2,4-二硝基氟苯柱前衍生HPLC检测树莓中游离氨基酸

目的：建立一种简单、快速、可同时检测22 种氨基酸的色谱方法,并利用此方法对树莓中的游离氨基酸进行检测和分析,为树莓的开发提供科学依据。方法：以2,4-二硝基氟苯为柱前衍生试剂,Hypersil ODS2色谱柱,流动相A为40 mmol/L pH 6.4乙酸钠溶液,流动相B为体积分数50%乙腈-水溶液,梯度洗脱,反相高效液相色谱,二极管阵列检测器,检测波长360 nm。结果：22 种氨基酸线性方程的相关系数范围为0.993 1～1.000 0,相对标准偏差在0.95%～4.98%之间,加标回收率为97.10%～103.39%,最低检出限为0.39～2.87 μg/mL,定量限为1.29～9.47 μg/mL。利用所建方法检测出树莓中含有14 种游离氨基酸,其中6 种人体必需氨基酸,占总氨基酸含量的29.36%。结论：所建方法操作简便、重复性好,树莓氨基酸含量丰富,有开发价值。     

异硫氰酸苯酯柱前衍生RP-HPLC法同时测定莲雾中15种游离氨基酸的含量

采用异硫氰酸苯酯(PITC)为柱前衍生化试剂,结合反相高效液相色谱法,建立了同时测定莲雾果实中15种游离氨基酸含量的方法。采用Atlantis T3柱为固定相,以0.1 mol/L乙酸钠缓冲液(pH6.5,含0.5%三乙胺)和乙腈-水(4:1)为流动相进行梯度洗脱,流速为1.0 mL/min,柱温35 ℃,紫外检测器波长254 nm。各氨基酸组分在2.0~500 mg/L范围内具有良好的线性关系,相关系数r均大于0.99,检出限为0.08~0.35 mg/L,定量限为0.35~1.25 mg/L,加标回收率为85.61%~106.68%,相对标准偏差RSD均小于5%。该方法使得15种氨基酸很好的分离,且准确度和精密度好,灵敏度和回收率高,适用于莲雾果实游离氨基酸含量的分析。     

Determination of 21 Free Amino Acids in Fruit Juices by HPLC Using a Modification of the 6-Aminoquinolyl-<Emphasis Type="Italic">N</Emphasis>-hydroxysuccinimidyl Carbamate (AQC) Method

The present paper reports the application of an optimized pre-column derivatization procedure with aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to the determination of 21 free amino acids in fruit juices. The method permitted the optimal separation of 21 amino acids and ammonium in 30 min. Excellent response linearity was obtained from 2 to 1,000 μM for all amino acids, except tryptophan, for which the linearity was 5–1,000 μM. The fluorescence detection limits ranged from 0.08 pmol (phenylalanine) to 0.69 pmol (cysteine), except for tryptophan (7.89 pmol), for a signal-to-noise ratio of 3. The relative standard deviations of peak areas ranged from 0.1 to 0.4 % for intraday analysis and from 2.1 to 5.0 % for interday analysis. The method was applied to analyze the free amino acids in six fruit juices, and the recoveries of the proposed method were 90.4 to 103.3 %. The total free amino acid content ranged from 56.97 mg L^?1 (apple) to 469.45 mg L^?1 (longan) in six fruit juices. Aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), and glutamine (Gln) were present in higher levels in most samples, whereas cysteine (Cys) was below the detection limit in the selected samples. Notable advantages of this method include its speed, ease, low cost, good repeatability, high sensitivity, excellent precision and accuracy, and anti-interference in high-sugar matrices.     

PITC柱前衍生高效液相色谱法测定黄酒中17种氨基酸方法研究

建立了柱前衍生高效液相色谱法同时测定黄酒中17种氨基酸的方法。方法采用异硫氰酸苯酯(PITC)为柱前衍生试剂,色谱柱为C18(4.6×250mm,5μm),乙酸钠-乙腈-水混合体系梯度洗脱,紫外检测器,检测波长254nm,40min内17种氨基酸得到良好的分离。在0.025～1.25mmol/L范围内其浓度与响应值具有良好的线性关系,相关系数大于0.99;样品的平均加标回收率在78.6～118.1%之间,相对标准偏差在0.45%～3.30%范围;检出限为0.259～0.925μmol/L,定量限为0.863～3.083μmol/L。相对其他柱前衍生测定氨基酸的方法而言,该方法在可操作、低成本、易推广等方面具有明显优势,可供黄酒中氨基酸测定方法行业标准制定提供参考。     

