高效液相色谱-串联质谱法测定卷烟中的儿茶酚

建立了一种高效液相色谱-串联质谱(HPLC-MS/MS)测定卷烟中儿茶酚的分析方法。样品经2.5 mol/L硫酸加热回流后用水蒸气蒸馏提取,C₁₈固相萃取柱富集净化后进行HPLC-MS/MS分析,采用甲醇-0.2%(v/v)的甲酸水溶液作为流动相进行梯度洗脱,以电喷雾负离子(ESI^-)扫描和多反应监测(MRM)模式对目标物进行定性和定量分析。目标物儿茶酚的含量在0.5~200 μg/kg时与峰面积呈良好的线性关系(r²=0.9989);在样品中添加高、中、低3个水平的标准品,其加标回收率在83.1%~98.6%之间,相对标准偏差(RSD)在1.9%~5.8%之间。应用本方法对6种市售卷烟样品进行了测试,结果从6种市售卷烟中均检出了儿茶酚。该方法操作简单、快速、灵敏度高,适用于卷烟中儿茶酚的检测。     

超高效液相色谱-串联质谱法定量分析烟叶中的12种类黄酮物质

类黄酮物质是烟草非常重要的次生代谢物。本研究建立了一种烟叶中12种类黄酮物质的定量分析方法,相对于传统方法增加了10种类黄酮物质的定量分析。采用甲醇-水-氯仿(5 : 2 : 2, v/v/v)对类黄酮物质进行提取和色素剔除,采用超高效液相色谱-串联质谱进行分析,整个仪器分析时间为13 min。该方法对所有被测类黄酮物质的线性相关系数(r²)均高于0.99,检出限在0.3~100 μg/L之间,定量限在1.2~400 μg/L之间,日内重复性在3.5%~7.4%之间,日间重复性在5.2%~11.4%之间,回收率在81.2%~111.9%之间,符合定量分析要求。将该方法应用于11种不同品种烟草的类黄酮物质检测,发现不同烟草之间的类黄酮物质含量相差很大,而结构相似的类黄酮物质在烟叶中的含量分布存在显著的正相关关系。     

Study of metabolite differences of flue‐cured tobacco from different regions using a pseudotargeted gas chromatography with mass spectrometry selected‐ion monitoring method

A pseudotargeted method based on gas chromatography and mass spectrometry with selected‐ion monitoring was established to investigate the metabolite differences of flue‐cured tobacco from three different growing regions. The mixed solvent of acetonitrile/isopropanol/water (3:3:2, v/v/v) was chosen as the optimal extraction system based on the good repeatability and extraction efficiency. A self‐developed software coupled with commercial software was used to establish the pseudotargeted method including 289 peaks and 47 groups. Multivariable statistical analysis indicated that tobacco samples can be obviously separated based on the geographical origins. On the basis of a Mann–Whitney U test, organic acids, phenols, and alkaloids had higher levels in Hunan province. In contrast, a large proportion of amino acids (including l ‐tyrosine, l ‐proline, and serine), sucrose, and linoleic acid were the highest in Yunnan province. Meanwhile, multiple metabolic pathways (including carbohydrate metabolism, tricarboxylic acid cycle, and nitrogen metabolism) were influenced by growing regions. Twenty‐eight differential metabolites, which had great contributions to the classification of tobacco samples of three growing regions, were further defined. The results demonstrated that the developed pseudotargeted method was a powerful tool to investigate the metabolic profiling of tobacco leaves and discriminate tobacco leaves of different growing regions.     

Liquid chromatography–mass spectrometry for analysis of a novel β2‐adrenoceptor agonist trantinterol and its metabolites in beagle dog urine

A liquid chromatography–tandem mass spectrometry method was developed for the identification of metabolites of trantinterol, a novel β₂‐adrenoceptor agonist, in beagle dog urine. The separation of metabolites was performed on a reversed‐phase C₈ column using 0.1% formic acid in water and methanol (70 : 30, v/v) as the mobile phase. The structural information and elemental information of metabolites were acquired by an electrospray ionization tandem mass spectrometer and a quadrupole time‐of‐flight mass spectrometer, respectively. A total of 13 metabolites were detected and characterized on the basis of their tandem MS/MS fragmentation patterns. The accurate masses of nine metabolites were determined and two metabolites were further confirmed by comparing with reference standards. The metabolic pathways of trantinterol in beagle dog are proposed. Copyright © 2009 John Wiley & Sons, Ltd.     

