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 saquinavir mesilate capsules is provided below for comment.

Saquinavirum mesilas capsulae
Saquinavir mesilate capsules (first draft)

Category. Antiretroviral (protease inhibitor).

Storage. Saquinavir mesilate capsules should be kept in a well-closed container, protected from light.

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

Additional information. Strength in the current WHO Model List of Essential Medicines: 200 mg of saquinavir.

REQUIREMENTS

Complies with the monograph for “Capsules” (Vol. 4, p.32*)

Saquinavir mesilate capsules contain not less than 90.0 % and not more than 110.0 % of the amount of C₃₈H₅₀N₆O₅ stated on the label.

Identity tests

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 8 volumes of dichloromethane R, 2 volumes of 2propanol R as the mobile phase. Apply separately to the plate 5 µl of each of the following 2 solutions in methanol: (A) shake a quantity of the contents of the capsules equivalent to 22 mg of saquinavir with 5 ml, filter, and use the clear filtrate; and (B) 5 mg saquinavir 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).

* 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 8 volumes of dichloromethane R, 2 volumes of 2propanol R as the mobile phase. Apply separately to the plate 5 µl of each of the following 2 solutions in methanol: (A) shake a quantity of the contents of capsules equivalent to 22 mg of saquinavir with 5 ml, filter, and use the clear filtrate; and (B) 5 mg saquinavir 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. 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. To a quantity of the contents of capsules equivalent to about 20 mg saquinavir mesilate add 100 ml of methanol R, shake, and filter. Dilute 5 ml of the filtrate to 100 ml with the same solvent. The absorption spectrum of resulting solution, when observed between 220 nm and 280 nm, exhibits one maximum at about 239 nm.

C. To a quantity of the contents of capsules equivalent to 50 mg of saquinavir mesilate add 10 ml of methanol R, shake to dissolve, and filter. Evaporate the filtrate to dryness under vacuum. Carry out the examination with the residue as described under ‘‘Spectrophotometry in the infrared region’’ (Vol. 1, p. 40*). The infrared absorption spectrum is concordant with the spectrum obtained in a similar way from saquinavir mesilate RS or with the reference spectrum of saquinavir mesilate.

* Refers to The International Pharmacopoeia

[Note from WHO Secretariat: Feedback on the applicability of this method would be much appreciated.]

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: 50 volumes of a mixture of 5 parts of acetonitrile and 2 parts of methanol, 15 volumes of phosphate buffer pH 3.4 and 35 volumes of purified water.

Mobile phase B: 70 volumes of acetonitrile, 15 volumes of phosphate buffer pH 3.4 and 15 volumes of purified water.

Prepare the phosphate buffer pH 3.4 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.

Prepare the following solutions using mobile phase A as diluent. For solution (1) mix the content of 20 capsules and transfer a quantity equivalent to about 0.025 g of saquinavir, accurately weighed, into a 50 ml glass-stoppered flask. Add about 40 ml mobile phase A, 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 filtered solution. For solution (2) dilute a suitable volume of solution (1) to obtain a concentration equivalent to 2.5 µg of per ml.

For the system suitability test: prepare solution (3) using 2 ml of solution (1) and 5 ml of sufuric acid (475 g/l), heat in a water bath at 100°C 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 220 nm.

Maintain the column temperature at 30°C.

Inject 20 µl of solution (3). The test is not valid unless the resolution between the peak due to saquinavir (retention time = about 21 minutes) and the peak of similar size with a retention time of about 0.45 relative to the saquinavir peak is not less than 14. The test is also not valid unless the resolution between two smaller peaks of similar size, eluted after the saquinavir peak and which are increasing during decomposition, is not less than 2. The ratio of the retention times of these two peaks relative to the saquinavir 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 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 that obtained with solution (2) (0.5 %). Not more than one peak is greater than half the area of the principal peak obtained with solution (2) (0.25 %). The sum of the areas of all peaks, other than the principal peak, is not greater than twice the area of the principal peak obtained with solution (2)

(1.0 %). Disregard any peak with an area less than 0.2 times the area of the principal peak in thechromatogram obtained with solution (2) (0.1 %).

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: 50 volumes of a mixture of 5 parts of acetonitrile and 2 parts of methanol, 15 volumes of phosphate buffer pH 3.4 and 35 volumes of purified water. Prepare the phosphate buffer by dissolving 4.88 g of anhydrous sodium dihydrogen phosphate in 800 ml of distilled water, adjust the pH to 3.4 by adding phosphoric acid (105 g/l) and dilute to 1000 ml with purified water.

Prepare the following solutions using the mobile phase as diluent. For solution (1) mix the content of 20 capsules and transfer a quantity equivalent to about 0.025 g of saquinavir, accurately weighed, into a 50 ml glass-stoppered flask. Add about 40 ml mobile phase, 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 filtered solution. For solution (2) use 0.5 mg of saquinavir RS per ml mobile phase.

Operate with a flow rate of 1.0 ml per minute. As a detector use an ultraviolet spectro- photometer set at a wavelength of 220 nm.

Maintain the column temperature at 30?C.

Inject 20 µl of solution (2) in six replicate injections into the chromatographic system. The relative standard deviation for the peak area of saquinavir 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 saquinavir.

Measure the areas of the peak responses obtained in the chromatograms from solutions (1) and (2), and calculate the percentage content of C₃₈H₅₀N₆O₅.

B. Mix the contents of 20 capsules and transfer a quantity equivalent to about 0.020 g of saquinavir, accurately weighed, to a 100 ml glass stopperd flask. Add about 50 ml of methanol R, sonicate for 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 100.0 ml with the same solvent. Measure the absorbance of this solution in a 1-cm layer at the maximum at about 239 nm. Calculate the content of C₃₈H₅₀N₆O₅ using an absorptivity value of 71.5 (A ^{1 %}_{1 cm}= 715).

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.

Potassium permanganate, basic (1 g/l) TS

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