Performance Liquid Chromatography-linear Ion Research Articles

Separation and identification of the synthetic cathinone isomers dipentylone and N-ethylpentylone using chromatographic and mass spectral characteristics

Synthetic cathinone isomers exhibit very similar structure and physicochemical properties, which makes them unable to be effectively separated and identified, therefore increasing the likelihood of misidentification. In this study, the chromatographic and mass spectral characteristics of a sample by gas chromatography-mass spectrometry (GC–MS) were highly like those of N-ethylpentylone. The powder, however, was finally identified as dipentylone (also known as N, N-dimethylpentylone) by high performance liquid chromatography-linear ion trap-orbitrap mass spectrometry (HPLC-LTQ-Orbitrap-MS) and nuclear magnetic resonance (NMR). Because of the possibility of misidentification, the collision-induced dissociation (CID) and electron ionization (EI) pathways of dipentylone and its isomer N-ethylpentylone were further examined to assist forensic laboratories in identifying the synthetic cathinone isomers in their case work. The separation of the two isomers was successfully achieved through ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS/MS). These results demonstrate that the methods developed in this study can be used to separate and identify the two isomers.

Integrated transcriptomics and lipidomics investigation of the mechanism underlying the gastrointestinal mucosa damage of Loropetalum chinense (R.Br.) and its representative component

BackgroundLoropetalum chinensis (R.Br) Oliv (Bhjm), a Chinese folk herbal medicine, was traditionally used in the treatment of wound bleeding and skin ulcers. A new drug named JIMUSAN granules used for gastrosia was developed by our group, and clinical trials have been approved. However, as the principal herb, the material basis and underlying mechanisms of Bhjm in attenuating gastrointestinal mucosa damage (GMD) remain to be systemically illuminated. PurposeAn integrated strategy was used to explore the therapeutic effects and mechanisms of Bhjm and ellagic acid (EA) on GMD zebrafish, using network pharmacology, transcriptomics, lipidomics, and real-time quantitative PCR (RT-qPCR) verification. MethodsFirst, network pharmacological analysis was used to infer the major effective constituents and targets of Bhjm. Ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap HRMS) and ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) were employed to identify the chemical constituents and quantify the different types of constituents. Second, zebrafish model of GMD was established by using 2,4,6-trinitrobenzenesulfonic acid (TNBS) to evaluate the efficacy of Bhjm and EA. The potential mechanism was examined by integrated transcriptomics and lipidomics analysis. Finally, validation tests were implemented using RT-qPCR. ResultsIn this study, targets indentified by network pharmacology were related to inflammation and mucosal damage. Ten representative components that interacted with these targets were simultaneously determined by UHPLC-MS/MS. Sixty four compounds were identified or tentatively characterized, most of which were flavonoids and polyphenols. Bhjm and EA alleviated mucosal damage and reduced inflammation in a TNBS-induced zebrafish GMD model, indicating that EA was the main active compounds. Eight common differentially expressed genes were downregulated by Bhjm and EA, as determined by transcriptomics analysis. Lipidomics analysis confirmed 12 differential lipids, including phosphatidylcholine (PC) and triglyceride (TG). Further network enrichment analysis demonstrated that differential lipid metabolism was regulated by klf4 and hist1h2ba, and were validated by RT-qPCR. ConclusionIn our study, the chemical profile of Bhjm was clarified. Moreover, the GMD repair effect and the mechanism of Bhjm and EA was comprehensively analyzed for the first time, involving inflammation and lipid metabolism. Collectively, these findings will be significantly helpful for deeply exploring the clinical application value of Bhjm.

