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EFFECT OF VARIOUS ANTICOAGULANTS ON AMLODIPINE BESYLATE AND VALSARTAN FOR SIMULTANEOUS ANALYSIS IN HUMAN PLASMA USING ULTRA PERFORMANCE LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY

Amlodipine besylate and valsartan are one of the fixed combination drugs used for antihypertensive therapy. To obtain plasma from blood, addition of anticoagulant is needed. Anticoagulant citrate is usually used in the analysis of plasma in vitro ; EDTA and heparin are used in vivo . This anticoagulant difference can interfere with the analysis. Thus, partial validation is needed. The study evaluated the effect of anticoagulant type (citrate, heparin, and EDTA) on the analysis of amlodipine besylate and valsartan in plasma using ultra performance liquid chromatography tandem mass spectrometry. This study involved six healthy volunteers. The chromatography used an Acquity â bridged ethylene hybrid C18 column (2.1 × 100 mm; 1.7 μm). The mobile phase consisted of 0.1 % formic acid in water–acetonitrile with gradient elution. The flow rate was 0.2 mL/minute and irbesartan was the internal standard. Aliquots were obtained by liquid-liquid extraction with ammonium acetate and ethyl acetate as the extraction solvent. The accuracy and precision of the analysis of citrate, heparin and EDTA plasma met the requirements of linear calibration curves at concentrations range of 0.2 to 10 ng/mL for amlodipine besylate and 5 to 6000 ng/mL for valsartan. Stability and recovery did not differ significantly (p > 0.05, ANOVA). The peak area ratio displayed significant differences (p < 0.05, Kruskal Wallis test) in the three types of plasma. Overall, the analysis using heparin produced better results than analyses using EDTA and citrate plasma.