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

GHENT UNIVERSITY

FACULTY OF PHARMACEUTICAL SCIENCES

LABORATORY OF PHARMACEUTICAL TECHNOLOGY

___________________________

Academic year 2002-2003

AN IN VITRO STUDY OF THE QUALITY OF ESSENTIAL DRUGS AVAILABLE ON THE RWANDAN MARKET

Thesis submitted to Ghent University, Belgium, in partial fulfilment of the

requirements for the degree of Master in Pharmaceutical Sciences (M. Pharm)

By

Pierre Claver KAYUMBA (B. Pharm.)

Faculty of Sciences and Technology, Department of Pharmacy, National University of Rwanda, Butare-Rwanda.

Promoters:

Prof. Dr. Apr. J.P. REMON

Prof. Dr. Apr. C. VERVAET

This work is dedicated to my lovely wife Verdiane and to our beloved children Audrey, Gabin and Jean Marc, for their love, patience, understanding and care.

Acknowledgements

At the end of this thesis, I would like to express my gratitude to the government of Rwanda (GOR) for the provision of a scholarship to support my studies in Belgium.

I wish to thank Prof.Dr. J.P. Remon, for accepting me in his laboratory and promoting this work. His encouragement, inspiration and moral support were very important in the accomplishment of this work.

I am also grateful to Prof.Dr. C. Vervaet for his advice and support towards the realisation of this work.

My appreciation goes to Prof.Dr. W. Baeyens and Prof.Dr. J. Demeester for their lectures, which were very useful in my research work.

Thanks to Pharm. D. Ameye for his guidance and assistance in the various analytical methods.

I am grateful to Dr. J.D. Ntawukuliryayo for his advice, kindness and sympathy in carrying out my study.

My gratitude goes to Pharm. E. Bienvenu for his critical comments, which contributed to the final form of this thesis.

My sincere thanks to the Laboratory of Pharmaceutical Technology for their hospitality and support. Especially, I thank D. Tensy, M. De Meyer, K. Wullaert, and B. Vandenbussche for their invaluable assistance.

I wish to thank my colleagues, master students A. Dukic, A. Eltraplsi and K. W. Mwamwitwa for their useful moral and technical support, advices and suggestions in the realisation of this work.

I extend special thanks to my wife Verdiane, my daughter Audrey, my sons Gabin and Jean Marc, for their love, courage, patience and understanding that made ever possible the realisation of this work.

Thanks to all Rwandan people resident in Ghent or Brussels, who have contributed to this thesis in many different ways.

Pierre Claver KAYUMBA

September, 2003

I. Introduction, Background, and Objectives

I.1 Introduction

The World Health Organization (WHO) passed in 1975 a resolution (WHO 28.66) which marked the birth of the Essential Drugs Concept (EDC). The aim was to solve the problem of accessibility to drugs by the population in developing countries. In most developing countries people lack access to drugs because they are expensive and the purchase capacity is very low. The idea behind the EDC is the recognition that only a few drugs are necessary for the treatment of the majority of the diseases facing the majority of the population. In 1977 a model list of Essential Drugs was established, the criteria of including a drug in the list were: established safety and efficacy, proven quality, constant availability and affordability. The WHO encouraged all nations to establish their own Essential Drug List based on the above criteria. The Rwandan government through the Ministry of Health established its first national essential drug list in 1991. The principe was that all drugs included should be, if possible, generics which are cheap and the health workers (governmental as well as private) were recommend to refer to that list when prescribing and dispensing. The list was reviewed in 1997 and 1999 and the last revision was this year.

Counterfeiting of pharmaceuticals and the proliferation of substandard drugs constitute a serious health risk to the consumers around the world. The WHO records show that problems of substandard and counterfeit drugs are on the increase as 50% of all reported cases occurred in the period 1993 to 1997. Most of these incidences (70%) were reported in developing countries. The report identified the cause of the poor quality of drugs: in about 50% of all cases the formulations did not contain any drug, 20% contained the wrong active ingredient and 10% the wrong amount of active ingredients. In another 5% of the reported incidences did the formulation contain the right active ingredient in the correct amount, but were judged substandard by failing in other quality tests. The antibiotics were the major pharmacological class of drugs with the largest incidence (60%) of counterfeiting (WHO, 2000). According the International Federation of Pharmaceutical Manufacturers Association (IFPMA) about 7% of all drugs being sold around the world in 1992 were of poor quality: being counterfeit or substandard.

In Rwanda there are no facilities for quality control of pharmaceuticals, no systematic monitoring of the quality of drugs on the market. This gross deficiency increases the risk that the importers of pharmaceuticals would go for cheap possibly low quality products because the substandard products would not be detected.

After the genocide, the Rwandan pharmaceuticals market is characterized by the presence of many generics from multisource suppliers and healthcare providers. Consequently clinicians and pharmacists are faced with selecting a product that gives the same clinical effect than that claimed to do so. In most of cases this selection is based on economical considerations and on the assumption that those dosage forms containing the same amount of active ingredient are the equivalent.

In addition, there are wide price differences between formulations containing the same amount of active ingredient (even more than 500%); subsequently patients with low purchasing power will go for cheap brands. With such differences in price it is essential to know if those brands are really pharmaceutically equivalent, or if there is a relationship between price and quality.

I. 2 Background

The quality of pharmaceutical products has been a major concern in many WHO forums. The existence of counterfeit and substandard drug preparations, which are of unacceptable quality, incited many studies about the quality of pharmaceuticals available in different countries. The quality of the pharmaceuticals in the market depends much on the manufacturer and purchaser's integrity. Through several studies done, it has been shown that the regular surveillance on the quality and bioavailability of the formulations marketed in a country is very important.