柱前衍生-HPLC法测定五指毛桃中16种游离氨基酸含量

目的:建立柱前衍生-高效液相色谱法同时测定不同产地、不同叶形五指毛桃Ficus hirta Vahl根中16种氨基酸含量,评价其质量差异。方法:采用高效液相色谱法,以异硫氰酸苯酯进行柱前衍生化,采用Ultimate Amino Acid色谱柱（4.6 mm×250 mm,5 μm）,以0.1 mol/L乙酸钠-乙腈为流动相,进行梯度洗脱,流速1.0 mL/min,柱温为40 ℃,检测波长为254 nm。结果:9个产地12份五指毛桃根中16种氨基酸线性关系良好,相关系数均大于0.9992,平均加样回收率介于98.1%~105.9%之间,RSD为0.67%~2.11%,L-天门冬氨酸、L-谷氨酸、L-丝氨酸、甘氨酸、L-组氨酸、L-精氨酸、L-苏氨酸、L-丙氨酸、L-脯氨酸、L-酪氨酸、L-缬氨酸、L-甲硫氨酸、L-异亮氨酸、L-亮氨酸、L-苯丙氨酸和L-赖氨酸的平均含量分别为3.308、3.424、2.750、2.978、1.302、4.549、2.778、3.989、3.818、1.327、3.732、0.395、2.925、4.549、2.873、3.305 mg/g。产地为漳州卫生职业学院的四种叶形五指毛桃根中氨基酸总含量由高到低为,三指浅裂>七指深裂>五指深裂>全缘;其余8个产地的五指毛桃根中16种氨基酸总含量也存在差异,含量最高的为广西钦州灵山县,其次为广东河源源城区源南镇,含量最低的为福建漳州龙海区程溪镇。结论:该方法能使16种氨基酸很好地进行分离,可靠准确,且加样回收率较高,可作为五指毛桃根中氨基酸含量的测定方法,并可以用氨基酸含量来评价五指毛桃质量。     

A Validated RP-HPLC-DAD Method for the Determination of l-Theanine in Tea

l-Theanine, an amino acid occurring in the leaves of the tea plant (Camellia sinensis) possesses several beneficial pharmacologic effects. Its effects on the central nervous system are most widely studied, in addition, other activities (potential anticancer, cardiovascular) also stress the significance of this compound. However, analytical methods for the quantification of l-theanine in tea based on the application of HPLC are scarce in the literature. Considering the chemical characteristics of the molecule (high polarity, lack of chromophore group), derivatization and the application of special stationary phases have been preferred for reliable analysis. Here, we report the development and validation of an eligible RP-HPLC-DAD method without the need of pre- or post-column derivatization and using a buffer-free mobile phase (chromatographic separation of l-theanine with water, eluting other compounds with water–acetonitrile gradient) for the determination of l-theanine. The elaborated method provides quick (analysis time 15 mins) and reliable (mean intraday precision 0.94 RSD%, interday precision 0.48 %, recovery >96.1 %, LOQ?=?0.38 mg theanine/g dry tea leaf weight) quantification of this amino acid in aqueous tea extracts.     

Analysis of free amino acids in cereal products

Free amino acids were extracted from cereal products using 50% ethanol to prevent solubilization of polysaccharides and other viscous polymers and to avoid starch gelatinization. The extracts were analyzed by GC after ion-exchange solid phase extraction and chloroformate derivatization using Ez-Faast technology (Phenomenex). Free amino acids in cereal products could be analyzed within 1 h of extraction and determination, with good separation between peaks and repeatable retention times. Relative correction factor for each amino acid was established. The matrix did not affect the results and the method was repeatable for most of the amino acids (coefficient of variation was in the order of 10%). Different fractions and products of wheat, rye, oats and barely were analyzed. The bran contained more free amino acids than did the other analysed fractions of cereals. Fermentation seemed to consume free asparagine and aspartic acid and to use or release other amino acids.     

