Liquid Chromatography Tandem Triple Quadrupole Research Articles

A comprehensive analytical strategy based on characteristic fragments to detect synthetic cannabinoid analogs in seized products and hair samples

Synthetic cannabinoids, one of the most widely abused new psychoactive substances (NPS), are now placed under national control generally in China. Due to continuous modification of synthetic cannabinoid structure, an ongoing dilemma in the forensic laboratory is that newly emerging substances cannot be detected by established methods. Thus, the screening methods for simultaneous detection of known or unknown substances have become research hotspots. In this study, the ultra high performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-QqQ-MS) with precursor ion scan (PIS) as acquisition mode was used for prescreening purposes of all possible synthetic cannabinoids-related substances. In detail, four common characteristic fragments, m/z of 144.0, 145.0, 135.1, and 109.0 corresponding to acylium-indole, acylium-indazole, adamantyl, and fluorobenzyl cation respectively, were selected for PIS mode, and their collision energies were optimized by 97 available synthetic cannabinoids standards with relevant structures. Those suspicious signals observed in the screening experiment were confirmed by ultra high performance liquid chromatography tandem quadrupole time-of-flight mass spectrometry (UPLC-QTOF-MS) via high-resolution MS and MS2 data obtained by full scan (TOF MS) and product ion scan mode. After methodological validation, the integrated strategy established above was applied to the screening and identification of the seized e-liquids, herbal blends and hair samples, confirming the presence of multiple synthetic cannabinoids in these samples. In particular, a novel synthetic cannabinoid was identified as 4 F-ABUTINACA, for which no relevant high-resolution mass spectrometry (HRMS) data has been retrieved until now, making this study the first to report the cleavage pattern of this compound in electrospray ionization (ESI) mass spectrometry. In addition, four other suspected by-products of the synthetic cannabinoids were found in the herbal blends and e-liquids, and their possible structures were also deduced via the information from high-resolution mass spectra.

Screening, characterization, and determination of suspected additives bimatoprost and latanoprost in cosmetics using NMR and LC-MS methods.

Recently, many new types of cosmetic illegal additives have been screened in the market. Most of the new additives were new drugs or analogues with very similar structures to other prohibited additives, which were difficult to be identified by liquid chromatography-mass spectrometry (LC-MS) only. Therefore, a new strategy is proposed, which is chromatographic separation combined with nuclear magnetic resonance spectroscopy (NMR) structural identification. The suspected samples were screened by ultra-high-performance liquid chromatography tandem high-resolution mass spectrometry (UPLC-Q-TOF-MS), followed by purification and extraction through silica-gel column chromatography and preparative high-performance liquid chromatography (HPLC). Finally, the extracts were identified unambiguously by NMR as bimatoprost and latanoprost, which were identified to be new cosmetic illegal additives in eyelash serums in China. Meanwhile, bimatoprost and latanoprost were quantified by high-performance liquid chromatography tandem triple quadrupole mass spectrum (HPLC-QQQ-MS/MS). The quantitative method demonstrated good linearity in the range of approximately 0.25-50ng/mL (R2 > 0.9992), with limit of detection (LOD) and limit of quantification (LOQ) values of 0.01 and 0.03mg/kg, respectively. The accuracy, precision, and reproducibility were confirmed to be acceptable.

Polygoni Multiflori Radix interferes with bile acid metabolism homeostasis by inhibiting Fxr transcription, leading to cholestasis.

Objective: To explore the possible mechanisms of cholestasis induced by Polygoni Multiflori Radix (PM). Methods: Low and high doses of water extract of PM were given to mice by gavage for 8weeks. The serum biochemical indexes of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glutamyltransferase (GGT) alkaline phosphatase (ALP) and so on were detected in the second, fourth, sixth, and eighth weeks after administration. At the end of the eighth week of administration, the bile acid metabolic profiles of liver and bile were screened by high-performance liquid chromatography tandem triple quadrupole mass spectrometry (HPLC-QQQ-MS/MS). Liver pathological changes were observed by hematoxylin and eosin staining. Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the mRNA transcription of the target genes and Western blotting (WB) was used to the detect target protein expression. Results: Biochemical tests results showed the values of ALP and GGT were two and three times greater than the normal values respectively, and the value of R was less than 2. Histopathology also showed that PM caused lymphocyte infiltration, a small amount of hepatocyte necrosis and nuclear fragmentation in mouse liver. The proliferation of bile duct epithelial cells was observed in the high group. These results indicated that PM may lead to cholestatic liver injury. HPLC-QQQ-MS/MS analysis with the multivariate statistical analysis revealed significant alterations of individual bile acids in liver and gallbladder as compared to those of the control group. RT-qPCR showed that the transcription of Fxr, Shp, Bsep, Bacs, Mdr2, and Ugt1a1 were downregulated and that of Cyp7a1, Mrp3, and Cyp3a11 was significantly upregulated in the treatment group. WB demonstrated that PM also markedly downregulated the protein expression of FXR, BSEP, and MDR2, and upregulated CYP7A1. Conclusion: PM inhibited the expression of FXR, which reduced the expression of MDR2 and BSEP, leading to the obstruction of bile acids outflow, and increased the expression of CYP7A1, resulting in an increase of intrahepatic bile acid synthesis, which can lead to cholestasis.

