Quantification of inositol phosphates using (31)P nuclear magnetic resonance spectroscopy in animal nutrition

A (31)P NMR method for quantitative determination of inositol phosphates in simple incubation samples of sodium phytate and Aspergillus niger phytase and in different types of complex samples, such as diets, digesta, and feces, is described. The inositol phosphates in complex samples were extracted with HCl, concentrated, and purified using freeze-drying and filtration and subsequently determined at pH 12.6 in aqueous solution using a (31)P NMR method. The (31)P NMR technique has as its main advantages over the HPLC techniques that it does not necessitate standards that may cause background matrix effects and that the spectra of inositol phosphates and orthophosphate appear in the same run without further sampling errors. The results of inositol hexaphosphate analysis with HPLC can be confirmed by this (31)P NMR method. Contents of inositol tetra-, tri-, di-, and monophosphate in the biological samples appear to be quantitatively not important. The (31)P NMR method can be applied for use in animal nutrition in general and studies of using phytase in diets for farm animals in particular, by measuring the content of inositol phosphates in feed ingredients, complete feeds, ileal contents, and feces of pigs and poultry.     

Determination of grayanotoxins in biological samples by LC-MS/MS

A rapid LC-MS/MS method was developed for the quantitative determination of grayanotoxins I, II, and III in rumen contents, feces, and urine. The grayanotoxins were extracted from solid samples with methanol. The methanol extract was diluted with water and cleaned up using a reversed phase solid phase extraction column. HPLC separation was performed by reversed phase HPLC using a gradient of water and methanol containing 1% acetic acid. Determination was by positive ion electrospray ionization and ion trap tandem mass spectrometry. Grayanotoxin I quantitation was based on fragmentation of the sodium adduct ion at m/z 435 to a product ion at m/z 375. Grayanotoxins II and III were quantitated on the basis of fragmentation of the ion at m/z 335 to the product ion at m/z 299. The method detection limits were 0.2 microg/g in rumen contents and feces and 0.05 microg/g in urine. Fortifications at the detection limits and 10 times the detection limits of bovine rumen contents, caprine feces, and ovine urine were recovered in the range 80-114%. The diagnostic utility of the method was tested by analyzing samples submitted to the veterinary toxicology laboratory.     

Determination of theanine in commercial tea by liquid chromatography with fluorescence and diode array ultraviolet detection

Two liquid chromatographic methods that involve precolumn derivatization with o-phthaladehyde (OPA) and phenylisothiocyanate (PITC) with fluorescence and diode array UV detection for the determination of theanine have been developed. The chromatographic separations were achieved by reverse-phase high-performance liquid chromatography using octadecyl columns and gradient elution. The methods were applied to evaluate the theanine content of commercial tea leaves. The coefficient of variation of the peak area repeatability for within day (n = 8) and between day (n = 8 over 10 days) was lower than 3% for both of the methods. The estimated limit of detection (LOD) and limit of quantitation (LOQ) for the OPA method was 0.12 and 0.35 microg theanine, respectively. The PITC method was 500-fold more sensitive with LOD and LOQ values of 0.25 and 0.75 ng, respectively. The theanine content of the commercial tea samples varied from 2-5 mg/g leaf. The overall % recoveries for these methods ranged from 93-99.3. The sensitivity and simplicity of the method render them suitable for use in quality control laboratories.     

Phytate Quantification using HPIC in the Presence of Iron and Aluminum

Phytate is an organic form of P that is difficult to analyze in complex matrices. To test if high concentrations of aluminum (Al) and iron (Fe) hinder accurate quantification of phytate in dairy manure and broiler litter when measured by high-performance ion chromatography (HPIC), researchers spiked dairy manure and broiler litter samples with Al, Fe, and phytate. Samples were alkaline extracted, acidified, and filtered, and then phytate spike recovery was analyzed with HPIC. High concentrations of Fe did not hinder phytate recovery in manure or litter samples. While phytate recovery was complete at typical manure and litter Al concentrations, high concentrations of Al inhibited phytate recovery in litter samples and in some manure samples. Overall, alkaline extraction of dairy manure and broiler litter and analysis with HPIC proved to be relatively accurate, fast, and cheap within normal Al and Fe ranges, compared to the commonly used nuclear magnetic resonance (NMR) method.     

