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 nelfinivir mesilate tablets is provided below for comment.

Nelfinavir mesilas compressi
Nelfinavir mesilate tablets (first draft)

Category. Antiretroviral (protease inhibitor).

Storage. Nelfinavir mesilate capsules should be kept in a tightly closed container, protected from light.

Labelling. The designation on the container of nelfinavir mesilate tables should state that the active ingredient is in the mesilate form, and the quantity should be indicated in terms of the equivalent amount of nelfinavir. Expiry date.

Additional information. Strength in the current WHO Model List of Essential Medicines: 250 mg of nelfinavir (as mesilate).

REQUIREMENTS

Complies with the monograph for “Tablets” (Vol. 4, P. 26*).

* Refers to The International Pharmacopoeia

Nelfinavir mesilate tablet contains not less than 90.0 % and not more than 110.0 % of C₃₂H₄₅N₃O₄S stated on the label.

Identity tests

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 5 µl of each of the following 2 solutions in methanol R: (A) shake the quantity of powdered tablets equivalent to about 21 mg of nelfinavir with 5 ml, filter, and use the clear filtrate; and (B) 5 mg nelfinavir mesilate 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).

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 5 µl of each of the following 2 solutions in methanol R: (A) shake the quantity of powdered tablets equivalent to about 21 mg of nelfinavir with 5 ml, filter, and use the clear filtrate; and (B) 5 mg nelfinavir mesilate 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 the plate with basic potassium permanganate (5 g/l) TS. Examine the chromatogram in daylight.

The principal spot obtained with solution A corresponds in position, appearance, and intensity with that obtained with solution B.

B. To a quantity of the tablets equivalent to about 20 mg of nelfinavir add 50 ml of methanol R, shake, and filter. Dilute 5 ml of the filtrate to 50 ml with the same solvent. The absorption spectrum of the resulting solution, when observed between 220 nm and 280 nm, exhibits one maximum at about 253 nm.

Related substances

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 base-deactivated octadecylsilyl silica gel for chromatography R (5 µm).

* Refers to The International Pharmacopoeia

Use the following conditions for gradient elution:

Mobile phase A: 27 volumes of acetonitrile R, 20 volumes of methanol R, 28 volumes of phosphate buffer pH 3.4 and 25 volumes of purified water.

Mobile phase B: 41 volumes of acetonitrile R, 31 volumes of methanol R and 28 volumes of phosphate buffer pH 3.4.

Prepare the phosphate buffer pH 3.4 by dissolving 4.88 g of anhydrous sodium dihydrogenphosphate in 800 ml of purified water, adjust the pH to 3.4 by adding phosphoric acid (105 g/l) and dilute it to 1000 ml with purified water.

For solution (1) weigh and powder 20 tablets, and transfer a quantity equivalent to about 0.10 g of nelfinavir, accurately weighed, into a 50 ml volumetric flask. Add about 20 ml of methanol R, sonicate for about 15 minutes, allow to cool to room temperature, and make up the volume using mobile phase A. Filter a portion of this solution through a 0.45 µm filter, discarding the first few ml of the filtered solution. For solution (2) dilute a suitable volume of solution A to obtain a concentration equivalent to 10.0 µg of nelfinavir per ml of mobile phase A. For solution (3) use 100 µg of methanesulfonic acid per ml of mobile phase A.

For the system suitability test: prepare solution (4) using 2 ml of solution (1) and 5 ml of sulphuric acid (475 g/l), heat carefully in a boiling water-bath for 30 minutes.

Operate with a flow rate of 1.0 ml per minute. As a detector use an ultraviolet spectro- photometer set at a wavelength of 225 nm.

Maintain the column at 35?C.

Inject 20 µl of solution (4). The test is not valid unless the resolution between the principal peak (retention time = about 27 minutes) and the peak with a retention time relative to the principal peak of about 0.2 is not less than 15. The test is also not valid unless the resolution between the last two peaks out of three peaks, which are growing during decomposition, is not less than 4.0. The ratio of the retention times of these two peaks relative to the principal peak is about 1.8 and 1.9 respectively. If necessary adjust the amount of acetonitrile in both mobile phases A and B, or adjust the gradient programme.

