Proteome analysis of Pithecellobium dulce seeds using two‐dimensional gel electrophoresis and tandem mass spectrometry

BACKGROUND: Pithecellobium dulce Benth. belongs to the Leguminosae family, which contains several members that are important components of human diets owing to their high protein content and quality. In this study the seed proteins from P. dulce were separated and identified using two‐dimensional gel electrophoresis (2‐DE) and mass spectrometry respectively. RESULTS: The 2‐DE protein map revealed a total of 317 distinct protein spots, including a cluster of about 12 proteins located in the region of pI 5–6 with molecular masses of 55–97 kDa that accounted for more than 50% of the total proteins. Ninety‐six of the most abundant protein spots were analysed using nano liquid chromatography/tandem mass spectrometry (LC/MS/MS), from which 27 were successfully identified through the query of acquired tandem mass spectral data used in MASCOT searching against a custom legume protein database. A further four proteins from the highly abundant protein cluster were putatively identified using mass spectrometry‐driven BLAST (MS‐BLAST) homology searches. CONCLUSION: This research has generated a 2‐DE proteome reference map for P. dulce seeds and used LC/MS/MS to characterise the proteins. The identification of proteins from P. dulce was carried out using the sequence database successively for MASCOT and MS‐BLAST homology‐based searches. Copyright © 2009 Society of Chemical Industry     

Fast analysis of glufosinate,glyphosate and its main metabolite,aminomethylphosphonic acid,in edible oils,by liquid chromatographycoupled with electrospray tandem mass spectrometry

ABSTRACT

A method has been developed for the rapid, specific, accurate, precise and sensitive determination of glufosinate, glyphosate and its major metabolite, aminomethylphosphonic acid, in edible oils, by liquid chromatography coupled to tandem mass spectrometry. Oils were extracted with acidified water (1% formic acid), and the extracts were directly injected into an LC using a Hypercarb column as the stationary phase. The analytes were eluted by a mobile phase of methanol and water containing 1% acetic acid, and they were ionised by electrospray ionisation in negative ion mode. The method was validated and limits of quantification ranged from 5 μg kg^?1 (aminomethylphosphonic acid) to 10 μg kg^?1 (glyphosate and glufosinate). Three concentrations (10, 50 and 100 μg kg^?1) were selected to perform recovery studies. Mean recoveries ranged from 81.4% to 119.4%. Intra and inter-day precision were lower than 19%. Different edible oils were analysed, and no residues of the studied herbicides were detected above limits of quantification.     

Determination of naturally occurring progestogens in bovine milk as their oxime derivatives using high performance liquid chromatography–electrospray ionization–tandem mass spectrometry

BACKGROUND: Hormones and hormone‐like substances which are present in the environment have been repeatedly accused of being the cause of most endocrine disruption. However, the possible role of endogenous hormones in food of animal origin deserves to be discussed as well. The relation between steroid hormones and several human health problems has been previously reported, such as prostate and breast cancer, perturbation of human reproduction and endocrine disruption on humans and wildlife. This research is particularly concerned with cow's milk, which contains a considerable amount of sex hormones. RESULTS: A liquid chromatography–tandem mass spectrometry method has been developed for the simultaneous detection and quantification of four naturally occurring steroid hormones in commercial bovine milk (pregnenolone (P₅), progesterone (P₄), 17‐hydroxypregnenolone (17‐OHP₅), 17‐hydroxyprogesterone (P₄)). Oxime derivatives of steroids were analyzed in positive ionization and multiple reaction monitoring mode. Methodology has been validated according to Decision 2002/657/EC criteria. CONCLUSION: This method has been successfully used in real samples. It is fast and easy‐handling and provides a useful tool for the assessment of progestogens in bovine milk. Copyright © 2010 Society of Chemical Industry     

Quantitative analysis of acrylamide in tea by liquid chromatography coupled with electrospray ionization tandem mass spectrometry

An effective sample preparation procedure was optimized and a liquid chromatography–tandem mass spectrometry (LC–MS/MS) was developed for the quantitative analysis of acrylamide in tea. [¹³C₃]-acrylamide was used as internal standard. Acrylamide was extracted at 25 °C for 20 min by 10 ml water followed by 10 ml acetonitrile, and then 4 g of magnesium sulfate and 0.5 g of sodium chloride were added to the above mixture under stirring thoroughly. In order to increase the response of acrylamide, 9 ml acetonitrile layer was taken and concentrated to 0.5 ml. Solid-phase extraction with an Oasis MCX cartridge was carried out for clean-up. The limit of detection (LOD) and limit of quantification (LOQ) were 1 and 5 ng/ml, respectively. The recovery efficiency of the extraction procedure ranged between 74% and 79%. The levels of acrylamide in 30 tea samples were less than 100 ng/g. Black, oolong, white and yellow tea samples had quite low acrylamide contents (<20 ng/g). Higher acrylamide levels occurred in baked, roasted, and one sun-dried green tea samples (46–94 ng/g).     

