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

Abstract

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.

Keywords: Diagnostics; High-throughput screening; LC–MS; Metabolomics.

Fig. 1

Schematic overview of SQUID. The upper panel demonstrates a conventional injection cycle relative to the SQUID isocratic injection approach. The lower panel demonstrates the resulting chromatographic peak spacing observed when using the conventional versus SQUID approaches. Black arrows indicate the pacing of sample injections under each methodology

Fig. 2

Normalization by isotope ratio resolves differential ion suppression. (a) 12 serial sample injections of healthy urine samples spiked with a 5000 nM unlabelled agmatine standard and a 500 nM [U-¹³C]agmatine standard to demonstrate the run-to-run variability in signal intensity across technical replicates. Bar plots show the effect before (b) and after (c) isotope correction to the internal standard. Isotope ratios were defined as the ¹²C/[U-¹³C]agmatine peak areas observed for each injection

Fig. 3

Application of SQUID for detecting polyamines in urine. To illustrate the potential utility of SQUID in high-throughput approaches to detecting microbes, we analyzed 191 human urine specimens provided by Alberta Precision Laboratories by LC–MS. Ninety-five of these samples were identified as culture-positive (≥ 10⁷ CFU/L of E. coli) and 96 samples were culture-negative controls (< 10⁷ CFU/mL). Upper quadrants (a/b) display extracted ion chromatograms for native ¹²C agmatine levels and lower quadrants (c/d) show the respective internal standard levels

Fig. 4

Analysis of in vitro microbial cultures for the presence for putrescine by SQUID. In vitro cultures of two bacterial species, P. aeruginosa and E. coli (n = 9 each), were analyzed by LC–MS across three consecutive technical replicates. SQUID analyses were tuned for the presence of ¹²C (a) and [U-¹³C]putrescine (b), a microbial polyamine produced by E. coli [40]. An internal standard of 500 nM [U-¹³C]putrescine was added to each sample prior to analysis. As expected, putrescine levels could distinguish P. aeruginosa from E. coli cultures. Blue/orange boxes highlight each technical replicate for the nine biological samples