Simultaneous determination of thirteen kinds of amino acid and eight kinds of acylcarnitine in human serum by LC–MS/MS and its application to measure the serum concentration of lung cancer patients

Easy‐to‐use early cancer detection methods based on metabolomics using serum samples have been developed recently. Among metabolites, amino acids and acylcarnitine are two of the most suitable candidates for diagnosing lung cancer. The purpose of the present study was to develop a novel, sensitive and specific liquid chromatography–tandem mass spectrometry (LC–MS/MS) method to simultaneously determine 13 amino acids and 8 acylcarnitines in lung cancer patients in serum. After derivatization, the 21 analytes were separated using a C₁₈ column with gradient elution program in 14 min, obtaining recovery within 90.4–113.8% and precision within 0.3–14.8%. The method was successfully applied in concentration determination of lung cancer patients and healthy controls. The results showed that the serum concentration of lung cancer patients were significant from those of healthy controls.     

Rapid characterization of the sucrose esters from oriental tobacco using liquid chromatography/ion trap mass spectrometry

Sucrose esters (SEs) from oriental tobacco are normally characterized by gas chromatography/mass spectrometry (GC/MS) after a long saponification and derivatization procedure. To simplify the process, a rapid method has been developed by using liquid chromatography coupled with electrospray ion trap mass spectrometry (LC/ESI-MSn). Using the characteristic fragmentation behavior of abundant SEs identified by GC/MS after purification by gel permeation chromatography (GPC) from cuticular waxes of green oriental tobacco leaf, two types of SEs from green and cured oriental tobacco were identified by MSn analysis. The first is one of three types reported formerly and has 13 SE homologues. However, the presence of unsaturation in one of the acyl substituents of this first type gave rise to a new series with three homologues. The other was found to be a new type and had three homologues. The proposed method enables the rapid and sensitive characterization of SEs from oriental tobacco.     

气相色谱-电子轰击离子源质谱法分析卷烟和烟叶中29种农药的残留

开展了卷烟和烟叶中有机氯、有机磷和拟除虫菊酯3类29种农药残留的气相色谱-电子轰击离子源质谱(GC-EI/MS)的分析方法研究。优化与选择了卷烟和烟叶样品的前处理条件,样品经正己烷-丙酮(体积比为1∶1)混合提取剂超声提取、Florisil硅土和中性氧化铝双净化剂固相萃取柱净化、二氯甲烷-正己烷(体积比为95∶5)混合洗脱剂洗脱和浓缩后,以磷酸三苯酯(TPP)为内标物,采用GC-EI/MS的选择离子监测方式(SIM)进行定性和定量分析。当样品的加标水平为20,50,100 μg/kg时,加标回收率为70%～110%,相对标准偏差在2%～8%之间;除了甲氰菊酯、氯菊酯和溴氰菊酯的方法检出限(LOD)分别为1.85,1.74与2.54 μg/kg外,其余的26种农药的LOD均小于0.8 μg/kg;线性范围为5.0～500.0 μg/kg,相关系数都大于等于0.9994。此分析方法已成功地应用于卷烟和烟叶样品中3类29种痕量农药残留的分析     

A liquid chromatography–tandem mass spectrometry method to simultaneously determinate dichlorvos and phoxim in tobacco

A simple pretreatment method with liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed and validated to simultaneously determine dichlorvos and phoxim in tobacco and soil matrices. Satisfactory linearity (R² ≥ 0.9991) of the method was obtained for both analytes. The limits of detection and limits of quantification for dichlorvos and phoxim in three matrices were 0.0015–0.006 and 0.005–0.02 mg/kg, respectively. Average recoveries were 78.24–92.21% for dichlorvos and 76.62–100.51% for phoxim in soil, green tobacco leaves and cured tobacco leaves. The intra‐ and inter‐day relative standard deviations were <6%. The established method was successfully applied for the residual analysis of dichlorvos and phoxim in real soil and tobacco samples. The results indicated that the established method could be used to detect trace amounts of dichlorvos and phoxim in tobacco. The data could also help the Chinese government establish maximum residue limits of dichlorvos and phoxim on tobacco and establish proper and safe use of dichlorvos and phoxim on tobacco plants in China.     