Even in developed countries where the pharmaceutical market is highly controlled and strictly regulated, it was possible to find substandard drugs in the market:

- The National Medicine Control Laboratory of Finland reported on the quality and bioequivalency of different brands of erythromycin tablets: the bioavailability of one brand being very low (Venho et al., 1987). In the same laboratory Eranko et al. (1990) noticed differences in bioavailability between different brands of nifedipine tablets. In all occasions the low availability brand had to be withdrawn from the market.

- In studies done in Canada involving 229 generic brands, 9% were identified to be of an unacceptable standard (Maddock, 1986).

In developing countries, Rwanda included, the control and regulation of pharmaceuticals is not very strict and there have been many reports of substandard as well as fake drugs on the market:

- Studies done in Nigeria to evaluate the quality of quinine tablets reported the presence of fake formulations (Sowumni et al., 1994).

- A report on the quality of pharmaceuticals in developing countries was made by Shakoor et al. (1997) on 81 drugs sampled from Nigeria and 15 from Thailand, antimalarials and antibiotics commonly used in these countries. They analyzed by HPLC the content of the active ingredient as well as the presence of impurities and degradation products. The results showed that 36% (25) of the samples from Nigeria and 40% (6) from Thailand did not comply with pharmacopoeia standards and 3 of the substandard samples from Nigeria (2 chloroquine and 1 amoxicillin) and 3 from Thailand (chloroquine) were fake. Through these observations the authors concluded that the major reason for substandard drugs in the developing countries was poor manufacturing practice.

- Sulfamethoxazole, an active pharmaceutical ingredient manufactured in India, was found to be of poor quality and rejected, but was deliberately being placed at the bottom of every fourth drum ready to be exported abroad (WHO, 1997).

- Recently, in the Laboratory of Pharmaceutical Technology of Ghent University, a study on the quality of essential drugs available on the Tanzanian market was done by Risha et al. (2002). They evaluated the in vitro availability and its stability under simulated tropical conditions of 22 formulations containing paracetamol, acetylsalicylic acid, chloroquine and sulfadoxine/pyrimethamine. They used methods specified in the USP 24 monographs of the respective drugs. All drugs analyzed passed the pharmacopoeia requirements for the drug content. However seven formulations failed to meet the USP 24 tolerance limits for dissolution. In addition five formulations failed to meet the USP 24 tolerance limits for dissolution after being subjected for six months to an accelated stability test under simulated tropical conditions (75 % RH, 40 °C). They concluded that the dissolution behaviour of 12 of the samples was not satisfactory.

They recommended the validation of the manufacturing process and the use of excipients with predetermined properties.

I.3 Objectives

Main objective

Since there are no reports about the quality of pharmaceuticals in Rwanda, this study was undertaken to evaluate the quality of some essential drugs marketed in Rwanda. The main objective of this study is to assess the quality of some essential drugs available on the Rwandan market through the USP 24 requirements. Furthermore to check their stability under simulated tropical conditions of the IV^th climatic zone (40C and 75%RH).

Specific objectives

· Determination of the drug content

· Determination of the in vitro drug dissolution

· Evaluation of the impact of accelerated stability testing (storage at 40°C, 75 % RH) on drug content and in vitro dissolution.

According to this study an acceptable formulation complies with the USP 24 specifications with the respect to the dissolution and amount of active ingredients. A stable product is defined as a product which shows no significant degradation or change in its physical and chemical properties and remains within the labelled specifications.

II. Quantitative drug analysis and evaluation of the influence of accelerated stability testing on the in vitro dissolution.

Immediate release solid dosage forms are routinely subjected to tests such as content uniformity, weight, friability, hardness and disintegration, tests mainly performed by manufacturers to assess batch-to-batch uniformity. As the efficacy and safety of a dosage form is dependent on the content of active ingredient, the test for drug content is recommended in pharmacopoeia monographs. The test which is often most associated with the assessment of in vivo performance is the in vitro dissolution test, because even when a formulation contains the right amount of drug it can fail to release the content at the site of absorption due to the poor dissolution.

- Dissolution tests are used to assess the dissolution properties of the drug itself in order to choose appropriate excipients for the formulation.

- Dissolution tests are a very important tool to ensure continuing product quality and performance after certain changes, such as changes in the formulation, the manufacturing process, the site of manufacture, and the scale-up of the manufacturing process (Guidance for industry, 1997).

- Clinical scientists rely on dissolution tests to establish an in vitro/in vivo correlation between drug release from the dosage form and drug absorption. The dissolution of an oral solid product can impact the rate and the amount of drug available for absorption and hence influence the therapeutic efficacy of the product. It is essential that the dissolution characteristics remain unchanged throughout the product shelf life.

- Generally in developing countries, where technology and other resources are limited to conduct an in vivo bioequivalence study, appropriate dissolution studies, such as profile comparison between the local generic product and the reference product under different test conditions may be used to assure product quality (Shah, 1998).

Stability of a pharmaceutical product means the maintenance of the quality defined in the specifications of the drug product up till the end of the manufacturer's stated shelf life. The quality of the drug product is determined by the content and purity of the active ingredient and by the organoleptic, physiochemical and microbiological properties (Grimm, 1986).

Stability tests are a series of tests designed to obtain information on the stability of pharmaceutical products, in order to define their shelf life and utilisation period under specified packaging and storage. Dissolution stability is an important tool to assess the quality of the product. Is therefore both the legal and ethical responsibility of the manufacturer to ensure that the product meets all the quality specifications during the shelf life period as long as it is stored under the conditions specified on the label.

For worldwide stability tests, the earth is divided into four climatic zones into which individual countries are assigned. Rwanda can be assigned to the climatic zone II (subtropical and Mediterranean climates, storage conditions 25°C/60% RH) (Grimm, 1998). If imported drug formulations have not been optimised for the corresponding climate zone, their effectiveness may be compromised during transportation or/and storage.

