Analysis and occurrence of deoxynivalenol (DON) in cocoa

The primary objective of this study was to establish a current situation assessment of the possible occurrence of deoxynivalenol in cocoa and cocoa products. Since there was no analytic method for determining DON in cocoa and cocoa products, a special method was developed. The applicability and consistency of the method was confirmed by performing recovery assays on various cocoa products. A special post-column derivatisation procedure was used to increase selectivity and raise sensitivity by a factor of 80. The method’s limit of detection (LOD) was thereby reduced to 7 μg/kg; the limit of quantification (LOQ) was 14 μg/kg. The method was used to test 230 samples for possible DON content, ranging from cocoa beans to cocoa bean shells, nibs, cocoa liquor and cocoa powders through to finished cocoa-based products. In the case of cocoa beans and cocoa bean shells, DON content close to the detection limit was only determined in isolated cases. No DON content was detected in nibs, cocoa liquor, cocoa powders and finished cocoa-based products. Analogous to ochratoxin A and aflatoxins, the results show DON is more likely to be found in cocoa bean shells. Separation of shells during cocoa processing can reduce potential DON contents. Since no DON was determined in the fractions relevant for chocolate production, these assays show it does not represent a considerable issue for the cocoa and chocolate industry.     

Effects of oral exposure of pigs to deoxynivalenol (DON) sulfonate (DONS) as the non-toxic derivative of DON on tissue residues of DON and de-epoxy-DON and on DONS blood levels

The Fusarium toxin deoxynivalenol (DON) is of outstanding importance in pig nutrition because of its frequent occurrence in cereal grains at levels high enough to cause adverse effects such as a decrease in feed intake and impairment of the immune system. Thus, simple decontamination procedures would be useful. The present study aimed to examine the effects of wet preservation of triticale contaminated with DON and zearalenone (ZON) with sodium metabisulphite (SBS) on the treatment-related non-toxic derivative of DON (DON-sulfonate, DONS), and on ZON and its metabolites in blood and various physiological specimens of piglets. The uncontaminated control triticale (CON) and the DON-contaminated triticale (FUS) were included in the diets either untreated or SBS treated (CON-SBS, FUS-SBS) and fed to piglets for 28 days starting from weaning. The diet concentrations for DON were 0.156, 0.084, 2.312 and 0.275 mg kg^?1, for DONS were <0.05, <0.05, <0.05 and 1.841 mg kg^?1, and for ZON were <0.001, 0.006, 0.017, and 0.016 mg kg^?1 for each of CON, CON-SBS, FUS and FUS-SBS, respectively. DONS was present in the blood of piglets fed the FUS-SBS at a median concentration of 15.5 ng ml^?1 (3–67 ng ml^?1), while the median DON concentration amounted to 2 ng ml^?1 (0–5 ng ml^?1) at the same time. The median DON concentration in the blood of piglets fed the FUS diet reached a median concentration of 10.5 ng ml^?1 (5–17 ng ml^?1). Moreover, the relative differences between the DON concentrations in other physiological specimens (muscle, liver, kidney, bile and urine) in piglets fed the FUS-SBS and the FUS diet were comparable with the blood DON concentration differences. Although these differences can be taken as an indication for DONS stability after absorption and distribution further studies examining DONS in these other physiological specimens directly are necessary to substantiate this conclusion. Moreover, ZON and α-zearalenol could only be detected in bile and urine where their levels were not influenced by the SBS treatment.     

