Analysis of metabolic markers of tea origin by UHPLC and high resolution mass spectrometry

Tea is an infusion made from the dried leaves of Camellia sinensis L. and is the second most consumed beverage in the world. It has been shown that factors such as fermentation methods, cultivar, geographical origin and season can affect the biochemical composition of tea. In this study, the biochemical composition of green, oolong and black commercial tea samples from around the world was studied using a non-targeted method utilising reversed phase ultra high pressure liquid chromatography (UHPLC) and high resolution mass spectrometry. Principal component analysis of green, oolong and black tea extracts clearly showed that fermented tea can be resolved from non-fermented tea. When the non-targeted data were combined with the supervised multivariate technique, partial least squares discriminant analysis, the method was able to clearly distinguish ‘country of origin’ within green tea and to a lesser extent within a black tea sample set, plus provide indicative marker ions for the country of origin. Many of the significant components detected in this study are unknowns, emphasising the importance of un-biased non-targeted analytical techniques. This study highlights the potential efficacy of non-targeted UHPLC–mass spectrometry when combined with multivariate statistics to differentiate fermented from non-fermented tea and provide potential indicators of provenance of tea samples for further examination.     

高分辨质谱库和特征组分诊断比值法快速筛查鉴别食品中非法添加黄芪

目的：建立了一种基于高分辨质谱库和特征组分诊断比值法筛查鉴别食品中非法添加黄芪的方法。方法：采用高效液相色谱-四极杆-飞行时间质谱仪构建黄芪4种特征组分高分辨质谱库进行初步筛查,结合特征组分诊断比值法,对不同产地和采摘时间的黄芪中药材模拟的配制酒、代用茶和饮料样品进行特征组分分析,以不同基质组间无显著性差异的特征组分峰面积诊断比值作为确证指标。结果：建立了黄芪4种特征组分高分辨质谱库,确定了黄芪中不同基质组间无显著性差异的1组特征组分峰面积诊断比值DR1 (毛蕊异黄酮苷/芒柄花苷),诊断比值非异常值范围分别1.86-2.71;并对实际样品配制酒、代用茶及饮料进行分析,1批次配制酒样品与黄芪4种特征组分高分辨质谱库匹配一致,且特征组分诊断比值DR1为1.98,在规定区间内,推断该配制酒样品中加入了黄芪中药材。结论：该方法准确、快捷、稳定性好,可快速鉴别食品中非法添加黄芪,为打击食品中非法添加黄芪违法行为提供技术支撑。     

基于高分辨质谱库和特征组分诊断比值法鉴别配制酒和代用茶中非法添加补骨脂

目的:建立一种基于高分辨质谱库筛查和特征比值法确证配制酒和代用茶中非法添加补骨脂的高效液相色谱检测方法。方法:采用高效液相色谱—四极杆—飞行时间质谱仪建立补骨脂中药材特征组分筛查库,结合液相特征组分诊断比值法,对不同采摘时间和产地的共20批次补骨脂模拟的补骨脂配制酒、代用茶样品进行特征组分分析,从13种特征成分中选出2组受产地、采摘时间、加工工艺等影响较小,不同基质组间无显著性差异的特征组分峰面积诊断比值作为确证指标。结果:建立了补骨脂中药材特征组分筛查库,选定出2组特征组分CoryliA/补骨脂酚和补骨脂素/异补骨脂素,绘制箱图确定CoryliA/补骨脂酚的非异常值范围为0.061~0.115,补骨脂素/异补骨脂非异常值范围为1.14~1.68。通过市售样品对建立的筛查和确证方法进行验证。结论:该方法简单高效,适用于配制酒和代用茶中非法添加补骨脂的检测。     

A High-Performance Liquid Chromatographic Method for Simultaneous Determination of 21 Free Amino Acids in Tea

Free amino acids are closely related to the savory taste and beneficial effects of tea, and high-performance liquid chromatography (HPLC) is the most widespread analytical approach for simultaneous determination of free amino acids in tea. However, the reported HPLC methods have drawbacks such as long run times, low resolution, or poor efficiency. In this study, a special amino acid analysis column was used to separate and verify 21 amino acids including l-theanine, the predominant amino acid in tea. The mobile phases, including the sodium acetate and acetic acid concentration in buffer B, and the pH and concentration of sodium acetate in buffer A were optimized. The elution gradients were optimized using DryLab 2000 Plus software. In this way, an online o-phthaldialdehyde precolumn derivatization HPLC-fluorescence detection method was developed for simultaneous determination of 21 amino acids. Comparison to other HPLC methods for simultaneous determination of free amino acids in tea showed that our method is easy (automated derivatization), quick (30 min), inexpensive, and green (using a minimum of solution). It has good resolution (≥1.8) and high selectivity (interpark time?≥?0.5 min). Free amino acids in six tea samples were analyzed. This work provides an HPLC method to simultaneously measure 21 amino acids in tea and potential in other food products.     

