Determination of afloqualone in human plasma using liquid chromatography/tandem mass spectrometry: Application to pharmacokinetic studies in humans

Two methods for determining the central-acting muscle relaxant afloqualone in human plasma were developed and compared using API2000 and API4000 liquid chromatography tandem mass spectrometry (LC/MS/MS) systems. In the API2000 LC/MS/MS system, afloqualone and the internal standard methaqualone were extracted from plasma using a methyl-tertiary ether. After drying the organic layer, the residue was reconstituted in a mobile phase (0.1% formic acid-acetonitrile:0.1% formic acid buffer, 80:20 v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 ml/min. The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 117 for afloqualone and methaqualone, respectively.Sample preparation for the API4000 LC/MS/MS system involved simple protein precipitation with an organic mixture (methanol:10% ZnSO₄ = 8:2). The ion transitions monitored in multiple reaction-monitoring mode were m/z 284 → 146 and 251 → 131 for afloqualone and methaqualone, respectively.In both assays, the coefficient of variation of the precision was less than 11.8%, the accuracy exceeded 91.5%, the limit of quantification was 0.5 ng/ml, and the limit of detection was 0.1 ng/ml for afloqualone. Two methods were used to measure the plasma afloqualone concentration in healthy subjects after a single oral 20-mg dose of afloqualone. During subsequent application of the methods, we observed that high-concentration plasma samples (>7 ng/ml) prepared using the protein precipitation method resulted in about 20% higher afloqualone concentrations than with plasma samples prepared using the liquid-liquid extraction method. We believe that this phenomenon was related to the cleanness of the sample and its chemical nature.