The biotransformation of oleuropein in rats

A highly sensitive and specific LC‐MS/MS method was developed to investigate the in vivo bio‐transformation of oleuropein in rat. Rat feces and urine samples collected after oral administration were determined by liquid chromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assay procedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat feces and urine with ethyl acetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at a flow rate of 0.50 mL/min on an RP‐C₁₈ column with a total run time of 31 min. This method was successfully applied to simultaneous determination of oleuropein and its metabolites in rat feces and urine. De‐glucosylation, hydrolysis, oxygenation and methylation were found to comprise the major metabolic pathway of oleuropein in rat gastrointestinal tract and three metabolites were absorbed into the blood circulatory system within 24 h after oral administration. Copyright © 2013 John Wiley & Sons, Ltd.     

Simultaneous determination of sibutramine and its active metabolites in human plasma by LC‐MS/MS and its application to a pharmacokinetic study

A simple and sensitive liquid chromatography–electrospray ionization–tandem mass spectrometry (LC‐ESI‐MS/MS) technique was developed and validated for the determination of sibutramine and its N‐desmethyl metabolites (M1 and M2) in human plasma. After extraction with methyl t‐butyl ether, chromatographic separation of analytes in human plasma was performed using a reverse‐phase Luna C₁₈ column with a mobile phase of acetonitrile–10 mm ammonium formate buffer (50:50, v/v) and quantified by ESI‐MS/MS detection in positive ion mode. The flow rate of the mobile phase was 200 μL/min and the retention times of sibutramine, M1, M2 and internal standard (chlorpheniramine) were 1.5, 1.4, 1.3 and 0.9 min, respectively. The calibration curves were linear over the range 0.05–20 ng/mL, for sibutramine, M1 and M2. The lower limit of quantification was 0.05 ng/mL using 500 μL of human plasma. The mean accuracy and the precision in the intra‐ and inter‐day validation for sibutramine, M1 and M2 were acceptable. This LC‐MS/MS method showed improved sensitivity and a short run time for the quantification of sibutramine and its two active metabolites in plasma. The validated method was successfully applied to a pharmacokinetic study in human. Copyright © 2011 John Wiley & Sons, Ltd.     

气相色谱-串联质谱技术分析烟草中的132种农药残留

利用气相色谱-串联质谱(GC-MS/MS)检测技术,建立了检测烟草中的132种农药残留的高灵敏度方法。分析过程中考察了不同萃取溶剂、不同缓冲盐体系、不同净化剂对目标物回收率的影响。最终确定烟草样品以乙腈进行提取,以N-丙基乙二胺(PSA)与碳18(C18E)的混合净化剂进行净化,氮气吹近干后用正己烷-丙酮(9:1, v/v)复溶,过有机滤膜后进行GC-MS/MS测定,内标法定量。132种农药在20～2000 μg/kg之间线性关系良好(r2 ＞0.99);所有农药的方法定量限(LOQ, S/N=10)均低于20 μg/kg;在50、200、500 μg/kg的加标水平下,除灭蚁灵及六氯苯回收率稍低外,其他农药的平均回收率为68.10%～123.15%,相对标准偏差(RSD)为1.79%～19.88%。对国际烟草科学研究合作中心(CORESTA)2012年共同实验的烟草样品进行检测,对比本方法与已有的标准方法,其结果一致性较好。该方法准确、可靠,灵敏度好,适用于烟草中132种农药残留的快速筛查与定性、定量分析。     

Identification and determination of the major constituents in Kai‐Xin‐San by UPLC‐Q/TOF MS and UFLC‐MS/MS method

