Performance Liquid Chromatography-quadrupole Time Research Articles

Effects of beetroot (Beta vulgaris) preparations on the Maillard reaction products in milk and meat-protein model systems

The effects of beetroots (Beta vulgaris) on the formation of Maillard reaction (MR) products possessing health, nutritional and sensory implications were studied. The effect of dried beetroot juice on the formation of Nε-(carboxymethyl)lysine (CML) and Nε-(2-furoylmethyl)-L-lysine (furosine) was determined in a milk model system. Beetroot juice reduced furosine formation more than CML, inferring that betalain compounds present in the juice are more effective in reducing the formation of MR products in the early stage than in the advanced stage of MR. Beetroot water extract was fractionated on Sephadex LH20 and obtained three beetroot fractions were used to assess their effect on the formation of heterocyclic amines in a meat-protein model system. Beetroot fraction possessing the highest antioxidant capacity and containing the highest betalain content reduced the amounts of 2-amino-1-methyl-6-phenylimidazo-[4,5-b]-pyridine (PhIP), 2-amino-3,8-dimethyl-imidazo-[4,5-f]-quinoxaline (MeIQx), and 2-amino-3-methylimidazo-[4,5-f]-quinoline (IQ) by approximately 60, 77 and 87%, respectively. Beetroot preparations were characterized by ultra performance liquid chromatography quadrupole time of flight mass spectrometry (UPLC–Q-TOF–MS/MS). The antioxidant activities of beetroot preparations were also evaluated by 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) radical cation (ABTS+) scavenging capacity, oxygen radical absorbance capacity (ORAC) and total phenolic compound assays. Our findings could be useful for creating novel source of functional ingredients exerting anti-carcenogenic and antiglycation activities.

Matrix solid-phase dispersion extraction followed by high performance liquid chromatography-diode array detection and ultra performance liquid chromatography–quadrupole–time of flight–mass spectrometer method for the determination of the main compounds from Carthamus tinctorius L. (Hong-hua)

A simple and low-cost method based on matrix solid-phase dispersion (MSPD) extraction, HPLC separation, diode array detection and UPLC–Q–TOF–MS have been developed for the determination of Hydroxysafflor yellow A (HSYA), Kaempferol and other main compounds in Carthamus tinctorius. The experimental parameters that may affect the MSPD method, including dispersing sorbent, ratio of dispersing sorbent to sample, elution solvent, and volume of the elution solvent were examined and optimized. The optimized conditions were determined to be that silica gel was used as dispersing sorbent, the ratio of silica gel to sample mass was selected to be 3:1, and 10mL of methanol: water (1:3, v:v) was used as elution solvent. The highest extraction yields of the two compounds were obtained under the optimized conditions. The method showed good linearity (r2≥0.999 2) and precision (RSD≤3.4%) for HSYA and Kaempferol, with the limits of detection of 35.2 and 14.5ngmL−1, respectively. The recoveries were in the range of 92.62–101.7% with RSD values ranging from 1.5 to 3.5%. At the meanwhile, there were 21 compounds in the extraction by MSPD method were identified by TOF–MS method to improve the quality control for safflower. Comparing to ultrasonic and soxhlet methods, the proposed MSPD procedure was more convenient and less time-consuming with reduced requirements on sample and solvent amounts. The proposed procedure was applied to analyze four real samples that were collected from different localities.

Determination of N6-(4-hydroxybenzyl) adenine riboside in rat plasma by ultra performance liquid chromatography-quadrupole time of flight mass spectrometry