液相色谱-串联质谱法测定猪肉及组织中沙丁胺醇的含量不确定度评定

目的分析液相色谱一串联质谱法检测猪肉及组织中沙丁胺醇的不确定度,探讨各因素对检验结果的影响。方法依据JJG1059.1-2012《测量不确定度评定与表示》和CNAS-GL06《化学分析中不确定度的评估指南》规定的方法和程序,分析影响测量不确定的来源,并对各不确定度分量进行了评估。结果合成各变量的不确定度,最终得到测定结果的扩展不确定度:(11.05±0.77)μg/kg(k=2)。结论影响检测结果不确定度的主要因素为回收率和标准曲线拟合,在实际测量过程严格控制实验条件可提高检测的准确性和可靠性。     

Multi-residue method for the analysis of pesticides in Arabica coffee using liquid chromatography/tandem mass spectrometry

Coffee is a major tropical agricultural commodity and represents a significant fraction of the economy of many countries. However, certain plant and animal species can damage coffee crops, affecting trade. A solution to this issue is the use of pesticides, some of which are harmful to human health and the environment. This work consisted of the development of a multi-residue method for the analysis of pesticides in coffee by using LC-MS/MS. The QuEChERS extraction procedure was used. The following analytical parameters were optimised: selectivity, analytical range, linearity, LOD, LOQ, precision (RSD%) and recovery of the method. The results showed that the method is selective, as they were linear in the range of 10.0–100.0 µg kg^?1. The sensitivity, recovery and precision were adequate for the multi-residue analysis of pesticides in coffee. The method was applied to the analyses of 15 Brazilian coffee samples.     

Multitoxin evaluation in fermented beverages and cork stoppers by liquid chromatography–tandem mass spectrometry

A total of twenty‐eight mycotoxins were surveyed in wine (red, white and rose), cider (white and rose) and their cork stoppers from eight countries. Toxins of different fungi genera were detected as follows: Alternaria (ATs: alternariol – AOH; alternariol methyl – AME) and Penicillium/Aspergillus (ochratoxin A – OTA; penicillic acid – PAC). Toxins and levels varied with the sample types and country of origin. Wine presented contamination of OTA, AOH and AME. OTA was detected in forty‐one wine samples with levels ranging from 0.01 to 0.86 μg L^?1, below EU legislation. AOH and AME were detected in thirty‐three and eight of wines samples, respectively, at levels from 0.2 to 13.3 μg L^?1, while no contamination was detected in ciders up to the method LOQs. Regarding the cork stoppers toxins detected, they were AOH, AME and PAC. Corks of red wine from different countries had levels of OAH and AME ranging from 5.0 to 101.0 and 2.5 to 5 μg g^?1, respectively. It is necessary to pay more attention on the corks processing and cork type used in the bottles as, different from the ordinary ones, the ground bark and compressed type did not have toxins detected.     

水果中噻虫嗪农药残留LC/MS/MS测定

目的建立一种简单、快速、灵敏的水果中噻虫嗪农药残留的液相色谱-串联质谱(liquid chromatography-mass spectrometry/mass spectrometry,LC-MS/MS)分析方法。方法称取水果样品5 g,加入乙腈20 m L在超声波振荡条件下提取,提取液使用20 mg石墨化炭黑(Carb)和60 mg N-丙基乙二胺(PSA)粉末进行分散固相萃取净化,经液相色谱质谱联用仪检测,外标法定量。结果噻虫嗪农药残留的色谱图分离效果良好,方法的检出限为0.3μg/kg,线性相关系数为0.9999,噻虫嗪在苹果、梨、桃中的添加水平为0.01、0.05、0.10 mg/kg,回收试验表明该方法平均回收率为88.9%~100.3%(n=6),相对标准偏差为1.98%~4.53%。结论该方法简单、快速、灵敏、净化效果好、回收率高,适合水果中噻虫嗪农药残留的检测和安全监控。     

