Category. Antiretroviral (Nucleoside/Nucleotide Reverse Transcriptase Inhibitor).

Storage. Emtricitabine and tenofovir disoproxil tablets should be kept in a tightly closed container.

Additional information. Strength in the current WHO Model list of essential medicines: 200 mg Emtricitabine and 300 mg Tenofovir disoproxil fumarate.

Requirements

Comply with the monograph for Tablets.

Definition. Emtricitabine and tenofovir disoproxil tablets contain Emtricitabine and Tenofovir disoproxil fumarate. They contain not less than 90.0% and not more than 110.0% of the amounts of emtricitabine (C₈H₁₀FN₃O₃S) and tenofovir disoproxil fumarate (C₁₉H₃₀N₅O₁₀P,C₄H₄O₄) stated on the label.

Manufacture. The manufacturing process and the product packaging are designed and controlled so as to minimize the moisture content of the tablets. They ensure that, if tested, the tablets would comply with a water content limit of not more than 50 mg/g when determined as described under 2.8 Determination of water by the Karl Fischer method, Method A, using about 0.5 g of the powdered tablets.

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 1.14.1 Chromatography, Thin-layer chromatography 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 the following three solutions in methanol R. For solution (A) disperse a quantity of powdered tablets to obtain a concentration of 5 mg of Emtricitabine per mL, filter and use the filtrate. For solution (B) use 5 mg of emtricitabine RS. For solution (C) use 7.5 mg of tenofovir disoproxil fumarate RS per mL. After removing the plate from the chromatographic chamber allow it to dry exhaustively in air or in a current of air. Examine the chromatogram in ultraviolet light (254 nm).

One of the two principal spots obtained with solution A corresponds in position, appearance and intensity with that obtained with solution B and the other one corresponds with that obtained with solution C.

A.2 Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography using the conditions described above under test A.1 but using silica gel R5 as the coating substance. Stain the plate with iodine vapour and examine the chromatogram in daylight.

One of the two principal spots obtained with solution A corresponds in position, appearance and intensity with that obtained with solution B and the other one corresponds with that obtained with solution C.

B. Carry out test B.1 or, where UV detection is not available, test B.2.

B.1 Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography using silica gel R6 as the coating substance and a mixture of 50 volumes of heptane R, 30 volumes of glacial acetic acid R and 20 volumes of dichloromethane R as the mobile phase. Apply separately to the plate 5 μl of each of the following two solutions in ethanol R. For solution (A) disperse a quantity of powdered tablets to obtain a concentration of 10 mg of Tenofovir disoproxil fumarate per mL, filter and use the filtrate. For solution (B) use 2 mg of fumaric acid R per mL. Develop the plate in an unsaturated tank over a path of 10 cm. After removing the plate from the chromatographic chamber allow it to dry exhaustively in air or in a current of air. Examine the chromatogram in ultraviolet light (254 nm).

One of the spots obtained with solution A corresponds in position, appearance and intensity with that obtained with solution B.

B.2 Carry out the test as described under 1.14.1 Chromatography, Thin-layer chromatography using the conditions described above under test B.1 but using silica gel R5 as the coating substance. Spray lightly with a 16 g/l solution of potassium permanganate R and examine the chromatogram in daylight.

One of the spots obtained with solution A corresponds in position, appearance and intensity with that obtained with solution B.

C. See the test described under “Assay”. The retention times of the principal peaks in the chromatogram obtained with solution (1) are similar to those due to emtricitabine, tenofovir disoproxil and to fumarate in the chromatogram obtained with solution (2).

Dissolution

Carry out the test as described under 5.5 Dissolution test for solid oral dosage forms using as the dissolution medium 900 mL of hydrochloric acid (~0.04 g/l) TS and rotating the paddle at 50 revolutions per minute. At 30 minutes withdraw a sample of 10 mL of the medium and filter. Allow the filtered sample to cool to room temperature and dilute if necessary (solution (1)). Prepare solution (2) using 0.22 mg of emtricitabine RS and 0.33 mg of tenofovir disoproxil fumarate RS per mL of dissolution medium. Determine the content of emtricitabine (C₈H₁₀FN₃O₃S) and tenofovir disoproxil fumarate (C₁₉H₃₀N₅O₁₀P,C₄H₄O₄) as described under “Assay” using solution (1) and solution (2).

For each of the six tablets tested calculate the total amount of emtricitabine (C₈H₁₀FN₃O₃S) and tenofovir disoproxil fumarate (C₁₉H₃₀N₅O₁₀P,C₄H₄O₄) in the medium from the results obtained using the declared content of emtricitabine (C₈H₁₀FN₃O₃S) in emtricitabine RS and the declared content of tenofovir disoproxil fumarate (C₁₉H₃₀N₅O₁₀P,C₄H₄O₄) in tenofovir disoproxil fumarate RS. For both substances the amount in solution for each tablet is not less than 80% of the amount stated on the label. If the amount obtained for one of the six tablets is less than 80% repeat the test using a further six tablets; the average amount for all 12 tablets tested is not less than 75% and no tablet releases less than 60%.

