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;...     

植物中吡咯里西啶生物碱的检测分析方法研究进展

吡咯里西啶类生物碱（Pyrrolizidine alkaloids,PAs）是植物次生代谢产生的一类天然毒素,其中大部分不饱和型PAs及其氮氧化物（Pyrrolizidine alkaloid N-oxides,PANOs）具有多种毒性作用,并可通过膳食补充剂或食物链的形式进行传递从而威胁人类健康。PAs已被发现广泛存在于茶叶、蜂蜜等多种食品基质中,其污染成为令人担忧的食品安全问题。鉴于此,本文详尽梳理了不同国家不同食品基质中PAs的污染水平与可能的污染路径,综述了近年来PAs检测的前处理方法和分析检测技术的最新进展及发展趋势,分析了不同食品加工技术或烹饪制备对PAs污染物稳定性的影响,旨在为食品中PAs的检测研究、安全风险评估及控制提供警示和参考依据。

     

超高效液相色谱-高分辨质谱法同时测定茶叶中15种吡咯里西啶类生物碱

目的 建立超高效液相色谱-高分辨质谱法同时测定茶叶中15种吡咯里西啶类生物碱(PAs)的分析方法.方法 以甲醇甲酸水溶液作为提取溶剂,经超声提取,采用阳离子交换(MCX)固相萃取柱净化,以4.0 mmol/L甲酸铵溶液(含0.1％甲酸)和甲醇为流动相梯度洗脱,HSS T3柱(50 mm×2.1 mm,1.7 μm)分离...     

超高效液相色谱-串联质谱法测定铁皮石斛中的吡咯里西啶生物碱

为了测定铁皮石斛中的吡咯里西啶生物碱(pyrrolizidine alkaloids,PAs)的含量,该研究建立了固相萃取结合超高效液相色谱-串联质谱法同时测定铁皮石斛中16种不饱和PAs的分析方法.样品经0.05 mol/L硫酸甲醇提取、MCX固相萃取小柱纯化富集,以甲醇和0.005 mol/L甲酸铵-0.1％甲酸水...     

QuEChERS结合高效液相色谱-串联质谱法检测甘草中吡咯里西啶生物碱与风险分析

目的 建立QuEChERS和高效液相色谱-串联质谱法(high liquid chromatography-tandem mass spectrometry, HPLC-MS/MS)测定甘草中吡咯里西啶生物碱(pyrrolizidine alkaloids, PAs)。方法 甘草样品经0.05 mol/L硫酸水QuEChERS自动样品制备系统振荡提取后,用混合型阳离子交换固相萃取小柱(PCX SPE)进行净化,以5 mmol/L甲酸铵水(含0.1%甲酸) -5 mmol/L甲酸铵甲醇为流动相,Agilent Proshell 120 EC-C18柱进行分离,利用三重四极杆检测器在多反应监测模式下进行检测。结果 利用建立的前处理方法对甘草中34种PAs做添加回收实验,在10、20和50 μg/kg 3个浓度下的回收率为71.3%~112.0%,相对标准偏差为0.29%~8.18%,满足检测要求。利用建立的提取和检测方法对172个甘草样品中34种PAs进行监测,阳性样品检出率为22.1%,PAs总含量在9.5~118 μg/kg之间。其中石松胺、石松胺 N-氧化物、天芥菜碱和大尾摇碱N-氧化物的检出率最高。结论 本研究开发的甘草中PAs的提取和检测方法快速、准确,可同时测定甘草中34种PAs阳性。所有监测的甘草样品中PAs总量低于欧盟限量标准。     

液相色谱-三重四极杆质谱法测定花茶中5种吡咯里西啶类生物碱

目的 建立液相色谱-三重四极杆质谱法(liquid chromatography-tandem mass spectrometry, LC-MS/MS) 同时测定花茶中5种吡咯里西啶类生物碱含量的分析方法。方法 将花茶样品均质粉碎后, 经0.1%甲酸水溶液浸泡, 涡旋, 超声提取, 高速离心后取上清液过固相萃取柱, 洗脱液氮吹吹干后用流动相复溶, 经ZORBAX Eclipse PLUS C18色谱柱(2.1 mm×100 mm, 1.8 μm)分离, 以0.1%甲酸溶液和乙腈为流动相进行梯度洗脱, 流速为0.30 mL/min, 柱温为30 ℃, 多反应监测模式测定, 外标法定量。结果 该方法线性关系良好, 相关系数为0.997~0.998; 方法的检出限(S/N=3)为3~5 μg/kg、定量限(S/N=10)为10~15 μg/kg; 在15、30、150 μg/kg添加水平下的平均回收率为89.69%~102.12%, 相对标准偏差为0.3%~5.4%。结论 该方法样品前处理简单快速、萃取效果好、灵敏度和选择性高, 适用于常规检测。     

