Identification of new diketopiperazines in roasted coffee

This paper reports on the identification of new diketopiperazines (DKPs) in roasted coffee. The roasted coffee was extracted with hot water and CHCl₃ to achieve both a cleanup and a concentration of the DKPs. To get rid of the huge caffeine peak in both HPLC and GC analysis, this extract was cleaned up again by ion-exchange chromatography on Dowex 50Ǽ followed by gel chromatography on Sephadex G10. The identification was accomplished by HPLC-ESI-MS, HPLC-ESI-MS/MS, GC-EI-MS and by comparison with synthesised reference compounds. In roasted coffee the following DKPs were identified: cyclo(pro-gly), cyclo(pro-ala), cyclo(phe-val), cyclo(phe-leu), and cyclo(phe-ile). Except for cyclo(pro-gly), where only one isomer can be formed, each DKP was present in both possible isomeric forms.     

一种新型抗氧化五肽的纯化、鉴定与表征

以黑鲨鱼皮为原料,利用蛋白酶酶解制备和分离纯化抗氧化多肽,并探究其抗氧化机理。以1,1-二苯基-2-苦肼基（1,1-diphenyl-2-picrylhydrazyl,DPPH）自由基清除率为主要指标,以水解度为辅助指标,优化酶解工艺条件。通过反相高效液相色谱和电喷雾四极杆飞行时间质谱分离纯化和鉴定氨基酸序列并探究其体外抗氧化活性。结果显示,最佳水解蛋白酶为碱性蛋白酶,最优酶解工艺为pH 8.0、酶添加量311 U/mL、底物质量分数3%、酶解温度45 ℃、酶解时间4.9 h,所得的酶解液DPPH自由基清除率为77.61%,水解度为14.21%。经反相高效液相色谱分离,得到组分F19的DPPH自由基清除能力最强。经鉴定,选择抗氧化活性高的总离子流色谱峰（保留时间为10.02 min左右）进行一级质谱和二级质谱分析,获得纯化的新型抗氧化五肽,其分子质量为461 Da,氨基酸序列结构为Pro-Gly-Gly-Thr-Met,其DPPH自由基清除率为75.01%（1.0 mg/mL）,氧自由基吸收能力（以Trolox当量计）为2490.01 μmol/g。新型五肽的Pro和Met在抗氧化中起到关键性作用。本研究为黑鲨鱼皮抗氧化五肽的抗氧化机理及其构效关系提供了一定理论依据。     

山西老陈醋源萎缩芽孢杆菌细菌素对病原菌群体感应的抑制研究

该实验考察了不同理化因子、山西老陈醋源萎缩芽孢杆菌（Bacillus atrophaeus）SAV-CP 297细菌素对5种病原菌（大肠杆菌、金黄色葡萄球菌、沙门氏菌、志贺氏菌、单增李斯特菌）产信号分子二酮哌嗪（DKPs）能力的影响，并将该细菌素应用于草莓防腐中，考察其对群体感应的抑制作用。结果表明，5种病原菌均能产生cyclo-（L-Pro-L-Gly）、cyclo-（L-Pro-L-Leu）和cyclo-（L-Pro-L-Phe）3种类型的DKPs。随NaCl含量增加，DKPs的种类和含量减少；随pH、温度升高，DKPs含量均呈先升高后下降趋势，其种类在pH升高时增加，温度升高时不变。B. atrophaeus SAV-CP 297细菌素可以明显抑制病原菌产DKPs的能力，从而延缓贮藏期间草莓的腐败速率，达到良好的保鲜效果。     

Screening method for the determination of 199 pesticides in agricultural products by gas chromatography/ion trap mass spectrometry (GC/MS/MS)

A screening method is described for determining 200 pesticides, except dimethipin, divided into four groups by means of gas chromatography/tandem mass spectrometry (GC/MS/MS) using an ion trap mass spectrometer equipped with automated gain control (AGC). The quantitation limit for 194 pesticides was 0.01 mg/kg on a crop basis, except for allidochlor, dimethoate, hexythiazox, methamidophos and triadimenol. The calibration curve of each pesticide was linear in the range of 0.04-5.0 microg/mL. One hundred and ninety-nine pesticides were added to matrix of potato, spinach, cabbage, apple, orange, soybean and unpolished rice at twice the limits of quantitation. The recoveries of 194 pesticides from all crops were satisfactory (50-150%) for screening purposes. Although some pesticides in apple and orange were not determined by selected ion monitoring (SIM) analysis at the limits of quantitation, all of them were identified by ion-trap GC/MS/MS at the same concentration. Thus, the ion trap GC/MS/MS technique is useful for the screening of residual pesticides present at low levels in agricultural products.     

