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-sulphosalicyclic 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.     

Simultaneous analysis of free amino acids and biogenic amines in honey and wine samples using in loop orthophthalaldeyde derivatization procedure

This work presents a RP-HPLC method for the simultaneous quantification of free amino acids and biogenic amines in liquid food matrices and the results of the application to honey and wine samples obtained from different production processes and geographic origins. The developed methodology is based on a pre-column derivatization with o-phthaldialdehyde carried out in the sample injection loop. The compounds were separated in a Nova-Pack RP-C(18) column (150 mm x 3.9 mm, 4 microm) at 35 degrees C. The mobile phase used was a mixture of phase A: 10 mM sodium phosphate buffer (pH 7.3), methanol and tetrahydrofuran (91:8:1); and phase B: methanol and phosphate buffer (80:20), with a flow rate of 1.0 ml/min. Fluorescence detection was used at an excitation wavelength of 335 nm and an emission wavelength of 440 nm. The separation and quantification of 19 amino acids and 6 amines was carried out in a single run as their OPA/MCE derivatives elute within 80 min, ensuring a reproducible quantification. The method showed to be adequate for the purpose, with an average RSD of 2% for the different amino acids; detection limits varying between 0.71 mg/l (Asn) and 8.26 mg/l (Lys) and recovery rates between 63.0% (Cad) and 98.0% (Asp). The amino acids present at the highest concentration in honey and wine samples were phenylalanine and arginine, respectively. Only residual levels of biogenic amines were detected in the analysed samples.     

Liquid chromatographic fluorescence determination of amino acids in plasma and urine after derivatization with phanquinone

Phanquinone (4,7-phenanthroline-5,6-dione) has been investigated as a pre-column derivatization fluorogenic reagent for liquid chromatographic determination of primary amino acids in biological samples. The derivatization reaction was carried out at 68 degrees C both in the presence of aqueous phosphate buffer (pH 8) for 30 min and without buffer for 60 min to allow the determination of basic amino acids (Orn, Lys, Arg). The resulting derivatives were separated under reversed-phase HPLC and detected at lambda(em) = 460 nm with lambda(ex) = 400 nm. The proposed method was validated and applied to the determination of a variety of amino acids directly in urine and after deproteinization with 5-sulfosalicylic acid in plasma samples. The detection and quantitation limits were found in the range 10-450 and 35-1400 fmol, respectively.     

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 degrees 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.     

Simultaneous determination of 23 amino acids and 7 biogenic amines in fermented food samples by liquid chromatography/quadrupole time-of-flight mass spectrometry

A novel liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS) method was developed for the simultaneous determination of 23 amino acids and 7 biogenic amines in food samples. These analytes were pre-column derivatized with dansyl chloride and then separated in an Acquity column (1.7 μm; 2.1 mm × 100 mm). The separation of 31 compounds including an internal standard was achieved within 25 min at a flow rate of 0.2 mL/min. The method linearity for each amino acid and biogenic amine had a relatively wide range with r(2)>0.99. The intra- and inter-day precision, expressed as relative standard deviation (RSD), ranged from 1.1 to 4.6% and from 2.0 to 11.2%, respectively. The limit of detection was between 0.005 and 0.4 μg/mL. With a simple dilution, recoveries of around 80-120% were obtained for most of the compounds. No significant matrix effect was observed, and the developed method was successfully applied to the analysis of amino acids and biogenic amines in beer, cheese and sausage samples.     

Quantitation of amino acids in plasma by high performance liquid chromatography: Simultaneous deproteinization and derivatization with 9-fluorenylmethyloxycarbonyl chloride

This paper, as a novelty to this field, presents the deproteinization and derivatization of plasma's free amino acids (PFAAs), simultaneously, in a single step, with the acetonitrile (ACN) containing 9-fluorenylmethyloxycarbonyl chloride (FMOC) reagent. Deproteinization and derivatization, were studied with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection, simultaneously. Model investigations have been carried out as a function of the FMOC concentration, reaction time and reaction conditions: with standard solutions, with human plasma samples in its initial condition and fortified with standard amino acids (excluding tryptophan because it co-elutes with the hydrolyzed FMOC). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 3.0 mM FMOC concentration, at pH 9; derivatization time = 20 min), and characterized with the relative standard deviation percentages of their responses (≤4.4%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 2.5 pmol, except for cystine, ornithine (5 pmol) and for the total of tyrosines (N-FMOC-tyrosine and N,O-FMOC-tyrosine 10 pmol).     

