Search

Recent studies have shown a strong correlation between ambient fine particulate matter (PM 2.5 ) exposure and diabetes risk, including abnormal lipid accumulation and systemic insulin resistance (IR). Hawthorn total flavonoids (HF) are the main groups of active substances in Hawthorn, which showed anti-hyperlipidemic and anti-hyperglycemic effects. Therefore, we hypothesized that HF may attenuate PM 2.5 -induced IR and abnormal lipid accumulation. Female C57BL/6 N mice were randomly assigned to the filtered air exposure (FA) group, concentrated PM 2.5 exposure (PM) group, PM 2.5 exposure maintained on a low-dose HF diet (LHF) group, and PM 2.5 exposure maintained on a high-dose HF diet (HHF) group for an 8-week PM 2.5 exposure using a whole-body exposure device. Body glucose homeostasis, lipid profiles in the liver and serum, and enzymes responsible for hepatic lipid metabolism were measured. We found that exposure to PM 2.5 impaired glucose tolerance and insulin sensitivity. In addition, triacylglycerol (TAG) in serum elevated, whereas hepatic TAG levels were decreased after PM 2.5 exposure, accompanied by inhibited fatty acid uptake, lipogenesis, and lipolysis in the liver. HF administration, on the other hand, balanced the hepatic TAG levels by increasing fatty acid uptake and decreasing lipid export, leading to alleviated systemic IR and hyperlipidemia in PM 2.5 -exposed mice. Therefore, HF administration may be an effective strategy to protect against PM 2.5 -induced IR and metabolic abnormalities of lipids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Rapid advances in front-end separation approaches and analytical technologies have accelerated the development of lipidomics, particularly in terms of increasing analytical coverage to encompass an expanding repertoire of lipids within a single analytical approach. Developments in lipid pathway analysis, however, have somewhat lingered behind, primarily due to (1) the lack of coherent alignment between lipid identifiers in common databases versus that generated from experiments, owing to the differing structural resolution of lipids at molecular level that is specific to the analytical approaches adopted by various laboratories; (2) the immense complexity of lipid metabolic relationships that may entail head group changes, fatty acyls modifications of various forms (e.g. elongation, desaturation, oxidation), as well as active remodeling that demands a multidimensional, panoramic view to take into account all possibilities in lipid pathway analyses. Herein, we discuss current efforts undertaken to address these challenges, as well as alternative form of "pathway analyses" that may be particularly useful for uncovering functional lipid interactions under different biological contexts. Consolidating lipid pathway analyses will be indispensable in facilitating the transition of lipidomics from its prior role of phenotype validation to a hypothesis-generating tool that uncovers novel molecular targets to drive downstream mechanistic pursuits under biomedical settings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Aims: The depot-specific differences in lipidome of visceral adipose tissue (VAT) and subcutaneous adipose tissue (SAT) reflect heterogeneity of white adipose tissue (WAT), which plays a central role in its distinct response to outside stimuli. However, the detailed lipidome of depot-specific WAT is largely unknown, especially the minor constitutes including phospholipid and sphingolipid., Materials and Methods: To investigate this field, we applied a high-coverage targeted lipidomics approach of VAT and SAT in male C57BL/6J mice to compare the basal level of their lipid profiles. Applying microarray and quantitative real-time polymerase chain reaction, we analyzed the transcriptome of twodepot-specific WAT and verified the differences in individual genes., Key Findings: In total, 342 lipid species from 19 lipid classes were identified. Our results showed the composition of TAG and FFA were different in length of chain and saturation. Interestingly, low abundance phospholipid, sphingolipid and cardiolipin were significantly higher in SAT. Lipid correlation network analysis vindicated that TAG and phospholipid formed distinct subnet and had more connections with other lipid species. Enriched ontology analysis