Targeted Ion Mobility and Liquid Chromatography-Mass Spectrometry Metabolomic Strategies for Glucose Quantitation in Exhaled Breath Condensate for Cystic Fibrosis Studies

MARÍA EUGENIA MONGE; JOSÉ J. PÉREZ; PRABHA DWIVEDI; MANSHUI ZHOU; ARLENE STECENKO; FACUNDO M. FERNANDEZ

Minneapolis

Conferencia; 61st ASMS Conference on Mass Spectrometry & Allied Topic; 2013

American Society for Mass Spectrometry

Introduction (119/120) Progressive lung disease and eventual respiratory failure accounts for almost 90% of morbidity and mortality in cystic fibrosis (CF). CF-related diabetes (CFRD) occurs in 50% of CF adults and causes acceleration in the decline in lung function. However, it is unknown why diabetes worsens CF lung disease. Our overarching hypothesis is that hyperglycemia in CFRD results in increased airway glucose levels that persist after systemic glucose values return to normal resulting in marked and prolonged redox imbalance and oxidative stress thus providing a mechanism whereby CFRD worsens lung disease. This work presents the development of targeted ion mobility and liquid chromatography-mass spectrometry metabolomic methods as key tools for testing this hypothesis by monitoring glucose in airway samples from humans. Methods (117/120) EBC samples were collected with an R-TubeTM. Samples and standards were spiked with glucose and urea internal standards; stored at -80 °C for 2 h, freeze-dried for 24 h; and reconstituted in acetonitrile:water 80:20 v/v (concentration factor=20). UHPLC TOF-MS analysis was performed using a Waters ACQUITY H Class system fitted with a Waters ACQUITY UPLC® BEH Amide column (1.0 × 100 mm, 1.7 µm), and coupled to a Xevo G2 TOF mass spectrometer (Waters Corporation). UHPLC-MS/MS analysis was performed with an Agilent 1290 Infinity system coupled to an Agilent 6430 Triple Quadrupole LC/MS system. Direct-infusion traveling wave ion mobility spectrometry (TWIMS) with TOF-MS detection was performed using a Synapt G2 High Definition Mass Spectrometry system (Waters Corporation). Preliminary Data (300 /300) UHPLC-QqQ MS/MS, UHPLC TOF-MS, and TWIMS TOF-MS-based methods were developed and evaluated for glucose quantitation in airway fluid using urea as the EBC dilution marker. Glucose quantitation in trace levels with high accuracy and precision was successfully achieved by all three approaches. Urea was analyzed in positive ion mode and glucose in negative ion mode. For glucose analysis the [M-H]- ion signal provided higher signal-to-noise ratio than the [M+Na]+ ion signal. Matrix effects were mitigated using isotopically-labeled glucose and urea as internal standards The UHPLC-QqQ-MS/MS method provided the largest linear range of 3 orders of magnitude for glucose quantitation. The LOQ achieved was 0.05 µM in the reconstituted EBC samples which was equivalent to 2.5 nM of glucose in EBC. Linear ranges of 2.5 and 2 orders of magnitude were obtained with the UHPLC-TOF-MS and TWIMS-MS methods, respectively. However, the TWIMS-MS method allowed for measurement of glucose at higher concentration (80 µM) than the other two methods. Method precision was determined by the relative standard deviation (RSD) of repeat measurements (n=5) of each standard used to build the calibration curves. The UHPLC-QqQ-MS/MS method had an average RSD of 7.5% (2-10%), except for 0.05 µM concentration level with an RSD of 15%; the UHPLC-TOF-MS method had an average RSD of 7.9% (4-19%), and the TWIMS-TOF-MS had an average RSD of 13.7% (8-20%). Though analytical figures of merit were similar between UHPLC-TOF-MS and UHPLC-QqQ-MS/MS methods, the latter provided the lowest limit of detection (LOD) for both glucose and urea (1.5 nM in EBC). Ion mobility strategies compared favorably in terms of sample throughput, but with a higher glucose LOD (5 nM in EBC), and results compared in terms of accuracy and precision. These analytical figures of merit demonstrate the applicability of these methods to EBC metabolite analysis for CF and CFRD research. Novel aspect (19/20) Mass spectrometric methods for quantitative analysis of trace glucose in EBC are reported and compared for the first time.