固相萃取-超高效液相色谱-串联质谱法测定菊花中15种吡咯里西啶生物碱毒素

目的 建立超高效液相色谱-串联质谱法(ultra performance liquid chromatography-tandem mass spectrometry, UPLC-MS/MS)测定菊花中15种吡咯里西啶生物碱毒素含量的分析方法。方法 样品中吡咯里西啶生物碱毒素采用0.1 mol/L硫酸水溶液提取, 通过Oasis PRiME MCX固相萃取小柱净化, 采用UPLC-MS/MS进行分析。采用Acquity UPLC BEH C18色谱柱分离, 以0.1%甲酸水-甲醇溶液为流动相进行梯度洗脱, 电喷雾离子源正离子电离, 多反应离子监测模式监测, 外标法定量。结果 15种吡咯里西啶生物碱毒素在一定浓度范围内线性关系良好, 相关系数(r2)均大于0.999; 定量限为0.50~10.00 μg/kg, 检出限为0.15~3.00 μg/kg; 阴性样品的加标回收率为82.8%~92.4%, 相对标准偏差为2.1%~4.9% (n=5)。结论 该方法准确度和精密度高, 耗时短, 能同时测定多种菊花样品中的多种吡咯里西啶生物碱毒素含量。     

茶叶中生物碱检测方法的研究进展

茶叶作为主要饮品之一, 对茶叶品质评定是必不可少的一个过程。茶叶的品质除了感官评定其色、香、味, 还可通过测定其茶多酚、氨基酸、矿物质、蛋白质、生物碱等活性成分的综合含量来评定。生物碱是茶叶中的主要活性成分之一, 不仅决定着茶叶的品质, 对人体也具有一定的有益作用。生物碱的快速、高效检测方法是茶叶品质评估和茶叶中生物碱提取及含量检测的前提和关键。本文参考了国内及国外对茶叶中生物碱研究方法的相关文献, 对茶叶生物碱(主要是咖啡碱、可可碱、茶叶碱)最主要的检测方法进行了综述。为进一步研究茶叶中生物碱及测定其含量, 评估茶叶的品质提供参考。     

氟啶虫酰胺在茶叶中的残留分析及膳食摄入风险评估

目的? 建立了茶叶中氟啶虫酰胺残留分析方法,并评价氟啶虫酰胺在茶叶中的膳食摄入风险。方法 茶叶的氟啶虫酰胺残留用乙腈提取,无水MgSO4和乙酸钠除水盐析。上清液经无水MgSO4、C18、N-丙基乙二胺石墨化炭黑吸附净化剂净化,用超高效液相色谱串联质谱法（ultra performance liquid chromatograghy tandem mass spectrometry,UPLC-MS/MS）分析,采用多反应离子监测模式（MRM）检测,正离子（ESI+）化,基质匹配外标法定量。根据残留试验结果和我国人群的膳食结构进行风险评估。结果? 氟啶虫酰胺在一定质量浓度范围内具有良好的线性关系（r≥0.9999）。茶叶在0.01、0.5、20和30 mg/kg添加水平下,茶叶（干）和（鲜）中的平均回收率在84%～94%之间,相对标准偏差在1.1%～5.3%之间。方法定量限（LOQ）为0.01 mg/kg。最终残留试验和消解试验表明氟啶虫酰胺在茶叶上的残留量受施药剂量和采收间隔期的共同影响,氟啶虫酰胺在各地鲜和干茶叶的半衰期分别为0.9~4.0 d和0.8~5.1 d之间,属于易降解农药。膳食评估的结果为:通过茶叶摄入氟啶虫酰胺在不同年龄段人群的估算每日摄入量在0.74~0.81 μg/kg bw/d之间,占日允许摄入量的1.05% ~ 1.16%;结合我国农药登记情况和现有残留限量,一般人群氟啶虫酰胺的国家估算每日摄入量为58.48 μg/kg bw/d,占日允许摄入量的83.5%。结论? 该方法前处理操作简单,准确度和灵敏度高,可用于茶叶上氟啶虫酰胺的残留检测。经评估,氟啶虫酰胺对一般人群不会产生不可接受风险。     