Development of polyclonal antibodies for the detection of Tribolium castaneum contamination in wheat grain

Two polyclonal antibodies (Pab) were developed for the detection of Tribolium castaneum, which is a stored product pest of medical and economical importance. Selected Pab anti- T. castaneumK51 showed low cross-reactivity to other stored product arthropods but revealed high reactivity to T. destructor, T. confusum, and partly to Tenebrio molitor. PTA-ELISA was used to detect adults, larva, and feces of T. castaneum in artificially contaminated grain samples. Calibration methods were applied to determine detection limits for each type of contaminants. Anti- T.castaneumK51 enabled detection of T. castaneum in grain samples; detection limits reached 60 and 640 individuals/kg of grain for larvae and adults, respectively, and 4 mg of feces/kg of grain. After recalculation, the detection limit for feces enables detection of 30 larvae after 5 days of feeding in optimal conditions. The main advantage of the developed assay is traceability of T. castaneum contamination, especially when the adults and larvae are removed from contaminated material, based on the detection of feces that persist in the grain.     

Simultaneous determination of ethidimuron, methabenzthiazuron, and their two major degradation products in soil

An analytical method has been developed for the quantification of two herbicides (ethidimuron and methabenzthiazuron) and their two main soil derivatives. This method involves fluidized-bed extraction (FBE) prior to cleanup and analysis by reverse-phase liquid chromatography with UV detection at 282 nm. FBE conditions were established to provide efficient extraction without degradation of the four analytes. (14)C-labeled compounds were used for the optimization of extraction and purification steps and for the determination of related efficiencies. Extraction was optimal using a fexIKA extractor operating at 110 degrees C for three cycles (total time = 95 min) with 75 g of soil and 150 mL of a 60:40 v/v acetone/water mixture. Extracts were further purified on a 500 mg silica SPE cartridge. Separation was performed on a C18 Purosphere column (250 mm x 4 mm i.d.), at 0.8 mL min(-1) and 30 degrees C with an elution gradient made up of phosphoric acid aqueous solution (pH 2.2) and acetonitrile. Calibration curves were found to be linear in the 0.5-50 mg L(-1) concentration range. Besides freshly spiked soil samples, method validation included the analysis of samples with aged residues. Recovery values, determined from spiked samples, were close to 100%. Limits of detection ranged between 2 and 3 microg kg(-1) of dry soil and limits of quantification between 8 and 10 microg kg(-1) of dry soil. An attempt to improve these performances by using fluorescence detection following postcolumn derivatization by orthophthalaldehyde-mercaptoethanol reagent was unsuccessful.     

基于可见-近红外光谱的鸡只粪便分类判别

鸡只粪便的性状是反映鸡只健康状况的重要特征之一,不同性状的鸡便往往与特定的疾病相关联。针对鸡只粪便性状主要依靠人工监测,存在速度慢且易发生交叉感染等问题,该研究提出一种基于可见-近红外光谱技术的鸡只粪便分类判别模型。首先,通过扫描4类典型的鸡只粪便样本（正常粪便、红血丝粪便、绿色粪便和饲料粪便）在400～900 nm波段范围的光谱数据,对每一类别的鸡便样本按随机性原则以3∶1划分为校正集和测试集。其次,分别采用多元散射校正、SG卷积平滑和标准差标准化进行数据预处理,并建立偏最小二乘判别分析模型,根据模型评价指标确定最优预处理方法。然后,使用主成分分析、竞争性自适应重加权采样、改进的混合蛙跳3种方法对预处理后的样本进行数据降维,并最终建立分类判别模型。结果表明：基于模型评价指标确定最优数据预处理方法后,再采用改进后的混合蛙跳降维方法建立的判别模型区分正常粪便、红血丝粪便、绿色粪便表现最优,测试集判别准确率分别为92.27%、92.59%、100%;而对于饲料粪便,所选3种降维方法建立的判别模型,其测试集准确率均可达100%。因此,通过可见-近红外光谱检测手段,结合特征波长优选与偏最小二乘判别分析,可以有效判别不同类型的鸡只粪便,为实现鸡病智能化监测提供技术支持。     

Amino acid analysis for meat protein evaluation

The Food Safety and Inspection Service procedure for determination of essential amino acid content of mechanically processed products from red meat animals and poultry is based on hydrolysis of a powder prepared by blending samples in acetone-chloroform. The hydrolysis procedure incorporates thioglycolic acid to prevent loss of tryptophan. Aliquots of prepared hydrolysates are injected into a liquid chromatographic system, using gradient elution on an ion-exchange column for separation. The system also uses post-column hypochlorite oxidation coupled with orthophthalaldehyde reagent and fluorescence detection. Modification of the elution program allows concurrent determination of tryptophan with minimal added cost. Chromatograms from beef, pork, and poultry products show adequate separation and quantitation of beta-alanine, 1-methyl-histidine, and 3-methyl-histidine, indicating that the procedure could be used to estimate muscle content of products. A colorimetric procedure for assay of hydroxyproline was introduced and validated as an adjunct method for protein quality estimation.     

