伊枯草菌素A发酵过程中游离氨基酸的HPLC分析

建立一种快速、高效测定游离氨基酸含量的异硫氰酸苯酯(PITC)柱前衍生高效液相色谱法,并利用此方法分析检测iturin A发酵过程中游离氨基酸的动态变化。以异硫氰酸苯酯(PITC)为衍生化试剂,采用Venusil-AA(4.6 mm×250 mm,5μm)氨基酸分析专用柱,并优化HPLC检测色谱条件。结果表明:梯度洗脱程序、流动相pH值、色谱柱温对分析时间、色谱峰分离及峰型具有重要影响。当最优色谱条件为:流动相A为0.1 mol/L无水乙酸钠缓冲溶液(pH6.4±0.1)-乙腈(66∶5),流动相B为乙腈-水(4∶1),流速1.0 mL/min,检测波长254 nm,色谱柱温40℃,梯度程序洗脱,35 min内可完全分离16种氨基酸,且各氨基酸在一定浓度范围内线性关系良好(R2均大于0.9986),加标回收率在83.84%-108.02%之间,RSD值均小于2.77%。该方法耗时短、操作简便、准确可靠,具有良好的精密度和稳定性。通过此方法研究分析伊枯草菌素A发酵过程中各游离氨基酸含量变化规律,发现其氨基酸浓度变化规律大致分为三类。     

应用不同柱前衍生法于HPLC测定西洋参中的氨基酸

通过微波水解衍生方法与柱前衍生方法测定西洋参中的氨基酸成分并进行对比,评价两种方法使用的合理性与应用价值。通过微波水解衍生及柱前衍生两种不同的衍生方法测定同一来源的西洋参药材中的氨基酸成分含量,比较两种实验方法的操作性、准确合理性及测定氨基酸成分含量。两种实验方法平均回收率及相对标准偏差为:柱前衍生法回收率90.1%~101.7%、RSD 0.9%~3.9%,微波水解衍生法90.1%~113.1%、RSD 1.1%~9.5%(除个别氨基酸在酸水解过程中易被破坏)。柱前衍生法主要测定游离氨基酸的含量,其中西洋参根中的精氨酸占总量的59%。微波衍生法明显缩短了氨基酸分析时间,实验操作上体现显著优势。柱前衍生法准确、可靠,测定产物稳定。测定西洋参干燥根中游离的精氨酸含量占游离氨基酸总量的一半以上,含量最高,为西洋参的化学成分与药理作用的进一步研究提供参考依据。     

冬虫夏草鲜品及干品游离氨基酸比较分析

采用异硫氰酸苯酯(PITC)柱前衍生高效液相色谱法,测定并比较烘干、晒干所得的冬虫夏草干燥样品及未经处理的鲜冬虫夏草样品的游离氨基酸含量。结果表明:3类样品均含有19种游离氨基酸,其中鲜冬虫夏草的游离氨基酸总量、药效氨基酸、呈味氨基酸及必需氨基酸含量均最高,显著高于晒干和烘干冬虫夏草(P0.01),晒干和烘干冬虫夏草之间无显著差异。冬虫夏草的游离氨基酸在晒干、烘干过程中会造成损失。该研究为冬虫夏草加工工艺的提升提供了科学依据。     

柱前衍生反相高效液相色谱法测定绞股蓝茶叶中17种游离氨基酸含量

建立2,4-二硝基氟苯柱前衍生化-反相高效液相色谱法测定绞股蓝茶叶中17种游离氨基酸的含量。以Phenomenex Gemini NX C18(4.6mm×250mm,5μm)为分析柱,采用梯度洗脱,流动相A为0.05mol·L-1乙酸钠(pH=6.4,含0.1%N,N-二甲基甲酰胺),流动相B为乙腈-水(1∶1,v/v),检测波长为360nm,柱温35℃;经方法学考察,该方法具有良好的稳定性和重现性。测定结果表明,绞股蓝茶叶中17种游离氨基酸总量为39.79mg·g-1,其中人体必需氨基酸占游离氨基酸总量的36.57%。从氨基酸含量考虑,绞股蓝茶叶具备一定的开发利用价值。     

