist used for its antiemetic and prokinetic

properties. Therefore, it is commonly used to treat nausea and vomiting associated

with conditions including; emetogenic drugs, radiation sickness, malignancy, labor

and infection. It is also used in combination with paracetamol for the relief of

migraine. It is commercially available under version trade names, Reglan (CFL),

Perinorm (IPCA), Segmet (Sigma), Vominorm (Cipla) etc.

Chapter – 2

11

MCP has the following molecular structure:

O

HN

OCH3

N

Cl

H2N

. HCl

MCP is a white crystalline powder with melting point 182-184 °C. Soluble in

water, acetone, alcohol and chloroform. Its aqueous solution is stable for a week at

room temperature (ca. 27 ± 2 °C).

Dapsone

Dapsone (DAP) chemically, 4, 4’-sulfonyl bis (benzene amine), is a potent

anti bacterial (leprostatic). It is most commonly used in combination with rifampicin

and clofazimine as multidrug therapy (MDT) for the treatment of Mycobacterium

leprae infections (leprosy). It is also used to treat dermatitis herpetiformis and other

skin conditions including lichen planus. It has the following structural formula.

S

O

O

H2N NH2

It is soluble in alcohol, methanol, and acetone and dilute hydrochloric acid,

practically insoluble in water. It melts at 175-176 °C.

Cisapride

Cisapride (CPD), chemically 4-amino-5-chloro-N-[1-[3-(4-fluorophenoxy)

propyl]-3-methoxy-4-piperidyl]-2, methoxy-benzamide is a parasympathomimetic,

which acts as a serotonin-5-HT4 agonist. It has been used to treat bowel constipation

Chapter – 2

12

and it used as an alternative to MCP in the management of diabetic gastroparlsis. It

has the following structure.

O

HN

O N

O

O

F

Cl

H2N

It is freely soluble in methanol and 2-propanol, CPD is commercially known as

Cisawal (Wallace), Cisapro (Zy Alidac), Ciza (Intas) and Unipride (Torrent).

p-Aminobenzoic acid

p-Aminobenzoic acid (PABA), 4-aminobenzoic acid is an important skin

protective agent and it is widely used as a UV filter in sun screen formulations. PABA

is a white crystalline solid freely soluble in ethanol and slightly soluble in water. It

melts at 187-188 °C and it has the following structure.

O

HO

NH2

Literature Survey

The pharmacological importance of these drugs resulted in an increasing need

for analytical methods for their detection and quantification. The important analytical

techniques recommended for the assay of studied drugs include, gas chromatography-

mass-spectrometry, HPLC, HPTLC, electron-capture gas-liquid chromatography,

spectrometry etc., most of the above techniques except spectrophotometry, require an

expensive experimental set-up.

Among the various optical methods, spectrophotometric methods for the

determination of micro quantities of drugs have received considerable attention due to

their simplicity, reliability, rapidity and availability of number of chromogenic

Chapter – 2

13

reagents. The general availability of modern spectrophotometers in most academic

and industrial laboratories, have also contributed to the increased use of the technique.

This is undoubtedly reflected in the great volume of literature on spectrophotometric

analysis of bio-active compounds. In the following paragraphs, a modest attempt is

made to briefly review some of the recently applied spectrophotometric methods for

the determination of metoclopramide hydrochloride [MCP], dapsone [DAP], cisapride

[CPD] and p-.amino benzoic acid [PABA].

Patel et al. [1] have developed two spectrophotometric methods for the

determination of MCP in tablets using 4-dimethyl amino benzaldehyde and alkaline

β-naphthol. The former method results a yellow colour Schiff’s base with a

maximum absorption at 438 nm and the second method include diazo-coupling with

alkaline β-naphthol to form red-dye with a maximum absorption at 553 nm. Beer’s

law range is 10-100 µg mL-1 and 1-10 µg mL

-1 for methods first and second,

respectively.

MCP was spectrophotometrically determined by the treatment of sample

solution with dichlorophenolindophenol [DCPIP] [2] and the resulting bluish violet

radical ion exhibit a maximum absorption at 654 nm. Omran and Ahmed [3] have

proposed benzylacetone [BAC] in alkaline medium as a coupling agent for the

simultaneous spectrophotometric determination of MCP and DAP. The formed azo-

dyes show maximum absorption at 411 nm and at 437 nm for MCP and DAP,

respectively.

A spectrophotometric procedure [4] was developed for the assay of MCP in

pure and dosage forms using 2-naphthol-3, 6-disulphonic acid, the reaction mixture

was heating for about 45s at 100 °C to form an orange coloured complex with λmax

490 nm. Linear range and molar absorptivity values of this method are found to be 1-

25 µg mL-1 and 3.71×10

3 L mol

-1 cm

-1, respectively. In addition 1.10- phenanthroline-

Fe(III) reagent [5] was used for the determination of MCP.

Revanasiddappa and Manju [6] have proposed acetyl acetone as a reagent for

the determination of MCP, DAP, CPD and PABA. The same authors developed a

method [7] for the determination MCP and DAP in pure and in dosage forms by the

Chapter – 2

14

diazo-coupling of these drugs with a coupling-agent dibenzoyl methane in an alkaline

medium.

Recently, a derivative spectrophotometric method [8] was developed for the

determination of DAP. Nagaraja et al. [9] have described two spectrophotometric

procedures for the assay of DAP. In the first method, iminodibenzyl (IDB) was used

as a coupling agent in alcoholic medium, and in the second method DAP was made to

react with N-bromosuccinimide and promethazine hydrochloride to give a green

product. Absorbances were measured at 570 nm and at 610 nm in first and second

methods, respectively. Beer’s law range is 0.1-2.5 µg mL-1 and 0.5-5.0 µg mL

-1,

respectively. The same authors have reported [10] resorcinol in sulphuric acid as a

diazo-coupling agent for the determination of dapsone and the formed azo-dye shows

a maximum absorption at 530 nm. Beer’s law range was 0.1-2.5 µg mL-1. In another

method [11], sodium 1,2- naphtoquinone-4-sulphonate in pH 6.98 buffer solution was

used as chromogenic reagent for dapsone determination. The formed pink coloured

species had a molar absorption coefficient value of 3.68 ×104 L mol

-1cm

-1 at 525 nm

and Beer’s law is obeyed from 0.4 to 10 µg mL-1.

Salvador et al. [12] have reported an indirect sequential injection

spectrophotometric method for p-amino benzoic acid determination in sun screen

formulations based on the reaction of it with hypochlorite in acidic medium

and subsequent determination of residual chlorine. Beer’s law is valid from

1 to 20 µg mL-1. Another sequential-injection analysis [13] was described for the

assay of PABA in sun screens by using 8-hydroxy quinoline as a coupling agent

[linear range 2-25 µg mL-1].

In addition to the above recent methods, several other spectrophotometric

methods have also been reported for the determination MCP, DAP, CPD and PABA.

Metoclopramide was spectrophotometrically determined through diazo-

coupling reaction with N- (1-napthyl)-ethylenediamine dihydrochloride (NEDA) [14],

thymol [15], resorcinol [16], 8-anilino-1-naphthalene sulphonic acid [17],

naphtha-2-ol [17] and chromotropic acid [18]. Colorimetrically [19] it was determined

after the reaction with nitrous acid to form a stable yellow nitroso compound with a

maximum absorption at 375 nm. It was determined through ion-pair formation with

Chapter – 2

15

thiocyanate and molybdenum(V) or cobalt(II) [20], bromothymol blue [21], and based

on charge-transfer complexes with chloranil or bromanil [22]. Other chromogenic

reactions use sodium vanadate [23], ammonium metavanadate [24], sodium 3,4-

dioxonaphthalene-1-sulphonate [25], ammonium reineckate [26], citric acid –acetic

anhydride [27], catechol [28], Folin-Ciocalteu reagent [29], p-dimethylamino

cinnamaldehyde [30] and 3-methyl benzothiazolin-2-one hydrazone (MBTH) [18]. A

flow-injection spectophotometric method [31] and UV procedures [32, 33] were also

developed for its determination.

