Ultra-performance Liquid chromatography-MS Research Articles

Efficient plasma metabolic fingerprinting as a novel tool for diagnosis and prognosis of gastric cancer: a large-scale, multicentre study

ObjectiveMetabolic biomarkers are expected to decode the phenotype of gastric cancer (GC) and lead to high-performance blood tests towards GC diagnosis and prognosis. We attempted to develop diagnostic and prognostic...

Broccoli seed extract rich in polysaccharides and glucoraphanin ameliorates DSS-induced colitis via intestinal barrier protection and gut microbiota modulation in mice.

Broccoli has received widespread attention because of its anti-inflammatory and antioxidant effects. The present study aimed to explore the composition of broccoli seed extract (BSE) and its effect on colitis induced by dextran sulfate sodium (DSS). BSE mainly comprises glucoraphanin and polysaccharides composed of arabinose, galactose, glucose and mannose. Animal experiments suggested that BSE intervention effectively reversed body weight loss, suppressed the levels of proinflammatory interleukin-6, tumor necrosis factor-α and interleukin-1β, and elevated the levels of anti-inflammatory interleukin-10 and the activities of superoxide dismutase and glutathione in DSS-induced colitis mice. According to histopathologic and immunohistochemical analysis of colon tissue, BSE intervention may repair the intestinal barrier by upregulating mRNA levels and the expression of tight junction proteins (claudin-1, occludin and zonula occludens-1). Gas chromatography-mass spectrometry (MS) analysis demonstrated that cecal short-chain fatty acids in mice with BSE administration were significantly increased compared with the model group. Sulforaphane and sulforaphane-N-acetylcysteine were only detected in BSE group mice by ultra-performance liquid chromatography-MS analysis. In addition, BSE intervention evidently increased the abundance of Alistipeds, Coriobacteriaceae UCG-002 and Bifidobacterium and decreased the abundance of Escheichia-Shinella, Lachnospiraceae others, Parabacteroides, Ruminococcaceae others and Turicibacter, which possibly promoted carbohydrate metabolism and short-chain fatty acid production. The present study aimed to elucidate the effect of BSE on colitis and found that BSE, as a novel food ingredient, has great potential for the improvement of colitis. © 2022 Society of Chemical Industry.

In vivo evaluation and mechanism prediction of anti-diabetic foot ulcer based on component analysis of Ruyi Jinhuang powder.

BACKGROUNDDiabetes is a metabolic disease with a high complication rate. Diabetic foot ulcers (DFUs) seriously affect the quality of life of patients. A total of 15%-20% of diabetic patients develop DFUs, which heal with difficulty over a long time and can result in amputation and disability. Traditional Chinese medicine has a unique effect in the treatment of skin ulcerative diseases. Ruyi Jinhuang powder (RHP) is one of the classic prescriptions in traditional Chinese medicine and is widely used in clinical practice.AIMTo verify the ability of RHP to promote wound healing by electron microscopy analysis in animal models and hematoxylin-eosin (HE) staining. The effective components of RHP were extracted and identified by gas chromatography-mass spectrometry (GC-MS), and the obtained chemical components were analyzed by network pharmacology methods to predict its therapeutic mechanism.METHODSSprague Dawley rats were injected with streptozotocin to establish the DFU model. HE staining was used to observe the wound tissue under an electron microscope. The chemical constituents of RHP were extracted first by supercritical fluid extraction and alcohol extraction, and then, GC-MS and ultra-performance liquid chromatography–MS were used to separately identify the chemical constituents. In addition, the "herb-component-target" link was established through the Traditional Chinese Medicine Systems Pharmacology database to obtain the target information, and the molecular docking of important components and key targets was performed in Discovery Studio software. Cytoscape software was used to visualize and analyze the relationship between the chemical composition, targets and Traditional Chinese Medicine network.RESULTSRHP promoted DFU healing in rats by affecting fibroblasts and nerve cells. A total of 89 chemical components were obtained by GC-MS. Network pharmacological analysis revealed that RHP was associated with 36 targets and 27 pathways in the treatment of DFU, of which the important components were luteolin, trans caryophyllene, ar-turmerone, palmitic acid, methyl palmitate, gallic acid, demethoxycurcumin, berberine, and rheic acid. The key targets were posttranscriptional silencing, topoisomerase II alpha, muscarinic acetylcholine receptor M2, interleukin 6, tumor necrosis factor and retinoic X receptor alpha, and the key pathways were the phosphoinositide 3-kinase-protein kinase B signaling pathway, neuroactive ligand–receptor interactions, and the forkhead box O signaling pathway.CONCLUSIONOur results indicated that RHP may play a role in the treatment of DFU through these target pathways by affecting insulin resistance, altering the nervous system and immune system, participating in inflammatory responses and regulating cell proliferation, differentiation and apoptosis through other specific mechanisms.

