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

II.2 Acetylsalicylic acid formulations

II.2.1 Material and equipment

Material

· Aspirin 500 mg tablets (B.J. International, India)

· Minasprin 300 mg tablets (Girlloh Pharmacy, Surendra Nagar, India)

· Saraprin 500 mg tablets (S&R pharmaceuticals, Rwanda)

· Aspirin 500 mg tablets (Bayer, Greece)

· Acetonitrile (Biosolve, The Netherlands)

· Acetylsalicylic acid (Sigma - Aldrich chemie, Germany)

· Salicylic acid (Ludeco-Belgium)

· Formic acid (Sigma - Aldrich chemie, Germany)

· 1-Heptanesulfonate sodium (Sigma - Aldrich chemie, Germany)

· Glacial acetic acid (Vel, Belgium)

· Potassium dihydrogen phosphate (Vel, Belgium)

· Orthophosphoric acid (Vel, Belgium)

· Sodium acetate anhydrous (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.2.2 Quantitative drug analysis

2.2.1 Methods

The amount of acetylsalicylic acid and salicylic acid and the dissolution rate for each formulation was determined using the methods described in USP 24.

· Mobile phase

1.36 g of potassium dihydrogen phosphate was weighed dissolved in distilled water to make 1L of solution having a concentration of 0.01M.

0.67 ml of orthophosphoric acid (H₃PO₄ , M.M: 98, 1.71 kg/l, 85%) were transferred to a 1L flask and distilled water was added to make a 0.01M solution. The above solutions were mixed in a ratio of 50:50 and the pH adjusted to 2.3 with orthophosphoric acid.

A mixture of the resulting solution, acetonitrile, and methanol in the portion of 70:25:5 respectively was used as mobile phase.

· Standard solution salicylic acid (SA)

30 mg of salicylic acid was accurately weighed and dissolved in mobile phase to make 100 ml of solution. The resulting solution had a salicylic acid concentration of 300 mg/l.

500ul of the above solution were diluted to 10 ml, to obtain a standard solution with a salicylic acid concentration of 15 mg/l.

· Standard solution acetylsalicylic acid (ASA)

100 mg of acetylsalicylic acid was accurately weighed and dissolved to make a 100.0 ml solution, from which 5 ml was diluted twice to obtain a standard solution having an acetylsalicylic acid concentration of 500 mg/l.

· Sample preparation

From each formulation 10 tablets were weighed and powdered. An accurately weighed portion of powder, equivalent to 100 mg of acetylsalicylic acid was dissolved in 20 ml of mobile phase. The mixture was vigorously shaken for about 10 minutes, and then filtered through a 0.2-um cellulose acetate filter (Sartorius, Goettingen, Germany).

1.0 ml from the filtrate was diluted to 10.0 ml with diluting solution. The final solution had a theoretical concentration of 500 mg/l acetylsalicylic acid, and was used for the determination of the acetylsalicylic acid and salicylic acid amount in the formulation analysed.

· Calibration curve

A calibration curve (peak area vs. acetylsalicylic concentration) y = 12151 ( 44) x + 2378 (1115) with a correlation coefficient (R²) of 0.9999 (0.0001) (n = 3) was constructed using standard solution concentrations from 100 to 500 mg/l. And for salicylic acid a calibration curve) y = 938 (28) x - 5015 (516) with a correlation coefficient (R²) of 0.9999 (0.0001) (n = 3) was constructed using standard solutions concentrations from 10 to 50 mg/l. The precision of the acetylsalicylic acid and salicylic acid determination was determined by calculating the relative standard deviation (RSD) of the peak area responses after repeated injections (n =3) of a mixture of acetylsalicylic acid and salicylic acid standard solution (500 : 50mg/l) a day and within three days.

The resolution factor (R) between acetylsalicylic and salicylic acid was calculated as

R= 2 (t₂ - t₁ ) / (w₁ + w₂)

With t₁ and w₁ being the retention time and baseline width of the acetylsalicylic peak, t₂ and w₂ the respective values for salicylic acid.

· Chromatographic conditions

Flow rate : 1.2 ml/min

Detection wavelength : 280 nm

Injection volume : 20ul

Temperature : Room temperature

· Procedure

Equal volumes of the acetylsalicylic acid standard and assay preparations were separately injected, the chromatograms were recorded and the major peaks integrated. The quantity Q, in mg, of aspirin in the portion of tablets taken was calculated by the formula:

Q = 200 C (r_u/r_s)

In which C is the concentration, in mg/ml, of acetylsalicylic acid in the standard preparation, r_u and r_s are the aspirin peak responses obtained from the assay and the standard preparation, respectively.

The quantity, in mg, of salicylic acid in the portion of tablets taken was calculated by the formula:

2000 (C/Q) (r_u/r_s)

In which C is the concentration, in mg/ml, of salicylic acid in the standard preparation, Q the quantity, in mg, of acetylsalicylic acid in the portion of tablets as determined above, r_u and r_s are the salicylic acid 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 %). 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.

2.2.2 Results

The RSD was 0.25 % within a day and 1.78% within three days, which complies with the USP 24 requirements (RSD should be less than 2%). The resolution between acetylsalicylic acid and salicylic acid peaks was 1.75, which means that those two compounds were well separated. The results of the drug content (Table 2.1) show that the B.J. International formulation failed to comply with the USP 24 specifications for acetylsalicylic acid content (90% - 110%). All formulations were compliant with the USP 24 specifications for salicylic acid limits (<0.3%) (Table 2.2). Upon 6 months of storage at 40°C and 75 % RH, only the Bayer formulation did not show a significant change. The B.J. International formulation was badly affected as almost 50% of the tablet was transformed into the powder. As a consequence the salicylic acid content increased and the acetylsalicylic acid content decreased dramatically.

Table 2.1 The acetylsalicylic acid 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 tablet

0 months 6 months

Bayer 99.4 94.3

B.J. International 87.0 59.0

Girlloh (Minasprin) 99.4 80.3

S&R (Saraprin) 91.7 -

Table 2.2 The salicylic acid content (expressed as a percentage of the acetylsalicylic acid labelled amount) before and after 6 months of stability testing at simulated tropical conditions.

Manufacturer % of salicylic acid

0 months 6 months

Bayer 0.00 0.24

BJ international 0.00 0.61

Girlloh (Minasprin) 0.01 0.24

S&R (Saraprin) 0.02 -

Containing 300 mg of acetylsalicylic acid per tablet.

Not analyzed for stability testing.