Development and validation of an UFLC-MS/MS method for enantioselectivity determination of d,l-thero-methylphenidate, d,l-thero-ethylphenidate and d,l-thero-ritalinic acid in rat plasma and its application to pharmacokinetic study

• One step liquid–liquid extraction method to extract d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH and d,l-threo-RA was developed to replace the time-consuming SPE method.
• This study is the first UFLC-MS/MS method to quantify d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH and d,l-threo-RA simultaneously, and a better chiral separation of d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH was achieved by UFLC-MS/MS optimization.
• The concentrations of d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH and d,l-threo-RA in rat plasma were determined by the proposed UFLC-MS/MS method after oral administration of 2 mg/kg d-threo-MPH and l-threo-MPH as well as in combination in rats (n = 6).

A chiral UFLC-MS/MS method was established and validated for quantifying d-threo-methylphenidate (d-threo-MPH), l-threo-methylphenidate (l-threo-MPH), d-threo-ethylphenidate (d-threo-EPH), l-threo-ethylphenidate (l-threo-EPH) and d,l-threo-ritalinic acid (d,l-threo-RA) in rat plasma over the linearity range of 1–500 ng/mL. Chiral separation was performed on an Astec Chirobiotic V2 column (5 μm, 250 × 2.1 mm) with isocratic elution using methanol containing 0.003% ammonium acetate (w/v) and 0.003% trifluoroacetic acid (v/v) at a flow of 0.3 mL/min. All analytes and IS were extracted from rat plasma by a one-step liquid–liquid extraction (LLE) method. The intra- and inter-run accuracies were within 85–115%, and the intra- and inter-run precision were <10% for all analytes. Extraction recoveries were 55–62% for d-threo-MPH, 54–60% for l-threo-MPH, 55–60% for d-threo-EPH, 53–57% for l-threo-EPH and 25–30% for d,l-threo-RA. The validated UFLC-MS/MS method successfully applied to the pharmacokinetic interaction study of oral d-threo-MPH and l-threo-MPH (alone or in combination) in female Sprague Dawley rats. The EPH was not detected in rat plasma following oral administrated MPH without EtOH. As far as it is known to the authors, this study is the first one step liquid–liquid extraction method to extract and UFLC-MS/MS method to quantify d-threo-MPH, l-threo-MPH, d-threo-EPH, l-threo-EPH and d,l-threo-RA simultaneously.