Rapid LC-MS assay for targeted metabolite quantification by serial injection into isocratic gradients

Liquid chromatography mass spectrometry (LC-MS) has emerged as a mainstream strategy for metabolomics analyses. One advantage of LC-MS is that it can serve both as a biomarker discovery tool and as a platform for clinical diagnostics. Consequently, it offers an exciting opportunity to potentially transition research studies into real-world clinical tools. One important distinction between research versus diagnostics-based applications of LC-MS is throughput. Clinical LC-MS must enable quantitative analyses of target molecules in hundreds or thousands of samples each day. Currently, the throughput of these clinical applications is limited by the chromatographic gradient lengths, which-when analyzing complex metabolomics samples-are difficult to conduct in under ~ 3 min per sample without introducing serious quantitative analysis problems. To address this shortcoming, we developed sequential quantification using isotope dilution (SQUID), an analytical strategy that combines serial sample injections into a continuous isocratic mobile phase to maximize throughput. SQUID uses internal isotope-labelled standards to correct for changes in LC-MS response factors over time. We show that SQUID can detect microbial polyamines in human urine specimens (lower limit of quantification; LLOQ = 106 nM) with less than 0.019 normalized root mean square error. Moreover, we show that samples can be analyzed in as little as 57 s. We propose SQUID as a new, high-throughput LC-MS tool for quantifying small sets of target biomarkers across large cohorts.