Mass spectral characterization of ergot alkaloids by electrospray ionization, hydrogen/deuterium exchange, and multiple stage mass spectrometry: Usefulness of precursor ion scan experiments

Six ergot alkaloids belonging to the lysergic acid derivatives (ergonovine (EGN) and methysergide hydrogen maleinate (MHM)) and peptide-type derivatives (ergocristine (EGR), ergotamine (EGT), ergocornine (EGC) and alpha-ergokryptine (EGK)) were studied by positive electrospray tandem mass spectrometry. The fragmentation mechanisms of these compounds were studied by collision-induced dissociation (CID) using triple quadrupole and ion trap mass spectrometers, and the nature of the major product ions further confirmed by hydrogen/deuterium (H/D) exchange experiments. A common abundant product ion at m/z 223 was characteristic of the two classes of ergot alkaloids. Therefore, a precursor ion scan of m/z 223 that triggers information data acquisition (IDA) in combination with CID experiments was used to identify other potential ergot alkaloids. Using this approach, it was possible to confirm the presence of ergosine, another peptide-type ergot alkaloid, in a rye flour extract at trace levels.     

Determination of ergot alkaloids from grains with UPLC‐MS/MS

A simple and rapid method for determining six ergot alkaloids and four of their respective epimers was developed for rye and wheat. The analytes were extracted from the sample matrix with ACN/ammonium carbonate solution. The extract was purified with a commercial push‐through SPE column (Mycosep^® 150 Ergot). After concentration and filtration steps, the final separation of the analytes was achieved with ultra‐performance LC‐MS/MS. The chromatographic separation of the ergot alkaloids was achieved in 4.5 min. The method performance proved satisfactory in the preliminary validation. The calculated LOQs were low ranging from 0.01 to 1.0 μg/kg for wheat and from 0.01 to 10.0 μg/kg for rye. At the concentration levels of 10, 50 and 200 μg/kg, the recoveries were between 80 and 120% in most cases and the within‐day repeatability (expressed as RSD) ranged between 1.3 and 13.9%. Despite the cleanup of the samples, some matrix effect was observed in the MS, highlighting the necessity of using matrix‐assisted standards. This is the first article to describe the application of the push‐through columns and ultra‐performance LC in the analysis of ergot alkaloids.     

Simultaneous separation of ergot alkaloids by capillary electrophoresis after cloud point extraction from cereal samples

A new and sensitive analytical methodology for ergot alkaloids (EA) determination from cereal samples based on cloud point extraction (CPE) prior to CE‐UV absorbance was developed. The methodology involves extraction under acid conditions and subsequent preconcentration by applying a simple, rapid and environmentally friendly low volume surfactant extraction procedure. After extraction, CE analysis was carried out by performing dilutions on preconcentrated surfactant rich phase, achieving a single peak or simultaneous alkaloids determination. A real preconcentration factor of 22 of total EA was obtained, demonstrating the efficiency of this methodology. The limits of detection were 2.6 and 2.2 μg/kg for ergotamine and ergonovine, respectively. Validation procedure revealed suitable linearity, accuracy and precision. The average extraction and clean‐up recoveries were compared with the theoretical values and were better than 92%. This method was successfully applied to the determination of EA in different varieties of commercial flour samples, two grain samples and one of the leading brands cereal‐based product for infant feeding. The high sensitivity achieved for EA determinations in real samples suggests CPE procedure as an interesting approach to improve CE‐UV visible detection limits. Moreover, the whole process could be considered as a contribution to green chemistry because nonorganic solvents were involved, demonstrating its great potential over conventional techniques.     

