Validated LC-MS/MS method for determination of Alverine and one of its hydroxy metabolites in human plasma along with its application to a bioequivalence study.

The present research work involves a first of its kind rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method developed and validated for simultaneous analysis of Alverine (ALV) and one of its hydroxy metabolites, para hydroxy Alverine (PHA) in human plasma. The analytes were extracted from the matrix using a simple solid-phase extraction procedure. Mebeverine was used as the internal standard for both analytes. A Kromasil C8 column provided chromatographic separation of analytes followed by detection with mass spectrometry. The method involves simple isocratic chromatography conditions and mass spectrometric detection in the positive ionization mode using an API 5000 MS/MS system. The proposed method has been validated with a linear range of 100-10,000 pg/mL for both ALV and PHA. The interrun and intrarun precision values are within 6.3%, 3.7% for ALV and 6.3%, 3.2% for PHA at LOQ levels. The intrarun accuracy in terms of % RE was within the range of -7.0% to -0.1% and -8.1% to -1.7% for ALV and PHA, respectively whereas the interrun accuracy was within the range of -5.1% to -0.5% for ALV and -8.6% to 0.4% for PHA, respectively. The overall recoveries for ALV and PHA were 83.5% and 86.2% respectively. Total elution time was about 4 min which allowed quantitation of more than 150 plasma samples per day. This validated method was used successfully for analysis of real samples from a bioequivalence study.