Matrix using SPE and LC-MS/MS

Abstract

Drug analysis from whole blood is gaining popularity due to a more complete measurement of analytes in the biological system. Historically, most drug screens have been performed using immunoassay and similar technologies. These techniques often lack specificity and sensitivity necessary for today's complex legal requirements. In this poster, we demonstrate a fast, robust technique for the screening of a broad range of drugs from whole blood utilizing SPE and tandem mass spectrometry. Forty-one compounds, which include opiates (both natural and synthetic), amphetamines, benzodiazepines, phenylpiperidines, a muscle relaxant, and some illicit drugs (PCP and cocaine metabolite) were tested. Utilizing advanced sample preparation techniques we simplify a complicated matrix to allow for a fast and successful multi-component analysis by LC-MS/MS. Six pretreatment options for whole blood were investigated. The volume of whole blood used was 0.25 mL. A 5% zinc sulfate and acetonitrile combination worked for most classes. Benzodiazepines had a better overall response with 10% MeOH in ACN. However, there was not a single pretreatment procedure that worked well for all classes of compounds. The supernatant was loaded onto the Strata-XC 30 mg/3 mL cartridges. Salts and most endogenous components were removed using a two-step, 0.1% formic acid and 30% methanol, wash. Analytes were eluted using 2 × 500 uL of ammoniated IPA/ethyl acetate solution. Samples were then acidified and evaporated to dryness at 40–45 °C under a gentle nitrogen stream. The dry residue was re-suspended in mobile phase and injected onto the column. The chromatographic separation was performed on a Kinetex 2.6 um Biphenyl 50 × 3.0 mm column. The best choice of mobile phase was 0.1% formic acid in water and 0.1% formic acid in methanol. The detection was achieved on an AB Sciex Triple Quad 4500 and/or a 4000 QTrap LC-MS/MS system. All analytes were detected under positive polarity and MRM scan function using two mass transitions. A MRM-IDA-EPI combination scan was used on a small sample set to verify the analyte purity. A detection limit of 10 ng/mL was achieved for all tested analytes using this method. Tramadol, propoxyphene, fentanyl, carisoprodol and meprobamate produced strong signals. The absolute lowest detection levels for these analytes have not yet been determined. Although a full calibration method was not vigorously tested, the upper end of the calibration range was deemed to be 1000 ng/mL. A quadratic fit with 1/× weighting factor was employed for calibration purposes. Early eluting analytes were well separated from the ion suppression zone by a retention factor of two, or by three times the dead volume. Isomeric/isobaric compounds were completely resolved by the biphenyl LC column by at least a factor of 2. A resolution value of 3.2 was obtained for morphine and hydromorphone. The described procedure provides a simple and reliable solution to screen a wide range of illicit and pain control compounds from a complicated matrix.