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A selective and rapid method has been developed to determine 15 antibiotic residues (eight tetracyclines and seven quinolones) in milk samples by capillary zone electrophoresis coupled with quadrupole time-of-flight mass spectrometry (CZE-Q-TOF-MS). The use of this hybrid mass spectrometer allowed obtaining full scan and full MS/MS spectra for quantification/confirmation purposes in a single run. In addition, solid phase extraction (SPE) using the new Oasis PRiME HLB cartridge was proposed for the extraction, achieving excellent results in terms of sample throughput. The proposed method was validated using whole cow milk as representative matrix. Good linearity was obtained (R2>0.99) for all the studied compounds. The precision, expressed as relative standard deviation (%, RSD), at two concentration levels (50 and 100μgkg-1) was below 13%. Recoveries obtained from goat milk, whole cow milk and semi-skimmed cow milk, at two concentration levels, ranged from 76 to 106%, while limits of quantification ranged from 1.5 to 9.6μgkg-1, being lower than the established maximum residue limits in the European legislation. Matrix effect was negligible in all cases, showing that with this new SPE sorbent cleanest extracts were obtained with a minimum number of steps in the sample treatment. Thus, the proposed SPE-CZE-Q-TOF-MS method is suitable for multiclass multiresidue monitoring in different types of milk samples.

In this study, 21 flavonol O-glycoside standards including flavonol mono-, di-, tri- and tetra-O-glycosides have been systematically studied by ultra-high performance liquid chromatography-electrospray ionization-quadrupole time-of-flight mass spectrometry (UPLC-ESI-QTOF-MS/MS) in the negative ionization mode to analyze their fragmentation patterns. Here for the first time, the Z23− fragment (corresponding to the loss of C-2” terminal sugar moiety) was observed in MS/MS spectra of flavonol 3-O-triglycosides. The intensity ratio of [Y0-H] −/Y0− was proposed as a criterion to distinguish the interglycosidic 1 → 2 and 1 → 6 linkages in flavonol 3-O-diglycosyl-7-O-monoglycosides. The established fragmentation behaviors have been successfully applied to characterization of flavonol glycosides in Viola tianschanica. A total of 30 flavonoid glycosides including 3 flavonol mono-O-glycosides, 10 di-O-glycosides, 10 tri-O-glycosides, 4 tetra-O-glycosides and 3 flavone di-C-glycosides were identified or tentatively identified on the base of their UV profiles, MS/MS data and/or by comparing with reference substances. Among these 15 flavonoid glycosides were reported from V. tianschanica for the first time.

Ultrahigh-performance liquid chromatography–quadrupole-time of-flight mass spectrometry (UHPLC–Q-TOF-MS) was widely used in identification of complex ingredients in traditional Chinese herbs and herbal medicinal preparations for its excellent performance. Yangxinshi tablet, a Chinese compound herbal medicinal formula, has excellent efficacy for the clinical treatment of cardiovascular diseases, but its active ingredients are unclear. In this study, a rapid and sensitive UHPLC–Q-TOF/MS and secondary mass spectrometry (MS2) method were developed to characterize the comprehensive ingredients in Yangxinshi tablet and rat plasma after drug administration. And finally a total of 178 constituents in the Yangxinshi tablet were identified effectively, and 39 parent molecules in rat plasma were rapidly characterized by matching the Yangxinshi tablet chemical library established by ourselves. Of which, seven groups of isomers were further distinguished according to their MS2 spectra and fragmentation ions. F...

Acidic glycosphingolipids (AGSLs), which mainly consist of ganglioside and sulfatide moieties, are highly concentrated in the central nervous system. Comprehensive profiling of AGSLs has historically been challenging because of their high complexity and the lack of standards. In this study, a novel strategy was developed to comprehensively profile AGSLs using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry. Ganglioside isomers with different glycan chains such as GD1a/GD1b were completely separated on a C18 column for the first time to our knowledge, facilitated by the addition of formic acid in the mobile phase. A mathematical model was established to predict the retention times (RTs) of all theoretically possible AGSLs on the basis of the good logarithmic relationship between the ceramide carbon numbers of the AGSLs in the reference material and their RTs. A data set was created of 571 theoretically possible AGSLs, including the ceramide carbon numbers, RTs, and high-resolution quasi-molecular ions. A novel fast identification strategy was established for global AGSL profiling by comparing the high-resolution quasi-molecular ions and RTs of the tested peaks to those in the data set of 571 AGSLs. Using this strategy, 199 AGSL candidates were identified in rat brain tissue. MS/MS fragments were further collected for these 199 candidates to confirm their identity as AGSLs. This novel strategy was employed to profile AGSLs in brain tissue samples from control rats and model rats with bilateral common carotid artery (2-VO) cerebral ischemia. Forty AGSLs were significantly different between the control and model groups, and these differences were further interpreted.

Chimonanthus nitensOliv. (CNO), having been studied and developed as the tea beverages, tea raw materials and preparations, an unique species in China, have been extensively used for treating colds and influenza for centuries. In the present study, a method based on the modified mass defect filter (MDF) was firstly developed and validated for comprehensive profiling of coumarins in the different parts of CNO via ultra-performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). The five-point screening approach based on MDF and the visual isotopic ion technique could rapidly screen the interested precursor ions. The fragmentation behavior of coumarins was systematically investigated and a total of 42 coumarins including 27 potential new ones were unambiguously or tentatively identified in the CNO. Collectively, the results obtained in the present work may provide useful information for future utilization of CNO.

Si-Miao-Wan (SMW), a tradiational Chinese medicinal formula consisting of Atractylodis Rhizoma, Phellodendri Chinensis Cortex, Coicis Semen, and Achyranthis Bidentatae Radix, has been used for the treatment of gout and gouty arthritis for many years. In the present study, a liquid chromatography quadrupole-time-of-flight mass spectrometry (LC-Q-TOF/MS) method was established to identify the multiple constituents of SMW and its metabolites in rat biological samples after oral administration. A total of 48 compounds in SMW, including 21 alkaloids, 12 organic acids, 2 terpenes, 3 lactones, 2 phytosterols, and 8 other compounds, were tentatively characterized with the diagnostic-ion filtering strategy. Based on the diagnostic ions applied to identify compounds in SMW, 28 prototype compounds and 10 metabolic compounds were detected in the biological samples. This was the first comprehensive drug metabolism investigation of SMW in rats. The developed method could be a useful means for identifying the multi-components in SMW and the metabolic components. The results may help explore the possible metabolic processes and mechanism of action for SMW in vivo.