Regarding the regulatory aspects, the WHO recommends an accelerated stability test under zone IV climatic conditions (storage conditions of 40 °C / 75 % RH) to be performed on all drugs intended for the global market (Matthews, 1999).

II.1. Amoxicillin formulations

II.1.1 Material and equipment

Materials

· Amoxyphar 250 mg capsules ( Labophar, Rwanda)

· Elymox 250 mg capsules (Elys chemical industries, Kenya)

· Amoxysha 500 mg capsules (Dilam, Canada)

· Amoxicillin (Alpha Pharma, Belgium)

· Acetonitrile (Biosolve, The Netherlands)

· Monobasic potassium phosphate (Vel, Belgium)

All these 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.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.

II.1.3 In vitro dissolution

1.3.1 Methods

· Preparation of dissolution medium

Distilled water was used as dissolution medium.

· Calibration curve

Stock solution

30 mg of amoxicillin standard powder was accurately weighed and dissolved into a required volume of dissolution medium to make a solution having a concentration of 300mg/l, used as stock solution.

Standard solutions

5, 10, 15, 20, and 25 ml from the stock solution were separately transferred to 25.0 ml volumetric flasks and diluted to volume using dissolution medium. The resulting standard solutions had concentrations of 60, 120, 180, 240 and 300 mg/l. Absorbances of the above standard solutions were spectrophotometrically measured at 272 nm.

A calibration curve (absorbance vs. amoxicillin concentration) y = 0.003x + 0.0017 with a correlation coefficient (R²) of 0.9999 was constructed.

· Dissolution testing

Dissolution profiles were determined using the USP basket method (Method 1) at a rotational speed of 100 rpm for capsules containing 250 mg, and using the USP paddle method (Method 2) at a rotational speed of 75 rpm for capsules containing 500 mg.

Each of 6 capsules was placed inside a dissolution vessel filled with 900 ml of dissolution medium maintained at 37 0.5°C. At different time intervals (10, 20, 30, 40, 50 and 60 minutes) 5 ml of samples were manually withdrawn, filtered, and analyzed spectrophotometrically at 272 nm for their amoxicillin concentration. Samples from 500 mg capsules were diluted twice before analysis. The amount of the drug dissolved was calculated by means of the above mentioned calibration curve.

1.3.2 Results

Table 1.2 shows the percentage dissolved within 60 minutes of dissolution testing and Figure 1.1 the different dissolution profiles. Before stability testing all drugs complied with the USP 24 dissolution requirements (not less than 80% of the labelled amount should dissolve within 60 minutes). The amount of drug released after 60 minutes of dissolution test was more than 90% for all formulations. The accelerated stability testing did not affect the dissolution profiles; the percentage released remained within the USP 24 limits for all formulations.

Table 1.2: Percentage amoxicillin dissolved within 60 minutes of dissolution testing before and after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 80 % released within 60 minutes.

Manufacturer % of the labelled amount released

0 month 3 months 6 months

Elys chemicals (Elymox) 99.9 94.5 91.3

Labophar (Amoxyphar) 96.7 96.3 96.7

Dilam (Amoxysha 500) 104.2 102.9 97.7

Figure 1.1: Dissolution profiles of amoxicillin formulations before and after 3 and 6 months storage at 40°C and 75 % RH.

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.

II.2.3 In vitro dissolution

2.3.1 Methods

· Preparation of dissolution medium

The dissolution medium consisted of 0.05M acetate buffer prepared as follows: 9 g of anhydrous sodium acetate was dissolved in 800 ml distilled water, 8.3 ml of glacial acetic acid was added. The resulting solution was diluted to 5.0L.

· Calibration curve

Stock solution

35 mg of acetylsalicylic acid reference powder was accurately weighed and transferred to a 100.0 ml volumetric flask. 1 ml of methanol was added, then about 50 ml of dissolution medium. The mixture was sonicated for about 2 min. The solution was diluted to 100.0 ml using the dissolution medium to obtain a stock solution with a concentration of 350 mg of acetylsalicylic acid / l.

Standard solutions

3, 4, 5, 7 and 9 ml were separately diluted to 10.0 ml using the dissolution medium; the resulting standard solutions had concentrations of 105, 140, 175, 245 and 315 mg/l acetylsalicylic acid, respectively. Absorbances of those solutions were spectrophotometrically measured at 265nm. A calibration curve (absorbance vs. acetylsalicylic acid concentration) y = 0.0027x + 0.0031 with a correlation coefficient (R²) of 0.9998 was constructed.

Dissolution testing

Dissolution profiles were determined using the USP basket method (Method 1). Each of 6 tablets was added to a basket fixed to a stirring shaft, placed inside a dissolution vessel (filled with 900 ml of dissolution medium maintained at 37°C 0.5°C) and rotated at a speed of 50 rpm. At different time intervals (5, 10, 15, 20, 25 and 30 min) 5ml filtered samples were manually withdrawn, diluted twice with dissolution medium and spectrophotometrically analysed at 265 nm. Concentrations were calculated from the above mentioned calibration curve.

2.3.2 Results

The dissolution profiles for each formulation before and after 3 and 6 months of accelerated stability testing are shown in Figure 2.1 and the percent drug released after 30 minutes in Table 2.3. Before stability testing the S&R formulation did not disintegrate, while others complied with the USP 24 requirements (not less than 80% dissolved within 30 minutes). After six months of stability testing, only the Bayer formulation remained compliant with the USP 24 requirements. The percentage released for Minasprin formulation decreased, however it remained compliant with the USP 24 requirements. The release rate of the B.J International formulation decreased dramatically.