Rapid determination of 22 functional components in cosmetics using ultra-high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry

建立了超高效液相色谱-线性离子阱/静电场轨道阱高分辨质谱法同时测定化妆品中的22种功效成分。样品采用甲醇超声提取,C18色谱柱(100 mm×2. 1 mm, 1. 8 μm)分离,以0.1%(v/v)甲酸水溶液和乙腈为流动相进行梯度洗脱。在正离子模式下,以保留时间和一级母离子精确质量数进行定量分析,以高能碰撞诱导解离获得的二级碎片离子精确质量数进行确证。结果表明,该方法线性关系良好,检出限(LOD)为0.003~2.01 mg/kg;定量限(LOQ)为0.02~4.36 mg/kg;水、乳、霜3种基质中3个添加水平的回收率范围为63.2%~125.1%,相对标准偏差为0.18%~10.9%。对标示含有烟酰胺、抗坏血酸葡糖苷、咖啡因、泛醇及甘草类(光果甘草根茎叶、甘草根、胀果甘草根)、麦冬根、人参根、黄芪根、虎杖根、苦参根、地黄根、积雪草、茶叶提取物的54批样品进行检测,标示单体功效成分的样品均有检出,标示不同植物提取物的46批样品中,24批检出植物提取物的功效成分。该方法简便快速、定性定量可靠,适用于化妆品中22种功效成分的定量测定。

Identification of the New Psychoactive Substance Eutylone.

To establish a method to identify unknown sample based on the combined use of Fourier transform infrared spectroscopy (FTIR), gas chromatography-quadrupole time-of-flight mass spectrometry (GC-QTOF-MS), ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry (UPLC-LTQ-Orbitrap MS) and 1H-nuclear magnetic resonance spectroscopy (1H-NMR) technique. The unknown sample was directly analyzed by FTIR. The unknown sample was dissolved in methanol solution containing internal standard SKF525A and the supernatant was detected by GC-QTOF-MS and UPLC-LTQ-Orbitrap MS. The unknown sample was dissolved in methanol-d4 solution for structural analysis of 1H-NMR. The characteristic absorption peaks of FTIR spectra obtained from unknown sample were 1 682 (C=O bond), 1 503, 1 488, 1 436, 1 363, 1 256, 1 092, 1 035, 935, 840 and 800 cm-1, the characteristic fragment ions (m/z) of GC-QTOF-MS were 86.096 4 (base peak), 58.065 1, 149.023 5, 121.028 6 and 65.038 6, the accurate mass [M+H]+ detected by UPLC-LTQ-Orbitrap MS was 236.127 7. The sample was identified as synthetic cathinone new psychoactive substance Eutylone by 1H-NMR. The method established in this study can be used for structural confirmation of Eutylone.

Mechanism of Huangqi Guizhi Wuwu Decoction in treatment of rheumatoid arthritis based on UPLC-LTQ-Orbitrap-MS, network pharmacology, and cell experiment

In this study, ultra-high performance liquid chromatography-linear ion trap/electrostatic field orbit trap combined-type mass spectrometry(UPLC-LTQ-Orbitrap-MS) was used to analyze the main active components of Huangqi Guizhi Wuwu Decoction(HQGZ). A total of 50 active components were identified from HQGZ and 108 potential targets of the components related to the treatment of rheumatoid arthritis were retrieved based on network pharmacology, including 87 key targets, followed by Gene Ontology(GO) term enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment of the targets. The result indicated that HQGZ may exert therapeutic effects mainly through the sphingolipid signaling pathway, tumor necrosis factor(TNF) signaling pathway, as well as the positive regulation of ribonucleic acid(RNA) polymerase Ⅱ promoter transcription, inflammatory response and other biological processes. At the same time, cell experiment was performed to verify the key proteins in the TNF signaling pathway. The results demonstrated that HQGZ significantly reduced the expression of caspase-3(CASP3), TNF, relaxed(RELA) protein, and IkappaB kinase beta(IKBKB) in fibroblast-like synoviocytes induced by TNF-α. The results of UPLC-LTQ-Orbitrap-MS, network pharmacology and cell experiment showed that the active components in HQGZ may inhibit inflammatory response and regulate immune function and cell apoptosis by modulating key proteins in TNF signaling pathway to treat rheumatoid arthritis.

