Rapid analysis of amino acids using pre-column derivatization

A new approach to the pre-column derivatization and analysis of amino acids is described. The method is based upon formation of a phenylthiocarbamyl derivative of the amino acids. The derivatization method is rapid, efficient, sensitive, and specific for the analysis of primary and secondary amino acids in protein hydorlyzates. The liquid chromatographic system allows for the rapid, bonded-phase separation with ultraviolet detection of the common amino acids with 12-min analysis time and a 1-pmol sensitivity.     

An improved chemical approach toward the C-terminal sequence analysis of proteins containing all natural amino acids.

An improved chemical method, capable of derivatizing all natural amino acids to their corresponding thiohydantoins, is described. This involves activation by acetyl chloride in TFA followed by derivatization with ammonium thiocyanate. Possible interference of reactive side chains was investigated by reacting N-acetylamino acids as well as several peptides with propionyl chloride instead of acetyl chloride. The products were characterized by PDMS mass spectrometry and 1H-NMR. This chemical method allows, for the first time, complete derivatization of N-acetylproline to proline thiohydantoin. Applying this chemistry to peptides with a C-terminal proline, the yields for formation of proline thiohydantoin were found to be up to 60%, depending on the peptide sequence. The previous inability to derivatize C-terminal proline to thiohydantoin was thought to stem from the fact that proline cannot form the oxazolonium ion required for efficient reaction with the thiocyanate ion. However, we have found mass spectrometric evidence for the existence of a proline oxazolonium ion, under basic as well as under acidic conditions. This improvement in derivatization of C-terminal amino acids including proline is a major step forward in the development of a general chemical C-terminal sequencing method that permits the C-terminal sequence analysis of proteins of any amino acid composition.     

Amino acid analysis using phenylisothiocyanate prederivatization: elimination of the drying steps

Results of experiments on the procedure for amino acid analysis via analysis of the phenylthiocarbamyl amino acids are reported. It was found that yields of some amino acids varied in the presence of salt and with changes in the vacuum drying steps. An improved procedure is described which includes a standard addition of salt to the hydrolysate before drying it; the redrying step is omitted and the post derivatization drying is replaced by a simple addition of heptane to the reaction mixture.     

A novel chiral thiol reagent for automated precolumn derivatization and high-performance liquid chromatographic enantioseparation of amino acids and its application to the aspartate racemase assay

A novel optically active thiol compound, N-(tert-butylthiocarbamoyl)-L-cysteine ethyl ester (BTCC), is synthesized as a chiral derivatization reagent. This compound and o-phthalaldehyde react with amino acid enantiomers to produce fluorescent diastereomers that are readily separable on a reverse-phase column by HPLC. Enantioseparation of acidic amino acids in particular is markedly improved using BTCC. In this study, the HPLC method for enantioseparation with the novel compound is applied to the aspartate (Asp) racemase assay. Derivatized D-Asp is eluted before the L-Asp derivative. Consequently, a small amount of D-Asp produced by the activity of racemase on a large quantity of L-Asp substrate may be quantified accurately, even at very low activity. Since the derivatization reaction proceeds rapidly at room temperature, a fully automated system is established for derivatization and sample injection. The automated method is practical and successfully applied to the archaeal Asp racemase assay. We presume that the procedure is additionally applicable to the enantioseparation of other amino acids, amino alcohols, and catecholamines.     

Polymeric 6-aminoquinoline, an activated carbamate reagent for derivatization of amines and amino acids by high-performance liquid chromatography

A new polymeric reagent containing the 6-aminoquinoline (6-AQ) tag was developed and applied for the off-line derivatization of amines and amino acids in high-performance liquid chromatography (HPLC). The synthesis and characterization of this polymeric reagent are described. An authentic external standard of a typical amine was synthesized and characterized for the determination of the derivatization efficiency. All amines had a derivatization efficiency higher than 50%; the derivatization of amino acids was performed under optimized phase-transfer catalysis reaction conditions. Derivatized amines and amino acids were separated under conventional reversed-phase conditions and determined by UV and FL detectors. To investigate the practical applications, this polymeric reagent was also used to derivatize protein hydrolysates.     