Cross-reactivity features of deoxynivalenol (DON)-targeted immunoaffinity columns aiming to achieve simultaneous analysis of DON and major conjugates in cereal samples

Immunoaf?nity columns (IACs) are a well-established tool in the determination of regulated mycotoxins in food and feed commodities. However, they also have the potential to become attractive pre-concentration and clean-up materials for the determination of masked (also called modified) mycotoxins, which have been recognised as important contributors to the toxicological hazard deriving from fungal spoilage of goods. However, the information available in the literature concerning the cross-reactivity of DON-IACs against the major conjugates (DON-3-G, 15-AcDON and 3-AcDON) is incomplete and often contradictory. We have carried out a detailed characterisation of the cross-reactivity of the four main IACs brands against DON and its conjugates as well as an assessment of the competition among the analytes. Only one IAC enabled the simultaneous analysis of all relevant DON forms while two missed 15-AcDON and the fourth one missed DON-3-G and 3-AcDON. In the case of the multivalent IAC, the analytes modified at the C-3 position compete for the antibody binding with preference for 3-AcDON (less spatially hindered) while DON-3-G has the more-hindered access to the active sites. Taking into consideration the levels of DON conjugates existing in real samples, the cross-reactivity of one DON-IAC allows a quantitative analysis of all of these analytes. Important but rather neglected aspects such as the continuous supply of IACs with identical characteristics, and of columns which are strictly blank, are also addressed in this paper.     

气相色谱内标法测定植物油中角鲨烯含量

通过内标类型和用量的选择,在已有方法基础上建立了一种内标法测定植物油中角鲨烯含量的气相色谱法方法。以角鲨烷为内标,样品经氢氧化钾-甲醇皂化,正己烷萃取,采用HP-5毛细管谱柱,程序升温气相色谱条件。结果表明,角鲨烯的平均回收率为95.5%~103.0%,精密度为1.42%~5.89%,检出限为0.05 mg/kg。该方法不仅回收率高,重现性好,而且灵敏度高,该方法可用于测定植物油中角鲨烯含量,为科学评价植物油中营养成分提供技术支撑。     

同位素内标高效液相色谱-串联质谱法测定粮食及其制品中赭曲霉毒素A、B和C

建立一种同位素内标高效液相色谱—串联质谱法同时测定粮食及其制品中赭曲霉毒素A、B和C的方法。样品用乙腈-水(84:16,V/V)提取,振荡离心后取上清液过赭曲霉毒素免疫亲和柱富集净化,经Waters ACQUITY BEH C₁₈(50 mm×2.1 mm,1.7 μm)进行液相色谱-串联质谱分析,采用多反应监测(MRM)、正离子模式和内标法定量,同时对线性范围、准确度、精密度和加标回收率等进行方法学验证。结果表明,在优化的条件下,赭曲霉毒素A、B和C在0.25~2.5 ng/mL线性范围内线性良好,决定系数均在0.999以上,本方法对赭曲霉毒素的检出限(LODs,S/N=3)和定量限(LOQs,S/N=10)分别为0.003~0.018、0.011~0.059 μg/kg;在2.0~10.0 μg/kg 三个加标水平下,方法的平均回收率为80.50%~107.08%,相对偏差(RSDs)介于0.14%~4.94%(n=6)。利用此方法对10个批次的粮食及其制品进行检测,发现1个批次的样品检出赭曲霉毒素A,含量为(0.35±0.01) μg/kg。此方法操作简便、灵敏度高,适用于粮食及其制品中赭曲霉毒素A、B和C的检测。     

Stability of DON and DON-3-glucoside during baking as affected by the presence of food additives

The mycotoxin deoxynivalenol (DON) is one of the most common mycotoxins of cereals worldwide, and its occurrence has been widely reported in raw wheat. The free mycotoxin form is not the only route of exposure; modified forms can also be present in cereal products. Deoxynivalenol-3-glucoside (DON-3-glucoside) is a common DON plant conjugate. The mycotoxin concentration could be affected by food processing; here, we studied the stability of DON and DON-3-glucoside during baking of small doughs made from white wheat flour and other ingredients. A range of common food additives and ingredients were added to assess possible interference: ascorbic acid (E300), citric acid (E330), sorbic acid (E200), calcium propionate (E282), lecithin (E322), diacetyltartaric acid esters of fatty acid mono- and diglycerides (E472a), calcium phosphate (E341), disodium diphosphate (E450i), xanthan gum (E415), polydextrose (E1200), sorbitol (E420i), sodium bicarbonate (E500i), wheat gluten and malt flour. The DON content was reduced by 40%, and the DON-3-glucoside concentration increased by >100%, after baking for 20 min at 180°C. This confirmed that DON and DON-3-glucoside concentrations can vary during heating, and DON-3-glucoside could even increase after baking. However, DON and DON-3-glucoside are not affected significantly by the presence of the food additives tested.     

