Ultra-performance liquid chromatography-tandem mass spectrometry: a novel challenge in multiresidue pesticide analysis in food

Potential of ultra-performance liquid chromatography (UPLC) separation strategy coupled with tandem (in space) mass spectrometric detection (MS/MS) in multiresidue pesticide analysis was critically assessed. Performance parameters such as number of theoretical plates, height of theoretical plate, peak symmetry and peak capacity were measured/calculated on the basis of data generated by analysis of apple extracts containing 17 (semi)polar pesticides representing various classes of active ingredients of widely used crop protective preparations. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) procedure provided improved chromatographic parameters resulting in significantly increased sample throughput including lower solvent consumption and lower limits of quantitation (LOQs) for most of target analytes compared to common method employing conventional high-performance liquid chromatography (HPLC) separation.     

Ion-trap tandem mass spectrometry for the determination of heterocyclic amines in food

Heterocyclic amines (HAs) are mutagenic compounds to which humans are regularly exposed through diet. Due to the high complexity of the sample matrix and the low level of concentration of HAs, sensitive and selective analytical methodologies are required. Here we describe a methodology based on liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry using an ion-trap to analyse HAs. The collision-induced dissociation parameters for tandem ion-trap spectrometric analysis of these mutagenic compounds were optimised, and the full scan MS-MS spectra were used for unequivocal identification of the analytes. For aminoimidazoazaarenes, the most abundant ions were derived from the loss of a methyl group and the breaking of the aminoimidazole moiety, while for carbolines the major product ions arose from the loss of ammonia and HCN. Moreover, the performance of the LC-atmospheric pressure chemical ionisation MS-MS method was evaluated. The good precision (RSD lower than 11%) and the low detection limits achieved (10-60 pg injected) allow the determination of HAs at low part-per-billion level (0.4-5.0 ng g(-1)) in a lyophilised meat extract.     

Evaluation of different liquid chromatography-electrospray mass spectrometry systems for the analysis of heterocyclic amines

Three liquid chromatography-electrospray ionisation (LC-ESI) MS systems are evaluated for the analysis of heterocyclic amines (HAs). The electrospray sources and analysers (ion trap, single quadrupole and triple quadrupole) have been compared in terms of performance and quality parameters. In all cases, a C8 reversed-phase column and (acetic acid-ammonium acetate 30 mM pH 4.5)-acetonitrile (ACN) as mobile phase were used. Ionisation source parameters, post-column addition and working conditions for each acquisition mode (full scan, product ion scan, selected ion monitoring, and multiple reaction monitoring) were optimised for each instrument. The MS-MS spectra obtained with the ion trap and the triple quadrupole systems were very similar in both fragment ions and relative abundances, except for carbolines that showed adduct formation in the ion trap. Quality parameters were established and good precision (relative standard deviations (R.S.D.) < 12%) and very low limits of detection were obtained, mainly when using the triple quadrupole (< 9 pg injected). The content of HAs in a lyophilised beef extract was determined using the three instruments in order to compare their applicability for routine HAs analysis.     

Determination of heterocyclic amines by liquid chromatography-quadrupole time-of-flight mass spectrometry

This paper discusses the applicability of coupling liquid chromatography (LC) to mass spectrometry (MS) using a time-of-flight (TOF) mass analyzer for the analysis of heterocyclic amines (HAs). Accurate mass measurement (<2 mDa) with both MS-MS and in-source CID MS-MS was carried out to confirm the elemental composition of some fragments previously reported. Some isobaric assignments (fragments containing N versus CH2 and NH3 versus CD2H) were distinguished by taking advantage of the resolution provided by the TOF mass analyzer. On the other hand, the LC-MS analysis of HAs in MS acquisition mode was also performed. Quality parameters of the method were established. The linearity range extended over three orders of magnitude, limits of detection were in the pg level and good short-term precision values (R.S.D., 1.2-8.0%) were obtained. The LC-ESI-TOF method was applied to the determination of HAs in a lyophilized meat extract and the results obtained were comparable to those given by MS-MS with triple quadrupole and ion trap instruments.     

