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

II.7 Sulfadoxine & Pyrimethamine formulations

II.7.1 Material and equipment

Material

· Orodar^® 525 mg tablets (sulfadoxine 500mg / pyrimethamine 25mg)

(Elys chemicals Industries, Kenya)

· Sulfadoxine 500mg / pyrimethamine 25mg (Labophar, Rwanda)

· Sulfadoxine (Indis, Belgium)

· Pyrimethamine (Sigma-Aldrich Chemie, Germany)

· Potassium dihydrogen phosphate (Vel, Belgium)

· Phenacetin (Sigma-Alidrich Chemie, Germany)

· Acetonitrile (Biosolve, The Netherlands)

· Glacial acetic acid (Merck Eurolab)

· Perchloric acid (UCB, Belgium)

All chemicals and reagents were at least of analytical grade.

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)

· Lambda 12 UV/VIS Spectrophotometer

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

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

II.7.2 Quantitative drug analysis

7.2.1 Methods

The amount of sulfadoxine and pyrimethamine and the dissolution rate for each formulation was determined using the methods described in USP 24.

· Mobile phase

A mixture of glacial acetic acid and water was made at the ratio of (1:50). 1200 ml from the above solution was mixed with 800ml of acetonitrile, and then 8ml of perchloric acid was added. The homogenized mixture was used as mobile phase.

· Internal standard:

120 mg of phenacetin was dissolved and diluted to 100.0 ml. 10 ml of that solution was diluted to 100.0 ml to obtain an internal solution having a concentration of 120 mg/l.

· Stock solution

550mg of sulfadoxine and 27 mg of pyrimethamine were separately weighed and dissolved in 35 ml of acetonitrile, mobile phase was added to 100.0 ml. 10 ml from the above solutions was diluted to 100.0 ml to obtain stock solution with concentrations of 550 mg/l for sulfadoxine and 27 mg/l for pyrimethamine, respectively.

Standard solutions

1, 2, 3, 5 and 6 ml from the stock solution were separately transferred into different flasks, 1 ml of internal standard was added, after which the solutions were diluted to 10.0 ml to obtain standard solutions having concentrations of 55, 110, 165, 275 and 330 mg/l for sulfadoxine. The pyrimethamine concentrations were 2.7, 5.4, 8.1, 13.5 and 16.2 mg/l.The internal concentration was always 12 mg/l of phenacetin.

· Sample preparation

From each formulation 10 tablets were weighed and powdered. An accurately weighed portion of powder, equivalent to 550 mg of sulfadoxine and 27 mg of pyrimethamine, was dissolved in 35 ml acetonitrile. The mixture was sonicated for about 25 minutes, diluted with mobile phase to 100.0 ml. The mixture was then filtered through a 0.2-um cellulose acetate filter (Sartorius, Goettingen, Germany).

From the filtrate 5 ml was transferred to a 100.0 ml flask, 1 ml of phenacetin solution (internal standard) was added and the volume was adjusted with mobile phase to make the assay preparation.

· Calibration curve

For sulfadoxine, a calibration curve (peak area of the sulfadoxine/phenacetin ratios vs. concentration) y = 0.0427 (0.0000) x + 0.2276 (0.0133) with a correlation coefficient (R²) of 0.9999 (0.0000) (n = 5) was constructed using standard solutions from 55 to 330 mg sulfadoxine / l.

For pyrimethamine, y = 0.0433 (0.0000) x - 0.0197 (0.0007) with a correlation coefficient (R²) of 0.9999 (0.0001) (n = 5) was constructed using standard solutions from 2.7 to 24.3 mg pyrimethamine / l.

The precision of the method was determined by calculating the relative standard deviation (RSD) of the peak area responses after repeated injections (n =5) of a sulfadoxine/pyrimethamine standard solution (275 and 13.5 mg/l, respectively) a day and within three days.

The resolution factors between sulfadoxine and phenacetin(R) and between phenacetin and pyrimethamine (R') were calculated from their respective peaks:

R= 2 (t₂ - t₁) / (w₁ + w₂)

With t₁ and w₁ being the retention time and baseline width of the sulfadoxine peak, t₂ and w₂, the respective values of phenacetin.

R' = 2 (t₃ - t₂ ) / (w₂+ w₃ )

With t₂ and w₂ being the retention time and baseline width of the phenacetin peak, t₃ and w₃ , the respective values pyrimethamine.

· Chromatographic conditions

Flow rate : 1.4 ml/min

Detection wavelength : 254 nm

Injection volume : 20 ul

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 quantity, Q, (in mg, of sulfadoxine in the portion of tablets taken was calculated by the following formula:

Q = 12.5 C (r _u / r _s)

In which C is the concentration, in mg/l, of sulfadoxine in the standard preparation, r_u and r_s the peak responses obtained from the assay preparation and the standard preparation, respectively.

The drug quantity, Q, (in mg, of pyrimethamine in the portion of tablets taken was calculated by the following formula:

Q = 0.2 C' (r' _u / r' _s)

In which C is the concentration, in mg/l, of pyrimethamine in the standard preparation, r'_u and r'_s the peak responses obtained form the assay preparation 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 %). The box was placed in an incubator maintained at 40°C 2°C. After 3 and 6 months, tablets were withdrawn from the incubator and evaluated for dissolution rate and their content in active ingredient.