Identification of anatoxins in blue-green algae food supplements using liquid chromatography-tandem mass spectrometry

Blue-green algae (cyanobacteria) in tablets and capsules, which are marketed as health food supplements, were investigated for the presence of neurotoxins related to anatoxin-a. These neurotoxins, which are nicotinic agonists, were investigated using isocratic micro-liquid chromatography-tandem mass spectrometry (micro-LC-MS-MS). The investigated compounds were anatoxin-a and homoanatoxin-a, together with their degradation products, dihydroanatoxin-a, epoxyanatoxin-a, dihydrohomoanatoxin-a and epoxyhomoanatoxin-a which were synthesized from the parent toxins. The analytes were extracted with methanol followed by isocratic chromatography on a micro C₁₈     

Toxins of cyanobacteria

Blue-green algae are found in lakes, ponds, rivers and brackish waters throughout the world. In case of excessive growth such as bloom formation, these bacteria can produce inherent toxins in quantities causing toxicity in mammals, including humans. These cyanotoxins include cyclic peptides and alkaloids. Among the cyclic peptides are the microcystins and the nodularins. The alkaloids include anatoxin-a, anatoxin-a(S), cylindrospermopsin, saxitoxins (STXs), aplysiatoxins and lyngbyatoxin. Both biological and chemical methods are used to determine cyanotoxins. Bioassays and biochemical assays are nonspecific, so they can only be used as screening methods. HPLC has some good prospects. For the subsequent detection of these toxins different detectors may be used, ranging from simple UV-spectrometry via fluorescence detection to various types of MS. The main problem in the determination of cyanobacterial toxins is the lack of reference materials of all relevant toxins. In general, toxicity data on cyanotoxins are rather scarce. A majority of toxicity data are known to be of microcystin-LR. For nodularins, data from a few animal studies are available. For the alkaloids, limited toxicity data exist for anatoxin-a, cylindrospermopsin and STX. Risk assessment for acute exposure could be relevant for some types of exposure. Nevertheless, no acute reference doses have formally been derived thus far. For STX(s), many countries have established tolerance levels in bivalves, but these limits were set in view of STX(s) as biotoxins, accumulating in marine shellfish. Official regulations for other cyanotoxins have not been established, although some (provisional) guideline values have been derived for microcystins in drinking water by WHO and several countries.     

Determination of Paralytic Shellfish Poisoning (PSP) toxins in dietary supplements by application of a new HPLC/FD method

Over the past years the importance of food additives and the development of so-called novel food increased permanently. Especially, the application of dietary supplements was on the rise. Then, more and more new products based on plants hitherto not used for human consumption were launched. Algae products containing valuable amounts of essential nutrients such as amino acids and trace elements play a decisive role. On the other hand, some algae including the blue-green algae (cyanobacteria) are capable of synthesizing harmful substances depending on species and provenience. Therefore, methods must be available to evaluate possible risks caused by toxins in algae-based dietary supplements. There are different groups of toxins related to marine algae and cyanobacteria. However, both marine algae and cyanobacteria are able to produce Paralytic Shellfish Poisoning (PSP) toxins which are potential neurotoxins. Hence, analytical methods for PSP determination have to be developed. The method for PSP toxin determination described below is based on ion-pair chromatography of the underivatized PSP toxins followed by post-column oxidation and fluorescence detection (FD). The determination of very low amounts of PSP toxins in different matrices of novel food is possible. In addition, the method allows to compare PSP profiles of various algae-based dietary supplements.     

电感耦合等离子体质谱同时测定螺旋藻保健品中16种元素含量

目的建立微波消解-电感耦合等离子体质谱法同时测定螺旋藻保健品中Na、Mg、Al、K、Ca、Cr、Mn、Fe、Co、Ni、Cu、Zn、As、Se、Cd、Pb等16种矿物元素含量的方法。方法螺旋藻保健品经处理后加入硝酸,通过微波消解,采用电感耦合等离子体质谱法对其中的16种矿物元素含量进行测定,通过在线添加内标溶液对结果进行实时校正。结果建立的方法 16种元素线性关系良好(r2≥0.99),检出限低,精密度在8.61%以内,加标回收率在86%～112%之间。结论该方法操作简单、灵敏度高、准确可靠,适用于螺旋藻保健品中多种矿物元素含量的同时测定。     