Capillary electrophoresis of phosphorylated amino acids with fluorescence detection

A rapid and sensitive capillary electrophoresis (CE) method coupled with fluorescence detection was developed for identification of protein phosphorylation by determination of phosphoamino acids. Naphthalene-2,3-dicarboxaldehyde (NDA), a fluorescence derivatization reagent, was used to label protein hydrolysate. The optimal derivatization reaction was performed with 3.5mM NDA, 40 mM NaCN and 20mM borate buffer (pH 10.0) for 15 min. The baseline separation of three phosphorylated amino acids could be obtained in less than 180 s with good repeatability by using 30 mM borate (pH 9.2) containing 2.0mM beta-cyclodextrin (beta-CD) as the running buffer. The detection limits for phosphothreonine, phosphotyrosine and phosphoserine were 7.0 x 10(-9)M, 5.6 x 10(-9)M and 7.2 x 10(-9)M, respectively (S/N=3). Also, the interference from other protein amino acids with large molar excess over that of phosphoamino acids was studied. With beta-casein as the analysis protein, this method was successfully validated.     

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry-based amino acid analysis

Amino acid analysis has been an integral part of analytical biochemistry for more than 50 years. However, its experimental design, which includes derivatization of amino acids followed by some kind of chromatographic separation, has not changed over the years. We have developed a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF)-based method for the quantitative analysis of amino acids. This method does not require any amino acid modification, derivatization, or chromatographic separation. The data acquisition time is decreased to several seconds for a single sample. No significant ion suppression effects were observed with the developed sample deposition technique, and the method was found to be reproducible. Linear responses between the amino acid concentration and the peak intensities ratio of corresponding amino acid to internal standard were observed for all amino acids analyzed in the range of concentrations from 20 to 300 microM, and correlation coefficients were between 0.983 (for arginine) and 0.999 (for phenylalanine). Limits of quantitation were between 0.03 microM (for arginine) and 3.7 microM (for histidine and homocysteine). This method was applicable to the mixtures of free amino acids as well as to HCl hydrolysates of proteins. Furthermore, we have shown that this method can be applied to other biologically important low-molecular weight compounds such as glucose.     

Analysis of Free Amino Acids in Microbially Colonized Sandstone by Precolumn Phenyl Isothiocyanate Derivatization and High-Performance Liquid Chromatography

A fast, sensitive method for extraction and analysis of soluble free amino acids from microbially colonized sandstone is described. After precolumn phenyl isothiocyanate (PITC) derivatization, the PITC-amino acids were identified and quantified by reversed-phase high-performance liquid chromatography. This kind of analysis could be used to elucidate the role and function of amino acids in the nutrition of epi- and endolithic microorganisms active in biological weathering processes.     

High-sensitivity amino acid analysis by derivatization with O-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: applications in protein structure determination

An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.     

Pre-column derivatization high-performance liquid chromatographic method for determination of cysteine, cysteinyl–glycine, homocysteine and glutathione in plasma and cell extracts

A sensitive high-performance liquid chromatographic method for quantification of sulphydryl and disulfide amino acids in human plasma using ultra violet spectrophotometric detection was developed. Precolumn derivatization with 5,5′-dithio-bis-nitrobenzoic acid (DTNB) and an optional pre-derivatization reaction with dithiothreitol allowed both quantitative reduction of disulfides for measurement of total amino acid levels and the measurement of the reduced forms. A dynamic range of 500 nmol/l–750 μmol/l allowed the major analytes of interest to be quantified in plasma without sample dilution. The assay is a sensitive and precise method for the determination of sulphydryl and disulfide amino acids in plasma and cell extracts.     

Studies on the coupling rates in liquid-phase peptide synthesis using competition experiments.

Competition experiments were carried out in order to determine relative reaction rates, Vrel, for the peptide-forming step. Using the liquid-phase method of peptide synthesis a model system was developed to investigate the influence of coupling method, amino component, derivatization and solvent upon Vrel. According to a standard procedure, Vrel for all 20 commonly occurring amino acids were established. A strong dependence of Vrel upon steric effects of the coupling components was found. The data obtained may serve as a guideline for optimizing the reaction conditions in peptide synthesis.     

A comparison of fluorescamine and naphthalene-2,3-dicarboxaldehyde fluorogenic reagents for microplate-based detection of amino acids.

