Monographs for antiretrovirals
Within the framework of the Procurement, Quality and Sourcing Project for HIV, Tuberculosis and Malaria (http://www.who.int/prequal), The International Pharmacopoeia is collaborating with manufacturers, independent analytical drug quality control laboratories, national and regional pharmacopoeial bodies, research, governments, and regulatory bodies to provide specifications and monographs for the following antiretroviral agents: abacavir, didanosine, efavirenz, indinavir, lamivudine, nelfinavir, nevirapine, ritonavir, saquinavir, stavudine, zidovudine. A draft for zidovudine is provided below for comment.
Zidovudinum Zidovudine (first draft)
C10H13N5O4
Relative molecular mass. 267.2
Chemical name. 1-[(2R,4S,5S)-4-azido-5-(hydroxymethyl)tetrahydrofuran-2-yl]-5-methyl-pyrimidine-2,4(1H,3H)-dione; 1-(3-azido-2,3-dideoxy-b-D-erythro-pentofuranosyl)-5-methyl-pyrimidine-2,4(1H,3H)-dione; 3'-azido-3'-deoxythimidine (AZT); CAS Reg. NO.30516-87-1.
Description. A white or almost white powder.
Solubility. Soluble in ethanol (~750 g/l) TS (ethanol (95 per cent) R), sparingly soluble in water.
Category. Antiretroviral (Nucleoside Reverse Transcriptase Inhibitor).
Storage. Zidovudine should be kept in a well closed container, protected from light.
[Note from Secretariat: USP revised the ‘storage conditions’ by adding the following sentence: “Store at 25?C, excursions permitted between 15?C and 30?C.” Definition for “room temperature” in the International Pharmacopoeia. “When nothing is mentioned, the storage of the substance is at room temperature.”]
Additional information. Zidovudine shows polymorphism.
REQUIREMENTS
Zidovudine contains not less than 97.0% and not more than 103.0% of C10H13N5O4, calculated with reference to the dried substance.
Identity test
Either tests A and B, or test C may be applied.
A. Carry out test A.1. or, where UV detection is not available, test A.2.
A.1. Carry out the test as described under ‘‘Thin-layer chromatography’’ (Vol. 1, p. 83*), using silica gel R6 as the coating substance and a mixture of 90 volumes of dichloromethane R, 10 volumes of methanol R and 3 volumes of glacial acetic acid R as the mobile phase. Apply separately to the plate 5 µl of each of 2 solutions in methanol R containing (A) 1 mg of the test substance per ml and (B) 1 mg of zidovudine RS per ml. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air or in a current of cool air. Examine the chromatogram in ultraviolet light (254 nm).
* Refers to The International Pharmacopoeia
The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.
A.2. Carry out the test as described under ‘‘Thin-layer chromatography’’ (Vol. 1, p. 83*), using silica gel R5 as the coating substance and a mixture of 90 volumes of dichloromethane R, 10 volumes of methanol R and 3 volumes of glacial acetic acid R as the mobile phase. Apply separately to the plate 5 µl of each of 2 solutions in methanol R containing (A) 1 mg of the test substance per ml and (B) 1 mg of zidovudine RS per ml. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air or in a current of cool air. Dip the plate in dilute basic potassium permanganate (1 g/l) TS. Examine the chromatogram in daylight.
* Refers to The International Pharmacopoeia
The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.
B. The absorption spectrum of a 15 µg/ml solution in methanol R, when observed between 210 nm and 300 nm, exhibits one maximum at about 266 nm; the specific absorbance (A 1%1cm) is between 360 to 398.
[Note from Secretariat: The specific absorbance range has been defined within +/-5% limits as agreed by the EC. Test B may be referred to the UV assay and not described here. Please comment.]
C. Carry out the examination as described under “Spectrophotometry in the infrared region” (Vol. 1, p. 40*). The infrared absorption spectrum is concordant with the spectrum obtained from zidovudine RS or with the reference spectrum of zidovudine.