Liquid chromatographic preparative method for isolating ergot alkaloids, using a particle-loaded membrane extracting disk

A liquid chromatographic method is described for the determination of ergot alkaloids in wheat. Ergonovine, ergotamine, ergocornine, alpha-ergocryptine, and ergocristine are extracted from wheat with methanol-0.25% concentrated H3PO4 (40 + 60) pH 2.2, cleaned up by using a solid-phase extraction (SPE) disk, and separated by reversed-phase liquid chromatography with fluorescence detection. Ergot alkaloids are basic compounds that form water-soluble salts in acidic aqueous solution. Because ergot alkaloid salts are positively charged, they can be easily and selectively trapped on a negatively charged strong cation-exchange SPE disk. A strong wash solvent, methanol-0.25% concentrated H3PO4 (40 + 60) was used to remove matrix interferences not bonded by ionic interactions with the cation-exchange column. The ergot alkaloids were eluted from the ion-exchange column by adjusting the pH of the elution solvents to slightly basic conditions (pH 9). The SPE disk concentrated and cleanly separated the ergot alkaloids from matrix interferences. Standard calibration curves for ergot alkaloids for the concentration range 0.1-2.0 microg/mL were linear. The SPE disk had a column capacity equivalent to about 1 g extracted wheat. At spiking levels of 2.3-46 ng/g for ergonovine and 20-400 ng/g for ergotamine, ergocornine, alpha-ergocryptine, and ergocristine, the mean recovery was 88.1% with a coefficient of variation (CV) of 5.33%. The recovery data ranged from 79.1 to 95.9%. Ergonovine had the lowest overall recovery and the largest CV. The method has an estimated reliable limit of detection and limit of quantitation of <5 and <20 ng/g, respectively, for each ergot alkaloid tested.     

Simultaneous determination of aflatoxins, ochratoxin A, and zearalenone in grains by new immunoaffinity column/liquid chromatography

The simultaneous determination of mycotoxins was performed in 3 steps: extraction, cleanup, and detection. For extraction, a mixture of acetonitrile-water (60 + 40, v/v) was proved appropriate. For cleanup, a new Afla-Ochra-Zea immunoaffinity column was used. After derivatization with trifluoroacetic acid, the mycotoxins aflatoxins, ochratoxin A (OTA), and zearalenone (ZEA) were determined simultaneously by liquid chromatography with fluorescence detection. The detection limits in different matrixes after cleanup with the new immunoaffinity column were very low: aflatoxins, 0.002-0.7 microg/kg; OTA, 0.07-0.25 microg/kg; ZEA, 1-3 microg/kg. The limits of determination were: aflatoxins, 0.25 microg/kg; OTA, 0.5 microg/kg; ZEA, 5 microg/kg. The recovery rates for aflatoxins, OTA, and ZEA for rye and rice were between 86 and 93% when a 0.5 g sample matter per immunoaffinity column was used.     

Application of low-pressure gas chromatography/tandem mass spectrometry to the determination of pesticide residues in tropical fruits

A multiresidue method has been developed for determining pesticide residues in the tropical fruits kiwi, custard apple, and mango. The intended purpose of the method is for regulatory analyses of commodities for pesticides that have established maximum residue limits. A fast and simple extraction method with cyclohexane-ethyl acetate (1 + 1, v/v) and a high-speed homogenizer was optimized. Pressurized liquid extraction was evaluated as an alternative automated extraction technique. The pesticide residues were determined by using low-pressure gas chromatography coupled to tandem mass spectrometry. The proposed methodology was validated for each matrix. Pesticide recoveries ranged from 70 to 110%, with repeatability relative standard deviations of < or = 18% at spiking levels of 12 and 50 microg/kg. The limits of quantitation were in the range of 0.03-6.17 microg/kg, and the limits of detection were between 0.01 and 3.75 microg/kg. Mango can be selected as a representative matrix for calibration on the basis of the results of a potential matrix effect study. The method was successfully applied to the determination of pesticide residues in real samples in Spain.     

