Busulfan EUROPEAN PHARMACOPOEIA 5.0

STORAGEIn an airtight container, protected from light, at atemperature of 2 °C to 8 °C.If the substance is sterile, store in an airtight, sterile,tamper-proof container.

LABELLINGThe label states :— the mass of peptide in the container,— where applicable, that the substance is free from bacterial

endotoxins.

IMPURITIESSpecified impurities : A, B, C, D, E.

A. X2 = D-His, X4 = L-Ser, X5 = L-Tyr : [2-D-histidine]buserelin,

B. X2 = L-His, X4 = D-Ser, X5 = L-Tyr : [4-D-serine]buserelin,

D. X2 = L-His, X4 = L-Ser, X5 = D-Tyr : [5-D-tyrosine]buserelin,

C. buserelin-(3-9)-peptide,

E. [1-(5-oxo-D-proline)]buserelin.

01/2005:0542

BUSULFAN

Busulfanum

C6H14O6S2 Mr 246.3

DEFINITIONBusulfan contains not less than 99.0 per cent and not morethan the equivalent of 100.5 per cent of butane-1,4-diyldi(methanesulphonate), calculated with reference to thedried substance.

CHARACTERSA white or almost white, crystalline powder, very slightlysoluble in water, freely soluble in acetone and in acetonitrile,very slightly soluble in alcohol.It melts at about 116 °C.

IDENTIFICATIONFirst identification: A.Second identification : B, C, D.

A. Examine by infrared absorption spectrophotometry(2.2.24), comparing with the spectrum obtained withbusulfan CRS.

B. Examine by thin-layer chromatography (2.2.27), usingsilica gel G R as the coating substance.Test solution. Dissolve 20 mg of the substance to beexamined in 2 ml of acetone R.Reference solution. Dissolve 20 mg of busulfan CRS in2 ml of acetone R.Apply separately to the plate 5 µl of each solution.Develop over a path of 15 cm using a mixture ofequal volumes of acetone R and toluene R. Dry theplate in a stream of hot air and spray with anisaldehydesolution R. Heat the plate at 120 °C. The principal spotin the chromatogram obtained with the test solution issimilar in position, colour and size to the principal spot inthe chromatogram obtained with the reference solution.

C. To 0.1 g add 5 ml of 1 M sodium hydroxide. Heat untila clear solution is obtained. Allow to cool. To 2 ml ofthe solution add 0.1 ml of potassium permanganatesolution R. The colour changes from purple throughviolet to blue and finally to green. Filter and add 1 mlof ammoniacal silver nitrate solution R. A precipitateis formed.

D. To 0.1 g add 0.1 g of potassium nitrate R and 0.25 g ofsodium hydroxide R, mix and heat to fusion. Allow tocool and dissolve the residue in 5 ml of water R. Adjust topH 1 to 2 using dilute hydrochloric acid R. The solutiongives reaction (a) of sulphates (2.3.1).

TESTS

Appearance of solution. Dissolve 0.25 g in 20 ml ofacetonitrile R, dilute to 25 ml with water R and examineimmediately. The solution is clear (2.2.1) and not moreintensely coloured than reference solution B7 (2.2.2,Method II).

Acidity. Dissolve 0.20 g with heating in 50 ml of ethanol R.Add 0.1 ml of methyl red solution R. Not more than 0.05 mlof 0.1 M sodium hydroxide is required to change the colourof the indicator.

Loss on drying (2.2.32). Not more than 2.0 per cent,determined on 1.000 g by drying in vacuo at 60 °C.

Sulphated ash (2.4.14). Not more than 0.1 per cent,determined on 1.0 g.

ASSAYTo 0.250 g add 50 ml of water R. Shake. Boil under a refluxcondenser for 30 min and, if necessary, make up to theinitial volume with water R. Allow to cool. Using 0.3 ml ofphenolphthalein solution R as indicator, titrate with 0.1 Msodium hydroxide until a pink colour is obtained.1 ml of 0.1 M sodium hydroxide is equivalent to 12.32 mgof C6H14O6S2.

STORAGEStore in an airtight container, protected from light.

01/2005:1847

BUTCHER’S BROOM

Rusci rhizomaDEFINITIONDried, whole or fragmented underground parts of Ruscusaculeatus L.

1138 See the information section on general monographs (cover pages)

EUROPEAN PHARMACOPOEIA 5.0 Butcher’s broom

Content : minimum 1.0 per cent of total sapogenins,expressed as ruscogenins [mixture of neoruscogenin(C27H40O4 ; Mr 428.6) and ruscogenin (C27H42O4 ; Mr 430.6)](dried drug).

CHARACTERS

Macroscopic and microscopic characters described underidentification tests A and B.