Table 2.3 Percentage of acetylsalicylic acid dissolved within 30 minutes of dissolution testing before and after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 80 % released within 30 minutes.

Manufacturer % of the labelled amount per tablet

0 months 3 months 6 months

Bayer 99.0 97.2 95.6

BJ international 84.7 71.8 34.3

Girlloh (Minasprin) 97.2 80.5 76.5

S&R (Saraprin) 5.1 - -

Not analyzed for 3 and 6 months.

Figure 2.1 Dissolution profiles of acetylsalicylic acid before and after 3 and 6 months of storage at 40°C and 75 % RH:

II.3 Sulfamethoxazole / Trimethoprim (Cotrimoxazole) formulations

II.3.1 Material and equipment

Material

· Batrimox 480 mg tablets (Sulfamethoxazole 400 mg / Trimethoprim 80 mg)

(S&R Pharmaceuticals, Rwanda)

· Unitrim 480 mg tablets(Sulfamethoxazole 400 mg / Trimethoprim 80 mg)

(Elys chemicals industries, Kenya)

· Bactiphar 480 mg tablets (Sulfamethoxazole 400 mg / Trimethoprim 80 mg) (Labophar, Rwanda)

· Sulfamethoxazole (Alpha pharma, Belgium)

· Trimethoprim (Alpha pharma, Belgium)

· Hydrochloric acid 37% (Merck/Eurolab, Darmstadt, Germany)

· Acetonitrile HPLC grade (Biosolve, The Netherlands)

· Glacial acetic acid 100% (Merck/Eurolab, Darmstadt, Germany)

· Triethylamine (Sigma chemicals, St Louis, USA)

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.3.2 Quantitative drug analysis

3.2.1 Methods

The amount of sulfamethoxazole and trimethoprim and the dissolution rate of both drug for each formulation were determined using the methods described in the USP 24.

· Mobile phase

A mixture of 650 ml distilled water, 250 ml acetonitrile, and 1 ml triethylamine was homogenized and allowed to equilibrate at room temperature. The pH of the above mixture was adjusted to 5.9 0.1 using diluted glacial acetic acid (10%). The resulting solution was diluted to 1.0 L to obtain the mobile phase.

· Standard solution

Separately, 160 mg of sulfamethoxazole and 32 mg of trimethoprim were accurately weighed and dissolved in methanol to give a 100.0 ml solution. The above solution had a concentration of 1600 mg/l and 320 mg/l of sulfamethoxazole and trimethoprim, respectively. 5.0 ml from the above solution was diluted to 50.0 ml to obtain standard solution with concentration of 160 mg/l and 32 mg/l of those two compounds, respectively.

· Sample preparation

From each formulation 10 tablets were weighed and finely powdered. An accurately weighed portion of powder equivalent to 160 mg of sulfamethoxazole was diluted with mobile phase to give 100.0 ml of suspension, sonicated for about 5 min and filtered through a 0.2-um cellulose acetate filter (Sartorius, Goettingen, Germany). 5.0 ml from the filtrate were diluted to 50.0ml and used as assay preparation.

· Calibration curve

A calibration curve (peak area vs. concentration) y = 64590 (122) x + 43448 (351) with a correlation coefficient (R²) of 0.9995 (0.0001) (n = 5) was constructed using standard solutions with sulfamethoxazole concentrations from 16 to 160 mg/l. For trimethoprim a calibration curve y = 31476 (1265) x + 2088 ( 509) with a correlation coefficient (R²) of 0.9979 (0.0025) (n = 5) was constructed using standard solutions with trimethoprim concentrations from 3.2 to 32 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 = 5) of a standard solution (160 mg/l sulfamethoxazole and 32 mg/l trimethoprim).

The resolution factor (R) between sulfamethoxazole and trimethoprim was calculated from their respective peaks:

R= 2 ( t₁ - t₂ ) / (w₁ + w₂)

With t₁ and w₁ being the retention time and baseline width of the sulfamethoxazole peak, t₂ and w₂, the respective values for trimethoprim.

· Chromatographic conditions

Flow rate : 0.8 ml/min

Detection wavelength : 254 nm

Injection volume : 20ul

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 quantities, Q, (in mg of sulfamethoxazole and trimethoprim in the portion of tablets taken) were calculated by the formula:

Q=1000 C (ru/r_s)

Whereby C is the concentration, in mg/ml, of sulfamethoxazole and trimethoprim in the standard preparation, r_u and r_s are the analyte corresponding 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 %). 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 in active ingredient.

3.2.2 Results

The RSD was 0.47 and 0.24 % within a day and 1.51 and 1.29 % within three days for sulfamethoxazole and trimethoprim, respectively; which complies with the USP 24 requirements (RSD should be less than 2%). The resolution factor R between sulfamethoxazole and trimethoprim was 8.02, which means that they were well separated. As shown in Table 3.1, the S&R formulation (Batrimox) failed to comply with USP 24 requirements in terms of drug content for sulfamethoxazole (93 - 107 % of the labelled amount of sulfamethoxazole and trimethorim).

There was no impact of stability testing on the drug content for the Elys formulation (Unitrim), while the drug content of both sulfamethoxazole and trimethoprim for the Labophar formulation (Bactiphar) decreased.

Table 3.1 The sulfamethoxazole and trimethoprim 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

Sulfamethoxazole

Elys Chemicals (Unitrim) 97.1 94.6

Labophar (Bactiphar) 97.2 92.8

S&R pharmaceuticals (Batrimox)* 91.6 -

Trimethoprim

Elys Chemicals (Unitrim) 99.6 97.0

Labophar (Bactiphar) 98.1 84.8

S&R pharmaceuticals (Batrimox)* 97.4 -

* Not analyzed for stability testing because it failed the dissolution test for both two

compounds immediately after purchase.

