Simple and rapid quantitative high-performance liquid chromatographic analysis of plasma amino acids

A simple and rapid high performance liquid chromatographic method for the determination of plasma amino acids was developed. The method uses minimal sample volume and automated online precolumn derivitization of amino acids with o-phthalaldehyde and fluorescent detection. Amino acids are separated by a simplified gradient without column heating. The assay is linear from 5 to 1000 micromol/L for all amino acids. Recovery of amino acids was between 91 and 108%, intra-assay coefficient of variation (CV) was 1-7%, and inter-assay CV was 2-12%. The simple sample preparation and minimal sample volume make the method useful for the quantitation of amino acids in both patient and experimental animal samples.     

LC-MS/MS 检测癫痫患者神经递质类氨基酸

目的:建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价.方法:选用AAA-C18柱色谱柱,以乙腈水(含有0.01%七氟丁酸、0.1%甲酸)为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测(MRM)扫描方式进行检测.疾病组与健康组的统计采用t检验和主成份分析.结果:疾病组和健康组氨基酸测定结果显示:Trp、GABA两组间没有显著性差异(P＞0.05),Arg、Glv、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异(P＜0.05),通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸.结论:试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价.     

Development of a protocol for the automated analysis of amino acids in brain tissue samples and microdialysates

An automated precolumn derivatisation method has been developed for the measurement of fourteen amino acids in brain tissue and microdialysate samples. The method involves labelling amino acids with naphthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide (CN⁻). The resulting highly stable N-substituted 1-cyanobenz[f]isoindole (CBI) derivatives were separated using a binary gradient elution profile and detected fluorometrically. The order of elution of the derivatised amino acids was confirmed by using liquid chromatography with fluorescence and mass spectrometric detection in tandem. Linear calibration plots were obtained for all amino acids in the range studied (0.2–12.5 μM). The limit of detection for CBI derivatives of amino acids was in the range 5–20 fmol (S/N=2) using a 5 μl injection volume. The method has been used for the measurement of amino acids in microdialysates from rat brain and tissue homogenates from different regions of mouse brain.     

冬虫夏草口服液游离氨基酸和微量元素的分析比较

目的：为了研究冬虫夏草口服液的有效成分和药用价值,测定冬虫夏草口服液中的游离氨基酸和微量元素的不同种类和含量。方法：采用安捷伦1200高效液相色谱仪,C18反相柱梯度分离,邻苯二甲醛一芴甲酯柱前自动衍生化,利用已知浓度氨基酸标准品进行外标法定量,根据已知浓度氨基酸标准品的峰面积与样品峰面积之比计算游离氨基酸的含量。结果：冬虫夏草口服液中含有23种游离氨基酸和18种微量元素,氨基酸总量在口服液中占0．892％,必需氨基酸在游离氨基酸总量中占53．1％;含量最高的游离氨基酸依次是赖氨酸、亮氨酸、精氨酸。结论：游离氨基酸是冬虫夏草口服液中重要的生物有效成分,为冬虫夏草口服液的药效提供了基础数据。     

Amino acids analysis using a monolithic silica column after derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F)

A fast HPLC method using a monolithic silica column was developed for the measurement of amino acids. The amino acids were pre-column derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) and separated on a monolithic silica column (MonoClad C18-HS, 250 mm × 3 mm I.D.). The separation of 19 NBD-amino acids was achieved within 18 min, which was only one-fifth of the time taken by the methods using a conventional particle-packed column, with the gradient elution of a mobile phase at the flow rate of 1.4 mL/min. The sensitivity was good with a limit of detection for the individual amino acids ranging from 2.94 to 53.4 fmol. The calibration curves for all the amino acids were found to be linear in the range of 200 fmol to 20 pmol with correlation coefficients of 0.997 or better. The analytical method was successfully applied to determine the amino acids in a mouse plasma sample.     