Assessment of the main pathogens associated with clinical and subclinical endometritis in cows by culture and MALDI-TOF mass spectrometry identification

Clinical endometritis (CE) and subclinical endometritis (SCE) are diseases that affect dairy cows during the puerperium, causing negative effects on the animals' milk production and fertility. The objective of this study was to assess the main bacteria related to cases of CE and SCE from uterine samples of dairy cows in Brazilian herds. Selective and differential media were used for isolation of aerobic and anaerobic bacteria and further MALDI-TOF mass spectrometry (MS) identification. A total of 279 lactating dairy cows with 28 to 33 d in milk from 6 commercial farms were evaluated. Initially, cows were classified in 3 groups: cytologic healthy cows (n = 161), cows with CE (n = 83), and cows with SCE (n = 35). Healthy animals presented 97 species, followed by the CE group with 53 identified species, and SCE cows presented only 21 bacterial species. We found a significantly higher isolation rate of Trueperella pyogenes in CE (26.5%) cows compared with healthy and SCE cows. Some anaerobic species were exclusively isolated from the CE group, even though they presented lower frequency. Interestingly, 18.1% of samples from CE cows and 40% of SCE cows were negative to bacterial isolation. Despite the use of culture-dependent methods instead of molecular methods, the present study enabled the identification of a complex community of 127 different species from 48 genera, composed of aerobic and anaerobic bacterial species among the 3 different animal groups. The method of sample collection, culture, and identification by MALDI-TOF MS were essential for the success of the analyses.     

应用液相色谱―串联质谱仪和四极杆―轨道阱高分辨质谱仪检测饲料中克伦普罗干扰的原因分析与确证技术研究

目的 探究液相色谱―串联质谱仪(liquid chromatography-tandem mass spectrometer, LC-MS/MS)检测饲料中克伦普罗时, 环天冬氨酰苯丙氨酸对试验干扰的原因, 在此基础上探究出应用LC-MS/MS分析的确证技术与方法。方法 应用四极杆―轨道阱高分辨质谱仪和LC-MS/MS对环天冬氨酰苯丙氨酸、克伦普罗对照品以及含有环天冬氨酰苯丙氨酸的饲料样品进行分析。结果 饲料中含有环天冬氨酰苯丙氨酸对克伦普罗的检测干扰的原因为二者母离子及2个子离子的质荷比很接近, 且二者离子丰度比也很接近。应用LC-MS/MS分析的确证技术与方法为梯度碎裂离子片段依次匹配比较法。结论 应用LC-MS/MS检测会遇到假阳性的问题, 这是串联质谱结构与方法原理决定的; 建议调整克伦普罗的2个子离子为263.07>168.04, 263.07>132.06; 用LC-MS的梯度碎裂离子片段依次匹配比较法准确、实用。     

白腐乳中呈味肽的分离与鉴定

目的:从白腐乳中寻找呈味肽,分析其氨基酸序列,人工合成肽样品,研究其滋味特性。方法:白腐乳提取液经过超滤、Sephedex G-15葡萄糖凝胶过滤层析,分离出3个呈味组分,通过感官评价筛选出呈鲜效果最优的组分,采用反相高效液相色谱对其进一步分离纯化,选择峰面积最大的组分,使用基质辅助激光解析电离-飞行时间质谱结合De Novo软件分析呈味肽的氨基酸序列,人工合成肽样品,对合成肽样品的呈味特性进行分析。结果:在白腐乳中发现并确定序列的肽链3条,其氨基酸序列分别为:Asp-Phe-Lys-Arg-Glu-Pro、Asp-Arg-Glu-Lys-Phe-AspGlu、Asp-Glu-Asp-Phe-Lys-Arg-Glu-Pro。其中肽Asp-Phe-Lys-Arg-Glu-Pro具有鲜味,肽Asp-Glu-Asp-Phe-Lys-ArgGlu-Pro兼具鲜味与酸味,肽Asp-Arg-Glu-Lys-Phe-Asp-Glu虽无明显特征滋味,但在味精溶液中则体现出较强的增鲜效果。结论:腐乳的鲜味不仅仅来自于谷氨酸等氨基酸成分,还与小分子肽类的呈鲜作用有关。     

