TY - JOUR
T1 - Development of a high-performance liquid chromatography method for warfarin detection in human plasma
AU - Chua, Yung An
AU - Abdullah, Wan Zaidah
AU - Gan, Siew Hua
PY - 2012
Y1 - 2012
N2 - Aim: Most of the high-performance liquid chromatography (HPLC) methods that have been developed to measure warfarin levels use the preextraction spiked internal standard method. We developed a new liquid-liquid extraction (LLE) and reversed-phase HPLC method that uses a postextraction spiked internal standard for the determination of plasma warfarin. Materials and methods: The effect of varying the 1) mobile phase pH, 2) type and composition of organic solvents, 3) type and concentration of buffer solutions, 4) column temperatures, 5) flow rates, 6) organic modifier, and 7) ultraviolet wavelengths were tested. Results: Optimum separation was achieved by using 30:70 (v/v) acetonitrile and potassium dihydrogen orthophosphate (0.01 M) at pH 6.5 without the addition of an organic modifier at 300 nm. The column temperature was fixed at 30 °C and the flow rate at 1.0 mL/min. Phenylbutazone was the most suitable internal standard. For LLE, the optimum plasma pH was 4.5, using 2 volumes of 2.5 mL of diethyl ether. The average retention times of warfarin and phenylbutazone were 7 and 11 min, respectively. Conclusion: The postextraction spiked internal standard method saved a significant amount of development time and gave an excellent recovery of nearly 90%. This method was successfully tested using spiked human plasma. On the whole, the developed method is simple, economical, and suitable for routine application.
AB - Aim: Most of the high-performance liquid chromatography (HPLC) methods that have been developed to measure warfarin levels use the preextraction spiked internal standard method. We developed a new liquid-liquid extraction (LLE) and reversed-phase HPLC method that uses a postextraction spiked internal standard for the determination of plasma warfarin. Materials and methods: The effect of varying the 1) mobile phase pH, 2) type and composition of organic solvents, 3) type and concentration of buffer solutions, 4) column temperatures, 5) flow rates, 6) organic modifier, and 7) ultraviolet wavelengths were tested. Results: Optimum separation was achieved by using 30:70 (v/v) acetonitrile and potassium dihydrogen orthophosphate (0.01 M) at pH 6.5 without the addition of an organic modifier at 300 nm. The column temperature was fixed at 30 °C and the flow rate at 1.0 mL/min. Phenylbutazone was the most suitable internal standard. For LLE, the optimum plasma pH was 4.5, using 2 volumes of 2.5 mL of diethyl ether. The average retention times of warfarin and phenylbutazone were 7 and 11 min, respectively. Conclusion: The postextraction spiked internal standard method saved a significant amount of development time and gave an excellent recovery of nearly 90%. This method was successfully tested using spiked human plasma. On the whole, the developed method is simple, economical, and suitable for routine application.
KW - HPLC
KW - Method development
KW - Postextraction
KW - Warfarin
UR - http://www.scopus.com/inward/record.url?scp=84864094474&partnerID=8YFLogxK
U2 - 10.3906/sag-1101-1476
DO - 10.3906/sag-1101-1476
M3 - Article
AN - SCOPUS:84864094474
SN - 1300-0144
VL - 42
SP - 930
EP - 941
JO - Turkish Journal of Medical Sciences
JF - Turkish Journal of Medical Sciences
IS - 5
ER -