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

II.4 Metronidazole formulations

II.4.1 Material and equipment

Material

· Elogyl 200mg tablets (Elys Chemicals Industries, Kenya)

· Metronidazole 250mg tablets (Holden Medica, The Netherlands)

· Metronidazole 250mg tablets (Labophar, Rwanda)

· Metronidazole (Alpha pharma, Belgium)

· Methanol (Biosolve, The Netherlands)

· Hydrochloric acid (Sigma Aldrich Chemie, Germany)

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.4.2. Quantitative drug analysis

4.2.1 Methods

The amount of metronidazole and the dissolution rate for each formulation were determined using the methods described in USP 24.

· Mobile phase

A degassed mixture of methanol and distilled water (20:80) was used as mobile phase.

· Standard preparation

An accurately weighed quantity (50 mg) of metronidazole standard was dissolved in mobile phase to obtain a 100.0 ml solution having a known concentration of 0.5 mg/ml, which was used as standard preparation.

· Assay preparation

From each formulation 10 whole tablets were transferred to a suitable sized volumetric flask, which when diluted with methanol yielded a solution having a concentration of 10 mg/ml. In case of Elys formulation (Elogyl) a 200.0 ml flask was used, while for the others 250.0 ml flasks were used. Methanol was added and the mixture shaken by mechanical means until the tablets were disintegrated. Methanol was added to volume. The mixture was allowed to stand until the insoluble material had settled. 5 ml of the clear supernate liquid was pipeted, diluted to 100.0 ml using mobile phase, mixed and filtered through a 0.2 um cellulose acetate filter (Sartorius, Goettingen, Germany). The resulting filtrate was used as assay preparation.

· Chromatographic conditions

Flow rate: 1ml/min

Detection wavelength: 254 nm

Injection volume: 20ul

Temperature: Room temperature

· Calibration curve

A calibration curve (peak area vs. concentration) y = 16582622 (133565) x + 73066 (9932) with a correlation coefficient (R²) of 0.9997 (0.0001) (n = 3) was constructed using standard solutions from 50 to 500 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 =3) of a metronidazole standard solution (500 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 metronidazole in the portion of tablets taken) was calculated by the formula:

Q = 10(L/D) C (ru/r_s)

Whereby L is the labelled amount, in mg, of metronidazole in each tablet, D is the concentration (mg/ml) of metronidazole in the assay preparation, C is the concentration (mg/ml) of the standard preparation, r_u and r_s are the metronidazole peak responses obtained from 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 of active ingredient.

4.2.2 Results

The RSD was 0.37 % within a day and 0.46% within three days, which complies with the USP 24 requirements (RSD should be less than 2%).

The results of the drug content (Table 4.1) show that all formulations complied with the USP 24 specifications for metronidazole content: 90% - 110% of the labelled amount.

Table 4.1: The metronidazole content (expressed as percentage of the labelled amount) before and after 6 months of storage at 40°C and 75 % RH.

Manufacturer % of the labelled amount per tablet

0 months 6 months

Elys chemicals 98.2 93.5

Labophar 98.6 97.2

Holden Medica 91.7 90.3