A metabolomics approach to characterize raw,pasteurized, and ultra-high temperature milk using ultra-performance liquid chromatography–quadrupole time-of-flight mass spectrometry and multivariate data analysis

We developed a metabolomics workflow using ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry to determine the effect of thermal treatment on milk composition and metabolites based on multivariate data analysis. We analyzed raw, pasteurized, and UHT milk samples. The samples were first centrifuged to remove the fat layer and mixed with methanol to precipitate proteins. Subsequently, the supernatant was analyzed by ultra-performance liquid chromatography coupled to quadrupole time-of-flight mass spectrometry in electrospray negative mode. Mass spectral data were acquired in MS^E mode, a technique whereby both precursor and fragment mass spectral are simultaneously acquired by alternating between low and high collision energy (CE) during a single analytical run, to enable metabolite identification. Based on multivariate data analysis, these markers were significantly affected by thermal treatment. Among the 8 potential markers, we identified 7 oxylipids (9-hydroxydecanoic acid, 12-hydroxydodecanoic acid, 2-hydroxymyristic acid, 3-hydroxytetradecanoic acid, 5-hydroxyeicosatetraenoic acid, 3-hydroxyhexadecanoic acid, and 10-hydroxyoctadecanoic acid) and 1 phospholipid (LysoPE, hexadecanoyl-lysophosphatidylethanolamine). The oxylipids seemed to be adequate for distinguishing UHT milk from raw and pasteurized milk. The structures of the 8 potential markers were identified and characterized using informatics software. Our metabolomics workflow provides a fast approach for the identification of various types of milk.     

Electrospray ionization mass spectrometry fingerprinting of essential oils: Spices from the Labiatae family

Polar components of the methanolic extracts of the essential oils of the spices Origanum dictamnus, Origanum vulgare, Origanum majorana and Rosmarinus officinalis, all four belonging to the Labiatae family, were investigated by direct infusion electrospray ionisation mass spectrometry (ESI-MS) both in the negative and positive ion modes. Characteristic ESI mass spectra with many diagnostic ions were obtained for the extracts of all four spices, serving for fast and reliable identification of these species. Tandem mass spectrometry (ESI-MS/MS), which often forms a series of fragment ions, and this additional MS dimension increases selectivity for authenticity and adulteration tests for spice essential oils. The MS technique also provides complementary information of component structures revealing the presence of important bioactive components.     

Quantification of (+)-catechin and (−)-epicatechin in coconut water by LC–MS

An analytical technique has been developed to detect trace amounts of both (+)-catechin and (−)-epicatechin in the coconut water extract. Both (+)-catechin and (−)-epicatechin in the coconut water were found for the first time by the solid-phase extraction, and they were further analysed using liquid chromatography (LC)–ion trap mass spectrometry (MS) equipped with positive atmospheric pressure chemical ionisation interface on multiple reaction monitoring mode. The limit of detection and quantification for (+)-catechin were 7.8 and 15.6 μg/ml, respectively, while those for (−)-epicatechin were 3.9 and 7.8 μg/ml, respectively. The average concentration of (+)-catechin and (−)-epicatechin in the coconut water was 0.344 and 0.242 μg/ml, respectively. The LC–MS/MS analysis accelerated the quantitative analysis of (+)-catechin and (−)-epicatechin in the coconut water extract with high accuracy, precision and recovery. Results obtained in this study will serve as quality control and useful reference for drug development.     

