固相萃取-高效液相色谱-串联质谱法测定番茄酱中棒曲霉素

采用固相萃取-高效液相色谱-串联质谱(SPE-HPLC-MS/MS)建立了番茄酱中的棒曲霉素的定性定量分析方法。样品经Strata-X固相萃取净化处理,使用Agilent XDB-C18色谱柱(50mm×2.1mm,1.8μm)、以乙腈-水为流动相梯度洗脱,在流速为0.12mL/min条件下分离,采用电喷雾离子化源(ESI)多反应监测(MRM)模式下检测。在最佳检测条件下,棒曲霉素在0.05～0.5mg/L检测范围内线性关系良好,相关系数大于0.9995;检出限为0.03mg/L、定量限为0.1mg/L,该方法的精密度小于1.5%,回收率在90.2%～95.6%之间。说明该法准确可靠,可应用于番茄酱中棒曲霉素含量的测定。     

氰基柱SPE-HPLC检测浓缩苹果汁中棒曲霉素的研究

借建立了SPE-HPLC检测浓缩苹果汁中棒曲霉素残留的方法.采用氰基柱固相萃取浓缩苹果汁中的棒曲霉素,经HPLC测定,样品的回收率为95.16%～120.89%,变异系数≤3.98%,最低检出浓度为0.003mg/kg.该方法较传统的LLE法简便、快速、准确,是处理浓缩苹果汁中棒曲霉素较理想的方法.     

超声辅助-分散液液微萃取/快速液相色谱-串联质谱法测定食用植物油中黄曲霉毒素

建立了超声辅助-分散液液微萃取(UA-DLLME)/快速液相色谱-串联质谱法(RRLC-MS/MS)测定食用植物油中黄曲霉毒素(AFB_1、AFB_2、AFG_1、AFG_2)的方法。先以分散剂溶解样品,再以萃取剂萃取,离心后底层溶液经脱脂上机,在电喷雾离子源正离子模式(ESI~+)下采用多反应监测模式(MRM)监测。对样品前处理方法进行了优化。结果表明,最优样品前处理条件为:1 g样品,2 mL石油醚为分散剂,1 000μL乙腈-水(体积比8∶2)为萃取剂,超声辅助萃取时间2 min,离心时间5 min,离心转速4 000 r/min。该方法的线性范围为0.625～30.0 ng/mL,相关系数均大于0.998,加标回收率为88.2%～101.5%,相对标准偏差均小于3.3%。该方法简便、快速、经济、准确度和精密度高且环境友好,适合食用植物油中黄曲霉毒素的定性和定量测定。     

响应面试验优化溶剂诱导萃取水系基质中棒曲霉素工艺及其检测分析

基于溶剂诱导萃取,使用响应面法优化水系基质中棒曲霉的提取工艺,并对4种不同水系基质的料液比(V/V)进行选择。在单因素试验基础上,以棒曲霉素转移率为指标,响应面法优化溶剂诱导萃取的条件,并在不同料液比的4种水系基质中进行加标回收实验。通过溶剂诱导分层,获得棒曲霉素的最佳萃取条件为乙腈-样品体积比7∶3、Na Cl添加量1.4?g/3 m L样品、提取温度34℃,此条件下,棒曲霉素转移率为90.47%。在此最优条件下,苹果基质的最优料液比为7∶3,浓缩苹果汁、酿酒葡萄和葡萄酒基质的最优料液比均为1∶9,加标回收率分别为91.08%、85.51%、88.74%、89.17%。同时与中华人民共和国出入境检验检疫行业标准SN/T 2008—2007《进出口果汁中棒曲霉毒素的检测方法高效液相色谱法》进行比较,结果显示该前处理方法溶剂使用量少、操作简单且重复性好。综上,响应面法优化溶剂诱导分相萃取棒曲霉素的结果可靠,为水系基质中棒曲霉素的萃取提供了一种全新、快速的前处理方法。     

高效液相色谱- 电喷雾三级四极杆质谱测定苹果、山楂、番茄制品中棒曲霉素的研究

建立了苹果、山楂、番茄制品中棒曲霉素总量的提取方法和液相色谱串联质谱确证方法。清汁样品用乙腈稀释后直接过MycoSep 228 真菌霉素多功能净化柱;浊汁、固体样品用果胶酶水解后,再用乙酸乙酯提取样品中棒曲霉素,浓缩后经MycoSep 228 柱净化,用Atlantis dC18 色谱柱分离,液相色谱串联质谱测定,电喷雾负离子(ESI －)多反应模式下监测;实验结果表明,样品中棒曲霉素浓度在1.0～200μg/kg 范围内时与其峰面积成良好线性关系,检测低限为2.0μg/kg,3 个加标水平下回收率为76.0%～95.3%,相对标准偏差为5.4%～11.2%。该方法选择性好,灵敏度高,定性可靠,可满足苹果、山楂、番茄制品中棒曲霉素测定的要求。     