The use of appropriate fluorometric derivatization procedures is of considerable importance for accurate determination of amino acids in biological samples and in metal-assisted peptide hydrolysis reactions. It is especially critical for the relative fluorescence intensities (RFI) of equal amounts of amino acids to be as similar as possible. While fluorescamine and naphthalene-2,3-dicarboxaldehyde (NDA) have proven to be excellent fluorogenic reagents for amino acid detection, the effects of various factors such as organic solvent, buffer, and pH have never been rigorously evaluated with respect to normalizing the relative fluorescence intensities of individual amino acids. To this end, here we describe optimized fluorescamine and NDA derivatization reactions that enhance the accuracy of microplate-based detection of amino acids. For both fluorescamine and NDA, we have shown that the RFI values of 16 of 19 amino acids are greater than 70%. Although determination of tryptophan is problematic, this difficulty is overcome by the addition of beta-cyclodextrin to the NDA reaction. In principle, the optimized fluorescamine and NDA microplate procedures reported here can be utilized as complementary techniques for the detection of 19 of 20 naturally occurring amino acids.     

Gas—liquid chromatography of isobutyl ester,N(O)-heptafluorobutyrate derivatives of amino acids on a glass capillary column for quantitative separation in clinical biology

A gas chromatographic method adapted to routine analysis has been developed for quantitative separation on glass capillary columns for free proteic and other known amino acids normally or abnormally found in physiological fluids. The procedure involves ion-exchange chromatography and isobutyl ester, N(O)-heptafluorobutyrate derivatization of free plasma and urine amino acid samples. Derivatized components were ascertained by combined gas chromatography—mass spectrometry. The use of glass for the capillary column is mandatory to achieve qualitative and quantitative analysis of the known occurring amino acids in urine and small plasma samples. Quantitative analysis of several types of human amino acid disorders are presented.     

Measurement of free amino acid levels in ultrafiltrates of blood plasma by high-performance liquid chromatography with automatic pre-column derivatization

Although there are many techniques available for the analysis of amino acids, deproteinization is still one of the major problems in the analysis of amino acids in physiological fluids. The method used to prepare the plasma and to remove the plasma protein has a marked effect on the final results. The most widely used method of deproteinization is precipitation with 5-sulphosalicylic acid followed by centrifugation to remove the precipitated protein. We have not had success in using this deproteinization agent for the analysis of plasma amino acids by a high-performance liquid chromatographic method with automatic pre-column o-phthaldialdehyde—3-mercaptopropionic acid and 9-fluorenylmethyl chloroformate derivatization because of the adverse effect of the sulphosalicyclic acid supernatant on the quantitation and separation. Ultrafiltration was used as an alternative method for the preparation of plasma samples in this experiment. The results were satisfactory for the analysis of plasma amino acids in 1500 samples during a period of four years. Some factors that might influence the results of the ultrafiltration were investigated.     

Application of amino acid analysis by high-performance liquid chromatography with phenyl isothiocyanate derivatization to the rapid determination of free amino acids in biological samples

An amino acid analysis by reversed-phase high-performance liquid chromatography after precolumn derivatization with phenyl isothiocyanate was adapted to the determination of free amino acids in plasma or other biological fluids and in tissue homogenates. Preparation of samples included deproteinization by 3% sulphosalicylic acid, and careful removal under high vacuum of residual phenyl isothiocyanate after derivatization. A Waters Pico-Tag column (15 cm long) was used, immersed in a water-bath at 38°C. In rat or human plasma, separation of 23 individual amino acids, plus the unresolved pair tryptophan and ornithine, was obtained within 13 min. Including the time for column washing and re-equilibration, samples could be chromatographed at 23-min intervals. Variability was tested for each amino acid by calculating the coefficients of variation of retention times (less than 1% in the average) and peak areas (less than 4% for both intra-day and inter-day determinations). The linearity for each standard amino acid was remarkable over the concentration range 3–50 nmol/ml. The mean recovery of amino acid standards added to plasma prior to derivatization was 97 ± 0.8%, except for aspartate (82%) and glutamate (81%). This method is rapid (almost three samples per hour can be analysed, more than in any other reported technique), with satisfactory precision, sensitivity and reproducibility. Therefore, it is well suited for routine analysis of free amino acids in both clinical and research work.     