气相色谱-质谱法检测食品中的丙烯酰胺

建立了一种用于食品中丙烯酰胺含量的气相色谱-质谱联用检测方法。通过水和甲醇提取食品中的丙烯酰胺,经蛋白变性净化后用溴水对其进行加成衍生化,再采用有机溶剂进行液液萃取,之后同三乙胺发生定量反应转化为性质更稳定的产物后由气相色谱-质谱联用仪检测,同位素内标法定量。该方法在0.02,0.05和0.2 mg/kg等3个添加水平下面粉和面包中丙烯酰胺的回收率处于80%和110%之间,相对标准偏差（RSD）不大于12.7%;在0.04～4.00 mg/L内呈现良好的线性关系;灵敏度高,最低检测限达到5 μg/kg;选择性好,能有效消除复杂基质带来的干扰。可作为常见样品中丙烯酰胺含量检测的确证方法。     

Application of hollow fiber supported liquid membrane extraction to the simultaneous determination of pesticide residues in vegetables by liquid chromatography/mass spectrometry

A new analytical procedure using a hollow fiber supported liquid membrane (HFSLM) has been developed for the simultaneous determination of pesticide residues in vegetables by liquid chromatography (LC) coupled with electrospray mass spectrometry (MS). The extraction technique requires minimal sample preparation and solvent consumption. Optimum extraction conditions have been evaluated with respect to sample pH, ionic strength, liquid membrane composition, extraction time, stirring rate and acceptor composition. The extraction method has been validated for matrices such as cucumber, tomato and pepper, indicating that cucumber can be selected as representative matrix for routine analysis of these food commodities. Linear ranges of pesticides in vegetable samples were 10 to 200 microg/kg, and the repeatability of the method was less than 20% for the lowest calibration point. The limits of detection ranged from 0.06 to 2.7 microg/kg and the limits of quantification from 0.2 to 9.0 microg/kg, which were low enough to determine the pesticide residues at concentrations below or equal to the maximum residue levels (MRLs) specified by European Union. The method was finally applied to the determination of more than 20 pesticides in market vegetable samples and the concentrations found in these samples were always lower than the MRLs. This new approach can be considered as a powerful alternative to the traditional extraction techniques.     

Evaluation of different sample treatments for determining pesticide residues in fat vegetable matrices like avocado by low-pressure gas chromatography-tandem mass spectrometry

A multi-residue method has been developed for determining 65 pesticide residues in greasy vegetable matrices such as avocado. Conventional organic solvent extraction assisted by a high-speed homogenizer was compared to pressurized liquid extraction (PLE) as extraction techniques. Following this, the lipophilic extract was purified using gel permeation chromatography (GPC). Alternative clean-up methods were also evaluated, as solid-phase extraction cartridges individually used and downstream coupled, but less effective lipophilic separation was archived. The pesticide residue determination was carried out using low-pressure gas chromatography coupled to tandem mass spectrometry (LP-GC-MS-MS), showing the applicability of this type of GC columns for the analysis of fat vegetable matrices. The proposed methodology was validated in avocado matrix. The recoveries were in the range 70-110%, with RSD values lower than 19%, at 12 and 50 microg/kg spiking levels. The limits of quantitation (LOQs) were in the range 0.04-8.33 microg/kg and the limits of detection (LODs) were between 0.01 and 2.50 microg/kg. All of them were lower than the maximum residue levels (MRLs) set by the European Union (EU) in avocado. The proposed method was evaluated analyzing pesticide residues in real avocado samples.     

Development and single-laboratory validation of a reversed-phase liquid chromatography-electrospray-tandem mass spectrometry method for identification and determination of acrylamide in foods

A rapid and accurate method using reversed-phase liquid chromatography-tandem mass spectrometry interfaced with electrospray was developed for determination of acrylamide in cooked food samples. A simplified sample treatment procedure using an extraction step with acidified water without cleanup was developed. A C18 column with an aqueous formic acid-methanol mixture as the mobile phase was used under isocratic conditions. The method was validated in-house for robustness, limits of detection (LOD) and quantitation (LOQ), linearity, recovery, and accuracy both on standard and baked-product and potato flour matrixes. Good results in the low ppb level were obtained for LOD (< 15 microg/kg) and LOQ (< 25 microg/kg) of acrylamide in samples. Excellent linearity (r2 = 0.999-1.000) was established over 2 orders of magnitude by performing statistical tests. The absence of both constant and proportional systematic errors demonstrated good method accuracy. Excellent results were obtained for intraday repeatability (RSD < 1.5%) and between-day precision (RSD < 5%). Extraction recoveries from food products were calculated in the 97 +/- 3-99 +/- 2% (n = 6) range with a labeled internal standard (13C3-acrylamide). The applicability of the method to determination of acrylamide in cooked food products was demonstrated.     