异硫氢酸苯酯法(PITC)分析氨基酸

<正> 自从1958年Moore等人建立氨基酸测定方法以来,氨基酸分析方法发展迅速,日新月异。除采用阳离子交换柱,茚三酮柱后反应测定氨基酸方法外,70年代起,各种采用柱前衍生,反相色谱法分离测定氨基酸方法相继问世,为氨基酸分析开辟了新的领域。异硫氢酸苯酯(PITC)法作为其中一种,以其灵敏度高(可检测,1Pmal),分析速度快(水解液为10分钟,生理体液40～60分     

柱前衍生反相高效液相色谱法测定马尾松(Pinus massoniana )针叶中γ-氨基丁酸和17种游离氨基酸

建立了自动在线柱前衍生反相高效液相色谱法同时测定γ-氨基丁酸(GABA)和17种游离氨基酸含量的方法.以邻苯二甲醛-9-芴基甲基氯甲酸酯(OPA-FMOC)为衍生试剂进行衍生,Agilent Hypersil AA-ODS-C18色谱柱分离,梯度洗脱,二极管阵列检测器检测,在19min内分离测定了马尾松苗木针叶中GABA 和17种游离氨基酸的含量.该方法测定氨基酸的回收率高于90.1%,精密度和重现性均较好(相对标准偏差为0.21%～2.81%),经测定,发现马尾松被马尾松毛虫取食后,所测18种氨基酸总量明显降低,从418.3μg · g-1降低到310.4μg · g-1鲜重.     

柱前衍生化HPLC同时测定霍山石斛中13种游离氨基酸含量

为建立柱前衍生化HPLC同时测定霍山石斛中13种游离氨基酸的方法。采用Waters XBridge C_(18)色谱柱(250 mm×4. 6 mm,5μm);流动相为0. 1 mol/L醋酸钠缓冲液-乙腈∶水(4∶1),梯度洗脱;流速1. 0 mL/min,柱温35℃;检测波长254 nm。在该色谱条件下霍山石斛中13种游离氨基酸分离较好,平均加样回收率为92. 7%~104. 3%,RSD 2. 5%。该方法简便、准确、重现性好,适用于霍山石斛中13种游离氨基酸的同时测定。     

DNFB柱前衍生化RP-HPLC测定大黄种子氨基酸的含量

采用柱前衍生RP-HPLC法测定大黄种子中氨基酸的含量。6 mol/L盐酸水解得到氨基酸,以2,4-二硝基氟苯(DNFB)为柱前衍生化试剂,梯度洗脱。色谱柱为Gemimi-NX C18(4.6 mm×250 mm,5μm);流动相A相为0.1 M乙酸钠水溶液,B相为乙腈-水(1∶1),柱温37℃,检测波长360 nm。结果表明,三种正品大黄种子中均含有17种氨基酸,总量在11.30%~19.26%。该方法灵敏、准确,有良好的重复性和稳定性。     

利用乙腈作为内标气相色谱法快速测定发酵液中的乳酸

建立了一种内标法准确、快速测定发酵液中乳酸含量的方法。色谱柱为2m×3mm(i.d.),内填GDX-103的不锈钢柱,FID检测器。内标为1.0%的乙腈溶液。实验结果表明,该方法的相对标准偏差小于1.0%(n=7),回收率在99%以上。方法具有前处理简单、干扰小、灵敏度高、分析速度快,线性测定范围宽等优点。     

昆虫体内多胺的高效液相色谱(HPLC)测定

建立丹磺酰氯柱前衍生HPLC快速测定昆虫体内多胺含量的方法。以C18(250mm×4.6mm,5μm)为固定相,甲醇和水为流动相,梯度洗脱,柱温40℃,流速1mL/min,荧光检测波长激发波长(Ex)280nm,发射波长(Em)515nm,测得腐胺(put)、亚精胺(spd)和精胺(spm)三者回收率分别为98.7%,99.2%和97.8%,回归方程线性良好(r值均大于0.99),分析时间为16min。该法简洁、快速、灵敏度高、重现性好,可有效分析昆虫及其他生物样品中微量多胺的含量。     

