Amino Acids In Biological Samples Research Articles

Spectrophotometric Study of Excitatory Amino Acids with Ortho-Phthalaldehyde

Background: Understanding the concentration of amino acids in biological samples is pivotal for numerous biochemical and clinical studies. Aspartic acid and glutamic acid, being excitatory amino acids, play crucial roles in the neurotransmission processes of the mammalian brain. Their precise quantification is essential for advancing our knowledge in neurochemistry and for the diagnosis of related disorders. Objective: This study aimed to develop a robust, accurate, and sensitive spectrophotometric method for quantifying aspartic and glutamic acids in solution, both in their native forms and when complexed with ortho-phthalaldehyde (OPA), a derivatization agent that enhances their absorbance properties. Methods: Utilizing a double-beam Hitachi 220 Spectrophotometer equipped with dual 1 cm silica cuvettes, we performed spectrophotometric analyses across a wavelength range of 165 to 1000 nm. Calibration curves for aspartic acid and glutamic acid, with and without OPA, were generated by plotting absorbance against concentration. Various solvents including methanol, ethanol, acetone, and acetonitrile were evaluated for their efficacy in dissolving the amino acids and their complexes. Linear regression analysis was employed to establish the relationship between concentration and absorbance, and the method's precision and accuracy were validated through replicate measurements at multiple concentrations. Results: The calibration curves exhibited excellent linearity for both amino acids, with and without OPA, across the tested concentration ranges. For aspartic acid, absorption maxima were observed at 201 nm (106,333.33 L mol^-1 cm^-1 molar absorptivity) and 229 nm (71,250 L mol^-1 cm^-1) for its OPA derivative. Glutamic acid showed absorption maxima at 198 nm (84,333.33 L mol^-1 cm^-1) and 216 nm (128,857.14 L mol^-1 cm^-1) for its OPA derivative. The correlation coefficients (R²) for the calibration curves ranged from 0.9954 to 0.9982, indicating a high degree of linearity and reliability of the method. Conclusion: The developed spectrophotometric method provides a reliable, precise, and sensitive approach for quantifying aspartic and glutamic acids. Its application is significant for biochemical analysis and has potential implications in clinical diagnostics, offering a promising tool for neurochemical studies.

Simultaneous Determination of One-Carbon Folate Metabolites and One-Carbon-Related Amino Acids in Biological Samples Using a UHPLC-MS/MS Method.

One-carbon folate metabolites and one-carbon-related amino acids play an important role in human physiology, and their detection in biological samples is essential. However, poor stability as well as low concentrations and occurrence in different species in various biological samples make their quantification very challenging. The aim of this study was to develop a simple, fast, and sensitive ultra-high-performance liquid chromatography MS/MS (UHPLC-MS/MS) method for the simultaneous quantification of various one-carbon folate metabolites (folic acid (FA), tetrahydrofolic acid (THF), p-aminobenzoyl-L-glutamic acid (pABG), 5-formyltetrahydrofolic acid (5-CHOTHF), 5-methyltetrahydrofolic acid (5-CH3THF), 10-formylfolic acid (10-CHOFA), 5,10-methenyl-5,6,7,8-tetrahydrofolic acid (5,10-CH+-THF), and 4-α-hydroxy-5-methyltetrahydrofolate (hmTHF)) and one-carbon-related amino acids (homocysteine (Hcy), methionine (Met), S-ade-L-homocysteine (SAH), and S-ade-L-methionine (SAM)). The method was standardized and validated by determining the selectivity, carryover, limits of detection, limits of quantitation, linearity, precision, accuracy, recovery, and matrix effects. The extraction methods were optimized with respect to several factors: protease-amylase treatment on embryos, deconjugation time, methanol precipitation, and proteins' isoelectric point precipitation on the folate recovery. Ten one-carbon folate metabolites and four one-carbon-related amino acids were detected using the UHPLC-MS/MS technique in various biological samples. The measured values of folate in human plasma, serum, and whole blood (WB) lay within the concentration range for normal donors. The contents of each analyte in mouse plasma were as follows: pABG (864.0 nmol/L), 5-CH3THF (202.2 nmol/L), hmTHF (122.2 nmol/L), Met (8.63 μmol/L), and SAH (0.06 μmol/L). The concentration of each analyte in mouse embryos were as follows: SAM (1.09 μg/g), SAH (0.13 μg/g), Met (16.5 μg/g), 5,10-CH+THF (74.3 ng/g), pABG (20.6 ng/g), and 5-CH3THF (185.4 ng/g). A simple and rapid sample preparation and UHPLC-MS/MS method was developed and validated for the simultaneous determination of the one-carbon-related folate metabolites and one-carbon-related amino acids in different biological samples.

2,5-Dihydroxyterephthalic Acid: A Matrix for Improved Detection and Imaging of Amino Acids.