of gene screened from LIPID MAPS and microarray suggested the differences were mainly involved in lipid metabolism, insulin resistance and inflammatory response., Significance: Our comprehensive lipidomics and transcriptomics analyses revealed differences in lipid composition and lipid metabolism of two depot-specific WAT, which would offer new insights into the investigation of heterogeneity of visceral and subcutaneous white adipose tissue., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author Bowen Li was employed by company LipidALL Technologies Ltd. All other authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Uncoupling protein 1 (UCP1) plays a key role in nonshivering thermogenesis and is involved in the pathogenesis of obesity. In a previous study, we generated adipocyte-specific UCP1 knock-in (UCP1-KI) pigs, which exhibited improved thermoregulatory ability and decreased fat deposition. To investigate whether UCP1 knock-in alters the lipid composition of adipose tissues, lipidomics of inguinal subcutaneous white adipose tissue (iWAT) and backfat from 6-month-old cold-treated UCP1-KI pigs and wild-type (WT) pigs were profiled. In addition, genome-wide RNA-sequencing of iWAT was performed to further study the genetic basis for lipid alterations. The results showed that iWAT and backfat from UCP1-KI pigs exhibited distinct lipidomic profiles, as the mild lipid alteration was observed in backfat of UCP1 knock-in pigs. Inguinal WAT from UCP1-KI pigs contained significantly decreased total triacylglycerol (p < 0.05), together with the downregulation of genes involved in fatty acid metabolism, suggesting the decreased lipogenesis in iWAT of UCP1-KI pigs. Significantly increased levels of total sphingolipids (p<0.05) were also observed in iWAT from UCP1-KI pigs. Notably, two mitochondrial-specific lipid species, cardiolipin CL72:8 (18:2) and CL74:9 (18:2), were found to be dramatically increased in iWAT from UCP1-KI pigs, suggesting enhanced mitochondrial function. This observation was further supported by the significant upregulation of numerous mitochondrial-related genes and significantly increased number of large mitochondria and mitochondrial cristae in iWAT of UCP1-KI pigs. Taken together, these data illustrate the specific role of UCP1 in lipid metabolism of fat tissues in pigs and provide new data for characterization of fat traits in UCP1-KI pigs., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Lipid homeostasis is important for executing normal cellular functions and maintaining physiological conditions. The biophysical properties and intricate metabolic network of lipids underlie the coordinated regulation of different lipid species in lipid homeostasis. To reveal the homeostatic response among different lipids, we systematically knocked down 40 lipid metabolism genes in Drosophila S2 cells by RNAi and profiled the lipidomic changes. Clustering analyses of lipids reveal that many pairs of genes acting in a sequential fashion or sharing the same substrate are tightly clustered. Through a lipid-gene regulatory network analysis, we further found that a reduction of triacylglycerol (TAG) is associated with an increase of phosphatidylinositol (PI) and lysophosphatidylinositol (LPI) or a reduction of hexosyl-ceramide (HexCer) and hydroxylated hexosyl-ceramide (OH-HexCer). Importantly, negative coregulation between TAG and LPI/PI, and positive coregulation between TAG and HexCer, were also found in human Hela cells. Together, our results reveal coregulations of TAG with PI/LPI and with HexCer in lipid homeostasis., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Development of rapid metabolomic methods poised for pathway discovery is expected to facilitate the identification of therapeutic candidates in the metabolomic approach to translational medicine. Using sphingolipid homeostasis as a prototype, we present herein an integrated method to facilitate a fast interrogation of altered sphingolipid (and phospholipid) metabolism associated with perturbed endolysosomal functions in mammalian systems. Constructed upon high performance liquid chromatography coupled to mass spectrometry, this method allows semi-quantitative measurements of more than 300 individual species within 20 min. The method was applied to investigate cardiac- and neural-specific developmental