正丙醇作内标气相色谱法测定白酒的酒精度

建立了用正丙醇作内标测定白酒酒精度的气相色谱方法。采用DB-624UI（60 m×0.32 mm×1.8 μm）毛细管色谱柱,氢火焰离子化检测器进行测定。该方法在乙醇体积分数为0.009 98%～0.099 80%范围内线性关系良好,相关系数R2=0.999 9。精密度试验相对标准偏差（RSD）为0.5%,重复性试验RSD为0.5%。加标回收率在96.51%～101.13%,RSD为0.2%～1.8%。结果表明,该方法前处理操作简单、快捷、准确度高、精密度好,适用于白酒酒精度的快速检测。     

LC-MS/MS method for the determination of the fungal pigment bikaverin in maize kernels as an indicator of ear rot

Bikaverin is a polyketide-derived pigment produced by multiple species of the fungus Fusarium, some of which can cause ear and kernel rot of maize. A method was developed for the analysis of bikaverin by high-performance liquid chromatography (LC) coupled to electrospray ionisation tandem mass spectrometry (MS/MS). The quantitative nature of the LC-MS/MS method was demonstrated over a range of concentrations of bikaverin in maize. For spike-recovery experiments utilising maize spiked with bikaverin to a level 5?µg?g^?1 of maize, the measured recovery (%) was 70.6?±?10.4. Based on the utilised method, the limit of detection (based on a signal-to-noise ratio (S/N)?>?3) was better than 0.5?µg?g^?1 from bikaverin spiked into uncontaminated ground maize. Further, the limit of quantitation (LOQ) was 3?µg?g^?1 (based on S/N?>?10) from bikaverin spiked into ground maize. The method was applied to assess contamination of maize with bikaverin following inoculation of developing maize ears with Fusarium verticillioides under agricultural field conditions.     

电感耦合等离子体质谱测定食盐中的铅含量

选用铑(103 Rh)作为内标物质,采用电感耦合等离子体质谱内标法和标准加入法分别对浓度为4%,8%,12%,16%,20%的食盐样品溶液中铅的加标回收率进行测定。试验结果表明:采用内标法测定时,当食盐样品浓度从4%增加至20%时,内标回收率从54.8%降低至5.1%,各浓度梯度高、中、低加标浓度样品的加标回收率在90%~112%;采用标准加入法测定时各浓度食盐样品基体对应的内标回收率基本保持在100%左右,各浓度梯度高、中、低加标浓度样品的加标回收率在91%~114%;采用电感耦合等离子体质谱内标法和标准加入法能够快速、准确地测定不同浓度食盐样品中的铅含量,而且食盐样品基体浓度从4%增加到20%并未对方法的准确性造成影响。     

气相色谱内标法测定食品中防腐剂脱氢乙酸、苯甲酸和山梨酸

选用正十一烷酸为内标,采用毛细管气相色谱内标法测定食品中防腐剂脱氢乙酸、苯甲酸和山梨酸,样品回收率均在96％～104％之间,标准线性关系良好,样品测定的变异系数小,脱氢乙酸为0.71％,苯甲酸为0.82％和山梨酸为0.62％;本方法具有简便、快速、准确的特点。     

