Inexpensive,Computer-Automated HPLC for Ion Exchange Separation and Quantification of Amino Acids in Physiological Fluids

Abstract

An inexpensive, computer-automated HPLC for separation and quantification of amino acids in physiological fluids is described. The system offers fully automated equipment control, data collection, processing and storage capabilities. The component nature of the system and the software flexibility permit extensive system modification, accomodating a wide variety of different separatory procedures which are not possible with many dedicated amino acid analysers. The system uses a lithium-based ion exchange column with post-column o-phthalaldehyde derivatization. A time of 128 minutes, including column regeneration, is required for separation of all amino acids through to arginine. The advantages of post-column derivatization over pre-column derivatization methods for post-separatory amino acid techniques are discussed. Accurate quantification of radiolabel in amino acids is demonstrated.     

Gas chromatography-mass spectrometry analysis of amino acid enantiomers as methyl chloroformate derivatives: application to space analysis

This work describes a GC-MS method for enantioselective separation of amino acids. The method is based on a derivatization reaction which employs a mixture of alkyl chloroformate-alcohol-pyridine, as reagents to obtain the N(O,S)-alkyl alkoxy carbonyl esters of amino acids. Various reaction parameters are investigated and optimized to achieve a reproducible derivatization procedure suitable for separation of amino acid enantiomers on Chirasil-L-Val chiral stationary phase. In particular, the following topics are investigated for 20 proteinogenic amino acids: (i) the proper reagent and reaction conditions to obtain the highest derivative yield; (ii) the amino acid reactivity and the MS properties of the obtained derivatives; (iii) the linearity and sensitivity of the analytical method; (iv) the retention behavior of the derivatives and their enantiomeric separation on the Chirasil-L-Val chiral stationary phase. By combining the resolution power of the Chirasil-L-Val column and the high selectivity of the SIM MS detection mode, the described procedure enables the enantiomeric separation and quantification of 16 enantiomeric pairs of amino acids. The procedure is simple and fast and reproducible. It displays a wide linearity range at ppb detection limits for quantitative determinations: these properties make this derivatization method a suitable candidate for amino acid GC-MS analysis on board of the spacecrafts in space exploration missions of solar system body environments.     

Amino Acid Profiling of Protein Hydrolysates Using Liquid Chromatography and Fluorescence Detection

Abstract

A liquid chromatography procedure is described for separating the amino acids in protein hydrolysates. The proteins are hydrolyzed with hydrochloric acid and an aliquot of the hydrolysate is derivatized with dansyl chloride reagent. The derivatization procedure takes only 2 minutes using a reaction temperature of 100°C. The dansylated amino acids are chromatographed using a reversed-phase C₈ column and a multi-step, nonlinear gradient elution solvent program which is readily achieved using a microprocessor-controlled liquid chromatograph. Chromatography is complete in approximately 40 min. The procedure is useful for characterizing proteins and may also be used to analyse intact dansylated polypeptides. Chromatograms showing the amino acid profile of chymotrypsin, albumin and histone are given.     

A fully automated amino acid analyzer using NBD-F as a fluorescent derivatization reagent

A fully automated amino acid analyzer using NBD-F (4- fluoro-7-nitro-2,1,3-benzoxadiazole) as a fluorescent derivatization reagent was developed. The whole analytical process was fully automated from derivatization, injection to HPLC separation and quantitation. The derivatization reaction conditions were re-evaluated and optimized. Amino acids were derivatized by NBD-F for 40 min at room temperature in the borate buffer (pH 9.5). The derivatives were separated within 100 min and fluorometrically detected at 540 nm with excitation at 470 nm. The detection limits for amino acids were in the range of 2.8-20 fmol. The calibration curves were linear over the range of 20 fmol to 20 pmol on column with the correlation coefficients of 0.999. The coefficients of variation were less than 5% at 3 pmol injection for all amino acids. Amino acids in rat plasma were determined by the proposed HPLC method.     

Resolution of amino acids by a native-cellulose column

Seventeen aliphatic, hydroxyl, sulfur-containing, aromatic, imino, and heterocyclic DL-amino acids were resolved without derivatization using a native-cellulose column. Chirality was assigned by either CD spectra or co-chromatography. Under these conditions, it is known that the hydrophobic interaction of amino acids with the hydroxyl groups on the cellulose played an important role in the separations. The cellulose column chromatographic system for resolution of amino acid enantiomers was combined with an amino acid analyzer. Thus, the DL-amino acids separated by the latter system were successively resolved by the former.     

