Farmaceutische Analyse

Toets Analyse van Bereidingen 20 augustus 2001

• Tijd: 14.00-17.00 uur
• Aantal deelvragen: 20, per deelvraag 0,5 punten
• Schrijf op elk vel uw naam en studentnummer
• Het gebruik van een niet-geannoteerde Binas is toegestaan
• Relevante gegevens, statistische tabellen en formules zijn als bijlage bijgevoegd

Geef bij elke vraag zonodig een berekening en verklaar altijd het antwoord, ook al wordt dit niet expliciet aangegeven !
Succes !

Raadpleeg bij de beantwoording van deze vragen de bijlage.

Raadpleeg ook de uitwerking van dit tentamen.

Vraag 1

Een ziekenhuisapotheker laat 0,5 mg colchicine capsules bereiden volgens 2 verschillende methoden. Bij bereiding 1 wordt colchicine eerst opgelost in dichloormethaan. De oplossing wordt uitgegoten over microkristallijn cellulose en vervolgens drooggewreven. Bij bereiding 2 wordt de colchicine direct met microkristallijne cellulose gemengd. Om te onderzoeken welke bereidingsmethode beter is, wordt de gehaltespreiding bepaald op zes capsules uit beide charges. De resultaten van het onderzoek zijn hieronder gegeven en opgegeven voor zuivere colchicine. In het ziekenhuis is het gebruikelijk statistische uitspraken te doen met een betrouwbaarheid van 95%.

Tabel 1: Gehalte colchicine (mg/caps)

a. Een analist wil de waarde van de zesde capsule uit bereiding 2 verwerpen omdat de Q-test dit uitwijst. Bent u het eens met de beslissing dat deze waarde verworpen wordt?
b. Bereken of de spreidingen in het gehalte van beide bereidingen significant van elkaar verschillen.
c. Verklaar het eventueel gevonden verschil in gehaltespreiding tussen beide bereidingen.
d. Bereken of de gemiddelde gehalten significant van de gedeclareerde waarde verschillen.
e. Verklaar eventuele afwijkingen tussen het gevonden en gedeclareerde gehalte.

Vraag 2
a.Schat welke pKa-waarde(n) colchicine heeft.
b. Waarom wordt bij de gehaltebepaling van de grondstof een mengsel van tolueen en azijnzuuranhydride als oplosmiddel gebruikt?
c. Leg uit of colchicine in dit preparaat d.m.v. een titratie te bepalen is, zoals dit voor de grondstof beschreven staat.

Vraag 3
a. Leg uit of het UV-spectrum van colchicine een pH-shift vertoont.
b. In zuur of alkali kan colchicine omgezet worden naar colchiceïne (zie "impurities). Leg uit of het UV-spectrum van deze stof een pH-shift vertoont.
c. Beschrijf een optimale UV-gehaltebepaling van colchicine in deze capsules. Welke opwerking, welke meetconcentratie en welke golflengte gebruikt u?
d. Moet u bij de UV-bepaling rekening houden met ontleding van colchicine? Zo ja, uit welk(e) gegeven(s) uit de monografie blijkt dit en hoe controleert u of de gehaltebepaling hierdoor beïnvloed wordt. De ontledingsproducten die eventueel onder invloed van licht kunnen ontstaan zijn de onzuiverheden C t/m E.

Vraag 4
a. Hoe verifieert u of dit preparaat colchicine bevat?
b. Moet een HPLC bepaling voor colchicine uitgevoerd worden met een neutraal, zuur of alkalisch loopmiddel, of maakt dit geen verschil?
c. Hoe bepaalt u de de identiteit van cellulose.
d. Hoe bepaalt u of er nog resten dichloormethaan zijn achtergebleven in de capsules bij bereidingswijze 1.

Vraag 5
Zinkoxide zetpillen 10 % (samenstelling zie bijlage)
a. Schets het vlekkenpatroon van de hulpstoffen van de zetpil er uit op een silicagel plaat met loopmiddel ethyleenchloride/ether 9:1 en detectie met ANS-reagens en benoem de vlekken.
b. Hoe kunt u de vetzuursamenstelling van de hulpstoffen van de zetpil analyseren?
c. Hoe bepaalt u de identiteit van het zinkoxide? (vermeld bepalingsmethode en opwerking)
d. Hoe bepaalt u het gehalte van het zinkoxide? (vermeld bepalingsmethode en opwerking)<

Bijlage

COLCHICINE
Colchicinum C₂₂H₂₅NO
M_r 399.4

DEFINITION
Colchicine contains not less than 97.0 per cent and not more than the equivalent of 102.0 per cent of (S)-N-(5,6,7,9-tetrahydro-1,2,3,10-tetramethoxy-9-oxobenzo[a]heptalen- 7-yl)acetamide, calculated with reference to the anhydrous, ethyl acetate-free substance.

