Isocratic High Performance Liquid Chromatographic Method for Determination of Metoprolol and Its Metabolite in Human Urine
An isocratic reversed phase high performance liquid chromatographic (HPLC) method has been developed for the determination of metoprolol (MET) and α-hydroxymetoprolol (α-OH MET) in human urine. Analytes were extracted using dichloromethane. The optimized separations were performed on a Purospher® STAR RP-18e LiChroCART® (250 x 4.6 mm, 5μm) HPLC column within 12 min by an isocratic elution with 25 mmol/L aqueous potassium dihydrogen phosphate buffer (pH 3.0) containing 15% (v/v) acetonitrile and 15% (v/v) methanol, and UV detection at 234 nm. The method was validated for linearity, accuracy, precision and specificity. The calibration curves for both analytes were linear over the range investigated (1.0-64.0 μg/mL) with correlation coefficients of 0.999. Relative standard deviation (RSD) values were below 2%. For all of the analytes, recoveries value from 98.36±1.68 to 101.32±1.36 % and the limits of detection for MET and α-OH MET were 0.22 and 0.14 μg/mL, respectively. The method developed has been demonstrated to be sensitive and reliable for the measurement of MET and α-OH MET simultaneously in human urine.
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