Inject 20 µl of solution (3).

Inject alternatively 20 µl each of solutions (1) and (2).

Measure the areas of the peak responses obtained in the chromatograms from solutions (1) and (2). In the chromatograms obtained with solution (1), the area of any peak, other than the principal peak, is not greater than two times the area of the principal peak obtained with solution (2) (1.0 %). Not more than two peaks are greater than the area of the principal peak obtained with solution (2) (0.5 %). Not more than one other peak is greater than 0.4 times the area of the principal peak obtained with solution (2) (0.2 %). The sum of the areas of all peaks, other than the principal peak, is not greater than four times the area of the principal peak obtained with solution (2) (2.0 %). Disregard any peak with retention time less than 5 min and any peak with an area less than 0.2 times the area of the principal peak in the chromatogram obtained with solution (2) (0.1 %). Disregard any peak due to methanesulfonic acid, corresponding to the principal peak in the chromatogram obtained with solution (3).

Assay

Either method A or method B may be applied.

A. 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 base-deactivated octadecylsilyl silica gel for chromatography R (5 µm).

* Refers to The International Pharmacopoeia

As the mobile phase, use a solution prepared as follows: 27 volumes of acetonitrile R, 20 volumes of methanol R, 28 volumes of phosphate buffer pH 3.4 and 25 volumes of purified water. Prepare the phosphate buffer by dissolving 4.88 g of anhydrous sodium dihydrogen phosphate in 800 ml of purified water, adjust the pH to 3.4 by adding phosphoric acid (105 g/l) and dilute to 1000 ml with purified water.

For solution (1) weigh and powder 20 tablets, and transfer a quantity equivalent to about 0.10 g of nelfinavir, accurately weighed, into a 50 ml volumetric flask. Add about 20 ml of methanol R, sonicate for about 15 minutes, allow to cool to room temperature, and make up the volume using the mobile phase. Filter a portion of this solution through a 0.45 µm filter, discarding the first few ml of filtered solution. For solution (2) use 2 mg of nelfinavir mesilate RS per ml prepared in the same manner.

Operate with a flow rate of 1.0 ml per minute. As a detector use an ultraviolet spectro- photometer set at a wavelength of 225 nm.

Maintain the column temperature at 35?C.

Inject 20 µl of solution (2) in six replicate injections into the chromatographic system. The relative standard deviation for the peak area of nelfinavir is not more than 2.0 %.

Inject alternatively 20 µl each of solutions (1) and (2) and record the chromatograms for 1.5 times the retention time of nelfinavir.

Measure the areas of the peak responses obtained in the chromatograms from solutions (1) and (2), and calculate the percentage content of C₃₂H₄₅N₃O₄S.

B. Weigh and powder 20 tablets. Transfer a quantity of the contents of tablets equivalent to about 20 mg of nelfinavir, accurately weighed, to a 50 ml volumetric flask. Add about 25 ml of methanol R, sonicate for about 5 minutes, allow to cool to room temperature, and make up the volume using the same solvent. Filter a portion of this solution through a 0.45 µm filter, discarding the first few ml of the filtrate. Dilute 5.0 ml of the filtrate to 50.0 ml with the same solvent. Measure the absorbance of this solution in a 1-cm layer at the maximum at about 253 nm against a solvent cell containing methanol R.

Calculate the content of C₃₂H₄₅N₃O₄S using an absorptivity value of 15.7 (A ^{1 %}_{1 cm} = 157).

Reagents

Silica gel for chromatography, octadecylsilyl, base deactivated

A very finely divided silica gel, pre-treated before the bonding of octadecylsilyl groups to minimize the interaction with basic compounds.

Methanesulfonic acid

Molecular formula: CH₄O₃S
Description: Colourless and corrosive liquid.
Solubility: Miscible with water.
Density (d): ~1.48.
Melting point:About 20°C.

Sodium dihydrogen phosphate, anhydrous

Molecular formula: NaH₂PO₄
Description: White powder, hygroscopic.
Storage: in an airtight container.

Potassium permanganate, basic (5 g/l) TS

A solution of potassium permanganate R containing about 5 g of KMnO₄ per litre of sodium hydroxide (1 mol/l).