Over the past decades, mass spectrometry technologies have been developed to obtain mass accuracies of one ppm or less. Of the newly developed technologies, quadrupole time-of–flight mass spectrometry (Q-TOF–MS) has emerged as being well suited to routine and high-throughput analyses of pharmaceuticals. Dietary supplements and functional foods have frequently been found to be contaminated with pharmaceuticals. In our continuous efforts to develop methodologies to protect public health against adulterated dietary supplements, we have constructed a mass spectral database for 21 H1-antihistamines encountered as adulterants by using liquid chromatography-electrospray ionization (LC-ESI)/Q-TOF–MS, and have proposed their possible collision-induced dissociation pathways. This database will be very useful for the rapid and accurate detection of H1-antihistamines (known) and their analogues (unknown) illegally added to dietary supplements as well as in other sample matrices.

This study reports an applicable analytical strategy of comprehensive identification and structure characterization of target components from Gelsemium elegans by using high-performance liquid chromatography quadrupole time-of-flight mass spectrometry (LC-QqTOF MS) based on the use of accurate mass databases combined with MS/MS spectra. The databases created included accurate masses and elemental compositions of 204 components from Gelsemium and their structural data. The accurate MS and MS/MS spectra were acquired through data-dependent auto MS/MS mode followed by an extraction of the potential compounds from the LC-QqTOF MS raw data of the sample. The same was matched using the databases to search for targeted components in the sample. The structures for detected components were tentatively characterized by manually interpreting the accurate MS/MS spectra for the first time. A total of 57 components have been successfully detected and structurally characterized from the crude extracts of G. elegans, but has failed to differentiate some isomers. This analytical strategy is generic and efficient, avoids isolation and purification procedures, enables a comprehensive structure characterization of target components of Gelsemium and would be widely applicable for complicated mixtures that are derived from Gelsemium preparations. Copyright © 2017 John WileySons, Ltd.

In this study, a novel metabolomics technique based on ultra-performance liquid chromatography-quadrupole-time of flight mass spectrometry in the MSE mode was used to investigate the milk metabolomics of healthy, subclinical, and clinical mastitis cows, which were classified based on somatic cell count and presentation of clinical symptoms. Meanwhile, univariate and multivariate statistical analyses were performed to identify the significant differences across the 3 groups. Compared with healthy milk samples, less glucose, d-glycerol-1-phosphate, 4-hydroxyphenyllactate, l-carnitine, sn-glycero-3-phosphocholine, citrate, and hippurate were detected in the clinical mastitic milk samples, whereas less d-glycerol-1-phosphate, benzoic acid, l-carnitine, and cis-aconitate were found in the subclinical mastitic milk samples. Meanwhile, the milk concentration of arginine and Leu-Leu increased in both the clinical and subclinical mastitis groups. Besides, less 4-hydroxyphenyllactate, cis-aconitate, lactose, and oxoglutarate were detected in the clinical than the subclinical mastitic milk samples, whereas the abundance of some oligopeptides (Leu-Ala, Phe-Pro-Ile, Asn-Arg-Ala-Ile, and Val-Phe-Val-Tyr) increased by over 7.95-fold. Our results suggest that significant variations exist across healthy and mastitis cows. The current metabolomics approach will help in better understanding the pathobiology of mastitis, although clinical validation will be required before field application.

The fruits of Siraitia grosvenorii are considered to be health-promoting because of the diversity of their bioactive ingredients. In the present study, a screening method, using high-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (HPLC-Q-TOF-MS) combined with a screening strategy, has been established. The technology was used to systematically screening the targeted metabolites, primarily from the complex matrix of S. grosvenorii. The compounds were then identified by their exact masses and characteristic fragment ions, in comparison with the fragmentation behaviors of 19 references. Finally, 122 compounds, including 53 flavonols and flavonol glycosides, 59 triterpene glycosides and 10 siraitic acid glycosides, were screened and identified in 10-, 50- and 80-day fruits, roots, stems and leaves of S. grosvenorii. 98 of them were reported for the first time. Additionally, the distribution of all identified components in different parts of the plant was determined and metabolic networks for flavonol and triterpene glycosides were proposed.

The characterization of unknown compounds is still a great challenge currently. A strategy for deduction of potential new phthalides through the characterization of isomers based on ultra-performance liquid chromatography coupled with quadrupole time of flight tandem mass spectrometry was proposed here to characterize the unknown compounds of Ligusticum chuanxiong Hort. (Chuanxiong). This proposed strategy consisted of four steps: (1) the high resolution MS data was collected, and the peaks were screened preliminarily by UNIFITM platform based on the in-house database; (2) the fragmentation patterns and the characteristic fragments were summarized based on the representative standards; (3) the target compounds were identified based on the fragmentation rules, standards comparison and false positive exclusion; (4) the unknown components were structurally characterized according to the accurate mass and fragmentation patterns analysis. This strategy was successfully applied to the identification and deduction of phthalides in Chuanxiong. A total of 81 phthalides were detected. Fifty-five known phthalides were identified, and 26 potential new phthalides were characterized. This research enriched the material basis of Chuanxiong, and provided a liquid chromatography tandem mass spectrometry-oriented method for the discovery of the potential new compounds.

Oxidation of aviation fuels prior to combustion affects the safe operation of high performance aircraft. This study reports the use of comprehensive two-dimensional gas chromatography (GC×GC) with accurate mass time-of-flight mass spectrometry (accTOFMS) to study oxidized species in complex thermally oxidized conventional and alternate aviation fuel matrixes. Tens of thousands of unique compounds were identified in the fuels, highlighting the need for comprehensive separations. GC×GC operation allowed a range of oxidized species to be isolated and identified, and in a conventional fuel, a series of homologous aldehydes and 2-ketones were found. 4-Methyl-2-hexanone and 1-pentanol were found to be strongly correlated with established methods for quantitatively assessing fuel stability.

A metabolomics method based on ultra high performance liquid chromatography with quadrupole time-of-flight mass spectrometry was developed to evaluate the influence of processing times on the quality of raw and processed Polygoni Multiflora Radix. Principal component analysis and partial least-squares discriminant analysis was used to screen the potential maker metabolites that were contributed to the quality changes. Then these marker metabolites were selected as variables in Fisher's discriminant analysis to establish the models that were used to distinguish the raw and processed Polygoni Multiflora Radix in the markets. Additionally, 36 compounds were identified. Twelve raw Polygoni Multiflora Radix samples and 23 processed Polygoni Multiflora Radix samples were distinguished. The results showed that the 12 raw Polygoni Multiflora Radix samples belonged to the group of processing time of 0 h, and two processed Polygoni Multiflora Radix samples were part of the group of processing times of 4 h, 12 samples belonged to group of processing times of 8 to 16 h, and nine samples were the group of processing times of 24 to 48 h. The results demonstrated that the method could provide scientific support for the processing standardization of Polygoni Multiflora Radix.