A spectrophotometric procedure [34] was developed for the determination of

dapsone [DAP] based on the oxidation of metol by chromium; the resulting

oxidation product was measured at 520 nm. In separate procedures, 3-aminophenol,

1-naphthylamine, salbutamol and clioquinol [35], 8-anilinonaphthalene-1-sulphonic

acid and resorcinol or beta-naphthol [36], 9-chloroacridine [37], NEDA [38], and

cresyl fast violet acetate [39] were some of the reagents proposed for the

spectrophotometric determination of dapsone.

Sastry et al. [40-42] have used MBTH-Fe(III), Fe(II)-1,10-phenanthroline,

chloranilic acid [chromogenic reagents] and Suprachen Violet 3B, Erioglaucine,

Naphthalene Blue, Tropaeolin 000, Wool Fast Blue BL and sulphonaphthalein dyes

[43] for the extractive spectrophotometric determination of cisapride and other basic

drugs. And also, a spectrofluorimetric [44] and derivative spectrophotometric [45]

procedures were reported in literature for the determination of cisapride in

pharmaceutical preparations. A flow-injection spectrophotometric method [46] was

developed for the determination of p-aminobenzoic acid in pharmaceuticals and in

biological fluids. Very few methods have been reported in literature for its

determination. It is appropriate to mention some of the recently reported methods

apart from spectrophotometric methods for the determination of studied drugs, such

as, voltammetric [47, 48], HPLC [49-56] and GC-MS [57].

The survey of chemical literature revealed that no attempt was made use of

citrazinic acid, imipramine hydrochloride and N-bromosuccinimide as the reagents for

the determination of studied drugs (MCP, DAP, CPD and PABA). In this chapter, the

author has presented her findings of the reaction of the above drugs with cited

reagents in three sections 2A, 2B and 2C. Spectrophotometric determination of the

Chapter – 2

16

MCP, DAP, CPD and PABA with CZA and MCP and DAP with IPH are presented in

Sections 2A and 2B, respectively. A titrimetric assay of MCP in pure as well as in

dosage forms employing NBS as the oxidimetric titrant, is presented in Section 2C.

Chapter – 2

17

Section –2A

Spectrophotometric determination of metoclopramide

hydrochloride, dapsone, cisapride and p - amino benzoic

acid with citrazinic acid

2A.1. Introduction

Citrazinic acid (CZA), chemically 2,6-dihydroxy isonicotinic acid or 1,1-

dihydro-6-hydroxy-2-oxopyridine-4-carboxylic acid. It is yellowish powder with a

greenish tinge. It is insoluble in water and slightly soluble in hot hydrochloric acid.

CZA is freely soluble in alkali hydroxide or carbonate solutions. Alkaline solutions

turn blue on standing. It has the following structure.

N

OHO

HO OH

2,6-dihydroxy isonicotinic acid

Kavlentis has used citrazinic acid as a reagent in the spectrophotometric

determination of uranium(VI) and iron(III) [58], and kinetic spectrophotometric

determination of iron(III), copper(II) and vanadium(V) [59]. Revanasiddappa and

Kiran Kumar [60] have used CZA as a coupling agent with p-amino acetophenone for

the determination of chromium. The same authors have used CZA as a coupling

agent for the determination of nitrite with p-aminoacetophenone [61] and p-

nitroaniline [62]. It has also been found application as the coupling agent in the

indirect spectrophotometric determination of chromium with p-nitroaniline. [63]

2A. 2. EXPERIMENTAL

2A. 2. 1. Apparatus

An Elico model CL-27 digital spectrophotometer with 1cm matched quartz cells was

used for all absorbance measurements.

Chapter – 2

18

2A. 2. 2. Reagents

All chemicals used were of analytical reagent grade.

Citrazinic acid (CZA, 0.1%): Prepared by dissolving 0.1g of the reagent (Fluka,

Switzerland) in 2 mL 4M NaOH and diluting to 100 mL with water.

Sodium nitrite (0.1%): It was prepared by dissolving 0.1g of sodium nitrite in 100

mL distilled water.

Sulphamic acid (2.0%): Freshly prepared by dissolving 2 g of sulphamic acid in 100

mL distilled water. Aqueous solutions of sodium hydroxide (4M) and hydrochloric

acid (1M) were used.

Standard solutions of MCP, DAP, CPD and PABA

The pharmaceutical grade chemotherapeutics were received from different

companies and were used as such. Separate aqueous solutions of MCP (IPCA

Laboratories Ltd., India) and PABA (Franco-Indian Pharma Ltd.) and methanol

solution of DAP (Intas Laboratories Ltd., India) and CPD (Intas Laboratories Ltd.,

India) were prepared. The concentration of these solutions is equal to 1000 µg mL-1.

Solutions of lower concentration were prepared by diluting the standard solutions.

2A.2.3. Standard procedure for the preparation of calibration graph

Different aliquots i,e 0.5 – 6.0 mL [50 µg mL-1 ] of MCP, 0.2 – 2.5 mL [50

µg mL-1] of DAP, 0.5 – 2.5 mL [50 µg mL

-1] of PABA or 0.3 – 1.8 mL [100 µg mL

-1]

of CPD were transferred into a series of 25 mL calibrated flasks. A volume of 1.5 mL

of 0.1 % sodium nitrite was added to each flask, followed by the addition of 1 mL of

1M HCl. After 3 min, 2 mL of 2 % sulphamic acid were added to each flask. Then,

volumes of 2 mL of 0.1 % CZA and 4 mL of sodium hydroxide solution were added

and the contents were diluted to the mark with distilled water and mixed well. After

10 min, the absorbance of the coloured azo-dye was measured at 465 nm for MCP, at

515 nm for DAP, at 500 nm for CPD and at 475 nm for PABA against the

corresponding reagent blank. The amount of drug was computed from the standard

calibration graph [Fig.2A.1] or regression equation.

Chapter – 2

19

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0 5 10 15

C on c i n µg mL-1

Absorbance [1 ]

[2]

[3]

[4]

Fig. 2A. 1. Beer’s law curves of [1] MCP [2] DAP [3] PABA and [4] CPD

2A. 2. 4. Recovery of drugs from synthetic mixtures

Two mixtures of each drug (MCP, DAP, CPD and PABA) with several

excipients whose compositions are given in Table 2A.2 were prepared. A portion of a

mixture containing 25 mg of each drug was accurately weighed. Three portions of 20

mL of distilled water were added and the mixture was thouroghly shaken for about 30

min to extract the drug from the powder before filtering the mixture. The residue after

filtration was washed with 20 mL of distilled water. The filtrate and washings were

then combined in a 100 mL calibrated flask and the volume was made up with

distilled water. An aliquot of this solution was treated as described in the Standard

Procedure 2A.2.3. The results are presented in Table 2A.1.

Conc in µµµµg mL-1

Chapter – 2

20

Table 2A. 1. Recovery of drugs from synthetic samples by the proposed method

Drug

Amount

present

(mg)

Excipients (mg) % Recovery

± SD Talc Dextrose Starch

Sodium

alginate Gelatin

Gum

acacia

MCP

100

80

150

200

250

350

150

200

100

75

75

100

50

75

99.8 ± 0.7

100.3 ± 0.5

DAP

50

75

250

200

200

250

180

250

125

100

50

75

100

50

101.3± 0.9

99.9 ± 0.3

CPD

100

120

150

250

200

150

250

150

100

75

75

100

150

100

100.1± 0.8

99.6 ± 0.5

PABA

80

120

300

200

250

300

200

150

120

80

75

50

125

75

99.8 ± 0.4

100.5 ± 0.6

* Average recovery from five experiments.

2A. 2. 5. Procedure for pharmaceutical preparations

An accurately weighed amount of powdered tablets (MCP, DAP or CPD)

equivalent to 25 mg was transferred into a 100 mL calibrated flask; about 50 mL of

water (methanol for DAP and CPD) were added and shaken thoroughly for about 30

min. The volume was made upto the mark with the respective diluents, mixed well

and filtered using a quantitative filter paper. Appropriate aliquots of the drug solution

were taken and the proposed Standard Procedure [2A. 2. 3] was followed for the

analysis of drug content.

For the analysis of an injection solution, the requisite volume was transferred

into a 100 mL calibrated flask and diluted to the mark with distilled water. The

solution was then treated as described above. The same drug samples were also

analyzed by the reference method [6] and the results are given in the Table 2A.2.