燕麦生物碱研究进展

燕麦是全球的主要谷物之一,燕麦生物碱是燕麦中的主要功能成分,目前已发现20余种,其中主要为生物碱A、B、C。燕麦生物碱具有抗氧化、降血脂、消炎止痒和抑制细胞增殖等多种生理活性,燕麦生物碱的开发利用日益成为国内外学者的研究热点,但目前对燕麦生物碱的研究还不够系统和规范,对其种类、生理活性的探索还有待进一步发展。本文从燕麦生物碱的组成成分、提取方法、检测方法及生理活性等方面进行综述,主要介绍溶剂提取法、超临界CO_2流体萃取法和层析法等提取方法,高效液相色谱法和高效液相色谱-质谱联用法等检测方法,旨在为燕麦生物碱产品的深加工及开发利用提供科学依据。     

Pyrrolizidine and tropane alkaloids in teas and the herbal teas peppermint,rooibos and chamomile in the Israeli market

Dehydro pyrrolizidine alkaloids (dehydro PAs) are carcinogenic phytotoxins prevalent in the Boraginaceae, Asteraceae and Fabaceae families. Dehydro PAs enter the food and feed chain by co-harvesting of crops intended for human and animal consumption as well as by carry-over into animal-based products such as milk, eggs and honey. Recently the occurrence of dehydro PAs in teas and herbal teas has gained increasing attention from the EU, due to the high levels of dehydro PAs found in commercially available teas and herbal teas in Germany and Switzerland. Furthermore, several tropane alkaloids (TAs, e.g. scopolamine and hyoscyamine) intoxications due to the consumption of contaminated herbal teas were reported in the literature. The aim of the present study was to determine the dehydro PAs and TAs levels in 70 pre-packed teabags of herbal and non-herbal tea types sold in supermarkets in Israel. Chamomile, peppermint and rooibos teas contained high dehydro PAs levels in almost all samples analysed. Lower amounts were detected in black and green teas, while no dehydro PAs were found in fennel and melissa herbal teas. Total dehydro PAs concentrations in chamomile, peppermint and rooibos teas ranged from 20 to 1729 μg/kg. Except for black tea containing only mono-ester retrorsine-type dehydro PAs, all other teas and herbal teas showed mixed patterns of dehydro PA ester types, indicating a contamination by various weed species during harvesting and/or production. The TA levels per teabag were below the recommended acute reference dose; however, the positive findings of TAs in all peppermint tea samples warrant a more extensive survey. The partially high levels of dehydro PAs found in teas and herbal teas present an urgent warning letter to the regulatory authorities to perform routine quality control analysis and implement maximum residual levels for dehydro PAs.     

Determination of pyrrolizidine alkaloids in tea,herbal drugs and honey

Honey was previously considered to be one of the main food sources of human pyrrolizidine alkaloid (PA) exposure in Europe. However, comprehensive analyses of honey and tea sampled in the Berlin retail market revealed unexpected high PA amounts in teas. This study comprised the analysis of 87 honey as well as 274 tea samples including black, green, rooibos, melissa, peppermint, chamomile, fennel, nettle, and mixed herbal tea or fruit tea. Total PA concentrations in tea ranged from < LOD to 5647 µg kg^?1, while a mean value of about 10 µg kg^?1 was found in honey samples. Additionally, herbal drugs were investigated to identify the source of PA in teas. Results suggest that PA in tea samples are most likely a contamination caused by co-harvesting of PA-producing plants. In some cases such as fennel, anise or caraway, it cannot be excluded that these plants are able to produce PA themselves.     