Amino acid analysis of peptide loading ratios in conjugate vaccines: a comparison of direct electrochemical detection and 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate pre-column derivatization methods

Amino acid analysis using direct electrochemical detection was compared with precolumn fluorescent derivatization using 6-aminoquinolyl- N-hydroxysuccinimidyl carbamate (AQC) for evaluation of the degree of covalent coupling of peptides to a carrier-protein complex derived from the bacteria Neisseria meningitidis. AQC derivatization was found to give superior sensitivity compared to electrochemical detection, with less interference from sample components such as carbohydrates or buffer salts. Hydrolysis time and temperature were optimized for maximal recoveries of the marker amino acid 6-aminohexanoic acid (epsilon-Ahx) and the unique amino acids S-dicarboxyethyl cysteine (SDCEC) and S-carboxymethyl homocysteine (SCHMC), which are generated upon the hydrolysis of the covalent linkage between the peptide and the carrier protein. Quantitation of these amino acids enabled the determination of the ratio of peptide to protein in the conjugate samples.     

Analysis of 26 amino acids in human plasma by HPLC using AQC as derivatizing agent and its application in metabolic laboratory

The present study reports the simultaneous analysis of 26 physiological amino acids in plasma along with total cysteine and homocysteine by high-performance liquid chromatography (HPLC) employing 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as precolumn derivatizing reagent. Separations were carried out using Lichrospher 100 RP-18e (5 μm) 250 × 4.0 mm column connected to 100 CN 4.0 × 4.0 mm guard column on a quaternary HPLC system and run time was 53 min. Linearity of the peak areas for different concentrations ranging from 2.5 to 100 pmol/μL of individual amino acids was determined. A good linearity (R ² > 0.998) was achieved in the standard mixture for each amino acid. Recovery of amino acids incorporated at the time of derivatization ranged from 95 to 106 %. Using this method we have established the normative data of amino acids in plasma, the profile being comparable to the range reported in literature and identified cases of classical homocystinuria, cobalamin defect/deficiency, non-ketotic hyperglycinemia, hyperprolinemia, ketotic hyperglycinemia, urea cycle defect and maple syrup urine disease.     

Measurement of picomoles of phosphoamino acids by high-performance liquid chromatography.

A reverse-phase, high-performance liquid chromatographic system (HPLC) is described that makes possible optimal resolution and quantitation of picomole levels of phosphoamino acids, both with or without the presence of a large excess of nonphosphorylated amino acids. The assay involves precolumn derivatization of an amino acid mixture with phenyl isothiocyanate (PITC) at room temperature, followed by separation of phosphoamino acids from other amino acids by HPLC. The liquid chromatography was carried out on a C18 reverse-phase column at pH 7.4 and 30 degrees C using gradient elution with eluent A as 157 mM sodium acetate containing 2% acetonitrile and eluent B as 60% acetonitrile in water. A uv absorption at 254 nm is employed for detection of the PITC-derivatized amino acids eluting from the column. Amino acids are eluted with baseline resolution in the following order: phosphoserine, phosphothreonine, aspartic acid, glutamic acid, and phosphotyrosine followed by other amino acids. The sensitivity is in the picomole range, and the separation time, injection to injection, is 36 min. Phosphoserine, phosphothreonine, and phosphotyrosine are resolved within the first 8 min. This procedure enables determination of as low as 5 pmol of nonradioactive phosphoamino acids in a 100-fold excess of amino acids, as is usually present in most phosphoproteins in the natural state. Phosphoamino acids in polypeptides separated by sodium dodecyl sulfate-polyacrylamide electrophoresis and transferred to polyvinylidene difluoride (PVDF) membrane, or protein samples directly blotted on the membrane, can also be analyzed by this procedure after acid hydrolysis of the proteins bound to the PVDF membrane.     

