Use of a multifunctional column for the determination of deoxynivalenol in grains, grain products, and processed foods

Deoxynivalenol (DON), also known as vomitoxin, belongs to a class of naturally occurring mycotoxins produced by Fusarium spp. DON, 12, 13-epoxy-3,7 trihydroxytrichothec-9-en-8-one, is one of the most frequently detected mycotoxins in agricultural commodities worldwide. A method consisting of extraction, filtration, column cleanup, and RP-HPLC-UV separation and quantitation was validated for the determination of DON in grains (rice and barley), grain products (whole wheat flour, white flour, wheat germ, and wheat bran), and processed foods (bread, breakfast cereals, and pretzels). A 25 g test portion was extracted with 100 mL acetonitrile-water (84 + 16, v/v). After blending for 3 min, the supernatant was applied to a multifunctional column (MycoSep 225). The purified filtrate (2 mL) was evaporated to dryness and redissolved in the mobile phase. The toxins were then subjected to RP-HPLC-UV analysis. The accuracy and repeatability characteristics of the method were determined. Recoveries of DON added at levels ranging from 0.5 to 1.5 microg/g for all test matrixes were from 75 to 98%. SD and RSD(r) ranged from 0.7 to 11.6% and 0.9 to 12.7%, respectively. Within-laboratory HorRat values were from 0.1 to 0.7 for all matrixes analyzed. The method was found to meet AOAC method performance criteria for grains, grain products, and processed foods. The identity of DON in naturally contaminated test sample extracts was confirmed by HPLC/MS/MS analysis.     

Determination of deoxynivalenol in cereals and cereal products by immunoaffinity column cleanup with liquid chromatography: interlaboratory study

An interlaboratory study was performed on behalf of the UK Food Standards Agency to evaluate the effectiveness of an immunoaffinity column cleanup liquid chromatographic (LC) method for the determination of deoxynivalenol in a variety of cereals and cereal products at proposed European regulatory limits. The test portion was extracted with water. The sample extract was filtered a applied to an immunoaffinity column. After being washed with water, the deoxynivalenol was eluted with acetonitrile or methanol. Deoxynivalenol was quantitated by reversed-phase LC with UV determination. Samples of artificially contaminated wheat-flour, rice flour, oat flour, polenta, and wheat based breakfast cereal, naturally contaminated wheat flour, and blank (very low level) samples of each matrix were sent to 13 collaborators in 7 European countries. Participants were asked to spike test portions of all samples at a range of deoxynivalenol concentrations equivalent to 200-2000 ng/g deoxynivalenol. Average recoveries ranged from 78 to 87%. Based on results for 6 artificially contaminated samples (blind duplicates), the relative standard deviation for repeatability (RSDr) ranged from 3.1 to 14.1%, and the relative standard deviation for reproducibility (RSDR) ranged from 11.5 to 26.3%. The method showed acceptable within-laboratory and between-laboratory precision for all 5 matrixes, as evidenced by HorRat values < 1.3.     

基于手性固定相的超高效液相色谱-串联质谱法检测小麦及其加工制品中的腈菌唑对映体

建立了手性超高效液相色谱-串联质谱检测小麦及其加工制品中腈菌唑对映体残留的分析方法.样品经乙腈提取,N-丙基乙二胺(PSA)和C18净化,手性色谱柱Lux Cellulose-1(150 mm×2.0 mm,3μm)分离,质谱电喷雾正离子扫描(ESI+),多反应监测(MRM)模式进行检测.为准确定量,考察了小麦籽粒及其...     

Determination of synthetic phenolic antioxidants in food by high-performance liquid chromatography

This review deals with HPLC method to be used for the determination of synthetic phenolic antioxidants added to various foods. Sample preparation, isolation techniques, separation systems as well as detection methods used in applied food analysis procedures are discussed.     