青海循化线辣椒中氨基酸含量分析

采用柱前衍生高效液相色谱法对循化线辣椒中精氨酸、丙氨酸、丝氨酸等17种氨基酸的含量进行测定。结果表明:线辣椒所含17种氨基酸中,必需氨基酸含量合理,利于人体吸收。     

两种柱前衍生反相高效液相色谱法测定血液和尿液中游离氨基酸

建立以PITC法和AQC法为柱前衍生试剂测定血液和尿液中游离氨基酸含量的测定方法。采用Waters-e2695操作系统,色谱柱为Shim-vp,ODS(250mm×4.6mm,5μm)(日本,岛津公司),以甲醇/乙腈/水和醋酸钠溶液(pH 6.5)为流动相,梯度洗脱。分别采用紫外和荧光检测器对血液和尿液中游离氨基酸进行含量测定。结果显示,两种衍生化方法灵敏度好、分离度高,具有良好的线性范围(r>0.990 0),准确度高(平均回收率为75.1%～127.0%),进样精密度好(RSD为0.12%～3.42%)。PITC法在尿液中游离氨基酸含量测定中显示了良好的测试准确性;而AQC法测定尿液中组氨酸、苏氨酸、脯氨酸超出线性范围,需要对尿样的前处理进行深入研究。     

High-sensitivity phenylthiohydantoin amino acid analysis using conventional and microbore chromatography

Reversed-phase microbore high-performance liquid chromatography was investigated for high-sensitivity analysis of phenylthiohydantoin (PTH) amino acids. A mixed nitrile alkylsilane bonded phase was developed and ternary gradient elution conditions were devised for resolution 150 × 4.6 mm I.D. column and transferred to a 150 × 1 mmI.D. microbore column. The performance of these columns was evaluated in terms of PTH amino acid resolution, enhanced sample detectability, and retention time precision. For this work a general purpose high-performance liquid chromatograph was modified to reduce extra column band broadening and a preformed gradient elution technique was developed to achieve rapid analysis times at microbore flow-rates. The microbore high-performance liquid chromatographic system is useful for high-sensitivity analysis of PTH amino acids in micro-sequencing applications.     

Rapid high-throughput assay for the measurement of amino acids from microdialysates and brain tissue using monolithic C18-bonded reversed-phase columns

A rapid precolumn high-performance liquid chromatography method based on fluorescence detection has been developed for the measurement of multiple amino acids from both ex vivo and in vivo biological samples using monolithic C18 columns. A mixture of 18 primary amino acids were derivatised with napthalene-2,3-dicarboxaldehyde (NDA) in the presence of cyanide. The resulting isoindole derivatives were resolved within 10 min using a linear binary gradient elution profile with Rs values in the range 1.2-9.0. The limit of detection (LOD) was found to be between 6.0 and 60 fmol for 5 microl injection with a signal to noise ratio of 3:1. The NDA derivatives were found to be stable for 9 h at 4 degrees C. This assay has been employed for the rapid analysis of amino acids from brain tissue and microdialysis samples. Examples of application of the method are given.     

LC-MS/MS检测癫痫患者神经递质类氨基酸

目的：建立液相色谱串联质谱同位素内标法检测神经递质类氨基酸并用于癫痫患者临床评价。方法：选用AAA-C18柱色谱柱,以乙腈水（含有0.01%七氟丁酸、0.1%甲酸）为流动相,采用梯度洗脱进行分离,血浆样品用iTRAQ-115衍生化试剂处理后,加入iTRAQ-114衍生化的氨基酸内标并进样,选用3200QTRAP型质谱仪的多重反应监测（MRM）扫描方式进行检测。疾病组与健康组的统计采用t检验和主成份分析。结果：疾病组和健康组氨基酸测定结果显示：Trp、GABA两组间没有显著性差异（P〉0.05）,Arg、Gly、Ser、Tau、Asp、Glu、EtN、两组间有显著性差异（P〈0.05）,通过PCA分析显示,疾病组与健康组之间差异明显,Asp、Glu、Ser等是引起差异的主要氨基酸。结论：试验方法灵敏、专属性强,并初步的用于癫痫患者体内氨基酸评价。     

4-Methyleneglutamic acid and 4-methyleneglutamine: isolation from extracts of peanut seedlings and determination by high-performance liquid chromatography

The non-protein amino acids, 4-methyleneglutamic acid and 4-methyleneglutamine, are isolated from aqueous extracts of peanut seedlings in good yield and high purity using a simple HCl-gradient elution from a column of cation-exchange resin followed, in some instances, by a gradient elution with acetic acid from a column of an anion-exchange resin. All of the 4-substituted glutamic acids commonly found in legume species are resolved by a combination of these two system. For analytical purposes, resolution of the acidic amino acids as their phenylthiocarbamoyl derivatives is achieved by HPLC but not by conventional ion-exchange amino acid analysis. Although 4-methyleneglutamine undergoes cyclic deamidation in acidic medium at a slower rate than glutamine, this reaction occurs to a significant extent at 22 degrees C but not a 4 degrees C during the cation-exchange chromatographic fractionation.     

