HPLC-MS/MS法检测动物源性食品中喹诺酮类药物的基质效应及其补偿措施研究

目的 研究高效液相色谱-串联质谱法（HPLC-MS/MS）测定动物源性食品中15种喹诺酮类化合物的基质效应及其补偿措施。方法 以鱼肉、猪肉和鸡肉作为典型基质,采用提取后加入法和动态多反应监测方式评价基质效应。结果 在三种基质中,60%以上化合物受到中等及强的基质效应,恩诺沙星、培氟沙星和达氟沙星受到强的基质增强效应;氟罗沙星受到强的抑制效应。前处理方法、基质种类、化合物种类以及质量浓度是影响喹诺酮类化合物基质效应的主要因素。结论 日常监测过程中,在不改变检测标准前处理的条件下,采取基质匹配标准曲线、同位素内标、稀释样品以及改善分离度并结合动态多反应监测方式能有效补偿喹诺酮类化合物的基质效应,提高其检测结果的可靠性和准确性。     

基于天然大分子的黄酮类化合物纳米颗粒的研究进展

黄酮类化合物是存在于天然食品中一类重要的多酚类化合物,在食品、药品、保健品和化妆品等行业具有极大的应用潜力。然而黄酮类化合物却普遍因为自身的溶解度低、稳定性和渗透性差,导致其生物利用度差,因而限制了其在食品中的应用。利用纳米技术,以天然生物大分子为基质制备黄酮类化合物的纳米颗粒,进而改善黄酮类化合物的理化性质,是一种很有前途的策略。本文综合近年来国内外研究,简单介绍了黄酮类化合物的结构与性质,在此基础上,例举了多种蛋白质和多糖基质的纳米颗粒及相关的研究成果,讨论其结合机理和适用性,并展望黄酮类化合物纳米颗粒在食品行业中应用的发展前景。旨在为黄酮类化合物天然大分子纳米颗粒的开发利用提供理论基础和依据。     

脱细胞猪真皮基质材料改性

分别研究了植物多酚、戊二醛和环氧化合物对脱细胞真皮基质材料的改性性能,对改性后的材料进行收缩温度、力学性能、自由氨基等方面的表征.结果表明:植物多酚对脱细胞真皮基质的抗撕裂能力和硬度都有很大的提高,并且有一定的增厚性;戊二醛改性后脱细胞真皮基质的收缩温度大大提高,但是抗撕裂能力减弱;环氧化合物改性后脱细胞真皮基质的拉伸强度大幅度提高,收缩温度也相应提高.     

食品中全氟化合物检测前处理技术研究进展

全氟化合物(PFCs)是一类在生活生产中应用广泛的含氟烃类化合物,因为其潜在的环境污染持久性、生物毒性和累积性,受到了越来越多的关注。由于其分布广泛,目前已经危害到了人类的食品安全方面。本文论述不同食品基质中PFCs的前处理方法,目前主要利用液液萃取和液固萃取的前处理方法,同时利用固相萃取消除复杂基质对PFCs检测准确性的影响。由于PFCs在食品基质中处于痕量水平,所以进一步提高前处理技术的简便性和准确性尤为重要。     

马齿苋黄酮类化合物在食品保鲜上的应用研究

试验研究了不同外界因素(p H、紫外光照射和温度)和不同食品基质对马齿苋黄酮类化合物抑菌效果的影响。结果表明:食品基质对各微生物菌体有一定的保护作用,在一定浓度范围内,能加速菌体的死亡。在p H值为7,紫外光照射30min和80℃处理30min的条件下,马齿苋黄酮类化合物对各菌的抑菌效果较佳。感官评价结果显示,在15天以内,马齿苋黄酮类化合物可保护食品的色泽和风味不发生变化,而且这种作用很稳定,可延长食品的保藏时间。     