II.3.3 In vitro dissolution

3.3.1 Methods

· Preparation of dissolution medium

98.64 ml of 37 % hydrochloric acid was diluted to 10.0 L with distilled water. The resulting 0.1 N solution was used as dissolution medium.

· Calibration curves of sulfamethoxazole and trimethoprim

Based on the HPLC method, the calibration curves mentioned in quantitative drug analysis were used for calculation of the amount of drug released. The same mobile phase, the same standard solutions and the same concentrations were used.

· Dissolution testing

Dissolution profiles were determined using the USP paddle method (Method 2). Each of 6 tablets was placed inside a dissolution vessel filled with 900 ml of dissolution medium maintained at 370.5°C stirred by paddles rotating at 75 rpm. At 10, 20, 30, 40, 50 and 60 minutes 5 ml samples were withdrawn, filtered, diluted 5 times and analysed for their contents of sulfamethoxazole and trimethoprim by UV at 254 nm after chromatographic separation.

Procedure

20 ul of each of the collected samples was injected onto the HPLC system and the corresponding peak areas were recorded. The content of each sample was calculated using the calibration curve.

3.3.2 Results

Table 3.2 shows the percentage drug dissolved and Figures 3.1 to 3.3 the dissolution profiles of different formulations analyzed. For sulfamethoxazole the Elys formulation (Unitrim) complied with the USP 24 requirements (not less than 70% of sulfamethoxazole and trimethoprim labelled amount should dissolve within 60 min), however the drug percentage released decreased after 6 months of storage at 40°C/ 75% RH. Labophar formulation (Bactiphar) released 45% of the drug, the S&R formulation (Batrimox) released only 15%. Those last two formulations did not disintegrate completely within 60 minutes. For trimethoprim, 90% of the labelled amount of Unitrim and 77.5% of Bactiphar were released within 60 min, which complies with USP 24, while Batrimox failed (only 35.4 % was released).

Table 3.2 Percentage of sulfamethoxazole and trimethoprim dissolved within 60 minutes of dissolution testing before, after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 70 % released within 60 minutes.

Manufacturer % of the labelled amount per tablet

0 months 3 months 6 months

Sulfamethoxazole

Elys Chemicals (Unitrim) 98.0 94.2 77.0

Labophar (Bactiphar) 45.0 38.5 25.8

S&R pharmaceuticals (Batrimox) 15.0 - -

Trimethoprim

Elys Chemicals (Unitrim) 95.1 92.2 90.2

Labophar (Bactiphar) 77.6 47.4 32.5

S&R pharmaceuticals (Batrimox) 35.4 - -

Figure 3.1 In vitro dissolution profiles of sulfamethoxazole and trimethoprim before stability testing

Figure 3.2 Dissolution profiles of sulfamethoxazole formulations before and after 3 and 6 months storage at 40°C and 75 % RH.

Figure 3.3 Dissolution profiles of trimethoprim formulations before and after 3 and 6 months of storage at 40°C and 75 % RH.

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

II.4.3 In vitro dissolution

4.3.1 Methods

· Preparation of dissolution medium

98.64 ml of 37% hydrochloric acid was diluted to 10.0L with distilled water. The resulting 0.1N hydrochloric acid solution was used as dissolution medium.

· Calibration curve

Stock solution

40 mg of metronidazole was accurately weighed, dissolved in dissolution medium and sonicated for about 5 min to give a 25 ml solution having a concentration of 1600 mg/l.

5 ml from this solution was diluted to 50.0 ml with dissolution medium to give a stock solution with a concentration of 160 mg/l.

Standard solutions

0.5, 0.75, 1, 2 and 3 ml of the stock solution were separately diluted with dissolution medium to 10.0 ml. The standard solutions obtained had concentrations of 8, 12, 16, 32 and 48 mg/l, respectively.

A calibration curve (absorbance vs. concentration) y = 0.0355x + 0.0114 with a correlation coefficient (R²) of 0.9998 was constructed.

· Dissolution testing

Dissolution profiles were determined using the USP basket method (Method 1). Each of 6 tablets was added to a basket connected to a stirring shaft which was placed inside a dissolution vessel filled with 900ml of dissolution medium maintained at 370.5°C. The rotation speed of the basket was 100 rpm. At 10, 20, 30, 40, 50 and 60 minutes, 5ml samples were withdrawn, filtered, diluted 20 times and analysed spectrophotometrically at 278nm.

4.2.2 Results

Table 4.2 shows the percentage dissolved within 60 minutes of dissolution testing and Figure 4.1 the different dissolution profiles. Before stability testing all drugs complied with the USP 24 dissolution requirements (not less than 80% of the labelled amount should dissolve within 60 minutes). The amount of drug released after 60 minutes of dissolution test was more than 90% for all formulations. The Holden Medica formulation did not withstand the storage at high temperature and high relative humidity: the percentage released being outside the specifications after 6 months.

Table 4.2: Percentage of metronidazole dissolved within 60 minutes of dissolution testing before and after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 80 % released within 60 minutes.

Manufacturer % of the labelled amount released

0 months 3 months 6 months

Elys chemicals (Elogyl) 97.8 99.8 88.7

Labophar 98.2 92.6 90.1

Holden medica 95.3 87.8 66.9

Figure 4.1: Dissolution profiles of metronidazole formulations before and after 3 and 6 months of storage at 40°C and 75 % RH.

II.5 Paracetamol formulations

II.5.1 Material and equipment

Material

· Cetamol 500 mg tablets (Regal pharmaceuticals, Kenya)

· Panadol 500 mg tablets (SmithKline Beecham, Kenya)

· Saramol 500 mg tablets (S&R Pharmaceuticals, Rwanda)

· Paracetamol (Ludeco, Belgium)

· Potassium dihydrogen phosphate (Vel, Belgium)

· Sodium hydroxide (Acros Organics, Belgium)

· Methanol-HPLC quality (Biosolve B, The Netherlands)

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.5.2 Quantitative drug analysis

5.2.1 Methods

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

· Mobile phase

A degassed mixture of distilled water and methanol (75:25) was used as mobile phase.