Green Pea (Pisum sativum L.) Hull Polyphenol Extracts Ameliorate DSS-Induced Colitis through Keap1/Nrf2 Pathway and Gut Microbiota Modulation.

As a processing by-product, green pea hull (GPH) was found to be rich in phenolic components in our previous studies. In this study, UHPLC-LTQ-OrbiTrap-MS (Ultra performance liquid chromatography-linear ion trap orbitrap tandem mass spectrometry) technique was used to quantify polyphenols, and DSS (sodium dextran sulfate)-induced colitis mouse model was established to explore the effect of GPH extracts on colitis. The results showed that quercetin and its derivatives, kaempferol trihexanside and catechin and its derivatives were the main phenolic substances in the extract, reaching 2836.57, 1482.00 and 1339.91 µg quercetin/g GPH extract, respectively; GPH extracts can improved inflammatory status, repaired colonic function, regulated inflammatory factors, and restored oxidative balance in mice. Further, GPH extracts can activate Keap1-Nrf2-ARE signaling pathway, regulate downstream antioxidant protease and gut microbiota by increasing F/B value and promoting the growth of Lactobacillaceae and Lachnospiraceae, and improve the level of SCFAs (short-chain fatty acids) to relieve DSS-induced colitis in mice. Therefore, GPH may be a promising dietary resource for the treatment of ulcerative colitis.

Rapid identification of chemical constituents of Rhodiola crenulata using liquid chromatography-mass spectrometry pseudotargeted analysis.

Rhodiola crenulata (R. crenulata), is a famous traditional Chinese medicine, with observable effects such as anti-high-altitude illness and fatigue resistance. Nevertheless, comprehensive and systematic structural identification of its components remains a challenge. In this study, a pseudotargeted analytical method, involving key fragment filtering by ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry and ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry, was developed for rapid detection and identification of the chemical constituents of R. crenulata. The process consists of three steps: (i) acquiring sufficient mass spectral data, (ii) constructing a key fragments schedule and discovering the substructures rapidly by pseudotargeted key fragment filtering, and (iii) further identification of the compound structures based on accurate masses, fragment ions, related literatures, and authentic standards. As a result, 104 compounds were identified and divided into five categories, among which three potentially new and 59 ones were reported for the first time in R. crenulata. These results indicated that the major types of components are flavanols and gallic acid derivatives, organic acids, alcohols and their glycosides, flavonoids and their glycosides. This study enhances the understanding of R. crenulata and provides a reference for rapid and comprehensive identification of constituents in other herbal medicines.

Identification of New Psychoactive Tryptamines 4-OH-MET and 4-AcO-DMT Using High Resolution Mass Spectrometry and Nuclear Magnetic Resonance Spectroscopy

Objective To detect the uncontrolled new psychoactive tryptamines involved in drug-related cases with high resolution mass spectrometry and nuclear magnetic resonance spectroscopy. Methods White and brown powder obtained in actual cases were extracted and analyzed by gas chromatography-quadrupole time-of-flight mass spectrometry （GC-QTOF-MS）, ultra-high performance liquid chromatography-linear ion trap quadrupole-orbitrap mass spectrometry （UPLC-LTQ-Orbitrap MS） and 1H-nuclear magnetic resonance spectroscopy （1H-NMR）. Results After detection by GC-QTOF-MS, the components of white powder showed main characteristic fragment ion peaks at m/z 218.141 0 （molecular ion peak）, 72.080 6 （base peak）, etc. After detection by UPLC-LTQ-Orbitrap MS, its protonated molecular ion was m/z 219.149 4. The main ions in the secondary mass spectrum under the collision-induced dissociation （CID） mode were m/z 160.076 3 and 72.080 8. After detection by GC-QTOF-MS, the components of brown powder showed main characteristic fragment ion peaks at m/z 246.135 7 （molecular ion peak）, 58.065 1 （base peak）, etc. After detection by UPLC-LTQ-Orbitrap MS, its protonated molecular ion was m/z 247.145 0. The main ions in the secondary mass spectrum under CID mode were m/z 202.087 1, 160.076 3 and 134.060 5. NIST 17 library retrieval and 1H-NMR confirmed that the white powder and brown powder contained new psychoactive tryptamines 4-OH-MET and 4-AcO-DMT, respectively. Conclusion GC-QTOF-MS, UPLC-LTQ-Orbitrap MS and 1H-NMR can be used together to identify unknown new psychoactive substances.