IDENTIFICATION

A. The rhizome consists of yellowish, branched, articulated,somewhat knotty pieces, cylindrical or subconical, about5 cm to 10 cm long and about 5 mm thick. The surfaceis marked with thin annulations about 1 mm to 3 mmwide, separated from one another ; rounded scars of theaerial stems are present on the upper surface. On thelower surface numerous roots, or their scars, occur ; theroots are about 2 mm in diameter and similar in colour tothe rhizome. The outer layer is easily detached, revealinga yellowish-white, very hard central cylinder.

B. Reduce to a powder (355). The powder is yellowish.Examine under a microscope using chloral hydratesolution R. The powder shows groups of sclereids fromthe ground tissue of the rhizome, variously-shaped cells,ranging from rounded to elongated or rectangular ; thewalls are moderately thickened and distinctly beaded, withlarge, rounded to oval pits. Fragments of the endodermiscomposed of a single layer of irregularly-thickened cells.Groups of rounded parenchymatous cells, thickened atthe corners, with small, triangular intercellular spaces ;thin-walled parenchyma containing raphides of calciumoxalate. Groups of thick-walled fibres and small vessels,up to about 50 µm in diameter, the walls showingnumerous small, slit-shaped pits.

C. Thin-layer chromatography (2.2.27)

Test solution. Introduce 1.0 g of the powdered drug (355)and 50 ml of dilute hydrochloric acid R into a 100 mlflask with a ground-glass neck. Heat on a water-bathunder a reflux condenser for 40 min. Allow to cool andextract the unfiltered mixture with 3 quantities, each of25 ml, of methylene chloride R. Combine the organicsolutions and dry over anhydrous sodium sulphate R.Filter and evaporate to dryness. Dissolve the residue in5 ml of methanol R.

Reference solution. Dissolve 1 mg of ruscogenins R and1 mg of stigmasterol R in methanol R and dilute to 5 mlwith the same solvent.

Plate : TLC silica gel plate R.

Mobile phase : methanol R, methylene chloride R(7:93 V/V).

Application : 10 µl, as bands.

Development : over a path of 15 cm.

Drying : in air.

Detection : spray with vanillin reagent R, dry the plate inan oven at 100-105 °C for 1 min and examine in daylight.

Results : see below the sequence of the zones present inthe chromatograms obtained with the reference solutionand the test solution. Furthermore, other weak zonesmay be present in the chromatogram obtained with thetest solution.

Top of the plate

Several zones of various colours

Stigmasterol : a violet zone A violet zone

_______ _______

A violet zone

Ruscogenins : a yellow zone A yellow zone (ruscogenins)

Several zones of various colours

_______ _______

Reference solution Test solution

TESTS

Foreign matter (2.8.2) : maximum 5 per cent.

Loss on drying (2.2.32) : maximum 12.0 per cent, determinedon 1.000 g of the powdered drug (355) by drying in an ovenat 100-105 °C for 2 h.

Total ash (2.4.16) : maximum 12.0 per cent.

Ash insoluble in hydrochloric acid (2.8.1) : maximum5.0 per cent.

ASSAYLiquid chromatography (2.2.29).Test solution. To 2.000 g of the powdered drug (355), add60 ml of ethanol R, 15 ml of water R and 0.2 g of potassiumhydroxide R. Extract under reflux on a water-bath for 4 h.Allow to cool and filter into a 100 ml volumetric flask. Rinsethe extraction flask and the residue in the filter with 3quantities, each of 10 ml, of ethanol R and add the rinsingsto the volumetric flask. Dilute to 100.0 ml with ethanol R.Introduce 25.0 ml of the solution into a round-bottomedflask fitted to a rotary evaporator and evaporate to dryness.Dissolve the residue in 10 ml of butanol R, add 3 ml ofhydrochloric acid R1 and 8 ml of water R. Heat under refluxon a water-bath for 1 h. Allow to cool and transfer the liquidinto a separating funnel, rinse the round-bottomed flask with2 quantities, each of 10 ml, of butanol R. Add the rinsingsto the separating funnel. Extract with 3 quantities, each of20 ml, of butanol R saturated with water R. Combine thebutanolic extracts and evaporate to dryness using a rotaryevaporator. Dissolve the residue in 20 ml of methanol R andtransfer to a 100 ml volumetric flask. Rinse the extractionflask with 20 ml, 20 ml and 10 ml of methanol R and addthe rinsings to the volumetric flask. Dilute to 100 ml withmethanol R.Reference solution. Dissolve 5.0 mg of ruscogenins R in100 ml of methanol R.Column :— size : l = 0.25 m, Ø = 4.6 mm,— stationary phase : octadecylsilyl silica gel for

chromatography R (5 µm).Mobile phase :— mobile phase A : water R,— mobile phase B : acetonitrile for chromatography R,

Time(min)

Mobile phase A(per cent V/V)

Mobile phase B(per cent V/V)

0 - 25 40 60

25 - 27 40 → 0 60 → 100

27 - 37 0 100

37 - 39 0 → 40 100 → 60

39 - 42 40 60

Flow rate : 1.2 ml/min.

