MSACL 2019 EU Abstract

Determination of D- and L-Lactic Acid in Urine by UPLC-MS with Electrospray Ionization Quantification

Juliane Fagotti (1), Fahmina Fardus-Reid (1), Margaret Kosek (2), Matthew R. Lewis (1) and Jonathan R. Swann (1)
(1) Imperial College London, London, UK (2) University of Virginia, VA, USA

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Juliane Fagotti (Presenter) Imperial College London

Presenter Bio: Juliane is a PhD student at Federal University of Parana (Brazil), currently working with metabolomic analysis at Imperial College London as a visiting student. The focus of her research is to investigate putative early-phase biomarkers of sleep disturbances related to Parkinson’s disease in an animal model. For that, she’s applying both targeted and untargeted approaches in order to comprehend the outspread of these conditions. In parallel, she is also working in the optimization of a method for determination of d-amino acids in biological fluids using LC-MS.
She was awarded a MSc in Physiology (2016) by Federal University of Parana and a BSc in Biology (2009) by State University of Parana, both in Brazil.

Relevant Financial Disclosures (within past 24 months) No relevant financial relationship(s) to disclose. Discussing Products? NO, presenter will NOT present educational content related to products/services of the ineligible company(ies) listed above. Conflict mitigation NOT REQUIRED.

Introduction: Lactic acid is an organic acid found in two enantiomeric forms: the L- and D-lactate stereoisomers. D-lactic acid is produced from bacteria inhabiting the gut and also from mammalian methylglyoxal metabolism. In humans, the majority of systemic D-lactate is derived from the bacterial metabolism of carbohydrate in the upper GI tract. In pathological states, intestinal permeability is increased elevating the uptake of bacterial D-lactate from the gut. Circulating D-lactate concentrations may therefore provide a useful diagnostic tool for bacterial infections, gut permeability and GI health and disease.
Many studies show significant concentrations of D-lactate in the urine with potential to reflect an overload or fluctuations due to pathologies. However, a huge variation in the concentrations are reported depending on the analytical method used. Existing methods also lack sensitivity and due to demands for prior sample clean-up steps or sample derivatization are not sufficiently high-throughput to assess large sample sets. Determining the physiological significance and diagnostic value of urinary D-lactate requires a sensitive high-throughput analytical method to generate large-scale data to correlate with pathophysiological conditions. Here, we have optimized a sensitive and specific method to fulfil both requirements to investigate D-lactate concentrations following different types of enteric infections in infants.
Methods: The assay was developed from the method of Henry et al. (2012) using 170 urine samples that were processed without derivatisation or dilution. Samples were vortex-mixed, centrifuged at 4°C to remove debris and 500 μL aliquoted into vials. Isotopically labeled internal standards sodium d4-D/L lactic acid and 13C-L-lactic acid were spiked before injecting onto a Waters Acquity UHPLC solvent management system. Chromatographic separation was achieved by the use of an Astec Chirobiotic™ R chiral column under isocratic conditions using 15% (v/v) 33.3 mMol/L ammonium acetate in H2O and 85% (w/w) acetonitrile. MS detection was performed with a Waters Xevo TQ-S tandem quadrupole instrument using negative electrospray mode.
Results: The lower limit of quantification of D-lactic acid was 0.0005 mMol/L for D-lactate and 0.001 mMol/L for L-lactate. Calibration curves were linear over the ranges of 0.001–0.4 for L-lactic and 0.0005–0.1 mMol/L for D-lactic acid. The mean nominal concentrations were 0.006526 and 0.060077 mMol/L respectively for D- and L-lactic in samples.
Discussion and Conclusions: Following optimization this UPLC/MS approach for D-lactate determination is suitable for large-scale epidemiological research, reporting very low quantification limits. The protocol was successfully applied to a human infant cohort and on-going analysis is being performed to validate the method and investigate the biological significance of urinary D-lactate with intestinal infections and gut permeability status.