Gas-phase ion-molecule reactions and laser -induced acoustic desorption mass spectrometry for complex mixture analysis and structural elucidation

Mass spectrometry (MS) has proven invaluable in the field of mixture analysis and structural elucidation of mixture components, due to its high sensitivity, selectivity and speed. Tandem mass spectrometry (MS/MS) utilizing collision-activated dissociation (CAD) has become the technique of choice for structural elucidation of unknown analytes. The coupling of MS with high performance liquid chromatography (HPLC) has allowed the trace level analysis of components in very complex mixtures. However, these experiments alone do not always allow for unambiguous identification of an unknown analyte in a mixture. The experiments described in this thesis were aimed at providing more detailed structural information of mixture components that is difficult to obtain by established methods. In the first section (chapters 3, 4 and 5), gas-phase ion-molecule reactions were developed and implemented on a commercially available linear quadrupole ion trap (LQIT) mass spectrometer (Finnigan LTQ) to make these methods more widely applicable to the pharmaceutical industry. First, the performance a reagent mixing manifold that allows ion-molecule reactions to be carried out in the LQIT was probed by examining an established ion-molecule reaction previously employed on a Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Next, a novel ion-molecule reaction was developed using the new setup that exploits the favored formation of sodium and ammonium ion adducts when oxygen-containing analytes are ionized via electrospray ionization (ESI). Finally, the modified instrument was coupled with an HPLC to demonstrate that LC-MS3 using ion-molecule reactions and collision-activated dissociation (CAD) provides a powerful technique for the analysis of low level drug metabolites and impurities in pharmaceutical mixtures. Further, these methods were automated using the data-dependent features of the instrument, allowing for detailed structural information of unknown analytes “on-the-fly.” The second section (chapters 6, 7 and 8) focus on the development of laser-induced acoustic desorption (LIAD) methodology for structural elucidation. First, the coupling of this technique with a LQIT is presented and LIAD experiments are compared to those performed in a FT-ICR. Next, the use of LIAD in studies on the gas-phase reactivity of phenyl radicals toward tetrapeptides is presented. Very interesting results were obtained on the susceptibility of the disulfide bond to radical attack, especially considering the important role these bonds play in defining proteins’ structures. Finally, the ability of LIAD-based methods to perform unparalleled characterization of petroleum fractions is presented. Also, the development of ClMn(H2O)+ chemical ionization (CI) on the LQIT is examined. The coupling of this CI method with LIAD on the LQIT is the initial step for future implementation of this methodology on a hydrid LQIT/FT-ICR instrument to provide one of the most powerful instruments for petroleum characterization in the industry

Data-Dependent Neutral Gain MS3: Toward Automated Identification of the N-Oxide Functional Group in Drug Metabolites

We report here an automated method for the identification of N-oxide functional groups in drug metabolites by using the combination of liquid chromatography/tandem mass spectrometry (LC/MSn) based on ion-molecule reactions and collision-activated dissociation (CAD). Data-dependent acquisition, which has been readily utilized for metabolite characterization using CAD-based methods, is adapted for use with ion-molecule reaction-based tandem mass spectrometry by careful choice of select experimental parameters. Two different experiments utilizing ion-molecule reactions are demonstrated, data-dependent neutral gain MS3 and data-dependent neutral gain pseudo-MS3, both of which generate functional group selective mass spectral data in a single experiment and facilitate increased throughput in structural elucidation of unknown mixture components. Initial results have been generated by using an LC/MSn method based on ion-molecule reactions developed earlier for the identification of the N-oxide functional group in pharmaceutical samples, a notoriously difficult functional group to identify via CAD alone. Since commercial software and straightforward, external instrument modification are used, these experiments are readily adaptable to the industrial pharmaceutical laboratory

Growth Indices, Anemia, and Diet Independently Predict Motor Milestone Acquisition of Infants in South Central Nepal

The acquisition of bipedal locomotion is an important aspect of gross motor development that ultimately impacts the cognition of young children. Evidence for associations between nutrition-related variables and walking acquisition exist; however, questions about the importance of weight-for-length and dietary factors and the independent contribution of anemia and growth on walking remain. We examined the effect of nutritional factors on the acquisition of walking in a cross-sectional cohort of 485 4- to 17-mo Nepali children adjusting for age, sex, caste, and socio-economic status (SES). Participants were identified from census data collected in one village development committee (VDC) in Sarlahi District and enrolled in a cross-sectional, community-based study between January and March 2002. Hemoglobin and erythrocyte protoporphyrin (EP) were measured at baseline using a heel prick technique. The mean hemoglobin concentration was 101 ± 12.5 g/L. 57 % were anemic (hemoglobin < 105 g/L), 2.1% were severely anemic (hemoglobin < 7.0 g/L), and 43% of the children had iron deficiency anemia (hemoglobin < 105 g/L; EP > 90 μmol/mol heme). Growth was delayed; 33.5% were stunted and 20.4% were wasted. Multivariate logistic models that controlled for age, sex, caste, and SES revealed that children with higher length-for-age and weight-for-length Z-scores, no anemia, and meat consumption walked at an earlier age than children with lower scores, anemia, and no meat consumption. We conclude that growth, anemia, and diet are independently associated with delays in the onset of bipedal locomotion among young Nepali children

Everolimus with Reduced Calcineurin Inhibitor Exposure in Renal Transplantation

Background Everolimus permits reduced calcineurin inhibitor (CNI) exposure, but the efficacy and safety outcomes of this treatment after kidney transplant require confirmation.Methods In a multicenter noninferiority trial, we randomized 2037 de novo kidney transplant recipients to receive, in combination with induction therapy and corticosteroids, everolimus with reduced-exposure CNI (everolimus arm) or mycophenolic acid (MPA) with standard-exposure CNI (MPA arm). The primary end point was treated biopsy-proven acute rejection or eGFR<50 ml/min per 1.73 m2 at post-transplant month 12 using a 10% noninferiority margin.Results In the intent-to-treat population (everolimus n=1022, MPA n=1015), the primary end point incidence was 48.2% (493) with everolimus and 45.1% (457) with MPA (difference 3.2%; 95% confidence interval, -1.3% to 7.6%). Similar between-treatment differences in incidence were observed in the subgroups of patients who received tacrolimus or cyclosporine. Treated biopsy-proven acute rejection, graft loss, or death at post-transplant month 12 occurred in 14.9% and 12.5% of patients treated with everolimus and MPA, respectively (difference 2.3%; 95% confidence interval, -1.7% to 6.4%). De novo donor-specific antibody incidence at 12 months and antibody-mediated rejection rate did not differ between arms. Cytomegalovirus (3.6% versus 13.3%) and BK virus infections (4.3% versus 8.0%) were less frequent in the everolimus arm than in the MPA arm. Overall, 23.0% and 11.9% of patients treated with everolimus and MPA, respectively, discontinued the study drug because of adverse events.Conclusions In kidney transplant recipients at mild-to-moderate immunologic risk, everolimus was noninferior to MPA for a binary composite end point assessing immunosuppressive efficacy and preservation of graft function