Analytical potential of 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein for the determination of amino compounds by capillary electrophoresis with laser-induced fluorescence detection

The analytical potential of a fluorescein analogue, 6-oxy-(N-succinimidyl acetate)-9-(2'-methoxycarbonyl) fluorescein (SAMF), for the first time synthesized in our laboratory, as a labeling reagent for the labeling and determination of amino compounds by capillary electrophoresis (CE) with laser-induced fluorescence (LIF) detection was investigated. Biogenic monoamines and amino acids were chosen as model analytes to evaluate the analytical possibilities of this approach. The derivatization conditions and separation parameters for the biogenic amines were optimized in detail. The derivatization was performed at 30 degrees C for 6 min in boric acid buffer (pH 8.0). The derivatives were baseline-separated in 15 min with 25 mM boric acid running buffer (pH 9.0), containing 24 mM SDS and 12.5% v/v acetonitrile. The concentration detection limit for biogenic amines reaches 8 x 10(-11) mol.L(-1) (signal-to-noise ratio = 3). The application of CE in the analysis of the SAMF-derivatized amino acids was also exploited. The optimal running buffer for amino acids suggested that weak acidic background electrolyte offered better separation than the basic one. The proposed method was applied to the determination of biogenic amines in three different beer samples with satisfying recoveries varying from 92.8% to 104.8%. Finally, comparison of several fluorescein-based probes for amino compounds was discussed. With good labeling reaction, excellent photostability, pH-independent fluorescence (pH 4-9), and the resultant widely suited running buffer pH, SAMF has a great prospect in the determination of amino compounds in CE.     

Value of high-performance liquid chromatographic analysis of amino acids in the determination of Panax ginseng radix extract effect in cultured neurons

The present research describes a reversed-phase high-performance liquid chromatographic (RP-HPLC) method that allows the determination of several amino acids in primary cultured cortical neurons of rats. The concentration of amino acids was determined by using pre-column derivatization with dansyl chloride and UV-diode array detection. Data show that Panax ginseng radix extract (GS) can modulate amino acid release in neurons. The levels of glutamate (Glu), aspartate (Asp), gamma-aminobutyric acid (GABA) and glycine (Gly) in the GS-treated groups were higher than in the non-treated groups dose-dependentwise. In this case, Glu and GABA were the most released amino acids (74.43% +/- 0.97 and 88.41% +/- 4.12 at ginseng dose 0.01 mg/ml after 1h from treatment, respectively). The values obtained in the determination of the analytical parameters (linearity, precision, limit of detection and accuracy) confirm the quality of the method. The average recoveries for intra and inter-day assay (n = 5) were 101.18 and 102.38 for Asp, 99.35 and 98.44 for Glu, 99.59 and 99.66 for Gly, and 100.06 and 100.37 for GABA. These data proved that the method yields accurate results, with RSD lower than 2.2%. The precision of the method was estimated on the basis of RSD of six injections at two different concentrations of amino acids. This technique is useful in studying the GS-mediated modulation of the dynamic equilibrium of amino acids and neurotransmission in neurons.     

Determination of free amino acids in biological samples: problems of quantitation

An automatic on-line high-performance liquid chromatographic method was developed to study the effects of various precipitating agents and delayed deproteinization procedures on the estimation of plasma levels of amino acids. The optimized method for analysis is based on pre-column derivatization with o-phthalaldehyde in the presence of 2-mercaptoethanol. The separation of 25 amino acids is accomplished within 45 min on a 5-microns C18 column, using a multi-step gradient with two solvents. The method is sensitive and reproducible, and the relationship between the fluorescence intensity and concentration is linear for each amino acid over a wide concentration range.     

HPLC Determination of Amino Acids by Pre-Column Fluorescence Derivatization with Carbazole-9-yl-Acetyl Chloride

Atpresentseparationsandquantitativedeterminationsofaminoacidsbymeansofnewfluorescencereagentsforpre-columnorpost-columnderivatizationinRP-HPLCarestillanactivefiled,developmentshavingbeensummarizedbySnyder'.MostaminoacidsdonotshowUVabsorptionin220-254urn,henceinordertoincreasedetectionsensitivityandimproveselectivity,generallyderivatizationreagentsareemployed.Phenylisothiocyanate(PITC)',OPAand3,5-dinitrobenzoylchloride3arewellknownderivatizationreagefltsforthedeterminationofaminocompounds…     

Precolumn derivatization reagents for high‐speed analysis of amines and amino acids in biological fluid using liquid chromatography/electrospray ionization tandem mass spectrometry