OPA柱前衍生反相高效液相色谱法测定氨基酸含量

建立了邻苯二甲醛（ＯＰＡ）手动柱前衍生反相高效液相色谱法测定样品中氨基酸含量的方法。以邻苯二甲醛（ＯＰＡ）／３－巯基丙酸（３－ＭＰＡ）为衍生试剂进行衍生,ＯＤＳ柱分离,３４０ｎｍ检测,在４０ｍｉｎ内１８种氨基酸全部得到基线分离。测定牛血清白蛋白（ＢＳＡ）的氨基酸组成和小鼠血清中的游离氨基酸,取得满意的结果。     

Simple and rapid quantitative determination of lysinoalanine and protein hydrolysate amino acids by high-performance liquid chromatography after derivatization with dansyl chloride

Summary A rapid and simple method for the determination of both lysinoalanine (LAL) and protein hydrolysate amino acids after derivatization with dansyl chloride (5-dimethylaminoaphtalene-1sulfonyl chloride) and separation with RP-HPLC (UV detection) is presented. LAL is analysed in less than 15 minutes and complete separation of 22 amino acids is achieved in less than 30 minutes using single linear gradients of solvents (phosphate buffer and acetonitrile). Quantitative results obtained by HPLC compare well with results of the ion-exchange chromatography (amino acid analyser). The importance of the duration of the derivatization reaction and of the excess of reagent is discussed. As examples, the results of the determination of LAL in two samples of base treated α-casein and 22 samples of soy protein and the results of the analysis of amino acids in two balanced diet mixtures are presented. Presented at the 15th International Symposium on Chromatography, Nürnberg, October 1984     

Solid-phase reagent containing the 3,5-dinitrophenyl tag for the improved derivatization of chiral and achiral amines, amino alcohols and amino acids in high-performance liquid chromatography with ultraviolet detection

A solid-phase reaction technique is described for improved derivatization of aliphatic amines, amino alcohols and amino acids. A polymeric activated ester is used for the immobilization of the 3,5-dinitrobenzoyl group, which can then be used for derivatizations of strong or weak nucleophiles, while avoiding solution-phase derivatization conditions. The reagent is easily prepared and can be regenerated after use to attain its original reactivity. The resulting chromatograms are free of system peaks due to excess derivatizing reagent, and sample handling is kept to a minimum. The reagent can be used in conjunction with both reversed- and normal-phase chromatography and can be used for off-line gas chromatographic or high-performance liquid chromatographic (HPLC) derivatizations. In addition, the reagent can be used on-line for derivatization in HPLC. Since the labelling reagent is a strong pi-acid, chiral substrates can be derivatized and separated on a Pirkle-type pi-donor column. The confirmation and quantitation of amphetamine in urine was accomplished using a polymer containing two labelling moieties, p-nitrobenzoyl and 3,5-dinitrobenzoyl. The derivatization and separation of chiral and achiral amines, amino alcohols and amino acids is described.     

Determination of aromatic and branched-chain amino acids in plasma by HPLC with electrogenerated Ru(bpy)3(3+) chemiluminescence detection

An HPLC method is described for the electrochemiluminescence (ECL) detection of amino acids, following cycloaddition reaction of their amino groups with divinyl sulfone (DVS), using electrogenerated tris(bipyridine)ruthenium(III). The derivatization reaction conditions were examined, with the optimum conditions found to be 40 mM DVS (pH 8.0) at 50 degrees C for 15 min. Detection limits for the 15 amino acids examined varied greatly (0.04-8.0 pmol) using a standard solution by flow injection analysis (FIA). These optimized conditions were used for HPLC determination of the amino acids in human plasma. A linear relationship was obtained up to 100 pmol on a column for aromatic and branched-chain amino acids. Recoveries of Tyr, Met, Val, Leu, Ile, Phe and Trp when added to human plasma (1 micromol/10 ml plasma, n=5) were 101.5+/-1.1, 99.0+/-1.2, 98.0+/-1.4, 101.1+/-1.6, 95.1+/-1.6, 99.2+/-1.5 and 97.7+/-1.3 % (mean+/-S.D.) respectively. The concentrations of the amino acids in the plasma are in good agreement with other published data.     

HPLC assay of underivatized free amino acids with column switching and evaporative light-scattering detection

In this communication the authors report the HPLC SEPARATION OF 20 amino acids without derivatization. Separation was achieved by column swithing using a C-8 reversed-phase column and a cationic ion-exchange column. Detection of the underivatized amino acids was accomplished by means of an evaporative laser light-scattering (ELS) detector. The influence of the temperature of the diffusion tube and nebuolzation gas pressure on detector response is reported. The relationship of peak areas vs. mass show a good log-log linearity within a 1:10 range. Detechtion limit is in the order of 200 pmol for most amino acids under the conditions used.     