Determination of metabolites of phloretin in rats using UHPLC-LTQ-Orbitrap mass spectrometry

Purpose: To study the metabolites of phloretin in vivo using ultra-high performance liquid chromatography linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap).
 Methods: After administration of phloretin (50 mg/kg; oral route) to six rats, blood samples were taken from each animal. Each sample was then subjected to solid-phase extraction to prepare it for chromatographic/spectroscopic analysis. Finally, each sample was analyzed using UHPLC-LTQOrbitrap with a negative-mode electrospray ionization source.
 Results: Based on mass measurements, chromatographic retention times, and MS2 fragmentation ions, we detected and identified phloretin and 16 metabolites of the drug in vivo in rats. Metabolic reactions of phloretin included glucosylation and glucuronide conjugation, diglucuronide conjugation, glucosylation and sulfate conjugation, sulfate conjugation, glucuronide conjugation, and glucosylation and hydroxylation.
 Conclusion: The findings provide a better understanding of phloretin metabolism and metabolites, and new information about their effective forms, pharmacological actions, metabolic fate, and toxic actions in vivo.

Diagnostic product ions-based chemical characterization and antioxidative activity evaluation of solid fermentation for Astragali radix produced by Paecilomyces cicadae

Studies on herbal medicines and fermentation products have become increasingly essential with the development of modern industry and technology. In order to verify that fermentation can bring about changes, Paecilomyces cicadae [Paecilomyces cicadae (Miquel.) Samson] was used to ferment Astragali radix [Astragalus membranaceus (Fisch.) Bge. var. mongho-licus (Bge.) Hsiao]. After solid fermentation for Astragali radix produced by Paecilomyces cicadae (SF-AP) was established, an efficient strategy based on ultra-high performance liquid chromatography-linear ion trap-Orbitrap mass spectrometry (UHPLC-LTQ-Orbitrap MS) was developed to screen and identify the chemical transformations in SF-AP and Astragali radix according to the acquired diagnostic product ions (DPIs). As a result, 114 compounds including 45 saponins and 69 flavonoids were finally identified and validated. Moreover, two kinds of antioxidative tests corresponding to the scavenging of DPPH· and ABTS·+ were applied to evaluate the antioxidative activity of Astragali radix before and after fermentation. The results demonstrated that some significant chemical transformations such as relative content fluctuations and structural isomerism owing to the occurrence of hydrolysis and conversion reactions and the antioxidative activity of SF-AP was much higher than that of the Astragali radix. This study could provide a new method for the utilization of Astragali radix and constructive guidance for the further research of fermented herbal medicines.

Serum metabonomics coupled with HPLC-LTQ/orbitrap MS and multivariate data analysis on the ameliorative effects of Bidens bipinnata L. in hyperlipidemic rats

