In-Vial Dual Extraction for Direct LC-MS Analysis of Plasma for Comprehensive and Highly Reproducible Metabolic Fingerprinting.

Metabolic fingerprinting of biological tissues has become an important area of research, particularly in the biomarker discovery field. Methods have inherent analytical variation, and new approaches are necessary to ensure that the vast numbers of intact metabolites present in biofluids are detected. Here, we describe an in-vial dual extraction (IVDE) method and a direct injection method that shows the total number of features recovered to be over 4500 from a single 20 μL plasma aliquot. By applying a one-step extraction consisting of a lipophilic and hydrophilic layer within a single vial insert, we showed that analytical variation was decreased. This was achieved by reducing sample preparation stages including procedures of drying and transfers. The two phases in the vial, upper and lower, underwent HPLC-QTOF analysis on individually customized LC gradients in both positive and negative ionization modes. A 60 min lipid profiling HPLC-QTOF method for the lipophilic phase was specifically developed, enabling the separation and putative identification of fatty acids, glycerolipids, glycerophospholipids, sphingolipids, and sterols. The aqueous phase of the extract underwent direct injection onto a 45 min gradient, enabling the detection of both polarities. The IVDE method was compared to two traditional extraction methods. The first method was a two-step ether evaporation and IPA resuspension, and the second method was a methanol precipitation typically used in fingerprinting studies. The IVDE provided a 378% increase in reproducible features when compared to evaporation and a 269% increase when compared to the precipitate and inject method. As a proof of concept, the method was applied to an animal model of diabetes. A 2-fold increase in discriminant metabolites was found when comparing diabetic and control rats with IVDE. These discriminant metabolites accounted for around 600 entities, out of which 388 were identified in available databases

Recoveries of HILIC and reversed phase internal standards in experiment 2.

<p>A) plot of intensity of reversed phase internal standards Heptsdecanoic acid (negative) and Tripentadecanoin (positive), B) plot of intensity of HILIC internal standards in positive ionisation mode, C) plot of intensity of HILIC internal standards in negative ionisation mode, D) average intensity and coefficient of variance of all internal standards.</p

Plots of sample mass in milligrams against intensity for 9 annotated metabolites.

<p>A) taurine B) hypoxanthine C) glutamate D) pantothenate E) aspartate F) glcosylceramide (36:1) G) phosphatidylethanolamine (38:4) H) ceramide (38:1) I) triglyceride (48:3).</p

Graphical representation of the experimental designs used.

<p>A) experiment 1, a single 18mg brain section was homogenised then 7 parallel extractions were performed on 50μl aliquots of homogenate. B) Experiment 2, brain sections ranging from 3–17mg were homogenised and extracted parallel.</p

Applied analytical pipeline.

<p>Shows the seven steps from tissue sectioning to IVDE and onto data processing and multivariate analysis of variation.</p

Measured metabolite features in the reversed phase method in experiment 1.

<p>Showing the number of metabolite peaks identified and their relative variability in 100%, 85% and 70% of 7 sample replicates.</p><p>^a percentage of samples a peak is detected in</p><p>^b coefficient of variance of peak intensity between samples.</p><p>Measured metabolite features in the reversed phase method in experiment 1.</p

Comparison of 3-HB levels in all groups at different time points in plasma and urine.

<p>Comparison of 3-HB levels in all groups at different time points in plasma and urine.</p

Summary of UTI biomarker studies.

<p>Summary of UTI biomarker studies.</p

Experimental design for UTI metabolomics study.

<p>Experimental design for UTI metabolomics study.</p

Direct Monitoring of Exogenous γ‑Hydroxybutyric Acid in Body Fluids by NMR Spectroscopy

γ-Hydroxybutyric acid (GHB) is a popular drug increasingly associated with cases of drug-facilitated sexual assault (DFSA). Currently, expanding procedures of analysis and having forensic evidence of GHB intake in a long term are mandatory. Up to now, most studies have been performed using GC/MS and LC-MS as analytical platforms, which involve significant manipulation of the sample and, often, indirect measurements. In this work, procedures used in NMR-based metabolomics were applied to a GHB clinical trial on urine and serum. Detection, identification, and quantification of the drug by NMR methods were surveyed, as well as the use of NMR-based metabolomics for the search of potential surrogate biomarkers of GHB consumption. Results demonstrated the suitability of NMR spectroscopy, as a robust nondestructive technique, to fast and directly monitor (detect, identify, and quantify) exogenous GHB in almost intact body fluids and its high potential in the search for metabolites associated with GHB intake