QuEChERS-超高效液相色谱-串联质谱法测定食品中5种罂粟壳生物碱基质效应

目的考察3种不同食品基质中罂粟碱、吗啡等5种生物碱类物质在超高效液相色谱-串联质谱法检测中的基质效应。方法用提取前与提取后空白溶液加标方法分析基质效应的影响,并配制不同浓度加标溶液,通过拟合标准曲线,比较斜率、截距等,评估5种生物碱成分测定的基质效应。结果固体、半固体基质样品基质效应较强,那可丁、蒂巴因、可待因易受基质效应影响,通过优化梯度洗脱程序、加入内标溶液、稀释样品等方法可有效降低或消除基质效应影响。结论基质效应与基质种类、化合物种类等有明显关系,本试验中基质效应的改进方法可为不同样品基质中罂粟壳生物碱的测定提供参考。     

QuEChERS-LC-MS/MS法测定果蔬中4种植物生长调节剂残留

为比较3种QuEChERS方法提取21类果蔬中氯吡脲、多效唑、烯效唑和噻苯隆4种植物生长调节剂的有效性,考察了乙二胺-N-丙基硅烷(PSA)、C_(18)吸附剂和石墨化碳黑(GCB)对这4种植物生长调节剂的净化效果。试验发现,采用加入乙酸盐缓冲盐的QuEChERS方法提取, PSA和C18吸附剂净化, 4种化合物有更高的回收率。以提取后添加法评估了21种果蔬基质中各目标化合物液相色谱-串联质谱(LC-MS/MS)分析的基质效应,柑橘基质对4种化合物的基质抑制效应最强。在0.2～100μg/kg加标浓度范围内4种化合物的回收率为71.0%～109%,相对标准偏差(RSD)为0.8%～9.5%(n=6),方法检出限为0.1～5μg/kg。该方法简便快速,灵敏度和准确性好,适用于果蔬中植物生长调节剂残留量的测定。     

硅胶固载离子液体基质固相分散法提取蜂房中的酚酸和黄酮类化合物

建立一种基于离子液体的基质固相分散-高效液相色谱法同时测定蜂房中的5种酚酸和黄酮类化合物。采用浸渍法制备硅胶固载离子液体吸附剂,并将其作为基质固相分散提取法的分散剂用于提取蜂房中的咖啡酸、阿魏酸、桑色素、白杨素和山奈素,通过高效液相色谱对目标化合物进行分离和测定。实验结果表明,硅胶固载离子液体-基质固相分散法的最佳提取条件为:以含10%[C6MIM]Cl的硅胶固载离子液体为分散剂,20mL正己烷为淋洗剂,15mL甲醇为洗脱剂,样品与分散剂质量比为1:4。各目标化合物在线性范围内呈现良好的线性关系(r0.9995),检出限和定量限分别为0.05~0.18μg/mL和0.17~0.59μg/mL,日内和日间精密度(RSD)分别低于5.48%和6.60%,样品加标回收率在83.73~95.32%之间。本法结合了离子液体和基质固相分散提取法的优势,具有提取时间短,有机溶剂和样品用量少等优点,本法可广泛应用于药用动植物中酚酸和黄酮类化合物的提取分析。     

HPLC-MSMS测定果蔬中有机磷类农药的基质效应

利用提取后添加法定量分析高效液相色谱-串联质谱法(HPLC-MSMS)检测24种有机磷农药时的基质效应。用基质效应因子法分析了四种果蔬中基质浓度对基质效应的影响,并采用配制不含基质标准工作溶液和含基质标准工作溶液的方法,通过拟合标准曲线,比较斜率,从而评估果蔬的基质效应。结果表明,在四种果蔬中基质效应均随着基质浓度的增加而增大;具有基质效应的化合物其基质标准曲线基本保持线性,只是与溶剂标准曲线相比斜率发生变化。     

岛津GCMS-TQ8040结合双柱系统单次同步分析植物源性食品中900种化学污染物(下)