An ultra performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-QTOF-MS) method was developed and validated for the determination of N6-(4-hydroxybenzyl) adenine riboside and its pharmacokinetics in rat plasma. The chromatographic conditions were optimized. The separation was performed on an Agilent ZORBAX SB-C18 column(150 mm x 3 mm, 1.8 μm) with a gradient elution of 0.2% (v/v) formic acid aqueous solution and acetonitrile as the mobile phases at a flow rate of 0.35 mL/min. The detection was accomplished in positive mode with electrospray ionization (ESI) by UPLC-QTOF-MS, and 6-benzylamino purine was used as the internal standard (IS). The results showed that the linear range of calibration curve was 0.625-160 ng/mL for N6-(4-hydroxybenzyl) adenine riboside in rat plasma with the correlation coefficient more than 0.99. The recoveries were 88.41%-108.26%. The limit of detection was 0.1 ng/mL. The intra-day and inter-day precisions (RSDs) were less than 6%, and intra-day and inter-day accuracies (REs, RE = (measured concentration-spiked concentration)/spiked concentration x 100%) were less than ±15%. The method is rapid, sensitive and accurate for the quantitation of N6-(4-hydroxybenzyl) adenine riboside, which can be used for the study of pharmacokinetics of N6-(4-hydroxybenzyl) adenine riboside.

Tissue-specific metabolite profiling of Turmeric by using laser micro-dissection, ultra-high performance liquid chromatography-quadrupole time of fight-mass spectrometry and liquid chromatography-tandem mass spectrometry.

Curcuma longa L. is recognized for its therapeutic and culinary uses both in Ayurveda and traditional Chinese medicine and is considered to be a boon to mankind. It has been extensively studied for its benefits and still continues to be an important drug with continued potential for further exploration and research. We studied the tissue-specific distribution of secondary metabolites to establish the validity of the use of rhizome samples from India and China, as substitutes for each other, based upon their metabolite profiles and curcumin contents. Laser microdissection was used for the isolation of microscopic tissues, such as cork, cortex and leaf-trace vascular bundles from rhizomes. Metabolite profiling was carried out by ultra-high performance liquid chromatography-quadrupole time of fight-mass spectrometry and curcumin content was estimated by a method validated as per the Harmonized Tripartite Guidelines. The cortex and cork revealed the presence of a higher number of secondary metabolites than in the leaf-trace vascular bundles. The curcumin contents in rhizome samples from both the countries, estimated with the help of a precise and accurate validated method, were found to be comparable. Based on the results, we conclude that turmeric rhizomes grown in India and China are qualitatively and quantitatively indistinguishable and therefore can be used as substitutes. The developed method can be widely applied for microscopic identification, authentication and analysis of the distribution of phytoconstituents in other botanical species of interest or of species with a significant commercial and therapeutic value.

The rapid discovery and identification of physalins in the calyx of Physalis alkekengi L.var.franchetii (Mast.) Makino using ultra-high performance liquid chromatography–quadrupole time of flight tandem mass spectrometry together with a novel three-step data mining strategy

Physalins, uniquely discovered from genus physalis, showed significant bioactivities in many aspects. It is therefore very important for the exploration of natural resources rich of physalins. However, there is no efficient approach for rapid discovery and identification of this class of compounds due to their structural complexity. To address the issue, the fragmentation pathways and correspondingly fragmentation rules of physalins in negative MS/MS mode were thoroughly investigated in this study using seven physalin standards. As a result, diagnostic ions for the rapid screening of physalins and classification of different types of physalins were determined based on their MS/MS fragmentation patterns. On top of that, an integrated approach using UHPLC–QTOF-MS/MS together with a novel three-step data mining strategy was developed for the systematic analysis of physalins in complex samples. Consequently, 46 physalins including 20 novel ones were efficiently discovered and identified from the crude extracts of Ph. alkekengi calyx. The present study laid a foundation for future study of different parts of Ph. alkekengi and other physalis species with regard to rapid discovery of novel physalins. In addition, this study provided a base for establishing a quality control method of the raw materials of Ph. alkekengi according to the profile of physalins.