· Standard preparation

An accurately weighed quantity of paracetamol (100 mg) was dissolved in mobile phase to make a 100 ml solution having a concentration of 1 mg/ml. From that solution 200 ul was diluted to 20.0 ml, and a resulting solution (0.01 mg/ml) was used as standard solution.

· Sample preparation

From each formulation, 10 tablets were weighed and finely powdered. An accurately weighed portion of powder, equivalent to 100 mg of paracetamol was diluted with mobile phase to make 100 ml of mixture, which was filtered through a 0.2um cellulose acetate filter (Sartorius, Goettingen, Germany). From the filtrate 1 ml was diluted to 100.0 ml with mobile phase.

· Chromatographic conditions

Flow rate : 1.5 ml/min

Detection wavelength : 243 nm

Injection volume : 20 ul

Temperature : Room temperature

· Calibration curve

A calibration curve (peak area vs. concentration) y = 94199 (1687) x - 16441 (2852) with a correlation coefficient (R²) of 0.9998 (0.0002) (n = 3) was constructed using standard solutions from 4 to 40 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 paracetamol standard solution (20 mg/l).

· Procedure

Equal volumes of standard and assay preparations were separately injected, the chromatograms were recorded, and the major peak integrated.

The drug quantity, Q, (in mg of paracetamol in the portion of tablets taken) were calculated by the formula:

Q = 10.000C( r_u/r_s)

Whereby C is the concentration (mg/l) of the standard preparation, r_u and r_s are the paracetamol 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 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 in active ingredient.

5.2.2 Results

The RSD was 0.78 % within a day and 1.56% within three days, which complies with the USP 24 requirements (RSD should be less than 2%).

The results of the drug content (Table 5.1) show that the S&R formulation did not comply with the USP 24 specifications for paracetamol content (90% - 110% of the labelled amount) after 6 months of storage under simulated tropical conditions (40°C and 75 % RH).

Table 5.1: The paracetamol 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

Regal Pharmaceuticals (Cetamol) 95.6 92.5

Smith Kline Beecham (Panadol) 95.0 93.8

S&R Pharmaceuticals (Saramol) 90.8 87.7

II.5.3 In vitro dissolution

5.3.1 Methods

· Preparation of dissolution medium

68 g of potassium dihydrogen phosphate was diluted to 10.0 L with distilled water. The pH was adjusted to 5.8 0.05 using a 0.2 N solution of sodium hydroxide.

· Calibration curve

Stock solution

200 mg of accurately weighed paracetamol was dissolved in mobile phase to obtain 100.0 ml. 10 ml from this solution were diluted to 100.0 ml. The resulting solution had a concentration of 200 mg/l. 20 ml of this solution was diluted to 100.0 ml to give a stock solution with a concentration of 40 mg/l.

Standard solutions

1, 2, 5, 8 and 10 ml of the stock solution were separately diluted to 10.0 ml using the dissolution medium to obtain standard solutions having concentrations of 4, 8, 20, 32 and 40 mg/l, respectively.

A calibration curve (absorbance vs. concentration) y = 0.0633x + 0.0294 with a correlation coefficient (R²) of 0.9999 was constructed.

· Dissolution testing

Dissolution profiles were determined using the USP paddle method (Method 2). Each of 6 tablets was placed inside a dissolution vessel filled with 900 ml of dissolution medium maintained at 37 0.5°C and rotated at a speed of 50 rpm.

At different time intervals (5, 10, 15, 20, 25 and 30 min) 5 ml filtered samples were manually withdrawn, diluted with dissolution medium (1:40) and spectrophotometrically analyzed at 243 nm. From the absorbance of each sample, the drug concentration was calculated by means of the calibration curve.

5.3.2 Results

Table 5.2 shows the percentage dissolved within 30 minutes of dissolution testing and Figure 5.1 the different dissolution profiles. Before stability testing all drugs complied with the USP 24 dissolution requirements (not less than 80% of the labelled amount should dissolve within 30 minutes). The amount of drug released after 10 minutes of dissolution test was more than 80% for the Cetamol and Panadol formulations. The Saramol formulation disintegrated into larger particles compared to the others two formulations. The accelerated stability testing did not affect the Cetamol and Panadol drug percentage released. The amount of drug released from Saramol formulation decreased, however it remained within USP 24 tolerance limits for dissolution testing.

Table 5.2: Percentage of paracetamol dissolved within 30 minutes of dissolution testing before and after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 80 % released within 60 minutes.

Manufacturer % of the labelled amount per tablet

0 months 3 months 6 months

Regal Pharmaceuticals (Cetamol) 95.6 94.6 90.4

Smith Kline Beecham (Panadol) 95.0 94.9 93.9

S&R Pharmaceuticals (Saramol) 90.8 90.1 84.3

Figure 5.1: Dissolution profiles of paracetamol formulations before and after 3 and 6 months of storage at 40°C and 75 % RH.

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.

6.2.2 Results

The RSD was 0.65 % within a day and 1.67% within three days, which complies with the USP 24 requirements (RSD should be less than 2%).

The results of the drug content (Table 6.1) show that all formulations complied with the USP 24 specifications for quinine sulfate content: 90% - 110% of the labelled amount of quinine sulfate. Whereas the content of the Labophar tablets was just above the lower limit of the required interval before stability testing, it failed after six months of storage at 40° C and 75% RH.