Cyanobacterial toxins--occurrence, biosynthesis and impact on human affairs

Mass developments of cyanobacteria ("blue-green algae") in lakes and brackish waters have repeatedly led to serious concerns due to their frequent association with toxins. Among these are the widespread hepatotoxins microcystin (MC) and nodularin (NOD). Here, we give an overview about the ecostrategies of the diverse toxin-producing species and about the genes and enzymes that are involved in the biosynthesis of the cyclic peptides. We further summarize current knowledge about toxicological mechanisms of MC and NOD, including protein phosphatase inhibition, oxidative stress and their tumor-promoting capabilities. One biotransformation pathway for MC is described. Mechanisms of cyanobacterial neurotoxins (anatoxin-a, homanatoxin-a, and anatoxin-a(s)) are briefly explained. We highlight selected cases of human fatalities related to the toxins. A special focus is given to evident cases of contamination of food supplements with cyanobacterial toxins, and to the necessary precautions.     

Persistent Organic Pollutants in Fish Oil Supplements on the Canadian Market: Polychlorinated Dibenzo-p-Dioxins, Dibenzofurans, and Polybrominated Diphenyl Ethers

ABSTRACT:  Canadians are interested in improving their diet through the consumption of fish oil food supplements, which are marketed to be rich in omega-3 fatty acids. Convenience samples of omega-3 enriched dietary supplements ( n  = 30) were collected in Vancouver, Canada, between 2005 and 2007. All of the omega-3 supplements were analyzed for polychlorinated dibenzo- p -dioxins/furans (PCDD/Fs) and polybrominated diphenyl ethers (PBDEs) and, although every sample was found to contain detectable residues of PBDEs, only 24 samples were found to have PCDD/F concentrations above the level of detection. PCDD/F concentrations ranged from 0.05 pg TEQ/g lipid to 45.7 pg TEQ/g lipid in salmon and shark oils, respectively. Maximum PBDE concentrations similarly were observed in shark oil (113 μg/kg lipid), however, most supplements had concentrations below 5 μg/kg lipid. Average PCDD/F and PBDE intake estimates, based on consumption of maximum supplement dose following product label recommendations, were 4.32 pg TEQ/d and 25.1 ng/d lipid, respectively.     

高效液相色谱-串联质谱法测定水产品中7种微囊藻毒素

建立了高效液相色谇串联质谱法同时测定水产品中7种微囊藻毒素（微囊藻毒素-RR、微囊藻毒素-YR、微囊藻毒素-LR、微囊藻毒素-LA、微囊藻毒素-LW、微囊藻毒素-LF、微囊藻毒素．LY）的分析方法。样品用90%醇水（P／g）溶液提取,经离心、固相萃取柱净化后,用C18柱,以0．1％甲酸乙腈．0．1％甲酸溶液为流动相,梯度洗悦分离,使用三重四级杆质谱检测器捡测,以保留时间及特征离子对定性,外标法定量。结果表明,7种微囊藻毒素在线性范围内线性关系良好,相关系数均不低于0．9990,方法的定量限（以信噪比为10计）为0．2-0．5μg／kg,添加水平为1．0-20μg／kg时,回收率为71．5-106％,相对标准偏差（n=6）为312～9．1％。该法前处理简单、回收率高、精密度好,适用于水产品中7种微囊藻毒素的测定。     

光化学衍生法结合HPLC测定食品和饲料中的黄曲霉毒素

黄曲霉毒素的急性毒性很强，是重要的致癌物质。本文采用光化学衍生法结合HPLC测定了食品和饲料中的黄曲霉毒素，研究了光化学衍生的效果。结果表明，经光化学柱后衍生，四种黄曲霉毒素的检测灵敏度都比较高，检出限可达到3．25pg。     

