Sensitive hydrophilic interaction liquid chromatography/tandem mass spectrometry method for rapid detection, quantification and confirmation of cathinone-derived designer drugs for doping control in equine plasma.

RATIONALE: Cathinone derivatives are new amphetamine-like stimulants that can evade detection when presently available methods are used for doping control. To prevent misuse of these banned substances in racehorses, development of a liquid chromatography/tandem mass spectrometry (LC/MS/MS) method became the impetus for undertaking this study. METHODS: Analytes were recovered via liquid-liquid extraction using methyl tert-butyl ether. Analyte separation was achieved on a hydrophilic interaction column using liquid chromatography and mass analysis was performed on a QTRAP mass spectrometer in positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM). Analyte identification was carried out by screening for a specified MRM transition. Quantification was conducted using an internal standard. Confirmation was performed by establishing a match in retention time and ion intensity ratios comparison. RESULTS: The method was linear over the range 0.2-50 ng/mL. The specificity was evaluated by analysis of six different batches of blank plasma and those spiked with each analyte (0.2 ng/mL). The recovery of analytes from plasma at three different concentrations was >70%. The limits of detection, quantification and confirmation were 0.02-0.05, 0.2-1.0 and 0.2-10 ng/mL, respectively. The matrix effect was insignificant. The intra-day and inter-day precision were 1.94-12.08 and 2.58-13.32%, respectively. CONCLUSIONS: The method is routinely employed in screening for the eleven analytes in post-competition samples collected from racehorses in Pennsylvania to enforce the ban on the use of these performance-enhancing agents in racehorses. The method is sensitive, fast, effective and reliably reproducible.

Highly selective isolation and separation of 25-hydroxyvitamin D and 3-epi-25-hydroxyvitamin D metabolites from serum.

BACKGROUND: Increasing health concerns related to vitamin D deficiency including Alzheimer's and immune diseases, along with various cancers, have heightened awareness of the nutrient. As the associated health concerns grow, so does the need for fast and accurate analytical testing for diagnostics and treatment. Established immunoassay methods have been challenged for accuracy caused largely by endogenous interferences. This has driven the interest in more specific LC-MS/MS methodology where specificity is gained through chromatographic and MS resolution. RESULTS: Herein, a pentafluorophenyl stationary phase is shown to provide superior selectivity for the separation of the closely related 25-hydroxyvitamin D(3) and 3-epi-25-hydroxyvitamin D(3) as compared with many methods reported in the literature. To increase robustness and reliability, a novel protein precipitation/phospholipid removal device is also utilized. The novel approach was applied to human samples with a comparison with established clinical LC-MS/MS services for measuring 25-hydroxyvitamin D in adults and for infants (<1 year old). CONCLUSION: The data showed a good correlation between the routine service for adults and infant patient samples and illustrated the need to resolve the epimers. The unique selectivity of the pentafluorophenyl phase combined with the selective protein depletion and phospholipid enable a fast, accurate and robust analysis of 25-hydroxyvitamin D and related forms, which are otherwise unattainable with commonly used sample preparation and reversed-phase HPLC approaches.