Table 6.1: The quinine 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 105.0 98.1

Labophar 90.2 86.4

Pharmakina 97.0 94.6

II.6.3. In vitro dissolution

6.3.1 Methods

· Preparation of dissolution medium

98.64 ml of 37% hydrochloric acid was diluted to 10.0 L with distilled water. The resulting 0.1N hydrochloric acid solution was used as dissolution medium.

· Calibration curve

Stock solution

40 mg of quinine sulfate was accurately weighed and transferred to a 25 ml volumetric flask and dissolved to volume using the dissolution medium. 1 ml from the above solution was diluted to 100.0 ml to give a stock solution with a concentration of 16 mg/l.

Standard solutions

4, 8, 10, 16 and 20 ml from the stock solution were separately diluted to 20.0 ml to give standard solutions with concentrations of 3.2, 6.4, 8.0, 12.8 and 16.0 mg/l.

A calibration curve (absorbance vs. concentration) y = 0.0925x + 0.0053 with a correlation coefficient (R²) of 0.9999 was constructed.

· Dissolution testing

Dissolution profiles were determined using the USP basket method (Method 1). Each of 6 tablets was added to a basket connected to a stirring shaft which was placed inside a dissolution vessel filled with 900ml of dissolution medium maintained at 37 0.5°C. The rotation speed was 100 rpm. At 10, 20, 30, 35, 40 and 45 min 5 ml samples were withdrawn, filtered, diluted (1:40) and spectrophotometrically analyzed at 248 nm.

6.3.2 Results

Table 6.2 shows the percentage dissolved within 45 minutes of dissolution testing and Figure 6.1 the different dissolution profiles. Before stability testing all drugs complied with the USP 24 dissolution requirements (not less than 75% of the labelled amount should dissolve within 45 minutes). The amount of drug released after 45 minutes of dissolution test was more than 80% for all formulations. The Elys formulation was affected by stability test conditions, the drug percentage released decreased from 103.2% to 41.8% after 6 months. For the others, the drug released remained within USP 24 tolerance limits for dissolution testing.

Table 6.2: Percentage of quinine dissolved within 30 minutes of dissolution testing before and after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 75 % released within 45 minutes.

Manufacturer % of the labelled amount per tablet

0 months 3months 6 months

Elys Chemicals 103.2 69.6 41.8

Labophar 88.2 85.6 85.0

Pharmakina 96.1 94.0 92.7

Figure 6.1 Quinine dissolution profiles before, after 3 and 6 months of storage at 40°C and 75 % RH.

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.

7.2.2 Results

The RSD was 0.68 % within a day and 1.57 % within three days, which complies with the USP 24 requirements (RSD should be less than 2.5 %). The resolution between sulfadoxine and phenacetin and between pyrimethamine and phenacetin was 2.3 and 1.9, respectively, which means that those three compounds were well separated.

The sulfadoxine and pyrimethamine contents for each formulation (Table 7.1) were within the USP 24 requirements (90 - 110 % of the labelled amount of both sulfadoxine and pyrimethamine). The stability test conditions did not affect the formulations because the drug content did not show significant change.

Table 7.1 The sulfadoxine and pyrimethamine 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

Sulfadoxine

Elys Chemicals (Orodar) 105.3 103.4

Labophar 100.0 98.9

Pyrimethamine

Elys Chemicals (Orodar) 105.5 101.5

Labophar 90.9 90.3

II.7.3 In vitro dissolution

7.3.1 Methods

· Preparation of dissolution medium

68 g of monobasic potassium phosphate was accurately weighed and dissolved in about 9 L of distilled water. The pH was adjusted to 6.8 using a 2 N sodium hydroxide solution and distilled water was added to 10.0 L.

· Calibration curves of sulfadoxine and Pyrimethamine

Using the HPLC method, the calibration curves mentioned in quantitative drug analysis were used for calculation of the amount of drug released. The same mobile phase, the same standard solutions and the same concentrations were used.

· Dissolution testing

Dissolution profiles were determined using the USP paddle method (Method 2). Each of 6 tablets was placed inside a dissolution vessel filled with 900ml of dissolution medium maintained at 370.5°C and stirred by paddles rotated at 75 rpm. At 5, 10, 15, 20, 25 and 30 min 5 ml samples were withdrawn, filtered, diluted 3 times and analyzed for their contents of sulfadoxine and pyrimethamine by UV at 254 nm after chromatographic separation.

Procedure

20 ul of each of the collected samples was injected onto the HPLC system and corresponding peak areas were recorded.

The content of each sample was calculated based on the calibration curves.

7.3.2 Results

Table 7.2 shows the percentage drug dissolved and Figures 7.1 and 7.2 the dissolution profiles of the different formulations analyzed.

Before stability testing, all formulations complied with the USP 24 requirements for sulfadoxine: not less than 60% of the sulfadoxine and pyrimethamine labelled amount should dissolve within 30 minutes. For pyrimethamine, the Labophar formulation failed (only 18 % was released within 30 minutes). Tablets from Labophar took about 10 minutes to disintegrate which delayed the dissolution.

Upon stability testing (storage at 40°C, 75 % RH), the Elys formulation (Orodar) remained within the USP 24 requirements for in vitro drug release. The tablets from Labophar did not disintegrate completely within the interval time.

Table 7.2 Percentage of sulfadoxine and pyrimethamine dissolved within 30 minutes of dissolution testing before and after 3 and 6 months of storage at 40°C and 75% RH. USP requirements: more than 60 % released within 30 minutes.

Manufacturer % of the labelled amount per tablet

0 months 3 months 6 months

Sulfadoxine

Elys Chemicals (Orodar) 100.0 97.7 97.0

Labophar 90.7 67.6 44.4

Pyrimethamine

Elys Chemicals (Orodar) 90.4 79.2 78.0

Labophar 17.8 11.9 4.9

Figure 7.1 Dissolution profiles of sulfadoxine before and after 3 and 6 months of storage at 40° C and 75 % RH.