Tentative identification,quantitation, and principal component analysis of green pu-erh,green, and white teas using UPLC/DAD/MS

Tea (Camellia sinensis L.), an important drink and a natural medicine for thousands of years, contains many health beneficial compounds. Growing season, geographical region, and fermentation methods create many variations in tea compositions, which contribute to each tea’s uniqueness. In this study, a simple, rapid, and efficient ultra-performance liquid chromatography (UPLC) method combined with diode array detector (DAD) and mass spectroscopic (MS) detection and chemometrics analysis was used to analyse three different types of teas (green pu-erh, green tea, white tea). Using the developed method, 68 compounds were identified and 54 were quantified based on retention times, UV spectra, and MS spectra by referencing to available standards and data in the literatures. The results showed the chemical differences between the tested teas. Principal component analysis (PCA) was applied to classify and distinguish between tea samples.     

不同采摘时期贵定鸟王茶鲜叶次生代谢产物差异分析

目的 分析不同采摘时期贵定鸟王茶的次生代谢产物差异.方法 基于代谢组学技术对不同采摘时期贵定鸟王茶的次生代谢产物进行测定,利用XCMS软件和METLIN数据库对代谢物进行定性分析,用SIMCA-P软件进行定量分析和差异化合物筛选.结果 茶树样本间分离趋势明显,且春茶和秋茶代谢物差异最大.不同采摘时期鸟王茶之间共筛选出1...     

基于液质联用代谢组学方法分析不同产地和表型的玛咖

本实验基于液质联用代谢组学分析方法对20种不同表型和产地玛咖进行分析，采用超高效液相色谱串联四级杆静电场轨道阱高分辨质谱（Dionex Ultimate 3000-Q Exactive Orbitrap），原始数据经过Compound Discoverer软件处理后通过SIMACA 14.1软件分析可能的标志性代谢物，然后对这些潜在的标志性代谢产物进行二级质谱分析，通过相关文献和数据库（HMDB或NIST）对其进行定性。结果表明按照产地分组的多元变量统计分析中，五个产地的玛咖在OPLS-DA中成功分组，丽江和香格里拉两个产地玛咖在PLS-DA和OPLS-DA中分组成功；三种表型的紫色、黄色和黑色玛咖在PLS-DA和OPLS-DA中分组成功。聚类分析表明黄色、黑色和紫色玛咖各分为不同三类，三种表型玛咖内活性成分差异较大，与OPLS-DA得分图结果相吻合。表明高分辨质谱结合代谢组学技术可用于玛咖不同表型及产地的鉴别。由于目前对于玛咖中代谢物质定性的数据库和文献较少，本实验只定性出黑色玛咖的标志性代谢产物N-苄基-15Z-烯二十四酰胺以及黄色和紫色玛咖标志性代谢产物3-甲氧基苯基乙酸。     

Determination of catechins and flavonol glycosides in Chinese tea varieties

A standardised profiling method based on high performance liquid chromatography combined with ultraviolet (UV) and mass spectrometric detection (MS) was established to analyse the phenolic compounds of selected tea varieties used for manufacturing of green, black and oolong teas. The composition and content of 24 tea constituents were analysed, including catechins, flavonol and flavones glycosides, phenolic acids and purine alkaloids. Each tea variety had a unique chemical profile. The compositions of catechins were lower in the tea varieties for green tea manufacturing, while the content of myricetin glycosides was the lowest in the tea variety for oolong tea manufacturing. The content of individual phenolic compounds in the selected tea varieties is highly variable. However, the content of total catechins is proposed to be helpful to classify tea according to the future application as non fermented green and fermented oolong or black tea.     