changes in sphingolipid regulation from the postnatal stage to reproductive senescence in mice, revealing that endogenous lysobisphosphatidic acids and specific complex glycosphingolipids are tightly co-regulated to foster concerted reductions in sphingolipid levels at distinct stages of postnatal development. Our lipidomic data suggest that such changing regulatory patterns in sphingolipid homeostasis is attributed to differential endolysosomal degradation of complex sphingolipids, which may be critical in ensuring efficient sphingolipid catabolism and organismal health at each stage of postnatal development., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Accurate quantitation is prerequisite for the sustainable development of lipidomics via enabling its applications in various biological and biomedical settings. In this review, the technical considerations and limitations of existent lipidomics technologies, particularly in terms of accurate quantitation; as well as the potential sources of errors along a typical lipidomic workflow that could ultimately give rise to quantitative inaccuracies will be addressed. Furthermore, the pressing need to exercise stricter definitions of terms and protocol standardization pertaining to quantitative lipidomics will be critically discussed, as quantitative accuracy may substantially impact upon the persevering development of lipidomics in the long run. This article is part of a Special Issue entitled: BBALIP&#95;Lipidomics Opinion Articles edited by Sepp Kohlwein., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Lipid droplet (LD) accumulation in hepatocytes is a typical character of steatosis. Hepatitis C virus (HCV) infection, one of the risk factors related to steatosis, induced LD accumulation in cultured cells. However, the mechanisms of which HCV induce LD formation are not fully revealed. Previously we identified cytosolic phospholipase A2 gamma (PLA2G4C) as a host factor upregulated by HCV infection and involved in HCV replication. Here we further revealed that PLA2G4C plays an important role in LD biogenesis and refined the functional analysis of PLA2G4C in LD biogenesis and HCV assembly. LD formation upon fatty acid and HCV stimulation in PLA2G4C knockdown cells was impaired and could not be restored by complementation with PLA2G4A. PLA2G4C was tightly associated in the membrane with the domain around the amino acid residues 260-292, normally in ER but relocated into LDs upon oleate stimulation. Mutant PLA2G4C without enzymatic activity was not able to restore LD formation in PLA2G4C knockdown cells. Thus, both the membrane attachment and the enzymatic activity of PLA2G4C were required for its function in LD formation. The participation of PLA2G4C in LD formation is correlated with its involvement in HCV assembly. Finally, PLA2G4C overexpression itself led to LD formation in hepatic cells and enhanced LD accumulation in the liver of high-fat diet (HFD)-fed mice, suggesting its potential role in fatty liver disease., (Copyright © 2017. Published by Elsevier B.V.)

Despite its importance as the leading cause of vascular dementia, the primary pathogenic mechanisms in subcortical ischemic vascular dementia (SIVD) have remained elusive. Because of the lack of approved therapeutic agents for SIVD, there is a pressing need to identify novel therapeutic targets. Comparative lipidomic analyses of SIVD and mixed dementia (i.e., SIVD and Alzheimer's disease, MixD) may also confer new insights pertaining to the possible interaction between neurodegenerative and vascular mechanisms in the pathogenesis of dementia. Liquid chromatography coupled to mass spectrometry was used to comprehensively analyze the lipidomes of white and gray matter from the temporal cortex of nondemented controls, SIVD, and MixD subjects. Detailed molecular profiles highlighted the pathologic relevance of gray matter sphingolipid fatty acyl chain heterogeneity in dementia. In addition, the levels of sulfatides and lysobisphosphatidic acids were progressively increased in the temporal cortex gray matter from control to SIVD to MixD. White matter phospholipid profiles indicated possible adaptive mechanisms (i.e., increased unsaturation) to chronic ischemia in SIVD and elevated membrane degradation in MixD., (Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.)