Hydrogen abstraction and decomposition of bromopicrin and other trihalogenated disinfection byproducts by GC/MS

Tribromonitromethane (bromopicrin), dibromochloronitromethane, bromodichloronitromethane, and trichloronitromethane (chloropicrin) have been identified as drinking water disinfection byproducts (DBPs). They are thermally unstable and decompose under commonly used injection port temperatures (200-250 degrees C) during gas chromatography (GC) or GC/mass spectrometry (GC/MS) analysis. The major decomposition products are haloforms (such as bromoform), which result from the abstraction of a hydrogen atom from the solvent bythermally generated trihalomethyl radicals. A number of other products formed by radical reactions with the solvent and other radicals were also detected. The trihalonitromethanes also decompose in the hot GC/MS transfer line, and the mass spectra obtained are mixed spectra of the undecomposed parent compound and decomposition products. This can complicate the identification of these compounds by GC/MS. Trihalomethyl compounds that do not have a nitro group, such as tribromoacetonitrile, carbon tetrabromide, methyl tribromoacetate, and tribromoacetaldehyde, do not decompose or only slightly decompose in the GC injection port and GC/MS transfer line. The brominated trihalomethyl compounds studied also showed H/Br exchange by some of their fragment ions. This H/Br exchange also makes the identification of these compounds in drinking water more difficult. The extent of H/Br exchange was found to depend on the mass spectrometer ion source temperature, and it is proposed that the internal surface of the ion source is involved in this process.     

Screening for Human Milk Amino Acids by HPLC-ESI-MS/MS

Amino acids have a determining effect on the nutritional status of the newborn, and their appropriate levels in breast milk are vital for this first stage of life. The amino acids tryptophan, arginine, glutamate, and taurine, for example, are suggested to have a positive effect on immune functions. The purpose of the present study was to develop a new method for the assay of amino acids in human milk by high-performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC-ESI-MS/MS) after hydrolysis. Breast milk samples were collected from 77 healthy mothers living in the community of Extremadura (Spain) with less than 2 months of lactation. The samples were then stored at −80 °C. The HPLC-ESI-MS/MS technique proved to be a sensitive and efficient tool for the assay of amino acids in breast milk. The method could be used for the qualitative screening of 40 underivatized amino acids, and for the assay of 20. The resulting data will serve to improve commercial infant formula milks and bring them closer to the reference standard represented by human milk.     

Evaluation of purine alkaloids and diketopiperazines contents in processed cocoa powder

 This paper describes the evaluation of purine alkaloids and diketopiperazines (DKPs) contents in 24 samples of processed non-alkalinized cocoa powder. The chemical data obtained were related to sensory evaluation and subjected to analysis of variance. During cocoa roasting, the content of DKPs can be increased, resulting in a negative influence on the sensorial quality of cocoa. A pronounced bitter metallic taste can originate from some of these compounds, and becomes stronger when these DKPs are combined with purine alkaloids. The cocoa roasting process at different temperatures (125, 130, 135, 140 and 145  °C) and durations (3, 10, 16 and 20 min) was evaluated by measuring the flavour indices, formol numbers and DKP contents in 24 samples of cocoa powder. The addition of reducing sugars was also evaluated in the same industrial processes in 27 other samples of cocoa powder. The higher the roasting temperature of the cocoa was, the more elevated the flavour index became, except at temperatures up to 140  °C, where DKPs were generated in large proportions. Received: 2 February 1999 / Revised version: 23 April 1999     

Amino acid composition and anti‐polyphenol oxidase of peptide fractions from sericin hydrolysate

Sericin hydrolysate (SH), prepared from enzymatic hydrolysis of sericin, was investigated for its antipolyphenol oxidase (PPO) properties. SH decreased PPO activity from both purified mushroom PPO and extracts from apple and eggplant, and retarded browning in fresh‐cut apple and eggplant. SH was a competitive inhibitor using catechol as a substrate. SH exhibited copper ion‐chelating power and reducing power abilities. Fractionation of SH using size exclusion chromatography resulted in four fractions, designated as F1, F2, F3 and F4, with PPO inhibition of 35.75%, 3.89%, 24.52% and 14.75%, respectively. Ser and Asp were major amino acids found in F1. Amino acid sequences in F1, as investigated by LC‐MS/MS using de novo sequencing, contained a high ratio of amino acids with chelating ability. Moreover, amino acids with reducing power ability and with antityrosinase ability were also identified in the sequences.     