Separation and quantification by high-performance liquid chromatography with light scattering detection of the main wheat flour phospholipids during dough mixing in the presence of phospholipase

Phospholipids (PL) are minor components of wheat flour involved in baking quality and exogenous phospholipids are used as emulsifiers giving better loaf volume and crumb grain. Few biochemical data are available on the phospholipid evolution during mixing, probably because of the time-consuming methods proposed for their extraction, separation and quantification. In the present study, the extraction, separation and quantification of the main wheat flour phospholipids were carried out. Total lipids (2% dry mass of wheat flour) were extracted from flour or dough by a mixture of chloroform-methanol-water (1:1:1 (v/v)). The phospholipids were separated from the lipid extract on silica cartridge by solid-phase extraction (SPE) procedure under a 1.5-4 mmHg vacuum, at a 0.8 mL min(-1) flow rate (1 mmHg = 133.322 Pa). The recovery of the lipid extract was 100%, whereas the SPE yield for the PLs was 50%. The resulting fraction was then submitted to HPLC with evaporative light scattering detection on a Diol stationary phase allowing the separation and quantification of each class of phospholipids, in less than 16 min. The developed method allowed to quantify the phospholipid amounts from eight wheat flours as well as their evolution during mixing in the presence of phospholipase.     

Determination of carbohydrates and sweeteners in foods and biologically active additives by high-performance liquid chromatography

A method for the determination of large amounts of carbohydrates (glucose, lactose, maltose, mannose, sucrose, and fructose) and sweeteners (xylitol and sorbitol) by reversed-phase liquid chromatography with refractive index detection without derivatization has been developed. The limits of determination for glucose, fructose, and sucrose in liquid samples were 0.1 g/L, and for xylite, lactose, maltose, mannose, and sorbite, 1 g/L. In solid samples the limits of determination for glucose, fructose, and sucrose were 0.1%, and for xylite, lactose, maltose, mannose, and sorbite, 0.6%. The method is applicable to the analysis of samples of wine, juice, honey, cookies, dairy products, and biologically active additives. The developed method for the determination of carbohydrates and sweeteners in foods and biologically active additives was certified in the Mendeleev Institute for Metrology (St. Petersburg).     

电感耦合等离子体发射光谱法分析小麦粉制品中的K、Na、Ca、Mg、Al、Ti

采用硝酸-高氯酸混合酸湿式消化处理样品,建立了ICP-OES 法同时测定小麦粉及制品中钾、钠、钙、镁、铝、钛6种元素的方法.方法优化了仪器参数,探讨了不同消解方法和光谱干扰扣除方法,建立方法的线性范围宽,相关系数均大于0.9999,钾、钠、钙、镁、铝、钛检出限分别为10.2、9.5、0.25、0.006、0.31、0....     

基于真菌毒素污染差异的液相色谱-串联质谱法鉴别板栗粉中掺假小麦粉

建立了分散固相萃取-超快速液相色谱-串联质谱法同时测定板栗粉和小麦粉中43种真菌毒素的方法,对48份板栗粉和80份小麦粉样品的污染状况进行调查,筛选出5种专属于小麦粉的标志性真菌毒素.样品采用84％(v/v)乙腈水溶液提取,提取液采用C18结合增强型脂质去除净化剂(EMR-Lipid)净化,采用响应曲面-中心组合设计优...     

Determination by perfusion reversed-phase high-performance liquid chromatography of the soybean protein content of commercial soybean products prepared directly from whole soybeans

The use of soybean flour as external standard for the determination of soybean proteins in soybean products directly prepared from whole soybeans is investigated. For that purpose a perfusion reversed-phase high-performance liquid chromatography method consisting of a linear binary gradient acetonitrile-water (both with 0.1% trifluoroacetic acid) in 3 min at a flow-rate of 3 ml/min, and a temperature of 60 degrees C is used. Samples dissolved in water are directly injected in the chromatographic system. The method is validated by evaluating detection limits, precision, and accuracy and applied to the quantitation of soybean proteins in soybean products directly prepared from whole soybeans.     