正双柱系统提升分析结果准确性实例实验发现,对于基质过于复杂的样品,在一根色谱柱上同时分析数百种化合物时,即便利用串联质谱技术,仍然存在少量目标组分因基质干扰而无法准确定量的现象;此外,由于化合物的性质不同,在不同色谱柱上获得的分离效果也不同,因此一根色谱柱难以满足每个目标物最佳的分离效果。通过岛津独有的质谱双柱系统(Twin Line MS System),利用两根色谱柱不同的分离特性,将目标物和复杂基质进行有效分离,消除化学基质干扰,实现目标组分的准确测定。     

基质辅助激光解吸电离飞行时间质谱定性分析啤酒中糖类条件的优化

通过选择不同基质(2,5-二羟基苯甲酸、super DHB、2,4,6-三羟基苯乙酮)和设置不同啤酒样品稀释倍数(从原液到1 000倍稀释),优化了MALDI TOF MS定性分析啤酒中糖类的试验条件。研究结果显示,3种基质中,2,4,6-三羟基苯乙酮应用于MALDI TOF MS对啤酒中糖类表征的能力优于另外两种基质。当以2,4,6-三羟基苯乙酮为基质,啤酒样品稀释10倍时,MALDI TOF MS可达良好的定性分析效果。     

Analysis of green kiwi fruit (Actinidia deliciosa cv. Hayward) proteinases by two‐dimensional zymography and direct identification of zymographic spots by mass spectrometry

BACKGROUND: Proteinases present in kiwi fruits are potentially allergenic enzymes belonging to the papain family of cysteine proteinases. Actinidin is a prominent kiwi enzyme. The study of kiwi proteinases is important for the follow‐up of fruit maturation, a deeper insight in the allergenic properties of individual proteins, and the application of kiwi proteinases for meat tenderisation and other industrial purposes. RESULTS: Kiwi crude extracts were analysed by two‐dimensional zymography on gelatin‐containing gels. The digestion by the reactivated proteolytic enzymes after electrophoresis resulted in insights into kiwi proteinases. A mixture of several enzyme isotypes with the same pI but different molecular mass was observed. Clear spots, corresponding to the proteolytic activities, were excised, digested with trypsin, and submitted to MALDI‐ToF mass spectrometry for protein identification. The most representative enzyme was actinidin. CONCLUSIONS: The innovative achievements of the present study are the: (1) two‐dimensional zymographic map of kiwi gelatinases without the need for extensive purification; and (2) direct identification of proteinase isotypes by means of direct MALDI‐ToF MS analysis of the zymographic spots. Copyright © 2010 Society of Chemical Industry     

Identification of four low molecular and water‐soluble proteins from grape (Vitis vinifera L.) seeds

Profiles of soluble proteins isolated from mature seeds of grape (Vitis vinifera L.) pomace were studied using two‐dimensional polyacrylamide gel electrophoresis (2D‐PAGE) coupled with matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI–TOF–MS). Two‐dimensional gels stained with Coomassie brilliant blue revealed more than fifty protein spots. Four abundant protein spots showing low molecular weight (Mr) and wide isoelectric point (pI) were analysed by MALDI–TOF–MS, resulting in their identification. Taken together, these results suggest that identified proteins may be linked to seed development and metabolism, but more instructive is that they have some potential functions for future food application. These results provide some insights into conversion of grape processing wastes into useful products or even as raw material for other industries.     

Food allergen analysis: A review of current gaps and the potential to fill them by matrix-assisted laser desorption/ionization