Rapid characterization of impurities in the bulk drug of nifedipine by high performance liquid chromatography-quadrupole time of fight mass spectrometry

Four impurities in the bulk drug of nifedipine were detected by means of high performance liquid chromatography coupled with quadrupole time of fight mass spectrometry (HPLC-QTOF MS). A Kromasil C18 HPLC column (250 mm x 4.6 mm, 5 microm) was used with methanol-water (60:40, v/v) as mobile phase. The column temperature was maintained at 35 degrees C with an ultraviolet (UV) detection wavelength of 235 nm for analysis. The eluates were detected with a UV detector and a quadrupole time of fight mass spectrometer in positive mode. The sample was infused into the mass spectrometer from the HPLC system through a T-junction with a splitting ratio of 3:1. The ion source temperature was set at 320 degrees C and the electrospray ionization (ESI) needle voltage was always set at 4 000 V. Nitrogen was used as the drying gas and nebulizer gas. The protonated molecular ions were selected for determining the structures, and their fragmentation pathways were deduced. Based on the fragmentation behavior of nifedipine, the structures of 3 impurities were identified and one of them was identified as 3-ethyl-5-methyl-2,6-dimethyl-4-(2-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate, which was an unknown impurity to the best of our knowledge. An other impurity was identified by the comparison of the retention time and tandem mass spectra with the standard substance. The four proposed structures were all confirmed by exact mass evidence. The results indicated that the described method can be effectively applied to perform the identifications in nifedipine and its impurities.

Identification of substituted cathinones: 3,4-Methylenedioxy derivatives by high performance liquid chromatography–quadrupole time of flight mass spectrometry

The potential of high performance liquid chromatography–high resolution mass spectrometry (HPLC–HRMS) for screening of synthetic cathinones in legal highs was examined. Samples were analysed by liquid chromatography coupled to hybrid quadrupole time-of-flight mass spectrometer (Q/TOF). Nanoelectrospray ionisation (nanoESI) was employed. MS and MS/MS spectra were acquired. Six 3,4-methylenedioxy derivatives: methylone, butylone, pentylone, MDPBP, MDPV and BMDP were detected and identified. The fragmentation pattern of 3,4-methylenedioxy derivatives in collision induced dissociation (CID) was derived and described, which will facilitate future screenings and identifications of new synthetic cathinones. For 3,4-methylenodioxy derivative cathinones the loss of neutral groups CH4O2, H2O, amines and imines is observed. The loss of water and the methylenedioxy group does not occur when cyclic amino group – pyrrolidynyl is present in the molecule. Phenyloxazole cations are formed when CH4O2 is lost. The formation of the metylenedioxybenzoyloxonium and allyldioxybenzoyloxonium ions is typical for 3,4-methylenodioxy derivatives, however, the formation of the former appears to be inhibited by the presence in the molecule of the group of atoms able to form very stable tropylium carbocation.

Metabolism studies of benzbromarone in rats by high performance liquid chromatography–quadrupole time of flight mass spectrometry

A high performance liquid chromatography–quadrupole time of flight mass spectrometry (HPLC–QTOF-MS) method was employed in investigation of benzbromarone metabolites in rat plasma, urine, feces and bile samples. Meanwhile, the metabolic pathways of benzbromarone in rats were discussed. The identification was achieved on a reversed-phase C18 column with mobile phase gradient method. The QTOF-MS was operated under full scan of MS or MS/MS in negative mode. The fragments were acquired by raising collision induced dissociation (CID) energy for speculating the structures of parent ions. According to the information from the chromatograms and mass spectra, 17 metabolites were obtained. Among them, the deoxidized phase I metabolites and an array of phase II metabolites—sulfate conjugates detected in the biological samples made the work more significant.

Novel software-based method to classify structurally similar compounds combined with high performance liquid chromatography–quadrupole time of flight mass spectrometry to identify complex components of herbal medicines

The components of herbal medicines (HMs) are usually extremely complex, belonging to hundreds of compound classes with diverse chemical and physical properties. Full characterization of HMs is hugely important in order to identify the individual chemical constituents and provide a first step toward determining which components are responsible for the therapeutic effects of a particular medical plant. In this study, a novel software-based approach was developed to classify structurally similar compounds, and this was combined with high performance liquid chromatography/quadrupole time-of-flight mass spectrometry (HPLC–QTOF-MS) to identify the individual components in an extract of Mentha haplocalyx. A total of 33 compounds were tentatively identified in samples of M. haplocalyx extract, including 9 new minor constituents reported for the first time. Semi-quantitative analysis of the extract sample was also carried out. Software validation and robustness tests were performed. The results of this study demonstrate the enormous potential of this strategy, using classification based on structural similarity together with HPLC–QTOF-MS, for the identification and quantification of complex components in HMs and related products.

