RESUMO
Characterization of endogenous metabolites and xenobiotics is essential to deconvoluting the genetic and environmental causes of disease. However, surveillance of chemical exposure and disease-related changes in large cohorts requires an analytical platform that offers rapid measurement, high sensitivity, efficient separation, broad dynamic range, and application to an expansive chemical space. Here, we present a novel platform for small molecule analyses that addresses these requirements by combining solid-phase extraction with ion mobility spectrometry and mass spectrometry (SPE-IMS-MS). This platform is capable of performing both targeted and global measurements of endogenous metabolites and xenobiotics in human biofluids with high reproducibility (CV 6 3%), sensitivity (LODs in the pM range in biofluids) and throughput (10-s sample-to-sample duty cycle). We report application of this platform to the analysis of human urine from patients with and without type 1 diabetes, where we observed statistically significant variations in the concentration of disaccharides and previously unreported chemical isomers. This SPE-IMS-MS platform overcomes many of the current challenges of large-scale metabolomic and exposomic analyses and offers a viable option for population and patient cohort screening in an effort to gain insights into disease processes and human environmental chemical exposure.
RESUMO
High throughput-solid phase extraction tandem mass spectrometry (HT-SPE/MS) is a fully automated system that integrates sample preparation using ultrafast online solid phase extraction (SPE) with mass spectrometry detection. HT-SPE/MS is capable of conducting analysis at a speed of 5-10 s per sample, which is several fold faster than chromatographically based liquid chromatography-mass spectrometry (LC-MS). Its existing applications mostly involve in vitro studies such as high-throughput therapeutic target screening, CYP450 inhibition, and transporter evaluations. In the current work, the feasibility of utilizing HT-SPE/MS for analysis of in vivo preclinical and clinical samples was evaluated for the first time. Critical bioanalytical parameters, such as ionization suppression and carry-over, were systematically investigated for structurally diverse compounds using generic SPE operating conditions. Quantitation data obtained from HT-SPE/MS was compared with those from LC-MS analysis to evaluate its performance. Ionization suppression was prevalent for the test compounds, but it could be effectively managed by using a stable isotope labeled internal standard (IS). A structural analogue IS also generated data comparable to the LC-MS system for a test compound, indicating matrix effects were also compensated for to some extent. Carry-over was found to be minimal for some compounds and variable for others and could generally be overcome by inserting matrix blanks without sacrificing assay efficiency due to the ultrafast analysis speed. Quantitation data for test compounds obtained from HT-SPE/MS were found to correlate well with those from conventional LC-MS. Comparable accuracy, precision, linearity, and sensitivity were achieved with analysis speeds 20-30-fold higher. The presence of a stable metabolite in the samples showed no impact on parent quantitation for a test compound. In comparison, labile metabolites could potentially cause overestimation of the parent concentration if the ion source conditions are not optimized to minimize in-source breakdown. However, with the use of conditions that minimized in-source conversion, accurate measurement of the parent was achieved. Overall, HT-SPE/MS exhibited significant potential for high-throughput in vivo bioanalysis.