Enantiomeric separation of ornithine in complex mixtures of amino acids by EKC with off-line derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate

A new analytical methodology was developed by EKC enabling the fast enantiomeric separation of Ornithine in complex mixtures of amino acids. A previous derivatization step with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) was achieved to enable the sensitive UV detection of amino acids as well as to make possible their interaction with the CDs employed as chiral selectors. A dual CD system containing an anionic and a neutral CD in phosphate buffer at acid pH showed a high resolving power allowing the enantiomeric separation of 18 protein amino acids and Orn. The method was applied to the analysis of fermented foods to investigate the extent of the presence of Orn enantiomers.     

Application of the 6-aminoquinolyl-N-hydroxysccinimidyl carbamate (AQC) reagent to the RP-HPLC determination of amino acids in infant foods

The validation of a pre-column derivatization procedure with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) to the determination of the amino acid content by RP-HPLC with fluorescence detection (lambda excitation 250 nm, lambda emission 395 nm) in milk-cereal based infant foods was carried out. The analytical parameters: linearity (0.0025-0.2mM), precision of the method (0.2-3.5% variation coefficients), accuracy (derivatization: 86-106% average recovery and method: 88.3-118.2% average recovery) and the limits of detection (0.016-0.367 microM) and quantification (0.044-1.073 microM) were determined. Glutamic acid, proline and leucine were the most abundant amino acid whereas the lowest contents corresponded to tyrosine and cysteine.     

Direct extract derivatization for determination of amino acids in human urine by gas chromatography and mass spectrometry

The purpose of this study was to develop a simple and accurate analytical method to determine amino acids in urine samples. The developed method involves the employment of an extract derivatization technique together with gas chromatography-mass spectrometry (GC-MS). Urine samples (300 microl) and an internal standard (10 microl) were placed in a screw tube. Ethylchloroformate (50 microl), methanol-pyridine (500 microl, 4:1, v/v) and chloroform (1 ml) were added to the tube. The organic layer (1 microl) was injected to a GC-MS system. In this proposed method, the amino acids in urine were derivatized during an extraction, and the analytes were then injected to GC-MS without an evaporation of the organic solvent extracted. Sample preparation was only required for ca. 5 min. The 15 amino acids (alanine, aspartic acid, cysteine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, tyrosine, tryptophan, valine) quantitatively determined in this proposed method. However, threonine, serine, asparagine, glutamine, arginine were not derivatized using any tested derivatizing reagent. The calibration curves showed linearity in the range of 1.0-300 microg/ml for each amino acid in urine. The correlation coefficients of the calibration curves of the tested amino acids were from 0.966 to 0.998. The limit of detection in urine was 0.5 microg/ml except for aspartic acid. This proposed method demonstrated substantial accuracy for detection of normal levels. This proposed method was limited for the determination of 15 amino acids in urine. However, the sample preparation was simple and rapid, and this method is suitable for a routine analysis of amino acids in urine.     

Determination of amino acids in foods by reversed-phase HPLC with new precolumn derivatives,butylthiocarbamyl, and benzylthiocarbamyl derivatives compared to the phenylthiocarbamyl derivative and ion exchange chromatography

New precolumn derivatizing reagents for analysis of amino acids by HPLC—butylisothiocyanate (BITC) and benzylisothiocyanate (BZITC)—reacted quantitatively with 22 standard amino acids and the amino acids in the acid hydrolysate of food and protein standard, bovine serum albumin (BSA), at 40°C for 30 min to yield butylthiocarbamyl (BTC) amino acids and at 50°C for 30 min to yield benzylthiocarbammyl (BZTC) amino acids. BTC and BZTC amino acids were successfully separated in 35 min on the reversed-phase Nova-Pak C18 column (30 cm × 3.9 mm, 4 μm). The optimum wavelengths for determination of BTC and BZTC derivatives were 240 nm and 246 nm, respectively. Analysis of the results obtained with BSA and food samples as BTC and BZTC derivatives showed good agreement with those determined as ion-exchange chromatography and data presented in the literature. The advantage of BITC reagent over the phenylisothiocyanate (PITC) and BZITC was that it had high volatility, so the excess reagent and by-products were easily removed in about 10 min, compared to about 1 h in the PITC and BZITC reagents. In the BTC and BZTC derivatives, cystine and cysteine were determined separately, but in the PTC amino acids derivatized with PITC reagent they were resolved into single peak.     