For modern food safety control are using techniques that allow to determinate a large number of components. So for determination of pesticide residues in fruits and vegetables commonly used methods of gas and liquid chromatography with time-of-flight mass-spectrometric detection. This system allows to carry out quantitative determination several hundreds of pesticides and their identification by the characteristic fragments of the mass spectrum. The main problem when using mass spectrometric detection is a matrix effect, which is caused by the influence of matrix components extracted with pesticides from the sample. In this work, attempts have been made to reduce the influence of the matrix in the analysis of pesticide residues by high performance liquid chromatography with time of flight mass spectrometry (HPLC / TOFMS).

An analytical method for the reliable screening and confirmation of 156 illegal drugs (58 erectile dysfunction drugs, 49 synthetic steroids, 26 anabolic steroids, and 23 anti-histamine drugs) in supplementary diets using ultra-high-performance liquid chromatography coupled with quadrupole/time-of-flight mass spectrometry (UHPLC-Q/TOF-MS) was developed. Various types of supplements (liquid, capsule, powder, pill and tablet) with complicated matrices were pretreated by simple liquid-liquid extraction. The wide scope of 156 target compounds was effectively determined within 15min in the positive ion mode, detecting the compounds at a sub-ppb level. Their MS/MS spectra were preferentially investigated to find diagnostic common ions according to the structural similarity of diverse adulterants. For the rapid screening of multiple classes of the target adulterants, extracted common ion chromatograms (ECICs) based on specific fragments of similar molecular moieties were attempted. A database including the elemental compositions, retention times, and MS/MS spectra was built for the confirmation of adulterants. The established method was validated in terms of the linearity, limits of detection (LOD), precision, and accuracy. The linear correlation coefficient and limit of detection ranged from 0.9880 to 1 and from 0.02 to 16.04ng/mL, respectively. The precision and accuracy of intra- and inter-day experiments for the spiked samples at the range of 0.2 and 16.0ng/mL were from 0.16 to 13.50% and 0.19-11.48%, respectively, with relative standard deviation. Mean recoveries ranged from 81.6 to 124.7%, and relative standard deviation was less than 9.20%. The screening and confirmation method demonstrated the usefulness of UHPLC-Q/TOF-MS combined with ECICs as a promising approach for the analysis of multi-class adulterants. Finally, the established method was successfully applied for the monitoring of several types of dietary supplements in routine analysis.

A supercritical CO2 ultra-performance convergence chromatography (UPC2) system was utilized with a quadrupole time-of-flight mass spectrometry (Q-TOF MS) to examine the triacylglycerol compositions of sunflower, corn and soybean oils. UPC2 provided an excellent resolution and separation for the triacylglycerols, while the high performance Q-TOF MS system was able to provide the molecular weight and fragment ions information for triacylglycerol compound characterization. A total of 33 triacylglycerols were identified based on their elementary compositions and MS2 fragment ion profiles, and their levels in the three oils were estimated. The combination of UPC2 and Q-TOF MS may determine triacylglycerol compositions for oils and fats, and provide sn-position information for fatty acids, which may be important for food nutritional value and stability.

Anabolic steroids have similar structures to testosterone, both of which promote the growth of muscle mass and increase strength. However, the side effects of anabolic steroid use may lead to heart attacks or strokes. Additionally, the excessive use of steroids inhibits the production of the sex hormones in the body via a negative feedback loop, which results in testicular atrophy in males and amenorrhea in females. Currently, the method of choice used to test for the presence of anabolic steroids is GC-MS. However, GC-MS methods require chemical derivatization of the steroid sample to ensure compatibility with the analytical method; therefore, analysis of many different samples is difficult and time consuming. Unlike GC-MS, the liquid chromatography-quadrupoletime of flight mass spectrometry (LC-Q-TOF-MS) method is suitable for many samples. Twenty-two different anabolic steroids were analyzed by LC-Q-TOF-MS with various collision energies (CE). Accurate mass spectral data were obtained using a Q-TOF-MS equipped with an electro-spray ionization source and operated in the positive MS/MS mode for several classes of steroids that are often the targets of testing. Based on the collected data, fragmentation pathways were carefully elucidated. The high selectivity and sensitivity of the LC-Q-TOF-MS instrument combined with these fragmentation pathways offers a new approach for the rapid and accurate screening of anabolic steroids. The obtained data from the 22 different anabolic steroids will be shared with the scientific community in order to establish a library to aid in the screening of illegal anabolic steroids.

RATIONALE Diacylgycerol-N-trimethylhomoserine (DGTS) and diacylglycerylhydroxymethyl-N,N,N-trimethyl-β-alanine (DGTA) are structural isomers that are the most commonly described betaine lipids in microalgae. The structural differentiation and precise identification of DGTS and DGTA in microalgae need to be established during mass spectrometry analysis. METHODS Total lipid was extracted from Amphora spp. with CHCl3 /CH3 OH (1:1, v/v). The qualitative analysis of DGTS and DGTA in Amphora spp. was carried out using Li+ /H+ dual mode by ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry operating in MSE mode (UPLC/QTOF MSE ). RESULTS Characteristic fragment ions [C10 H22 O5 N]+ at m/z 236.15 and [C7 H14 O2 N]+ at m/z 144.10 from the [M + H]+ precursor ion can be used for the qualitative analysis of both DGTA and DGTS, whereas the loss of m/z 87 and 74 from the [M + Li]+ precursor ion are specific for DGTS, and the loss of m/z 103 from the [M + Li]+ precursor ion is only for DGTA. As a result, 9 DGTSs and 16 DGTAs with different fatty acids were identified simultaneously in Amphora spp. Semi-quantitative analysis of DGTS and DGTA in Amphora spp. showed that the contents of DGTS ranged from 0.003 to 0.438 nmol mg-1 dw, and that of DGTA from 0.004 to 0.414 nmol mg-1 dw. CONCLUSIONS This is the first report to achieve the ambiguous structural identification of DGTS and DGTA by UPLC/QTOF MSE using dual Li+ /H+ adduct ion mode, which has remained a challenge in the past. It could provide new insights into their phylogeny and be helpful to characterize the natural phytoplankton communities as intact polar lipid biomarkers. Copyright © 2017 John Wiley & Sons, Ltd.