反相高效液相色谱法测定血浆游离氨基酸

本文报告了采用高效液相色谱法反相梯度洗脱,邻苯二甲醛和β-巯基乙醇柱前衍生化,荧光检测分血浆游离氨基酸。实验采用线性洗脱,在50分钟内可同时测定18种氨基酸,血浆样品的预处理简单,衍生化反应的时间仅需1分30秒,血浆样品的实际进样量少于1μl。本测定方法的精确度高,各个氨基酸保留时间的变异系数平均为0.89%±0.45%(SD),峰面积的变异系数平均为2.06%±1.76%(SD),各个氨基酸的浓度在15—150μmol/L的范围中,线性关系的相关系数平均为0.985±0.0305(SD)。准确性好,各个氨基酸的回收率平均为97.6%±5.1%(SD)。实验还讨论了氨基酸分离时溶液pH值、柱温、离心速度等因素对分析结果的影响。     

Chemometric evaluation of on-line high-pressure liquid chromatography in mammalian cell cultures: analysis of amino acids and glucose.

An on-line high-pressure liquid chromatography (HPLC) system capable of measuring amino acids and carbohydrates was used to study metabolism in mammalian cell culture systems. The HPLC method utilized anion-exchange chromatography followed by integrated pulsed amperometric detection. The method is capable of measuring 19 amino acids plus glucose with a complete method time of 65 min. In actual cell cultures, the method was shown to be useful for monitoring 17 amino acids plus glucose. The two amino acids that were not accurately monitored were arginine and lysine, possibly due to their elution near the void volume of the column. The HPLC system was used to study variability in metabolism across different cell culture processes, as well as the effect of glucose and glutamine limitation on a single cell culture process. Chemometric analysis was used to draw statistically meaningful conclusions from the highly correlated, multivariate data set that resulted from these experiments. Using chemometrics, variation between processes was linked to differences in uptake rates of seven amino acids. Similarly, lactate concentration, cell density, and aspartate uptake rate were linked to glucose and glutamine limitation. The effect of nutrient limitation on glutamate, alanine, and ammonium was also considered.     

Quantification of urinary zwitterionic organic acids using weak-anion exchange chromatography with tandem MS detection

A rapid and accurate quantitative method was developed and validated for the analysis of four urinary organic acids with nitrogen containing functional groups, formiminoglutamic acid (FIGLU), pyroglutamic acid (PYRGLU), 5-hydroxyindoleacetic acid (5-HIAA), and 2-methylhippuric acid (2-METHIP) by liquid chromatography tandem mass spectrometry (LC/MS/MS). The chromatography was developed using a weak anion-exchange amino column that provided mixed-mode retention of the analytes. The elution gradient relied on changes in mobile phase pH over a concave gradient, without the use of counter-ions or concentrated salt buffers. A simple sample preparation was used, only requiring the dilution of urine prior to instrumental analysis. The method was validated based on linearity (r2>or=0.995), accuracy (85-115%), precision (C.V.<12%), sample preparation stability (相似文献   

Automatic Edman microsequencing of peptides containing multiple unnatural amino acids

It is now routine using automatic Edman microsequencing to determine the primary structure of peptides or proteins containing natural amino acids; however, a deficiency in the ability to readily sequence peptides containing unnatural amino acids remains. With the advent of synthetic peptide chemistry, combinatorial chemistry, and the large number of commercially available unnatural amino acids, there is a need for efficient and accurate structure determination of short peptides containing many unnatural amino acids. In this study, 35 commercially available alpha-unnatural amino acids were selected to determine their elution profile on an ABI protein sequencer. Using a slightly modified gradient program, 19 of these 35 PTH amino acids can be readily resolved and distinguished from common PTH amino acids at low picomole levels. These unnatural amino acids in conjunction with the 20 natural amino acids can be used as building blocks to construct peptide libraries, and peptide beads isolated from these libraries can be readily microsequenced. To demonstrate this, we synthesized a simple tripeptide "one-bead one-compound" combinatorial library containing 14 unnatural and 19 natural amino acids and screened this library for streptavidin-binding ligands. Microsequencing of the isolated peptide-beads revealed the novel motif Bpa-Phe(4-X)-Aib, wherein X = H, OH, and CH3.     