不同方法检测食品中对羟基苯甲酸酯类防腐剂

目的建立高效液相色谱法(high performance liquid chromatography,HPLC)检测对羟基苯甲酸酯类防腐剂的方法。方法采用高效液相色谱法对样品中对羟基苯甲酸酯类进行测定,并比较此方法与标准中规定的气相色谱(gas chromatography,GC)法之间的差异。结果气相色谱法和液相色谱法测定对羟基苯甲酸酯类线性范围均良好,气相色谱法加标回收率在82.3%～111.3%之间,相对标准偏差(relative standard deviation,RSD)在1.8%～5.2%之间,高效液相色谱法加标回收率在85.4%～107.0%之间,RSD在0.2%～0.6%之间,且高效液相色谱法中对羟基苯甲酸甲酯类的检出限分别为0.04、0.06、0.09、0.16mg/kg,定量限分别为0.1、0.2、0.3、0.5mg/kg,均低于气相色谱法的检出限和定量限。结论 HPLC法具有简单快速、灵敏度高等优点,能够满足饮料类食品检测及安全的需要,为防腐剂的检测提供了实用的技术手段。     

MULTI-RESIDUE DETERMINATION AND CONFIRMATION OF TEN HETEROCYCLIC AMINES IN COOKED MEATS

Heterocyclic aromatic amines (HAAs) have been identified in various cooked meat products. HAAs are formed from pyrolysis of proteins and amino acids and are of interest because many of these primary arylamines are mutagens and rodent carcinogens. We developed a screening method for ten HAAs using liquid-liquid extraction, reverse-phase HPLC separation, and tandem electrochemical (ECD) and fluorescence (FLU) detectors. This detection system exhibited greater sensitivity (50-100 fold) for HAAs than the HPLC-UV methods. HPLC-FLU has detection limits of 100 pg to 1 ng for six HAAs, and HPLC-ECD detection limits of four HAAs were 250 pg to 1 ng. We also developed a confirmatory procedure for ten HAAs using HPLC coupled to positive ion APCI-MS. This technique has subnanogram detection limits. These methods were used to quantify HAAs in meat products at levels formed by typical Western cooking methods.     

荧光光谱分析技术在食品检测领域的研究进展

荧光光谱分析技术作为一种食品检测技术,具有便捷、灵敏、高效的特点及检测食品中复杂成分及痕量成分的优势。本文介绍了荧光光谱分析技术的基本原理,系统总结了现阶段应用于食品检测领域的多种荧光光谱分析技术,包括原子荧光光谱、二维相关荧光光谱、三维荧光光谱、前表面荧光光谱、（总）同步荧光光谱以及荧光光谱成像技术等,对荧光光谱分析技术应用于粮油、畜禽肉、水产品、乳制品、果蔬等不同类别食品检测的方法与效果进行了归纳与总结,并进一步分析了荧光光谱分析技术现阶段存在的问题以及对未来发展趋势的展望。     

液相色谱法与氨基酸分析法测定食品中甘氨酸的比较研究

建立对食品中游离甘氨酸经提取、净化等处理后,邻苯二甲醛衍生,经高效液相色谱测定分析,并将该方法与氨基酸分析仪测定甘氨酸的方法进行比较研究。通过对市售的液体乳、含乳饮料(品)类和植物蛋白饮料(品)类中游离甘氨酸分别应用两种方法进行测定,结果表明,氨基酸分析仪法测定甘氨酸具有测定结果稳定准确、检出限低和操作简单的特点,而液相色谱法(荧光检测器)测定甘氨酸检测时间快,检出限也能满足常规检测的需要,因此,在实际应用中,两种方法均可采用,可作为互相验证的方法。     

Determination of the level of benzo[a]pyrene in fatty foods and food supplements

A routine method was developed for the quantification of benzo[a]pyrene (BaP) in edible oils and food supplements. BaP is often taken as an indicator of the presence of polycyclic aromatic hydrocarbons. The method consists of on-line liquid chromatography clean-up followed by injection to an HPLC system with fluorescence detection. The method has good performance characteristics and gave good results in proficiency tests. From 2002 to 2004, about 1350 samples of oils and food supplements were analysed using this method to test the level of BaP. About 20% of the edible oils contained more than 1.2 µg kg^-1 BaP (which is the limit applied by the Dutch Food and Consumer Product Safety Authority until 1 April 2005, and includes measurement uncertainty). In the case of food supplements, more then 30% contained too high levels of BaP, ranging from 1.2 to 135 µg kg^-1.     