Monolithic supports for the characterization of commercial maize products based on their chromatographic profile. Application of experimental design and classification techniques

Modern analytical techniques based on the use of RP-HPLC with monolithic stationary phases and the application of experimental design and classification tools have been applied to the analysis of maize proteins. Solubilization conditions of maize proteins and separation conditions (temperature, detection wavelength, type and concentration of ion-pairing agent, and gradient) were optimized. The elution gradient was optimized by the application of experimental design techniques. The optimized method consisted of a linear binary gradient of water/acetonitrile/0.1% trifluoroacetic acid in three steps at a flow rate of 3 mL/min with a column temperature of 35 degrees C and UV detection at 280 nm. The developed method enabled the separation of maize proteins in an analysis time close to 8 min. Moreover, this is the first time that commercial maize products have been characterized by the use of multivariate classification techniques.     

Eine Methode zur quantitativen Bestimmung von Pentachlorphenol in Umweltproben

A method for the quantitative determination of pentachlorophenol in environmental samples An analytical method is presented for the rapid extraction, purification and quantitative detection by gaschromatography of pentachlorophenol in environmental samples (plant, animal and soil samples). The detection sensivity of this method gets up to 5 ppb or 10 pg absolute.     

Profiling LC-DAD-ESI-TOF MS method for the determination of phenolic metabolites from avocado (Persea americana)

A powerful HPLC-DAD-ESI-TOF MS method was established for the efficient identification of the chemical constituents in the methanolic extracts of avocado (Persea americana). Separation and detection conditions were optimized by using a standard mix containing 39 compounds belonging to phenolic acids and different categories of flavonoids, analytes that could be potentially present in the avocado extracts. Optimum LC separation was achieved on a Zorbax Eclipse Plus C18 analytical column (4.6×150 mm, 1.8 μm particle size) by gradient elution with water+acetic acid (0.5%) and acetonitrile as mobile phases, at a flow rate of 1.6 mL/min. The detection was carried out by ultraviolet-visible absorption and ESI-TOF MS. The developed method was applied to the study of 3 different varieties of avocado, and 17 compounds were unequivocally identified with standards. Moreover, around 25 analytes were tentatively identified by taking into account the accuracy and isotopic information provided by TOF MS.     

Analysis of organic acids and inorganic anions in different types of beer using capillary zone electrophoresis.

Two methods for the investigation of different types of beer by capillary zone electrophoresis are presented. The first separation system described in this work allows the quantitative analysis of beers with respect to their contents of low molecular mass anionic components using indirect ultraviolet detection as well as conductivity detection, providing relative standard deviation between 0.5 and 6.6% for the peak areas and excellent limits of detection (LOD) ranging from 0.02 mg L(-)(1) for chloride to 0.41 mg L(-)(1) for phosphate. The second method offers the possibility of fast determination of amino acids in beer samples without the necessity of any sample pretreatment. LODs obtained for the investigated solutes were found to be strongly dependent on their spectroscopical properties and in the range of 0.5-50 mg L(-)(1). Despite this restriction, this analytical method can be regarded as a suitable tool for the screening of beers with respect to their amino acid patterns.     

离心式微流控液相萃取法测定水和土壤中油和油脂

现场便携式快速分析仪测定液体固体中的各种污染物,越来越成为人们的实际需求。如水和土壤中的油和油脂（OG）,会引起环境污染,威胁公共健康。微流控设备,由于其试剂消耗低、成本低、分析时间短、所需能量和空间小、经济、环境安全,使得分析方法发生着潜在的革命性变化,几个环境样品的同时制备和分析都在一个集成光盘上进行的离心式微流控系统分析能力越来越为人们熟知。在磁力驱动下,通过＂可动磁铁＂的有效搅拌完成液相和液固萃取,磁力驱动样品制备单元集合固体分析的沉积/过滤,以及最终液体或固体样品的检测池于一个光盘上,对水和土壤中的OG进行萃取,以四氯乙烯代替氟里昂113和四氯化碳为萃取溶剂,实现了OG的红外法检测,检出限为11μg.mL-1。     

Liquid chromatographic determination of carbadox and desoxycarbadox in medicated feeds and in porcine gastrointestinal tract

A liquid chromatographic method for the assay of carbadox and desoxycarbadox in medicated feeds and porcine stomach and intestinal contents is described. Samples were extracted with dimethylformamide-water and cleaned up on an alumina column. The eluate was chromatographed using either gradient elution for simultaneous assay of both compounds or isocratic elution for carbadox only. Detection of carbadox by its native fluorescence yielded a sensitive and specific assay without interferences by metabolites or matrix components. The optimal UV absorption of desoxycarbadox was at 280 nm. Mean recoveries of carbadox in spiked feed and stomach contents were 104 and 97%, respectively; mean recovery of desoxycarbadox in stomach contents was 106%. The day-to-day reproducibility for carbadox in different feed samples and stomach contents samples had a coefficient of variation of 6-13%.     