CHARACTERS
A yellowish-white, amorphous or crystalline powder, very soluble in water, rapidly recrystallising from concentrated solutions as the sesquihydrate, freely soluble in alcohol and in chloroform.

IDENTIFICATION
First identification: B. Second identification: A, C, D.
A. Dissolve 5 mg in alcohol R and dilute to 100.0 ml with the same solvent. Dilute 5.0 ml of the solution to 25.0 ml with alcohol R. Examined between 230 nm and 400 nm (2.2.25), the solution shows two absorption maxima, at 243 nm and 350 nm. The ratio of the absorbance measured at 243 nm to that measured at 350 nm is 1.7 to 1.9.
B. Examine by infrared absorption spectrophotometry (2.2.24), comparing with the spectrum obtained with colchicine CRS. Examine the substances in the form of discs prepared as follows: dissolve the substance in 0.5 ml of chloroform R, sprinkle the solution on the potassium bromide R, mix thoroughly and evaporate the solvent by first placing in a current of air and then by heating at 80 °C for 60 min.
C. To 0.5 ml of solution S (see Tests) add 0.5 ml of dilute hydrochloric acid R and 0.15 ml of ferric chloride solution R1. The solution is yellow and becomes dark green on boiling for 30 s. Cool, add 2 ml of chloroform R and shake. The organic layer is greenish-yellow.
D.& Dissolve about 30 mg in 1 ml of alcohol R and add 0.15 ml of ferric chloride solution R1. A brownish-red colour develops.

TESTS

Solution S. Dissolve 0.10 g in water R and dilute to 20 ml with the same solvent.

Appearance of solution. Solution S is clear (2.2.1) and not more intensely coloured than reference solution GY3 (Method II, 2.2.2).

Acidity or alkalinity. To 10 ml of solution S add 0.1 ml of bromothymol blue solution R1. Either the solution does not change colour or it becomes green. Not more than 0.1 ml of 0.01 M sodium hydroxide is required to change the colour of the indicator to blue.

Specific optical rotation (2.2.7). Dissolve 50.0 mg in alcohol R and dilute to 10.0 ml with the same solvent. The specific optical rotation is - 235° to - 250°, calculated with reference to the anhydrous, ethyl acetate-free substance.

Related substances. Carry out the test protected from bright light.
Examine by thin-layer chromatography (2.2.27), using silica gel HF254 R as the coating substance.
Prepare the solutions immediately before use.
Test solution. Dissolve 50 mg of the substance to be examined in chloroform R and dilute to 5 ml with the same solvent.
Reference solution (a). Dilute 2 ml of the test solution to 100 ml with chloroform R.
Reference solution (b). Dilute 5 ml of reference solution (a) to 10 ml with chloroform R.
Apply separately to the plate 10 ml of each solution. Develop over a path of 15 cm using a mixture of 1 volume of concentrated ammonia R, 25 volumes of ethylene chloride R and 50 volumes of acetone R. Allow the plate to dry in air and examine in ultraviolet light at 254 nm. Any spot in the chromatogram obtained with the test solution, apart from the principal spot, is not more intense than the spot in the chromatogram obtained with reference solution (a) (2.0 per cent) and at most one such spot is more intense than the spot in the chromatogram obtained with reference solution (b) (1.0 per cent).

Chloroform. Not more than 500 ppm, determined by head-space gas chromatography (2.2.28, Method II).
Test solution. Dissolve 0.400 g of the substance to be examined in water R and dilute to 10.0 ml with the same solvent. In three suitable identical vials, introduce 1.0 ml of this solution and stopper the vials.
Reference solution. Dilute 5 ml of chloroform R to 10.0 ml with water R. In each of three suitable identical vials, place 10 ml of this solution, add 1.0 ml of the test solution and stopper the vials. The chromatography may be carried out using: - a fused-silica column, 50 m long and 0.32 mm in internal diameter, the internal wall of which is covered with a 5 mm layer of chemically bonded polydimethylsiloxane R, - nitrogen for chromatography R as the carrier gas at a flow rate of 4 ml per minute, - a flame-ionisation detector, programming the temperature of the column from 80 °C to 200 °C at a rate of 10 °C per minute and maintaining the temperature of the detector at 250 °C. Maintain each solution at 90 °C for 20 min, pressurise for 30 s and transfer onto the column at a temperature of 120 °C. Carry out a blank test using a vial containing 1 ml of water R. Perform each test three times. From the chromatograms obtained with the test solution and the reference solution, calculate the content (per cent m/m) of chloroform taking its density (2.2.5) to be 1.48 g per millilitre at 20 °C.