* Refers to The International Pharmacopoeia
If the spectra are not concordant, use zidovudine RS. Dissolve the sample in a small amount of ethanol (~750 g/l) TS (ethanol (95 per cent) R), evaporate to dryness and carry out the IR spectrum with the residue as mentioned above. Treat zidovudine RS in the same way. The infrared absorption spectrum is concordant with the spectrum obtained from zidovudine RS.
Melting range. 124-126?C.
Specific optical rotation. Use a 10 mg/ml solution in ethanol (~750 g/l) TS (ethanol (95 per cent) R) and calculate with reference to the dried substance; [?]D25?C = +60? to +63?.
Heavy metals. Use 1.0 g for the preparation of the test solution as described under “Limit test for heavy metals”, Procedure 2 (Vol. 1, p.118*). Determine the heavy metals content according to Method A (Vol. 1, p. 119*); not more than 20 µg/g.
[Note from Secretariat: additional information needed:
-The method used, for instance in the European Pharmacopoeia and Indian Pharmacopoeia, is more complex (combustion method). Please comment.
-Which solvent has to be used (e.g. ethanol 95% R) if procedure 2 is retained?]
Sulfated ash. Not more than 2 mg/g.
Loss on drying. Dry for 3 hours at 105?C; it loses not more than 5 mg/g.
[Note from Secretariat: The limit for ‘loss on drying’ is more stringent in this monograph than, for example, in the European Pharmacopoeia, USP and Indian Pharmacopoeia (0.5% instead of 1.0%). Please comment.]
Related substances
A. Carry out the test as described under “Thin-layer chromatography” (Vol. 1, p.8*), using silica gel R4 as the coating substance and a mixture of 90 volumes of dichloromethane R and 10 volumes of methanol R as the mobile phase. Apply separately to the plate 10 µl of each of the 2 solutions in methanol R containing (A) a mixture containing 0.1 mg per ml each of zidovudine RS and triphenylmethanol R and (B) 20 mg per ml of the test substance. Develop over a path of 12 cm. After removing the plate from the chromatographic chamber, allow it to dry exhaustively in air. Examine the chromatogram in ultraviolet light (254 nm).
s* Refers to The International Pharmacopoeia
[Note from Secretariat: This method is described in the USP. However, chloroform has been changed to dichloromethane in the mobile phase (International Pharmacopoeia policy).]
Any spot obtained with solution (B), other than the principal spot, is not more intense and not larger than the principal spot obtained with solution A (0.5%). Furthermore, the sum of intensities of the secondary spots obtained with solution B does not exceed 3.0%.
[Note from Secretariat: It is suggested that the last sentence above referring to the sum of spot intensities be deleted (difficult interpretation).]
Spray the plate with a mixture of 0.5 g of carbazole R in 95 volumes of ethanol (~750 g/l) TS (ethanol (95 per cent) R) and 5 volumes of sulfuric acid R, heat for 10 minutes at 120?C.
Any spot corresponding to triphenylmethanol R (Rvalue about 2.3 relative to the R value of zidovudine) is not more intense than the corresponding spot in solution A (0.5%). Any other spot obtained with solution B, other than the principal spot, is not more intense and not larger than the principal spot corresponding to zidovudine obtained with solution A (0.5%). Furthermore, the sum of intensities of the secondary spots obtained with solution (B) does not exceed 3.0%.
[Note from Secretariat: It is suggested that the last sentence above referring to the sum of spot intensities be deleted (difficult interpretation).]
B. Carry out the test as described under “High-performance liquid chromatography” (Vol. 5, p. 257*), using a stainless steel column (25 cm x 4.6 mm) packed with octadecylsilyl silica gel for chromatography R (Stationary phase A) (5µm) (Waters Hypersil BDS is suitable). As the mobile phase, use a mixture of 20 volumes of methanol R and 80 volumes of water.
