Sample preparation and determination of acetylcholine in corneal epithelium cells using liquid chromatography-tandem mass spectrometry

A sample preparation method with subsequent liquid chromatography (LC)-mass spectrometry (MS)-MS analysis for acetylcholine in corneal epithelium is developed. The sample preparation is developed with a focus on compatibility with the LC-MS-MS system and the stability of acetylcholine because acetylcholine esterase is present in the tissue. It appears that both acetylcholine as well as the internal standard (IS) used (acetyl-beta-methylcholine) have fragments at m/z values in the tandem MS spectrum, which correspond with the m/z values of fragments of endogenous substances. Acetylcholine and (3-carboxypropyl)triethylammonium both have 146-->87 and 146-->60 transitions. Acetyl-beta-methylcholine and an unknown compound both have 160-->101 and 160-->60 transitions. This makes it necessary to use a chromatographic step, which has a baseline separation between these endogenous compounds, acetylcholine, and the IS. The analytical procedure has linearity from 1 ng/mL (30 pg/mg corneal epithelium tissue) to at least 250 ng/mL (7.55 ng/mg corneal epithelium tissue). The limits of detection and quantitation are 15 and 45 pg on column, respectively. Relative standard deviation and bias values are within the range of acceptance for all concentration levels.     

Determination of aflatoxins in hazelnuts by various sample preparation methods and liquid chromatography-tandem mass spectrometry

A sensitive and reliable liquid chromatography-tandem mass spectrometric with electrospray ionization method for determining aflatoxins in hazelnuts has been developed. Three different extraction techniques, such as homogenization, ultrasonic extraction, and matrix solid phase dispersion have been tested and compared in terms of recovery, matrix effect, accuracy and precision. Ultrasound extraction was the most performing sample preparation method. Absolute recoveries for analytes and I.S. ranged from 93 to 101%. Accuracy and precision were calculated using matrix matched calibration, and ranged 91-102% and 2-11%, respectively. CC alpha and CC beta for aflatoxin B1 (EU limit=2 microg/kg) were 2.15 and 2.33 microg/kg, respectively. A ruggedness test performed on three other matrices demonstrated that sonication time was critical and a matrix matched calibration must be constructed for every sort of matrix.     

样品制备方法对高效液相色谱-串联质谱分析人乳内源肽的影响

人乳内源肽是乳蛋白在乳腺中被降解形成的具有生理功能的肽,是人乳的重要组成部分,研究人乳内源肽对于婴儿健康具有重要的意义.高效液相色谱-串联质谱(LC-MS/MS)联用技术的应用,促使人乳内源肽的研究取得了突破性的进展.人乳中内源肽含量低、干扰组分多,样品制备方法是影响分析结果的关键步骤.为了研究样品制备方法对分析结果的...     

基于QuEChERS提取方法优化的液相色谱-串联质谱法测定蔬菜中51种氨基甲酸酯类农药残留

采用气相色谱-质谱（GC-MS）全扫描结合NIST谱库检索方法分析6种蔬菜（番茄、青刀豆、大葱、青花菜、姜、胡萝卜）提取液中的基质干扰物,以蒸发残渣重量法探讨乙二胺N-丙基硅烷（PSA）、十八烷基硅烷（C18）及两者组合对6种蔬菜提取液基质干扰物的净化效果及吸附机理,考察了原创QuEChERS方法及AOAC 2007.01方法对蔬菜中51种氨基甲酸酯类农药提取的适用性,并建立了液相色谱-串联质谱法测定蔬菜中51种氨基甲酸酯类农药残留的方法。结果表明,C18与PSA组合进行分散固相萃取的净化效果最好;AOAC 2007.01方法适用于二氧威以外的50种农药残留的提取,而原创QuEChERS方法对二氧威残留的提取可获得满意结果。经电喷雾正离子电离及多反应监测模式来测定目标化合物,采用基质匹配标准溶液曲线法进行定量。结果表明：51种农药在6种基质中3个添加水平（10、20、100 μg/kg）的回收率为58.4%~126%,相对标准偏差为3.3%~26%;以信噪比（S/N）≥10计,久效威及杀螟丹的定量限（LOQ）为50 μg/kg,其他49种农药的LOQ为0.2~10 μg/kg。本文方法有效、灵敏,适用于不同蔬菜基质中51种氨基甲酸酯类农药残留的测定。     

A liquid chromatography-tandem mass spectrometry method for fluazifop residue analysis in crops

An LC-MS-MS assay is described for fluazifop residue analysis in crops. The residues are extracted with acidified organic solvent, the esters and conjugates are hydrolysed with 6 M hydrochloric acid, then the extracts are cleaned-up by solid phase extraction using C₂(EC) and Si cartridges in tandem. Quantitative analysis is performed by gradient liquid chromatography coupled to triple quadrupole mass spectrometer using atmospheric pressure chemical ionisation. All fluazifop-P-butyl, free fluazifop-P and any conjugates are quantified as fluazifop-P. The limit of quantification is 0.01–0.05 mg/kg depending on crop matrices. The clean-up method is also suitable for LC-UV analysis with a compromise in higher limit of quantification 0.05–0.2 mg/kg.     