Ethnopharmacological relevanceHyperlipidemia (HLP) is a prevalence chronic cardiovascular disease, which is treated by traditional Chinese medicine (TCM) in China. More and more attention has been paid to the application of metabolomics in the study of TCM. Bidens bipinnata L. (BBL), a folk medicine in contemporary China, has the efficacy in the treatment of hyperlipidemia (HLP) in China. However, little is known of the underlying mechanism of BBL. This research aimed to investigate ameliorative effects of BBL on hyperlipidemic rats and explore the mechanism by metabolomics method. Materials and methodsHyperlipidemic rats were established by high fat diet (HFD). Biochemical assay was used to evaluate the efficacy of BBL. A metabolomics approach based on high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry (HPLC-LTQ/orbitrap MS) was performed to analyze the serum biomarkers from model group, control group and BBL group. Principle component analysis (PCA) and partial least-squares discriminate analysis (PLS-DA) were utilized to identify differences of metabolic profiles in rats among the three groups. In order to identify possible pathways that were affected by HLP, the identified endogenous metabolites were analyzed by using MetaboAnalyst. In the network pharmacology study, our research group found that PPAR signaling pathway was the most important pathway of BBL in the treatment of HLP. Then, it was found that changes in the major metabolic pathways would affect the PPAR signaling pathway through comprehensive analysis based on KEGG database. Therefore, the expression of key genes in the PPAR signaling pathway was detected by real-time quantitative fluorescence PCR (RT-qPCR). ResultsSix metabolites, which showed a significantly restoring trend from HLP to normal condition, were regarded as potential biomarkers of BBL treatment. The levels of phosphorylcholine, mevalonic acid and leukotriene B4 (LTB4) increased significantly (P < 0.01) in hyperlipidemic rats, while the levels of linoleic acid, arachidonic acid (AA) and lysophosphatidylcholine (18:0) (Lyso PC (18:0)) decreased significantly (P < 0.01) in comparison with control rats. Those endogenous metabolites were chiefly involved in linoleic acid metabolism, AA metabolism and terpenoid backbone biosynthesis. According to the results of RT-qPCR analysis, the mRNA expressions of PPAR α, PPAR β and PPARγ in model group were difference compared with control group. And the expression difference could be regulated closer to normal level after BBL intervention. ConclusionsThe results of biochemical assay, serum metabolic pattern and RT-qPCR analysis showed that BBL could exert a significant improvement on lipid levels, liver function, renal function, as well as the mRNA expression level of PPAR signaling pathway.

Differences of antipyretic effect between Qingkailing soft capsule and hard capsule based on metabonomics

The antipyretic effects of Qingkailing soft capsules and hard capsules were compared based on metabonomics technology,so as to provide basis for clinical rational use and quality control evaluation of its preparations. By using ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry( UPLC-LTQ/Orbitrap),and multivariate tatistical methods such as principal component analysis( PCA),partial least squares-discriminate analysis( PLS-DA) and orthogonal signal correction partial least square discriminant analysis( OPLS-DA),the changes of plasma endogenous metabolites in rat fever model induced by dry yeast were studied,so as to look for biomarkers related to fever. Based on these results,the antipyretic effects of two types of Qingkailing preparations were compared. The results indicated that metabolic profiles of the experimental groups could be distinguished distinctly,and 8 endogenous metabolites showed differences as compared with the normal control group( P<0. 05),including nicotinic acid,choline,hippuric acid,phosphocholine,Lyso PC( 14 ∶ 0),Lyso PC [16 ∶ 1( 9 Z) ],Lyso PC( 18 ∶ 0) and Lyso PC [20 ∶ 3( 5 Z,8 Z,11 Z) ]. They could be regarded as biomarkers related to fever. Qingkailing soft capsule and hard capsule had different effects on the regulation of plasma metabolites in yeast-induced fever model rats. Qingkailing soft capsule had different degrees of callbacks on eight biomarkers,including significant callbacks on nicotinic acid( P<0. 05),hippuric acid( P< 0. 01),phosphocholine( P< 0. 05),and Lyso PC [20 ∶3( 5 Z,8 Z,11 Z) ]( P<0. 05),while hard capsule only had significant callbacks on nicotinic acid( P<0. 05),hippuric acid( P<0. 01),and phosphocholine( P< 0. 05),with no callbacks on choline,Lyso PC( 14 ∶ 0),Lyso PC [16 ∶ 1( 9 Z) ],Lyso PC( 18 ∶0),and Lyso PC [20 ∶3( 5 Z,8 Z,11 Z) ]. This indicated that Qingkailing soft capsule had better callback effect than hard capsule. In this study,Qingkailing soft capsules and hard capsules were used to intervene the changes of related biomarkers in yeast-induced fever rat models,and then the antipyretic effect and mechanism between these two kinds of capsules were compared. The two dosage forms played an antipyretic role mainly by regulating lipid metabolism and controlling inflammation. The callback effect of soft capsule on the potential biomarkers of lysophosphatidylcholine was significantly higher than that of hard capsule. The differences in antipyretic effect of Qingkailing soft capsule and hard capsule were expounded from the point of metabonomics,providing experimental data and theoretical basis for the selection of clinical dosage forms.

