Estimation of Amiloride and Furosemide in human plasma using high performance liquid chromatography

A novel, simple, sensitive and precise RP-HPLC method has been developed and validated for quantitative analysis of amiloride and furosemide in human plasma. Use of a Inertsil (250 mm × 4.6 mm) 5-μm particle, C18 column with methanol: 0.1%glacial acetic acid (43:57, v/v) of pH 5.05 adjusted with sodium hydroxide as isocratic mobile phase at a flow rate of 1.0 mL/min. The reconstituted method involved the protein precipitation with methanol, followed by centrifugation and clear supernatant solution injected in to the column enabled separation of the drug from plasma. The detection was carried out using a UV-PDA detector at 226 nm. The method was validated for linearity, accuracy (recovery), precision, specificity and robustness. The linearity of the method was excellent over the range 100-350 ng/mL (correlation coefficient 0.999) for amiloride and 800-2800 ng/mL (correlation coefficient 0.997) for furosemide. The retention time for amiloride and furosemide was found to be 4.36 + 0.3 and 10.2 + 0.3 min respectively. The limit of detection was established as 50 ng/mL for amiloride and 100 ng/mL for furosemide. The accuracy was observed 97.75-101.2 % for amiloride and for furosemide 98.93%-100.2%. The recovery observed for amiloride of low, middle and high quality control samples was found to be 73.51 %, 75.19 % , 80.01 % respectively and for furosemide was found to be 71.9 %, 79.5 % and 86.4 % respectively. The stability of amiloride and furosemide was excellent, with no evidence of degradation during sample proceeding.

Author(s): Maulik Kumar. R. Amin, Deepa R. Patel, Ravi J. Shah, Jinali M. Amin, Nilesh Jain and Deepak Kumar Jain

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