In order to have overall chemical material information of Kai‐Xin‐San (KXS), the reliable ultra‐high‐performance liquid chromatography quadrupole time‐of‐flight mass spectrometer (UHPLC–Q‐TOF‐MS) and ultra‐fast liquid chromatography mass spectrometer (UFLC‐MS/MS) methods were developed for the identification and determination of the major constituents in KXS. Moreover, the UHPLC–Q‐TOF‐MS method was also applied to screen for multiple absorbed components in rat plasma after oral administration of KXS. The UHPLC–Q‐TOF‐MS method was achieved on Agilent 6520 Q‐TOF mass and operated in the negative ion mode. Good separation was performed on a ZORBAX Eclipse Plus C18 column with a gradient elution at a flow rate of 0.2 ml/min. A total of 92 compounds in KXS were identified or tentatively characterized based on their exact molecular weights, fragmentation patterns, and literature data. A total of 26 compounds including 23 prototype components and three metabolites were identified in rat plasma after oral administration of KXS. Then, 16 major bioactive constituents were chosen as the benchmark substances to evaluate the quality of KXS. Their quantitative analyses were performed by a triple quadrupole tandem mass spectrometer (MS/MS) operating in multiple‐reaction monitoring mode(MRM). The analysis was completed with a gradient elution at a flow rate of 0.4 ml/min within 35 min. The simple and fast method was validated and showed good linearity, precision, and recovery. Furthermore, the method was successful applied for the determination of 16 compounds in KXS. All results would provide essential data for identification and quality control of active chemical constituents in KXS. Copyright © 2016 John Wiley & Sons, Ltd.     

Development and validation of a HPLC method for quantification of rivastigmine in rat urine and identification of a novel metabolite in urine by LC‐MS/MS

A sensitive, specific and accurate HPLC method for the quantification of rivastigmine (RSM) in rat urine was developed and validated. The method involves the simple liquid–liquid extraction of RSM and pyridostigmine as an internal standard (IS) from rat urine with tertiary methyl butyl ether. The chromatographic separation of RSM and IS was achieved with 20 mm ammonium acetate buffer (pH 6.5) and acetonitrile (65:35, v/v) delivered at flow‐rate of 1 mL/min on a Kromasil KR‐100. The method was in linear range from 50 to 5000 ng/mL. The validation was done as per FDA guidelines and the results met the acceptance criteria. The method was successfully applied for the quantification of RSM in rat urine. Besides method validation, we have identified two metabolites of RSM in urine. Both the metabolites were characterized by HPLC‐PDA and LC‐MS/MS and it was found that one metabolite is novel. Copyright © 2010 John Wiley & Sons, Ltd.     

Urinary metabolomics analysis reveals the effect of volatile oil from Angelica sinensis on LPS‐induced inflammation rats

Lipopolysaccharide (LPS)‐induced inflammation occurs commonly and volatile oil from Angelica sinensis (VOAS) can be used as an anti‐inflammatory agent. The molecular mechanisms that allow the anti‐inflammatory factors to be expressed are still unknown. In this paper, we applied gas chromatography–mass spectrometry (GC–MS) and high‐performance liquid chromatography–time‐of‐flight mass spectrometry (LC‐Q/TOF–MS) based on a metabolomics platform coupled with a network approach to analyze urine samples in three groups of rats: one with LPS‐induced inflammation (MI); one with intervention with VOAS; and normal controls (NC). Our study found definite metabolic footprints of inflammation and showed that all three groups of rats, MI, intervention with VOAS and NC have distinct metabolic profiles in urine. The concentrations of 48 metabolites differed significantly among the three groups. The metabolites in urine were screened by the GC–MS and LC‐Q/TOF–MS methods. The significantly changed metabolites (p < 0.05, variable importance in projection > 1.5) between MI, NC and VOAS were included in the metabolic networks. Finally, hub metabolites were screened, including glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate, which have high values of degree (k). the Results suggest that disorders of glycine, arachidonic acid, l ‐glutamate, pyruvate and succinate metabolism might play an important part in the predisposition and development of LPS‐induced inflammation. By applying metabolomics with network methods, the mechanisms of diseases are clearly elucidated.     