Chapter – 2

21

Table 2A. 2. Analysis of studied drugs in pharmaceutical dosage forms

a Average of five determination , b Tabulated value 2.78, c Tabulated value 6.39

Sample

Pharmaceutical

formulation

Amount

taken

(µµµµg mL-1)

Proposed

Amount

found

(µµµµg mL-1 )

Methoda

% Rec ±±±± SD

% C V

Reference

Method [6]

% Rec.±±±± SD

t-valueb

F-valuec

Perinorm

10 mg / tab

4.0

3.99

99.7 ± 0.3

0.08

99.5 ± .0.4

2.29

1.77

8.0 7.99 99.9 ± 0.5 0.06 100.1 ± 0.4 1.11 1.56

12.0 12.0 100.1 ± 0.4 0.03 99.8 ± 0.6 0.85 2.25

Reglan

10 mg / tab

4.0 3.98 99.5 ± 0.8 0.20 99.7 ± 0.6 0.96 1.77

8.0 7.98 99.8 ± 0.2 0.03 100.2 ± 0.2 0.66 1.00

MCP

12.0 11.99 99.9 ± 0.3 0.03 99.9 ± 0.5 0.49 2.77

Emenil

10 mg / tab

4.0

3.98

99.6 ± 0.8

0.20

99.9 ± 0.5

0.99

2.56

8.0 7.96 99.5 ± 0.4 0.05 100.2 ± 0.3 1.89 1.77

12.0 11.97 99.8 ± 0.5 0.04 99.7 ± 0.4 0.23 1.56

Reglan inj

5 mg / mL

4.0 4.00 100.0 ± 0.4 0.10 100.2 ± 0.3 1.62 1.77

8.0 7.98 99.8 ± 0.3 0.04 100.0 ± 0.4 1.15 1.77

12.0 12.0 100.1 ± 0.5 0.12 99.9 ± 0.4 0.99 1.56

Dapsone

25 mg / tab

2.0

1.99

99.6 ± 0.8

0.40

100.1 ± 0.5

1.89

2.56

3.0 2.97 99.0 ± 0.4 0.13 99.6 ± 0.5 1.29 1.56

4.0 3.99 99.8 ± 0.5 0.12 99.1 ± 0.7 0.84 1.96

DAP

Dapsone

100 mg/ tab

2.0

1.99

99.6 ± 0.8

0.40

99.8 ± 0.5

1.90

2.56

3.0 2.98 99.5 ± 0.4 0.13 100.1 ± 0.3 1.75 1.77

4.0 3.98 99.5 ± 0.5 0.12 99.9 ± 0.3 1.65 2.77

Cisawal

10 mg / tab

2.0

1.99

99.6 ± 0.4

0.20

99.8 ± 0.4

2.01

1.56

4.0 3.98 99.5 ± 0.7 0.18 98.9 ± 0.6 0.68 2.25

6.0 5.98 99.8 ± 0.4 0.07 100.1 ± 0.5 0.56 1.56

Ciza

10 mg / tab

2.0

1.99

99.6 ± 0.5

0.25

99.8 ±.0.6

2.28

1.56

CPD 4.0 4.00 100.1± 0.4 0.10 99.1± 0.7 1.09 2.25

6.0 5.98 99.7± 0.4 0.07 99.9 ± 0.4 1.11 1.56

Unipride

10 mg / tab

2.0

2.00

100.1 ± 0.8

0.40

99.9 ± 0.6

1.28

1.77

4.0 3.99 99.8 ± 0.6 0.15 99.5 ± 0.4 1.1 2.25

6.0 5.98 99.6 ± 0.4 0.07 99.9 ± 0.2 0.41 4.00

Chapter – 2

22

2A.3 Results and Discussion

The proposed method is based on the diazo-coupling reaction of the studied

drugs with citrazinic acid in an alkaline medium to give an orange red coloured azo-

dye with a maximum absorption in the range 465 – 515 nm. The absorption spectra

of the azo-dyes are depicted in Fig. 2A. 2.

Two steps are involved in the reaction that produces the coloured dye. In the

first step, these chemotherapeutics are treated with nitrite solution in acidic medium

and they undergo diazotisation to give the diazonium chloride ion. In the second step,

the diazonium ion is coupled with a coupling agent, citrazinic acid, to form an azo-

dye in an alkaline medium. The reaction system is represented in Scheme 1. Here,

MCP is used as the model compound, since the other compounds also behaved

similarly to it (Scheme 2A.1).

+ NO2

_

+ H+

N

++

H2O

N

OHO

HO OH

+

O

NH

OCH3

N

Cl

NH2

O

NH

OCH3

N

Cl

N

[A]

[A]

N

O

NH

OCH3

N

Cl

N

N

O OH

OHHO

Azo-dye

MCP

NaOH

CZA

Scheme. 2A. 1. Reaction pathway of MCP with CZA

2A. 3. 1. Absorption spectra

In order to establish the optimum wavelength for maximum absorption (λmax) of the

formed azo-dye, a known amount of the pure drug solutions [MCP – 10 µg mL-1,

DAP – 4 µg mL-1, PABA – 4 µg mL

-1and CPD – 28 µg mL

-1] were taken and the

Standard Procedure (2.A.2.3) was followed to develop coloured azo-dye of the

Chapter – 2

23

studied drugs. The absorption spectra of the formed dye and its reagent blank were

scanned in the region 400 – 600 nm against the corresponding reagent blank and

distilled water, respectively. The formed coloured azo-dye showed maximum

absorption at 465 nm for MCP, at 515 nm for DAP at 500 nm for CPD and at 475 nm

for PABA, and the reagent blank had negligible absorption at these wavelengths

(Fig 2A.2).

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

400 450 500 550 600

Wavelength in nm

Absorbance

[1]

[2]

[3]

[4]

Blank

Fig. 2A. 2. Absorption spectra of [1] MCP, [2] DAP, [3] PABA, [4] CPD and blank with

CZA

2A. 3. 2. Optimization of experimental parameters

The factors influencing the colour development, sensitivity and adherence to

Beer’s law were investigated and were reported below.

2A. 3. 2. 1. Effect of the reagent concentration

The effect of the CZA concentration and its volume on the absorbance of the

coloured dye was studied for the cited drugs at the above-specified wavelengths. The

maximum colour intensity was obtained with 0.5 – 5.0 mL of 0.1 % CZA solution for

all the drugs. A volume of 2 mL of 0.1 % CZA was found to be sufficient in a total

volume of 25 mL of the reaction mixture. Thus, the same volume was employed in

the subsequent studies. The results are presented in the Fig. 2A. 3.

Chapter – 2

24

0.272

0.28

0.288

0.296

0 1 2 3 4 5

Vol.of CZA in mL

Absorb

ance

Fig. 2A. 3. Effect of CZA on the absorbance of the azo-dye

Fig. 2A. 4. Effect of sodium nitrite on the absorbance of the azo-dye

2A. 3. 2. 2 Effect of sodium nitrite

The absorbance values were found to be constant in the volume range 0.5 – 4.0 mL of

0.1 % sodium nitrite under the optimum conditions. An optimum volume of 1.5 mL of

0.1 % NaNO2 was fixed and thus used in further studies and the excess of nitrite could

be removed by the addition of 2 mL of 2 % sulphamic acid otherwise, nitrite strongly

interferes with the method. The results are depicted in Fig.2A.4.

0.36

0.364

0.368

0.372

0.376

0 1 2 3 4 5

Vol.NaNO2in mL

Absorbance

Chapter – 2

25

2A. 3. 2. 3 Effect of acid

The diazotisation reaction was carried out at room temperature (ca. 25 ± 2 °C).

The hydrochloric acid concentration for diazotisation was investigated. A suitable

acidity as evident from the maximum absorbance and stability of the azo-dye formed

was found to be 1 mL of 1 M hydrochloric acid in a 25 mL of reaction mixture.

2A. 3. 2. 4 Effect of alkali

To develop a quantitative method based on this reaction, a study was

conducted to determine the most effective alkalies and optimum alkali concentration

to be used. Sodium hydroxide was found to be the most effective base compared to

sodium carbonate or ammonia. The orange coloured azo-dyes are unstable in

ammonia and do not give maximum colour intensity in sodium carbonate medium.