Simultaneous determination of six major ergot alkaloids and their epimers in cereals and foodstuffs by LC–MS–MS

This paper describes a new and rapid method for accurate quantification of the six ergot alkaloids, ergometrine, ergotamine, ergosine, ergocristine, ergocryptine, and ergocornine, by liquid chromatography–tandem mass spectrometry (LC–MS–MS). The six ergot alkaloids studied have been defined by the European Food Safety Authority (EFSA) as among the most common and physiologically active ones. In addition, the method enables the quantification of the corresponding six epimers (ergo-inines) of these ergot alkaloids. This is of considerable importance in terms of the differences in toxicity of the isomeric forms. The method involves extraction under alkaline conditions using a mixture of acetonitrile and ammonium carbonate buffer followed by a rapid clean-up using dispersive solid-phase extraction with PSA (primary secondary amine) and a short chromatographic LC-run (21 min) with subsequent MS–MS detection. The method was developed and validated using ten different cereal and food samples. The major strength of the new method compared with previously published techniques is the simplicity of the clean-up procedure and the short analysis time. The limits of quantification were 0.17 to 2.78 μg kg⁻¹ depending on the analyte and matrix. Recovery values for the 12 ergot alkaloids spiked into ten different matrices at levels of 5, 50, and 100 μg kg⁻¹ were between 69 and 105% for 85 of 90 recovery measurements made over six days. Measurement uncertainty values were highly satisfactory. At a concentration level of 5 μg kg⁻¹ the expanded measurement uncertainty ranged from ±0.56 to ±1.49 μg kg⁻¹, at a concentration level of 100 μg kg⁻¹ the expanded measurement uncertainty ranged from ±8.9 to ±20 μg kg⁻¹. Both LOQs and measurement uncertainties were dependent on the analyte but almost independent of the matrix. The method performance was satisfactory when tested in a mini-intercomparison study between three laboratories from three different countries. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

气相色谱-质谱法测定动物组织中三种1,2-二苯乙烯类药物的残留量

建立了动物组织中己烯雌酚(DES)、己二烯雌酚(DIS)和己烷雌酚(HS)残留量的气相色谱-质谱分析方法。动物组织样品在碱性条件下用乙酸乙酯提取,经碳酸钠溶液液-液分配净化,再用硅胶柱固相萃取净化,洗脱液均分成两份后经氮气吹干,分别用双三甲基硅基三氟乙酰氨(BSTFA)和七氟丁酸酐(HFBA)衍生,采用选择离子监测模式(SIM)进行测定,外标法定量。检出限为0.30 μg/kg(cis-DES),0.10 μg/kg(trans-DES和HS)和0.15 μg/kg(DIS)。在0.5~4.0 μg/kg添加水平,回收率为73.0%～86.5%,相对标准偏差(RSD)为1.0%～7.2%。衍生物的峰面积与样品浓度在10～1000 μg/L(DES和DIS) 和5～500 μg/L(HS)范围内呈良好的线性关系,线性回归系数大于0.99。     

Determination of residues of enrofloxacin and its metabolite ciprofloxacin in chicken muscle by capillary electrophoresis using laser-induced fluorescence detection

A method for the residue analysis of the veterinary antimicrobial agent enrofloxacin and its active desethyl metabolite ciprofloxacin in chicken muscle tissue has been developed and validated. The detection of the analytes was performed by laser-induced fluorescence (LIF) detection using a HeCd laser (lambda(ex) = 325 nm) providing an enhancement in sensitivity and selectivity compared to conventional UV detection. The assay has been validated with satisfying results. The limits of quantification for enrofloxacin and ciprofloxacin were 5 microg/kg and 20 microg/kg, respectively, with a fivefold preconcentration yielded by a sample clean-up with a simple liquid-liquid extraction procedure. Calibration graphs were linear from 5 to 1000 microg/kg for enrofloxacin and from 20 to 1000 microg/kg for ciprofloxacin. The assay allows the detection of contaminated muscle samples at the required maximum residue limit of the European Union, which is 100 microg/kg for the sum of enrofloxacin and ciprofloxacin.     