High-throughput multiclass method for antibiotic residue analysis by liquid chromatography-tandem mass spectrometry

A simple and rapid method has been developed for the residue analysis of 39 antibiotics (tetracyclines, quinolones, penicillins, sulfonamides and macrolides) in foodstuffs of animal origin. The method combines an effective extraction technique, which uses water-methanol as extracting solvent, with ultra-high-pressure liquid chromatography-tandem mass spectrometry, allowing both confirmation and quantification in a single chromatographic run. The multiresidue method has been validated in chicken muscle matrix according to European Union Decision 2002/657/EC. It has been implemented as a routine method in a Public Health Laboratory, instead of the five plates test and LC methods previously used.     

A liquid chromatography-tandem mass spectrometry method for fluazifop residue analysis in crops

An LC-MS-MS assay is described for fluazifop residue analysis in crops. The residues are extracted with acidified organic solvent, the esters and conjugates are hydrolysed with 6 M hydrochloric acid, then the extracts are cleaned-up by solid phase extraction using C2(EC) and Si cartridges in tandem. Quantitative analysis is performed by gradient liquid chromatography coupled to triple quadrupole mass spectrometer using atmospheric pressure chemical ionisation. All fluazifop-P-butyl, free fluazifop-P and any conjugates are quantified as fluazifop-P. The limit of quantification is 0.01-0.05 mg/kg depending on crop matrices. The clean-up method is also suitable for LC-UV analysis with a compromise in higher limit of quantification 0.05-0.2 mg/kg.     

Pattern profiling of the herbal preparation picroliv using liquid chromatography-tandem mass spectrometry

At present, the construction of chromatographic fingerprints of complex herbal preparations in combination with mass spectrometry plays an important role in their development and standardization as potential therapeutic agents. Picroliv, an extract from roots and rhizomes of Picrorhiza kurroa, is a herbal hepatoprotective developed by CDRI. We report for the first time pattern profiling of various constituents of picroliv along with a precise and accurate method to estimate relative concentration of major components in the preparation by liquid chromatography-tandem mass spectrometry. In total, 27 components could be detected in multiple reaction monitoring (MRM) mode out of which fourteen could be quantified in terms of their relative concentration. Seven components were structurally correlated and confirmed based on the fragmentation pattern and information available in literature. The detection was carried out using MRM in negative ionization mode with analytes quantified from the summed total ion value of their most intense molecular ion transitions. The separation of various components was achieved using a gradient elution on RP-18 column with acetonitrile and Milli-Q water as mobile phase at a flow rate of 1.0 ml/min. The method was validated in terms of linearity, accuracy and precision (within- and between-assay variation) for 5 days. Linearity range was different for various components depending upon their sensitivity and abundance in the herbal preparation. Within- and between-assay accuracy (%bias) and precision (%R.S.D.) were within acceptable limits. The method was successfully applied to detect and determine relative concentrations of various components in two different batches of picroliv.     

Automated sample preparation method for mass spectrometry analysis on recombinant proteins

A completely automated procedure for the purification and desalting of proteins with a polyhistidine purification tag prior to mass spectrometry analysis is presented. The system is ideal for rapid quality control and optimization studies and it provides researchers with a straightforward, reliable tool for studies of recombinant proteins. Forty-eight samples can be prepared within 4.5 h and only small cultivation and buffer volumes are needed. In this proof of concept, 19,000–35,000 Da recombinant proteins from both crude and clarified cell lysates were successfully prepared for subsequent analysis by electrospray ionization and matrix-assisted laser desorption/ionization mass spectrometry as well as by gel electrophoresis.     