High-performance liquid chromatography determination of pipecolic acid after precolumn ninhydrin derivatization using domestic microwave

A novel procedure to specifically quantify low amounts of pipecolic acid and structurally related compounds in several types of biological materials has been characterized. From crude extracts of various types of biological material, the first step was to clear all low-molecular-weight compounds containing primary amino groups by a treatment of nitrous acid. Using a microwave-assisted reaction, the remaining substances containing secondary amino groups were then derivatized with ninhydrin and made soluble in glacial acetic acid. The derivatives produced were resolved by reverse-phase HPLC and detected by spectrophotometry at 570nm. This procedure allowed more rapid determination of pipecolic acid since microwave heating shortened the time needed for derivatization compared with heating at 95 degrees C in a water bath. The complete analysis of the chromogens for pipecolic acid and related substances was achieved in 20min. Under such conditions, the detection threshold for pipecolic acid was about 20pmol. The suitability of the technique was assessed in various biological matrices known to contain significant amounts of this amino acid. The data obtained are in accordance with those available in the literature. To our knowledge, this is the first method using the ninhydrin reaction in a precolumn, microwave-assisted derivatization procedure for detection and determination of heterocyclic alpha-amino acids.     

Determination of amino acids in saliva using capillary electrophoresis with fluorimetric detection

In the present study a sensitive method for the quantification of main free amino acids in saliva using capillary electrophoresis with laser induced fluorescence detection was developed. As background electrolyte 20 mM borate buffer pH 9.5 was used. Amino acids were separated after derivatization with fluorescein isothiocyanate (FITC) and the conditions for derivatization were optimized. The main amino acids occurring in saliva (Pro, Ser, Gly and Glu) were separated in less than 7 min. The parameters of validation such as linearity of response, precision and detection limits were determined. The detection limits were obtained in the range from 0.1 to 2.4 nM. The developed method was employed for determination of amino acids in real saliva samples.     

New preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for fatty acids and derivatives

A preparation method for 9-anthryldiazomethane (ADAM) as a fluorescent labeling reagent for carboxylic acids is described. 9-Anthraldehyde hydrazone is oxidized with an organic oxidant, N-chlorosuccinimide, in an organic solvent such as ethyl acetate to give ADAM, and then the reaction mixture is directly used as the reagent solution for the derivatization of carboxylic acids. Both the oxidation and the derivatization reaction are carried out at room temperature, and an aliquot of the derivatization mixture is directly injected into a chromatograph. 9-Anthrylmethyl ester derivatives formed from ADAM and various carboxylic acids are sufficiently separated on a reversed-phase column and are sensitively detected fluorometrically. The present method was applied to the high-performance liquid chromatographic determination of long and short chain fatty acids, keto acids, and hydroxy acids.     

GC-MS analysis of amino acid enantiomers as their N(O,S)-perfluoroacyl perfluoroalkyl esters: application to space analysis

The target of the in-situ research of optical activity in extraterrestrial samples stimulated an extended investigation of a GC-MS method based on the derivatization of amino acids by using a mixture of perfluorinated alcohols and perfluorinated anhydrides. Amino acids are converted to their N(O,S)-perfluoroacyl perfluoroalkyl esters in a single-step procedure, using different combinations of the derivatization reagents trifluoroacetic anhydride (TFAA)-2,2,2-trifluoro-1-ethanol (TFE), TFAA-2,2,3,3,4,4,4-heptafluoro-1-butanol (HFB), and heptafluorobutyric anhydride (HFBA)-HFB. The derivatives obtained are analyzed using two different chiral columns: Chirasil-L-Val and gamma-cyclodextrin (Rt-gamma-DEXsa) stationary phases which show different and complementary enantiomeric selectivity. The mass spectra of the derivatives are studied, and mass fragmentation patterns are proposed: significant fragment ions can be identified to detect amino acid derivatives. The obtained results are compared in terms of the enantiomeric separation achieved and mass spectrometric response. Linearity studies and the measurement of the limit of detection (LOD) show that the proposed method is suitable for a quantitative determination of enantiomers of several amino acids. The use of the programmed temperature vaporiser (PTV) technique for the injection of the untreated reaction mixture is a promising method for avoiding manual treatment of the sample and decreasing the LOD.     

Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde

A sensitive and reproducible fully automated method for the determination of amino acids in plasma based on reversed-phase high-performance liquid chromatography and o-phthaldialdehyde pre-column derivatization is described. A 5-μm Spherisorb ODS 2 column (125 × 3 mm I.D.) was selected for routine determination. Over 40 physiological amino acids could be determined within 49 min (injection to injection) and 48 samples could be processed unattended. The coefficients of variation for most amino acids in plasma were below 4%. We were also able to measure trace amounts of amino acids in plasma normally not detected in a routine analysis. The results obtained with the method described compared favourably with those of conventional amino acid analysis (r = 0.997) and were in excellent agreement with those of other laboratories (r = 0.999).