分散液液微萃取-液相色谱法测定白葡萄酒中拟除虫菊酯类农药

分散液液微萃取法处理样品,结合高效液相色谱测定白葡萄酒中拟除虫菊酯类(氟氯氰菊酯、氯氰菊酯、氰戊菊酯、联苯菊酯)农药残留.二氯甲烷作萃取剂,乙醇作分散剂.色谱条件:反相C18色谱柱,甲醇∶乙腈∶水(10∶75∶15,体积比)为流动相,流速1.0mL/min,210nm紫外检测.在0.05μg/mL～50.00μg/mL范围内线性良好.相关系数均大子0.9993.平均回收率为83.20％～102.71％,相对标准偏差为1.47％～3.24％,检出限为1.00μg/L～2.00μ g/L.     

液液萃取-高效液相色谱法同时测定植物油中的毒黄素、路霉素和热诚菌素

通过优化样品前处理方法,建立了高效液相色谱法测定植物油中的毒黄素、路霉素和热诚菌素的含量.样品采用甲醇液液萃取、氮吹浓缩,经高效液相色谱仪分析检测,外标法定量.采用Ultimate AQ-C 18(4.6 mm×250 cm,5μm)色谱柱分离,流速为1.0 mL/min,流动相为0.1％甲酸-甲醇(90:10),检测...     

MSPD和SPE两种方法前处理苹果(汁)中棒曲霉素的比较研究

建立并比较了分别采用基质固相分散(MSPD)和氰基柱固相萃取(SPE)两种前处理技术,高效液相色谱(HPLC)检测苹果(汁)中棒曲霉素的方法.两种方法的回收率和变异系数均满足检测要求.结果表明,建立的两种方法简便、快速、准确,各有优缺点,不同的样品中棒曲霉素的前处理应采用不同的方法.     

离子液体分散液-液微萃取-高效液相色谱法测定牛奶中己烯雌酚

建立离子液体分散液-液微萃取与高效液相色谱-紫外检测器相结合测定牛奶中己烯雌酚含量的方法。考察萃取溶剂、抗黏剂、pH值、离子强度及温度对萃取效率的影响。结果表明,该法对样品中己烯雌酚的萃取灵敏度高,检出限为0.5 μg/L,相对标准偏差在2.80%～4.43%之间,平均回收率为93.7%～103.2%。该技术可以满足牛奶中己烯雌酚含量的测定。     

气相色谱-质谱联用快速检测葡萄酒中有机磷农药残留

该试验旨在建立微液液萃取葡萄酒中9种有机磷农药多残留的气相色谱-质谱联用,选择离子模式进行监测的检测方法.样品采用乙腈提取,无需净化.该方法在0.020～1.0 mg/L范围内线性关系良好.在3种不同添加浓度(0.050,0.20和0.50 mg/L)下,有机磷平均回收率范围在70％～120％,相对标准偏差小于15％....     

Exposure to patulin from consumption of apple-based products.

Patulin is a mycotoxin produced by species of Penicillium, Aspergillus and Byssochylamys. Several Scientific Committees classify patulin as mutagenic, embryotoxic and immunotoxic. It has been found as a natural contaminant of processed apple products and its presence may be indicative of the quality of fruit used in production. In this work, a method for the analysis of patulin is described, based on a simple liquid-liquid extraction with acetonitrile; patulin is analyzed using liquid chromatography with UV detection. Patulin identity was confirmed by GC-MS after its reaction with N-methyl-N-(trimethylsilyl)trifluoroacetamide. Fifty-three apple-containing products were analyzed and patulin was detected in 14 samples in a range 1.5-50.9 microg l(-1); six of which were above the maximum permitted level of the European Union. Based on these results and juice consumption by the Spanish adult population, patulin estimated intake was 0.42 ng kg(-1) body weight per day.     