RATIONALE Isoacteoside (ISAT), a phenylethanoid glycoside that acts as the principal bioactive component in traditional Chinese medicines, possesses broad pharmacological effects such as neuroprotective, antihypertensive and hepatoprotective activities. However, its pharmaceutical development has been severely limited due to the poor oral bioavailability. It is essential and significant to investigate related hurdles leading to the poor bioavailability of isoacteoside. METHODS Whole animal metabolism studies were conducted in rats, followed by metabolic mechanism including gastrointestinal stability, intestinal flora metabolism and intestinal enzyme metabolism employing the powerful method ultrahigh-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC/QTOF-MS/MS). RESULTS A simple, rapid and sensitive method has been developed which comprehensively revealed the underlying cause of poor bioavailability of ISAT in a metabolic manner. The prototype of ISAT and its combined metabolites have not been detected in plasma. Furthermore, the residual content of the parent compound in in vitro experiments was approximately 59%, 5% and barely none in intestinal bacteria, intestinal S9 and simulated intestinal juice at 6 h, respectively. CONCLUSIONS The present work has demonstrated that the factors causing the poor bioavailability of isoacteoside should be attributed to the metabolism. In general, the metabolism that resulted from intestinal flora and intestinal enzymes were predominant reasons giving rise to the poor bioavailability of ISAT, which also suggested that metabolites might be responsible for the excellent pharmacological effect of ISAT. Copyright © 2016 John Wiley & Sons, Ltd.

Pyrrolizidine alkaloids (PAs) and their and the corresponding N-oxides (PAs-ox) are genotoxic plant metabolites which can be present as unwanted contaminants in food products of herbal origin like tea and food supplements. PAs and PAs-ox come in a wide variety of molecular structures including many structural isomers. For toxicity assessment it is important to determine the composition of a sample and to resolve all isomeric PAs and PAs-ox, which is currently not possible in one liquid or gas chromatographic (LC or GC) run. In this study an online two dimensional liquid chromatography quadrupole time-of-flight mass spectrometry (2D-LC QToF-MS) method was developed to resolve isomeric PAs and PAs-ox. After comprehensive column and mobile phase selection a polar endcapped C18 column was used at pH 3 in the first dimension, and a cross-linked C18 column at pH 10 in the second dimension. Injection solvents, column IDs, flow rates and temperatures were carefully optimized. The method with column selection valve switching described in this study was able to resolve and visualize 20 individual PAs/PAs-ox (6 sets of isomers) in one 2D-LC QToF-MS run. Moreover, it was shown that all isomeric PAs/PAs-ox could be unambiguously annotated. The method was shown to be applicable for the determination and quantification of isomeric PAs/PAs-ox in plant extracts and could be easily extended to include other PAs and PAs-ox.

The dried root of Ardisia gigantifolia Stapf. (RAG) is mainly used as a Chinese folk medicine. A clear understanding of its major components is very important in their rational clinical use and pharmacological research. To find more of the major components in RAG, a high performance liquid chromatography coupled to electrospray ionization and quadrupole time-of-flight-mass spectrometry method (HPLC-ESI-QTOF-MS/MS) was proposed. The components were characterised based on their accurate mass and MS/MS data provided by the QTOF-MS as well as by comparison with information from the literature. In this study, 27 phytochemical components were tentatively characterised. Notably, out of the 27 components, 8 triterpenoid saponins are new and their chemical structures have been proposed for the first time in RAG. These results showed that the HPLC-ESI-QTOF-MS/MS method was a powerful tool for a comprehensive research of RAG, which could provide a meaningful basis for further quality control and pharmacological as well as toxicological research studies.

Isovitexin, a bioactive flavonoid constituent isolated from Desmodii Styracifolii, is considered an adjuvant for antiurolithiasis diseases. In this study, an ultra-high-performance liquid chromatography coupled with hybrid triple quadruple time-of-flight mass spectrometry method was developed to characterize and compare the metabolic profiling of isovitexin experimented on normal and kidney stone model rats. The comparative research indicated that 28 metabolites (18 phase I and 10 phase II) in normal rats and 33 metabolites (20 phase I and 13 phase II) in kidney stone model rats were initially identified. The results of relative quantitative determination reflected that the contents of metabolites produced by deglycosylation, reduction, and isomerization in kidney stone model rats were greater than those in healthy rats. Instead, the levels of oxidative and dehydrogenated metabolites in normal groups were higher than those in kidney stone model groups. The results of this study are valuable and important for understanding the metabolic process of isovitexin in clinical application, and especially the metabolism study in kidney stone model rats could provide a beneficial reference for the further search of effective substances associated with the treatment of kidney stones.

In past two decades, numerous lipidomics approaches based on mass spectrometry with or without liquid chromatography separation have been established for identification and quantification of lipids in plants. In this study, we developed an efficient and comprehensive lipidomics approach based on UPLC with an Acquity UPLCTM BEH C18 column coupled to TripleTOF using ESI in positive ion mode and MS/MSALL scan for data collection. Lipid extract was prepared to 2 mg/ml solution according to dry tissue weight and mixed with 13 kinds of internal standards including PA, PC, PE, and PG. Each analysis required single injection of 5–10 μl lipid solvent and completed in 32 min. A target method dataset was generated using the LipidView software for prediction of the accurate mass of target lipid species. The dataset was uploaded into the PeakView to create processing datasets to search target lipid species, which achieved batch data processing of multiple samples for lipid species‐specific identification and quantification. As proof of concept, we profiled the lipids of different tissues of rapeseed. Thirteen lipid classes including 218 glycerolipids were identified including 46 TAGs, 15 DAGs, 20 PCs, 24 PEs, 13 PGs, 14 PIs, 26 PSs, 12 PAs, 16 MGDGs, 16 DGDGs, 6 LysoPCs, 5 LysoPEs, and 5 LysoPGs. Together, our approach permits the analysis of glycerolipids in plant tissues with simplicity in sample analysis and data processing using UPLC‐TripleTOF.