Evaluation of the effects of notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF (CNS) on the activities of cytochrome P450 enzymes using a cocktail method in rats.

Notoginseng total saponins (NS), safflower total flavonoids (SF), and the combination of NS and SF, namely CNS, are used for the treatment of cardiovascular diseases in clinic. This study developed a cocktail assay involving seven cytochrome P450 (CYP) enzymes to elucidate the effect of NS, SF, and CNS on CYP enzymes and to explore the synergistic effect of CNS in terms of CYP enzymes. Ultra-performance liquid chromatography-MS and reverse-transcription polymerase chain reaction were applied to detect the activities and mRNA expression levels of CYP enzymes. SF exhibited inhibitory effects on CYP1A2, 2B1, 2E1, and 2C11 and induction effects on CYP2C19 and 2D4. NS exhibited induction effects on CYP1A2, 2B1, 2E1, 2C11, 2C19, and 2D4. CNS exhibited induction effects on CYP1A2, 2B1, 2E1, 2C19, and 2D4 and inhibitory effects on CYP3A1 in vivo. Moreover, mRNA expression results were consistent with pharmacokinetic results. Potential herb-drug interactions should be studied closely when SF, NS, or CNS with clinical drugs are metabolized by CYP1A2, 2B1, 2E1, 2C11, 2C19, 2D4, and 3A1. CNS could change the inhibition or induction effects of CYP compared to the NS group, which might be one of the causes for the synergistic effects of the combination of NS and SF.

Quantitative mass spectrometry imaging of amino acids with isomer differentiation in brain tissue via exhaustive liquid microjunction surface sampling–tandem mass tags labeling–ultra performance liquid chromatography–mass spectrometry

Mass spectrometry imaging (MSI) has been used for localization of various biomolecules in tissues, but it is still challenging to have absolute pixel-to-pixel quantitation of analytes and differentiation of isobaric ions by MSI. In this proof-of-concept study, we present a quantitative MSI method for amino acids with distinguishing their constitutional isomers by exhaustive liquid microjunction surface sampling (LMJSS)–tandem mass tags (TMT) labeling–ultra performance liquid chromatography (UPLC)–MS. TMT6 reagents were used to differentially isotopically label amino acids in extracts from 6 pixels of brain section, resulting in multiplexed analysis of 6 pixels and largely shortening the LC–MSI time. From the results, with TMT labeling, the MS signals of amino acids in brain tissue extract were enhanced by (3–141)-fold. The calibration curves of TMT-labeled amino acids had good linearity in both MS1 and MS2 detection, which is essential for quantitation. A new extraction solvent for LMJSS (10% hexafluoroisopropanol-40% methanol-0.5% acetic acid-water) was developed to improve the extraction efficiencies of polar amino acids from brain tissue. The results showed that the extraction efficiencies of amino acids from different tissue regions were in the range of 75–110% with the new solvent, which made LMJSS an exhaustive sampling. Due to the complete extraction of amino acids from tissue, TMT0-labeled amino acid standards were directly added into the extracts for absolute quantitation. Finally, UPLC–MS was coupled with LMJSS to successfully separate the isobaric labeled amino acids in each pixel, allowing separate imaging of them. The imaging results of amino acid standard pattern demonstrate 500 µm spatial resolution of the MSI method. The brain tissue imaging results showed that the new method enabled quantitative MSI of 11 amino acids including three pairs of isomers, and the quantitation results were highly comparable and correlated with that by traditional bulk extraction–LC–MS method (correlation coefficient = 0.97, the slope of the correlation curve = 0.96).

Microflow UPLC and high-resolution MS as a sensitive and robust platform for quantitation of intact peptide hormones.

Aim: Recent advances in microflow ultra performance liquid chromatography (UPLC) systems offer higher sensitivity with robustness to meet the routine bioanalytical demands. Modern high-resolution mass spectrometers (HRMS) enable the development of highly selective methods with broad dynamic range. Results: The quantitative performances of tandem quadrupole MS and HRMS were comprehensively compared using seven intact peptide hormones up to 9.4kDa. Results show comparable performance between two platforms in sensitivity, accuracy and linearity. For some peptides, HRMS provided lower background interference. The benefit of increased sensitivity using microflow UPLC was also demonstrated. Conclusion: HRMS is a versatile platform capable of both basic characterization and reliable quantitation in complex matrices. Microflow UPLC provides lower LLOQs than conventional flow systems, even with less sample volume injected.