烟草转基因内标准定量检测中竞争性模板的制备

以转基因烟草为材料，通过DNA提取、PCR扩增获得NOS终止子片段产物，以重叠延伸扩增法制备该片段的竞争性模板，并进行酶切验证。     

Simultaneous determination of 15 aminoglycoside(s) residues in animal derived foods by automated solid-phase extraction and liquid chromatography-tandem mass spectrometry

An automated method has been developed for the simultaneous quantification of 15 aminoglycosides in muscle, liver (pigs, chicken and cattle), kidney (pigs and cattle), cow milk, and hen eggs by liquid chromatography tandem mass spectrometry. Homogenized samples were extracted by monopotassium phosphate buffer (including ethylene diamine tetraacetic acid), and cleaned up with auto solid-phase extraction by carboxylic acid cartridges. The analytes were separated by a specialized column for aminoglycosides, and eluted with trifluoroacetic acid and acetonitrile. The decision limits (CCα) of apramycin, gentamycin, tobramycin, paromomycin, hygromycin, neomycin, kanamycin, sisomicin, netilmicin, ribostamycin, kasugamycin, amikacin, streptomycin, dihydrostreptomycin and spectinomycin were ranged from 8.1 to 11.8 μg/kg and detection capabilities (CCβ) from 16.4 to 21.8 μg/kg. High correlation coefficients (r(2)>0.99) of calibration curves for the analytes were obtained within linear from 20 to 1000 μg/kg. Reasonable recoveries (71-108%) were demonstrated with excellent relative standard deviation (RSD). This method is simple pretreatment, rapid determination and high sensitivity, which can be used in the determination of multi-aminoglycosides in complex samples.     

Mass spectrometric determination of waxy corn (Maize) starch in glutinous (waxy) rice cake by use of 13C/12C ratio

Glutinous (waxy) rice cake is a popular New Year food in Japan. Economic incentives exist for rice cake producers to adulterate the products and mislabel them. Stable carbon isotope ratios (¹³C/¹²C) in rice cake products were determined by mass spectrometry to detect such adulteration. Since the ¹³C/¹²C ratios in rice (δ¹³C value of ca - 25 ppt, parts per thousand are different from those of corn (maize) starch (ca ?9 ppt), the presence of added waxy corn (maize) starch in rice cake could be detected.     

同位素内标-高效液相色谱-串联质谱法同时测定小麦和玉米中19种真菌毒素

建立高效液相色谱-串联质谱法测定小麦和玉米中19种真菌毒素的检测方法。样品经乙腈∶水∶甲酸(80∶18∶2)溶液提取、离心、浓缩后,以C18色谱柱分离,甲醇-甲酸水溶液为流动相梯度洗脱,采用电喷雾正负离子(ESI+、ESI-)同时电离,多反应监测模式检测,同位素内标法定量。19种真菌毒素在一定含量范围内均具有良好的线性关系(R²≥0.992),定量限范围为0.12～30μg/kg,在低、中、高3个添加浓度水平下的回收率范围为61%～117%,相对标准偏差小于15%(n=6),满足日常检测方法性能要求。本方法操作简便、灵敏度高、重现性好,适用于小麦、玉米等粮食中多种真菌毒素的同时测定。     

气质联用内标法测定饮料酒中氨基甲酸甲酯和氨基甲酸乙酯

为同时检测酒精性饮料中的氨基甲酸甲酯（MC）和氨基甲酸乙酯（EC）含量,采用固相萃取方式对酒样进行前处理,D5-氨基 甲酸乙酯为内标,使用气相色谱串联质谱（GC-MS）定量测定含量。 结果表明,在10～400 μg/L范围内,EC的线性相关系数为0.999 6, MC的相关系数为0.996 1,二者检出限均为10 μg/L,MC加标回收率为103%～108%,RSD为0.50%;EC加标回收率为96%～104%,RSD 为3.79%;方法准确性良好。 MC含量测定结果RSD为3.0%,EC含量测定结果RSD为2.0%,方法精密度良好。 抽样检验结果表明,白酒 中未检出MC,其他酒精性饮料均含有一定量的MC和EC。     

