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 stavudine is provided below for comment.

Stavudinum Stavudine (first draft)

C₁₀H₁₂N₂O₄

Relative molecular mass. 224.2

Chemical name. 1-[(2R,5S)-5-(hydroxymethyl)-2,5-dihydrofuran-2-yl]-5-methylpyrimidine-2,4(1H,3H)-dione; 1-(2,3-dideoxy-ß-D-glycero-pent-2-enofuranosyl)-5-methylpyrimidine-2,4(1H,3H)-dione (D4T); CAS Reg. No.3056-17-5.

Description. A white to almost white powder.

Solubility. Soluble in water and ethanol (~750 g/l) TS (ethanol (95 per cent) R).

Category. Antiretroviral (nucleoside reverse transcriptase inhibitor).

Storage. Stavudine should be kept in a well closed container, protected from light.

Additional information. Stavudine shows polymorphism.

REQUIREMENTS

Stavudine contains not less than 97.0% and not more than 103.0% of C₁₀H₁₂N₂O₄, 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 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 2 ml of each of 2 solutions in methanol containing (A) 5 mg of the test substance per ml and (B) 5 mg of stavudine 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 67 volumes of dichloromethane R, 20 volumes of acetonitrile R, 10 volumes of methanol R and 3 volumes of ammonia (~260 g/l) TS as the mobile phase. Apply separately to the plate 2 µl of each of 2 solutions in methanol containing (A) 5 mg of the test substance per ml and (B) 5 mg of stavudine 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. Spray with vanillin/ sulfuric acid TS1. Heat the plate for a few minutes at 120?C. 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 the final solution prepared for the Assay, when observed between 210 nm and 300 nm, exhibits one maximum at about 266 nm; the specific absorbance (A ^1%_1cm) is between 412 and 458.

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 stavudine RS or with the reference spectrum of stavudine.

If the spectra are not concordant, use stavudine 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 stavudine RS in the same way. The infrared absorption spectrum is concordant with the spectrum obtained from stavudine RS.

Specific optical rotation. Use a 7 mg/ml solution and calculate with reference to the dried substance; _D^25?C = -39? to -45?.

Heavy metals. Use 1.0 g for the preparation of the test solution as described under “Limit test for heavy metals”, procedure 1 (Vol. 1, p. 118*). Determine the heavy metals content according to method A (Vol. 1, p. 119*); not more than 20 mg/g.

Sulfated ash. Not more than 1.0 mg/g.

Loss on drying. Dry for 3 hours at 105?C; it loses not more than 5 mg/g.

Related substances

(Note: Prepare the solutions immediately before use and maintain at 2-8?C until use)

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) (5mm) (Supelcosil LC-18-DB is suitable). As mobile phase A, use a mixture of 35 volumes of acetonitrile R and 965 volumes of a 0.77 g/l solution of ammonium acetate R. As mobile phase B, use a mixture of 250 volumes of acetonitrile R and 750 volumes of a 0.77 g/l solution of ammonium acetate R.

* Refers to The International Pharmacopoeia

Use the following gradient elution system:

Prepare the following solutions. For solution (1) use 0.5 mg of the test substance per ml. For solution (2) dilute 1.0 ml of this solution to 200 ml. For solution (3) Dilute 10 ml of solution (2) to 50 ml.

Operate with a flow rate of 2 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 254 nm.

Maintain the column temperature between 20-25?C.

Inject alternately 10µl each of solutions (1), (2), (3) and (4).

In the chromatogram obtained with solution (1), the following peaks are eluted at the following retention times ratio with reference to stavudine: impurity A = about 0.26; impurity B = about 0.49; impurity C = about 0.52; impurity D = about 0.69; impurity E = about 1.1 and impurity F = about 1.3.

[Note from WHO Secretariat: Details on solution (4) will be added as soon as the availability of the test mix has been confirmed.]

The test is not valid unless in the chromatogram obtained with solution (4) the resolution between the peaks corresponding to impurities B and C is greater than 1.5 and between impurity E and stavudine is greater than 1.5.

Measure the areas of the peak responses obtained in the chromatograms from solutions (1), (2) and (3) and calculate the content of related substances as a percentage. For the calculation of limit contents, multiply the peak area of impurity A by a correction factor of 0.69.

In the chromatogram obtained with solution (1), the area of the peak corresponding to impurity A is not greater than the principal peak in the chromatogram obtained with solution (2) (0.5%). For any other impurity, the peak area is not greater than the principal peak in the chromatogram obtained with solution (3) (0.1%). The sum of the areas of all the peaks, other than the principal peak, is not greater than twice the area of the principal peak in the chromatogram obtained with solution (2) (1.0%). Disregard any peak with an area less than 0.5 times the area of the principal peak in the chromatogram obtained with solution (3) (0.05%).

Assay: Weigh accurately about 25 mg of the test substance into a 50 ml volumetric flask. Add about 40 ml of water and shake to dissolve. Dilute to volume with water and mix. Dilute 3 ml of this solution to 100 ml with 0.1M HSO and mix. For the blank use 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 C₁₀H₁₂N₂O₄ using the absorptivity value of 43.5 (A ^1%_1cm= 435).

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 WHO Secretariat: Chemical structures will be included in the next version.]

A. Thymine
B. Thymidine epimer
C. Thymidine
D. Stavudine lactone
E. Stavudine anomer alpha
F. 3’,5’-anhydrothymidine

A typical chromatogram obtained for stavudine (Refer to the monograph text for chromatographic conditions in “Related substances”)