Xanthylium salts formation involved in wine colour changes

Summary The reaction of (+)-catechin in wine-like model solution was investigated. First appearance of colourless dimeric compounds consisting of two flavanol units linked by carboxy-methine bridge was observed. Their isolation and further incubation was found to yield two types of yellowish pigments showing visible absorption maxima at 440 and 460 nm, respectively. Mass spectroscopy (MS) spectral analysis showed that the first type were xanthylium salt pigments formed by dehydration of the colourless compounds followed by an oxidation process. The loss of a water molecule was shown to take place between two A ring hydroxyl groups of the colourless dimers. The second type were shown to be ester derivatives of the first ones. Thus ethylester of xanthylium salt was obtained and fully characterized by mass and nuclear magnetic resonance (NMR) spectroscopy. Esterification was found to involve the colourless compound before dehydration and thus a general scheme for xanthylium salt formation was postulated. The proposed scheme constitutes a new xanthylium formation pathway as up to now only anthocyanin-flavanol reactions were supposed to form xanthylium salt derivatives during wine ageing. This work also provides new support to the contribution of xanthylium salt in colour evolution observed during wine ageing which is generally expressed in an increase of absorption in the 400–500 nm, region of xanthylium salt absorption maxima.     

Thermal decomposition of vitamin C: An evolved gas analysis−ion attachment mass spectrometry study

Evolved gas analysis−ion attachment mass spectrometry (EGA−IAMS) was utilised to study the real-time non-isothermal decomposition of vitamin C. Dehydro-l-ascorbic acid, which has until this study been undetectable from the solid phase degradation of vitamin C, was observed as a decomposition product. While it is an important compound because it possesses some biological activity, dehydro-l-ascorbic acid is difficult to measure due to its chemical instability. In the present study using EGA−IAMS, we were able to detect dehydro-l-ascorbic acid from the thermal degradation of vitamin C. Our EGA−IAMS results obtained from the thermal decomposition of vitamin C were compared with a previous study employing pyrolysis-gas chromatography−mass spectrometry (Pyr-GC−MS). The observed quantitative and qualitative differences of the pyrolysis products obtained by the two techniques (EGA−IAMS vs. Pyr-GC–MS) are in part due to the difference in transportation time of the products out of the pyrolysis chamber.     

类芽孢杆菌发酵原料浸提液提升再造烟叶品质

为提升再造烟叶品质,利用烟草中分离的类芽孢杆菌制备静息细胞,对再造烟叶原料浸提液进行发酵处理;采用连续流动分析、气质联用仪（GC-MS）、热裂解-气相色谱质谱（Py-GC/MS）等方法,测定了发酵过程中浸提液中蛋白质、还原糖、氨基酸含量变化;分析了其主要挥发性香味成分;并利用发酵浸提液浓缩涂布制备再造烟叶样品,进行热裂解产物分析和感官评价。结果表明:①发酵浸提液和对照组中的蛋白质、还原糖含量在5 h内持续减少,而氨基酸含量先增高后降低,且发酵浸提液的蛋白质、还原糖含量低于对照,氨基酸含量高于对照;②发酵液中香味物质种类和总量较对照组均有所增加,其中糠醛、糠酮等致香组分增幅明显;③对比再造烟叶裂解产物,发现发酵浸提液浓缩后涂布样品裂解产生的γ-丁内酯、2,3-二氢-5-甲基呋喃、糠醛等典型烟气致香组分提高;④发酵液浓缩后涂布的再造烟叶样品感官品质较对照样品明显提升。利用类芽孢杆菌发酵烟草浸提液,对于提升再造烟叶的感官品质具有促进作用。      

Determination of fungicide residues in baby food by liquid chromatography-ion trap tandem mass spectrometry