The orange coloured azo dyes, stable in the optimum concentration of sodium

hydroxide solution leading to a maximum intensity and stability of colour was found

to be 4 mL of 4 M sodium hydroxide for all the studied drugs under optimum

conditions. The results are presented in Fig. 2A.5.

Fig. 2A. 5. Effect of alkali on the absorbance of the azo-dye

0.3

0.34

0.38

0 2 4 6 8

Vol. NaOH, mL

Absorbance

Chapter – 2

26

2A. 3. 2. 5 Effects of the temperature and time

The coloured azo-dyes developed rapidly after the addition of reagents and

attained maximum intensity after about 10 min at room temperature (25 ± 2 °C).

Cooling to 0 – 5° C was not necessary and the formed azo-dyes are stable for a period

of more than 2 h. Beyond this time, the absorbance values are gradually decreasing.

2A. 3. 2. 6 Order of addition of reagents

Reagents were added in the described sequence in order to achieve the

maximum sensitivity of the colour system. Any change in the order of addition of

reagents affects the formation of azo-dye and the sensitivity of the system.

2A. 3. 3 Calibration graphs and Analytical parameters

The calibration graphs for the studied drugs as obtained under optimum

conditions are shown in Fig-2A.1. Good linear relationships were obtained over the

concentration ranges given in Table.2A.3. The corresponding molar absorptivity

values from Beer’s law data and their Sandell’s sensitivity values are presented in

Table.2A.3. The slope, intercept, correlation coefficients, detection limit and

quantitation limit of the method are also given in Table .2A.3.

Chapter – 2

27

Table 2A. 3. Optical characteristics of the studied chemotherapeutics.

Parameter MCP DAP CPD PABA

Beer’s law limit

(µg mL-1)

1.0-12.0 0.4-5.0 1.2-7.2 1.0-5.0

Molar absorptivity

(L mol-1cm

-2). 10

4

1.92 4.52 3.15 2.15

Sandell’s sensitivity

(µg cm-2)

0.0184 0.0054 0.0147 0.0063

Correlation

coefficient [r]

0.9999 0.9999 0.9999 0.9999

Regression equation

[Y*]

Slope [b] 0.0522 0.1606 0.0575 0.1359

Intercept [a] 0.0088 0.0335 0.0292 0.0471

Detection limit [DL]

(µg mL-1)

0.1892 0.0210 0.0845 0.0277

Quantitation limit

[QL] (µg mL-1)

0.5734 0.0638 0 .2562 0.0841

*y = a + bx, where x is the concentration in µg mL-1

Interference studies

In order to evaluate the suitability of the proposed method for the analysis of

pharmaceutical preparations of the studied drugs, the interference of associated

excipients and diluents in dosage forms was investigated. Talc, starch, dextrose,

sodium alginate and gelatin in amounts far in excess of their normal occurrence in

pharmaceutical formulations do not interfere. The results presented in Table 2A.1

shows that there is good agreement between the amounts of drugs taken and found in

the presence of associated excipients and diluents.

Precision

The precision of the proposed method was evaluated by replicate analysis

(within-day and Between-day) of samples containing the studied drugs [MCP, DAP,

CPD and PABA] at three different concentrations. The low values of the coefficient

Chapter – 2

28

of variation (CVs) both at the low and high concentrations reflect the high precision

of the proposed method [Table-2A.4].

Table. 2A. 4. Within-day and between-day studies of chemotherapeutics.

Average value of five determinations carried out over five days.

Applications to the pharmaceuticals

The proposed method was successfully applied to the analysis of MCP, DAP

and CPD in pharmaceutical preparations. The results of an assay of Perinorm,

Regaln, Emenil, Cisawal, Ciza, Unipride and Dapsone tablets and injection solutions

are presented in [Table. 2A.2], compare favorably with the reference method [6]. A

statistical analysis of the results by Student’s t- and F- tests showed no significant

difference in accuracy and precision between the proposed and reference methods

[Table-2A.2].

Sample

Amount

taken

(µµµµg mL-1)

Within day

Amount

found ±±±± SD

(µµµµg mL-1 )

CV%

Amount

taken

(µµµµg mL-1)

Between day

Amount

found ±±±± SD

(µµµµg mL-1 )

CV%

MCP

4.0 3.91 ± 0.05 1.27 4.0 3.90 ± 0.05 1.28

8.0 7.56 ± 0.02 0.25 8.0 7.94 ± 0.04 0.50

12.0 12.05 ± 0.01 0.08 12.0 11.95 ± 0.03 0.25

DAP

2.0

1.99 ± 0.07

3.51

2.0

1.99 ± 0.08

4.02

3.0 2.97 ± 0.05 1.68 3.0 2.91 ± 0.05 1.71

4.0 3.91 ± 0.03 0.76 4.0 4.01 ± 0.03 0.74

CPD

2.0

1.97 ± 0.05

2.53

2.0

1.96 ± 0.08

4.08

4.0 3.89 ± 0.03 0.77 4.0 3.90 ± 0.04 1.02

6.0 5.92 ± 0.02 0.33 6.0 5.97±0.02 0.33

PABA

2.0

1.97 ± 0.06

3.04

2.0

2.01 ± 0.03

2.98

3.0 3.01 ± 0.05 1.66 3.0 2.96 ± 0.06 1.35

4.0 3.98 ± 0.03 0.75 4.0 3.99 ± 0.03 0.75

Chapter – 2

38

Section –2C

Titrimetric method for the determination of metoclopramide

hydrochloride using N-bromosuccinimide

2C. 1. Introduction

Titrimetry is one of the classical methods and finds the application despite and

advent of modern physico-chemical methods. In contrast, e.g, to HPLC, titration is an

absolute method. The titration takes place strictly stoichiometrically and normally

very rapidly. If a titrant with a known titer is used then the content of the sample can

be determined directly. In recent years, titrimetric methods have found wide

applications in the quantitative analysis of therapeutic agents. In the following

paragraphs, a brief review of the oxidimetric titrant N-bromosuccinimide is presented.

N-Bromosuccinimide [NBS] is a chemical reagent first synthesized by

Seliwanow [64]. It is a white solid, melts at 175-178 °C and is soluble in hot water

(1.47 g/100 mL at 25 °C). NBS has the following structural formula.

O

O

NBr

N-bromosuccinimide

It has been used as a valuable reagent for the determination of several

bioactive compounds of therapeutic interest. A brief survey of its analytical utility is

presented in the following paragraphs.

Very recently, NBS was used as a reagent for the spectrophotometric

determination of fluoroquinolone antibiotics [65]. Basavaiah et al. have described

titrimetric and spectrophotometric methods for the assay of salbutanol sulphate [66]

and metoprolol tartrate[67] in pharmaceuticals using NBS.

Chapter – 2

39

Recently, Al-Momani was reported flow injection spectrophotometric

procedure for the determination of meloxicam, piroxicam and tenoxicam [68] and

levofloxacin [69] with NBS.

NBS has been used as a reagent for the spectrofluorimetric determination of

paracetamol [70], a titrimetric determination of nizatindine in capsule [71],

spectrophotometric determination of famotidine [72] and dapsone [73]. A detailed

account of the analytical applications of NBS for the analysis of several organic

compounds is given in the monograph compiled by Mathur and Narang [74]. Up to

2001, the analytical utility of NBS was admirably discussed by Manju [75].

Aforementioned literature survey revealed that no attempt has been made use of NBS

as the oxidimetric titrant for the determination of MCP in pure and in dosage forms.

In this section, an indirect titrimetric assay of MCP with NBS is reported.

2C.2 EXPERIMENTAL

2C. 2. 1. Apparatus

Pre calibrated pipettes, burettes and measuring flasks (Borosil or Corning

make) were used.

2C.2.2 Reagents

All chemicals used were of analytical reagent grade.

N-Bromosuccinimide (NBS) [0.01 M]

0.01 M NBS [Loba Chemie., India] was prepared by dissolving about 1.8 g of

freshly crystal lined powder in 100 mL water with the aid of heat, and diluted to 1

liter with distilled water and standardized iodometrically.

Sodium thiosulphate (0.01M)

Prepared by dissolving 2.48 g of sodium thiosulphate in 1 liter distilled water

and standardized using pure potassium dichromate.