Metabolomics‐based parallel discovery of xenobiotics and induced endogenous metabolic dysregulation in clinical toxicology

Intoxication by xenobiotics triggers the perturbation of metabolic fingerprints in biofluids, including the accumulation of xenobiotic compounds and the dysregulation of endogenous metabolites. In this work, an untargeted metabolomics workflow was developed to simultaneously profile both xenobiotic and endogenous metabolites for the identification of the xenobiotic origin and an in‐depth understanding of the intoxication mechanism. This workflow was demonstrated in a real‐world clinical case. Plasma samples were collected from four intoxicated children and another three healthy children. Untargeted metabolomics analysis was performed using ultraperformance liquid chromatography (UPLC) coupled to a high‐resolution mass spectrometer (HRMS) with data‐independent MS^E acquisition. LC–MS^E data was processed using an untargeted metabolomics data interpretation workflow, in which the identities of xenobiotics and altered endogenous metabolic features were determined via database searching. Five xenobiotic chemicals and 19 endogenous metabolites were found to be dysregulated. Combined with the clinical evidence, penfluridol was confirmed as the xenobiotic toxin. Furthermore, a mechanistic hypothesis was developed to explain the dysregulation of the four endogenous acyl‐carnitines. This workflow can be readily applied to a wide range of clinical toxicology cases, offering a powerful and convenient means of simultaneous discovery of intoxication source and the understanding of intoxication mechanisms.     

Metabolic profile of rosmarinic acid from Java tea (Orthosiphon stamineus) by ultra‐high‐performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry with a three‐step data mining strategy

Rosmarinic acid (RA) is a caffeic acid derivative and one of the most abundant and bioactive constituents in Java tea (Orthosiphon stamineus), which has significant biological activities. However, relatively few studies have been conducted to describe this compound's metabolites in vivo. Therefore, an ultra‐high‐performance liquid chromatography coupled to quadrupole‐time‐of‐flight tandem mass spectrometry (UHPLC–QTOF–MS/MS) analysis with a three‐step data mining strategy was established for the metabolic profile of RA. Firstly, the exogenously sourced ions were filtered out by the MarkerView software and incorporated with Microsoft Office Excel software. Secondly, a novel modified mass detects filter strategy based on the predicted metabolites was developed for screening the target ions with narrow, well‐defined mass detection ranges. Thirdly, the diagnostic product ions and neutral loss filtering strategy were applied for the rapid identification of the metabolites. Finally, a total of 16 metabolites were reasonably identified in urine, bile and feces, while metabolites were barely found in plasma. The metabolites of RA could also be distributed rapidly in liver and kidney. Glucuronidation, methylation and sulfation were the primary metabolic pathways of RA. The present findings might provide the theoretical basis for evaluating the biological activities of RA and its future application.     

Enantioseparation of nornicotine in tobacco by ultraperformance convergence chromatography with tandem mass spectrometry

Nornicotine, an alkaloid constituent of tobacco, is a precursor to the carcinogen N‐nitrosonornicotine that is produced during the curing and processing of tobacco. Accumulating evidence reveals that nornicotine enantiomers have different neurochemical and behavioral effects. In the present study, an accurate and rapid method was developed for the enantioseparation of (R )‐(+)‐nornicotine and (S )‐(−)‐nornicotine enantiomers in tobacco by ultra‐performance convergence chromatography with tandem mass spectrometry. Chromatographic conditions were investigated to achieve the optimal resolution of two enantiomers. Results indicated that (R )‐(+)‐nornicotine and (S )‐(−)‐nornicotine could be separated within 5 min when ammonium hydroxide was added into the cosolvent, and the best resolution (R _s = 4.76) was achieved on a immobilized cellulose tris‐(3,5‐dichlorophenylcarbamate) chiral stationary phase. The proposed method was validated and was finally applied to analyze the compositions of (R )‐(+)‐nornicotine and (S )‐(−)‐nornicotine in three typical types of tobaccos (flue‐cured, burley, and oriental). It was found that, enantiomer fraction of nornicotine (the proportion of (S )‐(−)‐nornicotine in the nornicotine pool) in burley tobacco samples was relatively high and constant compared with flue‐cured and oriental tobaccos. The effective and rapid enantioseparation of nornicotine may help the understanding of alkaloid metabolites in different tobacco varieties and may also benefit pharmacological studies of alkaloid enantiomers.     