全自动氨基酸分析仪法测定4种贝类肌肉中谷胱甘肽和游离氨基酸含量

建立一种测定贝类肌肉中谷胱甘肽和游离氨基酸的全自动氨基酸分析仪法,以4种常见贝类为研究对象,采用0.02 mol/L盐酸溶液作为提取剂,体积分数5%磺基水杨酸溶液为蛋白沉淀剂,同时优化了缓冲溶液的组成及洗脱程序,并对该方法的线性范围、检出限、精密度以及回收率进行测定。结果表明:贝类肌肉中的谷胱甘肽和游离氨基酸有着较好的分离效果,线性关系良好,相关系数为0.999 1~0.999 9,检出限(RSN=3)为0.07~0.27μmol/L,加标平均回收率为86.40%~102.42%,日内和日间相对标准偏差分别为0.31%~0.73%和1.14%~2.60%。4种贝类肌肉组织中的游离氨基酸含量比较丰富,总游离氨基酸含量分别为1 396.39、1 016.04、911.15、287.01 mg/100 g,文蛤青蛤缢蛏牡蛎,主要的游离氨基酸为牛磺酸(Tau)、谷氨酸(Glu)、丙氨酸(Ala)、精氨酸(Arg);谷胱甘肽含量分别为103.20、82.53、61.77、33.37 mg/100 g,青蛤文蛤缢蛏牡蛎。该方法适用于贝类肌肉中谷胱甘肽和游离氨基酸含量的测定与分析。     

柱后衍生高效阳离子交换色谱法测定宁夏枸杞及枸杞花粉中氨基酸含量

采用酸水解、离子交换色谱分离及茚三酮柱后衍生法测定宁夏当年产枸杞及枸杞花粉中17种氨基酸含量,建立宁夏枸杞中蛋白水解氨基酸的测定方法。结果表明：枸杞干果中17种氨基酸总量为7.788%～8.820%,其中人体必需氨基酸占氨基酸总量的28.9%～30.0%,半必需氨基酸占氨基酸总量的4.5%～4.8%。枸杞鲜果中氨基酸总量为1.960%～2.036%,必需氨基酸占总氨基酸含量的29.2%～30.3%,半必需氨基酸约占总量的5.2%。枸杞花粉中氨基酸总量为21.62%～22.08%,人体必需氨基酸约占总量的37.9%,半必需氨基酸约占总量的4.9%。     

反相高效液相色谱法测定白屈菜种子中挥发油 成分分析及氨基酸含量

目的研究白屈菜种子中挥发油的化学成分并建立一种反相高效液相色谱法对白屈菜种子中游离氨基酸和水解氨基酸含量进行测定。方法采用气质联用的方法对白屈菜种子中的挥发油成分进行分析,采用反相高效液相色谱法,以异硫氰酸苯酯为衍生试剂进行柱前衍生,采用梯度洗脱的方法,检测白屈菜种子中游离氨基酸和水解氨基酸含量。结果挥发油中共鉴定出化学成分27个,其中亚油酸的含量最高可达到65%左右。氨基酸的检测浓度线性范围为0.0020~0.2700 mmol/L,r均≥0.9991。21种游离氨基酸的平均加样回收率在87.88%~100.10%之间,RSD在0.62%~2.58%之间(n=6);水解氨基酸的平均加样回收率在91.79%~100.03%之间,RSD在0.48%~1.97%之间(n=6)。结论白屈菜种子种中挥发油的主要成分为不饱和脂肪酸,本文建立的氨基酸测定方法经方法学验证适合白屈菜种子中游离氨基酸和水解氨基酸的含量测定。     

GC-FID测定黄酒中17种游离氨基酸

建立以N-(特丁基二甲基硅烷)-N-甲基三氟乙酰胺(MTBSTFA)为衍生试剂测定黄酒中17种游离氨基酸的气相色谱(GC)方法。比较了衍生温度、时间、及辅助试剂等对衍生效率的影响,确定N,N-二甲基甲酰胺(DMF)为辅助衍生试剂,衍生温度80℃,衍生时间60 min,可以定量检测黄酒中主要的游离氨基酸。各氨基酸在50～500 mg/L内线性良好(相关系数均大于0.99)。对黄酒样品的测定结果与高效液相色谱测定结果相比皮尔逊相关系数为0.998,样品的平均加标回收率为87.11%～119.47%。结果表明:GC-FID方法操作简便、准确、重现性好,适用于黄酒中17种游离氨基酸的分离检测。该测定方法将为开发气相-燃烧-同位素比率质谱(GC-C-IRMS)测定黄酒中氨基酸13C稳定同位素丰度以及分析黄酒酿造过程中的代谢实验提供研究基础。     

Separation and quantification of component monosaccharides of the tea polysaccharides from Gynostemma pentaphyllum by HPLC with indirect UV detection