食用植物油中痕量游离棉酚的超高效液相色谱-串联质谱测定

建立了超高效液相色谱-三重四级杆-串联质谱法(UPLC-ESI-MS/MS)测定食用植物油中游离棉酚的含量,以乙醇为提取溶剂提取食用植物油中的游离棉酚,采用Waters AQUITY UPLC BEH C18色谱柱进行分离,以乙腈-0.1%的甲酸水溶液作为流动相,梯度洗脱。在电喷雾离子源(ESI)负离子模式和多反应通道监测(MRM)模式下进行定量分析。结果表明:棉酚的质量浓度在0.005～5.0μg/mL范围内线性关系良好,方法检出限LOD (S/N≥3)为10μg/kg,定量限LOQ (S/N≥10)为50μg/kg。以0.25、2.5、25μg/g 3个不同棉酚浓度进行加标后测得平均加标回收率为80.3%～112.6%,相对标准偏差小于11%,精密度0.42%。该方法灵敏度高,测定结果准确,回收率稳定,适用于确证检测食用植物油中的棉酚残留。     

Characterization of phenolics of Sarcandra glabra by non-targeted high-performance liquid chromatography fingerprinting and following targeted electrospray ionisation tandem mass spectrometry/time-of-flight mass spectrometry analyses

In this study, we successfully characterised the phenolic profiles of Sarcandra glabra (Thunb.) Nakai by high-performance liquid chromatography (HPLC) fingerprinting analyses and mass spectrometry (MS) identification. We first established a specific and valid HPLC approach for fingerprint analysis of S. glabra based on HPLC–UV detection. Using several chemometric methods such as similarity evaluation and principal components analysis, we determined herb-markers peaks from many HPLC peaks. The structures of these herb-markers were further identified targetedly by electrospray ionisation tandem mass spectrometry (ESI-MS/MS)/time-of-flight mass spectrometry (TOF-MS) analyses. As results, four phenolics, including chlorogenic acid, caffeic acid, 4-O-glucopyranosyl rosmarinic acid and rosmarinic acid, were characterised as major herb-markers for the stems of S. glabra, while another three phenolics, including kaempferol-3-O-β-d-glucuronic acid, chlorogenic acid and rosmarinic acid, were characteristic components for the leaves. The compounds may be very useful for further phenolome analysis.     

Multiclass mycotoxin analysis in edible oils using a simple solvent extraction method and liquid chromatography with tandem mass spectrometry

ABSTRACT

A simple and rapid method for the simultaneous determination of 11 mycotoxins – aflatoxins B₁, B₂, G₁ and G₂; fumonisins B₁, B₂ and B₃; ochratoxin A; zearalenone; deoxynivalenol; and T-2 toxin – in edible oils was established using liquid chromatography tandem mass spectrometry (LC-MS/MS). In this study, QuEChERS (Quick, Easy, Cheap, Effective, Rugged and Safe), QuEChERS with dispersive liquid–liquid microextraction, and solvent extraction were examined for sample preparation. Among these methods, solvent extraction with a mixture of formic acid/acetonitrile (5/95, v/v) successfully extracted all target mycotoxins. Subsequently, a defatting process using n-hexane was employed to remove the fats present in the edible oil samples. Mass spectrometry was carried out using electrospray ionisation in polarity switching mode with multiple reaction monitoring. The developed LC-MS/MS method was validated by assessing the specificity, linearity, recovery, limit of quantification (LOQ), accuracy and precision with reference to Commission Regulation (EC) 401/2006. Mycotoxin recoveries of 51.6–82.8% were achieved in addition to LOQs ranging from 0.025 ng/g to 1 ng/g. The edible oils proved to be relatively uncomplicated matrices and the developed method was applied to 9 edible oil samples, including soybean oil, corn oil and rice bran oil, to evaluate potential mycotoxin contamination. The levels of detection were significantly lower than the international regulatory standards. Therefore, we expect that our developed method, based on simple, two-step sample preparation process, will be suitable for the large-scale screening of mycotoxin contamination in edible oils.     

高效液相色谱串联质谱检测食用植物油中的乙基麦芽酚

为了建立食用植物油中乙基麦芽酚检测的高效液相色谱串联质谱方法,食用植物油样品与二氯甲烷混合后,以1.5%NaOH水溶液做为提取溶剂,涡旋、离心后,用磷酸将上层提取液调至4～7,调节pH后的上清液用PLS固相萃取柱进行净化除杂。采用电喷雾电离源正离子多反应监测模式进行检测,采用溶剂匹配标准曲线外标法进行定量。结果表明：该方法在6.25～100 ng/mL质量浓度范围内线性关系良好(R²>0.999),方法定量限为25.0μg/kg,在3个加标水平下的平均回收率为82.9%～105.1%,相对标准偏差小于5%,重复性好。该方法准确、灵敏度高,能有效减少芝麻油的基质效应,适用于多种食用植物油中乙基麦芽酚的检测。     