Metabolic perturbations associated with the consumption of a ketogenic medium-chain TAG diet in dogs with idiopathic epilepsy

Consumption of diets containing medium-chain TAG (MCT) has been shown to confer neuroprotective effects. We aim to identify the global metabolic perturbations associated with consumption of a ketogenic diet (medium-chain TAG diet (MCTD)) in dogs with idiopathic epilepsy. We used ultra-performance liquid chromatography-MS (UPLC-MS) to generate metabolic and lipidomic profiles of fasted canine serum and made comparisons between the MCTD and standardised placebo diet phases. We identified metabolites that differed significantly between diet phases using metabolite fragmentation profiles generated by tandem MS (UPLC-MS/MS). Consumption of the MCTD resulted in significant differences in serum metabolic profiles when compared with the placebo diet, where sixteen altered lipid metabolites were identified. Consumption of the MCTD resulted in reduced abundances of palmitoylcarnitine, octadecenoylcarnitine, stearoylcarnitine and significant changes, both reduced and increased abundances, of phosphatidylcholine (PC) metabolites. There was a significant increase in abundance of the saturated C17 : 0 fatty acyl moieties during the MCTD phase. Lysophosphatidylcholine (17 : 0) (P=0·01) and PC (17:0/20:4) (P=0·03) were both significantly higher in abundance during the MCTD. The data presented in this study highlight global changes in lipid metabolism, and, of particular interest, in the C17 : 0 moieties, as a result of MCT consumption. Elucidating the global metabolic response of MCT consumption will not only improve the administration of current ketogenic diets for neurological disease models but also provides new avenues for research to develop better diet therapies with improved neuroprotective efficacies. Future studies should clarify the involvement and importance of C17 : 0 moieties in endogenous MCT metabolic pathways.

Identification, Isolation and Determination of Biomarkers for Quality Control of Bush Tea (Athrixia phyllicoides).

Athrixia phylicoides, known as "bush tea", grows abundantly in South Africa. An infusion of the leaves is used as a beverage and to treat a multitude of health conditions. The aim of this study was to investigate the chemical variation within A.phylicoides and to identify characteristic compounds for quality control. Samples from 12 locations in South Africa were analysed using ultra-performance liquid chromatography. Hierarchical cluster analysis of the aligned ultra-performance liquid chromatography-time-of-flight mass spectrometry data indicated two groups on the resulting dendrogram, representing 48 samples. Five marker compounds, identified through visual inspection and the construction of a discriminant analysis model, were evident on the ultra-performance liquid chromatography-MS profiles. Four of these compounds were isolated and identified, three as hydroxy methoxyflavones and the fourth as a coumarate, using nuclear magnetic resonance spectroscopy. An ultra-performance liquid chromatography-photodiode array method was developed and validated for the determination of the marker compounds using the isolates as standards. The limits of detection for the four compounds ranged from 0.92 - 2.50 µg/mL. Their recoveries at three concentration levels (1.00, 10.0, and 100 µg/mL) were between 97.0 and 101%, while acceptable intra- and inter-day precision was obtained as reflected by percentage relative standard deviation values below 2.24%. The concentrations of all the marker compounds were found to be higher in samples corresponding to Group 1 of the dendrogram than in those from Group 2. This may be attributable to differences in altitude, climate, and some edaphic factors. Identification of these marker compounds will make a valuable contribution towards the quality control and sustainable commercialisation of bush tea.

Optimized extraction of anthocyanins from Reid Fruits’ Prunus avium ‘Lapins’ cherries

The influence of process parameters on the extraction of anthocyanins from the edible portion of fresh, sweet cherry were investigated. The optimal extraction time and temperature were determined as 90 min and 37 °C, respectively. A solvent/solid ratio of 10 mL/g using 100% acidified solvent resulted in the greatest anthocyanin yield. No significant difference was observed between the use of methanol or ethanol as the extraction solvent. Ultra Performance Liquid Chromatography-MS analysis of the extract identified four anthocyanins, with cyanidin-3-rutinoside and peonidin-3-rutinoside accounting for over 95% of the anthocyanin content, while cyanidin-3-glucoside and pelargonidin-3-rutinoside accounted for the remaining 5%. 244 mg/100 g fresh weight total anthocyanins were determined in the fresh cherries using the optimal extraction conditions.