A reversed-phase high-performance liquid chromatographic (HPLC) method is described for the simultaneous determination of aldoses and uronic acids. The separation was carried out on a RP-C₁₈ column (4.6 mm i.d. × 250 mm, 5 μm, Venusil, USA) using precolumn derivatization with 1-phenyl-3-methyl-5-pyrazolone (PMP) and UV detection at 250 nm, and the 10 PMP derivatives of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose, arabinose and fucose were baseline separated within 40 min. Furthermore, the described method was applied to the quantitative analysis of component monosaccharides in the water-soluble polysaccharides extracted from Gynostemma pentaphyllum Makino tea and the result showed that the tea polysaccharide was a typical heteropolysaccharide and consisted of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, xylose, galactose and arabinose in the molar contents of 16.3, 10.3, 47.1, 5.6, 24.0, 128.4, 25.0, 101.4 and 71.1 μM, respectively. Quantitative recoveries of the component monosaccharides in the tea polysaccharide were in the range of 94.6–108.0% and the RSD values were lower than 4.9%. The results demonstrated that the proposed HPLC method was precise and practical for the analysis of the G. pentaphyllum tea polysaccharide.     

柱前衍生反相高效液相色谱法测定柞蚕蛹蜕中氨基酸

建立一种反相高效液相色谱同时测定柞蚕蛹蜕中17 种氨基酸的分析方法。以正亮氨酸为内标物,异硫氰酸苯酯(PITC)作为Venusil-AA 氨基酸分析专用柱柱前衍生试剂,采用二元梯度洗脱,波长254nm 处检测柞蚕蛹蜕中氨基酸含量。17 种氨基酸在0.5～0.0125μmol 的范围内,具有良好的线性关系,加样回收率在96.2%～101.9%。柞蚕蛹蜕中氨基酸含量为21.9%,其中精氨酸含量最高。人体必需氨基酸占总氨基酸的33.62%。柱前衍生反相高效液相色谱法简单、高效,可以用于柞蚕蛹蜕氨基酸含量测定。     

PMP柱前衍生化HPLC法测定黄秋葵多糖的单糖组成

采用1-苯基-3-甲基-5-吡唑啉酮为柱前衍生化试剂,结合反相高效液相色谱法,建立同时测定黄秋葵多糖中8?种单糖组分的方法。采用InfinityLab Poroahell C18色谱柱为固定相,流动相为磷酸盐缓冲液（0.1?mol/L,pH?6.85）-乙腈（82∶18,V/V）,流速为1.0?mL/min,柱温25?℃,紫外检测器,波长250?nm。各单糖组分的线性范围较宽,相关系数均大于0.999,检出限为0.13～0.45?mg/kg,定量限为0.43～1.49?mg/kg,相对标准偏差小于5%,加标回收率为94.51%～106.44%。方法灵敏度高、准确性好、实用可靠,适用于黄秋葵多糖的单糖组分分析。同时测定从黄秋葵中顺序提取的热水浸提物（hot buffer soluble solution,HBSS）、螯合剂浸提物（chelating agent soluble solution,CHSS）和碱提物（dilute alkaline soluble solution,DASS）的单糖组成,确定其单糖组分的物质的量比。HBSS中,甘露糖∶鼠李糖∶葡萄糖醛酸∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=0.1∶7.0∶0.4∶5.8∶0.9∶14.6∶1.5;CHSS中,鼠李糖∶半乳糖醛酸∶半乳糖∶阿拉伯糖=4.7∶61.8∶9.6∶5.3;DASS中,甘露糖∶鼠李糖∶半乳糖醛酸∶葡萄糖∶半乳糖∶阿拉伯糖=9.5∶5.4∶1.3∶10.8∶2.4∶7.6。     

2-(4-Amino)-Phenyl-1-Hydrogen-Phenanthrene [9,10-d] Imidazole as a Novel Fluorescent Labeling Reagent for Determination of Fatty Acids in Raspberry

A novel method has been established for the rapid separation and determination of free fatty acids from 37 different varieties of raspberry. In this study, a new fluorescent labeling reagent for fatty acids, 2-(4-amino)-phenyl-1-hydrogen-phenanthrene [9, 10-d] imidazole (PIA), has been synthesized and successfully applied to the high-performance liquid chromatography (HPLC) determination of fatty acids in raspberry. The novel method has been optimized by HPLC with fluorescence detection and online mass spectrometry identification (HPLC–FLD–MS/MS). The 22 main fatty acids (FAs) present in raspberry were derivatized by PIA and separated on a reversed-phase Hypersil GOLD column with gradient elution. The main experimental parameters affecting extraction efficiency and derivatization yield were investigated and optimized by response surface methodology (RSM) combined with Box–Behnken design (BBD). Under the optimum conditions, the method was successfully applied for the analysis of 22 fatty acids in 37 different varieties of raspberry. Good linear correlations were observed for all fatty acids with correlation coefficients of > 0.9978. Limits of detection and quantification (LOD and LOQ) were in the range of 0.12 to 0.49 ng/mL and 1.07 to 2.81 ng/mL, respectively. Furthermore, the results indicated that the raspberries were rich in fatty acids, but the contents of the fatty acids varied among the different varieties.     