Rationale Hepatocellular carcinoma (HCC) is a highly malignant disease for which the development of prospective or prognostic biomarkers is urgently required. Although metabolomics is widely used for biomarker discovery, there are some bottlenecks regarding the comprehensiveness of detected features, reproducibility of methods, and identification of metabolites. In addition, information on localization of metabolites in tumor tissue is needed for functional analysis. Here, we developed a wide-polarity global metabolomics (G-Met) method, identified HCC biomarkers in human liver samples by high-definition mass spectrometry (HDMS), and demonstrated localization in cryosections using desorption electrospray ionization MS imaging (DESI-MSI) analysis. Methods Metabolic profiling of tumor (n = 38) and nontumor (n = 72) regions in human livers of HCC was performed by an ultrahigh-performance liquid chromatography quadrupole time-of-flight MS (UHPLC/QTOFMS) instrument equipped with a mixed-mode column. The HCC biomarker candidates were extracted by multivariate analyses and identified by matching values of the collision cross section and their fragment ions on the mass spectra obtained by HDMS. Cryosections of HCC livers, which included both tumor and nontumor regions, were analyzed by DESI-MSI. Results From the multivariate analysis, m/z 904.83 and m/z 874.79 were significantly high and low, respectively, in tumor samples and were identified as triglyceride (TG) 16:0/18:1(9Z)/20:1(11Z) and TG 16:0/18:1(9Z)/18:2(9Z,12Z) using the synthetic compounds. The TGs were clearly localized in the tumor or nontumor areas of the cryosection. Conclusions Novel biomarkers for HCC were identified by a comprehensive and reproducible G-Met method with HDMS using a mixed-mode column. The combination analysis of UHPLC/QTOFMS and DESI-MSI revealed that the different molecular species of TGs were associated with tumor distribution and were useful for characterizing the progression of tumor cells and discovering prospective biomarkers.

Semiliquidambar cathayensis Chang was a traditional medicinal plant and used to treat rheumatism arthritis and rheumatic arthritis for centuries in China with no scientific validation, while only 15 components were reported. Thus, a rapid, efficient, and precise method based on ultra-high-performance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry (UHPLC-Q-TOF-MS/MS) was applied in both positive- and negative-ion modes to rapidly analysis the main chemical compositions in S. cathayensis for the first time. Finally, a total of 85 chemical compositions, including 35 alkaloids, 12 flavonoids, 7 terpenoids, 5 phenylpropanoids, 9 fatty acids, 7 cyclic peptides, and 10 others were identified or tentatively characterized in the roots of S. cathayensis based on the accurate mass within 5 ppm error. Moreover, alkaloid, flavonoid, phenylpropanoid, and cyclic peptide were reported from S. cathayensis for the first time. This rapid and sensitive method was highly useful to comprehend the chemical compositions and will provide scientific basis for further study on the material basis, mechanism and clinical application of S. cathayensis roots.

Sealants, incorporated in the lids of food cans to ensure the can is hermetically sealed, are formulated from a wide variety of compounds. These compounds and associated non-intentionally added substances (NIAS) could migrate to the food contained in the can. In this work, ion mobility quadrupole time-of-flight mass spectrometry coupled to ultra-high performance liquid chromatography (UHPLC-IM-QTOF-MS) has been used to obtain ion mobility filtered extracted ion chromatograms. Subsequently, accurate mass precursor ions and their fragments have been used to identify the compounds migrating from the sealant to the content of the cans. Moreover, the correlation between the collision cross-section (CCS) values and m/z of the compounds was used to increase the level of confidence of the identification. Seven compounds were found to have migrated to the food simulants. The compounds bis(2-hydroxy-3-tert-butyl-5-methylphenyl)dicyclopentane,1-tetradecanesulfonic acid, 1-pentadecanesulfonic acid, 1-hexadecanesulfonic acid and naphthalene-2-sulfonic acid (whose migration was over the specific migration limit established by the European Regulation 10/2011/EU) were identified as NIAS in the food simulants studied.

Inherent complexity of plant metabolites necessitates the use of multi-dimensional information to accomplish comprehensive profiling and confirmative identification. A dimension-enhanced strategy, by offline two-dimensional liquid chromatography/ion mobility-quadrupole time-of-flight mass spectrometry (2D-LC/IM-QTOF-MS) enabling four-dimensional separations (2D-LC, IM, and MS), is proposed. In combination with in-house database-driven automated peak annotation, this strategy was utilized to characterize ginsenosides simultaneously from white ginseng (WG) and red ginseng (RG). An offline 2D-LC system configuring an Xbridge Amide column and an HSS T3 column showed orthogonality 0.76 in the resolution of ginsenosides. Ginsenoside analysis was performed by data-independent high-definition MSE (HDMSE) in the negative ESI mode on a Vion™ IMS-QTOF hybrid high-resolution mass spectrometer, which could better resolve ginsenosides than MSE and directly give the CCS information. An in-house ginsenoside database recording 504 known ginsenosides and 58 reference compounds, was established to assist the identification of ginsenosides. Streamlined workflows, by applying UNIFI™ to automatedly annotate the HDMSE data, were proposed. We could separate and characterize 323 ginsenosides (including 286 from WG and 306 from RG), and 125 thereof may have not been isolated from the Panax genus. The established 2D-LC/IM-QTOF-HDMSE approach could also act as a magnifier to probe differentiated components between WG and RG. Compared with conventional approaches, this dimension-enhanced strategy could better resolve coeluting herbal components and more efficiently, more reliably identify the multicomponents, which, we believe, offers more possibilities for the systematic exposure and confirmative identification of plant metabolites., Graphical abstract Image 1, Highlights • A dimension-enhanced strategy was proposed by 2D-LC/IM-QTOF-HDMSE. • New dimensions of tR in 1D-HILIC and CCS were provided. • An in-house ginsenoside database and a ginsenoside compound library were reported. • Automated peak annotation was achieved by UNIFI™. • 323 ginsenosides, including 125 unknown, were characterized.

Lycium barbarum fruit (Goji berry) have been used as a traditional Chinese medicine (TCM) with its outstanding biological and pharmacological activities. Spermidine alkaloids are a major class of bioactive constituents in goji berry, nevertheless, detailed information related to its identification remains scarce. In this study, chemical profiling of spermidines in goji berry was carried out by ultrahigh-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). Four structure types of standards were used to study the comprehensive fragmentation rules of spermidines. Different types of spermidines were identified by distinctive MS/MS fragment ions. Noticeably, it was first proposed that the co-existence of fragment ions at m/z 220 and 222 was the key characteristic for distinguishing spermidine isomers. According to the structural feature of spermidines, a quick, convenient, highly selective strong cation exchange solid-phase extraction (SCX-SPE) combined with RP-LC procedure was developed for selective enrichment and the MS detection compatibility. A total of 41 out of 58 spermidines were tentatively characterized using the established method, of which 26 were reported for the first time from goji berry. This study provides guidelines and references for the identification of spermidines in natural products.

