A rapid microwave-assisted derivatization process for the determination of phenolic acids in brewer’s spent grains

A simple and fast method for the TMS derivatization of phenolic acids in a closed vial using microwave irradiation followed by GC/MS analysis is proposed. A full factorial design was used to investigate the effects of two independent variables, namely, time and power of microwave irradiation, on the yield of silylation reaction. The optimal conditions were tested against the classical heating derivatization procedure. No significant differences were found between the classical heating and microwave-assisted derivatization. Chromatographic separation of the nine phenolic acids examined was achieved in 16 min. The mass spectral fragmentation patterns of the derivatives obtained by the proposed method were identical to those from the classical heating. Four different batches of brewer’s spent grain were extracted and analyzed for the total phenolic acid content. Significant differences between the batches of spent grains were found for all analytes. The total phenolic acid content varied between 2688 and 4884 μg/g.     

高效液相色谱-荧光检测法测定红肉及其加工肉中唾液酸含量

采用高效液相色谱-荧光检测法对红肉及其加工肉中两种唾液酸N-乙酰神经氨酸（N-acetylneuraminic acid,Neu5Ac）和N-羟乙酰神经氨酸（N-glycolylneuraminic acid,Neu5Gc）进行定性和定量分析。利用单因素试验对衍生化与样品酸解条件进行优化,并借助超声缩短酸解时间。结果表明,以盐酸作为酸解试剂,超声辅助酸解30 min,4,5-亚甲二氧基-1,2-邻苯二胺盐作为衍生化试剂,衍生化试剂浓度为13 mmol/L,样品与衍生化试剂比值为1∶1,衍生化时间120 min时,检测效果最佳。Neu5Ac和Neu5Gc在0.1～10 μg/mL范围内线性良好,回收率为91.2%～119.7%,检出限分别为0.003 mg/kg和0.01 mg/kg,重复性的相对标准偏差（relative standard deviation,RSD）为0.7%～1.8%,精密度RSD分别为1.4%和1.2%。本方法具有灵敏度高、分析时间短、重复性及准确性好等特点。     

高效液相色谱法检测食品中17种游离氨基酸

目的使用异硫氰酸苯酯衍生-高效液相色谱法对食品中17种游离氨基酸进行检测。方法对17种游离氨基酸及其苯胺硫甲酰基衍生物稳定性的分析,研究游离氨基酸的最佳衍生条件。同时,对盐沉淀法和C_(18)固相萃取(solid phase extraction, SPE)小柱法2种样品除杂方法的加标回收率进行测定。结果胱氨酸加热干燥5h后,回收率为68.46%±4.01%,热稳定性差;其它16种氨基酸加热1～5h后回收率均在90%～110%之间,热稳定性较好。胱氨酸的苯胺硫甲酰基衍生物,6h后回收率仅为11.04%±1.65%,稳定性差;其它氨基酸的苯胺硫甲酰基衍生物24 h后回收率均大于90%,比较稳定。另外,前处理中使用C_(18) SPE除杂时,样品中游离氨基酸的加标回收率均大于70%,而使用水杨酸和三氯乙酸除蛋白后苯胺硫甲酰基衍生物降解严重。结论除胱氨酸外,其余16种氨基酸的热稳定性均较好,且生成的苯胺硫甲酰基衍生物比较稳定,可以通过异硫氰酸苯酯衍生进行定量分析。相比于水杨酸和三氯乙酸沉淀法, C_(18) SPE柱是游离氨基酸除杂方法的首选,而无机盐影响异硫氰酸苯酯与氨基酸衍生,因此此方法不适用于含盐样品的衍生。     

邻苯二甲醛柱前衍生化时间对反相高效液相色谱法测定稻米氨基酸含量的影响

目的确定反相高效液相色谱法(reversed-phase high performance liquid chromatography, RP-HPLC)测定稻米氨基酸含量最适柱前衍生化时间。方法以氨基酸标准品以及稻米氨基酸提取物为对象,对柱前衍生化时间与氨基酸检测结果之间的关系进行研究。结果邻苯二甲醛(O-phthalaldehyde, OPA)和3-巯基丙酸与氨基酸的衍生化反应进行到15 min时,氨基酸的峰面积达到最大值,不同OPA-氨基酸衍生化产物的稳定性存在很大的差异。在15～300min的衍生化反应中,酪氨酸和苯丙氨酸衍生物的峰面积保持在非常稳定的水平上;而侧链含有羟基的丝氨酸和苏氨酸对衍生化时间特别敏感,衍生化时间超过15min后,其衍生物的峰面积开始快速下降;当衍生化时间在15～60min之内时,15种氨基酸中,只有酪氨酸、苯丙氨酸、精氨酸、谷氨酸、天冬氨酸、赖氨酸、亮氨酸、异亮氨酸和丙氨酸9种氨基酸的衍生物峰的面积下降幅度小于5%。结论柱前衍生化时间对稻米氨基酸衍生物峰面积的检测值较为敏感,衍生化时间控制在15 min之内能有效减小实验误差。     