高效液相色谱-二极管阵列检测法测定小麦粉及面粉改良剂中福美双

福美双是重要的二硫代氨基甲酸酯(DTC)杀菌剂,在小麦中使用限量以1 mg/kg二硫化碳(CS₂)计。目前我国相关检测方法是针对二硫代氨基甲酸酯一类的化合物,二硫代氨基甲酸酯通过与酸反应生成CS₂,采用光谱法或色谱法测定CS₂,间接实现二硫代氨基甲酸酯测定。该方法无法特异性实现对福美双的检测,因此开展小麦粉中福美双检测方法的研究具有重要意义。研究建立了高效液相色谱-二极管阵列检测(HPLC-DAD)测定小麦粉及面粉改良剂中福美双的分析方法。小麦粉及面粉改良剂样品用乙腈溶剂提取后,经涡旋、振荡、冰水浴超声和静置后取上清液过滤,供高效液相色谱测定。采用ZORBAX plus-C18色谱柱(150 mm×4.6 mm, 5 μm)分离,以水-乙腈为流动相洗脱分析,在波长280 nm下检测。实验优化了提取溶剂及其体积、振荡超声条件、色谱柱、检测波长、流动相等条件。该方法采用保留时间和紫外光谱图定性,外标法定量。该方法在线性范围内(0.30~30.0 μg/mL)线性关系良好,相关系数(r²)为0.99999。对小麦粉及面粉改良剂进行1.5、3.0、15 mg/kg 3个水平的加标回收试验,福美双的回收率为89.6%~98.3%,相对标准偏差为1.6%~3.9%(n=6)。方法的检出限和定量限分别为0.5 mg/kg和1.5 mg/kg。该方法采用溶剂提取,操作简单,分析时间短,特异性好,具有精密度高、重复性好、检出限低等特点,适用于小麦粉及面粉改良剂中福美双快速、准确的定量检测。     

Determination of vitamin B12 in food products by liquid chromatography/UV detection with immunoaffinity extraction: single-laboratory validation

A fast and simple method to determine vitamin B12 in foods is presented. The method allows, in addition to the determination of added cyanocobalamin, the determination of natural vitamin B12 forms, making it also applicable to nonfortified products, especially those that are milk-based. Vitamin B12 is extracted in sodium acetate buffer in the presence of sodium cyanide (100 degrees C, 30 min). After purification and concentration with an immunoaffinity column, vitamin B12 is determined by liquid chromatography with UV detection (361 nm). The method has been validated in analyses of a large range of products: milk- and soy-based infant formulas, cereals, cocoa beverages, health care products, and polyvitamin premixes. The method showed appropriate performance characteristics: linear response over a large range of concentrations, recovery rates of 100.8 +/- 7.5% (average +/- standard deviation), relative standard deviation of repeatability, RSDr, of 2.1%, and intermediate reproducibility, RSDiR, of 4.3%. Limits of detection and quantitation were 0.10 and 0.30 microg/100 g, respectively, and correlation with the reference microbiological assay was good (R2 = 0.9442). The proposed method is suitable for the routine determination of vitamin B12 in fortified foods, as well as in nonfortified dairy products. It can be used as a faster, more selective, and more precise alternative to the classical microbiological determination.     

Optimization of a chemical modifier in the determination of selenium by graphite furnace atomic absorption spectrometry and its application to wheat and wheat flour analysis.

A method for the determination of total selenium in wheat and wheat flour using graphite furnace atomic absorption spectrometry (GFAAS) with palladium/ascorbic acid as a chemical modifier was studied. The effects of nickel nitrate, palladium/ascorbic acid, and palladium/magnesium nitrate as chemical modifiers on the sensitivity in the determination of selenite, selenate and selenomethionine by GFAAS were compared. The palladium/ascorbic acid modifier was used for the determination of total selenium in wheat and wheat flour, because the oxidation states of the selenium ion are not important in the determination. The detection limit was estimated to be 1 microg L(-1) (calculated as 3sigma of the blank); the calibration curve was linear for the concentration range 5 - 50 microg L(-1) and the recovery range was 96.66 - 101.80%. The optimal ashing and atomizing temperatures were 1300 degrees C and 2250 degrees C, respectively. The proposed method was successfully applied to the determination of total selenium in wheat and wheat flour.     