Meibomian gland dysfunction (MGD) is a leading cause of evaporative dry eye and ocular discomfort characterized by an unstable tear film principally attributed to afflicted delivery of lipids to the ocular surface. Herein, we elucidated longitudinal tear lipid alterations associated with disease alleviation and symptom improvement in a cohort of MGD patients undergoing eyelid-warming treatment for 12 weeks. Remarkably, eyelid-warming resulted in stark reductions in lysophospholipids (P < 0.001 for lyso-plasmalogen phosphatidylethanolamine, lysophosphatidylcholine, and lysophosphatidylinositol), as well as numerous PUFA-containing diacylglyceride species in tears, accompanied by significant increases in several PUFA-containing phospholipids. These changes in tear lipidomes suggest that eyelid-warming leads to diminished activity of tear phospholipases that preferentially target PUFA-containing phospholipids. In addition, treatment led to appreciable increases (P < 0.001) in O-acyl-ω-hydroxy-FAs (OAHFAs), which are lipid amphiphiles critical to the maintenance of tear film stability. Longitudinal changes in the tear lipids aforementioned also significantly (P < 0.05) correlated with reduced rate of ocular evaporation and improvement in ocular symptoms. The foregoing data thus indicate that excess ocular surface phospholipase activity detrimental to tear film stability could be alleviated by eyelid warming alone without application of steroids and identify tear OAHFAs as suitable markers to monitor treatment response in MGD., (Copyright © 2014 by the American Society for Biochemistry and Molecular Biology, Inc.)

As current diagnostic markers for dry eye syndrome (DES) are lacking in both sensitivity and specificity, a pressing concern exists to develop activity markers that closely align with the principal axes of disease progression. In this study, a comprehensive lipidomic platform designated for analysis of the human tear lipidome was employed to characterize changes in tear lipid compositions from a cohort of 93 subjects of different clinical subgroups classified based on the presence of dry eye symptoms and signs. Positive correlations were observed between the tear levels of cholesteryl sulfates and glycosphingolipids with physiological secretion of tears, which indicated the possible lacrimal (instead of meibomian) origin of these lipids. Notably, we found wax esters of low molecular masses and those containing saturated fatty acyl moieties were specifically reduced with disease and significantly correlated with various DES clinical parameters such as ocular surface disease index, tear breakup time, and Schirmer's I test (i.e., both symptoms and signs). These structure-specific changes in tear components with DES could potentially serve as unifying indicators of disease symptoms and signs. In addition, the structurally-specific aberrations in tear lipids reported here were found in patients with or without aqueous deficiency, suggesting a common pathology for both DES subtypes.

The tear film covers the anterior eye and the precise balance of its various constituting components is critical for maintaining ocular health. The composition of the tear film amphiphilic lipid sublayer, in particular, has largely remained a matter of contention due to the limiting concentrations of these lipid amphiphiles in tears that render their detection and accurate quantitation tedious. Using systematic and sensitive lipidomic approaches, we validated different tear collection techniques and report the most comprehensive human tear lipidome to date; comprising more than 600 lipid species from 17 major lipid classes. Our study confers novel insights to the compositional details of the existent tear film model, in particular the disputable amphiphilic lipid sublayer constituents, by demonstrating the presence of cholesteryl sulfate, O-acyl-ω-hydroxyfatty acids, and various sphingolipids and phospholipids in tears. The discovery and quantitation of the relative abundance of various tear lipid amphiphiles reported herein are expected to have a profound impact on the current understanding of the existent human tear film model.

