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

II.6 Quinine formulations

II.6.1 Material and equipment

Material

· Quinine sulfate 300 mg tablets (Pharmakina, Dem. Rep. of Congo)

· Quinine sulfate sugar-coated 300 mg tablets (Elys Chemicals, Kenya)

· Quinine (base) 300 mg tablets (Labophar, Rwanda)

· Quinine sulfate dihydrate 99 % (Acros Organics, Belgium)

· Methane sulfonic acid (Acros Organics, Belgium)

· Diethylamine (Vel, 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, Germany)

· Lambda 12 UV/VIS Spectrophotometer

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

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

II.6.2 Quantitative drug analysis

6.2.1 Methods

The amount of quinine and the dissolution rate for each formulation was determined using the method described in USP 24 monogrphs.

· Mobile phase

The mobile phase consisted of a filtered and degassed mixture of water, acetonitrile, methane sulfonic acid, and diethylamine solution (860:100:20:20). The pH was adjusted to 2.6 with a diethylamine solution .

The methanesulfonic acid solution was prepared as follows: 35 ml of methanesulfonic acid was added to 20 ml of glacial acetic acid and the mixture was diluted to 500.0 ml with distilled water.

Diethylamine solution: 10 ml of diethylamine was diluted to 100.0 ml with distilled water.

· Standard preparation

20 mg of quinine sulfate, accurately weighed, was transferred to a 100 ml volumetric flask, dissolved and diluted to volume with mobile phase. The resulting solution was used as standard preparation.

· Assay preparation

From each formulation 10 tablets were weighed and finely powdered.

An accurately weighed portion of powder, equivalent to about 160 mg of quinine sulfate, was dissolved in about 80 ml of methanol and mechanically shaken for about 30 minutes, then diluted to 100 ml. The mixture was filtered through a 0.2 um cellulose acetate filter (Sartorius, Goettingen, Germany). The first 10 ml were discarded. 3 ml of the filtrate was diluted to 25 ml with mobile phase to obtain an assay preparation with concentration of 192 mg/l.

· Chromatographic conditions

Flow rate : 1 ml/min

Detection wavelength : 235 nm

Injection volume : 20 ul

Temperature : Room temperature

· Calibration curve

A calibration curve (peak area vs. concentration) y = 38643219 (5716) x + 78532 (2321) with a correlation coefficient (R²) of 0.9997 (0.0000) (n = 3) was constructed using standard solutions from 0.1 to 1.0 g/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 =3) of a quinine sulfate standard solution (200 mg/l).

· 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 the sum of quinine sulfate and dihydroquinine sulfate in the portion of tablets taken) was calculated by the formula:

Q = (2500/3)C (r _{b, u} +r _{d, u})/( r _{b, s} +r _{d, s)}

In which C is the concentration, in mg/ml, of quinine sulfate in the standard preparation, r _{b, u} and r _{b, s} are the peak responses of quinine obtained from the assay preparation and the standard preparation, respectively, r_{d, u} and r _{d, s} are the peak responses of dihydroquinine obtained from the assay and the standard preparation, respectively.

· Stability testing

A part of the tablets was stored in a sealed box above 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 of active ingredient.