This paper presents the development of a new LC-MS method for the determination of pyrimethamine, sulphadoxine and artesunate in human plasma. The analytes were extracted by liquid-liquid extraction with ethyl acetate. The analytes and internal standard artemether were separated on a Phenomenex C18 column using gradient elution with 20mM ammonium acetate (mobile phase A) and methanol (mobile phase B) both containing 0.5% acetic acid at a flow rate of 0.8 ml/min. The calibration curves were constructed over the range of 5-30 ng/ml for pyrimethamine, 50-300 ng/ml for sulphadoxine and 25-150 ng/ml for artesunate respectively. Within day and between-day precision and accuracy did not exceed 8%. All the three analytes were found to be stable in plasma samples with no evidence of degradation during three freeze-thaw cycles and three months storage in -20 °C. According to the validation results, the proposed method was found to be specific, accurate, precise and could be applied to the simultaneous quantitative analysis of pyrimethamine, sulphadoxine and artesunate in human plasma.
Key words: Pyrimethamine, Sulphadoxine, Artesunate, Artemether, LC-MS, Plasma, Validation.
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