Liquid chromatography tandem mass spectrometry determination of maduramycin residues in the tissues of broiler chickens

Maduramycin is a polyether ionophoric coccidiostat used to prevent coccidiosis in poultry at a prescribed concentration over a certain time interval. Due to public health concerns about the presence of coccidiostat residues in poultry, the aim of the present study was to determine the level of maduramycin residues in the tissues of broiler chickens fed commercially produced feed containing 5 mg kg^?1 of maduramycin in complete feed throughout the 5-day withdrawal period (WP). The residues were investigated by liquid chromatography (LC) coupled with electrospray ionisation (ESI) tandem mass spectrometry (MS/MS). The limit of detection (LOD) and limit of quantification (LOQ) of the method were 0.3 and 0.8 µg kg^?1, respectively. The average recovery based on matrix-fortified calibrations for chicken tissues was 90%. Maduramycin was found to be rapidly distributed in all tissues. The highest concentrations of maduramycin residues were found in the heart followed by the skin, liver, gizzard, kidneys and, finally, muscle (thigh and breast). On day 5 of the WP, residue concentrations of maduramycin did not decline below the LOQ of the method. Our results emphasize the need to establish a maximum residue limit (MRL) for maduramycin to control its residue levels in edible tissues from chickens before slaughter.     

液相色谱-串联质谱法检测食用油脂中多环芳烃

建立了有机溶剂萃取、硅胶固相萃取柱净化、液相色谱-串联质谱法(LC-MS/MS法)测定食用油脂中EPA 16种多环芳烃的检测方法。EPA 16种多环芳烃的定量限分别为0.02~0.43μg/kg,回收率为86.5%~104.6%,日内精密度小于6%,日间精密度小于5%。在40个受测油脂样品中,EPA 16种多环芳烃的含量范围为11.68~146.06μg/kg。对照我国GB 2716规定,所有受测样品中苯并(a)芘含量均不超过≤10μg/kg的限量标准。然而,8个油样的苯并(a)芘含量超过了欧盟≤2μg/kg的限量标准,10个油样的PAH4含量超过了欧盟≤10μg/kg的限量标准。     

Assessment of liquid chromatography–tandem mass spectrometry approaches for the analysis of ceftiofur metabolites in poultry muscle

The use of cephalosporin antibiotics in veterinary practice is likely to play an important role in the development of β-lactam-resistant bacteria. To detect off-label cephalosporin antibiotic usage, an analytical method is needed that, besides the native compound, also detects their active metabolites. In this paper, the applicability of three approaches for the quantitative analysis of ceftiofur using LC–MS/MS is assessed, viz. (A) analysis of ceftiofur, desfuroylceftiofur and/or desfuroylceftiofur cystein disulfide, (B) derivatisation of ceftiofur metabolites to desfuroylceftiofur acetamide and (C) chemical hydrolysis using ammonia, to produce a marker compound for ceftiofur. We found that approach A was not suited for quantitative analysis of total ceftiofur concentration or for effectively detecting off-label use of ceftiofur. Approach B resulted in adequate quantitative results, but was considered a single compound method because it depends on cleavage of a thioester group, which is present in only a limited number of cephalosporin antibiotics. Approach C showed adequate quantitative results but, in contrast to approach B, it is applicable to a range of cephalosporin antibiotics. Therefore, it is applicable as a broad quantitative screening of cephalosporin compounds in poultry tissue samples to indicate off-label use of cephalosporins in poultry breeding. Based on this study, it was concluded that approach C is the most suitable to detect off-label use of a range of cephalosporin antibiotics.     

超快速液相色谱-串联质谱法测定粮食及食用油中的黄曲霉毒素

应用超快速液相色谱―串联质谱法技术,建立粮食及食用油中黄曲霉毒素的检测方法,并对宁夏市售小麦粉、玉米制品和食用油进行分析。样品经乙腈―水(20︰80)提取,免疫亲和柱净化;以甲醇–0.1%甲酸水溶液为流动相,用Atlantis T3色谱柱分离,以电喷雾正离子模式进行质谱测定。采用该方法,黄曲霉毒素B1、B2、G1和G2可在6分钟内完成检测,分别以小麦粉、玉米制品和食用油为加标基质,三个加标水平下的平均回收率为75.6%~94.1%,相对标准偏差小于10.0%,检出限(S/N=3)为0.05μg/kg,定量限(S/N=10)为0.15μg/kg。该方法操作快速简单、重现性好,可用于粮食中黄曲霉毒素B1、B2、G1和G2的检测。