Rapid analysis of chemical components in Zizyphi Spinosae Semen by UHPLC-LTQ-Orbitrap-MS

This study aimed to analyze the chemical components of Zizyphi Spinosae Semen by using ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ-Orbitrap-MS). The chromatographic separation of the components was achieved on a Waters Acquity UPLC BEH-C₁₈ column (2.1 mm×50 mm, 1.7 μm), with acetonitrile-water (0.1% formic acid) as mobile phase at a flow rate of 0.3 mL·min⁻¹. Electrospray ionization-(ESI) source was applied and operated in negative ion mode. The parent ion list (PIL)-MS₂ and high energy collision dissociation (HCD) techniques were used to identify triterpene saponins in Zizyphi Spinosae Semen. Through analysis of the mass spectrometry data and literature data, 24 compounds and 4 unknown compounds were identified and inferred, including 21 flavonoids, 5 saponins and 2 triterpenoids. The use of UHPLC-LTQ-Orbitrap-MS technology provide an efficient and rapid analytical method for the qualitative analysis and quality control of the chemical components in Zizyphi Spinosae Semen. It can also provide reference data for the research on the material basis of pharmacodynamics and the resources development and utilization.

Rapid determination of 66 antibiotics in cosmetics using ultra high performance liquid chromatography-linear ion trap/Orbitrap mass spectrometry

A rapid screening and determination method, in conjunction with a database for confirmation, was established based on 66 antibiotic compounds using ultra-high performance liquid chromatography-linear ion trap/Orbitrap high resolution mass spectrometry (UPLC-LTQ/Orbitrap MS). The analytes were extracted with acetonitrile using ultrasonic extraction. The separation was performed on a C18 column (100 mm×2.1 mm, 1.8 μm) with a gradient elution of 0.1% (v/v) formic acid and acetonitrile. The retention time and accurate mass of the precursor ion were used for rapid screening in positive ionization mode, with the fragment ions obtained by higher energy collisional dissociation used for confirmation. The results indicated that each compound showed good linearity with a correlation coefficient of R2>0.99. The limits of detection (LODs) were in the range 2-4 μg/kg, and the limits of quantification (LOQs) were in the range 5-10 μg/kg. The average recoveries at three levels (1LOQ, 10LOQ, and 30LOQ) were between 58.2% and 119.1%, and the relative standard deviations (RSDs) were between 1.03% and 11.9%. The method is simple, rapid, reliable, and accurate, which is suitable for rapid screening and determination of the 66 antibiotic compounds in cosmetics.