固相微萃取-气相色谱法和气相色谱-质谱法测定茶叶中氟虫腈及其代谢物残留

基于固相微萃取-气相色谱(SPME-GC)建立了检测茶叶中氟虫腈及其代谢物(脱亚硫酰基氟虫腈、硫化氟虫腈、氟虫腈砜、酰胺氟虫腈)残留的分析方法。实验中采用85 μm聚丙烯酸酯(PA)萃取头,萃取温度为60 ℃,萃取缓冲体系的pH值为9。当添加水平为2～10 μg/kg时,回收率在71.2%～109.3%之间,相对标准偏差(RSD)在1.2%～7.1%之间。方法的检出限(LOD)和定量限(LOQ)分别在0.3～1.2 μg/kg和1.0～4.0 μg/kg之间。对所建立的方法采用气相色谱-质谱(GC-MS)进行确证,结果令人满意。本方法灵敏度、精密度和LOD均符合残留分析要求,具有快速、灵敏度高的特点,适合于茶叶中氟虫腈及其代谢物残留的痕量分析。     

Ergot alkaloids in Norwegian wild grasses: a mass spectrometric approach

Ergot alkaloids are mycotoxins which are produced among fungi in the family Clavicipitaceae. Poisoning with ergot alkaloids is an important veterinary problem in animal husbandry and has recently also been recognised in wild animals. While the poisoning syndrome observed in domestic animals such as cattle, horses and sheep is usually caused by endophyte-infected grass, the recently observed ergotism among Norwegian cervids is probably due to infection of wild grasses with Claviceps. Mass spectrometry is today the method of choice for the rapid qualitative and quantitative determination of many natural compounds. This study uses tandem quadrupole mass spectrometry as well as ion trap mass spectrometry in connection with electrospray(+) ionisation for the quantification, screening and fragmentation of ergot alkaloids in extracts from Claviceps sclerotia that had been picked from wild grasses from several locations in Norway. Ergotamine, ergovaline, ergonovine and ergocryptine were available as standards and were quantified in the extracts, while ergocrystine, ergocornine, ergonine/ergosine, lysergic acid and lysergol were identified on the basis of their molecular weights and semi-quantified. Ergocrystine dominated the alkaloid spectrum of most extracts. Levels of the quantified alkaloids were in the range 0.2-9300 microg/g. Several unknown ergot alkaloids were found in the extracts. MS(n) experiments identified some as simple lysergic acid amide derivatives, while othes are probably related to ergocrystine and ergocryptine by dehydration, dehydrogenation and/or amino acid substitution at R(1) of the peptide moiety.     

固相萃取-高效液相色谱法测定动物肉组织中磺胺类药物的残留

建立了一种快速、灵敏、环保的固相萃取-反相高效液相色谱同时分析动物肉组织中5种磺胺类药物残留的方法。将样品加入到盛有无水硫酸钠的离心管中,再用乙酸乙酯提取;提取液经氨基固相萃取柱净化后,用1.5%（体积分数）乙酸乙醇溶液洗脱。洗脱液用高效液相色谱分离,二极管阵列检测器检测,外标法定量。5种磺胺类药物的线性关系良好,磺胺二甲基嘧啶(SM2)、磺胺间甲氧嘧啶(SMM)、磺胺甲唑(SMZ)的线性范围均为30～5000 μg/L,磺胺二甲氧嘧啶(SDM)、磺胺喹啉(SQ)的线性范围均为60～5000 μg/L。2种动物肉组织(鸡肉、猪肉)中5种磺胺类药物的加标回收率在73.2%至97.3%范围内,当添加水平为50 μg/kg时,加标回收率的相对标准偏差在2.5%至11.6%范围内;SM2,SMM和SMZ的检测限（S/N=3）和定量限（S/N=10）分别为3 μg/kg和10 μg/kg,SDM和SQ的检测限和定量限分别为7 μg/kg和25 μg/kg。     

Étude par chromatographie liquide à haute pression des alcaloïdes de l''ergot

High-performance liquid chromatography of ergot alkaloid.