Figure 7.2 Dissolution profiles of pyrimethamine before and after 3 and 6 months of storage at 40° C and 75 % RH.

III. Discussion

The assay results on the drug content showed that there are some substandard drugs on Rwandan market. Before stability testing 1 acetylsalicylic acid was found substandard. After stability testing, 1 acetylsalicylic acid more, 1 cotrimoxazole, 1 paracetamol, 1 quinine were found substandard. In total about 23.8 % (5/21) of the sampled drugs were found substandard.

This result is similar to those obtained by Shakoor et al. (1997) on pharmaceuticals from Nigerian and Thai markets and to those obtained by Kibwage et al. (1992) on drugs from the Kenyan market, except that no fake drug was found in this study.

Among those that passed the drug content test, 1 acetylsalicylic acid, 2 cotrimoxazole, 1 sulfadoxine / pyrimethamine failed the initial dissolution test. After stability testing, 2 acetylsalicylic acid, 1 metronidazole and 1 quinine failed the dissolution test. In total about 38 % (8/21) of the sampled drugs failed the dissolution test. The findings are similar to those obtained by Risha et al. (2002) on the in vitro evaluation of the quality of essential drugs on the Tanzanian market, where 29% of the samples that passed the assay test, failed the initial dissolution test.

Dramatic changes in the dissolution behaviour of some formulations have been observed after storage at high temperature and high relative humidity. However it was not possible to determine the exact cause of the failure, as the composition of the formulation was not known.

These failures can not be attributed to a single manufacturer and it was observed that different drug formulations from the same manufacturer had different characteristics for drug content as well as for dissolution rate: acetylsalicylic acid tablets from S&R Pharmaceuticals did not disintegrate, while paracetamol tablets released more than 80% within 10 minutes. Cotrimoxazole tablets from Labophar failed the dissolution requirements for sulfamethoxazole, while metronidazole and quinine formulations complied with the pharmacopoeia.

The above observations might be the results of the fact that the manufacturers do not practice the Good Manufacturing Practices (GMP) principles. The ingredients used may be of inferior quality or they do not validate their manufacturing process.

Dissolution stability can be influenced by several factors. Important among them are the manufacturing process, formulation variables (e.g. physiochemical properties of the active and inactive ingredients), storage conditions and packaging.

Any one of the above factors acting alone or in combination may alter the characteristics of the product. Based on literature data one can speculate about the possible causes of the changes in dissolution rate seen after stability testing.

For example the solubility, hygroscopicity and thermal characteristics of the active component and excipients (including coating materials) are critical parameters that influence dissolution profiles, hence its stability. During storage under high humidity conditions, the active drug may dissolve and recrystallize and in the processes alter the release characteristics of the tablet. A tablet can absorb moisture, in such circumstances the original interparticulate bonds formed in the compact will be replaced by the new bonds, possibly resulting in a tablet having a different porosity and pore structure and, hence, having a different in vitro release pattern compared with the original. Some manufacturers such as Labophar and S&R Pharmaceuticals did not include a desiccant into the packaging containers, while it is known that desiccants absorb the moisture and reduce the humidity in the container, thus contribute to the dissolution stability of the product.

The initial moisture level of the finished product also impacts the dissolution. The tablets with a higher moisture level are more apt to change during aging than those prepared from compounds containing low moisture.

Fillers or diluents in the formulation are usually viewed as inert excipients. Whereas this is true for the most part, some fillers by their hygroscopic nature, provide the necessary moisture for reaction to occur and thereby promote chemical or physical changes in the product. Others act as adsorbents that interfere with the liberation of the drug from the dosage form (Murthy et al., 1993).

Specific interactions between the active ingredient and a component of formulation have been reported to result in slower dissolution under accelerated storage conditions. When phenylbutazone was prepared by direct compression with lactose and microcrystalline cellulose as diluents and the tablets were stored in paper bags at 40°C and 90% RH for 14 weeks, a significant reduction in the dissolution rates of phenylbutazone was observed. This was attributed to the reaction between lactose and the drug based on the appearance of a new endothermic peak at 220°C that was not related to the melting point of lactose and phenylbutazone, which are 200 and 107°C, respectively (Murthy et al., 1993).

During dissolution experiments involving immediate release products, gum-type binders may form a viscous gel barrier in and around the tablet, thereby inhibiting disintegration of the dosage form and causing subsequent delay in drug release (Murthy et al., 1993).

The swelling capacity of the disintegrant is an important property that determines the outcome of the dissolution after storage. For example maize starch looses its capacity to swell on aging or after exposure to high humidity and temperature (Risha et al. (2002). Dissolution behavior of tablets manufactured with this type of starch will decrease progressively with aging or during accelerated stability testing.

The dissolution rate of Quinine sugar-coated tablets manufactured by Elys Chemicals (Kenya) decreased dramatically; probably the cause is the coating material. Several examples cited in literature suggest that enteric- and sugar-coated products are more sensitive to the effect of humidity than uncoated products.(Murthy et al., 1993).

IV. Conclusion and recommendations

The in vitro study of the 21 formulations of 7 essential drugs available on Rwandan market has shown that most formulations meet the USP 24 requirements in term of drug content. Some among them fail to meet dissolution requirements, others were not able to withstand storage at high temperature and high humidity.

Based on our findings we recommend:

· A systematic evaluation of essential drug formulations available on the Rwandan market.

· The registration of each commercially available drug, documenting its specifications, and most importantly the verification of these specifications.

· To perform (if possible) an in-vivo study because the observed changes in the dissolution profiles during storage are not necessarily indicative of impaired bioavailability.

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