A sensitive LC-MS/MS method-based pharmacokinetic study of fifteen active ingredients of Yindan Xinnaotong soft capsule in rats and its potential mechanism in the treatment of cardiovascular diseases

Yindan Xinnaotong soft capsule (YDXNT) is a commonly used Chinese herbal preparation for the clinical treatment of coronary disease. However, there is a lack of pharmacokinetic studies on YDXNT, and its active ingredients and their mechanism in the treatment of cardiovascular diseases (CVD) are still unclear. In this study, 15 absorbed ingredients in rat plasma after oral administration of YDXNT were quickly identified based on liquid chromatography tandem quadrupole time-of-flight mass spectrometry (LC-QTOF MS), and then a sensitive and accurate quantitative method based on ultra-high performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-QQQ MS) was established and validated for simultaneous determination of the 15 ingredients of YDXNT in rat plasma, which was then applied to the pharmacokinetic study. Different types of compounds showed various pharmacokinetic characteristics, for instance, ginkgolides with higher maximum plasma concentration (Cmax), flavonoids presenting concentration–time curve with double peaks, phenolic acids with shorter time to reach maximum plasma concentration (Tmax), saponins with long elimination half-life (t1/2) and tanshinones showing fluctuant plasma concentration. Then the measured analytes were regarded as effective compounds and their potential targets and mechanism of action were predicted by constructing and analyzing the compound-target network of YDXNT and CVD. Those potential active compounds of YDXNT interacted with targets such as MAPK1 and MAPK8, and molecular docking showed that the binding free energies of 12 ingredients with MAPK1 were less than −5.0 kcal/mol, indicating that YDXNT intervened in the MAPK signaling pathway to display its therapeutic effect on CVD.

Characterization of Ginsenosides from the Root of Panax ginseng by Integrating Untargeted Metabolites Using UPLC-Triple TOF-MS

To compare the chemical distinctions of Panax ginseng Meyer in different growth environments and explore the effects of growth-environment factors on P. ginseng growth, an ultra-performance liquid chromatography-tandem triple quadrupole time-of-flight mass spectrometry (UPLC-Triple-TOF-MS/MS) was used to characterize the ginsenosides obtained by ultrasonic extraction from P. ginseng grown in different growing environments. Sixty-three ginsenosides were used as reference standards for accurate qualitative analysis. Cluster analysis was used to analyze the differences in main components and clarified the influence of growth environment factors on P. ginseng compounds. A total of 312 ginsenosides were identified in four types of P. ginseng, among which 75 were potential new ginsenosides. The number of ginsenosides in L15 was the highest, and the number of ginsenosides in the other three groups was similar, but it was a great difference in specie of ginsenosides. The study confirmed that different growing environments had a great influence on the constituents of P. ginseng, and provided a new breakthrough for the further study of the potential compounds in P. ginseng.

A Rapid Determination of 30 Prohibited Pesticide Residues in Lycii Fructus by UPLC-MS/MS