应用单磺酰氯柱前衍生法测定食品中的牛磺酸

提出柱前衍生高效液相色谱法测定饮料中的牛磺酸。在碱性条件下,用单磺酰氯作为衍生化试剂,以乙腈－醋酸钠缓冲溶液（ｐＨ＝７．２）为流动相。在２９０ｎｍ处进行紫外检测。     

Integration of Rapid Derivatization and Gradient Elution Techniques for Enhanced High-Performance Liquid Chromatography Analysis of Key Amino Acids in Wheat Flour

ABSTRACT A rapid pre-column derivatization process before ultraviolet chromatographic analysis using a Pico-Tag C18 column was developed in combination with modified gradient elution techniques for the analysis of key amino acids (proline and glutamine) in wheat flour. With use of a vacuum concentrator at 45°C, the pre-column derivatization process was significantly accelerated before entering a reverse-phase high-performance liquid chro-matography. When running at an optimized scheme—in which the gradient elution started at a flow rate of 1 mL/ min and then gradually increased to 1.3 mL/min in 22 min, followed by reset to 1 mL/min at 45 min—the profiled mobile phase mixture was able to yield chromatograms with desirable retention factor and resolution. Keywords: proline, glutamine, glutamic acid, reverse-phase HPLC, resolution     

高效液相色谱法测定乳酸菌发酵液中γ-氨基丁酸

通过对衍生试剂浓度、衍生时间、衍生温度、检测波长、流动相比例与柱温条件的选择,建立了一种高效液相色谱（HPLC）法灵敏检测发酵液中γ-氨基丁酸（GABA）含量的方法。以邻苯二甲醛为衍生化试剂,衍生时间5 min,衍生温度为室温,色谱条件为：检测波长228 nm,流动相为乙腈-20 mmol/L结晶乙酸钠溶液（21∶79,V/V）,柱温30℃,流速0.8 m L/min。结果表明,在γ-氨基丁酸含量0.05μg/m L～0.50 mg/m L范围内线性关系良好（相关系数R2=0.999 4）,平均加标回收率为91.87%～105.58%,精密度试验结果相对标准偏差（RSD）为0.55%～3.74%(n=5),检出限为0.02μg/m L。采用该方法检测发酵液中γ-氨基丁酸含量,其含量范围在0.157～0.369 mg/m L之间。该方法操作简单、灵敏、准确可靠,适用于发酵液中γ-氨基丁酸含量的检测。     

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。     

柱前衍生化HPLC法测定叶游离氨基酸成分及风味评价

建立柱前衍生化HPLC法测定柊叶游离氨基酸的方法，对其营养价值和风味进行评价。柱前衍生化HPLC法以异硫氰酸苯酯为衍生试剂，色谱条件为:C₁₈色谱柱（4.6 mm×250 mm，5μm），醋酸钠缓冲液-乙腈（93:7）和乙腈-水（4:1）为流动相，进行梯度洗脱，流速为1.0 mL/min，柱温40℃，检测波长为254 nm。结果表明，18种氨基酸均呈现良好的线性关系（R2>0.9990），柊叶游离氨基酸的平均回收率分别为98.3%~105.6%（RSD为0.63%~3.11%，n=6）。所建立的方法稳定、灵敏、重现性好，可用于柊叶游离氨基酸的定量分析。柊叶含有18种氨基酸及8种人体必需氨基酸，必需氨基酸总量为38.85%，接近于WHO/FAO模式值（40.0%），各种必需氨基酸组分达到WHO/FAO氨基酸成人模式的需求；鲜味氨基酸和甜味氨基酸的含量分别为10.73和5.20 mg/g，占呈味氨基酸总含量的22.58%和46.59%；鲜味氨基酸Asp和甜味氨基酸Thr的含量较高，分别为3.75 mg/g和3.33 mg/g，鲜味氨基酸Asp和芳香族氨基酸Cys的RCT值分别为125.00和20.50，对风味贡献最高。总之柊叶氨基酸种类齐全，比例合理，呈味氨基酸含量丰富，具有极大开发价值。