Validation of a liquid chromatographic-tandem mass spectrometric method for the determination of loperamide in human plasma

On-line LC-MS/MS system using atmospheric pressure chemical ionization (APCI) has been developed for the determination of loperamide. The separation is obtained on a octadecylsilica based stationary phase using a mobile phase consisting in a mixture of methanol and 5 mM ammonium acetate solution (25:75, v/v). Clonazepam is used as internal standard (IS). The MS/MS ion transitions monitored are m/z 477 → 266 and 316 → 270 for loperamide and clonazepam, respectively. The most appropriate regression model of the response function as well as the limit of quantitation were first selected during the pre-validation step. These latter criteria were then assessed during the formal validation step. The limit of quantitation (LOQ) was around 50 pg/ml for loperamide. The method was also validated with respect to recovery, precision, trueness, accuracy and linearity.