Food allergy remains a public health, business, and regulatory challenge. Risk analysis (RA) and risk management (RM) of food allergens are of great importance and analysis for food allergens is necessary for both. The current workhorse techniques for allergen analysis (enzyme linked immunosorbent assay [ELISA] and real-time polymerase chain reaction) exhibit recognized challenges including variable and antibody specific responses and detection of species DNA rather than allergen protein, respectively. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) enables protein identification, with potential for multiplex analysis and traceability to the System of International units (SI), aiding global measurement standardization. In this review, recent literature has been systematically reviewed to assess progress in LC–MS/MS and define the potential and benefits of matrix-assisted laser desorption/ionization–time-of-flight MS (MALDI–ToF-MS) technology for allergen analysis. MALDI–ToF-MS of initially intact protein is already applied to verify in silico-derived peptide sequences for LC–MS/MS analysis. We describe the origins of MALDI and its future perspectives, including affinity bead-assisted assays coupled to MALDI. Based on the proliferation of reliable and reproducible MALDI-based clinical applications, the technique should emulate the detection capability (sensitivity) of established allergen detection techniques, whilst reducing technical support and having equivalent multiplexing potential to competing techniques, for example, LC–MS/MS and ELISA. Although unlikely to offer inherent SI traceability, MALDI-based allergen analysis will complement existing MS approaches for allergens. Affinity bead-MALDI appears capable of higher throughput at lower cost per sample than almost any existing technique, enabling repeated sub-sampling as a way to reduce representative sampling issues.     

Beer fingerprinting by Matrix-Assisted Laser Desorption-Ionisation-Time of Flight Mass Spectrometry

A method allowing parallel fingerprinting of proteins and maltooligosaccharides directly from untreated beer samples is presented. These two classes of compounds were detected by Matrix-Assisted Laser Desorption-Ionisation-Time of Flight-Mass Spectrometry (MALDI-TOF-MS) analysis of beer mixed with 2,5-dihydroxybenzoic acid solution. The maltooligosaccharide profiles acquired from the MALDI sample spot center were not found characteristic for beers of different source and technology. On the other hand, according to profiles containing protein signals acquired from crystals formed on the border of the MALDI sample spot, we were able to distinguish beer samples of the same brand produced by different breweries. The discriminatory abilities of the method were further examined on a set of 17 lager beers, where the fingerprints containing protein signals enabled resolution of majority of examined brands. We propose MALDI-TOF-MS profiling as a rapid tool for beer brewing technology process monitoring, quality control, and determination of beer authenticity.     

Proteome analysis of the wort boiling process

A comprehensive proteome map was constructed of brewers wort. The map consisted of protein identification on two-dimensional gel electrophoresis (2DE) images and identified 63 out of 202 protein spots, which were categorized into 20 protein species. To analyze the modification of protein Z during wort boiling, protein Z spots on 2DE gel of the sweet wort, the boiled wort and the trub were analyzed by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF-MS) and then the spectra were compared. The analysis identified several specific signals detected only in the trub, suggesting that specific modification occurred in the precipitated protein Z during wort boiling. The protein Z spot on the 2DE gel of the precipitate was further analyzed by liquid chromatography mass spectrometry/mass spectrometry. The analysis identified low molecular weight fragment (1.3 kDa) derived from wound induced protein (barwin) in the protein Z spot of the trub. These results suggested that protein Z was precipitated by binding with comparatively small size specific fragment(s) derived from sweet wort protein, i.e., barwin during wort boiling. Our results and understandings have application for quality assurance and control in commercial brewing practice, and development of DNA markers for malting barley breeding.     

金属有机骨架衍生氧化物材料作为MALDI-TOF MS基质用于氨基酸分析

目的:为了拓展基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）在小分子检测领域的应用,本文合成了三种金属有机骨架衍生氧化物,作为MALDI-TOF MS新型基质,用于高效分析氨基酸。方法:本文以三种金属有机骨架材料（MOFs）为前驱体,通过TiO₂纳米涂层修饰,并经过煅烧得到其金属氧化物,用作MALDI质谱基质,研究其在氨基酸检测方面的优势,探究基质浓度对检测结果的影响,考察材料的重现性和定量分析能力。结果:三种MOF-衍生金属氧化物材料相比其前驱体MOFs和TiO₂ NPs,分析氨基酸（Pro、Ile、His和Try）时具有信噪比高、离子强度高和重现性好的优点;其中Cr₂O₃@TiO₂表现最佳,基质浓度为0.1 mg·mL?1,定量检测Ile和His时线性范围为0.001~0.1 mg·mL?1。结论:本文制备的MOF-衍生金属氧化物材料作为MALDI-TOF MS基质,实现了对4种氨基酸的同时精确检测,具有在氨基酸及小分子代谢物检测方面进一步的拓展和应用价值。     