Colchiceine. Dissolve 50 mg in water R and dilute to 5 ml with the same solvent. Add 0.1 ml of ferric chloride solution R1. The solution is not more intensely coloured than a mixture of 1 ml of red primary solution, 2 ml of yellow primary solution and 2 ml of blue primary solution (Method II, 2.2.2) (about 0.2 per cent).

Ethyl acetate. Not more than 6.0 per cent m/m of ethyl acetate, determined by gas chromatography (2.2.28) using ethanol R as internal standard.
Internal standard solution. Dilute 1.0 ml of ethanol R to 100.0 ml with water R. Dilute 10.0 ml of this solution to 50.0 ml with water R.
Test solution. Dissolve 0.10 g of the substance to be examined in water R. Add 5.0 ml of the internal standard solution and dilute to 10.0 ml with water R.
Reference solution. Dissolve 1.0 ml of ethyl acetate R in water R and dilute to 100.0 ml with the same solvent. To 1.0 ml of this solution add 5.0 ml of the internal standard solution and dilute to 10.0 ml with water R.
The chromatographic procedure may be carried out using: - a glass or stainless steel column 1.5 m long and 4 mm in internal diameter, packed with silanised diatomaceous earth for gas chromatography R impregnated with 10 per cent m/m of macrogol 1000 R, - nitrogen for chromatography R as the carrier gas at a flow rate of 30 ml per minute, - a flame-ionisation detector, maintaining the temperature of the column at 75 °C, that of the injection port at 130 °C and that of the detector at 150 °C.
From the chromatograms obtained with the test solution and the reference solution, calculate the content (per cent m/m) of ethyl acetate, taking its density (2.2.5) to be 0.901 g per millilitre at 20 °C.

Water (2.5.12). Not more than 2.0 per cent, determined on 0.500 g by the semi-micro determination of water.

Sulphated ash (2.4.14). Not more than 0.1 per cent, determined on 0.5 g.

ASSAY
Dissolve 0.250 g with gentle heating in a mixture of 10 ml of acetic anhydride R and 20 ml of toluene R. Titrate with 0.1 M perchloric acid determining the end-point potentiometrically (2.2.20).
1 ml of 0.1 M perchloric acid is equivalent to 39.94 mg of C22H25NO6.

STORAGE
Store in a well-closed container, protected from light.

IMPURITIES

Cochiceïne

Impurity A

Impurity B

Impurity C

Impurity D

Impurity E

UV- spectrum van colchicine in methanol:
maxima:
242 nm A1% = 775
350 nm A1% = 427

Samenstelling Zinkoxide zetpillen 10%

TRIGLYCERIDES, MEDIUM-CHAIN
Triglycerida saturata media

DEFINITION
Medium-chain triglycerides are obtained from the oil extracted from the hard, dried fraction of the endosperm of Cocos nucifera L. or from the dried endosperm of Elaeis guineensis Jacq. They consist of a mixture of triglycerides of saturated fatty acids, mainly of caprylic acid (C₈H₁₆O₂) and of capric acid (C₁₀H₂₀O₂). They contain not less than 95 per cent of saturated fatty acids with 8 and 10 carbon atoms.

CHARACTERS
A colourless or slightly yellowish, oily liquid, practically insoluble in water, miscible with alcohol, with methylene chloride, with light petroleum and with fatty oils.

HARD FAT
Adeps solidus

DEFINITION
Hard fat consists of a mixture of triglycerides, diglycerides and monoglycerides of saturated fatty acids, mainly of lauric acid (C₁₂H₂₄O₂), of myristic acid (C₁₄H₂₈O₂), of palmitic acid (C₁₆H₃₂O₂) and of stearic acid (C₁₈H₃₆O₂), which may be obtained either by esterification of fatty acids of natural origin with glycerol or by transesterification of natural fats. Each type of hard fat is characterised by its melting point, its hydroxyl value and its saponification value. It contains no additives.

CHARACTERS
A white or almost white, waxy, brittle mass, practically insoluble in water, freely soluble in ether, slightly soluble in ethanol. When heated to 50 °C, it melts giving a colourless or slightly yellowish liquid.