This is the first report of quadrupole time-of-flight (Q-TOF) mass spectrometric identification of the hemoglobin (Hb) subunits, α, β, δ and γ peptides, derived from enzymatic-digestion of proteins in the early unknown peaks of the cation exchange chromatography of Hb. The objectives were to identify the unknown high performance liquid chromatography (HPLC) peaks in healthy subjects and in patients with β-thalassemia (β-thal). The results demonstrate the existence of pools of free globin chains in red blood cells (RBCs). The α-, β-, δ- and γ-globin peptides were identified in the unknown HPLC peaks. The quantification and role of the free globin pool in patients with β-thal requires further investigation. Identification of all types of Hb subunits in the retention time (RT) before 1 min. suggests that altered Hbs is the nature of these fast-eluting peaks. Relevancy of thalassemias to the protein-aggregation disorders will require review of the role of free globin in the pathology of the disease.

Mycophenolate mofetil is an antiproliferative immunosuppressive agent. Since its clinical efficacy and safety highly depend on the quality, the stability, and impurity profiles of mycophenolate mofetil are paid ever-increasing attention. However, there are few published studies reporting the complete characterization of both the process-related substances and degradation products in mycophenolate mofetil. In the present study, a highly specific and efficient liquid chromatography coupled with quadrupole-time of flight mass spectrometry method was developed for the separation and identification of all the potential impurities in mycophenolate mofetil. According to the ICH Q1A (R2) guideline, the forced degradation studies were conducted to elucidate the stability and degradation pathways of mycophenolate mofetil. A total of 15 related substances, including the process-related substances and stress degradation products were characterized by the established hyphenated method, 11 of them have not been reported before. In view of the synthetic route and degradation pathways of mycophenolate mofetil, the origins and formation mechanisms of these related substances were discussed. Based on the obtained stability and impurity profiles, key points of the manufacturing process were proposed to deliver mycophenolate mofetil with high purity.

Gangliosides, widely distributed in tissues and body fluid, have been connected to the therapy of cancer and brain related diseases. The complexity of the gangliosides structures with different polar moieties coexisting, a carbohydrate moiety and a ceramide chain, make it a great challenge in separation and analysis science. This study aimed to develop a strategy on the basis of high-accuracy data collected by an offline two-dimensional (2D) supercritical fluid chromatography (SFC) × reversed phase liquid chromatography (RPLC)/quadrupole time-of-flight (Q-ToF) system, and to integrate an in-house library with self-developed software for fast screening and identification of gangliosides from a complex sample (swine brain extract). Subsequent positive-mode MS/MS was used to validate the identified gangliosides. Finally, 153 gangliosides were separated and 79 of them were identified by the in-house library and self-developed software, 4-fold more than those by manual identification (18 gangliosides). Among the identified ones, 20 were detected in swine brain for the first time. This study established an offline 2D SFC × RPLC system and provided a new method for fast screening and automatic identification of gangliosides in complex mixtures. It will be conducive to further study of biological functions of gangliosides.

Grape byproducts are rich sources of polyphenols with powerful antioxidant and health-promoting effects. The impact of supplementing chicken diets with grape byproducts on plasma and thigh meat concentrations of phenolic metabolites was evaluated by analyzing samples by high-performance liquid chromatography quadrupole time of flight mass spectrometry. Chickens were fed three experimental diets: Control diet, Control+8% grape pomace, and Control+0.1% grape seed extract. In plasma, 32 phenolic metabolites were identified, some of which were conjugated catechin/epicatechin metabolites exclusively identified in chickens fed diets enriched in grape byproducts. Also, these chickens showed significantly higher plasmatic concentrations of 21 phenolic metabolites. In thigh meat, 14 phenolic metabolites were identified, but no differences were found between diets. Higher plasmatic tocopherol was found when supplementing diets with grape byproducts, while no changes were observed in meat. Thus, supplementing chicken diets with grape byproducts leads to a significant increase in the circulation of phenolic metabolites and tocopherol., Authors thank the CAM for financially supporting Project MEDGAN-CM S2013/ABI2913 and MINECO and CSIC for financially supporting Project Intramural 2014470E07.

We developed a highly sensitive and accurate differential method for analyzing chiral and achiral carboxylic acids in Japanese commercial beers, using ultra high-performance liquid chromatography–quadrupole time-of-flight mass spectrometry (UHPLC–Q-TOF-MS), based on chiral derivatization combining three isotopic chiral reagents: 12C-DMT-3(S)-Apy, 13C2-DMT-3(S)-Apy, and d6-DMT-3(S)-Apy. By combining these reaction solutions and analyzing the resulting mixture, simultaneous comparative analyses of the three samples could be achieved while offsetting the matrix effects on the samples. Using this approach, it was possible to differentiate the beers according to the type (draft, low-malt, and non-alcoholic), manufacturer, and storage conditions (temperature and storage period), based on the concentrations of carboxylic acids in the beers. Furthermore, we identified α-ketoglutaric acid as a new marker for determining the storage condition of Japanese beers. The proposed method would provide insights into the identification of markers in various fields, such as in biological samples, as well as food and beverages.

Xiaojin Capsule, a classic traditional Chinese medicine formula, has been used to treat mammary cancer, thyroid nodules, and hyperplasia of the mammary glands. However, its systematic chemical information remained unclear, which hindered the interpretation of the pharmacology and the mechanism of action of this drug. In this research, an ultra high performance liquid chromatography coupled with a quadrupole time-of-flight mass spectrometry method was developed to identify the complicated components and metabolites of Xiaojin Capsule. Two acquisition modes, including the MSEnergy mode and fast data directed acquisition mode, were utilized for chemical profiling. As a result, 156 compounds were unambiguously or tentatively identified by comparing their retention times and mass spectrometry data with those of reference standards or literature. After the oral administration of Xiaojin Capsule, 53 constituents, including 24 prototype compounds and 29 metabolites, were detected in rat plasma. The obtained results were beneficial for a better understanding of the therapeutic basis of Xiaojin Capsule. A high-resolution and efficient separation method was firstly established for systematically characterizing the compounds of Xiaojin Capsule and the associated metabolites in vivo, which could be helpful for quality control and pharmacokinetic studies of this medicine.