Purification and characterisation of an acid protease from the Aspergillus hennebergii HX08 and its potential in traditional fermentation

A protease AP3 from Aspergillus hennebergii HX08 was purified by ammonium‐sulphate precipitation, followed by anion‐exchange chromatography and gel filtration. The molecular weight of acid protease AP3 was 33 kDa (SDS‐PAGE and MALDI‐TOF‐MS). The protease AP3 was identified as an acid protease with MALDI‐TOF/TOF tandem MS. Its optimal temperature and pH were 60°C and 4.0, respectively. Its K _max and V _max were 57.92 mg/mL and 32.57 U/mL, respectively. The enzyme was active over a broad pH and temperature range (pH 3.0–5.0 and 30–60°C), and exhibited high activity and stability in 2–12% (v /v) ethanol solvent. Subsequent studies suggest that the enzyme presents a relatively high substrate affinity to wheat protein (98% of total activity). Its application to solid‐state fermentation of wheat flour with Saccharomyces cerevisiae could increase the hydrolysis degree of wheat protein (28.26%) and amino acid nitrogen concentration of fermented grains (34.21%). Additionally, enhanced S. cerevisiae biomass (37.09%) and alcohol concentration (38.29%) were also observed during the process. Volatile compounds analysis of fermented grains by headspace solid‐phase micro‐extraction and GC‐MS revealed more flavour compounds. These results suggest its potential in food and alcohol industries. Copyright © 2017 The Institute of Brewing & Distilling     

Off-line liquid chromatography-MALDI by with various matrices and tandem mass spectrometry for analysis of glycated human serum albumin tryptic peptides

Advanced glycation end-product (AGE)/peptides, arising from in vivo digestion of glycated proteins, are biologically important compounds, due to their reactivity against circulating and tissue proteins. For information on their possible structure, in vitro glycation of HSA and its further enzymatic digestion were performed. The resulting digestion product mixture was analysed directly by MALDI MS with various matrices [2,5-dihydroxy benzoic acid (DHB) and alpha-cyano-4-hydroxy cinnamic acid (CHCA)]. Alternatively, offline microbore LC prior to MALDI analysis was used, and showed that 63% of the free amino groups prone to glycation are modified, indicating the contemporary presence of unglycated peptides. This result proves that, regardless of the high glucose concentration employed for HSA incubation, glycation does not go to completion. Further studies showed that the collisionally activated decomposition of singly charged glycated peptides leads to specific fragmentation pathways, all related to the condensed glucose molecule. These unique product ions can be used as effective markers to establish the presence of a glucose molecule within a peptide ion.     

Analysis of glycated proteins by mass spectrometric techniques: qualitative and quantitative aspects.

The analysis of protein glycation poses a difficult challenge due to the complex nature of the reaction. Of the several methods developed for the qualitative and quantitative evaluation of the glycation reaction between proteins and reducing sugars, soft ionization mass spectrometry is the most direct and reliable. In this paper we review the major mass spectrometric methods (ESI and MALDI mass spectrometry) used in the study of protein glycation. We also tested the assumption that limited glycation has little or no effect on the ionization potential of proteins and that the distribution profile of molecular ion peaks of different glycoforms in a mass spectrum reflect their solution composition in a mixture. The results confirm the validity of the above assumption under dilute solution conditions (0.2-0.6 g/ml total protein). A comparison of ESI and MALDI mass spectrometry in the analysis of protein glycation showed that both methods provide qualitative and quantitative analytical results, but the choice of instrument depends on the nature of the sample to be analysed, the level of accuracy and the type of information that is required.