Prohibited pesticide residues have become one of the main factors affecting the quality and safety of Lycii Fructus, However, rarely studies focus on the rapid determination of these residues. Here, a total of 30 kinds of prohibited pesticide residues were determined by ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-MS/MS) in five different process ways. Pretreatment methods, chromatographic separation and detection conditions in mass spectrometry were all optimized accordingly. Among the five different pretreatment methods, the first and third solid phase extraction failed to provide high recoveries of sulfosulfuron compounds (both lower than 60%). Recovery of chlorphenamidine by the Quick Easy Cheap Effective Rugged and Safe multiresidue method (QuEChERS) was lower than 60%, which did not meet the requirements of trace determination. The concentrations of 30 prohibited pesticides residues treated by straightforward and solid phase extraction showed good linearity in their corresponding ranges, with correlation coefficients over 0.99. The average recoveries of straightforward ranged from 78.13% to 110.9%, while RSD ranged from 1.3% to 16.9%, albeit poor purification was observed. The recovery yield from solid phase extraction was between 67.75% and 103.08% with RSD value from 0.8% to 14.0%, which met the requirements of trace determination, this method has good precision and stability. These results could be employed to other Traditional Chinese Medicines (TCMs) in detecting prohibited pesticide residues.

Visual authentication of steroidal saponins in Allium macrostemon Bge. and Allium chinense G. Don using MALDI-TOF imaging mass spectrometry and their structure activity relationship

Allium macrostemon Bge. (AMB) and Allium chinense G. Don (ACGD), both named Allii Macrostemonis Bulbus or Xiebai in Chinese, belong to the same genus. They are both used as edible vegetables and medicinal herbs for the treatment of atherosclerosis. So far, comparative analysis of the spatial metabolomes and platelet aggregation function of steroidal saponins has rarely been performed. In this study, high mass resolution matrix-assisted laser desorption/ionization time-of-flight imaging mass spectrometry (MALDI-TOF MSI) were performed on the fresh bulbs of these two herbs for the spatial distribution of steroidal saponins, and ultra-high-performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC/TQD-MS) was used to quantify their content levels. Platelet inhibitory activity induced by arachidonic acid, adenosine diphosphate and collagen were fully investigated. A total of 53 differential variables with compounds were identified or tentatively characterized between AMB and ACGD samples by UHPLC/TQD-MS. Furthermore, these steroidal saponins were almost distributed in tunic and outer scale regions of AMB, while they were rich in tunic and whole leaf scale, and rarely in developing flower bud of ACGD bulbs. Quantitative results demonstrated several saponins were the unique components for AMB (macrostemonoside I, timosaponin B-II, macrostemonoside F, etc.) and ACGD (chinenoside I, chinenoside II, chinenoside IV, etc.). Moreover, spirostanol saponins exhibited stronger inhibitory platelet activity than furostanol saponins, and the structure activity relationship was further summarized. In conclusion, high-mass resolution MALDI full-scan MSI provides abundant spatial distribution information of steroidal saponins in AMB and ACGD. And we finished the discussion of their activity characteristics. These findings would benefit the understanding of safety and effectiveness for their edible and medicinal use.

Conduction of a chemical structure-guided metabolic phenotype analysis method targeting phenylpropane pathway via LC-MS: Ginkgo biloba and soybean as examples

The phenylpropane pathway (PPP) is one of the most extensively investigated metabolic routes. This pathway biosynthesizes many important active ingredients such as phenylpropanoids and flavonoids that affect the flavor, taste and nutrients of food. How to elucidate the metabolic phenotype of PPP is fundamental in food research and development. In this study, we designed a structural periodical table filled with 103 metabolites produced from PPP. All of them especially the 62 structural isomers were qualified and quantified with high resolution and sensitivity via multiple reaction mode in liquid chromatography tandem triple quadrupole mass spectrometry. Ginkgo biloba and soybean were used as samples for the practical application of this method: The delicate spatial-temporal metabolic balance of PPP from ginkgo biloba has been first elucidated; It is first confirmed that the salt and draught stresses could redirect the biosynthesis trend of PPP to produce more isoflavones in soybean leaves.

A novel single step solid-phase extraction combined with bromine derivatization method for rapid determination of acrylamide in coffee and its products by stable isotope dilution ultra-performance liquid chromatography tandem triple quadrupole electrospray ionization mass spectrometry

This work aimed to establish a novel determination method for acrylamide in coffee and its products by ultra-performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Acrylamide in samples were prepared by a single-step solid-phase extraction clean-up using mixed mode sorbents. The bromine derivatization efficiency of acrylamide and its internal standard were improved at an acidic condition. After derivation, the retention capability of acrylamide and its resistance to interference were significantly improved. The limit of detection (LOD) and the limit of quantification (LOQ) were 1.2 and 4 μg/kg for roasted and instant coffees, while they were 0.24 and 0.8 μg/kg for ready-to-drink coffees. The average recoveries for acrylamide ranged from 99.3 to 102.2% in coffee and its products. All the results showed that the developed method was simple, quick, specific and suitable for screening and determination of acrylamide in batch samples of coffee and its products.