基于咖啡碱和氨基酸中碳氮稳定同位素比率的不同年份普洱生茶真实性认证

采用咖啡碱和氨基酸中碳氮稳定同位素比率分析方法（气相色谱-燃烧-同位素比值质谱,GC-C-IRMS）开展不同年份普洱生茶真实性认证研究。化学计量学分析表明,基于茶叶咖啡碱和氨基酸中碳氮稳定同位素比率结合化学计量学模型可以对不同年份普洱生茶进行有效区分,预测和验证准确率均达到80%以上,其中BP-ANN认证性能最优,预测和验证准确率均达到100%;进一步筛选和鉴定,得到不同年份普洱生茶的3种化学标记,包括δ15N_Thea、δ13C_Glu和δ15N_Glu。因此,基于GC-C-IRMS分析的咖啡碱和氨基酸中碳氮稳定同位素比率分析方法可以用于不同年份普洱生茶的真实性认证。本研究初步建立了普洱生茶产品认证的指标体系及技术体系,也为其他食品的认证研究提供研究思路和方法参考。     

基于咖啡碱和氨基酸中碳氮稳定同位素比率的茶叶地理溯源

采用咖啡碱和氨基酸中碳氮稳定同位素比率分析方法,基于气相色谱-燃烧-同位素比值质谱（gas chromatography combustion isotope ratio mass spectrometry,GC-C-IRMS）开展不同产地茶叶地理溯源技术研究。结果表明,基于GC-C-IRMS特异性化合物稳定同位素比值分析方法可以用于茶叶地理溯源;化学计量学分析表明,本方法可以对不同产地茶叶进行有效区分,预测和验证准确率均达到85%以上,BP人工神经网络认证性能最优,预测和验证准确率均达到100%;经过进一步筛选和鉴定,得到不同产地秀芽的2 种特征标识化合物——δ15N丙氨酸和δ13C茶氨酸。本研究可为茶叶地理溯源提供一定技术支撑,有利于茶叶产品的认证体系的建立和完善。     

茶叶中农药残留检测方法研究进展

茶叶中农药残留问题受到越来越多的关注,现代仪器分析技术在农残检测中的应用也在不断创新。本文主要综述了茶叶中农药残留检测方法的研究状况,对茶叶中农残检测样品前处理方法和检测技术进行了分析和比较。目前茶叶中农药残留检测方法主要有气相色谱法(gas chromatography,GC)、高效液相色谱法(high performance liquid chromatography,HPLC)、色谱-质谱联用法和酶联免疫吸附测定法(enzyme-linked immunosorbent assay,ELISA)。本文分析了不同检测方法使用的局限性和优越性,为不同性质的茶叶样品农残检测方法的选择提供了依据,旨在为茶叶中农药残留检测新方法的研究提供理论参考。     

基于UHPLC-Q-TOF/MSE技术分析武夷岩茶的化学成分

利用超高效液相色谱-四极杆-飞行时间质谱法（UHPLC-Q-TOF/MSE）结合UNIFI数据分析平台在线鉴定了武夷岩茶中104个化合物,包括茶多酚、茶色素、生物碱、氨基酸和脂质。通过主成分分析（PCA）及正交偏最小二乘判别分析（OPLS-DA）发现了水仙和肉桂的化学成分具有明显差异,并筛选出19个显著差异性成分,其中水仙样品中表儿茶素、槲皮素-3-O-葡萄糖基-鼠李糖基-(对香豆酰基-阿拉伯糖基)葡萄糖苷、茶黄素-3-没食子酸酯等成分的相对含量较高;肉桂样品中5-O-对香豆酰基奎宁酸、表没食子儿茶素-3-O-(3-O-甲基)-没食子酸酯、杨梅素-3-O-半乳糖苷、山柰酚-3-O-葡萄糖苷等成分的相对含量较高。此方法可用于区分水仙和肉桂成品茶。该研究了解了武夷岩茶的化学组成并阐明了其两个主栽品种水仙和肉桂的化学成分差异性,对合理开发武夷岩茶的食用及药用价值具有指导意义,为武夷岩茶品种的鉴别提供科学依据。     

Spot urine collection: A valid alternative to total urine collection for metabolomic studies in dairy cattle