Chapter – 2

40

Others: Potassium iodide [10 %], 5M acetic acid and starch indicator [1%] was

prepared in the usual way.

Standard solution of MCP

Pharmaceutical grade MCP was received from IPCA Laboratories Ltd., India

as gift and was used as received. A stock solution containing 1000 µg mL-1 of drug

was prepared daily by dissolving accurately weighed amount of MCP in distilled

water. Solutions of lower concentration were prepared by diluting the stock solutions

with distilled water.

2C. 2. 3. Determination of drug in pure form

A 10 mL aliquot of pure drug solution containing 1-13 mg of MCP was

accurately measured and transferred into a 100 mL conical flask. The solution was

acidified by adding 5 mL of 5M acetic acid followed by the addition of 0.01 M NBS.

The content was mixed and kept aside for 15 min. Then, 5 mL of 10 % potassium

iodide was added to the flask and the liberated iodine was titrated against sodium

thiosulphate (0.01M) using starch indicator towards the end point. A blank titration

was performed under identical conditions using all the reagents except the drug. The

amount of the drug was calculated from the equation,

(A-B) MwM

Amount of drug found (mg) = N

where, A = Volume of thiosulphate solution used in blank titration.

B = Volume of thiosulphate solution used in sample titration.

Mw = Relative molecular mass of MCP

M = Molarity of thiosulphate

2C. 2. 4. Recovery of drugs from synthetic mixture

The mixtures of MCP with several excipients whose composition are given in

Table 2C.1 were prepared. A portion of a mixture containing 100 mg MCP was

accurately weighed. Three portions of 20 mL of distilled water were added and the

mixture was shaken thoroughly for about 30 min to extract the drug from powder

before filtering the mixture. The residue was then washed with 10 mL of distilled

Chapter – 2

41

water. The filtrate and washings were then combined in a 100 mL calibrated flask and

the volume was made upto the mark with distilled water. An aliquot of this solution

was analysed by following the above Procedure 2C. 2. 3. The results are presented in

Table 2C.1.

Table 2C.1: Analysis of drugs from various excipients

Name of

the

compound

Amount

present

(mg)

Excipients (mg) %

Recovery

± SD

Talc

Dextrose Starch Sodium

alginate

Gelatin Gum

acacia

MCP 75 150 50 150 75 50 75 99.7 ± 0.56

50 200 100 100 50 75 100 99.0 ± 0.78

Average recovery from five experiments.

2C.2.5 Analysis of MCP in pharmaceutical formulations

Tablets: An accurately weighed amount of powdered tablets equivalent to 100 mg

was transferred, extracted and analyzed as described under recovery of drugs from

synthetic mixtures in Section 2C.2.4.

Injections: For analysis of injection solution, an appropriate volume of the sample

containing 100 mg of the drug was transferred into a 100 mL calibrated flask and

diluted to the mark with distilled water. The drug content in the diluted solution was

determined as described above. The samples were also analyzed by the reference

method of British Pharmacopoeia [76] and the results are presented in Table 2C.2

Table 2C.2: Analysis of MCP in pure drug samples.

Formulation % Recovery ± SD

t-value b F-value

c

Reference method Proposed methoda

Perinorm [10 mg/tab] 100.20 ± 0.55 99.90 ± 0.37 2.0 2.20

Reglan [10 mg/tab] 99.80 ± 0.91 100.50 ± 0.50 1.7 3.31

Emenil [10 mg/tab] 100.10 ± 0.68 100.40 ± 0.90 1.4 1.75

Perinorm [5 mg/mL] 99.50 ± 0.75 99.90 ± 0.49 1.09 2.34

Reglan [5 mg/mL] 100.50 ± 0.40 101.0 ± 0.81 1.24 4.10

a Average of five determination , b Tabulated value 2.78, c Tabulated value 6.39

Chapter – 2

42

2C. 3. Results and Discussion

This method is based on the oxidation of MCP by a known excess of NBS and

followed by the determination of unreacted NBS iodometrically. NBS is found to

react quantitatively with MCP in acetic acid medium and the amount of NBS reacted

corresponds to the drug content. Considering the available literature [74] and

stoichiometry, it may be postulated that the MCP undergoes oxidation at suitable

positions. The possible oxidation process based on the literature reports, is as shown

in the following Scheme 2C.1

O

O

N Br

N-bromosuccinimide

+ 3

O

HN

OCH3

N

Cl

H2N

O

BrN

OCH3

N

Cl

H2NBr

Br

Scheme 2C.1. Probable reaction between MCP and NBS

2C. 3. 2. Optimization of experimental variables

In order to establish the optimum conditions for the quantitative estimation of

MCP, the effects of acids and time were studied, and reported in the following

paragraphs.

2C. 3. 2. 1. Effect of acid

At laboratory temperature (ca 25 ± 2 °C), the rate of the reaction was found to

depend on the nature of the acid and its concentration. Different acids such as

hydrochloric acid, sulphuric acid and acetic acid were tried as reaction medium to

obtain reproducible and stoichiometric results. Non-stoichiometric results obtained in

a hydrochloric acid medium as well as sulphuric acid medium Volume of 2-10 mL of

5M acetic acid gave reproducible results and sharp end point in a total volume of

about 25 mL reaction mixture. Thus, a volume of 5 mL of 5M acetic acid was used in

all subsequent work.

Chapter – 2

43

2C. 3. 2. 2. Effect of time

The oxidation reaction was found to be complete and quantitative in 15

minutes and contact times upto 30 min had no effect on the stoichiometry and the

results. Beyond 30 min, a small amount of NBS consumed but without producing any

definite stoichiometry. Hence, it is necessary to terminate the oxidation step at the end

of the 15th min to obtain the reproducible results.

2C. 3. 2. 3. Mole ratio

Under the optimum conditions, three moles of NBS were required for the

complete oxidation of MCP (1:3, drug: NBS), which is conformity, with the scheme

of the oxidation process presented in Scheme 2C.1.

2C. 3. 2. 4. Range of determination

Under the experimental conditions, 10 mL of drug solution containing the

amounts ranging from 1-13 mg of MCP was titrated. It was observed that inaccurate

results (error > 5%) were obtained outside the above limits and do not obtain a sharp

end point beyond the upper limit.

2C. 3. 2. 5. Precision of the method

The precision of the developed method was assessed from the results of

replicate analysis on pure drug at three different concentrations (low, medium and

high). The low values of CVs at both the low and high concentration reflect the high

precision of the method. The results are given in Table 2C.2

2C. 3. 2. 6. Application to pharmaceutical preparations

The applicability of the proposed method for the assay of the different

pharmaceutical formulations containing MCP was examined. The results were

statistically compared with those obtained by the reference method of British

Pharmacopoeia [76].The t-test and F-tests were carried out, which showed that the

proposed method and other reported methods are of comparable accuracy and

precision. The results are given in Table 2C.2

Chapter – 2

44

Conclusions

The methods presented in this chapter have been validated and successfully

used for the determination of selected therapeutics (MCP, DAP, CPD and PABA) in

their pure form and in pharmaceutical formulations, with good accuracy and

precision. Both spectrophotometric methods (2A and 2B) are based on diazo-coupling

reaction utilizing citrazinic acid and imipramine hydrochloride as the coupling agents

shows high sensitivity and simplicity of the methods, and cooling to 0 – 5 °C was not

necessary for diazotization. The formed azo-dyes are stable for a sufficient interval of

time, which makes the methods more practicable.

In section 2C, the developed titrimetric procedure for the assay of MCP using

NBS as the oxidimetric titrant is simple, inexpensive, accurate and more precise than

the other instrumental methods. The methods developed are compared reported

methods and are presented in Table. 2C. 4. The proposed procedures offer the

advantage of simplicity, rapidity and low cost.