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。     

Retention studies of acrylamide for the design of a robust liquid chromatography-tandem mass spectrometry method for food analysis

A wide range of solid phases for SPE (solid-phase extraction) (n=14) and HPLC (n=9) were compared regarding the chromatographic retention of acrylamide. For SPE, a hydroxylated polystyrene-divinylbenzene copolymer phase (ENV+) gave the strongest retention. Twenty millilitre of water per gram solid phase could be passed with less than 5% loss of acrylamide from the column, thus enabling significant enrichment of food extracts. Other polymer phases gave varying degrees of retention, while silica bonded phases gave low retention. For HPLC, columns were evaluated both in reversed-phase and aqueous normal-phase (hydrophilic interaction chromatography) modes. The best retention was obtained with a phase comprising porous graphitic carbon (Hypercarb), giving a k-value of 4 with water as the mobile phase. Based on these investigations, a method for analysis of acrylamide in food using liquid chromatography-tandem mass spectrometry was designed to meet the demands of a collaborative validation trial. A comparative investigation of solid phases has not been published earlier. Thus, the paper should provide a base for new method developments regarding clean-up, enrichment and chromatography of acrylamide. In addition, the detailed standard operating procedure (SOP) method, as used in a collaborative validation trial, is provided as an electronic supplement (www.elsevier.com).     

液相色谱-串联质谱法检测食品中的多种易滥用着色剂

建立了硬糖、果酱、液态奶、果汁中酸性红52、红色2G、喹啉黄、专利蓝、酸性红26、柠檬黄、靛蓝、胭脂红、诱惑红、日落黄、亮蓝、苋菜红等12种易滥用着色剂残留量的液相色谱-串联质谱分析方法。样品用水溶液稀释提取,经聚酰胺固相萃取柱净化后,在Agilent XDB-C18色谱柱分离,以20 mmol/L乙酸铵溶液和乙腈为流动相进行梯度洗脱。质谱采用电喷雾负离子(ESI~)模式电离,多反应监测(MRM)模式检测,基质匹配标准溶液外标法定量。易滥用着色剂在0.5～50 mg/L范围内呈线性相关,相关系数(r)均大于0.99,其定量限(信噪比大于10)为0.5 mg/kg,检出限(信噪比大于3)为0.1 mg/kg。各种基质样品在0.5、5和50 mg/kg添加水平时,易滥用着色剂的回收率范围为62.6%～115.3%,相对标准偏差(RSDs)为2.6%～26.3%,可以满足食品中易滥用着色剂含量的检测需要。     

Ultra-performance liquid chromatography/mass spectrometry of intact proteins

Given that numerous small molecule applications of ultra-performance liquid chromatography (UPLC) have been published, efforts were made to examine the potential of UPLC to enhance the separation of intact proteins. Beginning with typically employed conditions, column temperature and organic solvent were optimized followed by an HPLC vs. UPLC comparison. When applied to a mixture of 10 protein standards, the optimized method yielded improved chromatographic resolution, enhanced sensitivity, and a threefold increase in throughput. Subsequent cell lysate analysis demonstrated no compromise in chromatographic or mass spectral data quality at 1/3 of the original run time.     

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.     

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.     

Ultra-performance liquid chromatography coupled to quadrupole-orthogonal time-of-flight mass spectrometry

Ultra-performance liquid chromatography (UPLC) utilizes sub-2 microm particles with high linear solvent velocities to effect dramatic increases in resolution, sensitivity and speed of analysis. The reduction in particle size to below 2 microm requires instrumentation that can operate at pressures in the 6000-15,000 psi range. The typical peak widths generated by the UPLC system are in the order of 1-2 s for a 10-min separation. In the present work this technology has been applied to the study of in vivo drug metabolism, in particular the analysis of drug metabolites in bile. The reduction in peak width significantly increases analytical sensitivity by three- to five-fold, and the reduction in peak width, and concomitant increase in peak capacity, significantly reduces spectral overlap resulting in superior spectral quality in both MS and MS/MS modes. The application of UPLC/MS resulted in the detection of additional drug metabolites, superior separation and improved spectral quality.     