气相色谱内标法测定奶粉及乳饮料中的DHA和EPA

奶粉及乳饮料中提取的脂肪经皂化处理后生成游离脂肪酸，其中的长碳链不饱和脂肪酸(DHA和EPA)经甲酯化后挥发性提高。所有这些脂肪酸可以用色谱柱有效分离，用氢火焰离子化检测器检测，并使用内标法定量。实验结果表明该方法线性范围宽，DHA和EPA标准曲线相关系数分别为0．99972和0.99983，DHA和EPA的回收率分别为98．50～101．50％和97．34～100．92％，相对标准偏差均小于2．0％，方法的重现性好。     

Preparation of [13 C 3]-melamine and 13 C 3]-cyanuric acid and their application to the analysis of melamine and cyanuric acid in meat and pet food using liquid chromatography-tandem mass spectrometry

For the determination of melamine and cyanuric acid the labelled internal standards [¹³ C ₃]-melamine and [¹³ C ₃]-cyanuric acid were synthesized using the common substrate [¹³ C ₃]-cyanuric chloride by reaction with ammonia and acidified water, respectively. Standards with excellent isotopic and chemical purities were obtained in acceptable yields. These compounds were used to develop an isotope dilution liquid chromatography/mass spectrometry (LC/MS) method to determine melamine and cyanuric acid in catfish, pork, chicken, and pet food. The method involved extraction into aqueous methanol, liquid–liquid extraction and ion exchange solid phase clean-up, with normal phase high-performance liquid chromatography (HPLC) in the so-called hydrophilic interaction mode. The method had a limit of detection (LOD) of 10 µg kg^?1 for both melamine and cyanuric acid in the four foods with a percentage coefficient of variation (CV) of less than 10%. The recovery of the method at this level was in the range of 87–110% and 96–110% for melamine and cyanuric acid, respectively.     

气质联用-内标法测定豆类中脂肪酸含量及因子分析

对市售9种豆类(鹰嘴豆、黄豆、青豆、花芸豆、扁豆、豌豆、绿豆、黑豆、红豆)中的脂肪酸组分进行含量测定,以十一烷酸甘油三酯为内标物,采用气相色谱-质谱联用技术对各个样品中脂肪酸甲酯组分进行分析,辅助NIST检索工具鉴定各脂肪酸组分。建立内标法计算豆类中脂肪酸组分含量的方法。结果表明,9种豆类样品脂肪酸组分及含量均有差异,其中青豆的总脂肪含量最高,鹰嘴豆的不饱和脂肪酸含量最高。通过软件分析相关性,其中各组分间除十五烷酸外,都具有很好的显著相关性,主成分分析获得前2个主成分可以解释全部变异的98%。聚类分析将9种豆类归入4大类。该方法前处理简便快捷,结果准确度高,分析时间较短,可为内标法测定脂肪酸含量的研究提供借鉴。     

Development and application of a heterologous internal standard for polymerase-chain-reaction-based detection of Campylobacter jejuni and Campylobacter coli in foods

 A heterologous internal standard, termed "mimic", was developed for the polymerase chain reaction (PCR)-based detection of Campylobacter jejuni and Campylobacter coli in food. Mimic was designed to contain a heterologous DNA fragment of plasmid pUC18, flanked by a primer binding site, identical to the bacterial target DNA. Application of mimic in the PCR permitted its co-amplification together with the bacterial DNA with similar efficiency. As the length of the amplified products differed, they were easily detectable by agarose gel electrophoresis. The presence or absence of the mimic PCR product was indicative of the efficacy of the PCR. The use of approximately 60 mimic molecules per reaction was optimal for determining the reliability of the diagnostic PCR assays without decreasing the detection limit. This system for the detection of the two species of Campylobacter was successfully applied in routine food surveillance. Received: 4 January 1999