Urine is a highly suitable biological matrix for metabolomics studies. Total collection for 24-h periods is the gold standard as it ensures the presence of all metabolites excreted throughout the day. However, in animal studies, it presents limitations related to animal welfare and also due to alterations of the metabolome originating from the use of acid for preventing microbial growth or microbial contamination. In this study, we investigated whether spot urine collection is a practical alternative to total collection for metabolomic studies in lactating cows. For this purpose, we collected urine samples from 4 lactating Holstein cows fed 4 diets in a 4 × 4 Latin square design. Urine was collected for 24 h using a collecting device (i.e., total collection) or collected once per day 4 h after the morning feeding (i.e., spot urine collection). Dietary treatments differed by the amount of nitrogen content (high vs. low) and by the nature of the energy (starch vs. fiber). Urine metabolome was analyzed by 2 untargeted complementary methods, nuclear magnetic resonance and hydrophilic-interaction liquid chromatography (HILIC) coupled to a time-of-flight mass spectrometer, and by 1 targeted method, HILIC–tandem mass spectrometry. Although sampling technique had an effect on the abundance of metabolites detected, spot urine samples were equally capable of showing differences in urine metabolome than samples from total collection. When considering nitrogen levels in the diet, the robustness and precision for discriminating high- and low-nitrogen diets was equally achieved with both sampling techniques. A total of 22 discriminant metabolites associated with the N level of diets were identified from untargeted HILIC coupled to a time-of-flight mass spectrometer (n = 9) and nuclear magnetic resonance (n = 11), and 2 from targeted HILIC–tandem mass spectrometry. Alternatively, starch or fiber in the diet induced less changes in the metabolome that were not clearly discriminated independently of the sampling technique. We concluded that spot urine collection can successfully reveal differences in the urine metabolome elicited by dietary N levels and be used as a substitute of total urinary 24-h collection for metabolomic studies.     

UPLC-Q Exactive轨道阱高分辨质谱法测定小鼠血液中的NNN及其7种代谢物

为了准确测定小鼠血液中的N-亚硝基降烟碱(NNN)及其代谢物,通过对色谱、质谱条件的优化,建立了以NNN-d₄和HPB-d₄为内标,用乙腈沉淀蛋白后,直接经超高效液相色谱(UPLC)仪器的亲水相互作用色谱(HILIC)柱进行分离,通过全扫描/数据依赖二级质谱扫描(FS/dd MS2)测定小鼠血浆中NNN及其7种代谢物的UPLC-Q Exactive轨道阱高分辨质谱法。结果表明:①NNN及其7种代谢物的检出限为0.019~0.067 ng/m L,回收率在78.0%~117.5%之间,日内精密度为0.47%~8.38%。②该方法适合于小鼠血浆中NNN及其7种代谢物的同时检测。      

酿造酱油与配制酱油鉴别方法研究进展

以氯丙醇、铵盐、乙酰丙酸、氨基酸作为鉴别酿造酱油与配制酱油的指标,采用同位素稀释气相色谱-质谱联用法、顶空衍生化固相微萃取法等检测酸水解植物蛋白液(HVP)的氯丙醇,采用凯氏定氮法、近红外光谱法检测酱油中的铵盐与氨基酸,采用液相色谱-质谱法、气相色谱法等检测HVP中的乙酰丙酸,采用高效液相色谱法、氨基酸自动分析仪检测酱油中的氨基酸。     

游离氨基酸检测方法及其应用

游离氨基酸是动植物体中重要的活性成分和风味物质,随着现代科学技术的进步,游离氨基酸的检测方法多样且应用研究进展迅速,汇总整理各类检测方法及其相关应用不可或缺。综述了常用于检测分析游离氨基酸的分光光度法、离子色谱-积分脉冲安培法、氨基酸分析仪法、高效液相色谱法和液相色谱-串联质谱法5种方法,以及2种新技术,分析了各检测方法的原理及优缺点,并对各检测方法列举了相关应用研究。现代新型技术的发展和推广,多元化离子化方式、高分辨质谱、多级串联质谱等技术将会使游离氨基酸分析的灵敏度、准确度、分析速度及自动化程度均提高到一个崭新的水平,选择性好、准确度高、前处理简便的检测手段将是游离氨基酸检测分析技术的未来发展趋势。     

茶叶中吡咯里西啶生物碱检测技术、污染水平及健康风险研究进展

吡咯里西啶生物碱（pyrrolizidine alkaloids,PAs）是一类植物次级代谢产物,其中不饱和PAs及大部分PAs的氮氧化物具有多种毒性作用。PAs已被发现广泛存在于各类茶叶中,且成为导致我国茶叶出口受阻的主要污染物之一。本文综述了茶叶中PAs检测的前处理方法和色谱-质谱检测技术的发展,并展望其发展趋势;总结了不同国家茶叶PAs污染水平;分析了茶叶中PAs种类、不同茶叶的PAs差异以及茶叶与其他食品或农产品中PAs差异;解析了茶叶中PAs对人体的健康风险。我国茶叶PAs污染水平监测与风险评估研究相关数据处于缺失状态,急需开展茶叶中PAs污染来源与风险评估研究。     