高效液相色谱-串联质谱法快速测定食品中的抗倒胺残留量

建立了水果、蔬菜、茶叶、蜂蜜、粮谷和动物源性食品中抗倒胺残留的高效液相色谱-串联质谱(HPLC-MS/MS)分析方法。样品经乙腈提取,混合使用乙二胺-N-丙基硅烷和十八烷基硅烷键合相基质分散净化后,用HPLC-MS/MS检测和确证,外标法定量。质谱分析采用电喷雾电离,正离子扫描,多反应监测模式。该方法通过建立基质标准曲线消除基质效应,抗倒胺在1～100 μg/kg范围内具有良好的线性关系,相关系数在0.998～0.999之间;样品中添加5、10、50 μg/kg的标准品,其添加回收率在85.2%～112.4%之间,相对标准偏差均小于8.5%;检出限(LOD)在0.08～1.64 μg/kg之间,定量限(LOQ)在0.30～5.48 μg/kg之间。实验结果表明,该方法提取效果好,具有良好的灵敏度、回收率和重复性。     

Doping control analysis of methoxyphenamine using liquid chromatography-tandem mass spectrometry

Methoxyphenamine (o-methoxy-N,alpha-dimethylphenethylamine, Orthoxine) used in earlier times as a bronchodilator is prohibited in sports according to the regulations of the World Anti-Doping Agency (WADA). The drug and several of its metabolites are commonly analysed in doping control screening assays using gas chromatography-mass spectrometry requiring extraction from urine specimens. A complementary method employing liquid chromatography-atmospheric pressure chemical ionisation-tandem mass spectrometry and direct injection of urine aliquots was developed, which provided a fast and sensitive alternative to confirm the presence of the prohibited compound and degradation products in sports drug testing samples. In particular, the chromatographic separation of the active drug from isomeric compounds such as the designer drug p-methoxymetamphetamine (PMMA) was of particular interest to unambiguously identify the applied substance and was accomplished using a C6-phenyl reverse-phase column with isocratic elution. The established procedure was validated for methoxyphenamine with regard to specificity, limit of detection (0.7 ng mL(-1)), intraday- and interday precision (2.5-5.8% and 10.8-16.2%, respectively) and its applicability was demonstrated with an authentic doping control sample which tested positive for the prohibited compound early in 2008.     

Confirmatory analysis of acetylgestagens in plasma using liquid chromatography-tandem mass spectrometry

A confirmatory method has been developed and validated for the determination of chlormadinone acetate (CMA), megestrol acetate (MGA), melengestrol acetate (MLA) and medroxyprogesterone acetate (MPA) in bovine and porcine plasma. Analytes are extracted from plasma samples using matrix-assisted liquid-liquid extraction (LLE) on Extrelut NT columns followed by C18 solid-phase extraction (SPE). Analytes were analysed using liquid chromatography-tandem mass spectrometry (LC-MS/MS), and quantification was performed using matrix-matched calibration standards in combination with deuterated internal standards. In accordance with Commission Decision 2002/657/EC, two ion transitions were monitored for each analyte. Decision limits (CCalpha) were estimated by analysing 20 blank plasma samples and ranged from 0.1 to 0.2 ng mL(-1). Detection capabilities (CCbeta) were estimated using 20 plasma samples fortified at 0.5 ng mL(-1) and were <0.5 ng mL(-1). In the range 0.5-2 ng mL(-1), the mean intra-laboratory reproducibility of the analytes ranged from 6 to 18% (%R.S.D.). Analytes were shown to be stable in fortified plasma samples for >8 months when stored at -20 degrees C.     

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.     

Application of liquid chromatography-tandem mass spectrometry to the analysis of benzodiazepines in blood

A sensitive and selective high-performance liquid chromatography/atmospheric pressure chemical ionisation tandem mass spectrometry (HPLC/APCI-MS/MS) method for the simultaneous detection of 18 benzodiazepines and metabolites in human blood is described. The procedure utilises butyl chloride extraction at alkaline pH followed by reversed-phase liquid chromatography. The technique is suitable for screening analyses and confirmation of identity of the benzodiazepines at their lowest reported therapeutic concentrations using 500 microL of blood. The method has been successfully applied in forensic cases involving low concentrations of benzodiazepines.     