Amino acids (AAs), which are low-molecular-weight (low-MW) metabolites, serve as essential building blocks not only for protein synthesis but also for maintaining the nitrogen balance in living systems. In situ detection and imaging of AAs are crucial for understanding more complex biological processes. Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) is a label-free mass spectrometric imaging technique that enables the simultaneous detection and imaging of the spatial distribution and relative abundance of different endogenous/exogenous compounds in biological samples. The excellent efficiency of MALDI-MSI is attributed to the choice of the MALDI matrix. However, to the best of our knowledge, no matrix has been specifically developed for AAs. Herein, we report a MALDI matrix, 2,5-dihydroxyterephthalic acid (DHT), which can improve the detection and imaging of AAs in biological samples by MALDI-MS. Our results indicated that DHT exhibited strong ultraviolet-visible (UV-vis) absorption, uniform matrix deposition, and high vacuum stability. Moreover, the matrix-related ion signals produced from DHT were reduced by 50 and 71.8% at m/z < 500 compared to the commonly used matrices of 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA), respectively, in their respective organic solvents. In terms of quantitative performance, arginine, glutamic acid, glutamine, and proline can be detected with limits of detection of 6, 4, 6, and 4 ng/mL, respectively, using the DHT as the matrix. Using DHT as the matrix, all 20 protein AAs were successfully detected in human serum by MALDI-MS, whereas only 7 and 10 AAs were detected when DHB and CHCA matrices were used, respectively. Furthermore, 20 protein AAs and taurine were successfully detected and imaged in a section of edible Crassostrea gigas (oyster) tissue for the first time. Our study demonstrates that using DHT as a matrix can improve the detection and imaging of AAs in biological samples by MALDI-MS.

Targeted Quantification of Amino Acids by Dansylation.

Quantification of amino acids in biological samples is a critical tool for studying metabolism. Although many methods for amino acid analysis exist, important considerations include ease of sample preparation, dynamic range, reproducibility, instrument availability, and throughput. Here, we present a simple, rapid, and robust method for the analysis of amino acids by chemical derivatization and liquid chromatography-mass spectrometry (LC-MS). We provide a detailed protocol for the analysis of 20 proteinogenic amino acids in biological samples which will enable straightforward implementation on modern LC-MS instruments.

Compound-specific Nitrogen Isotope and Radiocarbon Analyses of Amino Acids in Biological Samples

Compound-specific Nitrogen Isotope and Radiocarbon Analyses of Amino Acids in Biological Samples

Accurate determination of amino acids by quadruple isotope dilution-reverse phase liquid Chromatography-Tandem mass spectrometry after derivatization with 2-Naphthoyl chloride

The determination of amino acids in biological samples is central to the diagnosis of inherited metabolic disorders and also gives significant information about the metabolisms in the cells and living body. The development of analytical method for reliable quantification of amino acids in biological samples is still challenging because of the polar nature of amino acids and complex nature of biological samples causing a high degree of interferences during analysis. In the present study, a pre-column derivatization method using 2-naphtoyl chloride combined with liquid chromatography-tandem mass spectrometry method was developed for the determination of 17 amino acids in human serum and urine matrices. Low detection limits were obtained in the range of 0.015 – 0.266 µmol kg−1 and acceptable recovery results were obtained in human serum and urine samples. Isotopically labelled (15N labelled) amino acids were spiked to standards and samples before derivatization to compensate for the analytical errors in the whole procedure. The combination of quadrupole isotope dilution strategy with the derivatization based reversed phase chromatography allowed to improve method accuracy and precision.

Ultrasound‐assisted low‐density‐solvent‐based solvent‐demulsified dispersive liquid–liquid microextraction for the quantification of amino acids in biological samples

AbstractAn ultrasound‐assisted low‐density‐solvent‐based solvent‐demulsified dispersive liquid–liquid microextraction followed by high‐performance liquid chromatography with ultraviolet detection method has been established for the purpose of microextraction and recognition of tyrosine, phenylalanine and tryptophan from plasma and urine samples. For the preconcentration of analytes, 10 mL of sample solution was placed in test tube. A blend of 200 μL 1‐dodecanol and 100 μL acetonitrile were quickly injected into the sample solution and then shaken for 4.8 min using ultrasonic irradiation. The addition of demulsifier into the formed emulsion results in rapid separation of the mixture into two phases. The central composite design was used for optimization of microextraction conditions. Under the optimized conditions, the calibration graphs of tyrosine, phenylalanine and tryptophan were linear in the range of 1–200 μmol L−1. Limits of detection and quantification were 0.17–0.32 and 0.64–1.09 μmol L−1, respectively. This procedure was successfully used to determine target analytes in spiked plasma and urine samples; the relative mean recoveries ranged from 84.7 to 96.4%.

Switchable polarity solvents for preconcentration and simultaneous determination of amino acids in human plasma samples

The application of a mixture of amidine and alcohol as a switchable polarity solvent for the microextraction of 19 amino acids in biological samples.

Complementary Cell-Free Translational Assay for Quantification of Amino Acids.