Identification of ten new designer drugs by GC‐MS, UPLC‐QTOF‐MS, and NMR as part of a police investigation of a Danish Internet company

The ability of forensic laboratories to detect and identify unknown compounds is highly important since new, non-controlled designer drugs are appearing on the market with increasing frequency. In this study, the combined use of gas chromatography-mass spectrometry (GC-MS) and ultra performance liquid chromatography-quadrupole time of flight-mass spectrometry (UPLC-QTOF-MS) was used for screening of new unknowns. In one large seizure from a Danish Internet company, ten different drugs were identified. Several of the compounds were seized for the first time in Denmark. The GC-MS and UPLC-QTOF-MS analyses were supplemented by nuclear magnetic resonance (NMR) spectra for the structural elucidation of p-fluoroamphetamine, mephedrone (4-methylmethcathinone), flephedrone (4-fluoromethcathinone), PPP (α-pyrrolidinopropiophenone), MDPV (3,4-methylenedioxypyrovalerone), Bk-MBDB (2-methylamino-1-(3,4-methylenedioxyphenyl)butan-1-one), pFBT (3-(pfluorobenzoyl)-tropane), and JWH-073 (1-butyl-3-(1-naphthoyl)indol), whereas methylone (3,4-methylenedioxymethcathinone) and N-ethylcathinone matched electron impact-mass spectrometry (EI-MS) library spectra and therefore the screenings were considered sufficient. EI-MS spectra and the proposed main fragmentation patterns are presented as well as QTOF-MS exact masses and fragments and NMR chemical shifts. For the β-ketophenylethylamines (mephedrone, flephedrone, PPP, MDPV, Bk-MBDB, methylone, and N-ethylcathinone) some general fragmentation patterns observed in the EI-MS and QTOF-MS spectra are further discussed and compared to other β-ketophenylethylamines.

Urinary Metabolite Markers of Precocious Puberty

The incidence of precocious puberty (PP, the appearance of signs of pubertal development at an abnormally early age), is rapidly rising, concurrent with changes of diet, lifestyles, and social environment. The current diagnostic methods are based on a hormone (gonadotropin-releasing hormone) stimulation test, which is costly, time-consuming, and uncomfortable for patients. The lack of molecular biomarkers to support simple laboratory tests, such as a blood or urine test, has been a long standing bottleneck in the clinical diagnosis and evaluation of PP. Here we report a metabolomic study using an ultra performance liquid chromatography-quadrupole time of flight mass spectrometry and gas chromatography-time of flight mass spectrometry. Urine metabolites from 163 individuals were profiled, and the metabolic alterations were analyzed after treatment of central precocious puberty (CPP) with triptorelin depot. A panel of biomarkers selected from >70 differentially expressed urinary metabolites by receiver operating characteristic and logistic regression analysis provided excellent predictive power with high sensitivity and specificity for PP. The altered metabolic profile of the PP patients was characterized by three major perturbed metabolic pathways: catecholamine, serotonin metabolism, and tricarboxylic acid cycle, presumably resulting from activation of the sympathetic nervous system and the hypothalamic-pituitary-gonadal axis. Treatment with triptorelin depot was able to normalize these three altered pathways. Additionally, significant changes in the urine levels of 4-hydroxyphenylacetic acid, 5-hydroxyindoleacetic acid, indoleacetic acid, 5-hydroxytryptophan, and 5-hydroxykynurenamine in the CPP group suggest that the development of CPP condition may involve an alteration in symbiotic gut microbial composition.