A method for the analysis of ergot alkaloids has been developed by high-performance liquid chromatography. A silica column was used for preparation of ergotinine and ergotoxine from ergot. Separation of the individual ergot alkaloids was successfully accomplished on a column of Hitachi Gel No. 3011-O (porous polystyrene modified by hydroxymethyl) with an eluent of n-hexane—ethanol—triethylamine (70:30:0.5, v/v). The method was applied to the analysis of -ergocryptinine, ergocorninine, ergocristinine, -ergocryptine, ergocornine and ergocristine in ergot and dihydroergocryptine ( + β), dihydroergocornine and dihydroergocristine in pharmaceutical tablets.     

加速溶剂萃取-液相色谱-质谱/质谱法分析动物组织中的壬基酚、辛基酚和双酚A

建立了测定内分泌干扰物质烷基酚、双酚A的液相色谱-电喷雾串联质谱(负离子模式)分析方法,优化了样品前处理方法。以二氯甲烷作提取溶剂,采用加速溶剂萃取法萃取动物组织样品,萃取液用500 mg OASIS氨基固相萃取柱进行浓缩净化。对流动相组分和流动相添加剂对质谱的离子化效率进行了考察,测得3种化合物在高、中、低3个添加水平的回收率为88%~101%,相对标准偏差小于15%;双酚A、壬基酚和辛基酚的方法检出限分别为0.3, 0.05和0.1 μg/kg。对从北京市场上采集的27份动物组织样品进行检测,结果表明壬基酚广泛存在于各种动物源性食品中,检出含量为0.49~55.98 μg/kg,其中鱼肉组织中都检出壬基酚,而且其含量也较高(9.13~55.98 μg/kg)。     

Aflatoxin M1 determination in cheese by liquid chromatography-tandem mass spectrometry

A new method for determining aflatoxin M1 (AFM1) in cheese by liquid chromatography-tandem mass spectrometry has been developed. Two methodologies were compared for sample extraction. The first one involves sample extraction with dichloromethane for hard, aged cheese or acetone for fresh cheese and includes a preliminary matrix solid-phase dispersion-extraction step before solid-phase extraction (SPE) clean-up by a Carbograph-4 cartridge. The second method uses a water/methanol solution (90:10, v/v) extraction at 150 degrees C before clean-up. The average recoveries of AFM1 from samples spiked at levels of 0.25-0.45 microg/kg, were 81-92% and the precision (RSD) ranged from 3 to 7% with the first method, whilst the average recoveries were 79-84%, and RSD ranged from 7 to 15% for the second method. Due to different matrix effect, the quantification limits were 0.019-0.025 microg/kg in the first case and 0.048-0.143 microg/kg in the second one, depending on cheese typology.     

免疫亲和柱净化-柱后光化学衍生-高效液相色谱法同时检测粮谷中的黄曲霉毒素、玉米赤霉烯酮和赭曲霉毒素A

建立了同时检测粮谷中黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A的免疫亲和柱净化-柱后光化学衍生-高效液相色谱方法。样品经过甲醇-水(体积比为80∶20)提取,通过免疫亲和柱富集和净化,采用Waters Nova-Pak色谱柱(3.9 mm i.d.×150 mm,4 μm),以甲醇、乙腈和1%的磷酸溶液为流动相,梯度洗脱,柱后光化学衍生、改变波长荧光检测。黄曲霉毒素(B1、B2、G1和G2)、玉米赤霉烯酮和赭曲霉毒素A检出限分别为0.24,4.0和0.5 μg/kg,标准曲线的线性范围分别为0.24~6.0,4.0~100.0和0.5~40.0 μg/L;在小麦、玉米、黑麦样品中,平均加标回收率为70.8% ~94.0%,相对标准偏差为2.79% ~9.38%。