Nelfinavir mesilas
Nelfinavir mesilate

Molecular formula: C₃₂H₄₅N₃O₄S,CH₄O₃S

Relative molecular mass: 663.9

Graphic formula:

Chemical name: (3S,4aS,8aS)-N-(1,1-dimethylethyl)-2-[(2R,3R)-2-hydroxy-3-[(3-hydroxy-2-methylbenzoyl)amino]-4-(phenylsulfanyl)butyl]decahydroisoquinoline-3-carboxamide methanesulfonate CAS Reg. No. 159989-65-8.

Description: White or almost white powder.

Solubility: Practically insoluble in water and soluble in methanol R.

Category: Antiretroviral (protease inhibitor).

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

Additional information: Nelfinavir mesilate is hygroscopic.

REQUIREMENTS

General requirement: Nelfinavir mesilate contains not less than 98.0 % and not more than 101.0 % of C₃₂H₄₅N₃O₄S.CH₄O₃S, calculated with reference to the dried substance.

Identity tests

Either tests A and B or test C may be applied.

A. TLC: to be done.

B. The absorption spectrum of a 40 mg/ml solution in methanol R, when observed between 220 nm and 280 nm, exhibits one maximum at about 253 nm; the specific absorbance (A ^1%_1cm) calculated with reference to the dried substance is 124 to136.

C. Carry out the examination as described under “Spectrophotometry in the infrared region” (Vol. 1, p. 40¹). The infrared absorption spectrum is concordant with the reference spectrum of nelfinavir mesilate.

¹ Refers to The International Pharmacopoeia.

Specific optical rotation: Use 10.0 mg/ml solution in methanol R and calculate with reference to the dried substance; [α]_D 20 °C = -110° to -125°.

Heavy metals: Use 1.0 g in 30 ml of methanol R for the preparation of the test solution as described under “Limit test for heavy metals”, Procedure 2, (Vol. 1, p.118¹); determine the heavy metals content according to method A (Vol. 1, p.118¹); not more than 20 μg/g.

¹ Refers to The International Pharmacopoeia.

Sulfated ash: Not more than 1.0 mg/g. (Vol. 1, p.123¹).

¹ Refers to The International Pharmacopoeia.

Loss on drying: Weigh 1.000 g, dry to constant mass at 100 °C; it loses not more than 30 mg/g.

Related substances: Carry out the test as described under “High-performance liquid chromatography” (Vol. 5, p. 257¹), using a stainless steel column (25 cm x 4.6 mm) packed with base-deactivated octadecylsilyl silica gel for chromatography R (5μm) (Note: Hypersil BDS C18 is suitable.)

¹ 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.

Prepare the following solutions using mobile phase A as diluent. For solution (1) use 2.0 mg of nelfinavir mesilate per ml. For solution (2) dilute a suitable volume of solution (1) to obtain a concentration equivalent to 10.0 μg of nelfinavir mesilate per ml. For solution (3) use 100 μg of methanesulfonic acid per ml.

For the system suitability test: prepare solution (4) using 2 ml of solution (1) and 5 ml of sulfuric acid (475 g/l), heat carefully in a water bath at 90 °C for 110 minutes.

Operate with a flow rate of 1 ml per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of about 225 nm.

Maintain the column at 35 °C.

Inject 20 ml of solution (4). The test is not valid unless the resolution factor between the principal peak 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 factor 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.

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) and calculate the content of related substances as a percentage. 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 %). The sum of the areas of all peaks, other than the principal peak, is not greater than two times the area of the principal peak obtained with solution (2) (1.0 %). Disregard any peak with an area less than 0.04 times the area of the principal peak in the chromatogram obtained with solution (2) (0.02%). Ignore any peak due to methanesulfonic acid, corresponding to the principal peak in the chromatogram obtained with solution (3).

Assay: Dissolve about 0.50 g, accurately weighed, in 50 ml of methanol R and titrate with sodium hydroxide (0.1 mol/l) VS, determine the end point potentiometrically. Perform a blank determination and make the necessary correction. Each ml of sodium hydroxide (0.1 mol/l) VS is equivalent to 66.39 mg of C₃₂H₄₅N₃O₄S.CH₃SO₃H.

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 R

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

A typical chromatogram obtained for nelfinavir mesilate (Refer to the monograph text for chromatographic conditions in “Related substances” ¹)

¹ Refers to The International Pharmacopoeia.

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

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