Simultaneous Determination of Levamisole, Mebendazole, and the Two Metabolite Residues of Mebendazole in Poultry Eggs by High-Performance Liquid Chromatography–Tandem Mass Spectrometry

The quantitative determination of levamisole (LMS), mebendazole (MBZ), and the two metabolites of MBZ, 5-hydroxymebendazole (HMBZ) and 2-amino-5-benzoylbenzimidazole (AMBZ), in poultry eggs (hen, duck, and goose) was achieved with high-performance liquid chromatography–tandem triple quadrupole mass spectrometry (HPLC–MS/MS). Samples were pretreated by liquid–liquid extraction and solid-phase extraction (LLE–SPE) to extract the target compounds, and an Oasis MCX SPE column was used for purification. Determination was performed on an Xbridge C18 column with 0.1% formic acid aqueous solution and acetonitrile as mobile phases. LMS, MBZ, HMBZ, and AMBZ were detected in a triple-quadrupole mass spectrometer with ESI in positive mode and quantified with an external standard. In blank eggs, the target analyte concentrations were within the limits of quantification (LOQs)—25 μg/kg (LMS) and 150 μg/kg (MBZ, HMBZ, and AMBZ)—and the matrix-matched calibration curves had good linearity (R2 ≥ 0.9990). In the same concentration range, the average recoveries of the target analytes were 85.98–97.38% (n = 6); the relative standard deviation (RSD), intraday RSD, and interday RSD ranged from 2.06 to 4.22%, 1.40 to 5.85%, and 2.34 to 6.32%, respectively. The limits of detection (LODs) ranged from 0.03 to 0.33 µg/kg, and the LOQs ranged from 0.08 to 1.00 µg/kg. Experimental verification showed that the HPLC–MS/MS method exhibited high specificity and sensitivity for quantitative analyses of egg samples. This study provides a rapid, efficient, and sensitive method for the simultaneous detection of LMS, MBZ, HMBZ, and AMBZ residues in foods of animal origin.

Synthesis of superparamagnetic MnFe2O4/mSiO2 nanomaterial for degradation of perfluorooctanoic acid by activated persulfate.

In this paper, magnetic MnFe2O4/mSiO2 nanocomposites were successfully synthesized, and the activation performance of the materials for persulfate was evaluated by the degradation efficiency of perfluorooctanoic acid. The structure of the catalyst was proved to be a core-shell structure by several characterization methods. The mesoporous silicon coating can effectively avoid the agglomeration of MnFe2O4 and at the same time increase the contact area with the reactants. A comparison of different catalyst addition conditions demonstrates that MnFe2O4/mSiO2 can effectively activate the persulfate. The optimal reaction conditions were investigated by several key influencing factors. It was experimentally demonstrated that about 90% of PFOA (10mg·L-1) could be decomposed under the conditions of 0.4g·L-1 MnFe2O4/mSiO2 and PS, pH 5.68, and 25°C within 4h; the defluorination rate reached 58.33%. In addition, the cyclability and stability tests demonstrated that MnFe2O4/mSiO2 is a stable material that can be recycled. Furthermore, XPS characterization and radical scavenging experiments demonstrated that sulfate radicals (SO4·-) and hydroxyl radicals (OH) play a major role in the reaction of MnFe2O4/mSiO2 activated PS. Subsequently, the degradation products were detected by high-performance liquid chromatography tandem triple quadrupole mass spectrometry, indicating that the degradation of PFOA is a gradual process of defluorination and decarbonization in the presence of free radicals. Finally, the metal leaching rate is tested to prove that the material meets environmental requirements while reacting efficiently. In conclusion, this study shows that MnFe2O4/mSiO2 is an easily recoverable and highly efficient and stable material that has great potential for PS activation to treat organic pollutants in water.

