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

II.1.2 Quantitative drug analysis

1.2.1 Methods

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

· Standard preparation

160 mg of amoxicillin was accurately weighed and dissolved in about 80 ml of diluent. The resulting solution was diluted to 100.0 ml to give a solution with an amoxicillin concentration of 1600 mg/l. 7.5ml from the above solution were diluted to 10.0 ml with diluent to give a standard solution with an amoxicillin concentration of 1200 mg/l.

· Sample preparation

The content of 10 capsules was removed as complete as possible and accurately weighed. A portion equivalent to 240 mg of anhydrous amoxicillin was dissolved in about 180 ml of diluent.The suspension was mixed, sonicated and diluted to 200.0 ml, then filtered through a 0.2um cellulose acetate filter (Sartorius, Goettingen, Germany). The filtrate was used as assay preparation.

· Calibration curve

A calibration curve (peak area vs. amoxicillin concentration) y = 29147 (298)x + 2709 (59) with a correlation coefficient (R²) of 0.9996 (0.0001) (n = 3) was constructed using standard solutions from 60 to 300 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 an amoxicillin standard solution (120 mg/l).

· Diluent preparation

13.6 g of monobasic potassium phosphate (KH₂PO₄) was dissolved in 2000 ml of distilled water, the pH adjusted to 5.0 0.1 by using a 45% (w/w) aqueous solution of potassium hydroxide.

· Mobile phase

The mobile phase consisted of a degassed mixture of diluent and acetonitrile in a ratio of 94:6 (v/v).

· Chromatographic conditions

Flow rate: 1.4 ml/min

Detection wavelength: 230 nm

Injection volume: 20ul

Temperature: Room temperature

Equal volumes of standard and assay preparations were separately injected, the chromatograms were recorded, and the major peak integrated. The quantity, in mg, of anhydrous amoxicillin in the portion of capsules taken was calculated by the formula:

0.2 CP( r_u/r_s )

In which C is the concentration, in mg/ml, of amoxicillin in the standard preparation, P is the stated amoxicillin content in ug/mg, r_u and r_s are the amoxicillin peak responses obtained from the assay and the standard preparation, respectively.

A part of the capsules was stored in a sealed box above a saturated solution of sodium chloride (RH 75% 5 %). The sealed box was placed in an incubator maintained at 40°C.

1.2.2 Results

The relative standard deviation (RSD) of the chromatographic method was 0.24 % within a day and 1.36% within three days, which complies with the USP 24 requirements (RSD should be less than 2%) and proving the precision of the method.

The results of the drug content (Table 1.1) show that all formulations complied with USP 24 specifications for amoxicillin content (90% - 120% of the labelled content).

Table 1.1: The amoxicillin content (expressed as a percentage of the labelled amount) before and after 6 months of stability testing at simulated tropical conditions.

Manufacturer % of the labelled amount per capsule

0 months 6 months

Elys chemicals (Elymox) 102.4 100.8

Labophar (Amoxyphar) 103.7 101.6

Dilam (Amoxysha 500) 100.8 98.4

Containing 500 mg amoxicillin per capsule.