Sensitive liquid chromatography-tandem mass spectrometry method for quantification of hydrochlorothiazide in human plasma

A simple, rapid, sensitive and specific liquid chromatography-tandem mass spectrometry method was developed and validated for quantification of hydrochlorothiazide (I), a common diuretic and anti-hypertensive agent. The analyte and internal standard, tamsulosin (II) were extracted by liquid-liquid extraction with diethyl ether-dichloromethane (70:30, v/v) using a Glas-Col Multi-Pulse Vortexer. The chromatographic separation was performed on a reversed-phase column (Waters symmetry C18) with a mobile phase of 10 mm ammonium acetate-methanol (15:85, v/v). The protonated analyte was quantitated in negative ionization by multiple reaction monitoring with a mass spectrometer. The mass transitions m/z 296.1 solidus in circle 205.0 and m/z 407.2 solidus in circle 184.9 were used to measure I and II, respectively. The assay exhibited a linear dynamic range of 0.5-200 ng/mL for hydrochlorothiazide in human plasma. The lower limit of quantitation was 500 pg/mL, with a relative standard deviation of less than 9%. Acceptable precision and accuracy were obtained for concentrations over the standard curve ranges. A run time of 2.5 min for each sample made it possible to analyze a throughput of more than 400 human plasma samples per day. The validated method has been successfully used to analyze human plasma samples for application in pharmacokinetic, bioavailability or bioequivalence studies.     

Rapid liquid chromatography-tandem mass spectrometry method for quantification of ziprasidone in human plasma

A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the determination of ziprasidone (ZIP) in human plasma was developed. ZIP and N-methyl ziprasidone as internal standard (IS) were extracted from alkalinized plasma using tert- butyl methyl ether. Separation was performed isocratically on a C8 column with 90% acetonitrile containing 2 mmol/L ammonium acetate as a mobile phase with a total run time of 2.5 min. MS/MS transitions of m/z 413 --> 194 and m/z 427 --> 177 of the analyte and internal standard were used for quantification. Confirmatory ions of m/z 413 --> 177 and m/z 427 --> 180 were collected as well. The calibration curve based on peak-area ratio was linear up to at least 200 ng/mL with a detection limit of 0.1 ng/mL. The method showed satisfactory reproducibility with a coefficient of variation of less than 5%. The method was successfully applied to the analysis of ZIP in spiked human plasma.     

A high-performance liquid chromatography-tandem mass spectrometry method for quantitation of nitrogen-containing intracellular metabolites

A comprehensive method of quantifying intracellular metabolite concentrations would be a valuable addition to the arsenal of tools for holistic biochemical studies. Here, we describe a step toward the development of such method: a quantitative assay for 90 nitrogen-containing cellular metabolites. The assay involves reverse-phase high-performance liquid chromatography separation followed by electrospray ionization and detection of the resulting ions using triple-quadrupole mass spectrometry in selected reaction monitoring mode. For 79 of the 90 metabolites, the assay is linear with a limit of detection of 10 ng/mL or less. Using this method, 36 metabolites can be reliably detected in extracts of the bacterium Salmonella enterica, with the identity of each metabolite confirmed by the presence, on growing of the bacteria in (13)C-glucose, of a peak corresponding to the isotope-labeled form of the compound. Quantitation in biological samples is performed by mixing unlabeled test cell extract with (13)C-labeled standard extract, and determining the (12)C/(13)C-ratio for each metabolite. Using this approach, the metabolomes of growing (exponential phase) and carbon-starved (stationary phase) bacteria were compared, revealing 16 metabolites that are significantly down-regulated and five metabolites that are significantly up-regulated, in stationary phase.     

Streamlined sample preparation procedure for determination of perchlorate anion in foods by ion chromatography-tandem mass spectrometry

A rapid, sensitive, and specific method was developed for the determination of perchlorate anion in foods. The foods included high moisture fruits and vegetables, low moisture foods (e.g. wheat flour and corn meal), and infant foods. Improvements to existing procedures were made in sample preparation that reduced sample test portion size from 100 to 5 or 10 g, extraction solvent volume from 150 to 20-40 ml, and replaced blending extraction-vacuum filtration and their associated large glassware with a simple shakeout-centrifugation in a small conical tube. Procedures common to all matrices involved: extraction, centrifugation, graphitized carbon solid phase extraction (SPE) cleanup, and ion chromatography-tandem mass spectrometry (IC-MS/MS) analysis. A Waters IC-Pak Anion HR column (4.6 mm × 75 mm) was eluted with 100 mM ammonium acetate in 50:50 (v/v) acetonitrile/water mobile phase at a rate of 0.35 ml/min. A triple stage quadrupole mass spectrometer, equipped with electrospray ionization (ESI) in the negative ion mode, was used to detect perchlorate anion. An ¹⁸O₄-labeled perchlorate anion internal standard was used to correct for any matrix effects. The method limit of quantitation (LOQ) was: 1.0 μg/kg in fruits, vegetables, and infant foods; 3.0 μg/kg in dry products. Fortified test portions gave 80-120% recoveries. Determination of incurred perchlorate anion residues agreed well with results for comparable commodities or products analyzed by published methods.     