A rapid and sensitive method was developed for quantitative profiling of wax esters (WEs) in human tear lipidome. Individual WE species was separated by liquid chromatography and detected by electrospray ionisation mass spectrometry using specific multiple reaction monitoring (MRM) scanning. Palmitoyl palmitate and in-house synthesized wax esters (13)C18:1(oleic acid-1,2,3,7,8,9,10-(13)C7)C26:0 were used as internal standards for quantitation of WEs containing saturated and unsaturated fatty acids (FA), respectively. The limit of detection was approximately 70 nmol/L. The linearity range of the liquid chromatography (LC)-MRM detection for WEs was about three orders of magnitude. Quantitative analyses of 141 individual WE in the human tear lipidome demonstrated that species comprising FA18:1 and FA16:1 each accounted for 47.7% and 24.0% (molar%) of total WE, while fatty alcohols in WEs of human tears ranged from 17 carbons to 32 carbons with predominant species represented by C24, C25 and C26., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Apolipoprotein E (ApoE) is important in facilitating the transport of lipids (cholesterol, phospholipids, and sulfatides) and plays a fundamental role in normal lipid metabolism. High cholesterol levels increases the risk of developing Alzheimer's disease. In this study, we investigated the effects of a high-fat high cholesterol (HFHC) diet on brain lipid profiles in 95 young and aged APOE ε3 and ε4 knock-in mice to determine whether diet leads to altered brain levels of a number of glycerophospholipids, sphingolipids, cholesterol precursors, cholesterol, cholesterol oxidation products, and cholesterol esters. The results in this study revealed significant changes in lipid levels. The HFHC-enriched diet influenced the levels of cholesterol esters. A sharp increase in cholesterol ester levels, particularly in the aged APOE ε4 diet-enriched group, might be suggestive of abnormal acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT) activity and/or levels. Age exerts appreciable effects on the brain lipidome, especially with regard to polar lipid species., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The level of cholesterol in host cells has been shown to affect viral infection. However, it is still not understood why this level of regulation is important for successful infection. We have shown in this study that dengue virus infection was affected when the cholesterol intake in infected cells was disrupted using a cholesterol transport inhibitor, U18666A. The antiviral effect was found to result from two events: retarded viral trafficking in the cholesterol-loaded late endosomes/lysosomes and suppressed de novo sterol biosynthesis in treated infected cells. We also observed an additive antiviral effect of U18666A with C75, a fatty acid synthase inhibitor, suggesting dengue virus relies on both the host cholesterol and fatty acid biosynthesis for successful replication., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The human lipodystrophy gene product Berardinelli-Seip congenital lipodystrophy 2/seipin has been implicated in adipocyte differentiation, lipid droplet (LD) formation, and motor neuron development. However, the molecular function of seipin and its disease-causing mutants remains to be elucidated. Here, we characterize seipin and its mis-sense mutants: N88S/S90L (both linked to motoneuron disorders) and A212P (linked to lipodystrophy) in cultured mammalian cells. Knocking down seipin significantly increases oleate incorporation into triacylglycerol (TAG) and the steady state level of TAG, and induces the proliferation and clustering of small LDs. By contrast, overexpression of seipin reduces TAG synthesis, leading to decreased formation of LDs. Expression of the A212P mutant, however, had little effect on LD biogenesis. Surprisingly, expression of N88S or S90L causes the formation of many small LDs reminiscent of seipin deficient cells. This dominant-negative effect may be due to the N88S/S90L-induced formation of inclusions where wild-type seipin can be trapped. Importantly, coexpression of wild-type seipin and the N88S or S90L mutant can significantly reduce the formation of inclusions. Finally, we demonstrate that seipin can interact with itself and its mutant forms. Our results provide important insights into the biochemical characteristics of seipin and its mis-sense mutants, and suggest that seipin may function to inhibit lipogenesis.