Human Tear Fluid Lipidome: From Composition to Function

We have explored human aqueous tear fluid lipidome with an emphasis to identify the major lipids. We also address the physiological significance of the lipidome. The tears were analysed using thin layer chromatographic, enzymatic and mass spectrometric techniques. To emphasize the physiological aspect of the lipidome, we modelled the spreading of the non-polar tear fluid lipids at air-water interface in macroscopic scale with olive oil and egg yolk phosphatidylcholine. Based on enzymatic analysis the respective concentrations of choline-containing lipids, triglycerides, and cholesteryl esters were 48±14, 10±0, and 21±18 µM. Ultra performance liquid chromatography quadrupole time of flight mass spectrometry analysis showed that phosphatidylcholine and phosphatidylethanolamine were the two most common polar lipids comprising 88±6% of all identified lipids. Triglycerides were the only non-polar lipids detected in mass spectrometric analysis i.e. no cholesteryl or wax esters were identified. The spreading experiments show that the presence of polar lipids is an absolute necessity for a proper spreading of non-polar tear fluid lipids. We provide evidence that polar lipids are the most common lipid species. Furthermore, we provide a physiological rationale for the observed lipid composition. The results open insights into the functional role of lipids in the tear fluid and also aids in providing new means to understand and treat diseases of the ocular surface.

Serum and Urine Metabolite Profiling Reveals Potential Biomarkers of Human Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a common malignancy in the world with high morbidity and mortality rate. Identification of novel biomarkers in HCC remains impeded primarily because of the heterogeneity of the disease in clinical presentations as well as the pathophysiological variations derived from underlying conditions such as cirrhosis and steatohepatitis. The aim of this study is to search for potential metabolite biomarkers of human HCC using serum and urine metabolomics approach. Sera and urine samples were collected from patients with HCC (n = 82), benign liver tumor patients (n = 24), and healthy controls (n = 71). Metabolite profiling was performed by gas chromatography time-of-flight mass spectrometry and ultra performance liquid chromatography-quadrupole time of flight mass spectrometry in conjunction with univariate and multivariate statistical analyses. Forty three serum metabolites and 31 urinary metabolites were identified in HCC patients involving several key metabolic pathways such as bile acids, free fatty acids, glycolysis, urea cycle, and methionine metabolism. Differentially expressed metabolites in HCC subjects, such as bile acids, histidine, and inosine are of great statistical significance and high fold changes, which warrant further validation as potential biomarkers for HCC. However, alterations of several bile acids seem to be affected by the condition of liver cirrhosis and hepatitis. Quantitative measurement and comparison of seven bile acids among benign liver tumor patients with liver cirrhosis and hepatitis, HCC patients with liver cirrhosis and hepatitis, HCC patients without liver cirrhosis and hepatitis, and healthy controls revealed that the abnormal levels of glycochenodeoxycholic acid, glycocholic acid, taurocholic acid, and chenodeoxycholic acid are associated with liver cirrhosis and hepatitis. HCC patients with alpha fetoprotein values lower than 20 ng/ml was successfully differentiated from healthy controls with an accuracy of 100% using a panel of metabolite markers. Our work shows that metabolomic profiling approach is a promising screening tool for the diagnosis and stratification of HCC patients.

Analysis of Quinocetone and its Four Metabolites in Swine Liver by Ultra–performance Liquid Chromatography Quadrupole Time–of–flight Mass Spectrometry

A new method using ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC/ Q-TOF-MS) was developed for determination of quinocetone (QCT) and its major metabolites, 1–desoxyquinocetone (1– DQCT), 4–desoxyquinocetone (4–DQCT), 1,4–bisdesoxyquinocetone (DQCT) and methyl–3–quinoxaline–2–carboxylic acid (MQCA) in swine liver. Tissue samples were extracted by a two-step method. Firstly, all analytes except MQCA were extracted with ethyl acetate. Then a hydrochloric acid solution was added in the remained sediment for hydrolysis of combined MQCA. The MQCA in the acid extract was transferred to ethyl acetate by liquid-liquid extraction (LLE) approach. All the ethyl acetate extract were combined, evaporated, resolved in a phosphate buffer, and cleaned up using a Oasis MAX cartridges. The chromatographic separation of all the analytes was achieved in less than 5 min using UPLC. The identification and confirmation by accurate mass measurement and their different fragmentation pathways were performed on ESI-Q-TOF-MS (MS/MS). The quantitation was carried out in TOF mode using narrow window extracted ion chromatogram of each compound. In the range of 1-100 µg·kg–1, the calibration curve equations of MQCA, QCT, 4-DQCT, 1-DQCT and DQCT were y=0.7878x+10.49, y=4.656x+42.244, y=5.5825x+24.919, y=8.491x+21.195 and y=10.733x+160.72, respectively. And the relative coefficients of all calibration curves were higher than 0.98. The limit of detection and the limit of quantification were 1.4–4.8 µg·kg –1 and 4.6–15.9 µg·kg–1, respectively. The established method was validated for determination of incurred swine liver samples in an actual residue study.