Analysis of Amino Acids in Soy Isolates and Navy Beans Using Precolumn Derivatization with Phenylisothiocyanate and Reversed-Phase High Performance Liquid Chromatography

Amino acid analysis of three legume samples using precolumn phenylisothiocyanate (PITC) derivatization and reversed-phase high performance liquid chromatography (RP-HPLC) was compared with literature data or that obtained by the conventional ion-exchange chromatographic (IEC) method. The results indicated that the amino acid profiles obtained by PITC derivatization and RP-HPLC compared fairly well with literature data for soy isolates, and fairly with that obtained by the IEC method for navy beans. Average coefficient of variability of all amino acids determined by the PITC method was 6%. It was not essential to remove lipids (2.1%) in navy beans prior to acid hydrolysis and PITC derivatization for the analysis of amino acids.     

柱前衍生高效液相色谱法测定水产品中羟脯氨酸含量

建立水产品中羟脯氨酸含量的柱前衍生高效液相色谱测定方法。样品经酸水解,丹酰氯衍生,C1 8柱分离,紫外检测器检测。结果表明,羟脯氨酸与其他氨基酸较好分离,在0.5～20μg/mL范围内线性关系良好(r>0.9999),检出限0.5μg/mL,平均回收率80.2%～103%,RSD范围3.2%～5.6%。该方法灵敏、准确,适用于水产品中羟脯氨酸含量的测定。     

眼氨肽注射液中氨基酸的含量测定

目的测定眼氨肽注射液中氨基酸的含量。方法用AccQ.Tag方法对该注射液中氨基酸进行衍生化处理,反相HPLC分离衍生产物,用紫外监测器以外标法定量检验衍生化物。结果用该法测得的6种氨基酸含量符合规定。结论所用方法结果准确可靠,可用于眼氨肽注射液中氨基酸的含量测定。     

盐酸水解方法对鸡肉中氨基酸含量的影响

摘 要: 目的 研究盐酸水解过程中, 水解时间和添加巯基乙醇和苯酚保护剂对鸡肉中18种氨基酸含量的影响, 确定获得鸡肉氨基酸最高含量的盐酸水解方法。方法 以30个鸡胸肌(3种鸡、每种10个)为实验对象, 设计6个水解时间, 研究盐酸水解时间对鸡肉样品中18种氨基酸含量的影响, 确定获得氨基酸最高含量的盐酸水解时间。在上述研究的基础上, 随机选择6个鸡胸肌(3种鸡、每种2个)为实验对象, 设置3个水解时间, 研究巯基乙醇和苯酚对鸡肉样品中18种氨基酸含量的影响, 最终确定获得鸡肉中18种氨基酸最高含量的盐酸水解方法。结果 缬氨酸(valine, Val)和异亮氨酸(isoleucine, Ile)的最佳水解时间为24或26 h, 两水解时间无显著性差异(P>0.05); 其余16种氨基酸的最高含量均在22 h; Ile的含量在盐酸水解法水解24 h显著高于20 和22 h(P<0.05), 但在苯酚+盐酸水解法水解22h含量最高; 天冬氨酸(aspartate, Asp) 的含量在巯基乙醇+盐酸水解法水解22 h显著高于20 和24 h(P<0.05), 但在盐酸水解法水解22h含量最高; 甘氨酸(glycine, Gly)、丙氨酸 (alanine Ala)、酪氨酸(tyrosine, Tyr) 和苯丙氨酸 (phenylalanine, Phe)的含量在苯酚+盐酸水解法水解22 h显著高于20 h(P<0.05), 但水解22与24 h之间无显著性差异(P>0.05), 其中, Gly在苯酚+盐酸水解法水解22 h含量最高, Ala在盐酸水解法水解22 h含量最高, Tyr和Phe在巯基乙醇+盐酸水解法水解22 h含量最高; Val在盐酸水解法水解24 h含量最高, 脯氨酸(proline, Pro)和赖氨酸 (lysine, Lys)在苯酚+盐酸水解法水解22 h含量最高, 亮氨酸 (leucine, Leu) 在巯基乙醇+盐酸水解法水解22 h含量最高; 其余氨基酸在不同水解方法及水解时间中均无显著性差异(P>0.05), 但都在盐酸水解法水解22 h时含量最高。结论 巯基乙醇和苯酚对鸡肉中的含硫氨基酸无保护作用, 获得鸡肉中18种氨基酸最高含量的盐酸水解方法是盐酸水解法水解22 h。     