The proteomic makeup of lipid droplets (LDs) is believed to regulate the function of LDs, which are now recognized as important cellular organelles that are associated with many human metabolic disorders. However, factors that help determine LD proteome remain to be identified and characterized. Here we analyzed the phospholipid and protein composition of LDs isolated from wild type (WT) yeast cells, and also from fld1Δ, cds1, and ino2Δ mutant cells which produce 'supersized' LDs. LDs of fld1Δ and WT cells exhibited similar phospholipid profiles, whereas LDs of cds1 and ino2Δ strains had a higher (cds1) or lower (ino2Δ) percentage of phosphatidylcholine than those of WT, respectively. Unexpectedly, the presence of most known LD resident proteins was greatly reduced in the LD fraction isolated from cds1 and ino2Δ, including neutral lipid hydrolases. Consistent with this result, mobilization of neutral lipids was seriously impaired in these two strains. Contrary to the reduction of LD resident proteins, the Hsp90 family molecular chaperones, Hsc82 and Hsp82, were greatly increased in the LD fractions of cds1 and ino2Δ strains without changes at the level of expression. These data demonstrate the impact of LD phopholipids and size on the makeup of LD proteome., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Direct measurement of various sterols in crude lipid extracts in a single experiment from limited biological samples is challenging. Current mass spectrometry (MS) based approaches usually require chemical derivatization before subjecting to MS analysis. Here, we present a derivatization-independent method for analyzing various sterols, including cholesterol and its congeners, using liquid chromatography and atmospheric pressure chemical ionization mass spectrometry. Based on the specific tandem mass spectrometry pattern of cholesterol, multiple reaction monitoring (MRM) transitions were used to quantify free cholesterol and its fatty acyl esters. Several cholesterol oxidation products could also be measured using the upfront liquid chromatography separation and specific MRM transitions. The method was validated alongside established enzymatic assays in measuring total cholesterol. As a proof of concept, we analyzed plasma sterols in rabbits administrated with a high cholesterol diet (HCD) which is a classical atherosclerotic model. Free cholesterol, cholesterol esters, 7-hydroxycholesterol, and 7-ketocholesterol were elevated in plasma of rabbits on HCD. This method could also serve as an excellent tool for quantitative analysis of other sterols such as ergosterol and sitosterol in other organisms beside mammalian. In Saccharomyces cerevisiae, our results indicated dramatic increases of the ratio of ergosterol esters to free ergosterol in both yeh2Δ and tgl1Δ cells, which are consistent with the function of the respective enzymes., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Here, we present an improved method for sensitive profiling of lipids in a single high-performance liquid chromatography-electrospray ionization-quadrupole time of flight mass spectrometry experiment. The approach consists of i) sensitive isocratic elution, which takes advantage of C18 column material that is resistant to increased pH values induced by piperidine, ii) chemometric alignment of mass spectra followed by differential analysis of ion intensities, and iii) semiquantitative analysis of extracted ion chromatograms of interest. A key advantage of this method is its wide applicability to extracts that harbor lipids of considerable chemical complexity. The method allows qualitative and semiquantitative analysis of fatty acyls, glycerophospholipids (such as glycerophosphatidylinositols, glycerophosphatidylserines, and glycerophosphatidylcholines in brain extracts), phosphatidylinositol mannosides, acylated glycerophospholipids, sphingolipids (including ceramides and gangliosides in brain extracts), and, for the first time with ESI, prenols and mycolic acids (MAs). MAs are targets in antimycobacterial therapy, and they play an important immunomodulatory role during host-pathogen interactions. We compared high-resolution mass spectra of MAs derived from Mycobacterium bovis Bacille Camette-Guérin during entry into nonreplicative conditions induced by oxygen deprivation (hypoxic dormancy). Although the overall composition is not drastically altered, there are pronounced differences in individual MAs. alpha-MAs accumulate during entry into dormancy, whereas a subpopulation of keto-MAs is almost entirely eliminated. This effect is reversed upon resuscitation of dormant mycobacteria. These results provide detailed chemical information with relevance to drug development and immunobiology of mycobacteria.

Ulam raja (Cosmos caudatus) is used traditionally for improving blood circulation. In this study, it was found that ulam raja had extremely high antioxidant capacity of about 2,400 mg l-ascorbic acid equivalent antioxidant capacity (AEAC) per 100 g of fresh sample. Antioxidant peaks in extract of ulam raja were firstly characterized using free radical spiking test through high performance liquid chromatography coupled with mass spectrometry (MS). Upon reaction with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals, intensities of antioxidant peaks will be significantly reduced. HPLC/MS(n) was further applied to elucidate the chemical structures of antioxidant peaks characterized in the spiking test. More than twenty antioxidants were identified in ulam raja, and their chemical structures were proposed. The major antioxidants in ulam raja were attributed to a number of proanthocyanidins that existed as dimers through hexamers, quercetin glycosides, chlorogenic, neo-chlorogenic, crypto-chlorogenic acid and (+)-catching. High content of antioxidants antioxidants contained in ulam raja could be partly responsible for its ability to reduce oxidative stress.