TPN171 is a novel potent pyrimidinone phosphodiesterase type 5 (PDE5) inhibitor with high selectivity and long duration of action. It has been used to treat patients suffering from pulmonary arterial hypertension and entered phase I clinical trials in 2016. Considering the potential therapeutic value of TPN171, its metabolism in humans is necessary to be elucidated during early-stage of drug development. This study aimed to establish a rapid and reliable method based on ultra-performance liquid chromatography/quadrupole time-of-flight mass spectrometry for the characterization of TPN171 metabolites in human plasma, urine and stool samples. A total of 17 metabolites, including 14 from phase I and 3 from phase II metabolic reactions, were identified and characterized. TPN171 was found to be the predominant component in all plasma, urine and stool samples. Seven proposed metabolites were validated by comparing with synthetic reference standards. N-demethylation, O-depropylation, N-oxidation and dehydrogenation were demonstrated to be the main metabolic pathways of TPN171 in humans, yielding metabolites M4, M3, M7-2 and M5-3, respectively. Notably, M5-3 (a dehydrogenation product) and M3 (an O-depropylation product) were the main metabolites in human plasma while M5-3 (produced via dehydrogenation) and M7-2 (produced via N-oxidation) were the major metabolites in human urine. Besides, O-depropylation product M3 and N-demethylation product M4 were the main metabolites in human stool. In overall, this study assessed the metabolic fate of TPN171 in humans, which may yield considerably benefits for subsequent studies focusing on TPN171 metabolism and development of other PDE5 inhibitors.

The aquatic ecosystems are dynamic environments often affected directly or indirectly by a myriad of anthropogenic contaminants that need to be properly identified. In this study, liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-QqTOF-MS) suspected-screening was applied to mussels and riverine sediment both, non-spiked and spiked with a mixture of 32 pharmaceuticals. Three data acquisition methods –sequential window acquisition of all theoretical fragment-ion spectra (SWATH), in fix (FSWATH) and variable (VSWATH) window modes and Information Dependent Acquisition (IDA)– were compared to determine the most suitable acquisition technique. The results obtained in the spiked samples showed that the two SWATH modes enable to obtain the MS/MS spectrum of a higher number of compounds (up to 27 with FSWATH and 25 with VSWATH) than IDA (up to 19) in sediment and mussel. The different data acquisition modes were also tested in non-spiked samples to verify the results obtained in the spiked ones. Importantly, all the methods are able to detect the MS/MS spectrum of several contaminants in the samples when analysed against a database of >600 compounds. Up to 7 contaminants were tentatively detected with IDA, 15 with FSWATH and 17 with VSWATH. Most pollutants were pesticides and pharmaceuticals, being of particular interest the presence of ibuprofen and acetaminophen in mussels., This work has been supported by the Spanish Ministry of Science, Innovation and Universities and the ERDF (European Regional Development Fund) through the project Eco2RISK-DDS (CGL2015-64454-C2-1) and by the Generalitat Valenciana through the project ANTROPOCEN@ (PROMETEO/2018/155).

The method for identification and quantification of imidazoles in atmospheric aerosol particles with an aerodynamic diameter up to 2.5 μm (PM2.5) is scarce, and the existing method focus on only a few imidazoles. With the goal of measuring more imidazoles, especially some previously unidentified ones, we developed a screening workflow based on data-dependent acquisition (DDA) auto MS/MS with a preferred targeted list containing 421 imidazoles using ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS). To enable our method to effectively and accurately detect as many imidazoles as possible, we optimized and validated the method based on specificity, limit of detection (LOD), limit of quantification (LOQ), linearity, accuracy, precision and matrix effects using 20 imidazole standards with different functional groups. The method exhibited excellent performance with LOD and LOQ of 0.5–2 ng/mL and 1.5–6 ng/mL, respectively, and spiked recoveries ranging from 64.7 to 98.7% with standard deviations less than 16.0%, and with relatively shorter analytical time. The established method was then used to screen imidazoles in 37 ambient PM2.5 samples. Ten targeted imidazoles were identified and quantified using imidazole standards, while five suspected imidazoles were identified without standards, and three imidazoles have not been reported before. Concentrations of the 10 targeted imidazoles ranged from 0.13 to 0.42 ng/m3. The established method enabled us to identify a wide range of imidazoles in ambient aerosol particles with and without using standards.

Enrichment of mortar mixtures by the addition of natural organic materials has a long history. Already in ancient times it has been empirically proved that proteinaceous additives significantly increase mortar durability, strength or plasticity. The knowledge of the protein occurrence in historical mortars would be very useful for the restoration work. However, some of historical recipes did not survive till these days or it is difficult to interpret them if they have survived, and, therefore,we are not able to prepare all kinds of mortars of an appropriate quality nowadays. The goal of this work was to identify and relatively quantify proteins contained in the model mortars samples enriched with four different protein additives (animal glue, blood, egg, and milk). For this purpose, aged mortar samples were analysed by LC-ESI-Q-TOF MS (Liquid Chromatography and ElectroSpray Ionization Quadrupole Time of Flight Mass Spectrometry) and the relative content of proteins was quantified by label-free technique. Number of semi-specific and non-specific peptides were detected in different ageing times and preferential sites in non-specific peptide were found. Principal component analysis (PCA) applied on MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization – Time of Flight) spectra was also used to differentiate between protein additives in mortars. Peptides that are stable in the prepared model mortars and characteristic for the individual protein additives were found and together with the results of relative quantification they confirmed the possibility of unambiguous identification of protein additives in century-old mortars that brings the opportunity to make an improved hypothesis about the old traditional recipes. By the mass spectrometric methods milk additive was identified in historical mortar from Swedish church dated back to 14th century.

Human milk lipids are an important energy source and essential nutrients for the growth and development of infants. The UPLC/Q-TOF-MS was used to qualitatively and quantitatively analyze human milk lipids. Totally, 411 species of lipids were identified, in which the content of OPL was generally higher than that of OPO; SM (75.38 mg/L, 40.39%), PE (51.12 mg/L, 27.39%) and PC (40.10 mg/L, 21.49%) had the highest contents among polar lipids, mainly including SM42:2:2 (22.24 mg/L), PE36:2 (C18:0-C18:2, 21.39 mg/L) and PC36:2 (C18:0-C18:2, 19.80 mg/L). In human milk, TAG56:7 (137.14 mg/L), TAG56:8 (59.49 mg/L), TAG58:8 (65.90 mg/L) and TAG58:9 (49.99 mg/L) were the main sources of AA and DHA; PE was an important source of AA and DHA in polar lipids; and linoleic acyl in glycerides and phospholipids had higher contents than other polyunsaturated fatty acyls. These results provided the scientific basis for the simulation of human milk at molecular level.

