SPECTROPHOTOMETRIC DETERMINATION AND STABILITY STUDIES OF ARTEMETHER IN ARTEMETHER-LUMEFANTRINE SUSPENSIONS MARKETED IN ZARIA, NIGERIA

ABSTRACT

The increasing use of artemether-lumefantrine combination as an effective treatment for resistant malaria demands the need for analytical methods for the quality control of these drugs in tablets and suspensions. Though some UV Spectrophotometric methods have been developed for quantification of artemether in various biological fluids and formulations, they require strenuous heating conditions which is a limitation. This limitation coupled with non-availability of HPLC hence the need to develop and validate a simple method for the quantification of artemether in paediatric suspensions. In this work, we report the method developed by reacting artemether solution in methanol with concentrated HCl for 30 minutes to obtain an α,β-unsaturated ketone which was scanned with a UV Spectrophotometer. The method developed obeyed Beers law in the range 20 – 120 µg/ml, slope (y); 0.01 0, intercept (x) 0.193, correlation coefficient (r) 0.9987, λmax 260 nm, precision (% CV); 2, Accuracy (% Er); 2.67 and a recovery of 97%. The detection and quantification Limit (µg/ml) are 0.14 and 0.58 respectively. The developed method was successfully applied in the assay of five brands of artemether-lumefantrine suspensions with 98-101.6% content, and comparison of the means of the assay results of the method and the IP (2008) method showed no statistically significant difference (P<0.05). Stability studies of the standard artemether suspension prepared and five different brands of artemether/lumefantrine powders for suspension was carried out by extracting artemether with methanol from the suspensions and analyzing it using the developed method. The content of artemether over 14 days study period ranged between 98.5-102% and this showed that the suspensions are stable under ambient conditions for up to 14 days after reconstitution with bottled table water. The results of the study suggested that the developed method could be used interchangeably in analysis, and that co-formulation of artemether with lumefantrine has no effect on the stability of artemether.

CHAPTER ONE

INTRODUCTION

1.2 General Introduction

Malaria is a public health problem (Loset and Kaur, 2009; Karuna et al., 2014). It is an important cause of morbidity and mortality in children and adults in tropical countries. About half of the world’s population is at risk of this mosquito borne parasitic disease (WHO, 2010).

Malaria is caused by the protozoan parasite Plasmodium and transmitted by mosquitoes (Shah and Patel, 2012). Five species of the parasite have been shown to infect humans: P. falciparum, P. vivax, P. ovale, P. malariae and P. knowlesi. While they share a basic life-cycle, certain distinctive features relate to the virulence of each species. P. falciparum causes the most severe manifestations of malaria including coma, anaemia and multi organ failure. The severity of P. falciparum infection has been attributed to the relatively high parasitemia during infection and to the adherence of P. falciparum infected erythrocytes to the endothelium of capillaries and venules, a process known as sequestration (Dondorp, 2008)

The main treatments for malaria were inexpensive “monotherapies” such as chloroquine (Price and Douglas, 2009). Unfortunately, the malaria parasite quickly developed resistance to many of these monotherapies, including amodiaquine, chloroquine, mefloquine, quinine sulphadoxine and pyrimethamine (WHO, 2006). One of the cornerstones of control programs today is the early diagnosis of malaria and its treatment with highly effective drugs. New combination therapies containing artemisinin derivatives are central to this approach, providing practical treatment regimens with high cure rates and transmission-blocking potential (Dondorp, 2008; Price and Douglas, 2009).

Artemisinin based combination therapy (ACT) is increasingly being advocated as promising treatment (Arun and Smith, 2011). ACT is based on the use of two drugs with different modes of action: The rationale is that the short-acting but highly potent artemisinin derivative delivers a rapid and effective reduction in parasite biomass, with the remaining parasites being removed by the intrinsically less active but more slowly eliminated partner drug (Price and Douglas, 2009; Arun and Smith, 2011). The most common combinations are artemether-lumefantrine, artesunate-amodiaquine, artesunate-sulfadoxine-pyrimethamine, artesunate-mefloquine, and dihydroartemisinin-piperaquine. World Health Organization (WHO) recommends the ACTs as first line therapy for falciparum malaria in endemic areas (WHO, 2010).