Quantification of major milk proteins using ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry and its application in milk authenticity analysis

An authenticity-based method was developed to detect economically motivated adulteration (EMA) in milk. To achieve this goal, 46 peptides released from trypsin digested milk proteins were identified and marker peptides were further selected based on their digestibility and stability characteristics to allow further quantifications of the milk proteins in the original samples using an isotope-dilution ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQMS). Furthermore, to detect EMA in milk samples, 22 authentic milk powder samples were analyzed using the isotope-dilution UPLC-TQMS, and an authenticity threshold value --70.26 g/100 g as the content of all five major milk proteins to total protein—was established to evaluate milk samples with high adulteration risks. Finally, the threshold value was validated using soy protein adulterated milk samples. Results showed that the reported authenticity-based method was sensitive enough to detect the tampering of milk samples with as little as 10 % soy protein added. It is clear that this newly developed authenticity-based detection method is a powerful tool to detect unknown adulteration risks in milk samples. • Selected 5 marker peptides from 46 identified peptides based on their digestibility and stability characteristics. • Established and validated an isotope-dilution UPLC-TQMS method to quantitatively analyze major milk proteins. • Developed a milk authenticity threshold value to detect milk adulterations. • Validated the threshold value using artificially adulterated milk samples.

Quality Evaluation of Taxilli Herba from Different Hosts Based on Simultaneous Determination of Multiple Bioactive Constituents Combined with Multivariate Statistical Analysis.

Taxilli Herba (TAXH) is an important traditional Chinese medicine with a long history, dating from the Eastern Han Dynasty to the present times. However, the active constituents in it that parasitize different hosts vary, affecting its clinical efficacy. Given the complexity of the host origins, evaluating the quality of TAXH is critical to ensure the safety and effectiveness of clinical medication. In the present study, a quantitative method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC-QTRAP-MS/MS) was established, which simultaneously determined the content of 33 active constituents, including 12 flavonoids, 4 organic acids, 12 amino acids, and 5 nucleosides in 45 samples. Orthogonal partial least squares discriminant analysis (OPLS-DA) was employed to classify and distinguish between TAXH and its adulterants, Tolypanthi Herba (TOLH). A hierarchical clustering analysis (HCA) was conducted combined with a heatmap to visually observe the distribution regularity of 33 constituents in each sample. Furthermore, gray relational analysis (GRA) was applied to evaluate the quality of samples to get the optimal host. The results demonstrated that TAXH excelled TOLH in quality as a whole. The quality of TAXH parasitizing Morus alba was also better, while those that were parasitic on Cinnamomum camphora and Glyptostrobus pensilis had relatively poor quality. This study may provide comprehensive information that is necessary for quality control and supply a scientific basis for further exploring the quality formation mechanism of TAXH.

Effects of Xian-Ling-Gu-Bao capsule on the gut microbiota in ovariectomized rats: Metabolism and modulation

Xian-Ling-Gu-Bao capsule (XLGB) has been proven to prevent and treat osteoporosis. However, as a long-term oral formula, XLGB's effects on the metabolic capacity, structure and function of gut microbiota have yet to be elucidated in ovariectomized (OVX) rats. Our objectives were to evaluate the capacity of gut microbiota for metabolizing XLGB ingredients and to assess the effect of this prescription on gut microbiota. Herein, an integrated analysis that combined ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF-MS) and ultrahigh-performance liquid chromatography tandem triple quadrupole mass spectrometry (UPLC-TQD-MS) was conducted to determine the metabolic capacity of gut microbiota. The effects of XLGB on gut microbiota were explored by metagenomic sequencing in OVX rats. Fecal samples from each group were collected after intragastric administration for three months. In total, 64 biotransformation products were fully characterized with rat gut microbiota from the OVX group and the XLGB group. The deglycosylation reaction was the main biotransformation pathway in core structures in the group that was incubated with XLGB. Compared with the OVX group, different biotransformation products and pathways of the XLGB group after incubation for 2 h and 8 h were described. After three months of feeding with XLGB, the domesticated gut microbiota was conducive to the production of active absorbed components via deglycosylation, such as icaritin, psoralen and isopsoralen. Comparisons of the gut microbiota of the OVX and XLGB groups showed differences in the relative abundances of the two dominant bacterial divisions, namely, Firmicutes and Bacteroidetes. The proportion of Firmicutes was significantly lower and that of Bacteroidetes was significantly higher in the XLGB group. This result demonstrated that XLGB could provide a basis for the treatment of osteoporosis by regulating lipid and bile acid metabolism. In addition, the increase in Lactobacillus, Bacteroides and Prevotella could be an important factor that led to easier production of active absorbed aglycones in the XLGB group. Our observation provided further evidence of the importance of gut microbiota in the metabolism and potential activity of XLGB.