Reactions of chlorogenic acid and quercetin with a soy protein isolate--influence on the in vivo food protein quality in rats

Plant phenolic compounds are known to interact with proteins producing changes in the food (e. g., biological value (BV), color, taste). Therefore, the in vivo relevance, especially, of covalent phenol-protein reactions on protein quality was studied in a rat bioassay. The rats were fed protein derivatives at a 10% protein level. Soy proteins were derivatized with chlorogenic acid and quercetin (derivatization levels: 0.056 and 0.28 mmol phenolic compound/gram protein). Analysis of nitrogen in diets, urine, and fecal samples as well as the distribution of amino acids were determined. Depending on the degree of derivatization, the rats fed with soy-protein derivatives showed an increased excretion of fecal and urinary nitrogen. As a result, true nitrogen digestibility, BV, and net protein utilization were adversely affected. Protein digestibility corrected amino acid score was decreased for lysine, tryptophan, and sulfur containing amino acids.     

New starch preparations resistant to enzymatic digestion

BACKGROUND: New starch preparations were produced by thermolysis of potato starch in the presence of inorganic (hydrochloric) and organic (citric and tartaric) acids under controlled conditions. The starch preparations were physicochemically and structurally characterised and analysed for their resistance to enzymatic digestion in vitro. RESULTS: The content of resistant fraction in dextrin D1, obtained by heating starch acidified with hydrochloric and citric acids, determined by the AOAC 2001.03 and pancreatin‐gravimetric methods was similar (～200 g kg^?1). In the case of dextrin D3, obtained by heating starch acidified with hydrochloric and tartaric acids, the result of determination by the pancreatin‐gravimetric method was almost four times higher than that obtained with the AOAC 2001.03 method. The enzymatic tests revealed that dextrin D3 obtained with excess tartaric acid can be classified as RS4, which can only be partially determined by enzymatic‐gravimetric methods. Tartaric acid at high concentration had a significantly stronger influence on starch hydrolysis than citric acid. This was confirmed by chromatographic analysis of dextrins and chemical investigation of the reducing power. CONCLUSION: The results confirmed the possibility of applying dextrins, prepared under specific conditions, as soluble dietary fibre. Copyright © 2011 Society of Chemical Industry     

邻氨基苯酚衍生气相色谱-质谱分析不饱和脂肪酸

以邻氨基苯酚为脂肪酸的衍生化试剂,柱前衍生气相色谱-EI质谱分析不饱和脂肪酸,邻氨基苯酚将不饱和脂肪酸羧基化学修饰成含氮杂环,从而避免了链烯基中碳碳双键在EI源中的移动,并使质谱呈现显示双键位置的规范信息。通过解析不饱和脂肪酸邻氨基苯酚衍生产物的EI质谱图,得到了不饱和脂肪酸中碳碳双键的定位规则,从而为分析不饱和脂肪酸精细结构提供了手段。用脂肪酸邻氨基苯酚衍生气相色谱-EI质谱法分析了葵子油脂肪酸,鉴定出2种不饱和脂肪酸,确定了脂肪酸中碳碳双键的位置,结果与文献一致。     

A High-Performance Liquid Chromatographic Method for Simultaneous Determination of 21 Free Amino Acids in Tea

Free amino acids are closely related to the savory taste and beneficial effects of tea, and high-performance liquid chromatography (HPLC) is the most widespread analytical approach for simultaneous determination of free amino acids in tea. However, the reported HPLC methods have drawbacks such as long run times, low resolution, or poor efficiency. In this study, a special amino acid analysis column was used to separate and verify 21 amino acids including l-theanine, the predominant amino acid in tea. The mobile phases, including the sodium acetate and acetic acid concentration in buffer B, and the pH and concentration of sodium acetate in buffer A were optimized. The elution gradients were optimized using DryLab 2000 Plus software. In this way, an online o-phthaldialdehyde precolumn derivatization HPLC-fluorescence detection method was developed for simultaneous determination of 21 amino acids. Comparison to other HPLC methods for simultaneous determination of free amino acids in tea showed that our method is easy (automated derivatization), quick (30 min), inexpensive, and green (using a minimum of solution). It has good resolution (≥1.8) and high selectivity (interpark time?≥?0.5 min). Free amino acids in six tea samples were analyzed. This work provides an HPLC method to simultaneously measure 21 amino acids in tea and potential in other food products.     

