An in vitro study of the quality of essential drugs available on the rwandan market

II.3 Sulfamethoxazole / Trimethoprim (Cotrimoxazole) formulations

II.3.1 Material and equipment

Material

· Batrimox 480 mg tablets (Sulfamethoxazole 400 mg / Trimethoprim 80 mg)

(S&R Pharmaceuticals, Rwanda)

· Unitrim 480 mg tablets(Sulfamethoxazole 400 mg / Trimethoprim 80 mg)

(Elys chemicals industries, Kenya)

· Bactiphar 480 mg tablets (Sulfamethoxazole 400 mg / Trimethoprim 80 mg) (Labophar, Rwanda)

· Sulfamethoxazole (Alpha pharma, Belgium)

· Trimethoprim (Alpha pharma, Belgium)

· Hydrochloric acid 37% (Merck/Eurolab, Darmstadt, Germany)

· Acetonitrile HPLC grade (Biosolve, The Netherlands)

· Glacial acetic acid 100% (Merck/Eurolab, Darmstadt, Germany)

· Triethylamine (Sigma chemicals, St Louis, USA)

Equipment

· Incubator: U-60 (Memmert, Analis, Namen, Belgium)

· Column: Lichrospher 100 RP-C 18 e (5um), 250X4 mm

(Merck-Hitachi, Darmstadt, Germany)

· Detector: L-7400 UV detector (Merck-Hitachi, Darmstadt, Germany)

· Pump: L-7100 pump (Merck-Hitachi, Darmstadt, Germany)

· Integrator: D-7000 integrator (Merck-Hitachi, Darmstadt, Germany)

· Software Package `HPLC System Manager'

(Merck-Hitachi, Darmstadt, Germany)

· Lambda 12 UV/VIS Spectrophotometer

(Perkin Elmer UV/VIS, Perkin Elmer, Norwalk, USA)

· Dissolution equipment (VK 7000, Vankel Technology, Cary, NC, USA)

II.3.2 Quantitative drug analysis

3.2.1 Methods

The amount of sulfamethoxazole and trimethoprim and the dissolution rate of both drug for each formulation were determined using the methods described in the USP 24.

· Mobile phase

A mixture of 650 ml distilled water, 250 ml acetonitrile, and 1 ml triethylamine was homogenized and allowed to equilibrate at room temperature. The pH of the above mixture was adjusted to 5.9 0.1 using diluted glacial acetic acid (10%). The resulting solution was diluted to 1.0 L to obtain the mobile phase.

· Standard solution

Separately, 160 mg of sulfamethoxazole and 32 mg of trimethoprim were accurately weighed and dissolved in methanol to give a 100.0 ml solution. The above solution had a concentration of 1600 mg/l and 320 mg/l of sulfamethoxazole and trimethoprim, respectively. 5.0 ml from the above solution was diluted to 50.0 ml to obtain standard solution with concentration of 160 mg/l and 32 mg/l of those two compounds, respectively.

· Sample preparation

From each formulation 10 tablets were weighed and finely powdered. An accurately weighed portion of powder equivalent to 160 mg of sulfamethoxazole was diluted with mobile phase to give 100.0 ml of suspension, sonicated for about 5 min and filtered through a 0.2-um cellulose acetate filter (Sartorius, Goettingen, Germany). 5.0 ml from the filtrate were diluted to 50.0ml and used as assay preparation.

· Calibration curve

A calibration curve (peak area vs. concentration) y = 64590 (122) x + 43448 (351) with a correlation coefficient (R²) of 0.9995 (0.0001) (n = 5) was constructed using standard solutions with sulfamethoxazole concentrations from 16 to 160 mg/l. For trimethoprim a calibration curve y = 31476 (1265) x + 2088 ( 509) with a correlation coefficient (R²) of 0.9979 (0.0025) (n = 5) was constructed using standard solutions with trimethoprim concentrations from 3.2 to 32 mg/l. The precision of the method was determined by calculating the relative standard deviation (within a day and within three days) of the peak area responses after repeated injections (n = 5) of a standard solution (160 mg/l sulfamethoxazole and 32 mg/l trimethoprim).

The resolution factor (R) between sulfamethoxazole and trimethoprim was calculated from their respective peaks:

R= 2 ( t₁ - t₂ ) / (w₁ + w₂)

With t₁ and w₁ being the retention time and baseline width of the sulfamethoxazole peak, t₂ and w₂, the respective values for trimethoprim.

· Chromatographic conditions

Flow rate : 0.8 ml/min

Detection wavelength : 254 nm

Injection volume : 20ul

Temperature : Room temperature

· Procedure

Equal volumes of standard and assay preparations were separately injected, the chromatograms were recorded and the major peaks integrated. The drug quantities, Q, (in mg of sulfamethoxazole and trimethoprim in the portion of tablets taken) were calculated by the formula:

Q=1000 C (ru/r_s)

Whereby C is the concentration, in mg/ml, of sulfamethoxazole and trimethoprim in the standard preparation, r_u and r_s are the analyte corresponding peak responses obtained from the assay and the standard preparation, respectively.

· Stability testing

A part of the tablets was stored in a sealed box containing a saturated solution of sodium chloride (RH 75 5 %). This box was placed in an incubator maintained at 40 2°C. After 3 and 6 months, tablets were withdrawn from the incubator and evaluated for dissolution rate and their content in active ingredient.