Quantification of patulin in fruit leathers by ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA)

Patulin is a mycotoxin commonly found in certain fruit and fruit products. For this reason many countries have established regulatory limits pertaining to, in particular, apple juice and apple products. Fruit leathers are produced by dehydrating fruit puree, leaving a sweet product that has a leathery texture. A recent report in the literature described the detection of patulin at substantial levels in fruit leathers. To investigate this further, an ultra-high-performance liquid chromatography-photodiode array (UPLC-PDA) method was developed for the sensitive detection of patulin in fruit leathers. Investigations were also made of the suitability of direct analysis in real time-mass spectrometry (DART-MS) for detection of patulin from the surface of fruit leathers. Results indicated DART-MS was insufficiently sensitive for quantification from the surface of home-style apple leathers, although patulin spiked onto the surface of leather or peel could be detected. The UPLC-PDA method was used to determine the fate of patulin during the preparation of home-made fruit leathers. Interestingly, when a home-style process was used, the patulin was not destroyed, but rather increased in concentration as the puree was dehydrated. The UPLC-PDA method was also used to screen for patulin in commercial fruit leathers. Of the 36 products tested, 14 were above the limit of detection (3.5 μg kg^–1) and nine were above the limit of quantification (12 μg kg^–1). Positive samples were confirmed by UPLC-MS/MS. Only one sample was found above the US regulatory limit for single-strength apple juice products (50 μg kg^–1). These results suggest patulin can be concentrated during preparation and can be found in fruit leathers. The limited survey suggests that patulin is fairly prevalent in such commercial products, but that the levels are usually low.     

拟粉红锁掷孢酵母降解展青霉素的机制

展青霉素(PAT)是一类主要由曲霉属和青霉属等真菌产生的有毒次级代谢产物,主要污染水果及其制品,严重威胁着人类的健康。为了寻找一种安全高效的PAT脱毒方法,近年来采用生物法清除PAT已成为主要的研究方向。作者研究了拟粉红锁掷孢酵母(Sporidiobolus pararoseus)对PAT降解的影响及其降解机制。结果显示,S.pararoseus可以显著降低苹果伤口处PAT的积累量,体外实验发现S.pararoseus与PAT(5μg/mL)共同培养18 h,PAT可以被S.pararoseus完全降解。S.pararoseus对PAT的降解作用既不是酵母细胞壁的吸附作用,也不是细胞的吸收作用,而是细胞正常代谢下产生的胞内酶对PAT具有降解作用。本研究有助于了解S.pararoseus降解PAT的作用机制,为真菌毒素的生物降解提供了新的理论依据。     

Comprehensive Review of Patulin Control Methods in Foods

The mycotoxin, patulin (4‐hydroxy‐4H‐furo [3, 2c] pyran‐2[6H]‐one), is produced by a number of fungi common to fruit‐ and vegetable‐based products, most notably apples. Despite patulin's original discovery as an antibiotic, it has come under heavy scrutiny for its potential negative health effects. Studies investigating these health effects have proved inconclusive, but there is little doubt as to the potential danger inherent in the contamination of food products by patulin. The danger posed by patulin necessitates its control and removal from foods products, creating a demand for handling and processing techniques capable of doing so, preferably at low cost to industry. With this being the case, much research has been devoted to understanding the basic chemical and biological nature of patulin, as well as its interaction within foods and food production. While past resarch has elucidated a great deal, patulin contamination continues to be a challenge for athe food industry. Here, we review in depth the past research on patulin with an emphasis upon its influence within the food industry, including its regulation, health effects, biosynthesis, detection, quantification, distribution within foods, and control, during the various stages of apple juice production. Finally, key areas where future patulin research should focus to best control the patulin contamination problem within the food industry are addressed.     

CONTAMINATION OF PATULIN IN CLEAR APPLE JUICE IN MASHHAD, IRAN

Patulin is a frequent contaminant of moldy and rotten apples and apple products. The aim of this study was to evaluate patulin contamination in 58 apple juices collected from a retail market in Mashhad during winter and spring of 2006.
Samples were assayed for patulin by high-performance liquid chromatography. Fifty-four samples were positive for patulin at levels that ranged from 10.5 to 121.8 µg/L, and six samples had patulin levels higher than 50 µg/L. The overall mean of patulin concentration was 29.2  ±  19.5 µg/L. Forty-eight samples had patulin concentration between 5 and 50 µg/L. Although the mean concentration of patulin samples was lower than Iranian maximum tolerated level of 50 µg/L, contamination of 10% of the samples at levels higher than 50 µg/L indicated the need for improving production techniques by the industry.

PRACTICAL APPLICATIONS

Studies have shown that the concentration of patulin may exceed the determined limits in apple juice and in other fruit products. Its presence can be a potential threat to the health of consumers, particularly children. The results may help us in understanding what should be the level of patulin in apple juice. The awareness of the apple industry of patulin contamination in fruit and the implementation of improved techniques for the production of apple products with reduced patulin concentrations have contributed to the quality of apple juice that are available on the Iranian market.     