Development of a combined SEM and ICP-MS approach for the qualitative and quantitative analyses of metal microparticles and sub-microparticles in food products

An integrated approach based on the use of inductively coupled plasma mass spectrometry (ICP-MS) and scanning electron microscopy (SEM) for the qualitative and quantitative analyses of metal particles in foods was devised and validated. Different raw materials and food products, like wheat, durum wheat, wheat flour, semolina, cookies, and pasta were considered. Attention was paid to the development of sample treatment protocols for each type of sample to avoid potential artifacts such as aggregation or agglomeration. The analytical protocols developed followed by ICP-MS and SEM investigations allowed us the quantitative determination and the morphological and dimensional characterization of metal nano- and microparticles isolated from the raw materials and finished food products considered. The ICP-MS method was validated in terms of linearity (0.8–80 μg/g and 0.09–9 μg/g for Fe and Ti, respectively), quantification limits (0.73 μg/g for Fe and 0.09 μg/g for Ti), repeatability (relative standard deviation (RSD) % equal to 10% for Fe and 20% in a wheat matrix as an example), and extraction recoveries (93 ± 2–101 ± 2%). Validation of the scanning electron microscopy–energy dispersive X-ray spectroscopy (SEM-EDS) measurements was performed working in a dimensional range from 1 to 100 μm with an estimated error in the size determination equal to 0.5 μm. ICP-MS data as well as SEM measurements showed a decrease in the concentration of metal particles from wheat to flour and from durum wheat to semolina samples, thus indicating an external contamination of grains by metal particles. These findings were confirmed by environmental SEM analysis, which allowed investigation of particles of lower dimensions. Generally, the largest number of particles was found in the case of iron and titanium, whereas particles of copper and zinc were only occasionally found without any possibility of quantifying their number.     

Multiplex liquid chromatography-tandem mass spectrometry for the detection of wheat,oat, barley and rye prolamins towards the assessment of gluten-free product safety

Celiac patients should feel confident in the safety of foods labelled or expected to be gluten-free. In this context, a targeted proteomic approach based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) technique was proposed to assess the presence of celiotoxic cereals, namely wheat, oats, barley and rye, in raw and processed food products. To this aim, unique marker peptides were properly selected in order to distinguish between the different cereal types. A revised cocktail solution based on reducing and denaturing agents was exploited for prolamin extraction from raw and processed food; in addition, defatting with hexane was carried out for sample clean-up, allowing to largely reduce problems related to matrix effect. Method validation on fortified rice flour showed good analytical performance in terms of sensitivity (limits of detection in the 2–18 mg kg⁻¹ range). However, poor trueness was calculated for self-made incurred bread (between 3 and 30% depending on the peptide), probably due to baking processes, which reduce gluten extractability. Thus, it is evident that in the case of processed foods further insights into sample treatment efficiency and reference materials for protein calibration are required to obtain accurate gluten determination. Finally, the developed method was applied for the analysis of market food products, offering the possibility to discriminate among cereals, with good agreement with labelled ingredients for gluten-containing foodstuffs.     

高效液相色谱-串联质谱法测定酵素食品中的匹可硫酸钠

建立了高效液相色谱-串联质谱法测定酵素食品中新型减肥类非法添加物匹可硫酸钠的分析方法。针对现有方法不适用的果冻、软糖型样品，开发了新的前处理方法。样品经水提取、聚酰胺净化，使用Thermo Accucore RP-MS色谱柱（100 mm×2.1 mm，2.6 μm），以乙腈-10 mmol/L乙酸铵（15：85，v/v）等度洗脱，流速0.3 mL/min，柱温35℃，多反应监测（MRM）模式下质谱检测，外标法定量。结果表明，匹可硫酸钠在5~500 μg/L范围内线性关系良好，相关系数（r）为0.9999，方法检出限为0.05 mg/kg。匹可硫酸钠在各基质样品中的加标回收率为89.2%~111.8%，相对标准偏差为2.5%~10.4%。按上述方法检测152批次酵素食品，检出阳性样品58批次，检出率为38.2%。该法准确、灵敏，可用于酵素食品中匹可硫酸钠的测定。     