Pyrrolizidine alkaloids in food and feed on the Belgian market

Pyrrolizidine alkaloids (PAs) are widely distributed plant toxins with species dependent hepatotoxic, carcinogenic, genotoxic and pneumotoxic risks. In a recent European Food Safety Authority (EFSA) opinion, only two data sets from one European country were received for honey, while one feed data set was included. No data are available for food or feed samples from the Belgian market. We developed an LC-MS/MS method, which allowed the detection and quantification of 16 PAs in a broad range of matrices in the sub ng g^?1 range. The method was validated in milk, honey and hay and applied to honey, tea (Camellia sinensis), scented tea, herbal tea, milk and feed samples bought on the Belgian market. The results confirmed that tea, scented tea, herbal tea and honey are important food sources of pyrrolizidine alkaloid contamination in Belgium. Furthermore, we detected PAs in 4 of 63 commercial milk samples. A high incidence rate of PAs in lucerne (alfalfa)-based horse feed and in rabbit feed was detected, while bird feed samples were less contaminated. We report for the first time the presence of monocrotaline, intermedine, lycopsamine, heliotrine and echimidine in cat food.     

Pyrrolizidine alkaloids: their occurrence in Spanish honey collected from purple viper’s bugloss (Echium spp.)

The incidence and concentration of pyrrolizidine alkaloids (PAs) from Echium spp. plant have been defined in 103 Spanish honey samples. Each sample was examined to determine total pollen content, the percentage of Echium spp. pollen, and simultaneous measurements of PAs and their N-oxides concentrations by the HPLC-ESI/MS method to identify the potential origin of PAs in honey. PAs were found in 94.2% of the raw honey samples analysed, in the range of 1–237 µg kg^?1 (average concentration = 48 µg kg^?1). The PA pattern was clearly dominated by echimidine, lycopsamine and their N-oxides, representing the 97.8% of total ∑PAs, and only echimidine and echimidine-N-oxide surpassed the 87% of total ∑PA content. Others PAs, seneciphylline and heliotrine-N-oxide, appear to be reported in a lower incidence and concentration (average of 3 and 1 µg kg^?1, respectively). The Pearson Chi-squared test (p ≤ 0.01) confirms the non-correspondence between pollen plants and PA content. This study was also realised to generate a dataset in order to evaluate the potential risk of Spanish honeys containing PA plants belonging to the genera Echium.     

Investigation of the presence and significance of theanine in the tea plant

Theanine was determined in black teas and tea plants by a combination method of thin-layer chromatography (t.l.c.) and densitometry. It was shown that in 20 samples of black tea from different qualities and sources the amount varied from 0.33 to 1.59 g 100 g^?1 dry wt. The highest quality black teas possessed the lowest amount of theanine, but it is subject to chemical degradation during black tea manufacture. The investigation of physiological function of theanine in the tea plant showed that during germination the theanine level reached a maximum after 45 days. This indicates that at this period of the growth of the tea plant theanine acted as a source of nitrogen and as a starting point for the synthesis of the carbon skeletal compounds of the tea plant. Theanine was found to exist in all parts of the tea plant but it accumulated more in young and active tissues and also in younger plants, which emphasises its metabolic role in the tea plant. The distribution of theanine in the shoots indicated that the first leaf was the principal site for the synthesis of polyphenolic compounds from theanine.     

Inter-laboratory comparison study for pyrrolizidine alkaloids in animal feed using spiked and incurred material

Pyrrolizidine alkaloids (PAs) are hepatotoxic metabolites produced by plants. PAs in animal feed can cause acute or chronic intoxications in animals and can be transferred to milk. An inter-laboratory comparison study among 12 laboratories, using their own methods of analysis, was conducted for the detection and quantification of PAs in animal feed. The participants were asked to quantify PAs in a blank test sample, a blank test sample to be spiked with a provided spiking mixture of seven PA standards, and a test sample contaminated with common groundsel (Senecio vulgaris). Ten of the participating laboratories used an LC-MS/MS method, one used an LC-ToF-MS method, and one used a GC-MS method. None of the laboratories reported false-negative samples, while two laboratories reported false-positive results in the blank sample. z-scores were calculated for each laboratory for seven PAs in test samples B and C. z-scores varied considerably between laboratories for the concentrations of the free bases and less for the N-oxides, probably due to the lower levels of the free bases as compared with the N-oxides in the contaminated feed. Questionable or unsatisfactory results for the z-scores were obtained for 8% of the cases for the spiked sample and for 12% of the incurred sample. Three laboratories scored consequently positive or negative results. No preferred method for quantification of PAs in feed could be identified within the methods used for this study due to the relatively small number of participants. It was concluded that this inter-laboratory study shows that the methods used for PA detection need further development for accurate estimation of PAs in contaminated feed.