A rapid analytical method for amines and amino acids was developed, involving derivatization with the novel reagent 3‐aminopyridyl‐N‐hydroxysuccinimidyl carbamate (APDS), followed by reversed‐phase high‐performance liquid chromatography and electrospray ionization tandem mass spectrometry (HPLC/ESI‐MS/MS). More than 100 different analytes with amino groups, including amino acids in biological fluids such as mammalian plasma, could be measured within 10 min. The analytes were easily derivatized with APDS under the mild conditions. Selective reaction monitoring of ESI‐MS/MS in positive mode was carried out to include the transitions of all of the protonated molecular ions of analytes derivatized with APDS to the common fragment at m/z 121, which was derived from the amino pyridyl moiety of the reagent. We evaluated the retention time precision, the quantification limits, the linearity, the intra‐ and inter‐day precisions and the accuracy of 22 typical amino acids found in biological fluids, by analyzing a standard amino acid mixture and rat plasma. The intra‐day relative standard deviations (RSDs) of the retention times of the 22 amino acids and their internal standards were within 0.9% and the inter‐day RSDs were less than 1.1%, except for asparagines, with an RSD of 1.9%. The intra‐day and inter‐day RSDs of amino acid analyses in rat plasma were within 8.0% and 4.5%, respectively. The method, which facilitates the amino acid analysis of more than 100 samples in a day, represents an alternative to traditional amino acid analysis techniques, such as chromatography using postcolumn derivatization by ninhydrin. Copyright © 2009 John Wiley & Sons, Ltd.     

Analysis of amino acids and biogenic amines in biological tissues as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate derivatives by high-performance liquid chromatography. A deproteinization study

The extraction of ornithine, lysine, putrescine, cadaverine, 1,7-diaminoheptane, spermidine and spermine from biological tissues was optimized for HPLC quantitation as their o-phthalaldehyde/ethanethiol/fluorenylmethyl chloroformate (OPA/ET/FMOC) derivatives. In applying perchloric acid deproteinization two approaches have been followed: (i) deproteinization with subsequent neutralization by potassium hydroxide and lyophilization, and (ii) deproteinization without neutralization and lyophilization. Neutralization and lyophilization resulted in the loss of free biogenic amines. HPLC analysis of ornithine (Orn), lysine (Lys), putrescine (Put), cadaverine (Cad), 1,7-diaminoheptane (Dah), spermidine (Spd) and spermine (Spm) content of biological tissues as their OPA/ET/FMOC derivatives was performed in the supernatant of perchloric acid-deproteinized samples (model solutions and tissues) with an average reproducibility of < or =2.6% relative standard deviation (RSD), including recovery of sample treatment and chromatography.     

Automated capillary liquid chromatography for simultaneous determination of neuroactive amines and amino acids

A method for the separation and quantitative determination of neuroactive amino acids (aspartate, glutamate, citrulline, arginine, glycine, taurine, gamma-aminobutyric acid) and neuroactive amines (noradrenaline, dopamine and serotonin) in a single chromatographic analysis is presented. The method is based on pre-column derivatization with o-phthalaldehyde and tert.-butyl thiol, on-column preconcentration and separation using 50 microns I.D. packed capillary columns, and detection by amperometry. Mass limits of detection are 80-900 amol for all neurotransmitters with RSDs of 0.71 and 4.6% or better for retention time and peak area, respectively. The method was demonstrated by application to the determination of neurotransmitters in microdialysis samples collected from striatum of live rats and tissue samples extracted from butterfly brains.     

High-performance liquid chromatographic analysis of free amino acids in fruit juices using derivatization with 9-fluorenylmethyl-chloroformate

A simple, rapid, and reliable reversed-phase high-performance liquid chromatographic method for the analysis of 16 amino acids of main interest in commercial fruit juices (pear, orange, grapefruit, pineapple, peach, and apricot) is described. No sample cleanup is required. The pH of the fruit juices is adjusted to alkaline value (8.5) using 200 mM borate buffer, then amino acid is converted to stable derivatives using 9-fluorenylmethyl-chloroformate. The excess of derivatization reagent is removed by a hydrophobic amine, 1-amino-adamantane hydrochloride. The derivatization procedure is simple, fast, and described in detail. Amino acids are detected at 263 nm and eluted within 35 min. The calibration, precision (< or = 6.1%), and recovery (102% +/- 4%) of the method are reported. The conditions of separation are optimized; however, serine partially overlapped with aspartic acid. The amino acid profile of fruit juices is consistent with data from the literature.     

Complete separation of ephedrines by graphite-layer open-tubular (GLOT) column

Summary A reversed phase high performance liquid chromatographic (RP-HPLC) method for the analysis of amino acids in kelp, using precolumn derivatization, is described. Phenylisothiocyanate (PITC) was used as the reagent for derivatization. The kelp samples were prepared by microwave hydrolysis in only 5 min; seventeen PTC amino acids were separated after hydrolysis and derivatization within 12 min. The coefficients of variation were >1.94 and the correlation coefficients for concentration versus response were >0.999 for all derivatives. The ratio of branched amino acid (BAA) to aromatic amino acid (AAA) was also studied. The method has the advantage of shorter hydrolysis and analysis times with optimum separation. In addition, it gives high repeatability of retention times and peak areas for all the amino acids present.     

