Metaxalone estimation in biological matrix using high-throughput LC–MS/MS bioanalytical method

► High-throughput bioanalytical method was developed and validated adhering to global regulatory norms. ► We examined metaxalone in blood, plasma and in hemolyzed/lipemic plasma for effect of matrix component interference. ► Achieved mean recovery >78% for both metaxalone and its internal standard and intra- and inter-day precisions and accuracy were all within 6%. ► We have found that matrix effect was negligible though the method is sensitive and demonstrated high extraction efficiency. Metaxalone is a skeletal muscle relaxant, an approved drug for pain relief. Published bioanalytical methods lacked detailed stability evaluation in blood and plasma. An accurate, precise, high-throughput tandem mass spectroscopic method has been developed and validated. Following solid phase extraction (SPE), metaxalone and the internal standard metaxalone-d3 were extracted from an aliquot of 200μL of human plasma. Chromatographic separation achieved on an Ascentis Express C18 column (50mm×4.6mm i.d., 2.7μm particle size) with mobile phase is a mixture of 10mM ammonium acetate buffer (pH 4.5)–methanol–acetonitrile (20:50:30, v/v/v), at an isocratic flow rate of 0.7mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer by multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The mass transitions of metaxalone and metaxalone-d3 were m/z 222.3→161.2 and m/z 225.3→163.3, respectively. The linear calibration curves were obtained in the concentration range of 0.105–10.081μg/mL (r2≥0.99) with a lower limit of quantification (LLOQ) of 0.105μg/mL. The intra- and inter-day precisions and relative error were all within 6%. Despite achieving high mean recovery (>78%), no interference peaks or matrix effects were observed. Detailed stability exercises including drug stability in blood, hemolyzed, lipemic and normal plasma were conducted to extend the method applicability in vast majority of clinical studies using 800mg metaxalone extended release oral dosage form.