Analysis of bromate in drinking water using liquid chromatography-tandem mass spectrometry without sample pretreatment

An analytical method for determining bromate in drinking water was developed using liquid chromatography-tandem mass spectrometry (LC-MS/MS). The (18)O-enriched bromate was used as an internal standard. The limit of quantification (LOQ) of bromate was 0.2 μg/L. The peak of bromate was separated from those of coexisting ions (i.e., chloride, nitrate and sulfate). The relative and absolute recoveries of bromate in two drinking water samples and in a synthesized ion solution (100 mg/L chloride, 10 mg N/L nitrate, and 100 mg/L sulfate) were 99-105 and 94-105%, respectively. Bromate concentrations in 11 drinking water samples determined by LC-MS/MS were <0.2-2.3 μg/L. The results of the present study indicated that the proposed method was suitable for determining bromate concentrations in drinking water without sample pretreatment.     

QuEChERS结合高效液相色谱-串联质谱法测定饲料中的18种β-兴奋剂

建立了饲料中克仑特罗、莱克多巴胺、喷布特罗、妥布特罗等18种β-兴奋剂的QuEChERS结合高效液相色谱-串联质谱的检测方法。饲料样品加水分散后经4%(v/v)氨水乙腈提取,加入25 mg十八烷基硅烷(C18)和50mg N-丙基乙二胺(PSA)吸附剂分散固相萃取净化后,以高效液相色谱-串联质谱进行定性和定量分析。采用Agilent Zorbax Eclipse XDB-C18(50 mm×4.6 mm,1.8μm)分析柱,以甲醇-0.1%(v/v)甲酸水溶液为流动相进行梯度洗脱,串联质谱在多反应监测(MRM)正离子模式下进行检测,基质外标法定量。结果表明,18种待测物在质量浓度为5~200μg/L范围内线性关系良好,相关系数为0.9912~0.9995;在0.05、0.1、0.5 mg/kg 3个浓度加标水平下,饲料中18种β-兴奋剂的平均回收率为78.4%~107.1%,相对标准偏差(RSD)为3.5%~12.3%,定量限(以信噪比≥10计)均为0.05 mg/kg。该方法准确、灵敏,前处理简单,可作为饲料中克仑特罗等18种β-兴奋剂筛选和确认的检测方法。     

High-throughput sample preparation and analysis using 96-well membrane solid-phase extraction and liquid chromatography-tandem mass spectrometry for the determination of steroids in human urine

A 96-well solid-phase extraction (SPE) system is used to rapidly prepare human urine samples for high-throughput quantitative analysis of two steroids, equilenin and progesterone, by liquid chromatography-tandem mass spectrometry using deuterated estrone as the internal standard. We define high-throughput here as analysis of 384 samples in a 24 h period. A total of 384 samples and standards were extracted by an individual in one day and subsequently analyzed within a 24 h period. The inter- and intratray accuracy and precision obtained over the course of these injections was within 8% coefficient of variation when analyzed by atmospheric pressure chemical ionization mass spectrometry using positive ion detection. A semiautomated sample processing workstation was used to add internal standard and then process 96 samples at a time. The recovery of the analytes from the SPE was approximately 85%. The accuracy and precision obtained was comparable to that ordinarily obtained using manual sample preparation techniques.     

土壤中典型人工甜味剂的前处理方法优化及高效液相色谱-串联质谱分析

优化了同时提取土壤中4种典型人工甜味剂的前处理与净化方法,并结合高效液相色谱-串联质谱（HPLC-MS/MS）技术,建立了一种快速高效的分析方法。样品采用25 mL 0.01 mol/L醋酸-醋酸钠溶液（pH 4）提取2次,每次提取20 min;萃取样品进一步采用CNW Poly-Sery PWAX固相萃取小柱净化浓缩;最后采用HPLC-MS/MS进行测定。在干燥土壤中添加1、10、100 μg/kg标准品时,4种甜味剂的平均回收率为86.5%~105%,日内精密度（RSD）≤5.94%,日间精密度（RSD）≤6.53%;在1~100 μg/kg范围内的线性关系良好（r²>0.995）;方法的检出限为0.01~0.21 μg/kg,定量限为0.03~0.70 μg/kg。利用该方法对天津某污水灌溉的农田土壤进行了分析测定,结果表明该方法快捷可靠,可用于土壤中人工甜味剂的环境调查。     