Preparative-scale isolation of isotopically labeled amino acids

Over 10 g of individual ²H, ¹⁵N-labeled amino acids was resolved and recovered on a laboratory-scale ion-exchange system from a crude bacterial protein hydrolyzate derived from 20 g of lyophilized cells. The 17 amino acids (cystine was not isolated) were recovered containing less than 1.0% of other contaminating amino acids except for proline (4.0%). The aromatic and basic amino acids were isolated on a dual-column carrier displacement system (390-ml resin bed volume) while most of the neutral and acidic amino acids were separated on a pyrazolium chloride elution system (560-ml resin bed volume). The two remaining overlapping pairs were resolved on small carrier displacement columns. In addition, the overlapping fractions from adjacent peaks of the pyrazolium chloride elution system represent only 3.5% (0.37 g) of the total sample.     

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.     

Determination of D-amino acids labeled with fluorescent chiral reagents, R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles, in biological and food samples by liquid chromatography

D-Amino acids in food and biological samples labeled with R(-)- and S(+)-4-(3-isothiocyanatopyrrolidin-1-yl)-7-(N, N-dimethylaminosulfonyl)-2,1,3-benzoxadiazoles (DBD-PyNCS) were separated by reversed-phase chromatography and detected fluorometrically at 550 nm (excitation at 460 nm). DL-Amino acids were efficiently labeled at 55 degrees C for 20 min in basic medium. The resulting thiocarbamoyl-amino acids were resolved by an isocratic elution using water:30% methanol in acetonitrile (72:28) containing 0.1% trifluoracetic acid as mobile phase for hydrophilic amino acids and gradient elutions using sodium acetate buffer (pH 5. 2)/acetonitrile as gradient solvent mixture for hydrophobic amino acids, respectively. The detection limits (S/N = 3) of DL-amino acids tested were in the range of 0.16-0.75 pmol. The proposed method was applied to determine the D-amino acid(s) in milk, cream, fermented dairy products (yogurt and yakult), tomato products (juice, puree, and catchup), fermented beverages (beer and red wine), and human urine. The existence of D-amino acid(s) was demonstrated in all the samples tested. Furthermore, the identification of the D-amino acid(s) was performed using both isomers of DBD-PyNCS and by on-line HPLC-electrospray ionization-MS.     

Amino acid analysis by high-performance liquid chromatography after derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide

Amino acids are quantitatively determined by precolumn derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide and reversed-phase high-performance liquid chromatography with photometric detection at 340 nm. Excellent chromatographic resolution of a mixture of the derivatives of 20 amino acids including proline and cystine is achieved within 110 min by linear gradient elution with acetonitrile in 13 mM trifluoroacetate plus 4% (by vol) tetrahydrofuran. The limit of detection is 50 pmol. Amino acid analyses of acid hydrolysates of the several proteins gave results equivalent to those obtained by conventional ion-exchange-based amino acid analysis. The simplicity of the procedure allows its use on any multipurpose high-performance liquid chromatographic system.     

Determination of dimethylamine in biological samples by high-performance liquid chromatography

A reversed-phase HPLC method for the quantification of dimethylamine in serum and urine is presented. Dimethylamine (DMA) is converted into a stable fluorescent product by precolumn derivatization with fluorenylmethylchloroformate. The DMA derivative is resolved from derivatives of other amines and amino acids by gradient elution with a total run-time of 15 min. The lower limit of determination in biological samples is 0.1 μmol/1. Recoveries from spiked serum samples were 99–107%. Within- and between-run precision were better than 6%. Concentrations of DMA in serum from normal human subjects (n=8) and from continuous ambulatory peritoneal dialysis patients (n=15) were 3.3±1.5 and 29.2±12.1 μmol/1, respectively.