Simultaneous measurement of phenylalanine and tyrosine in phenylketonuric plasma and dried blood by high-performance liquid chromatography

Phenylketonuria (PKU) is a disorder characterized by an interruption in the conversion of phenylalanine to tyrosine, a reaction catalyzed by phenylalanine hydroxylase (PAH). Animal models of PKU used in this study were induced by daily subcutaneous injections of pups with alpha-methylphenylalanine plus phenylalanine in utero and postnatally from day 4 to day 14. Dry blood and plasma were utilized to measure phenylalanine concentration in PKU rats. The results indicated that the concentration of phenylalanine is higher and more stable in plasma than dry blood. Precolumn derivatization of dried blood and plasma free amino acids were conducted with phenylisothiocyanate (PITC). The phenylthiocarbamyl (PTC) derivatives were separated on a reversed-phase C-18 column (15 cm x 4.6 mm). A gradient high-performance liquid chromatography method with two eluents, 0.1 M sodium acetate buffer and 100% acetonitrile was developed to facilitate the separation of nine amino acids within 11 min. Tyrosine and phenylalanine eluted the column at 5.4 and 9.4 min, respectively. This method provides a quick and reliable technique for neonatal screening.     

A targeted metabolomic protocol for short-chain fatty acids and branched-chain amino acids

Research in obesity and metabolic disorders that involve intestinal microbiota demands reliable methods for the precise measurement of the short-chain fatty acids (SCFAs) and branched-chain amino acids (BCAAs) concentration. Here, we report a rapid method of simultaneously determining SCFAs and BCAAs in biological samples using propyl chloroformate (PCF) derivatization followed by gas chromatography–mass spectrometry (GC–MS) analysis. A one-step derivatization using 100 μL of PCF in a reaction system of water, propanol, and pyridine (v/v/v = 8:3:2) at pH 8 provided the optimal derivatization efficiency. The best extraction efficiency of the derivatized products was achieved by a two-step extraction with hexane. The method exhibited good derivatization efficiency and recovery for a wide range of concentrations with a low limit of detection for each compound. The relative standard deviations of all targeted compounds showed good intra- and inter-day (within 7 days) precision (<10 %), and good stability (<20 %) within 4 days at room temperature (23–25 °C), or 7 days when stored at ?20 °C. We applied our method to measure SCFA and BCAA levels in fecal samples from rats administrated with different diet. Both univariate and multivariate statistical analysis of the concentrations of these targeted metabolites could differentiate three groups with ethanol intervention and different oils in diet. This method was also successfully employed to determine SCFA and BCAA in the feces, plasma and urine from normal humans, providing important baseline information of the concentrations of these metabolites. This novel metabolic profile study has great potential for translational research.     

Improved method for the measurement of large neutral amino acids in biological matrices

A high-performance liquid chromatographic method for measuring neutral amino acids in rat sera, brain tissues, and perfusates was developed by using o-phthalaldehyde sulfite as a pre-column derivatization reagent. With the present method, it was possible to separate the neutral amino acids within a single run in 25 min, while the acidic amino acids were eluted near or at the solvent front. The recovery was above 88.8% with a relative standard deviation (RSD) below 4.2%. The within- and between-day assay reproducibility for the determination of rat serum amino acids showed RSDs below 1.35 and 7.61%, respectively. In the present study, the neutral amino acids were assayed with high sensitivity, accuracy and good reproducibility in a relatively short time and on a small sample size.