Development of an ultrarapid one-step fluorescence immunochromatographic assay system for the quantification of microcystins

Microcystins are a family of potent toxic oligopeptides produced by freshwater cyanobacteria genera and have been a great threat to the welfare of humans and animals. There has been a great demand for developing a fast and convenient analytical method to detect microcystins. Recently, direct competition ELISA using monoclonal or polyclonal antibody has become the prevailing method for detecting microcystins. In this study, we report rapid quantification methods of microcystins using fluorescence for a detection signal and a lateral-flow-type immunochromatography as a separation system. The assay systems consist of a test strip housed in a disposable cartridge and a portable laser-fluorescence scanner. The components of a test strip are as follows: a nitrocellulose membrane, a sample pad, an absorption pad, and a backing card. A fluorescence scanner was designed to fit the cartridge and to quantify the distribution of the fluorescence intensity along the strip. When the calibration curve for an antibody-immobilized system was determined, a good linearity was displayed in the range from 125 to 2000 pg/mL of microcystin-LR. The linear-regression coefficient (R) was 0.938 between relative fluorescence intensity and the microcystin concentration. The limit of detection was determined to be 95.38 pg/mL. We then designed another biosensor system by changing an experimental format from the competition type to the inhibition type. When compared to the antibody-immobilized system, the antigen-immobilized assay detected a lower level of microcystin but did not discern microcystin-LR above 1000 pg/mL. The detection of limit for the antigen-immobilized system was 47.23 pg/mL. The linear regression coefficient (R) in the antigen-immobilized system equaled to 0.927. The reproducibility in the antigen-immobilized system was good through the entire range. The reproducibility in the antibody-immobilized system was relatively poor when compared to a MC-immobilized system. However, it still registered in the acceptable range of 7.32-9.91% except for the extreme ends of the MC concentration. Finally, surface water was tested to check for potential matrix interference. The calibration curve displayed a similar pattern as did those for other matrixes, including PBS and tap water, although its sensitivity was a little less due to the interference with certain components in the surface water. Overall, either of the biosensor systems can be used as a useful on-site detection tool for checking drinking water or surface water for microcystins. The laser-fluorescence scanner we developed is relatively small, transportable, and easy to use. Thus, the samples can be analyzed for microcystins at the test site using a real-time base within 15 min without having to bring the samples back to the laboratory.     

Free amino acids in botanicals and botanical preparations

ABSTRACT:  Numerous studies were carried out about aminoacidic composition of vegetable proteins, but information about the free amino acid pool and the role of these substances is very incomplete. The aim of this paper was to contribute to the scarce knowledge concerning the composition of free amino acids in botanicals and botanical preparations widely used as food, in dietary supplements, and in pharmaceutical products. This work studied the composition of free amino acids, identified the major components of 19 species of plants, and evaluated the influence of different types of extraction on the amino acid profile. Amino acids were determined using an automatic precolumn derivatization with fluorenylmethyl-chloroformate and reversed-phase liquid chromatography with fluorescence and ultraviolet detection. The amounts of total free amino acids varied widely between plants, from approximately 12 g in 100 g of Echinacea pallida extract to less than 60 mg in the same amount of Coleus forskohlii, Garcinia cambogia , and Glycine max. In 13 plants arginine, asparagine, glutamine, proline, and γ-aminobutyric acid were the free amino acids found in preponderant quantities. The levels of free amino acids above the quantification limit in 36 assayed samples of botanicals, extracts, and supplements are shown.     

多壁碳纳米管分散固相萃取净化在测定水产品中3 种微囊藻毒素的应用及与固相萃取法的比较

建立多壁碳纳米管净化-超高效液相色谱-串联质谱法对水产品中3?种微囊藻毒素残留量的测定方法。水产品用80%甲醇溶液提取,经多壁碳纳米管净化后,采用电喷雾正离子多反应监测模式进行分析。对不同性质碳纳米管进行考察,并对净化条件进行优化。将优化后的分散固相萃取条件与采用HLB柱固相萃取的净化方式进行比较,两者在1～50?μg/L范围内呈现良好的线性,线性相关系数均大于0.99,3?种微囊藻毒素的检出限为0.1～0.2?μg/kg。在不同浓度水平下进行加标实验,平均回收率在88.7%～95.5%之间,相对标准偏差不大于4.2%。该方法适用于水产品中微囊藻毒素的痕量检测。     