3 种有机酸在不同pH值条件下酸沉处理对米糠蛋白结构的影响

以传统碱溶酸沉制备米糠蛋白所用的盐酸为对照,采用酒石酸、苹果酸、柠檬酸3?种有机酸分别在pH?4.0、3.5、3.0、2.0条件下酸沉制备米糠蛋白,研究有机酸酸沉对米糠蛋白结构的影响。结果表明,同种有机酸在不同pH值条件下酸沉,当酸沉pH值下降时,米糠蛋白总巯基含量和α-螺旋相对含量无显著变化,游离巯基含量和无规卷曲相对含量上升,β-折叠和β-转角相对含量下降,内源荧光强度和表面疏水性先增加后下降。相比盐酸酸沉,有机酸在相同pH值条件下酸沉制备米糠蛋白的总巯基、二级结构组成和表面疏水性无显著差异,但游离巯基含量下降、内源荧光强度升高。分子质量分布、粒径分布及电泳结果表明,较低pH值条件下的酸沉可导致米糠蛋白结构发生部分解离和聚集,但没有形成新的亚基和高分子质量聚集体。     

Sequential hydrolysis of swine carcass samples and determination of amino acid concentrations using pre‐column derivatization with phenyl isothiocyanate

Comparing amino acid (AA) retention levels in pig carcass to true ileal digestible AA intake provides an estimate of the marginal efficiency of AA utilization. Accurate analysis of AA levels in the carcass samples is critical. However, the standard 24 h of hydrolysis does not always provide maximum AA values. A study was carried out to investigate the effect of hydrolysis time on AA measurements in pig carcass. Correction factors to standardize AA levels to 24 h of hydrolysis were also determined. Ground carcass samples were hydrolysed with 6 mol litre⁻¹ hydrochloric acid (HCl) in a 110 °C oven for nine different time periods. Pre‐column derivatization with phenyl isothiocyanate (PITC) was used to determine AA concentrations in all of the samples. Hydrolysis time significantly affected (P < 0.001) AA levels. The highest levels (P > 0.05) of valine, isoleucine, serine, glycine, threonine, alanine, arginine, proline, histidine and phenylalanine were not observed with 24 h hydrolysis. Therefore, correction factors and sequential hydrolysis curves are important for these amino acids. In conclusion, the effect of hydrolysis time should be considered in amino acids analysis. © 2000 Society of Chemical Industry     

柱前衍生反相高效液相色谱法测定新疆特色食品馕中氨基酸含量

目的建立柱前衍生反相高效液相色谱法(reversed-phase high performance liquid chromatography,RP-HCLP)测定新疆特色食品馕中17种氨基酸含量的检测方法。方法通过优化酸水解参数,并以6-氨基喹啉-N-羟基琥珀酰亚胺基甲酸酯为衍生试剂,用乙腈为流动相A,水为流动相B,磷酸盐缓冲液溶液为流动相C进行梯度洗脱,柱温37℃条件下用AccQ-Tag色谱柱(3.9 mm×150 mm, 4μm)进行分离,采用反相-高效液色谱法荧光检测器(激发波长250 nm、发射波长395 nm)进行测定。结果当取样量为100 mg、水解液加入体积为10 mL、水解温度为115℃、水解时间为24 h时,水解效果最好。17种氨基酸线性回归方程的相关系数在0.9985以上(n=3),检出限为0.011~0.058mg/L,回收率为81.6%~116.2%,相对标准偏差为0.41%~6.94%(n=3)。结论该方法操作简单、灵敏度高、线性关系和回收率好,适用于新疆特色食品馕中氨基酸含量的测定。     