Chapter – 2

45

2C. 4. Comparison of the proposed method with other spectrophotometric

methods

Reagent

Range,

µg mL-1

References Remarks

MBTH 2-24 [18] Less sensitive

Catechol 10-40 [28] Less sensitive

Suprachen Violet 3B and 2.5-4 [41] Extractive and time

consuming

Tropaeolin 000 2.5-25 [41]

9-Chloroacridine 20-200 [37] Time consuming and less

sensitive

Ammonium molybdate and

thiocyanate

1-20 [20]

Extractive and less sensitive

Chloranil or bromanil 40-160 [22] Requires heating at 80º C

NaVO3 40-160 [23] Less sensitive and requires

heating

2-naphthol-3,6 disulphonic

acid

1-250 [4] Requires heating at 100º C

and less sensitive

p-dimethylamino

cinnamaldehyde

5-30 [30] Derivative and less sensitive

p-dimethylamino

cinnamaldehyde and

phosphoric acid

160-560 Less sensitive

Bromocresol

green,bromothymol blue and

bromocresol purple

2-10 Less sensitive

8-hydroxy quinoline 2-25 [13] Less sensitive

Citrazinic acid

Imipramine hydrochloride

Titrimetric

1-12 [MCP]

0.4-5[DAP]

1-5 [PABA]

1.2-7.2[CPD]

0 -5.0 [MCP]

0 - 4.0 [DAP]

1 -13 mg

Proposed

methods

Highly sensitive,

inexpensive, rapid and

facile

Chapter – 2

46

REFERENCES

1. S. A. Patel, C. N. Patel and M. M. Patel, Indian J. Pharm. Sci., 2006, 68, 397.

2. S. E. Marwa, S. Abdalla, M.N. Elbolkiny, and M.K. Hawa, J. Drug Research,

2004, 25, 107.

3. Orman, A. Ahamed, Chem. Pharm. Bull., 2005, 53, 1498.

4. A. A. Kazi, T. Aman, A. Mumtaz, K. Shams and M. Aslam, Proc. Pakistan

Acad. Sci., 2004, 41, 113.

5. S.E. Marwa, S. Abdalla, M.N. Elbolkiny, and M. Kalil Hawa, Scientia

Pharmaceutica, 2004, 72, 73.

6. H.D. Revanasiddappa and B. Manju, Drug Dev. Ind. Pharm., 2002, 23, 517.

7. H.D. Revanasiddappa and B. Manju, J. Pharm. Biomed. Anal., 2001, 25, 631.

8. M.I. Toral, A. Tassara, C. Soto and P. Richter, J. AOAC. Inter’l., 2003, 86,

241.

9. P. Nagaraja, H.S. Yathirajan, K.R. Sunitha and R.A. Vasantha, Anal .Lett.,

2002, 35, 1531.

10. P. Nagaraja, K.R. Sunitha, R.A. Vasantha, and H.S. Yathirajan, Indian Drugs,

2001, 38, 489.

11. H.Y. Wang, L.X. Xu, Y. Xiao and J. Han, Spectrochimica Acta , Part A, Mol.

Biomol. Spectro., 2004, 60A, 2933.

12. A. Salvador, A. Chisvert, A. Rodriguez and J.G. March, Anal. Chim. Acta,

2003, 493, 233.

13. A. Chisvert, J.V. Izquierdo and A. Salvador, Anal. Bioanal. Chem., 2002,

374, 963.

14. D.N. Shingbal and K.V. Sawant, Indian Drugs, 1982, 19, 239.

15. O.S. Kamalapurakar and J.J. Chudasama, Indian Drugs, 1983, 20, 298.

16. D.M. Shingbal and S.V. Joshi, Indian Drugs, 1984, 21, 517.

17. S.S. Zarapker and S.R. Mehara, Indian Drugs, 1989, 26, 357.

18. P.G. Ramappa, Somashekar and H.D. Revanasiddappa, Indian Drugs, 1999,

36, 381.

Chapter – 2

47

19. D.M. Shingbal and S.D. Naik, Indian Drugs, 1981, 18, 441.

20. F.M. Abdel-Gawad and N.M. El-Guindi, Anal. Lett., 1995, 28, 1437.

21. D.M. Shingbal and V.S. Velingkar, Indian Drugs, 1988, 25, 529.

22. A.E. El-Gendy, Spectrosco. Lett., 1992, 25, 1297.

23. P.G. Ramappa and C. Shivakumara, East. Pharm., 1990, 33, 149.

24. S. Singh, S. Shukla and I.C. Shukla, J. Inst. Chem (India)., 1990, 62, 126.

25. D.M. Shingbal and H.S. Kudchadkar, Indian Drugs, 1987, 25, 75.

26. R.G. Bhatkar and S.K. Chodankar, East. Pharm., 1981, 24, 125.

27. J. Emmanuel and Yogyanarayanan, East. Pharm., 1981, 24, 133.

28. C.S.P. Sastry, D.S. Mangala and B.G. Rao, J. Inst. Chem (India)., 1984,

56,182.

29. G.R. Rao, A.B. Avadhanulu and D.K. Vasta, East. Pharma, 1990, 33, 147.

30. A.B. Moussa, J. Pharm. Biomed. Anal., 2000, 23, 1045.

31. M.R. Herrero, A.M. Romero and J.M. Calatayud, Talanta, 1998, 47, 223.

32. S. Raghuveer, B.E. Rao, C.M.R. Srivastava and D.K. Vasta, East. Pharm.,

1992, 35, 125.

33. Z. Chen and X. Wang, Yaowa-Fenxi-Zazhi, 1992, 12, 113 through Anal.

Abstr., 1993, 55, 5G171.

34. R. Ramakrishna, P. Siraj and C.S.P. Sastry, Indian J. Pharm. Sci., 1979, 41,

200.

35. R.T. Sane, V.K. Shastri, P.G. Anaokar and V.G. Nayak, Indian Drugs, 1982,

19, 198.

36. S.S. Zarapker, S.J. Vaidya and R.V. Lele, Indian Drugs, 1988, 26, 115.

37. I. Shoukrallah, A. Sakla and R. Wintersteiger, Pharmazie, 1990, 45, 675.

38. K.T. Shetty, P.M. Naik and P.R. Mahadevan, Indian J. Clin. Biochem., 1990,

5, 101.

39. C.S.P. Sastry, K.R. Srinivas and K.M.M.K. Prasad, Anal. Lett., 1996, 29,

1329.

Chapter – 2

48

40. C.S.P. Sastry, Y. Srinivas and P.V.S. Rao, Microchim. Acta.,1997, 126, 63

41. C.S.P. Sastry, Y. Srinivas and P.V.S. Rao, Talanta, 1997, 44, 517.

42. C.S.P. Sastry, S.G. Rao and K.R. Srinivas, Indian Drugs, 1998, 35, 594.

43. H.H. Abdina, Alexandrin. J. Pharma. Sci, 2000, 14, 75.

44. M.I.G. Martin, C.G. Perez and M.A.B. Lopez. Anal. L. H., 1994, 27, 1713.

45. E.M. Hassan, M.E.M. Hagga and H.I. Al-Johar, J. Pharm. Biomed. Anal.,

2001, 24, 659.

46. M.I. Evgen’ev, S.Y. Garmonov and L.S. Shakirova, J. Anal. Chem, 2000, 55,

696.

47. O.A. Farghaly, M.A. Taher, A.H. Naggar and A.Y. El-Sayed, J. Pharm. Biom.

Anal., 2005, 38, 14.

48. E. Satana, B. Uslu, and S.A. Ozkan, Pharmazie, 2002, 57, 501.

49. S. Kwadijik and J.S. Torano, Biomed. Chrom., 2002, 16, 203.

50. Y.L. Chen, H. Junga, X.Y. Jiang and W. Naidong, J. Separation Sci., 2003,

26, 1509.

51. S. Huang, M. Zhang, and K. Chen, Huaxue Fenxi Jiliang, 2003, 12, 21.

52. J.E. Belgaied and H. Trabelsi, J. Pharm. Biomed. Anal., 2003, 33, 991.

53. D.B. Kiss, K.B. Nemes and I. Klebovich, Chromatographia, 2003, 57, 47.

54. M.E. Abdel-Hamed and D. Sharma, J. Liq. Chrom. and Related Tech., 2004,

27, 641.

55. H. Lamparczyk, A. Chmielewska, L. Konieczna, A. Plenis and P.K. Zarzycki,

Biomed. Chrom., 2001, 15, 513.

56. D.J. Schakel, D. Kalsbeek and K.Boer, J. Chrom. A., 2004, 17, 127.

57. K.W. Riggs, A. Szieitz, D.W. Rurak, A.E. Mutlib, F.S. Abbot and J.E.

Axelson, J. Chrom. Biomed. Appl., 1994, 660, 315.