Quantitative analysis of mutagenic heterocyclic aromatic amines in cooked meat using liquid chromatography-atmospheric pressure chemical ionisation tandem mass spectrometry

Five mutagenic heterocyclic aromatic amines (HAAs) were quantified from meat extracts, and grilled and pan fried bacon samples using stable isotopically labeled internal standards. These compounds were isolated from the matrices by a tandem solid-phase extraction procedure, followed by separation on reversed-phase liquid chromatography (HPLC) and quantified by atmospheric pressure chemical ionization tandem mass spectrometry (APCIMS-MS). Tandem mass spectrometry (MS-MS) acquisition was done in selected reaction monitoring (SRM) mode to provide a high degree of sensitivity and selectivity for accurate quantification of HAAs. The detection and quantification limits of these HAAs approached 0.015 and 0.045 microg/kg (part-per-billion), respectively, with only 4 g of meat. The HAA levels ranged widely from 0.045 to 45.500 microg/kg, and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx) was the predominant HAA found in these samples. The amount of HAAs formed was highly dependent upon the type of meat and method of preparation. An intralaboratory comparison of the extraction procedure showed that estimates of these HAAs obtained by three different individuals at HAA levels below 2 microg/kg were within 5% with coefficients of variation below 19%, indicating the robustness of the analytical method. Moreover, because all of these HAAs from this class of molecules undergo facile cleavage at the N-methylimidazole moiety under collision-induced dissociation (CID) conditions, MS-MS analysis in the constant neutral loss mode of [M+H]+-15 enabled the identification of two other HAAs, 2-amino-3-methylimidazo[4,5-f]quinoxaline (IQx) and 2-amino-1,7,9-trimethylimidazo[4,5-g]quinoxaline (7,9-DiMeIgQx), which have rarely been reported in cooked meats.     

Use of liquid chromatography-tandem mass spectrometry for the analysis of pentoxifylline and lisofylline in plasma

A method was developed for the quantitation of pentoxifylline [1-(5-oxohexyl)-3,7-dimethylxanthine] and a primary active metabolite, lisofylline [1-(5-hydroxyhexyl)-3,7-dimethylxanthine], using high-performance liquid chromatography (HPLC)-tandem mass spectrometry. This method was developed in order to overcome problems encountered with HPLC-ultraviolet detection. The operating parameters of the electrospray interface (PE SCIEX, TurboIon Spray) and lens voltages of the triple-quadrupole detector (PE SCIEX 365) were optimized in positive ion mode to obtain the best sensitivity of the analytes. Collision-induced dissociation was used to produce fragment ions, and multiple reaction monitoring was used to quantitate pentoxifylline (m/z 279/181) and lisofylline (m/z 263/181). Dichloromethane was used to extract the drug, metabolite, and the internal standard (3-isobutyl-1-methylxanthine) from plasma. A reverse-phase C8(2) 150 x 1.0 mm HPLC column was used to resolve all three compounds in less than 6 min. Calibration curves were generated using peak area and were linear from 1 to 1000 ng/mL (R(2) > 0.99). The small sample volume, ease of extraction, and sensitivity provide advantages over more conventional methods of quantitation.     

超声提取-超高效液相串联质谱分析沉积物中15种抗生素

在现有方法基础上对沉积物中磺胺类、喹诺酮类、大环内脂类和四环素类抗生素的提取、富集、净化以及仪器分析方法进行了优化。以EDTA-Mcllvaine缓冲溶液与乙腈(V:V,1:1)混合液作为提取液,利用超声波细胞破碎仪进行超声提取,串联强阴离子交换柱(SAX)和HLB固相萃取柱进行固相萃取(SPE),通过超高效液相/串联质谱(UPLC-MS/MS)测定沉积物中抗生素的含量。抗生素基质加标回收率在56.4%~110%,相对标准偏差为1.1%~24.3%,方法检出限0.0055~0.716 ng/g。本方法有效地提高了沉积物中抗生素的提取效率,并应用于实际样品的测定中。     

High performance liquid chromatography —Electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) for the analysis of heterocyclic aromatic amines (HAA)

Summary Sensitive and selective detection of the sixteen most abundant heterocyclic aromatic amines (HAA) has been achieved by application of high performance liquid chromatography-electrospray tandem mass spectrometry (HPLC-ESI-MS-MS) in combination with selected reaction monitoring (SRM). Detection limits between 0.1 and 50 ng mL^–1 were established by use of HAA model solutions.