* Refers to The International Pharmacopoeia
Prepare the following solutions. For solution (1) prepare 1 mg/ml solution of the test substance in the mobile phase. For solution (2) dilute 1.0 ml of solution (1) to 5 ml with the mobile phase. For solution (3) dissolve 2 mg of zidovudine impurity C (thymine R) in 10 ml of methanol R. Then dilute 2 ml to 20 ml with the mobile phase. For solution (4), dissolve 2 mg of impurity B RS (1-(3-chloro-2,3-dideoxy-ß-D-erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione) in 10 ml of mobile phase. Mix 5 ml of this solution with 5 ml of solution (2) into a 10-ml volumetric flask. For solution (5) dilute 2 ml of solution (4) to 20 ml with the mobile phase. For solution (6) dilute 0.5 ml of solution (1) to 100 ml with the mobilephase.
Operate with a flow rate of 1.2 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 265 nm.
Inject alternately 10 µl each of solutions (1), (3), (5) and (6). Record the chromatogram for 4 times the retention time of zidovudine in solution (1).
The retention times ratio with reference to zidovudine are about 0.26 for zidovudine impurity C (thymine R), 1 and 1.18 for zidovudine related impurity B (1-(3-chloro-2,3-dideoxy-ß-D-erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione). The test is not valid unless the resolution factor between the peaks corresponding to zidovudine and zidovudine impurity B obtained with solution (5) is greater than 2.
Measure the areas of the peak responses obtained in the chromatograms from solutions (1), (3), (5) and (6).
In the chromatogram obtained with solution (1) the area of the peak corresponding to zidovudine impurity C (thymine R) is not greater than the area of the principal peak obtained with solution (3) (2.0%). The area of the peak corresponding to zidovudine impurity B RS is not greater than the area of the corresponding peak in the chromatogram obtained with solution (5) (1.0%). The area of any other peak, other than the principal peak, is not greater than the area of the peak obtained with solution (6) (0.5%). The sum of the areas of all peaks, other than the principal peak, obtained with solution (1) is not greater than 6 times the area of the principal peak obtained with solution (6) (3.0%). Disregard any peak with an area less than 0.1 times the area of the principal peak obtained with solution (6) (0.05%).
Assay. Weigh accurately about 40 mg of sample into a 200 ml volumetric flask. Add about 160 ml of a mixture consisting of 20 volumes of methanol R and 80 volumes of water and dissolve by using an ultrasonic bath. Dilute to volume with the same solvent and mix. Dilute 5 ml of this solution to 50 ml with 0.1M H2SO4 and mix. For the blank, use 5 ml of a mixture consisting of 20 volumes of methanol R and 80 volumes of water diluted to 50 ml with 0.1M H2SO4.
Measure the absorbance of a 1-cm layer of the final solution at a maximum about 266 nm against a solvent cell containing the blank. Calculate the content of C10H13N5O4 using the absorptivity value of 38.0 (A 1%1cm= 380).
[Note from Secretariat: The UV wavelength has been changed from 267 to 266 nm to be consistent with identity test B. The specific absorbance has been experimentally determined by 2 different laboratories. It would be good to have additional experimental feedback to confirm this value. Otherwise the use of a reference substance is an alternative. Please comment.]
Impurities
The following list of known and potential impurities that have been shown to be controlled by the tests in this monograph is given for information.
Note from Secretariat: Chemical structures to come.
A. 1-[(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione
B. (1-(3-chloro-2,3-dideoxy-b-D-erythro-pentofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione)
C. 5-methylpyrimidine-2,4(1H,3H)-dione (thymine)
D. triphenylmethanol
Reagents
Carbazole R.
Dibenzopyrolle
C12H9N.
A commercially available reagent of suitable grade.
Melting point. about 245?C.
Potassium permanganate, basic, dilute (1 g/l) TS
A solution of potassium permanganate R containing about 1 g of KMnO4 per litre of sodium hydroxide (1 mol/l).
Thymine R.
5-methylpyrimidine-2,4(1H,3H)-dione; C5H6N2O2.
A commercially available reagent of suitable grade.
Description. Short needles or plates.
Solubility. Slightly soluble in cold water, soluble in hot water. It dissolves in dilute solution of alkali hydroxide.
Triphenylmethanol R.
Triphenylcarbinol; C19H16O.
A commercially available reagent of suitable grade.
Description. Colourless crystals.
Solubility. Practically insoluble in water, freely soluble in ethanol (~750 g/l) TS (ethanol (95 per cent) R).
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