Quantification of major royal jelly proteins using ultra performance liquid chromatography tandem triple quadrupole mass spectrometry and application in honey authenticity

Major royal jelly proteins (MRJPs) have been used as endogenous biomarkers for honey authentication. However, no study on quantification of MRJPs in honey authentication was reported. In order to develop a new honey authenticity method based on quantitative analysis of MRJPs in honey, trypsin-digested peptides from MRJPs were examined by mass spectrometry (MS). Three peptides, YNGVPSSLNVISK, TLQMIAGMK, and LTVAGESFTVK were selected as specific markers for MRJP1, MRJP 2 and MRJP 3(MRJP1∼3), respectively. An ultra performance liquid chromatography-triple quadrupole MS (UPLC-TQMS) method used for quantification of MRJPs in honey was developed and validated. A database on the MRJP1∼3 contents of honey established by quantification of 70 authentic samples showed a potential application in honey authenticity identification. The analysis results of 10 commercial honey samples by UPLC-TQMS with reference to the MRJP1∼3 database of authentic honey were compared and confirmed by widely used isotope ratio mass spectrometry, a AOAC Official Method. In conclusion, the results showed our method, a proteomic-based UPLC-TQMS method, has a high accuracy, sensitivity and specificity for honey authenticity.

Simultaneous determination and quantitation of hypolipidemic drugs in fingerprints by UPLC-Q-TRAP/MS

An ultra-performance liquid chromatography tandem triple quadrupole compound linear ion trap mass spectrometry (UPLC-Q-TRAP/MS) method was developed and validated for the detection of hypolipidemic drugs in fingerprints. 13 hypolipidemic drugs were well separated by the gradient elution of 0.01% formic acid in water and methanol at a flow rate of 0.4 mL/min within 11 min. The analytes were detected in positive (ESI+) and negative (ESI-) modes and scanned using scheduled multiple reaction monitoring-information dependent acquisition-enhanced product ion (SMRM-IDA-EPI) for best selectivity and sensitivity. The calibration curves showed good linearity in the range of 0.050–50.000 ng/patch with coefficients (r2) higher than 0.9904 for all analytes. Meantime, the LODs and LLOQs were in ranges of 0.001–0.034 and 0.003–0.050 ng/patch. The accuracies, intra-day and inter-day precision ranged from −13.3 to 0.3%, 1.1–10.4% and 3.7–14.5%, respectively. The recoveries ranged from 79.9 to 114.8%, while the absolute and relative matrix effects were in the range of 83.0–107.2% and 2.2–9.7%. By comparing the non-spiked fingerprints from healthy volunteers with the fingerprints obtained from patients, demonstrated that the method was competent for determination and quantitation of hypolipidemic drugs in fingerprints.

Pharmacokinetics and biotransformation investigation in beagle dog of active compounds from naoxintong capsule

Naoxintong Capsule (NXTC), a standardized herbal medicine, has been widely applied in treating cardiovascular and cerebrovascular diseases with remarkable efficacy. However, the efficacy contributing components of NXTC are unclear, and the in vivo absorption and metabolism processes of NXTC remain largely obscured. In this study, using beagle dog as model species, we have identified and tentatively characterized 25 prototype and 15 catabolites of NXTC in beagle dog plasma by ultra-fast liquid chromatography/quadrupole-time-of-flight tandem mass spectrometry (UFLC-Q-TOF-MS/MS). We have proposed the in vivo bio-transformation pathways of these absorbed constituents. In addition, for six crucial components, we have developed a quantitative method and conducted plasma pharmacokinetic study of these six components by rapid resolution liquid chromatography tandem triple quadrupole mass spectrometry (RRLC-QQQ-MS/MS). In conclude, our study provided comprehensive insights into the understanding of the plasma absorbed components profiling of NXTC as well as their in vivo transformation behaviors, which would be of great value for identifying efficacy contributing critical components as well as mechanism related investigations of NXTC in the future.