High throughput residue analysis of paraquat and diquat involving hydrophilic interaction liquid chromatographic separation and mass spectrometric determination

A selective, sensitive and robust LC-MS/MS method is reported for the determination of the residues of paraquat and diquat in various fruit matrices, including grape, apple and pomegranate. The extraction with acidified water (0.1 M HCl) at 80°C (15 min) offered superior recoveries for both analytes with a significantly lower matrix effects as compared to the extraction with acidified methanol by the methods reported in the existing literature. The optimised HPLC conditions on hydrophilic interaction liquid chromatography (HILIC) columns, when coupled with electrospray ionisation-tandem mass spectrometry, offered their limit of quantification at 0.01 mg kg^?1. The analysis on an XBridge HILIC column required a thorough optimisation of the gradient programme to induce chromatographic separation and minimise matrix effects. This was not necessary when a CORTECS HILIC column was used, which provided selective and sensitive analysis within 5 min runtime using isocratic flow. Isotopically labelled internal standards corrected the recoveries of both analytes within 70–120% (RSD < 20%). For the first time, the applications of high resolution accurate mass analysis in the ‘time of flight – multiple reaction monitoring’ mode have been demonstrated as a complementary means of targeted screening of these compounds at 0.01 mg kg^?1 level. The method has a strong potential for applications in both official control and by those involved in food production for checking compliance with the EU MRLs.     

基于UPLC-Q-Exactive Orbitrap-MS分析不同足火方式对绿茶中叶绿素降解的影响

探索不同足火方式（远红外足火、微波足火、提香机足火、理条足火、六角辉锅足火）对绿茶色泽和叶绿素降解的影响。建立基于超高效液相色谱-四极杆-静电场轨道阱高分辨质谱联用技术分析茶叶中叶绿素组分及其降解衍生物方法,可在20 min内检测19 种叶绿素及降解组分,并较好分离6 对差向异构体,且预处理简单。将该方法用于5 种不同足火方式所制绿茶中叶绿素及其降解组分的检测分析,并结合正交偏最小二乘法判别分析及单因素方差分析,探究不同足火方式对绿茶叶绿素降解的影响。结果显示,微波足火所制绿茶色泽感官品质和色泽属性均优于其他足火方式。微波足火所制得绿茶中叶绿素a、叶绿素b、叶绿素a’、叶绿素b’等含量最高,而降解产物焦脱镁叶绿酸a、焦脱镁叶绿酸b、焦脱镁叶绿素a、焦脱镁叶绿素b含量最低,表明微波足火过程中绿茶发生异构化、脱镁降解反应、脱羧甲基化反应的程度均低于其他足火方式,故微波足火更有利绿茶中叶绿素的保留。本研究可为绿茶品质提升和定向化加工提供理论基础和技术指导。     

霉变六堡茶中真菌群落鉴定及其代谢产物的变化

为了解霉变六堡茶中真菌群落及其代谢产物的变化,利用Illumina高通量测序技术平台,对高温高湿环境仓储获得的霉变六堡茶样本（GGZ）和作为对照样本（BDL）的竹筐仓储的正常六堡茶进行真菌多样性分析,采用超高效液相色谱串联质谱（UPLC-MS/MS）对样本中的代谢物进行分析,然后对显著差异代谢物对应的通路进行代谢物集富集分析,并对差异代谢物与差异微生物进行关联分析。结果表明,霉变六堡茶丰度较高的菌为两门[Ascomycota（子囊菌门）和Basidiomycota（担子菌门）]、两属[Aspergillus（曲霉真菌属）和Wallemia（节担菌属）],BDL组丰度较高的菌主要为Ascomycota的Aspergillus和Penicillium（青霉菌属）。GGZ组与BDL组相比,A. ruber（赤曲霉）和W. sebi水平显著（P<0.05）上调,两者为导致六堡茶霉变的优势真菌种群。此外,霉变茶叶中黄酮类化合物含量较高,氨基酸代谢发生显著变化,咖啡因代谢下调。霉变六堡茶中,具有一定细胞毒性和遗传毒性的2-羟基异己酸的含量上调,与下调的Penicillium负相关。这些数据揭...