58. E. Kavlentis, J. Chem. Soc. Dalton Trans., 1990, 484.

59. E. Kavlentis, Analusis, 1989, 17, 217.

60. H.D. Revanasiddappa and T.N. Kiran Kumar, J. Anal. Chem., 2001, 56, 1084.

Chapter – 2

49

61. H.D. Revanasiddappa and T.N. Kiran Kumar, Fresenius Environ. Bull., 2001,

10, 781.

62. H.D. Revanasiddappa and T.N. Kiran Kumar, Chem. Anal. (Warsaw), 2003,

48, 759.

63. H.D. Revanasiddappa and B.P. Dayananda, Anal. Chem., An Indian Journal,

2006, 2, 113.

64. T. Seliwanow, Ber. Dev. Chem. Ges., 1893, 26, 423.

65. H. Askal, I. Refaat, I. Darwish and M. Marzouq, Chem. Pharm. Bull., 2007,

55, 1551.

66. K. Basavaiah, B.C. Somashekar and V. Ramakrishna, Acta Pharm., 2007, 57,

87.

67. K. Basavaiah and B.C. Somashekar, E-J. Chem., 2007, 4, 117.

68. I.F. Al-Momani, Anal. Sci., 2006, 22, 1611.

69. I.F. Al-Momani, Anal. Lett., 2006, 39, 741.

70. H.M. Abdel-Wadood, N.A. Mohamed and F.A. Mohamed, J. AOAC Inter’l.,

2005, 88, 1626.

71. F.A. El-Yazbi, A.A. Gazy, H. Mahgoub, M.A. El-sayed and R.M. Youseef, J.

Pharm. Biomed. Anal., 2003, 31, 1027.

72. A.S. Amin, S.A. Shama, I.S. Ahmed and E.A. Gouda, Anal. Lett., 2002, 35,

1851

73. P. Nagaraja, H.S. Yathirajan, K.R. Sunitha, R.A. Vasantha, Anal. Lett., 2002,

35, 1531.

74. N. K. Mathur and C.K. Narang, “The Determination of Organic Compounds

with N-Bromosuccinimide and Allied Reagents”, Academic Press, New York,

1975.

75. B. Manju, Thesis “Analytical studies on some chemotherapeutic agents”,

University of Mysore, 2001.

76. British Pharmacopoeia, H. M. Stationery Office, London, Vol. I and II,

2003, pp.1010.

Chapter – 2

29

Section-2B

Diazocoupling method for the determination of

metoclopramide hydrochloride [MCP] and dapsone

[DAP] using imipramine hydrochloride

2B.1. Introduction

Imipramine [IPH] chemically, 10,11-dihydro-N,N-dimethyl-5H-dibenz [b,f]

azepine-5-propanamine hydrochloride. Imipramine hydrochloride crystals from

acetone (m.p -174-175 °C). It acquires a yellow to reddish discolouration under the

influence of light and it has the following structure:

It is freely soluble in water, less soluble in alcohol and sparingly soluble in acetone.

An examination of literature review is revealed that, no researchers used it as a

reagent for the analysis of either metal ions or pharmaceutical samples by

spectrophotometry. In this section, the author has utilized imipramine as the coupling

agent for the determination of MCP and DAP through diazo-coupling reaction.

2B.2. EXPERIMENTAL

2B. 2.1. Apparatus

Instruments used are described in Section 2A.2.1.

2B. 2. 2. Reagents

All chemicals used were of analytical reagent grade.

Imipramine hydrochloride (IPH, 0.5%): It was prepared by dissolving 0.5 g of IPH

(Max Pharma Ltd. India) in 100 mL distilled water.

Sodium nitrite (0.1%), sulphamic acid (2.0 %), 1M and 6 M HCl were used.

Standard solution

Preparation of pure drug samples of MCP and DAP is described in Section 2A.2.2.

N

N

Chapter – 2

30

2B. 2. 3. Preparation of calibration graph

Accurately measured volumes of drug solutions equivalent to 0 -5.0 µg mL-1 and

0 - 4.0 µg mL-1 final solution of MCP and DAP, respectively, were transferred into a

series of 10 mL calibrated flasks. Then, a volume of 1mL of 0.1% sodium nitrite

solution was added to each flask followed by 0.5 mL of 1 M hydrochloric acid. After

3 min, 1 mL of 2% sulphamic acid was added to each flask. A volume of 2 mL of

0.5% IPH solution was added and the contents were diluted to the mark with 6 M

hydrochloric acid and mixed well. After 5 min, the absorbance of the coloured azo

dye was measured at 570 nm for both MCP and DAP against the corresponding

reagent blank. The amount of drug was computed from the standard calibration

graphs [ Fig 2B.1].

0

0.1

0.2

0.3

0.4

0.5

0.6

0 2 4 6

Conc in µg mL-1

Ab

sorb

an

ce

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0 2 4 6

Conc in µg mL-1

Ab

sorb

an

ce

(a) (b)

Fig 2B. 1. Beer’s law curves of (a) MCP and (b) DAP

2B.2.4 Procedure for pharmaceutical preparations

Twenty tablets of MCP and DAP were weighed and finely powdered. An

accurately weighed amount, equivalent to 20 mg of the respective drug was

transferred into a 100 mL calibrated flask and dissolved in 75 mL of distilled water

and the contents were thoroughly shaken for about 30 min and completed to volume

with distilled water (methanol was used for DAP), and then filtered. Appropriate

Chapter – 2

31

aliquots of the drug solution were taken and the above procedure [2B.2.3] was

followed for analyzing the drug content.

To analyze the injection solution, the requisite volume was transferred to a

100 mL standard flask and diluted to the mark with distilled water. The drug content

in the diluted solution was determined as described above. For comparison, the

reference method [6] was employed to determine the drug content in the same

pharmaceutical samples and the results of the analysis are given in Table 2B.1

Chapter – 2

32

Table 2B.1. Results of assay of metoclopramide and dapsone in dosage forms.

a) Average of five determinations; b) Tabulated t - value 2.78; c) Tabulated F value 6.39.

Sa

mp

le

Ph

arm

ace

uti

ca

l

form

ula

tion

Proposed Methoda Reference

Method[ 6 ]

t-valueb

F-valuec Amount

taken

(µµµµg mL-1

)

Amount

found

(µµµµg mL-1

)

% Rec ±±±± SD % Rec.±±±± SD

MC

P

Perinorm

10 mg / tab

1.0

0.99

99.6 ± 0.50

100.2 ± 0.40

2.30

2.10

2.0 1.99 99.5 ± 0.30 99.8 ± 0.40 1.15 1.70

3.0 2.99 99.6 ± 0.10 100.1 ±.0.10 0.80 1.17

Reglan

10 mg / tab

1.0

0.99

99.8 ± 0.30

99.9 ± 0.20

0.23 1.59

2.0 1.99 99.5 ± 0.20 99.9 ± 0.30 0.89 2.25

3.0 3.00 100.0 ± 0.90 100.3 ± 0.70 0.49 1.65

Emenil

10 mg / tab

1.0

1.00

100.0 ± 0.55

99.8 ±. 0.50

1.26

1.21

2.0 1.99 99.4 ± 0.56 99.8 ± 0.49 1.06 1.30

3.0 2.99 99.6 ± 0.58 99.8 ± 0.50 1.47 1.29

Perinorm

5 mg / mL

1.0

0.99

99.5 ± 0.38

99.9 ± 0.19

0.23

3.20

2.0 1.99 99.5 ± 0.44 100.0 ± 0.34 0.24 1.67

3.0 2.99 99.6 ± 0.53 99.8 ± 0.47 1.29 1.27

Reglan inj

5 mg / mL

1.0

0.99

99.7 ± 0.90

99.5 ±.0.50

0.78

1.25

2.0 1.99 99.5 ± 0.53 99.9 ± 0.47 1.29 1.27

3.0 3.00 100.0 ± 0.61 99.9 ± 0.48 1.25 1.61

Dapsone

25 mg / tab

1.0

1.00

100.0±0.55

99.8 ± 0.50

1.26

1.21

2.0 1.99 99.5±0.40 100.2 ± 0.58 2.30 2.10

3.0 2.99 99.6±0.13 100.1 ± 0.12 1.80 1.10

DA

P

Dapsone

100 mg/ tab

1.0

0.99

99.9±0.60

99.8±0.48

1.25

1.61

2.0 1.99 99.9±0.58 99.9±0.51 1.47 1.29

3.0 3.00 100.0±3.0 99.7±0.46 1.75 1.37

Chapter – 2

33

2B.3 Results and Discussion

The proposed method involves the diazo-coupling reaction of MCP or DAP

with IPH in an acidic medium to form a violet coloured azo-dye with a maximum

absorption at 570 nm for both the drugs [Fig 2B.2].

