Use of refractometry and colorimetry as field methods to rapidly assess antimalarial drug quality

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Abstract

The proliferation of counterfeit and poor-quality drugs is a major public health problem; especially in developing countries lacking adequate resources to effectively monitor their prevalence. Simple and affordable field methods provide a practical means of rapidly monitoring drug quality in circumstances where more advanced techniques are not available. Therefore, we have evaluated refractometry, colorimetry and a technique combining both processes as simple and accurate field assays to rapidly test the quality of the commonly available antimalarial drugs; artesunate, chloroquine, quinine, and sulfadoxine. Method bias, sensitivity, specificity and accuracy relative to high-performance liquid chromatographic (HPLC) analysis of drugs collected in the Lao PDR were assessed for each technique. The HPLC method for each drug was evaluated in terms of assay variability and accuracy. The accuracy of the combined method ranged from 0.96 to 1.00 for artesunate tablets, chloroquine injectables, quinine capsules, and sulfadoxine tablets while the accuracy was 0.78 for enterically coated chloroquine tablets. These techniques provide a generally accurate, yet simple and affordable means to assess drug quality in resource-poor settings.

Introduction

Pharmaceutical counterfeiting and production of substandard drugs continue to burden the quality of health care worldwide. The World Health Organization has reported up to 25% of medicines consumed in developing countries are counterfeit or substandard [1]. These countries are most susceptible to this serious public health problem because of insufficient resources and infrastructure necessary to monitor and preserve drug quality. Since approximately 40% of the world's population is at risk of malaria [2], antimalarial drugs have become a particular favorite of counterfeiters [3], [4], [5], [6], [7]. The ability to identify counterfeit or poor quality pharmaceuticals is a critical component of a drug quality assurance system. Product quality is typically evaluated using the specifications and methods described in pharmacopeias, but the analytical techniques used, generally involve sophisticated instrumentation such as high-performance liquid chromatography (HPLC), spectrophotometers and dissolution apparatus. These instruments usually require periodic maintenance, highly trained personnel and a controlled laboratory environment for proper functioning. Unfortunately, these resources are often lacking in countries where counterfeit drugs are common. Since counterfeit drugs have been increasingly recognized as a significant public health issue, more inspection and random testing are required in these financially poor countries. Until, more effective testing procedures are implemented, simple and affordable field methods provide a practical means of rapidly monitoring drug quality. Field methods should be robust, inexpensive, portable, simple to conduct and reasonably accurate. Minimal use of toxic or flammable reagents is desirable.

Drug quality assessments initially include organoleptic inspection of the product followed by a determination of the amount of proper active pharmaceutical ingredients (API) present in the sample by commonly used basic tests such as colorimetry and thin-layer chromatography (TLC). Colorimetric techniques aid in the identification of particular active ingredients by making use of color changes produced by specific chemical reactions. The color changes are usually rapid and easily discernable. Quantitative measurements of active ingredient concentration as a function of color absorbance can be made using a portable battery-powered photometer. Although less specific than TLC, colorimetry does not require a separation phase requiring flammable or toxic organic solvents. A number of colorimetric tests for antimalarials and other essential drugs have been published [8], [9], [10], [11], [12].

Common physical properties of matter (bulk properties) such as weight, density, refractive index, viscosity, crystal morphology and solubility can be used to identify counterfeits [13]. The equipment required for measuring these properties (e.g. balance, refractometer, hydrometer, magnifying glasses or microscopes) are relatively inexpensive, portable, and rugged. For example, it has been shown that a simple refractometer can be used to monitor tampering of controlled substances by measuring the refractive index of a drug solution [14], [15].

The objective of this study was to evaluate the use of two techniques, refractometry and colorimetry as separate and complimentary field methods to rapidly assess the quality of chloroquine injectables, chloroquine (EC) enteric-coated tablets, quinine capsules, sulfadoxine/pyrimethamine (SP) tablets and artesunate tablets. The previously reported colorimetric tests have been adapted for use as an adjunct to the refractive index methods. The adaptations include a modification of the extraction component for each technique. Extracts of the active ingredients from each solid dosage form or injectable solutions were subjected to refractive index (RI) measurements and a specific colorimetric assay. The results were compared with high-performance liquid chromatographic (HPLC) analysis.

Section snippets

Reagents and apparatus

All reagents used were of analytical-reagent grade and deionized water used for all aqueous solutions. Reference standards were 97–103% purity and prepared at 25 mg ml−1. All dye solutions were freshly prepared.

Artesunate reference standard (gift from Mepha, Aesch-Basel, Switzerland) was prepared in isopropanol–methanol (1:9, v/v). Reagents for the colorimetric test include 1.1 M acetic acid, 1N sodium hydroxide, and an aqueous solution of 5 mg ml−1 fast red TR salt, dye content ∼20% (Sigma, St.

HPLC analysis

The HPLC analyses were used as reference methods in assessing the colorimetric and refractometric techniques. Therefore, evaluation of the HPLC methods in terms of accuracy, precision, and linearity are shown in Table 1. The accuracy of the methods ranged from 92 to 106% for all drugs at all concentrations. Precision was satisfactory (<15%) for chloroquine, quinine, and S/P.

The variability for the artesunate preparations was higher (≤20%) than the other antimalarials, possibly resulting from

Conclusions

Highly accurate and specific analysis methods require sophisticated and expensive instrumentation as well as experienced personnel for operation and maintenance. In many circumstances, these resources are not available, resulting in inadequate oversight of drug quality. As a consequence, counterfeit and poor quality drugs will continue to proliferate. Although, the accuracy and selectivity of simple and inexpensive techniques are less than more sophisticated techniques, these methods have the

Acknowledgements

This work was supported by funding from the Opportunistic Diseases Working Group US Centers for Disease Control and Prevention and USAID Amazon Malaria Initiative (AMI) project. The collection of antimalarial drugs from Laos was funded by the British Embassy–Bangkok Small Grants Scheme and the Wellcome Trust of Great Britain as part of the Wellcome Trust-Mahosot Hospital-Oxford Tropical Medicines Research Collaboration. We are grateful to all those who collected the samples, the British Embassy

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