Heat-stabilised rice bran consumption by colorectal cancer survivors modulates stool metabolite profiles and metabolic networks: a randomised controlled trial.

Rice bran (RB) consumption has been shown to reduce colorectal cancer (CRC) growth in mice and modify the human stool microbiome. Changes in host and microbial metabolism induced by RB consumption was hypothesised to modulate the stool metabolite profile in favour of promoting gut health and inhibiting CRC growth. The objective was to integrate gut microbial metabolite profiles and identify metabolic pathway networks for CRC chemoprevention using non-targeted metabolomics. In all, nineteen CRC survivors participated in a parallel randomised controlled dietary intervention trial that included daily consumption of study-provided foods with heat-stabilised RB (30 g/d) or no additional ingredient (control). Stool samples were collected at baseline and 4 weeks and analysed using GC-MS and ultra-performance liquid chromatography-MS. Stool metabolomics revealed 93 significantly different metabolites in individuals consuming RB. A 264-fold increase in β-hydroxyisovaleroylcarnitine and 18-fold increase in β-hydroxyisovalerate exemplified changes in leucine, isoleucine and valine metabolism in the RB group. A total of thirty-nine stool metabolites were significantly different between RB and control groups, including increased hesperidin (28-fold) and narirutin (14-fold). Metabolic pathways impacted in the RB group over time included advanced glycation end products, steroids and bile acids. Fatty acid, leucine/valine and vitamin B6 metabolic pathways were increased in RB compared with control. There were 453 metabolites identified in the RB food metabolome, thirty-nine of which were identified in stool from RB consumers. RB consumption favourably modulated the stool metabolome of CRC survivors and these findings suggest the need for continued dietary CRC chemoprevention efforts.

Induction of Apoptosis in Human Cancer Cells Through Extrinsic and Intrinsic Pathways by Balanites aegyptiaca Furostanol Saponins and Saponin-Coated SilverNanoparticles.

The aim of this investigation is to examine the anticancer activities of Balanites aegyptiaca fruit extract with its biogenic silver nanoparticles (AgNPs) against colon and liver cancer cells. B. aegyptiaca aqueous extract was fractionated according to polarity and by biosynthesized AgNP. The cytotoxicity of the extract, semi-purified fractions, and the AgNPs was examined on noncancerous cell lines. The safer fraction was subjected to ultra-performance liquid chromatography-MS to identify the major active constituents. The anticancer activities of the nontoxic doses of all the used treatments were tested against HepG2 and CaCo2 cells. The nontoxic dose of the B. aegyptiaca (0.63mg/ml) extract showed high anti-proliferative activities against HepG2 and CaCo2 with a percentage of 81 and 77%, respectively. The butanol fraction was safer than the other two fractions with 46.3 and 90.35% anti-proliferative activity against Caco2 and HepG2 cells, respectively. The nontoxic dose of AgNPs (0.63mg/ml) inhibits both HepG2 and Caco2 cells with a percentage of 84.5 and 83.4%, respectively. In addition, AgNPs regulate the expression of certain genes with folding higher than that of crude extract. Saponin-coated AgNPs showed great abilities to select the most anticancer ingredient(s) from the B. aegyptiaca extract with a more safety pattern than the polarity gradient fractionation.

An overview of recent developments in metabolomics and proteomics – phytotherapic research perspectives

ABSTRACTIn recent years, medicinal plants have gained much attention as potential source of bioactives for the development of novel herbal drugs for primary healthcare. However, phytotherapeutic mechanisms of action of phytomedicines need to be explored comprehensively. Out of the available strategies for the said purpose, metabolomics is one, which results in the production of inclusive metabolite profiles providing a clear understanding of diagnostic changes in the levels of metabolites, leading to therapeutic monitoring of drug targets through the elucidation of metabolomic pathways. On the other hand, proteomics is also a powerful strategy to deal with systematic protein expression analysis and analyze different biomarkers compared to metabolomics during drug treatment. Currently, 1H nuclear magnetic resonance spectroscopy, mass spectrometry (MS), Fourier transform infrared spectroscopy, gas chromatography equipped with mass spectrometry, liquid chromatography coupled with MS and ultra-performance liquid chromatography-MS coupled with multivariate statistical techniques, such as principle component analysis, partial least square and orthogonal-partial least square are among the widely applicable analytical tools for metabolite profiling, whereas two-dimensional electrophoresis and matrix-assisted laser desorption ionization time-of-flight -MS are the most accepted analytical methods for proteomic biomarker investigation. The present review summarizes the recent developments and perspectives of the biomarker investigation strategies so as to elaborate their imperative role for sustainable herbal drug developments.