Quantitative analysis of phytate globoids isolated from wheat bran and characterization of their sequential dephosphorylation by wheat phytase

Wheat phytase was purified to investigate the action of the enzyme toward its pure substrate (phytic acid - myo-inositol hexakisphosphate) and its naturally occurring substrate (phytate globoids). Phytate globoids were purified to homogeneity from wheat bran, and their nutritionally relevant parameters were quantified by ICP-MS. The main components of the globoids were phytic acid (40% w/w), protein (46% w/w), and several minerals, in particular, K > Mg > Ca > Fe (in concentration order). Investigation of enzyme kinetics revealed that K(m) and V(max) decreased by 29 and 37%, respectively, when pure phytic acid was replaced with phytate globoids as substrate. Time course degradation of phytic acid or phytate globoids using purified wheat phytase was followed by HPIC identification of inositol phosphates appearing and disappearing as products. In both cases, enzymatic degradation initiated at both the 3- and 6-positions of phytic acid and end products were inositol and phosphate.     

A new approach to the study of glucosinolates by isocratic liquid chromatography. Part I. Rapid determination of desulfated derivatives of rapeseed glucosinolates.

Liquid chromatographic (LC) analysis of desulfated derivatives of rapeseed glucosinolates has been carried out under isocratic elution conditions with different CN-bonded stationary phases. The effects of the eluant composition (water, acetonitrile, and methanol) with the stationary phase (Zorbax CN, Lichrospher CN, and Ultrasphere CN) and temperature (20 and 50 degrees C) are described. An isocratic LC method performed at room temperature using a Lichrospher CN column and water as mobile phase is proposed. The chromatographic analysis can be done in less than 12 min, and it is easier and less expensive than the traditional gradient mode. Four commercial samples of rapeseed containing various quantities of other cruciferous seeds (wild mustard and stinkweed) as an admixture have been analyzed to determine the total glucosinolate content. Relative standard deviations of repeatability of the isocratic and gradient LC methods ranged from 0.4 to 1.7% and from 2.7 to 4.7%, respectively. Comparison of the results showed good agreement between the 2 methods (beter than 98%).     

Complete amino acid analysis in hydrolysates of foods and feces by liquid chromatography of precolumn phenylisothiocyanate derivatives

The amino acid analysis method using precolumn phenylisothiocyanate (PITC) derivatization and liquid chromatography was modified for accurate determination of methionine (as methionine sulfone), cysteine/cystine (as cysteic acid), and all other amino acids, except tryptophan, in hydrolyzed samples of foods and feces. A simple liquid chromatographic method (requiring no derivatization) for the determination of tryptophan in alkaline hydrolysates of foods and feces was also developed. Separation of all amino acids by liquid chromatography was completed in 12 min compared with 60-90 min by ion-exchange chromatography. Variation expressed as coefficients of variation (CV) for the determination of most amino acids in the food and feces samples was not more than 4%, which compared favorably with the reproducibility of ion-exchange methods. Data for amino acids and recoveries of amino acid nitrogen obtained by liquid chromatographic methods were also similar to those obtained by conventional ion-exchange procedures.     

Liquid chromatographic determination of aflatoxins, ochratoxin A, and zearalenone in grains, oilseeds, and animal feeds by post-column derivatization and on-line sample cleanup

A liquid chromatographic method using on-line sample cleanup, reverse flow analytical column loading, gradient elution, and postcolumn derivatization with iodine permits direct, rapid determination of aflatoxins B1, B2, G1, and G2, as well as ochratoxin A and zearalenone. Limits of quantitation are 5 ppb for the aflatoxins and ochratoxin A and 30 ppb for zearalenone. This procedure performs well as a multimycotoxin screen for cereal grains and oilseeds, with more limited success in complete animal feeds.     

Organic acids from leaves,fruits, and rinds of Garcinia cowa

Organic acids in fresh leaves, fruits, and dried rinds of Garcinia cowa (G. cowa) were determined by high-performance liquid chromatography. Fresh leaves, fruits, and dried rinds were extracted with water at 120 degrees C for 20-30 min under 15 lbs/in(2) pressure. Also, dried rinds were extracted with solvents (acetone and methanol) using a Soxhlet extractor at 60 degrees C for 8 h each. The samples were injected to HPLC under gradient elution with 0.01 M phosphoric acid and methanol with a flow rate of 0.7 mL/min using UV detection at 210 nm. The major organic acid was found to be (-)-hydroxycitric acid present in leaves, fruits, and rinds to the extent of 1.7, 2.3, and 12.7%, respectively. (-)-Hydroxycitric acid lactone, and oxalic and citric acids are present in leaves, fruits, and rinds in minor quantities. This is the first report on the composition of organic acids from G. cowa.