Automated precolumn derivatization system for analyzing physiological amino acids by liquid chromatography/mass spectrometry

An automated method for high‐throughput amino acid analysis, using precolumn derivatization high‐performance liquid chromatography/electrospray mass spectrometry (HPLC/ESI‐MS), was developed and evaluated. The precolumn derivatization step was performed in the reaction port of a home‐built auto‐sampler system. Amino acids were derivatized with 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate, and a 3 μm Wakosil‐II 3C8‐100HG column (100 × 2.1 mm i.d.) was used for separation. To achieve a 13 min cycle for each sample, the derivatization and separation steps were performed in parallel. The results of the method evaluation, including the linearity, and the intra‐ and inter‐precision, were sufficient to measure physiological amino acids in human plasma samples. The relative standard deviations of typical amino acids in actual human plasma samples were below 10%. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of conditions for the HPLC determination of plasma amino acids using precolumn derivatization

For the determination of free amino acids in plasma, the conditions for precolumn derivatization of the amino acids and the chromatographic separation were examined. The isoindole products, formed by the reaction of the primary amino acids with orthophthalaldehyde (OPA), were readily separated by RPLC and detected spectrofluorometrically using an excitation wave-length of 300 nm and an emission cut-off filter of 440 nm. Since the sensitivity of this method permits determination of amino acids in the femtomole range, the analysis can be performed on samples as small as 10 μl of filtered plasma or serum. The separation is achieved in approximately 35 minutes with good precision for the majority of the amino acids.     

Sensitive analysis of amino acids in injection liquor by reversed-phase HPLC

A convenient derivatization method of amino acids with l-fluoro-2,4-dimtrobenzene as reaction reagent and a separation system were described. The derivative amino acids were separated on a specific chemically bonded phase column with a simple linear gradient elution consisting of aqueous buffer and methanol. The eluate was detected by common ultraviolet absorption detector at 360 nm. The detection limits of amino acids were as low as 10 picomole. This method has been successfully applied to assay amino acid injection liquor used in hospital. It has good repro-ducibility and precision. The procedures avoid the requirements of particular derivative equipment and analyzer employed in conventional amino acid analysis.     

Automated phenylthiocarbamyl amino acid analysis of carboxypeptidase/aminopeptidase digests and acid hydrolysates

A fully automated exopeptidase digestion procedure for the partial determination of N- and C-terminal peptide/protein sequence is described. The digestion of various substrates with aminopeptidase M, carboxypeptidase A, P or Y was accomplished with the Varian 9090 autosampler's robotic automix routines. The released free amino acids, in addition to free amino acids from acid hydrolysates, were derivatized with phenylisothiocyanate in an automated fashion and subsequently chromatographed on a C18 column for separation and quantitation. The advantages of automating this precolumn phenylisothiocyanate derivatization are the virtual elimination of sample manipulation errors and very reproducible data due to the precise control of the reaction conditions both of which, facilitate the interpretation of the exopeptidase reaction kinetic data.     

Simultaneous separation and quantitation of amino acids and polyamines of forest tree tissues and cell cultures within a single high-performance liquid chromatography run using dansyl derivatization

The objective of the present study was to develop a rapid HPLC method for simultaneous separation and quantitation of dansylated amino acids and common polyamines in the same matrix for analyzing forest tree tissues and cell cultures. The major modifications incorporated into this method as compared to previously published HPLC methods for separation of only dansyl amino acids include: use of a 10 cm column to reduce the total run time by approximately 15 min; modification of the dansyl derivatization process and gradient profile to elute amino acids and common polyamines within the same run; addition of steps for column cleaning within each run; shorter re-equilibration time; and finally, column cleaning and physically reversing the column at the end of a loop of samples. These changes improved peak resolution and increased column longevity by several-fold. Over 1000 foliar samples from mature forest trees could be analyzed with the same column as compared to only 200-250 samples before the incorporation of these changes. This method eluted 22 amino acids within 40 min plus all three common polyamines between 44 and 47 min. The total run time is 53.6 min for amino acids only and 55.6 min for both amino acids and polyamines.     

Determination of amino acid neurotransmitters in rat hippocampi by HPLC‐UV using NBD‐F as a derivative

A simple, rapid and accurate high‐performance liquid chromatography method with ultraviolet–visible detection was developed for the determination of five amino acid neurotransmitters – aspartate, glutamic acid, glycine, taurine and γ‐aminobutyric acid – in rat hippocampi with pre‐column derivatization with 4‐fluoro‐7‐nitrobenzofurazan. Several conditions which influenced derivatization and separation, such as pH, temperature, acetonitrile percentage mobile phase and flow rate, were optimized to obtain a suitable protocol for amino acids quantification in samples. The separation of the five neurotransmitter derivatives was performed on a C₁₈ column using a mobile phase consisting of phosphate buffer (0.02 mol/L, pH 6.0)–acetonitrile (84:16, v/v) at a flow rate of 1.0 mL/min with the column temperature at 30°C. The detection wavelength was 472 nm. Without gradient elution, the five neurotransmitter derivatives were completely separated within 15 min. The linear relation was good in the range from 0.50 to 500 µmol/L, and the correlation coefficients were ≥0.999. Intra‐day precision was between 1.8 and 3.2%, and inter‐day precision was between 2.4 and 4.7%. The limits of detection (signal‐to‐noise ratio 3) were from 0.02 to 0.15 µmol/L. The established method was used to determine amino acid neurotransmitters in rat hippocampi with satisfactory recoveries varying from 94.9 to 105.2%. Copyright © 2013 John Wiley & Sons, Ltd.     