Tenofovir monosoproxil.¹ Carry out the test as described under 1.14.1 Chromatography, High-performance liquid chromatography using the conditions given under “Assay”.

¹ The structure of tenofovir monosoproxil is given in the monograph on Tenofovir disoproxil fumarate.

After preparation keep the solutions at about 6 °C or use an injector with cooling.

Prepare the following solutions using a mixture of 20 volumes of acetonitrile R and 80 volumes of water R as a diluent. For solution (1) disperse a quantity of the powdered tablets containing about 100 mg of Tenofovir disoproxil fumarate, accurately weighed, in 100 mL of the diluent and filter. For solution (2) dilute a suitable volume of solution (1) to obtain a concentration of 5 µg of Tenofovir disoproxil fumarate per mL. For solution (3) heat carefully 1 mg of tenofovir disoproxil fumarate RS per mL of water R in a boiling water-bath for 20 minutes.

Operate with a flow rate of 1.0 mL per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of 280 nm.

Maintain the column temperature at 35 °C.

Inject 20 μl of solution (3). The peak due to tenofovir monosoproxil elutes at a relative retention of about 0.9 with reference to tenofovir disoproxil (retention time about 18 minutes).

Inject separately 20 μl each of solutions (1) and (2). The test is not valid unless in the chromatogram obtained with solution (1) three principal peaks are shown and the resolution between these peaks is at least 5.

In the chromatogram obtained with solution (1) the area of any peak due to tenofovir monosoproxil is not greater than ten times the area of the principal peak obtained with solution (2) (5.0%).

Assay

Carry out the test as described under 1.14.1 Chromatography, High-performance liquid chromatography using a stainless steel column (25 cm x 4.6 mm) packed with base-deactivated particles of silica gel the surface of which has been modified with chemically-bonded octadecylsilyl groups (5 μm).

The mobile phases for gradient elution consist of a mixture of Mobile phase A and Mobile phase B using the following conditions:

Mobile phase A:	5 volumes of potassium dihydrogen phosphate (27.2 g/l) TS and 95 volumes of water R.
Mobile phase B:	70 volumes of acetonitrile R, 5 volumes of potassium dihydrogen phosphate (27.2 g/l) TS and 25 volumes of water R.

Time (min)	Mobile phase A (% v/v)	Mobile phase B (% v/v)	Comments
0–9	93	7	Isocratic
9–15	93–0	7–100	Linear gradient
15–19	0	100	Isocratic
19–19.1	0–93	100–7	Return to initial composition
19.1–30	93	7	Re-equilibration

After preparation keep the solutions at about 6 °C or use an injector with cooling.

Prepare the following solutions using a mixture of 20 volumes of acetonitrile R and 80 volumes of water R as a diluent. For solution (1) weigh and powder 20 tablets. Disperse a quantity of the powder containing about 10 mg of Tenofovir disoproxil fumarate, accurately weighed in 100 mL of the diluent, and filter. For solution (2) use 0.1 mg of tenofovir disoproxil fumarate RS and 66.7 µg of emtricitabine RS per mL of diluent. If necessary adapt the concentration of solution (2) according to the ratio of Emtricitabine and Tenofovir disoproxil fumarate in the tablets. For solution (3) use 0.02 mg of fumaric acid R per mL of water R.

Operate with a flow rate of 1.0 mL per minute. As a detector use an ultraviolet spectrophotometer set at a wavelength of 280 nm.

Maintain the column temperature at 35 °C.

Inject separately 20 µl each of solutions (1), (2) and (3).

The test is not valid unless in the chromatograms obtained with solutions (1) and (2) the three principal peaks that elute at the following retention times are shown: fumarate (about 2.5 minutes); emtricitabine (about 9 minutes); tenofovir disoproxil (about 18 minutes). The test is also not valid unless the resolution between these peaks is at least 5.

Measure the areas of the peak responses obtained in the chromatograms from solutions (1) and (2) and calculate the content of emtricitabine (C₈H₁₀FN₃O₃S) and tenofovir disoproxil fumarate (C₁₉H₃₀N₅O₁₀P,C₄H₄O₄) in the tablets using the declared content of emtricitabine (C₈H₁₀FN₃O₃S) in emtricitabine RS and the declared content of tenofovir disoproxil fumarate (C₁₉H₃₀N₅O₁₀P,C₄H₄O₄) in tenofovir disoproxil fumarate RS.