Patulin biodegradation by marine yeast Kodameae ohmeri

Patulin contamination of fruit- and vegetable-based products had become a major challenge for the food industry. Biological methods of patulin control can play an important role due to their safety and high efficiency. In this study, a strain of marine yeast with high patulin degradation ability was screened. The yeast was identified as Kodameae ohmeri by the BioLog identification system and partial 26S rRNA gene sequencing. The degradation products of patulin were identified as (E)- and (Z)-ascladiol through HPLC and LC-TOF/MS. High patulin tolerance at 100 μg ml^–1 and a high degradation rate at 35°C at a pH between 3 and 6 indicates the potential application of K. ohmeri for patulin detoxification of apple-derived products.     

Determination of patulin in apple and quince products by GC–MS using C5–7 patulin as internal standard

A reliable gas chromatographic mass spectrometric method has been validated for the determination of trace levels (<10 μgL⁻¹) of patulin in apple products and quince jam. The method was based on extraction of patulin with ethyl acetate-hexane, alkalinisation and silylation with N,O-bis-trimethylsilyltrifluoroacetamide with 1% of trimethylchloro-silane. The accurate determination of patulin was achieved by employing commercial ¹³C_5–7 patulin labelled as an internal standard, which allowed compensating target analyte losses and enhancement or suppression matrix effects. Limits of detection and quantification of method using real samples were 0.4 and 1.6 μgkg⁻¹, respectively. Recoveries of patulin from samples spiked at 8–50 μgkg⁻¹ levels ranged between 71% and 89%. The repeatability of measurements (expressed as relative standard deviation) was lower than 16%. The method was successfully applied to the determination of patulin in apple fruit and apple products including juice, cider and baby food, and also in quince fruit and quince jam. A new PCR system for the detection of Penicillium expansum in samples containing highly degraded DNA was developed which permitted the detection of the mould in 2/3 of the samples containing patulin, including juices and jams.     

水果及其制品中展青霉素的研究进展

展青霉素是一种毒性极强的真菌毒素,广泛存在于水果及其制品中,严重危害人类健康。本文综述了近年来水果及其制品中的展青霉素的研究进展,介绍了展青霉素的相关性质,分析了展青霉素的生物合成及降解机制,并论述了展青霉素的检测方法及相关微生物对展青霉素的脱除效果。      

Development of real-time PCR methods to quantify patulin-producing molds in food products

Patulin is a mycotoxin produced by different Penicillium and Aspergillus strains isolated from food products. To improve food safety, the presence of patulin-producing molds in foods should be quantified. In the present work, two real-time (RTi) PCR protocols based on SYBR Green and TaqMan were developed. Thirty four patulin producers and 28 non-producers strains belonging to different species usually reported in food products were used. The patulin production was tested by mycellar electrokinetic capillary electrophoresis (MECE) and high-pressure liquid chromatography-mass spectrometry (HPLC-MS). A primer pair F-idhtrb/R-idhtrb and the probe IDHprobe were designed from the isoepoxydon dehydrogenase (idh) gene, involved in patulin biosynthesis. The functionality of the developed method was demonstrated by the high linear relationship of the standard curves constructed with the idh gene copy number and Ct values for the different patulin producers tested. The ability to quantify patulin producers of the developed SYBR Green and TaqMan assays in artificially inoculated food samples was successful, with a minimum threshold of 10 conidia g⁻¹ per reaction. The developed methods quantified with high efficiency fungal load in foods. These RTi-PCR protocols, are proposed to be used to quantify patulin-producing molds in food products and to prevent patulin from entering the food chain.     

Patulin contamination in fruit derivatives,including baby food,from the Spanish market

The aim of this study was to provide a current overview on patulin (PAT) contamination of apple products in Spain. A second aim was to provide some data on the effect of processing stages of fruit juice concentrates on patulin levels. None of the analysed samples contained PAT concentrations exceeding the maximum levels fixed by the EU. Pear and apple concentrates showed the highest levels (up to 126 μg/kg). After milling, patulin levels did not exhibit important differences, except for at the concentration stage where a greater than fourfold mean increase was recorded. Moreover, a decrease was observed in those fruit juices that were decolourised after ultrafiltration. Thus, patulin presence in fruit products might not pose a health risk for the average consumer, although studies on patulin intake by populations consuming high amounts of fruit derivatives might be of importance.