Stilbene synthase gene transfer caused alterations in the phenylpropanoid metabolism of transgenic strawberry (Fragaria x ananassa).

The gene encoding stilbene synthase is frequently used to modify plant secondary metabolism with the aim of producing the self-defence phytoalexin resveratrol. In this study, strawberry (Fragaria×ananassa) was transformed with the NS-Vitis3 gene encoding stilbene synthase from frost grape (Vitis riparia) under the control of the cauliflower mosaic virus 35S and the floral filament-specific fil1 promoters. Changes in leaf metabolites were investigated with UPLC-qTOF-MS (ultra performance liquid chromatography-quadrupole time of flight mass spectrometry) profiling, and increased accumulation of cinnamate, coumarate, and ferulate derivatives concomitantly with a decrease in the levels of flavonols was observed, while the anticipated resveratrol or its derivatives were not detected. The changed metabolite profile suggested that chalcone synthase was down-regulated by the genetic modification; this was verified by decreased chalcone synthase transcript levels. Changes in the levels of phenolic compounds led to increased susceptibility of the transgenic strawberry to grey mould fungus.

Comparative study of an estradiol enzyme-linked immunosorbent assay kit, liquid chromatography–tandem mass spectrometry, and ultra performance liquid chromatography–quadrupole time of flight mass spectrometry for part-per-trillion analysis of estrogens in water samples

Estrogens have been often identified as the major contributors to the endocrine-disrupting activity observed in environmental waters. However, their analysis in these, sometimes very complex, matrices is still challenging due to the very low detection limits and the selectivity required for their reliable determination at the very low concentrations at which they are physiologically active. In this work, a polyclonal enzyme-linked immunosorbent assay (ELISA) kit for 17-β-estradiol analysis, high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) based on triple-quadrupole analyzer (QqQ), and a newly developed method based on ultra performance liquid chromatography–quadrupole time of flight mass spectrometry (UPLC–Q-TOF-MS) have been evaluated in terms of performance for the rapid screening, quantitative analysis, and unequivocal identification of some selected, environmentally relevant estrogens in different water matrices, including urban wastewater, river water, and ground water, after solid phase extraction. Compounds quantified and/or identified included the estrogens 17-β-estradiol, estrone, 17-α-ethynyl estradiol and estriol, and the isoflavones daidzein, genistein, and biochanin A. Except for a moderate overestimation using the ELISA kit, especially in the analysis of complex wastewater samples, results obtained by all the investigated techniques were in very good, general agreement. The instrumental sensitivity achieved increased in the order: UPLC–Q-TOF-MS < polyclonal ELISA kit < HPLC–MS/MS (QqQ). Direct analysis of water samples by using the ELISA kit permitted to reach a limit of detection of 2.5 ng L −1. However, using an appropriated sample pretreatment method detection limits at nanogram to picogram per liter levels can be obtained with all techniques and the risk for matrix effects is minimized. In terms of selectivity, both HPLC–MS/MS (QqQ) and UPLC–Q-TOF-MS show outstanding performance, but the latter allows, in addition, shorter analysis times (16 min vs. 45 min) and the identification of non-target, unknown compounds. The identification of unknown compounds is based on the accurate mass measurements for the precursor and product ions, that permit the elemental compositions calculation and the chemical structures to be identified searching against different databases.