微孔草籽油脂肪酸的HPLC-MS分析

采用柱前衍生-高效液相色谱荧光检测法(HPLC-FLD)分析了超临界CO2萃取得到的微孔草籽油的脂肪酸成分。用荧光衍生试剂1-[2-(对甲苯磺酸酯)乙基]-2-苯基咪唑[4,5-f]9,10-菲(TSPP)作为柱前衍生试剂,对39种饱和与不饱和脂肪酸标准品进行衍生,经梯度洗脱实现了39种脂肪酸衍生物完全分离,使用外标法定量,建立了同时测定17种脂肪酸绝对含量的方法。结果表明,微孔草籽油中油酸、亚油酸、亚麻酸相对含量分别是29.09%、23.85%、20.25%;不饱和脂肪酸占总脂肪酸的相对含量为73.19%。     

The Control of Selected Micro‐Organisms During the Malting Process

The micro‐organisms associated with malt have an important influence on quality parameters. Although it was not possible to eliminate the growth of micro‐organisms entirely during the malting process, it was possible to control particular groups by applying some simple procedures. A three steep schedule was associated with increased fungal growth which was not observed when a two steep schedule was used. Fungal proliferation could also be reduced by the use of hop beta‐acids. The use of sodium hypochlorite, potassium hydroxide, hydrochloric acid and hop beta‐acids reduced or eliminated E. coli. The most effective means of controlling total micro‐flora was an alternate acid/alkaline wash. There was no evidence that a correlation existed between growth site and microbial loading for the years studied. Furthermore, whilst many of the barley associated micro‐organisms were able to secrete brewing related enzymes, the activities of these were too low to have any significant effect during mashing.     

OPA-FMOC在线柱前衍生高效液相色谱法测定不同产地燕窝中氨基酸含量

通过邻苯二甲醛-9-芴甲基氯甲酸酯(O-phthalaldehyde-9-fluorenylmethyl chloroformate,OPA-FMOC)柱前衍生高效液相色谱法,优化得到快速检测燕窝氨基酸的方法,并对10种不同来源燕窝中的氨基酸进行了定性和定量分析。结果表明,该法在14 min内可完成对22种氨基酸的分析,且在5~200 nmol/mL浓度范围内决定系数R2均大于0.998,检出限0.4~4 nmol/mL,回收率80%以上。该方法简便快速,灵敏度高,对高蛋白质食品中氨基酸的快速测定具有借鉴作用。     

Simultaneous determination of mono- and dicarboxylic acids, omega-Oxo-carboxylic acids, midchain ketocarboxylic acids, and aldehydes in atmospheric aerosol samples

This paper describes a method for the simultaneous determination of monocarboxylic acids (C6-C34), dicarboxylic acids (C2-C24), omega-oxo-carboxylic acids (C2-C9), ketocarboxylic acids (pyruvic and pinonic acid), and select aldehydes (glyoxal, methylglyoxal, and nonanal) in atmospheric particles. Quantification of these compounds gives information on important chemical characteristics of aerosols for source apportioning of aerosol organics and for studying atmospheric processes leading to secondary organic aerosol formation. These target analytes were determined as their butyl ester or butyl acetal derivatives using gas-chromatography mass spectrometry. The method is modified from a method described by Kawamura. Kawamura's original method involved a water extraction step, which practically restricted the method to the determination of only those compounds that are water-soluble. Our method eliminates the water extraction step and combines extraction and derivatization of the target compounds in one step. A mixture of hexane/butanol/BF3 simultaneously derivatizes the polar function groups (i.e., -COOH, -C=O) and extracts the target analytes from the aerosol filter substrates. A prominent advantage of our method is improved recoveries for the more volatile analytes in the target compound classes as a result of eliminating the water evaporation step. Recoveries better than 66% were obtained for the target analytes, including the relatively volatile ones. This improvement for the light species has allowed detection of a new midchain ketocarboxylic acid, 4-oxopentanoic acid, which would have escaped detection by the Kawamura method because of its high susceptibility to evaporative loss. Examples are presented to demonstrate the use of this method in analysis of ambient aerosol samples.     

Mobile Phases in Reverse-Phase HPLC for the Determination of Bitter Peptides in Cheese

A cheese extract and a standard solution with a mixture of peptides and amino acids were used to evaluate the separations of reverse-phase HPLC with alternative mobile phases to be used in conjunction with sensory evaluation. Acceptable resolution of components was observed when acetonitrile was replaced with absolute ethanol and trifluoroacetic acid was replaced with food-grade hydrochloric acid. The average hydrophobicity and molecular weight of the peptides and amino acids were used to construct a linear regression equation with corrected retention times. Suspected bitter compounds were not confined to any one section of the chromatogram based on the established regression model.