Implementation of a single quad MS detector in high-throughput transdermal research of plant extracts

In this study, a new type of single quadrupole mass spectrometric detector was implemented in transdermal research. The local skin pharmacokinetic properties of the plant N-alkylamides (NAAs) pellitorine and anacycline, present in an Anacyclus pyrethrum extract, and spilanthol, present in a Spilanthes acmella extract were investigated. This single quad MS detection method showed great advantages compared to the traditional UV detector. The NAAs could be identified and quantified in the samples with an ultra performance liquid chromatography (UPLC)–single quad MS detection system, even if they were not separated, which is a requirement when using an UV–detector. Another advantage of the UPLC–MS system is that lower limit of detection values could be obtained allowing a more accurate and precise determination of the experimental lag time in the in vitro skin permeation experiments. To conclude, this single quad MS detector coupled to UPLC is a useful analytical tool with improved performance compared to high performance liquid chromatography (HPLC)–UV for biomedical-pharmaceutical purposes in transdermal research.

Determination of perfluorocompounds in popcorn packaging by pressurised liquid extraction and ultra-performance liquid chromatography–tandem mass spectrometry

The development and characterisation of a method based on reverse-phase ultra-performance liquid chromatography (UPLC) coupled to a quadrupole-time of flight mass spectrometer (Q-TOF-MS) with negative electrospray ionisation (ESI) to determine perfluorinated compounds (PFCs) in packaging is presented in this paper. Analytes were quantitatively recovered from packaging with methanol in only one PLE cycle of 6min at 100°C. The UPLC allowed the successful separation of the studied PFCs in less than 4min. The whole method presented good precision, with RSDs below 8%, LODs from 0.6 to 16ngg−1; and excellent recovery values, around 100% in all cases, were achieved. The PLE–UPLC–MS method was applied to the analysis of popcorn packaging for microwave cooking. Besides the most commonly studied PFCs: PFOA and PFOS, the presence of other perfluorocarboxylic acids (PFCAs) in popcorn packaging is evidenced in this work.

Simple and rapid analysis of amatoxins using UPLC–MS–MS

Amatoxins, such as α-amanitin and β-amanitin, are highly toxic bicyclic octapeptides present in Amanita mushrooms. We present a simple and rapid sensitive determination of α-amanitin, β-amanitin, and phalloidin in body fluids (serum, plasma, and urine) using an ultraperformance liquid chromatography (UPLC)–tandem mass spectrometry (MS–MS) system. After extraction from serum, plasma, and urine samples by an Oasis HLB column, the three compounds were subjected to an UPLC–MS–MS system with an electrospray ionization interface. All three compounds were completely separated within 4.5 min. The calibration curves for serum, plasma, and urine samples showed good linearities in the range of 2–420 ng/ml, using virginiamycin B as internal standard. Their detection limits were as low as 0.5–1.5 ng/ml. The present method was validated; the coefficients of variation (CV) were: intraday for serum, plasma, and urine samples, less than 9.9, 13.2, and 11.5 %, respectively; interday for serum, plasma, and urine samples, less than 15.0, 10.4, and 15.3 %, respectively. The influence of matrix effects, especially in urine, was observed in human and rat samples; however, the difference in matrix effects between individuals were low. Therefore, each calibration curve was prepared for each biological specimen type. Furthermore, we succeeded in determining the compounds in rat urine samples, obtained 6 or 24 h after intraperitoneal administration. The present method is applicable in forensic and clinical toxicology because of its simplicity and rapidness with high sensitivity.

Ultra performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometric procedure for qualitative and quantitative analyses of nortriterpenoids and lignans in the genus Schisandra

An ultra performance liquid chromatography (UPLC) coupled with quadrupole time-of-flight mass spectrometry (QTOF-MS) procedure is designed for the first simultaneous analysis of nortriterpenoids and lignans in Schisandra samples. The method consists of three individual mass spectrometric experiments, including the full scan MS, MS/MS experiment and in-source collision induced dissociation (CID) MS/MS, which enable the identification of diagnostic fragmentation pathways of nortriterpenoids and lignans. As such, a total of 6 nortriterpenoids and 10 lignans were unequivocally identified, and one nortriterpenoid and 20 lignans were tentatively identified from different Schisandra samples within 12.5 min. In addition, 6 nortriterpenoids and 10 lignans were quantified in 48 samples of S. chinensis and S. sphenanthera using an extract ion chromatogram (XIC) of the full scan MS experiment. Dataset obtained from UPLC–MS was processed with principal component analysis (PCA) and orthogonal partial least squared discriminant analysis (OPLS-DA) to compare the difference between the two Schisandra species.

Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry

Metabolism has an essential role in biological systems. Identification and quantitation of the compounds in the metabolome is defined as metabolic profiling, and it is applied to define metabolic changes related to genetic differences, environmental influences and disease or drug perturbations. Chromatography-mass spectrometry (MS) platforms are frequently used to provide the sensitive and reproducible detection of hundreds to thousands of metabolites in a single biofluid or tissue sample. Here we describe the experimental workflow for long-term and large-scale metabolomic studies involving thousands of human samples with data acquired for multiple analytical batches over many months and years. Protocols for serum- and plasma-based metabolic profiling applying gas chromatography-MS (GC-MS) and ultraperformance liquid chromatography-MS (UPLC-MS) are described. These include biofluid collection, sample preparation, data acquisition, data pre-processing and quality assurance. Methods for quality control-based robust LOESS signal correction to provide signal correction and integration of data from multiple analytical batches are also described.

Rapid separation and characterization of active flavonolignans of Silybum marianum by ultra-performance liquid chromatography coupled with electrospray tandem mass spectrometry

Ultra-performance liquid chromatography (UPLC) interfaced with the electrospray ionization (ESI) tandem mass spectrometer (MS n ) was developed for the simultaneous determination of silychristins A ( 1) and B ( 2), silydianin ( 3), silybins A ( 4) and B ( 5), and isosilybins A ( 6) and B ( 7), major bioactive flavonolignans in silymarin, a herbal remedy derived from the milk thistle Silybum marianum. In this study, the seven major active flavonolignans including the diastereomers 1/ 2, 4/ 5, and 6/ 7 were completely separated using UPLC with an ACQUITY UPLC C 18 column and a MeOH/water/formic acid mobile phase system. The collision-induced dissociation (CID) MS n spectra of these flavonolignans were studied systematically using ESI-MS. The results with the present methodology show that UPLC–MS n can be useful for general screening of active natural products from plant extracts and for the specific quality control of silymarin.

Chemometric analysis of urine fingerprints acquired by liquid chromatography‐mass spectrometry and capillary electrophoresis: Application to the schistosomiasis mouse model

Urine fingerprints from Schistosoma mansoni infected and control animals were acquired with ultra performance liquid chromatography-MS (UPLC-MS) and compared with the urine fingerprints obtained by CE by applying the same set of multivariate analysis tools. Principal component analysis of the aligned data provided a time trajectory where the infection was observed after 30 days with UPLC-MS and CE. Two main markers describing infected and control, respectively - phenyl acetyl glycine (PAG) and hippurate - were selected to illustrate the use of orthogonal partial least-square discriminant analysis in determining the discriminatory confidence. PAG was found to be significantly related to the disease (high covariance and correlation), whereas hippurate was found to be nonsignificant as an indicator. Orthogonal partial least-square discriminant analysis models were validated for sensitivity and specificity. Multivariate data analysis derived from two different detection systems showed that CE-UV and UPLC-MS found equivalent results. This work gives additional mechanistic insight into the progress of the S. mansoni infection; the biochemical role and specificity of PAG as a biomarker is yet to be determined.

Global metabolic profiling procedures for urine using UPLC–MS

The production of 'global' metabolite profiles involves measuring low molecular-weight metabolites (<1 kDa) in complex biofluids/tissues to study perturbations in response to physiological challenges, toxic insults or disease processes. Information-rich analytical platforms, such as mass spectrometry (MS), are needed. Here we describe the application of ultra-performance liquid chromatography-MS (UPLC-MS) to urinary metabolite profiling, including sample preparation, stability/storage and the selection of chromatographic conditions that balance metabolome coverage, chromatographic resolution and throughput. We discuss quality control and metabolite identification, as well as provide details of multivariate data analysis approaches for analyzing such MS data. Using this protocol, the analysis of a sample set in 96-well plate format, would take ca. 30 h, including 1 h for system setup, 1-2 h for sample preparation, 24 h for UPLC-MS analysis and 1-2 h for initial data processing. The use of UPLC-MS for metabolic profiling in this way is not faster than the conventional HPLC-based methods but, because of improved chromatographic performance, provides superior metabolome coverage.