基于整体材料的微流控芯片反相液相色谱-串联质谱平台用于蛋白质分析

微流控芯片高效液相色谱-串联质谱系统具有高通量、高灵敏度等优点,已成为生物样品分析的热点领域之一。本文在玻璃芯片上以甲基丙烯酸十二酯(LMA)和三羟甲基丙烷三甲基丙烯酸酯(TMPTMA)为单体,制备了以聚丙烯酸酯整体材料为固定相的捕集柱和分离柱。通过在芯片通道末端连接细内径的毛细管作为芯片-质谱接口,并以常规的液相色谱泵和微阀控制流体,构建了芯片反相液相色谱-电喷雾串联质谱(RPLC-ESI-MS/MS)平台,并将其用于分析牛血清白蛋白(BSA)的酶解产物。经过3次平行分析,BSA的序列覆盖率分别为39.37%、37.89%和34.10%(相对标准偏差为7.3%)。采用不同批次制作的芯片构建RPLC-ESI-MS/MS平台,对BSA酶解产物进行分析,其序列覆盖率相当。上述结果表明,该平台具有灵敏度高和重现性好等优点,有望用于蛋白质样品的快速分离和高灵敏度鉴定。     

Applicability of ultra-performance liquid chromatography-tandem mass spectrometry for heroin profiling

The applicability of ultra- performance liquid chromatography tandem-mass spectrometry (UPLC-MS/MS) for heroin profiling is described. The coupling of the high separation power of UPLC with the highly selective and sensitive detection of MS/MS is well suited for heroin profiling. An Acquity UPLC BEH C18 1.7 microm particle column (100 mm x 2.1mm) with binary gradients containing 1% formic acid (pH 2.0) or 10 mM ammonium bicarbonate (pH 10.0)/acetonitrile mixtures was investigated for the profiling. For MS/MS detection, an atmospheric pressure positive electrospray source was employed with multiple reaction monitoring (MRM). MRMs for individual basic impurities were generated for heroin profiling using low and high pH mobile phases, while MRMs for neutral impurities were generated using a high pH mobile phase. Compared to a pH 2.2 mobile phase, the use of a pH 10 mobile phase allowed for significantly greater sample loading, major selectivity differences, and lower MRM sensitivity. UPLC-MS/MS allowed for the highly selective and sensitive detection of many of the targeted solutes in seized heroin exhibits. Basic impurities detected included morphine, codeine, noscapine, papaverine and the previously unreported solutes reticuline, reticuline monoacetate (2 products), reticuline diacetate, narceine, codamine, laudanidine, cryptopine, laudanosine, and norlaudanosine. Neutral impurities found included N,3,6-triacetylnormorphine, N-acetylnorcodeine, N-acetylnornarcotine, 3,6-dimethoxy-4-acetyloxy-5-[2-(N-methylacetamido)]-ethylphenanthrene, and cis-n-acetylanhydronornarceine. The detection of these impurities, at levels as low as 10(-6)% w/w should allow for greatly enhanced heroin profiles.     

比较纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱用于自顶向下蛋白质组学

自顶向下蛋白质组学的一个重要难题是缺乏与质谱可以在线连用并且可以提供高效蛋白质分离的液相分离技术。毛细管区带电泳与纳升反相色谱都可以与质谱在线连用,并且在复杂蛋白质样品分析方面也都有了显著的提升。在这里,我们首次比较了先进的纳升反相色谱-串联质谱与毛细管区带电泳-串联质谱平台用于自顶向下蛋白质组学分析。相对于纳升反相色谱-质谱而言,毛细管区带电泳-质谱可以将标准蛋白质样品的消耗量降低10倍,而且保持与纳升反相色谱-质谱相当的蛋白质信号强度。有意思的是,与毛细管区带电泳-质谱相比,纳升反相色谱-质谱可以获得更高的蛋白质分子的气相价态。这个现象可能是由于反相流动相中的高浓度乙腈使得蛋白质变性的更加充分。从1微克的大肠杆菌蛋白质样品中,毛细管区带电泳-串联质谱可以鉴定到159个蛋白质和513个蛋白质变体,而纳升反相色谱-串联质谱仅鉴定到105个蛋白质和277个蛋白质变体。当将大肠杆菌蛋白质的上样量提高到8微克时,纳升反相色谱-串联质谱可以鉴定到245个蛋白质和1004个蛋白质变体。由于纳升反相色谱-串联质谱具有比毛细管区带电泳-串联质谱更高的上样量与更宽的分离窗口,当蛋白质样品量不受限制时,纳升反相色谱-串联质谱具有明显的优势。但是,在痕量样品分析方面,毛细管区带电泳-串联质谱具有更大的潜力。