Rapid determination of 15 nutrients in rice by ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry

A rapid method was developed for the simultaneous determination of 15 nutrients containing eight vitamin E, six γ-oryzanols and β-carotene in rice by ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UHPLC-LTQ/Orbitrap HRMS). The analytes were extracted by methanol containing 0.05% (v/v) 2,6-di-tert-butyl-4-methylphenol (BHT) under the ultrasonic condition, then separated by a Poroshell 120 PFP column (150 mm×3.0 mm, 2.7 μm) with a gradient elution program using 0.1% (v/v) formic acid aqueous solution and methanol containing 0.1% (v/v) formic acid as mobile phases. The detection was performed using an LTQ/Orbitrap HRMS detector with full scan in positive ion mode. Fifteen nutrients were simultaneously separated within 13 min. Furthermore, matrix effects of the white and brown rices were investigated. The correlation coefficients (r) were ≥ 0.9950 in the linear ranges of the 15 nutrients. The limits of detection (LODs, S/N=3) and limits of quantification (LOQs, S/N=10) of the 15 nutrients were 0.2-1.8 μg/L and 0.7-6.1 μg/L, respectively. At the three spiked levels, the recoveries were 73.2%-101.5% with the relative standard deviations (RSDs) ranging from 1.1% to 5.0% (n=3). The method is accurate, efficient and reliable. It is suitable for the simultaneous determination of various nutrients in rice.

Liquid chromatography-electrospray linear ion trap mass spectrometry analysis of targeted neuropeptides in Tac1−/− mouse spinal cords reveals significant lower concentration of opioid peptides

Tachykinin and opioid peptides play a central role in pain transmission, modulation and inhibition. The treatment of pain is very important in medicine and many studies using NK1 receptor antagonists failed to show significant analgesic effects in humans. Recent investigations suggest that both pronociceptive tachykinins and the analgesic opioid systems are important for normal pain sensation. The analysis of opioid peptides in Tac1−/− spinal cord tissues offers a great opportunity to verify the influence of the tachykinin system on specific opioid peptides. The objectives of this study were to develop an HPLC–MS/MRM assay to quantify targeted peptides in spinal cord tissues. Secondly, we wanted to verify if the Tac1−/− mouse endogenous opioid system is hampered and therefore affects significantly the pain modulatory pathways. Targeted neuropeptides were analyzed by high performance liquid chromatography linear ion trap mass spectrometry. Our results reveal that EM-2, Leu-Enk and Dyn A were down-regulated in Tac1−/− spinal cord tissues. Interestingly, Dyn A was almost 3 fold down-regulated (p<0.0001). No significant concentration differences were observed in mouse Tac1−/− spinal cords for Met-Enk and CGRP. The analysis of Tac1−/− mouse spinal cords revealed noteworthy decreases of EM-2, Leu-Enk and Dyn A concentrations which strongly suggest a significant impact on the endogenous pain-relieving mechanisms. These observations may have insightful impact on future analgesic drug developments and therapeutic strategies.

Identification of ustiloxins in false smut balls of rice based on high performance liquid chromatography-high resolution mass spectrometry

A sensitive method was developed for the simultaneous identification of five ustiloxins in the false smut balls of rice by high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (HPLC-LTQ/Orbitrap MS). The samples were extracted with deionized water under ultrasonic condition for 10 min, then purified by a strong cation exchange column (PCX). The ustiloxins were separated on an Xselect HSS T3 column (150 mm x 2.1 mm, 3.5 μm) by using 0.1% (v/v) formic acid water solution and methanol as mobile phases with gradient elution at a flow rate of 0.3 mL/min. The full scan range was m/z 200-1 000. The confirmatory analysis of the target compounds was carried out by the accurate mass of quasi-molecular ion, isotope abundance ratio and qualitative fragments. The results showed that the five ustiloxins (A, B, C, D and F) were identified from the false smut balls with mass accuracy less than 1 x 10(-6) (1 ppm) and the absolute values of the deviation of isotope abundance ratio were not more than 3.3%. The product ions were consistent with the theoretical fragment mode. The recoveries were 90% to 105%. This method is accurate and sensitive for the simultaneous identification of the five ustiloxins, which can provide technical means for the research of the ability in toxin producing by Ustilaginodea virens.