Current European Commission Directives on foods for infants and young children places emphasis on the control of pesticide residues at levels below 10 μg kg(-1). In the present work, a liquid chromatography electrospray ionisation ion trap tandem mass spectrometry (LC-Ion Trap-MS/MS) has been developed for the multiresidue of 10 multiclass fungicides (carbendazim, thiabendazole, imazalil, tridemorph, triadimefon, bitertanol, prochloraz, flutriafol, myclobutanil and diphenylamine) in fruit-based baby food. The developed method is based on a simple sample treatment (QuEChERS), which consists of a liquid-liquid extraction using acetonitrile, followed by a clean-up step based on dispersive solid-phase extraction with primary secondary amine (PSA). Subsequent identification and quantitation was accomplished by liquid chromatography/electrospray tandem mass spectrometry using an ion-trap mass spectrometer in the product ion scan MS/MS mode. Matrix effects were evaluated in LC-MS and LC-MS/MS mode experiments, obtaining a reduction of these effects when working in MS/MS mode for most of the analytes. Limits of detection (LOD) were between 0.5 and 3.0 μg kg(-1) depending on the pesticide studied, all being within European Union regulations for baby food. Finally, the proposed method was applied to 25 baby food samples obtained from local supermarkets. Imazalil, thiabendazole and carbendazim were detected in the studied samples. However, none of the samples tested were found to be upper the EU standard.     

Identilication of the novel cycloaliphatic brominated flame retardant 1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane in Canadian Arctic beluga (Delphinapterus leucas)

1,2-dibromo-4-(1,2-dibromoethyl)cyclohexane (TBECH) is used primarily as an additive flame retardant. Technical grade TBECH consists of near equimolar amounts of two (of a possible four) diastereoisomers: rac-(1R,2R)-1,2-dibromo-(4S)-4-((1S)-1,2-dibromoethyl)cyclohexane ((alpha-TBECH) and rac-(1R,2R)-1,2-dibromo-(4S)-4-((1R)-1,2-dibromoethyl)cyclohexane (beta-TBECH). The two other possible isomers, gamma- and delta-TBECH, appear in the technical mixture when heated at temperatures above 120 degrees C. Careful selection of GC-capillary column length was critical in resolution of the two main diastereoisomers. Column lengths of 60 or 30 m (0.25 microm film thickness) resulted in incomplete separation of the alpha- and beta-isomers, while on a 10 m column, the isomers were baseline separated. The gamma- and delta-isomers could not be resolved on any column length in this study. Increased injector port temperature induced thermal conversion of the alpha- and beta-isomers to gamma- and delta-TBECH. Electron impact ionization (EI) was used to provide specificity because no characteristic ions in the electron capture negative ionization (ECNI) mass spectrum of TBECH were evident. In EI, the dominant ions in the mass spectrum corresponded to a concomitant loss of HBr and Br from the molecular ion; the biggest peak in this ion cluster (m/z 266.9208) was used for quantitation and the second biggest peak (m/z 264.9227) was used for confirmation. Beluga (Delphinapterus leucas) blubber extracts of animals from the Canadian Arctic (n=29) were analyzed using low resolution (LR) MS and high resolution (HR) MS run at a resolving power of 10,000. beta-TBECH was the only isomer observed in the samples and was detected in 17 samples. The LRMS technique appeared to overestimate beta-TBECH concentrations compared to HRMS, suggesting a small interference arose at the nominal mass monitored. This potential interference also led to some false positive and negative values (n=7) based on the expected ion ratio of the quantitation and confirmation ions. Observed concentrations of the beta-isomer as measured by HRMS ranged from 1.1 to 9.3 ng/g (lipid weight).     

Determination of gellan gum by capillary electrophoresis and CE–MS

Intact gellan gum (0.1% m/v) was detectable by capillary electrophoresis (CE) with UV detection. Characteristic tetrasaccharide fragments, prepared with a newly characterised gellan-degrading enzyme, provided a clearer signal that was detectable in complex food products containing other polysaccharides. Food products spiked with gellan gum could be analysed reproducibly with high accuracy and specificity by CE–ESI–MS, which is recommended as the technique of choice. Gellan gum declared as a fruit flavour drink ingredient could not be identified by CE–ESI–MS. When added to the product at the start of sample preparation, before enzyme treatment, the gum was readily detectable, demonstrating that the method was compatible with this sample type. Possible explanations for the negative results are that gellan gum was used as a trace component, with other texturing agents; that its declaration was precautionary only; or that the product contained a chemically modified form. Further work will establish whether modified gellan gums can be similarly analysed.