Algae in food: a general review

Abstract

Algae are common all over the Earth. Due to their rich chemical composition and content of bioactive substances they have been used in many fields of industry. Their gelling, thickening and stabilizing properties have led to the development of such products as agar, alginate and carrageenan. Moreover, algae are used in the food industry as food supplements and an addition to functional food. Algae are also added to meat products, such as pasty, steaks, frankfurters and sausages, as well as to fish, fish products, and oils, to improve their quality. Cereal-based products, such as pasta, flour and bread, are another group of products enriched with algae. Due to their properties algae may also be used for construction of fermented functional food. Fermented products containing algae are, most of all, dairy products, such as cheese, cream, milk deserts, yoghurt, cottage cheese, and processed cheese. Combination of fermented products offering a high content of lactic acid bacteria with algae possessing biologically active metabolites of natural origin allows not only to compose products with a high content of nutrients, but also to create a brand new segment of fermented food.     

Determination of phytoestrogens in dietary supplements by LC-MS/MS

Labelling data quantifying the exact content of individual phytoestrogen analytes in dietary supplements are generally poor. As these products are commonly used in the management of menopause symptoms, any clinical benefits would be dependent on the exact dosage of isoflavones received. Well-established extraction procedures and updated isotope dilution mass spectrometry liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) have been used to accurately quantify the concentrations of ten common isoflavones in 35 dietary supplement samples on sale in the UK, Canada and Italy. Concentration-specific ionization suppression is described for biochanin A and formononetin. All supplements contained phytoestrogens. The soya isoflavones (genistein, daidzein, glycitein) were present in all products and the majority also contained the red clover isoflavones (biochanin A, formononetin) and some the Kudzu isoflavones (daidzein, puerarin). The content of total isoflavones per dose ranged from <1 to 53 mg. Trace amounts of coumestrol were found in six products. Other less common analytes, the prenylnaringenins (6-prenylnaringenin, 8-prenylnaringenin, 6,8-diprenylnaringenin) were not found in any of the products. Only 14 of 35 supplements were found to deliver more than or equal to 40 mg day(-1) of aglycone isoflavones, a consensus dose value recognized as delivering therapeutic benefit. Eleven did not match label claims. Six delivered less than 10 mg day (-1) of isoflavones. There has been little improvement in the overall quality of industry labelling in the five years since this was last investigated. Consequently, the public, retailers and healthcare professionals should consider using standardized isoflavone supplements, which are supported by analytical measurements.     

Survey of nutritional supplements for selected illegal anabolic steroids and ephedrine using LC-MS/MS and GC-MS methods, respectively.

Several studies have highlighted that nutritional supplements may contain undeclared substances that are banned by the International Olympic Committee (IOC)/World Anti-Doping Agency (WADA). This paper describes a qualitative liquid chromatography coupled with tandem mass spectrometry detection (LC-MS/MS) method to detect anabolic androgenic steroids (4-androsten-3,17-dion, 4-oestren-3,17-dion, 5alpha-androsten-17beta-ol-3-one, boldenone, nandrolone, nandrolone decanoate, testosterone, and testosterone decanoate) and ephedrine in food supplements. The products are dissolved in methanol and analysed by gas chromatography-mass spectrometry (GC-MS). The methanolic solution was added to testosterone-d(3), evaporated to dryness, mixed with NaOH and extracted with n-pentane:diethylether (9:1). LC-MS/MS analyses were performed in selected reaction monitoring (SRM) on an ion-trap equipped with an atmospheric pressure chemical ionization (APCI) probe operating in positive-ion mode. The method was applied to 64 nutritional supplements. A total of 12.5% of the nutritional supplements analysed contained banned substances not declared on the label (anabolic steroids and ephedrine). Detection limits were in the range 1-25 ng g(-1).     

改良柱前荧光胺衍生化-高效液相色谱法测定猪肉中11种磺胺类药物残留

建立改良的磺胺类药物与荧光胺反应的衍生化方法,并应用于猪肉中11种磺胺类药物的高效液相色谱-荧光法测定.选用0.1 mol/L KH2PO4溶液(pH 3.0)0.8 mL、0.3 mg/mL荧光胺溶液0.2 mL与磺胺类药物发生衍生化反应,4℃避光反应40 min,得到磺胺-荧光胺衍生产物.该衍生化方法稳定、可靠,衍...