High performance liquid chromatography and capillary electrophoresis in the analysis of soybean proteins and peptides in foodstuffs

The increasing interest in functional and healthy food products has promoted the use of soybean in the manufacture of foods for human consumption. Soybean basic products (soybeans, textured soybean, soybean flour, soybean protein concentrate and soybean protein isolate) as well as soybean derivatives (soybean dairy-like products, soybean drinks with fruits, meat analogues, etc.) are commercially available. In addition, due to the interesting nutritional and functional properties of soybean proteins, they are usually employed as ingredient in the elaboration of a large number of food products such as bakery or meat products among others. In spite of the good characteristics of soybean proteins, their addition to some products is forbidden or allowed up to a certain limit. Therefore, analytical methodologies to achieve the determination of soybean proteins in foods are necessary in order to make possible adequate quality control and to prove that legal regulations controlling their addition are accomplished. However, this is not an easy task due to the diversity and complexity of the food matrices and the technological treatments to which some of these foods are submitted during their elaboration. This article presents for the first time a comprehensive review on the analytical methodologies developed using HPLC and CE to characterize soybeans and to analyse soybean proteins in meals. Moreover, the use of HPLC and CE in the characterization of soybean protein fractions and their hydrolyzates, and a study of their relationships to nutritional, functional and biomedical properties are included. Finally, the application of proteomic methodologies in soybean food technology is also reviewed.     

Determination of St. John's wort components in dietary supplements and functional foods by liquid chromatography

St. John's wort (Hypericum perforatum L.) preparations, a top-selling botanical dietary supplement used primarily as an antidepressant, has recently been used as an ingredient in some food products sold as functional foods. A rapid extraction technique followed by a liquid chromatographic (LC) method was developed to determine 4 characteristic bioactive compounds (pseudohypericin, hypericin, hyperforin, and adhyperforin) from St. John's wort in dietary supplements and functional foods to which it was added. Solid samples, including dried leaf/flower mixture, dietary supplement capsules, tea bags, puff and snack bar, were extracted with methanol by sonication. Noncarbonated, fruit-flavored drinks were centrifuged and mixed with methanol. Compounds were then determined by isocratic, reversed-phase LC with UV detection at 2 wavelengths and further identified or confirmed by photodiode array spectra and LC/mass spectrometry. Within-laboratory method variations (% RSD) were satisfactory. Very low amounts, if any, of the 4 components were found in drink and puff samples, and none was found in the snack bar. The methods developed provide a useful means for the determination of St. John's wort components in dietary supplements and functional foods.     