Enantioseparation and stereoselective dissipation of the novel chiral fungicide pydiflumetofen by ultra-high-performance liquid chromatography tandem mass spectrometry

Pydiflumetofen is a novel and efficient broad-spectrum chiral fungicide consisting of a pair of enantiomers. A simple and sensitive chiral analytical method was established to determine the enantiomers of this chiral fungicide in food and environmental samples by ultra-high-performance liquid chromatography tandem triple quadrupole mass spectrometry (UHPLC-MS/MS) using QuEChERS method coupled with octadecylsilane-dispersive solid-phase extraction (C18-dSPE) as extraction procedure. The specific optical rotation and the absolute configuration of the enantiomers were identified by polarimetry and electronic circular dichroism (ECD). The elution order of the pydiflumetofen enantiomers on Lux Cellulose-2 was S-(−)-pydiflumetofen and R-(+)-pydiflumetofen. The average recoveries of eleven matrices ranged from 71.3% to 107.4%. The intraday relative standard deviations (RSDs) were less than 11.8%, and the interday RSDs were less than 12.6% for the two enantiomers. Stereoselective dissipation in pakchoi and soil were observed: S-(−)-pydiflumetofen was degraded faster than R-(+)-pydiflumetofen in pakchoi, causing the enantiomer fraction (EF) of the enantiomers to change from 0.50 to 0.42 in 7 days. However, R-(+)-pydiflumetofen was degraded faster than S-(−)-pydiflumetofen in soil, causing the EF of the enantiomers to change from 0.49 to 0.52 in 21 days. This study provides a method for monitoring pydiflumetofen enantiomer residues, which is crucial for improving risk assessments and the development of chiral pesticides.

Simultaneous Quantitative Analysis of Multiple Biotransformation Products of Xian-Ling-Gu-Bao, a Traditional Chinese Medicine Prescription, with Rat Intestinal Microflora by Ultra-Performance Liquid Chromatography Tandem Triple Quadrupole Mass Spectrometry.

Xian-Ling-Gu-Bao (XLGB), a famous traditional Chinese medicine prescription consisted of six herbal medicines, was used for prevention and treatment of osteoporosis in China. As an oral formulation, the multiple components contained in XLGB were inevitably biotransformed by the intestinal microflora before absorption via the gastrointestinal tract. However, the dynamic profiles of biotransformation products of XLGB remain unknown. In this paper, a rapid and sensitive ultra-performance liquid chromatography tandem triple quadrupole mass spectrometry method was developed for the simultaneous quantitative analysis of multiple biotransformation products of XLGB with rat intestinal microflora. For 10 selected quantitative compounds, all calibration curves revealed good linearity (r2>0.99) within the sampling ranges considered. The whole intra- and inter-day precisions (as relative standard deviation) of all analytes were <13.5%, and the accuracies (as relative error) were in the range from -11.3 to 11.2%. The lower limits of quantification were 20, 10, 5, 20, 2, 2, 2, 5, 2 and 2ng/mL for sweroside, timosaponin BII, epimedin C, asperosaponin VI, psoralen, isobavachin, icariside II, timosaponin AIII, isobavachalcone and icaritin, respectively. The matrix effects, extraction recoveries and stabilities were all satisfactory. Meanwhile, dynamic profiles of 21 additional biotransformation products were also monitored by their area-time curves. The analytical method was successfully applied to describe dynamic profiles of 31 biotransformation products of XLGB and the recipes with removal of a definite composed herbal medicine (Anemarrhenae Rhizoma or Rehmanniae Radix).

Dynamic Variations in Multiple Bioactive Constituents under Salt Stress Provide Insight into Quality Formation of Licorice.

The demand for licorice and its natural product derivatives in domestic and foreign market is considerably huge. The core production areas of licorice are covered with salinity and drought land in northwestern China. Studies have shown that suitable environmental stress can promote the accumulation of glycyrrhizin and liquiritin to improve its quality as medicinal materials. However, there are few reports on other bioactive constituents of licorice, not to mention their dynamic accumulation under stressed conditions. To explore the quality formation of licorice from the perspective of salt influence, a reliable method based on ultra-fast liquid chromatography tandem triple quadrupole mass spectrometry (UFLC–MS/MS) was established for simultaneous determination of sixteen bioactive constituents, including triterpenoids, flavonoids, chalcones and their glycosides. Physiological experiments were performed to investigate salt tolerance of licorice under different salinity treatments. The expressions of crucial genes (bAS and CHS), key enzymes of triterpenoid and flavonoid synthesis, were also tested by qRT-PCR. Our study found that 50 mM NaCl treatment (low stress) was the most favorable to promote the accumulation of bioactive constituents in the long term, without harming the plants. Flavonoid accumulation of non-stressed and low-stressed groups became different in the initial synthesis stage, and glycosyltransferases may have great influence on their downstream synthesis. Furthermore, bAS and CHS also showed higher levels in low-stressed licorice at harvest time. This work provides valuable information on dynamic variations in multiple bioactive constituents in licorice treated by salt and insight into its quality formation under stressed conditions.