The drug distribution network in Nigeria is in a state of chaos because it consists of open markets that act as major source of purchase to pharmacy stores, hospitals, wholesalers, retailers, medicine stores and pharmaceutical manufacturers (Erhun et al., 2001). Most importers supply drugs to open drug markets because they make more profit from there. The lack of strict monitoring and regulatory mechanisms allows for easy access to legitimate channels of distribution, making counterfeiting an appealing source of illicit revenue (Finlay, 2011).

Olugbenga, (2014) demonstrated that profits accruing to pharmaceutical industries are so huge that it attracts a lot of prospective investors, both genuine and fake, a situation that renders it very susceptible to fraud and corruption, especially through the distribution network as products are transported across international boundaries and distributed in many countries in order to reach the final consumer.

The WHO defines counterfeit drugs as drugs that have been deliberately or fraudulently mislabeled with respect to identity and or source (WHO, 2011). The products could include incorrect ingredients, may misstate the amount of the active ingredients, or are manufactured under circumstances that lack quality control. Counterfeit drugs in Nigeria include preparations without active ingredients, toxic preparations, expired drugs that are relabelled, drugs issued without complete manufacturing information and drugs that are unregistered with the National Agency for Food and Drug Administration and Control (NAFDAC) (Akinyandenu, 2013). Current estimate suggests that 10% of prescription drugs sold worldwide are counterfeits, fake or contaminated, and in parts of Africa and Asia, the figures exceed 50% (Erhun et al., 2001).

The loose control system in the Nigerian economy has contributed to the circulation of fake and counterfeit drugs in the country. A major function of NAFDAC is the regulation and control of imported products. This is done by having inspectors at various airports and seaports. Registration of pharmaceuticals is a criterion that must be passed before any drug is released into the Nigerian market. A condition for registration is the analysis and testing of the drug to ensure quality and safety. Unfortunately, the forensic laboratory, which is the major public laboratory for the purpose of quality control analysis, is not adequately equipped to cope with the volume of work, particularly for analysis of imported/smuggled drugs. These loose control systems are exploited by counterfeiters to manufacture, import and distribute fake and adulterated products. (Chinwendu, 2008)

The high cost of drugs allows for the proliferation of counterfeit drugs in Nigeria and poses a major challenge to public health. Most genuine drugs are expensive and counterfeiters take advantage to supply cheap fake drugs to consumers, especially those who cannot afford the high priced good quality version in the legal sector (Chinwendu, 2008).

The proliferation of fake drug has led to treatment failures, organ dysfunction or damage, worsening of chronic disease conditions and death of many Nigerians (Bate et al., 2009). In 1947, fourteen children were reported dead after being administered chloroquine phosphate injections and in 1990, 109 children died after being administered fake paracetamol (Aluko, 1994; Bate et al., 2009). In 1995, the Nigerian supply of 88,000 Pasteur Merieux and SmithKline Beecham meningitis vaccines to Niger republic during an epidemic resulted in about 2,500 deaths after vaccination. In 2004, three Nigerian hospitals reported cases of adverse reactions from the use of contaminated infusions produced by four Nigerian companies. It was established that the infusions were heavily contaminated with microorganisms and 147 of the 149 brands of screened water for injection were found to be unsterile (Akinyandenu, 2013). In November 2008, 34 Nigerian children, aged 4 months to 3 years died and more than 50 were hospitalised with severe kidney damage after taking the drug “My Pikin” (“my child” in local pidgin), a teething mixture containing paracetamol (Aluko, 1994; Akinyandenu, 2013). The outbreak was due to the use of diethylene glycol (DEG) as a solvent for the paracetamol. DEG was present because of inadvertent or deliberate substitution of propylene glycol, a less toxic compound than DEG, widely used in the pharmaceutical industry (Akinyandenu, 2013).

In Nigeria today, drugs are still sold in open markets, car parks, unlicensed chemists and shops, on buses, ferries and almost in any gathering of people. Most of the products sold in such and similar places are exposed to adverse weather conditions that can affect their quality, apart from the fact that they are mostly adulterated or substandard (Olugbenga, 2014).

