Craig Aurand

Coupling Desorption Electrospray Ionization with Solid-Phase Microextraction for Screening and Quantitative Analysis of Drugs in Urine

Direct analysis of silica C(18)-coated solid-phase microextraction (SPME) fibers using desorption electrospray ionization mass spectrometry (DESI-MS) for the purpose of analyzing drugs from raw urine is presented. The method combines a simple, inexpensive, and solvent-less sample preparation technique with the specificity and speed of DESI-MS and MS/MS. Extraction of seven drugs from raw urine is performed using specially designed SPME fibers coated uniformly with silica-C(18) stationary phase. Each SPME device is inserted into unprocessed urine under gentle agitation and, then, removed, rinsed, and analyzed directly by DESI-MS (MS/MS). Rapid screening over a wide mass range is afforded by coupling the method with a time of flight (TOF) mass spectrometer while quantitative analysis is performed using selected reaction monitoring (SRM) using a triple quadrupole mass spectrometer. The performance of the SPME DESI-MS/MS method was evaluated by preparing calibration standards and quality control (QC) samples of the seven drug compounds from urine over a range from 20 to 1000 ng/mL, with the exception of meprobamate which was prepared from 200 to 10000 ng/mL. The calibration curves constructed for each analyte had an R(2) > 0.99. The range of precision (%CV) and accuracy values (% bias) for low QC samples was 1-11% and 3-38%, respectively. Precision and accuracy values for high QC samples range from 0.9 to 8% and -31 to -8%. Results from urine specimens of actual exposure to drugs screened using the SPME DESI-MS/MS method showed good agreement with the conventional immunoassays and GC/MS analysis. Liquid desorption of the SPME fiber followed by LC/MS/MS also showed good agreement with the SPME DESI-MS/MS method.

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 Alzheimers 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.