Dissipation,residues and risk assessment of spirotetramat and its four metabolites in citrus and soil under field conditions by LC‐MS/MS

A modified Quick, Easy, Cheap, Effective, Rugged and Safe (QuEChERS) method for the simultaneous determination of spirotetramat and its four metabolite residues in citrus, peel, pulp and soil was developed and validated by liquid chromatography with tandem mass spectrometry (LC‐MS/MS). The samples were extracted with acetonitrile (1%, glacial acetic acid, v/v) and purified using primary secondary amine and octadecylsilane. The limit of detection was 0.01–0.13 mg/kg, whereas that of quantification was 0.02–0.40 mg/kg for spirotetramat and its metabolites. The average recoveries of spirotetramat, spirotetramat‐enol, spirotetramat‐mono‐hydroxy, spirotetramat‐enol‐glucoside and spirotetramat‐ketohydroxy in all matrices were 73.33–107.91%, 75.93–114.85%, 76.44–100.78%, 71.46–103.19% and 73.08–105.27%, respectively, with relative standard deviations < 12.32%. The dissipation dynamics of spirotetramat in citrus and soil followed first‐order kinetics, with half‐lives of 2.3–8.5 days in the three sampling locations. The terminal residues of spirotetramat in four matrices at the three locations were measured below the 1.0 mg/kg maximum residue limit set by China, and residues were found to be concentrated on the peel. The risk assessment of citrus was evaluated using risk quotients. The risk quotient values were found to be significantly <1, suggesting that the risk to human health was negligible when using the recommended doses of spirotetramat in citrus. These results could provide guidance for the safe and proper application of spirotetramat in citrus in China.     

液相色谱-电喷雾离子阱串联质谱同时分析烟草中的20种游离氨基酸

建立了液相色谱-电喷雾离子阱串联质谱(LC-ESI-IT-MS/MS)同时分析烟草中20种游离氨基酸的方法。烟草样品经萃取后过滤直接进样,无需进行衍生和固相萃取等其他前处理步骤。液相色谱采用HyPURITY C18反相色谱柱(200 mm×2.1 mm, 5 μm),采用1%(体积分数,下同)乙腈水溶液(含0.1%九氟戊酸)和90%乙腈水溶液(含0.1%九氟戊酸)为流动相进行梯度洗脱。结果表明,20种氨基酸的检出限(LOD)为0.01～0.05 μmol/L (S/N=3),线性相关系数均大于0.9977,峰面积测定的相对标准偏差(RSD)为0.78%～4.93%。该方法分析效率、灵敏度和选择性高,已成功应用于多种烟草样品中氨基酸的分析测定。     

A robust and rapid liquid chromatography tandem mass spectrometric method for the quantitative analysis of 5‐azacytidine

The DNA methyltransferase inhibitor 5‐azacytidine is being evaluated clinically as an oral formulation to treat various solid tumors. A sensitive, reliable method was developed to quantitate 5‐azacytidine using LC‐MS/MS to perform detailed pharmacokinetic studies. The drug of interest was extracted from plasma using Oasis MCX ion exchange solid‐phase extraction 96‐well plates. Chromatographic separation was achieved with a YMC J'sphere M80 C₁₈ column and isocratic elution with a methanol–water–formic acid (15:85:0.1, v/v/v) mobile phase over a 7 min total analytical run time. An AB Sciex 5500 triple quadrupole mass spectrometer operated in positive electrospray ionization mode was used for the detection of 5‐azacytidine. The assay range was 5–500 ng/mL and proved to be accurate (97.8–109.1%) and precise (CV ≤ 9.8%). Tetrahydrouridine was used to stabilize 5‐azacytidine in blood/plasma samples. With the addition of tetrahydrouridine, long‐term frozen plasma stability for 5‐azacytidine at ?70°C has been determined for at least 323 days. The method was applied for the measurement of total plasma concentrations of 5‐azacytidine in a cancer patient receiving a 300 mg oral daily dose. Copyright © 2015 John Wiley & Sons, Ltd.