Rapid screening and identification of 22 allergenic disperse dyes in ecological textiles by high performance liquid chromatography- linear ion trap/orbitrap mass spectrometry

A rapid screening method based on high performance liquid chromatography-linear ion trap/orbitrap high-resolution mass spectrometry (HPLC-LTQ/Orbitrap MS) for 22 disperse dyes in ecological textiles has been established. The target compounds were extracted by pyridine/water (1:1, v/v) by shaking extraction in 90 degrees C water bath. The extracts were then separated by a CAPCELL PAK C18 column (100 mm x 2.0 mm, 5 μm) using gradient elution with acetonitrile-5 mmol/L ammonium acetate containing 0.01% (v/v) formic acid as mobile phases, and finally analyzed by HPLC-LTQ/Orbitrap in positive and negative ESI modes. The retention time and accurate mass of parent ion were used for fast screening of 22 disperse dyes, while the confirmatory analysis was obtained by fragments generated by collision-induced dissociation (CID) MS/MS. Target analysis exhibited high mass accuracy (< 5 x 10(-6)). Each target showed a good linearity in its own concentration range and the correlation coefficient was higher than 0.99. The LOQs were 0.125-2.5 mg/kg. Except for Disperse Yellow 49, the average recoveries of most disperse dyes at three spiked levels were 65%-120%, and the relative standard deviations (n = 6) were less than 15%. The method was applied for screening 40 different kinds of textiles, and Disperse Orange 37/76 was detected in one of them. With high selectivity and strong anti-jamming ability, this method is simple, rapid, accurate, and it can be used for the inspection of disperse dyes in textiles.

Direct analysis of 38 polyphenols in wine by ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry

Wine has been described previously as a rich source of polyphenols. However, an accurate screening of its complete phenolic profile is still lacking. In the present work, the analysis of 38 polyphenols in wine using ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UPLC-LTQ/Orbitrap MS) was explored. Wines were directly detected. The sample was loaded onto a Thermo Hypersil Gold C18 column (100 mmx 2.1 mm, 1.9 µm) using a gradient elution of acetonitrile/water containing 0. 1% (v/v) formic acid for the separation. UPLC-LTQ/Orbitrap mass spectrometer acquired full scan MS date for quantification, and data dependent MS2 product ion spectra for identification and/ or confirmation. The regression coefficients (R2) for the calibration curves (two orders of magnitude up to the lowest calibration level) in the study were ≥ 0.99. The limits of detection for the 38 compounds were 0.002-0.50 mg/kg. The average recoveries at three spiked levels were in the range of 90%-102% with the relative standard deviations (RSDs) of 0.51%-2.56%. Mass errors were always ≤ 5 ppm. This procedure was then successfully applied to the analysis of the polyphenols in wines.

Screening and confirmation of 24 hormones in cosmetics by ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry

A method of ultra high performance liquid chromatography-linear ion trap/orbitrap high resolution mass spectrometry (UPLC-LTQ/Orbitrap MS) was established to screen and confirm 24 hormones in cosmetics. Various cosmetic samples were extracted with methanol. The extract was loaded onto a Waters ACQUITY UPLC BEH C18 column (50 mm x 2.1 mm, 1.7 microm) using a gradient elution of acetonitrile/water containing 0.1% (v/v) formic acid for the separation. The accurate mass of quasi-molecular ion was acquired by full scanning of electrostatic field orbitrap. The rapid screening was carried out by the accurate mass of quasi-molecular ion. The confirmation analysis for targeted compounds was performed with the retention time and qualitative fragments obtained by data dependent scan mode. Under the optimal conditions, the 24 hormones were routinely detected with mass accuracy error below 3 x 10(-6) (3 ppm), and good linearities were obtained in their respective linear ranges with correlation coefficients higher than 0.99. The LODs (S/N = 3) of the 24 compounds were < or = 10 microg/kg, which can meet the requirements for the actual screening of cosmetic samples. The developed method was applied to screen the hormones in 50 cosmetic samples. The results demonstrate that the method is a useful tool for the rapid screening and identification of the hormones in cosmetics.