基于超高效液相色谱-高分辨质谱的非衍生化法测定面粉和燕麦中草甘膦及氨甲基膦酸残留

建立了固相萃取-超高效液相色谱-高分辨质谱（UPLC-HRMS）快速准确测定面粉和燕麦中残留草甘膦（GLY）及其代谢物氨甲基膦酸（AMPA）的分析方法。面粉和燕麦样品经水涡旋和超声提取,用混合阳离子交换固相萃取（MCX）小柱净化与乙腈沉淀蛋白质后,以5 mmol/L乙酸铵水溶液（pH=10.5）和乙腈溶液作为流动相在Dikma Polyamino HILIC色谱柱（150 mm×2.0 mm,5 μm）上进行梯度洗脱与分离,采用电喷雾电离源、负离子模式和平行反应监测（PRM）模式下,内标法定量分析。系统优化了液相色谱与高分辨质谱等仪器条件和样品前处理条件对GLY及其代谢物AMPA测定的影响,并比对了不同分析方法的基质效应,研究了进样系统残留。实验结果表明,GLY和AMPA在5.0~100.0 μg/L范围内呈现良好的线性关系（线性相关系数R²>0.999）,检出限分别为0.005和0.05 mg/kg;低（0.1 mg/kg）、中（0.5 mg/kg）、高（2.0 mg/kg）3个添加水平下,GLY和AMPA的加标回收率分别为93.8%~115%和89.8%~110%,相对标准偏差均小于10%。基质效应实验结果表明,利用同位素内标物能有效降低方法的基质抑制效应（基质效应参数|η|<3%）;进样系统的残留率小于1.0%。本方法与文献报道的衍生化法方法进行比对,结果表明,两种检测方法与靶值的相对偏差分别为2.19%和3.07%。将该方法用于弗帕斯（FAPAS）能力验证样品的测定（编号为09122,燕麦中GLY的测定）,结果满意,测定值与真值之间的偏离程度（z值）=0.2。FAPAS质控样品（编号为T09119QC,面粉中GLY的测定）检测结果显示本方法的准确度为102.2%。该方法具有快速、简便、灵敏和准确等优点,适合面粉与燕麦样品中GLY及其代谢物AMPA的日常监测。     

Effect of Coconut and Chestnut Flour Supplementations on Texture,Nutritional and Sensory Properties of Baked Wheat Based Bread

Wheat bread, produced by the single-phase method, is a common food consumed all over the world. Due to changes in lifestyle and nutritional trends, alternative raw materials are sought to increase the nutritional value and improve the taste of daily consumed products. Additionally, customers seek a wide variety of foods, especially when it comes to basic foods. Nuts, such as coconuts or chestnuts, might provide an attractive flavour with benefits to the nutritional quality. In this study, the effect of substituting wheat flour with coconut or chestnut flour (flour contribution level: 5, 10, 15, 30, 50% w/w), was evaluated in terms of the breads specific volume, texture, colour, nutritional composition, and dietary fibre fraction contents. Moreover, a sensory evaluation was conducted to assess potential consumer acceptance. Based on the consumer’s perception, the overall acceptance of bread with 15% w/w of coconut and chestnut flour was in privilege compared to the control sample. As a result, taking all of the tested parameters into account, the breads with 5, 10, and 15% supplementation of chestnut or coconut flour were still of good quality compared to the wheat bread and their fibre content was significantly higher.     

Determination of zearalenone in botanical dietary supplements, soybeans, grains, and grain products by immunoaffinity column cleanup and liquid chromatography: single-laboratory validation

The accuracy, repeatability, and reproducibility characteristics of a method for measuring levels of zearalenone (ZON) in botanical root products, soybeans, grains, and grain products were determined by an AOAC single-laboratory validation procedure. Replicates of 10 test portions of each powdered root product (black cohosh, ginger, ginseng), brown rice flour, brown rice grain, oat flour, rice bran, soybeans, and wheat flour at each spiking level (ZON at 0, 50, 100, and 200 microg/kg) were analyzed on 3 separate days. Test samples were extracted with methanol-water (75 + 25, v/v). The extracts were centrifuged or filtered, diluted with phosphate-buffered saline (PBS) containing 0.5% Tween 20, and filtered; the filtrates were applied to an immunoaffinity column containing antibodies specific for ZON. After the column was washed with methanol-PBS (15 + 85, v/v) containing 0.5% Tween 20 and then with water, the toxin was eluted from the column with methanol, and the eluate was diluted with water. The eluate containing the toxin was then subjected to RPLC with fluorescence detection. All commodities that were found to contain ZON at < 10 microg/kg were used for the recovery study. The average within-day and between-days recoveries of ZON added at levels of 50-200 microg/kg ranged from 82 to 88% and from 81 to 84%, respectively, for all test commodities. The total average of within- and between-day SD and RSDr values for all test commodities ranged from 2.5 to 7.3 microg/kg and from 4.6 to 6.2%, respectively. HorRat values were <1.3 for all matrixes examined. The tested method was found to be acceptable for the matrixes examined.