Two steps are involved in the reaction that produces the coloured azo-dye. In

the first step, MCP or DAP are treated with nitrite solution in acidic medium,

undergoes diazotisation to give the diazonium chloride ion. In the second step, the

diazonium chloride couples with a new coupling agent, IPH in an acidic medium to

form an violet coloured azo-dye. IPH has highest electron density at second and

eighth positions, which permits electrophilic substitution by the diazonium ion. The

reaction can be represented as in Scheme 2B.1. Here, MCP is used as the model

compound, since the DAP behaved similarly to it

NO2

_

+ H+

O

NH

OCH3

N

Cl

NH2

+

N

++

H2O

O

NH

OCH3

N

Cl

N

[A]

[A] +

N

R'

R' : CH2CH2CH2N CH3

CH3

N

R'

R' : CH2CH2CH2N CH3

CH3

O

NH

OCH3

N

Cl

NH2

N NHCl

IPH]Azo-dye

MCP

Scheme 2B.1: Proposed diazo-coupling reaction of MCP with IPH

Chapter – 2

34

2B.3.1 Absorption spectra

The general procedure for the determination of MCP and DAP [2B.2.3] was

followed for the formation of azo-dye [MCP- 4 µg mL-1 and DAP- 2.5 µg mL

-1] and

the wavelength of maximum absorption was determined by recording the absorbance

of the azo-dye in the wavelength range 450-650 nm. The absorbance of the reagent

blank against distilled water was also scanned in the same wavelength range.

Absorption spectra [Fig. 2B.2] were obtained by plotting the absorbance values

against wavelength. The azo-dye showed a maximum absorption at 570 nm for both

MCP and DAP and the reagent blank had negligible absobance at this wavelength.

0

0.1

0.2

0.3

0.4

0.5

450 490 530 570 610 650

Wave length in nm

Ab

sorb

an

ce (a)

(b)

blank

Fig .2B. 2: Absorption spectra of (a) MCP and (b) DAP with IPH

2B. 3. 2. Optimization of experimental parameters

Experimental conditions were optimized at 570 nm by studying the influence

of the following parameters with solutions containing a fixed concentration of drugs

2B.3. 2.1 Effect of the reagent

The effect of the concentration of IPH was studied by measuring the

absorbance at 570 nm for solution containing a fixed concentration of drugs and

varying amounts of IPH. A volume of 0.5% solution in a total volume of 10 mL was

found to be sufficient. The results are presented in Fig. 2B.3.

Chapter – 2

35

0

0.1

0.2

0.3

0.4

0 1 2 3

Vol of IPH in mL

Ab

sorb

an

ce

Fig. 2B. 3: Effect of IPH on the absorbance of azo-dye.

2B.3.2.4 Effect of reaction time

The diazotisation was carried out at room temperature (25 ± 2 °C), and 3 min

was the minimum time required for the diazotisation. No cooling (0 – 5 °C) was

required for the diazotisation. A time of 5 min was required for completion of

coupling reaction. The formed azo-dye was stable for more than 3 h.

2B.3.2.5 Effects of acid and nitrite

Different acids such as hydrochloric acid, sulphuric acid, phosphoric acid and

acetic acid were tested for diazotization reaction .HCl medium was the best and 0.5

mL of 1M HCl was used for this purpose. When sulpuric acid was used as a diluent

high blank colour was obtained, with the use of acetic acid very slow colour

development occur and when phosphoric acid was used, the produced dye is less

stable. But, 6 M the formed azo-dye was highly intense and stable in 6 M HCl

medium. Thus, hydrochloric acid found to be necessary for full colour development

and hence 6 M HCl was selected as a diluent for further studies.

Under the optimum conditions, a volume of 1 mL of 0.1 % solution of sodium

nitrite was found to be sufficient in a 10 mL reaction mixture, and the residual sodium

nitrite could be removed by the addition of 1 mL of 2 % sulphamic acid.

Chapter – 2

36

2B. 3 .3. Linearity and Optical characteristics

Under the experimental conditions, standard calibration curves for MCP and

DAP were constructed by plotting absorbance versus concentrations. Linear

relationships were obtained in the concentration range 0.5-5.0 µg mL-1and 0.5-4.0 µg

mL-1 for MCP and DAP, respectively [Fig 2B.1]. The molar absorptivity values from

Beer’s law data and their Sandell’s sensitivity values are presented in Table 2B. 2.

The slope, intercept, correlation coefficient, detection limit and quantitation limit of

the method are also given in Table 2B.2.

Table. 2B. 2. Optical characteristics

Parameter MCP DAP

Linear range

(µg mL-1)

0 -5.0 0 - 4.0

Molar absorptivity

(L mol-1cm

-2)

4.5×104 2.96×10

4

Sandell’s sensitivity (µg cm-2) 0.0078 0.0055

Correlation coefficient [r] 0.9999 0.9999

Regression equation [Y*]

Slope [b] 0.1074 0.1291

Intercept [a] 0.0275 -0.0099

Detection limit [DL] (µg mL-1) 0.0144 0.0132

Quantitation limit [QL] (µg mL-1) 0.0437 0.0402

*y = a + bx, where x is the concentration in µg mL-1

2B.3.3 Recovery Experiments

To assess the selectivity of the method, the interference of those species

accompanying the studied drugs (MCP and DAP) in pharmaceutical formulations was

studied. Samples were prepared by mixing known amount of the drug with various

amounts of common excipients such as talc, starch, dextrose, sodium alginate, gum

acacia and gelatin. The analysis of these laboratory prepared samples was carried out,

and the recovery values were determined. No interference was found from the added

excipients. The results are presented in Table 2B. 3.

Chapter – 2

37

Table 2B.3. Recovery of metoclopramide hydrochloride and dapsone from various

excipients by the proposed method

Nam

e of

the

com

pou

nd

Am

ou

nt

Pre

sen

t (m

g)

Excipients (mg)

Recovery

%

Talc

dextrose Starch Sodium

alginate

Gelatin Gum

acacia

MCP 75 300 400 200 80 50 100 98.7

50 250 350 250 100 75 75 99.0

DAP 75 250 300 250 100 75 100 98.8

50 300 350 200 75 50 75 99.2

* Average recovery from five experiments.

2B. 3. 4. Precision

The precision of the proposed method was determined by analyzing five

replicate samples containing MCP and DAP at three different concentrations.

[Table 2B.4] The intra day precision showed a CV of 1.6 or 0.2% at low

concentration. The inter day precision evaluated over a period of five days showed a

CV of 1.2 or 0.3% at the low concentration. The low values of both the intra and inter

day CVs at the low concentrations reflect the high precision of the proposed method.

The results are given in Table 2B.4.

Table 2B.4. Intra – day and Inter – day precision of the assay of MCP and DAP.

*Average of five determinations carried out over five days.

Sample

in pure

form

Intra - day Inter-day

Analyte

taken

(µg mL-1

)

Analyte

found *

(µg mL-1

) ± SD

CV %

Analyte

taken

(µg mL-1

)

Analyte

found *

(µg mlL-1

) ± SD

CV %

MCP

1

2

3

0.98 ± 0.07

1.96 ± 0.04

2.97 ± 0.04

1.65

0.31

0.22

1

2

3

0.89 ± 0.05

1.94 ± 0. 04

2.95 ± 0.03

1.18

0.60

0.27

DAP

1

2

3

0.99 ± 0.03

1.96 ± 0.03

3.10 ± 0.02

0.63

0.36

0.15

1

2

3

0.98 ± 0.05

1.82 ± 0.02

2.95 ± 0.03

2.40

1.90

0.63