The increasing use of artemether-lumefantrine combination as an effective treatment for resistant malaria demands the need for simpler and accurate analytical methods for the quality control of these drugs in tablets and suspension dosage formulations to complement the existing methods used for determining either artemether or lumefantrine in various pharmaceutical and biological matrices (Arun and Smith, 2011).

UV-visible Spectrophotometric methods are the instrumental methods which are commonly used in industrial and research laboratories because of their simplicity, accuracy, precision and low cost (Raza et al., 2003; Raza et al., 2005). The act of identifying materials based on their color was probably one of the earliest examples of qualitative molecular absorption spectrophotometry. Also, the first recognition that color intensity can be the indicator of concentration was probably the earliest application of employing molecular absorption spectroscopy for quantitative determination. The first measurements were made by using the human eye as the detector and undispersed sunlight or artificial light as the light source. Later it was found that the accuracy and the precision could be improved by isolating specific frequencies of light using optical filters. Further improvement of the measurement came with the use of prism and grating monochromators for wavelength isolation. Photoelectric detectors were soon developed, but were quickly replaced with phototubes and photomultiplier tubes. The development of solid state microelectronics has now made available a wide range of detector type which if coupled with the computers; provide highly sophisticated readout electronic systems (Marczenko, 2000).

The artemisinins lack strongly absorbing chromophores as a result, artemisinin and its derivatives absorb weakly in the low wavelength region and this makes their quantification difficult. The available UV Spectrophotometric methods for the analysis of artemether make use of its HCl decomposition product. This acid decomposition product of artemether has been described as an α, β unsaturated decalone and absorbs at a wavelength of 254nm (Thomas et al., 1992; Arun and Smith 2011). Though this product absorbs strongly at the said wavelength, it requires very vigorous conditions for its formation.

The International Pharmacopoeial (IP) method for the assay of artemether (both as the pure sample and in formulations) requires the addition of 1M ethanolic HCl solution to an aliquote of artemether in ethanol solution followed by heating at 55˚C for five hours (IP, 2008).

Another method developed by Shrivastava et al., (2008) requires heating at 60 ˚C for three hours. Green et al., (2001) have also described a method for the assay of Artemether and other artemisinins by the reaction of the acid decomposition product with a dye to yield a coloured derivative which absorbs at 420nm. This method requires a period of one hour for the formation of the product prior to reaction with the dye.

1.2 Statement of Research Problem

Evidence abounds on the circulation of poor quality drugs in tropical areas of the world, (Nigeria inclusive) and counterfeiting of drugs is also a major concern in these parts of the world (Awofisayo et al., 2010). Lack of readily availabile and accessible sophisticated equipments (e.g HPLC) required for analysis of drugs is also a major concern. Thus, simpler accessible analytical methods are needed to monitor the quality of diverse brands of ACTs in the market.

1.3 Justification for the Study

Proliferation of various brands of ACTs have led to the presence of fake and substandard products in several underdeveloped countries such as Nigeria. Although some UV Spectrophotometric methods have been developed for quantification of artemether in various biological fluids, the strainous heating conditions required make them uneconomical and may limit their application in routine analysis.

Also very few studies have been carried out on the stability of artemether/ lumefantrine FDC suspensions to check for the quality and stability over the period of use after reconstitution of the powder with water, thus there is need to determine the quality and stability of the drugs especially the suspensions.

1.4 Aim of the Research

The aim of this work is to develop a simple, accurate cost effective and sensitive UV spectrophotometric method of analysis for the determination of artemether in pure and combination dosage formulations and to apply it in the assay and determination of the stability profile of some selected brands of the suspension.

1.5 Specific Objectives

1. Development and validation of UV spectrophotometric method for the determination of artemether by using the acid decomposition product of the drug.

2. Apply the method in assaying different brands of artemether/lumefantrine powders suspension.

3. Assay different brands of artemether/lumefantrine powders for suspension using the IP method and compare the results with the developed method.

4. Determine the stability of the reconstituted suspensions over a period of 14 days.

1.6 Research Hypothesis

The developed UV method can be used to assay and determine the stability profile of the suspensions.