Quantitative liquid chromatographic determination of cefatrizine in serum and urine by fluorescence detection after post-column derivatization

A fast, specific and sensitive high-performance liquid chromatographic procedure for the determination of cefatrizine, an orally active cephalosporin, in serum and urine is proposed. The drug is determined by the internal standard method, using cephradine as the internal standard. The separation is carried out on a reversed-phase column, filled with octadecylsilane chemically bonded microparticles. The eluent is a mixture of acetonitrile with 0.025 M sodium phosphate buffer (pH 7). Quantitation is effected by fluorescence detection of the fluorophores formed after post-column derivatization with fluorescamine in a packed-bed reactor. The chromatographic conditions and the conditions for the post-column derivatization are discussed. The method has been applied to serum and urine samples, which were analysed after deproteinization with trichloroacetic acid and injection of the clear supernatant. The accuracy and reproducibility of the procedure were investigated by the determination of the cefatrizine content in spiked serum and urine samples.     

柱前衍生-超高效液相色谱法测定3种奶粉中18种氨基酸

近年来羊奶粉和骆驼奶粉备受消费者青睐,它们具有潜在的低致敏性,因此成为牛乳不耐受人群尤其是婴幼儿的母乳替代品,其营养价值备受关注.牛奶粉、羊奶粉和骆驼奶粉中氨基酸含量的比较研究鲜有报道.利用酸水解得到游离氨基酸,选择6-氨基喹啉-N-羟基琥珀酰亚胺氨基甲酸酯(AQC)进行柱前衍生,超高效液相色谱分离并检测,外标法定量....     

Coupling of sequential injection with liquid chromatography for the automated derivatization and on-line determination of amino acids

The principle of sequential injection (SI) was exploited to develop a fully automated pre-column derivatization procedure combined on-line to liquid chromatography (LC). Using SI-LC derivatization 14 amino acids were determined fluorimetrically in pharmaceuticals with o-phthaldialdehyde (OPA) as the derivatization reagent. The SI system was used for the handling of samples and reagents, on-line mixing and introduction to the LC injection system. Chemical (pH and reagents concentrations) and instrumental variables (sample and reagent volumes, reaction time and flow rate) were optimized to attain the highest reaction yield and detector signal. Reversed phase chromatographic resolution of 14 amino acids was achieved within 35 min using gradient elution. The automated operation of the coupled SI-LC system resulted in very satisfactory performance. The method was applied for the simultaneous determination of amino acids in pharmaceutical formulations.     

Potential of fermentation profiling via rapid measurement of amino acid metabolism by liquid chromatography-tandem mass spectrometry

Monitoring amino acid metabolism during fermentation has significant potential from the standpoint of strain selection, optimizing growth and production in host strains, and profiling microbial metabolism and growth state. A method has been developed based on rapid quantification of underivatized amino acids using liquid chromatography-electrospray tandem mass spectrometry (LC-MS-MS) to monitor the metabolism of 20 amino acids during microbial fermentation. The use of a teicoplanin-based chiral stationary phase coupled with electrospray tandem mass spectrometry allows complete amino acid analyses in less than 4 min. Quantification is accomplished using five isotopically labeled amino acids as internal standards. Because comprehensive chromatographic separation and derivatization are not required, analysis time is significantly less than traditional reversed- or normal-phase LC-based amino acid assays. Intra-sample precisions for amino acid measurements in fermentation supernatants using this method average 4.9% (R.S.D.). Inter-day (inter-fermentation) precisions for individual amino acid measurements range from 4.2 to 129% (R.S.D.). Calibration curves are linear over the range 0-300 microg/ml, and detection limits are estimated at 50-450 ng/ml. Data visualization techniques for constructing semi-quantitative fermentation profiles of nitrogen source utilization have also been developed and implemented, and demonstrate that amino acid profiles generally correlate with observed growth profiles. Further, cellular growth events, such as lag-time and cell lysis can be detected using this methodology. Correlation coefficients for the time profiles of each amino acid measured illustrate that while several amino acids are differentially metabolized in similar fermentations, a select group of amino acids display strong correlations in these samples, indicating a sub-population of analytes that may be most useful for fermentation profiling.     

Simultaneous determination of cysteine, cystine and 18 other amino acids in various matrices by high-performance liquid chromatography

An extraction protocol and a reversed-phase high-performance liquid chromatographic method for the simultaneous determination of cysteine, cystine and 18 other amino acids in biological and vegetable samples are described. Among the different methods proposed for amino acid determination, phenylisothiocyanate was used as the reagent for derivatization. Chromatograms obtained in the analysis of standard solutions and actual samples are reported, together with regression equation, correlation coefficient (> 0.999 for all), limit of detection and recoveries (between 86 and 102% for all the examined matrices) for each amino acid. Practical protocol and method applications in normal patients and patients affected by different pathologies, and in algal products are discussed.