Thomas A. Swift

Drug Screening in Medical Examiner Casework by High-Resolution Mass Spectrometry (UPLC–MSE-TOF)

Postmortem drug findings yield important analytical evidence in medical examiner casework, and chromatography coupled with nominal mass spectrometry (MS) serves as the predominant general unknown screening approach. We report screening by ultra performance liquid chromatography (UPLC) coupled with hybrid quadrupole time-of-flight mass spectrometer (MS(E)-TOF), with comparison to previously validated nominal mass UPLC-MS and UPLC-MS-MS methods. UPLC-MS(E)-TOF screening for over 950 toxicologically relevant drugs and metabolites was performed in a full-spectrum (m/z 50-1,000) mode using an MS(E) acquisition of both molecular and fragment ion data at low (6 eV) and ramped (10-40 eV) collision energies. Mass error averaged 1.27 ppm for a large panel of reference drugs and metabolites. The limit of detection by UPLC-MS(E)-TOF ranges from 0.5 to 100 ng/mL and compares closely with UPLC-MS-MS. The influence of column recovery and matrix effect on the limit of detection was demonstrated with ion suppression by matrix components correlating closely with early and late eluting reference analytes. Drug and metabolite findings by UPLC-MS(E)-TOF were compared with UPLC-MS and UPLC-MS-MS analyses of postmortem blood in 300 medical examiner cases. Positive findings by all methods totaled 1,528, with a detection rate of 57% by UPLC-MS, 72% by UPLC-MS-MS and 80% by combined UPLC-MS and UPLC-MS-MS screening. Compared with nominal mass screening methods, UPLC-MS(E)-TOF screening resulted in a 99% detection rate and, in addition, offered the potential for the detection of nontargeted analytes via high-resolution acquisition of molecular and fragment ion data.

Multi-drug and Metabolite Quantification in Postmortem Blood by Liquid Chromatography–High-Resolution Mass Spectrometry: Comparison with Nominal Mass Technology

High-resolution mass spectrometry (HRMS) is being applied in postmortem drug screening as an alternative to nominal mass spectrometry, and additional evaluation in quantitative casework is needed. We report quantitative analysis of benzoylecgonine, citalopram, cocaethylene, cocaine, codeine, dextromethorphan, dihydrocodeine, diphenhydramine, 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine, hydrocodone, hydromorphone, meperidine, methadone, morphine, oxycodone and oxymorphone in postmortem blood by ultra-performance liquid chromatography (UPLC)-MS(E)/time-of-flight (TOF). The method employs analyte-matched deuterated internal standardization and MS(E) acquisition of precursor and product ions at low (6 eV) and ramped (10-40 eV) collision energies, respectively. Quantification was performed using precursor ion data obtained with a mass extraction window of ± 5 ppm. Fragment and residual precursor ion acquisitions at ramped collision energies were evaluated as additional analyte identifiers. Extraction recovery of >60% and matrix effect of <20% were determined for all analytes and internal standards. Defined limits of detection (10 ng/mL) and quantification (25 ng/mL) were validated along with a linearity analytical range of 25-3,000 ng/mL (R(2) > 0.99) for all analytes. Parallel UPLC-MS(E)/TOF and UPLC-MS/MS analysis showed comparable precision and bias along with concordance of 253 positive (y = 1.002x + 1.523; R(2) = 0.993) and 2,269 negative analyte findings in 159 postmortem cases. Analytical performance and correlation studies demonstrate accurate quantification by UPLC-MS(E)/TOF and extended application of HRMS in postmortem casework.

Effects of the polyamine spermine on binding of follicle-stimulating hormone to membrane-bound immature bovine testis receptors

The effect of the polyamine, spermine, on the interaction of human 125I-labeled FSH with membrane-bound receptors derived from bovine calf testes has been examined. Concentrations of spermine less than 0.01 M resulted in a slight but insignificant (P greater than 0.10) enhancement of FSH concentrations of 0.01 M and above caused a progressive reduction of FSH binding. Membrane receptors incubated in the presence of spermine at concentrations inhibitory to human 125I-FSH binding (0.01-0.04 M) resulted in an 8-50% decrease in the apparent FSH receptor concentration and a 10-65% decrease in the affinity constant as determined by computerized analysis of the isothermic ligand-binding data. Within the temperature range 4-20 degrees C, simultaneous addition of spermine (0.025 M) increased the reversibility of human 125I-FSH binding approx. 10% (P less than 0.005). Delayed addition of spermine (0.01-0.04 M) resulted in a dose-related dissociation of human 125I-FSH already bound to its receptor (P less than 0.05). However, preincubation of membrane receptors with spermine (0.002-0.04 M) at 4 degrees C or 34 degrees C followed by washing and addition of human 125I-FSH, resulted in an increase in hormone binding (P less than 0.05) over that of controls. If membrane receptor was incubated at 34 degrees C with spermine in the absence of radioligand, the usual loss of hormone binding was reduced (P less than 0.05), while membrane receptor incubated with spermine at 4 degrees C exhibited hormone binding greater (P less than 0.05) than that observed before treatment. Thus, the mechanism of inhibition of human 125I-FSH binding to membrane receptors appears to be correlated with an increase in reversibility of the membrane receptor-human 125I-FSH complex and is expressed as a decrease in the calculated receptor concentration and affinity constant of that interaction. Second, spermine appears to stabilize the membrane receptor in the absence of ligand, presumably through a membrane effect. These data suggest that spermine, and possibly other polyamines, which are endogenous to eukaryotic cells and undergo increases in concentration following stimulation by trophic hormone may play a role in the modulation of the ligand-membrane receptor interaction, in part, through direct effects on the membrane and/or the receptor.