The high demand of fresh-like products to meet the fruits and vegetables serving encouraged the implementation of non-thermal food processing techniques, such as high pressure processing (HPP) and pulsed electric fields (PEF), with low impact on nutritional components. The aim of the present work is to evaluate the application of HPP and PEF techniques as useful decontamination tool for aflatoxins (AFs) reduction in grape juice. Spiked grape juice samples with AFs treated by PEF or HPP were extracted using dispersive liquid-liquid microextraction (DLLME) and determined by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS-IT). Reduction percentages of 14–29% have been obtained with both HPP and PEF treatments even higher reductions were obtained for AFB2 and AFG1 under PEF treatment, reaching reduction of 72 and 84% respectively. Results obtained in grape juice samples differed slightly from those obtained in water controls highlighting the matrix effect. Both HPP and PEF techniques showed measurable impact on AFs levels. Furthermore, an AFB2 degradation product obtained after PEF has been identified by quadrupole time of flight mass spectrometry detector (qTOF-MS) and its toxicological endpoints predicted by Pro Tox-II web server.

The World Anti-Doping Agency (WADA) publishes yearly their prohibited list, and sets a minimum required performance limit for each substance. To comply with these stringent requirements, the anti-doping laboratories have at least two complementary methods for their initial testing procedure (ITP), one using gas chromatography - mass spectrometry (GC-MS) and the other using liquid chromatography-MS (LC-MS). Anabolic androgenic steroids (AAS) have in previous years consistently been listed as the most frequently detected class of compounds. Over the last decade, evidence has emerged where a longer detection time is attained by focusing on sulfated metabolites of AAS instead of the conventional gluco-conjugated metabolites. Despite a decade of research on sulphated AAS using LC-MS, no LC-MS ITP has been developed that combines this class of compounds with the other mandatory targets. Such combination is essential for economical purposes. Recently, it was demonstrated that the direct injection of non-hydrolysed sulfates is compatible with GC-MS. Using this approach and by taking full use of the open screening capabilities of the quadrupole time of flight MS (QTOF-MS), this work describes for the first time a validated ITP that allows the detection of non-hydrolysed sulfated metabolites of AAS while, simultaneously, remaining capable of detecting a vast range of other classes of compounds, as well as the quantification of endogenous steroids, as required for an ITP compliant with the applicable WADA regulations. The method contains 263 compounds from 9 categories, including stimulants, narcotics, anabolic androgenic steroids and beta-blockers. Additionally, the advantages of the new method were illustrated by analysing excretion samples of drostanolone, mesterolone and metenolone. No negative effects were observed for the conventional markers and the detection time for mesterolone and metenolone increased by up to 150% and 144%, respectively compared to conventional markers.

Animal feeding method is a crucial factor in influencing meat quality. Consumers would preferentially select meat obtained from pasture-fed animals. In this study, an untargeted metabolomic and lipidomic method based on ultra-performance liquid chromatography quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) combined with chemometric analysis was utilized to investigate the differences between meat from free-range and intensively-fed sheep/goats. Distinct separation between these two kinds of sheep/goats meat obtained were identified by principal component analysis. Analysis of variance, fold change and orthogonal projection to latent structures discriminant analysis were then conducted to determine specific potential markers. A total of 46 potential markers were selected according to online chemical databases. The support vector machine (SVM) method was used to process the responses of the selected potential markers, and the results of metabolomics and lipidomics from an additional 59 samples revealed the discrimination rate of 89.3% and 98.3%. These findings provided a basis for differentiation of meat from sheep/goats fed in the two methods.

For controlling pesticide residues in food and ensuring food safety, multiresidue methods that can monitor a wide range of pesticides in various types of foods are required for regulatory monitoring. In this study, to demonstrate the applicability of liquid chromatography&ndash, quadrupole time-of-flight mass spectrometry (LC&ndash, QTOF-MS) for quantitative and confirmatory analysis of pesticide residues in cereal grains and legumes, the LC&ndash, QTOF-MS method using full-scan acquisition was validated for 151 pesticides in brown rice, soybeans, and peanuts at a spiked level of 0.01 mg/kg. With the exception of 5 out of 151 target pesticides, sufficiently high signal intensities were obtained at 0.005 &mu, g/mL (corresponding to 0.01 mg/kg). Trueness was in the range 70&ndash, 95%, with intra- and inter-day precisions below 16% and 24%, respectively, with the exception of 7 pesticides in brown rice, 10 pesticides in soybeans, and 9 pesticides in peanuts. No interfering peaks were observed near the retention times of the target pesticides. Furthermore, information on accurate fragment-ion masses obtained by a data-independent acquisition enabled unambiguous confirmation. The results suggest that the LC-QTOF-MS method is suitable for pesticide residues&rsquo, analysis of cereal grains and legumes, and can be utilized for regulatory routine analysis.

Rationale Koumine is one of the major components of total alkaloids from Gelsemium. Koumine possesses a variety of interesting pharmacological effects, including anti-tumor, anti-inflammatory, and anxiolytic activities. It might be a promising lead drug because of its pharmacological activities and mild toxicity. However, little information is available on the metabolism of koumine. Methods A rapid and accurate high-performance liquid chromatography/quadrupole-time-of-flight (HPLC/QqTOF) mass spectrometry method was applied to characterize koumine metabolites. Multiple scans of koumine metabolites, which were formed in rat liver S9, were automatically performed simultaneously through auto MS/MS mode acquisition in only a 30-min analysis. The structural elucidation of these metabolites was performed by comparing their changes in accurate molecular masses and product ions with those of the parent drug or metabolites. Results As a result, a total of eleven metabolites of koumine were identified, of which nine new metabolites were found. The present results showed that the N-demethylenation, hydrogenation and the oxidation were the three main metabolic pathways of koumine. Conclusions This was the first investigation of in vitro metabolism of koumine in rat liver S9 using a sensitive and specific HPLC/QqTOF-MS method. The possible metabolic pathways of koumine were tentatively proposed based on the structural elucidations of these metabolites. This work may be useful in the in vivo metabolism of koumine in animals and humans. Copyright © 2016 John Wiley & Sons, Ltd.