Analysis of amino acids as formamidene butyl esters by electrospray ionization tandem mass spectrometry.

Amino acid formamidene butyl esters are optimally prepared by heating amino acids with dimethylformamide dimethylacetal (DMF-DMA) for 2 minutes at 65 degrees C and then with n-butanol/hydrogen chloride for 15 minutes at 65 degrees C. The formamidene butyl esters of simple alpha-amino acids and beta-amino acids afford between 1.1 and 20 times the signal intensity of the corresponding butyl esters during electrospray ionization tandem mass spectral analysis. Complex alpha-amino acids, such as ornithine, arginine and citrulline, and gamma-amino acids are better analyzed as butyl esters. Secondary alpha-amino acids, such as proline and sarcosine, give a mixture of two iminium salts with the DMF-DMA derivatization method. A derivative incorporating two molecules of the amino acid predominates at higher derivatization temperatures. Contrary to a previous report, N-formylamino acids were not detected. The presence of secondary amino acids in amino acid mixtures, derivatized as formamidene butyl esters, affords derivatives that incorporate two different amino acids. The new formamidene butylation method is unlikely to replace the butylation procedure used by neonatal blood spot screening programs for amino acid disorders, since a much poorer response was obtained with formamidene butyl esters of arginine and citrulline, important in the diagnosis of arginase deficiency and citrullinaemia.     

Resolution of amine enantiomers using precolumn derivatization with dansyl-L-proline and reversed-phase liquid chromatography

A derivatization procedure was developed for converting enantiomeric amino acids, amino alcohols and amines into diastereoisomers for resolution by liquid chromatography. Dansyl-L-proline (DLP) was used as a chiral reagent for the precolumn derivatization of many enantiomeric compounds bearing primary and secondary amino groups. The diastereoisomeric amides that were formed in the presence of diethyl phosphorocyanidate can be resolved efficiently on a conventional reversed-phase column. The resolution was optimized by varying the mobile phase. Intramolecular hydrogen bonding of the diastereoisomeric amide is shown to be very important for the efficient resolution of enantiomers. The detection limits for DLP derivatives were 4 × 10^?14?5 × 10^?14 mol.     

Gas-liquid chromatographic analysis of protein amino acids as N-isobutyloxycarbonylamino acid methyl esters.

A practical method for the quantitative determination of protein amino acids by gas-liquid chromatography (GLC) is described. All of the common protein amino acids except arginine can be readily converted into their N-isobutyloxycarbonyl (N-isoBOC) methyl ester derivatives by a simple procedure involving isobutyloxycarbonylation with isobutyl chloroformate in aqueous medium, followed by methylation with diazomethane. Arginine was converted into N-isoBOC ornithine methyl ester by treatment with arginase, followed by the above derivatization procedure. The resulting N-isoBOC methyl esters of the amino acids have good GLC properties. Complete resolution of the derivatives of 20 protein amino acids was achieved by using a dual-column system consisting of a 0.65% Poly-A-101A column and a 0.70% FFAP-Poly-A-101A (1:1, w/w) column. The reproducibility of response was found to be good for derivatives carried through the entire chemical and chromatographic procedure. The calibration graphs were linear and showed no statistical bias. The results of recovery experiments with synthetic mixtures containing known amounts of the amino acids were satisfactory, the recoveries ranging from 94.3 to 106.2%.     

Analysis of cysteine-containing proteins using precolumn derivatization with <Emphasis Type="Italic">N</Emphasis>-(2-ferroceneethyl)maleimide and liquid chromatography/electrochemistry/mass spectrometry

N-(2-Ferroceneethyl)maleimide (FEM) is introduced as an electroactive derivatizing agent for thiol functionalities in proteins. Using appropriate reaction conditions, the derivatization is completed within five minutes and no unspecific labeling of free amino functions is observed. Liquid chromatography/electrochemistry/mass spectrometry was used to detect the reaction products. The reagent is a useful tool for determining the number of free thiol groups or the total number of free and disulfide-bound thiol groups in proteins. The electrochemical cell provides additional information, because the increase in mass spectrometric response upon electrochemical oxidation of the neutral ferrocene to the charged ferrocinium groups is monitored. The method was successfully applied to the analysis of native proteins and their tryptic digests. Dedicated to Prof. Werner Engewald on the occasion of his 70th birthday.