Major antioxidants of aqueous ethanol extract from Lady's Finger (Hibiscus esculentus Linn) were systematically investigated in this study. Firstly, high-performance liquid chromatography (HPLC) was applied to identify antioxidant peaks in a sample by spiking the sample extract with 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radical, which was prepared from manganese dioxide and ABTS. Secondly, in order to identify the elution period of major antioxidant peaks, the antioxidant capacities of different fractions from solid-phase extraction (SPE) were measured, and the chromatograms of fractions were also recorded. Lastly, multiple mass spectrometry (MS(n)) was used to elucidate the possible chemical structures of antioxidants, and nuclear magnetic resonance (NMR) was further applied for structure confirmation. The major antioxidant compounds in lady's finger were identified to be quercetin derivatives and (-)-epigallocatechin using HPLC-MS and HPLC-MS(n) (n = 2-4) techniques. It was found that about 70% of total antioxidant activity was contributed by four quercetin derivatives. The structures of major antioxidants, which were isolated by semi-preparative RP-HPLC with two tandem C18 columns, were further confirmed using UV-vis absorption spectroscopy and 13C NMR spectra. Quercetin 3-O-xylosyl (1''' --> 2'') glucoside, quercetin 3-O-glucosyl (1''' --> 6'') glucoside, quercetin 3-O-glucoside and quercetin 3-O-(6''-O-malonyl)-glucoside were first identified and characterized as major antioxidants in lady's finger.

Our previous study indicated that star fruit (Averrhoa carambola L.) is a very good source of natural antioxidants. However, it was still not clear which compounds were responsible for its antioxidant properties. The purpose of this study is to separate and identify compounds that contribute to total antioxidant activity in star fruit using HPLC and mass spectrometry (MS). HPLC coupled with a diode array detector (DAD) was used to characterise antioxidant peak in the juice or residue extract through spiking with free radicals. By analysing the antioxidant capacity and chromatograms of fractions from solid phase extraction, main antioxidants were attributed to phenolic compounds. The peaks were identified as L-ascorbic acid, (-)epicatechin and gallic acid in gallotannin forms. Other antioxidant peaks were further investigated using HPLC-ESI-MS-MS. Identification was confirmed with electronspray ionisation (ESI) MS-MS spectra of pure standards and singly-linked proanthocyanidins from pycnogenol. The major antioxidants were initially attributed to singly-linked proanthocyanidins that existed as dimers, trimers, tetramers and pentamers of catechin or epicatechin.

A high-performance liquid chromatographic (HPLC) separation method with photo-diode array detection has been developed for the simultaneous determination of organic acids and phenolic compounds in juices and drinks. The chromatographic analysis of organic acids and phenolic compounds was carried out after their elution with sulphuric acid solution (pH 2.5) and methanol from C18 stationary phase. The mobile phase employed was sulphuric acid solution working at a flow-rate of 0.35 ml min(-1) for the whole run, while methanol was linearly increased to 0.45 ml min(-1) from 15 to 75 min followed by a 5-min isocratic elution. Ten organic acid acids were eluted in 30 min and 21 phenolic compounds, which include phenolic acids and flavonoids, were eluted in the following 50 min. Target compounds were detected at 215 nm. The repeatability (n=3) and between day precision of peak area (n=3) were all within 5.0% RSD. The within-day repeatability (n=3) and between-day precision (n=10) of retention times were within 0.3 and 1.6% relative standard deviation (RSD), respectively. The accuracy of the method was confirmed with an average recovery ranging between 85 and 106%. The method was successfully used to measure a variety of organic acids and phenolic compounds in juices and beverages. This method could also be used to evaluate the authenticity, spoilage or micronutrient contents of juices.