General Notices (1) apply to all monographs and other texts 1139

Butyl parahydroxybenzoate EUROPEAN PHARMACOPOEIA 5.0

Detection : spectrophotometer at 203 nm.Injection : 20 µl.Retention time with reference to neoruscogenin (retentiontime = about 16 min) : ruscogenin = about 1.2.System suitability : reference solution :— resolution : minimum 1.5 between the peaks due to

neoruscogenin and ruscogenin.Calculate the percentage content of sapogenins expressedas ruscogenins (neoruscogenin and ruscogenin) in thetest solution by comparing the areas of the peaks in thechromatograms obtained with the test solution and thereference solution.

01/2005:0881

BUTYL PARAHYDROXYBENZOATE

Butylis parahydroxybenzoas

C11H14O3 Mr 194.2

DEFINITIONButyl 4-hydroxybenzoate.Content : 98.0 per cent to 102.0 per cent.

CHARACTERSAppearance : white or almost white, crystalline powder orcolourless crystals.Solubility : very slightly soluble in water, freely soluble inalcohol and in methanol.

IDENTIFICATIONFirst identification : A, B.Second identification : A, C, D.A. Melting point (2.2.14) : 68 °C to 71 °C.B. Infrared absorption spectrophotometry (2.2.24).

Comparison : butyl parahydroxybenzoate CRS.C. Examine the chromatograms obtained in the test for

related substances.Results : the principal spot in the chromatogram obtainedwith test solution (b) is similar in position and size tothe principal spot in the chromatogram obtained withreference solution (b).

D. To about 10 mg in a test-tube add 1 ml of sodiumcarbonate solution R, boil for 30 s and cool (solution A).To a further 10 mg in a similar test-tube add 1 ml ofsodium carbonate solution R ; the substance partlydissolves (solution B). Add at the same time to solution Aand solution B 5 ml of aminopyrazolone solution Rand 1 ml of potassium ferricyanide solution R and mix.Solution B is yellow to orange-brown. Solution A isorange to red, the colour being clearly more intense thanany similar colour which may be obtained with solution B.

TESTS

Solution S. Dissolve 1.0 g in alcohol R and dilute to10 ml with the same solvent.

Appearance of solution. Solution S is clear (2.2.1) andnot more intensely coloured than reference solution BY6(2.2.2, Method II).

Acidity. To 2 ml of solution S add 3 ml of alcohol R, 5 ml ofcarbon dioxide-free water R and 0.1 ml of bromocresol greensolution R. Not more than 0.1 ml of 0.1 M sodium hydroxideis required to change the colour of the indicator to blue.

Related substances. Thin-layer chromatography (2.2.27).Test solution (a). Dissolve 0.10 g of the substance to beexamined in acetone R and dilute to 10 ml with the samesolvent.Test solution (b). Dilute 1 ml of test solution (a) to 10 ml withacetone R.Reference solution (a). Dilute 0.5 ml of test solution (a) to100 ml with acetone R.Reference solution (b). Dissolve 10 mg of butylparahydroxybenzoate CRS in acetone R and dilute to10 ml with the same solvent.Reference solution (c). Dissolve 10 mg of propylparahydroxybenzoate R in 1 ml of test solution (a) anddilute to 10 ml with acetone R.Plate : suitable octadecylsilyl silica gel with a fluorescentindicator having an optimal intensity at 254 nm as thecoating substance.Mobile phase : glacial acetic acid R, water R, methanol R(1:30:70 V/V/V).Application : 2 µl.Development : over a path of 15 cm.Drying : in air.Detection : examine in ultraviolet light at 254 nm.System suitability : the chromatogram obtained withreference solution (c) shows 2 clearly separated principalspots.Limits :— any impurity : any spot in the chromatogram obtained

with test solution (a), apart from the principal spot, is notmore intense than the spot in the chromatogram obtainedwith reference solution (a) (0.5 per cent).

Sulphated ash (2.4.14) : maximum 0.1 per cent, determinedon 1.0 g.

ASSAYTo 1.000 g add 20.0 ml of 1 M sodium hydroxide. Heat atabout 70 °C for 1 h. Cool rapidly in an ice bath. Prepare ablank in the same manner. Carry out the titration on thesolutions at room temperature. Titrate the excess sodiumhydroxide with 0.5 M sulphuric acid, continuing the titrationuntil the second point of inflexion and determining theend-point potentiometrically (2.2.20).1 ml of 1 M sodium hydroxide is equivalent to 194.2 mg ofC11H14O3.

IMPURITIES

A. R = H: 4-hydroxybenzoic acid,

B. R = CH3 : methyl 4-hydroxybenzoate,

C. R = CH2-CH3 : ethyl 4-hydroxybenzoate,

D. R = CH2-CH2-CH3 : propyl 4-hydroxybenzoate.

1140 See the information section on general monographs (cover pages)