In this study, we present a simple and economical method that enables rapid quantification of amino acids based on their polymerization into a signal-generating protein. This method harnesses amino acid-deficient cell-free protein synthesis systems that generate fluorescence signals in response to exogenous amino acids. When premixed with assay samples containing the amino acids in question, incubation of the cell-free synthesis reaction mixture rapidly resulted in the production of sfGFP, the fluorescence intensity of which was linearly proportional to the concentration of the amino acids. The assay method achieved a limit of detection as low as ∼100 nM and was successfully applied to the quantification of disease-related amino acids in biological samples. Compared with standard methods in current use that require chemical derivatization of amino acids and chromatographic equipment, the complementation assay method developed in this work enables the direct translation of amino acid titer into measurable biofluorescence intensity in a much shorter period, providing a more affordable and flexible option for the quantification of amino acids.

Chemiluminescence Detection of Serine, Proline, Glycine, Asparagine, Leucine, and Histidine by Using Corresponding Aminoacyl-tRNA Synthetases as Recognition Elements

Analysis of the concentration of free amino acids in biological samples is useful in clinical diagnostics. However, currently available methods are time consuming, potentially delaying diagnosis. Therefore, the development of more rapid analytical tools is needed. In this study, a chemiluminescence detection method for amino acids was developed, and the conditions for the enzyme reaction and assay were examined. For the recognition of each amino acid (here, serine, proline, glycine, asparagine, leucine, and histidine), the corresponding aminoacyl-tRNA synthetase (aaRS) was employed, and multiple enzymatic reactions were combined with a luminol chemiluminescence reaction. This method provided selective quantification from 1 to 20μM for serine, proline, glycine, and leucine; 1 to 60μM for asparagine; and 1 to 150μM for histidine. This assay, which utilized aaRSs for the detection of amino acids, could be useful for simple and rapid analysis of amino acids in clinical diagnostics.

Towards the chiral metabolomics: Liquid chromatography–mass spectrometry based dl-amino acid analysis after labeling with a new chiral reagent, (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl)pyrrolidine-2-carboxylate, and the application to saliva of healthy volunteers

A novel triazine-type chiral derivatization reagent, i.e., (S)-2,5-dioxopyrrolidin-1-yl-1-(4,6-dimethoxy-1,3,5-triazin-2-yl) pyrrolidine-2-carboxylate (DMT-(S)-Pro-OSu), was developed for the highly sensitive and selective detection of chiral amines and amino acids by UPLC–MS/MS analysis. The enantiomers of amino acids were easily labeled with the reagents at room temperature within 40min in an alkaline medium containing triethylamine. The diastereomers derived from proteolytic amino acids, except serine, were well separated under isocratic elution conditions by reversed-phase chromatography using an ODS column (Rs=1.2–9.0). dl-Serine was separated by use of an ADME column which has relatively higher polar surface than the conventional ODS column. The characteristic product ions, i.e., m/z 195.3 and m/z 209.3, were detected from all the diastereomers by the collision-induced dissociation of the protonated molecule. A highly sensitive detection on the amol–fmol level was obtained from the selected reaction monitoring (SRM) chromatogram. The chiral amines (e.g., adrenaline and noradrenaline) labeled with DMT-(S)-Pro-OSu were also well separated and sensitively detected by the present procedure. The method using DMT-(S)-Pro-OSu was used for the determination of dl-amino acids in the human saliva from healthy volunteers. Various l-amino acids were identified in the saliva. Furthermore, d-alanine (d-Ala) and d-proline (d-Pro) were also detected in relatively high concentrations (>5%). The ratio was higher in male saliva than in female saliva. However, the difference in the ratio of d-Ala for one day was not very high and the effect of foods and beverage seemed to be negligible. Based on the results using l-Ala-d3, the d-Ala in saliva seemed to be produced due to the racemization with some enzymes such as racemase. The racemization reaction was reversible, i.e., d-Ala-d3 was also racemized to l-Ala-d3 in saliva. Thus, care should be taken during the analysis of dl-amino acids in saliva. The present method using DMT-(S)-Pro-OSu may be applicable for the determination of chiral amine metabolomics, because the resulting derivatives produce the same product ions without relation to the compounds and show highly sensitive detection in the SRM mode of MS/MS. Consequently, DMT-(S)-Pro-OSu seems to be a useful chiral derivatization reagent for the determination of amines and amino acids in biological samples.

High Performance Liquid Chromatography Fluorescence Method for the Determination of Seleno-Amino Acids in Ovine Blood Plasma

This study was performed to validate an effective high performance liquid chromatography-fluorescence detection method for the separation of seleno-amino acids and sulfur amino acids in biological samples using pre-column derivatization with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate. Standard calibration and standard addition procedures were developed, but the quantification of seleno-amino acids was carried out by a second method. The coefficient of variation for precision was < 15% and accuracies were 96 to 98%. Recoveries in the extraction method were between 63 and 99%. This method was applied to the contribution of seleno-amino acids containing methylselenocysteine and selenomethionine in plasma tissue of lambs during infusion study. The method demonstrated good selectivity, sensitivity, precision, and accuracy.

Robust Method for Investigating Nitrogen Metabolism of 15N Labeled Amino Acids Using AccQ•Tag Ultra Performance Liquid Chromatography-Photodiode Array-Electrospray Ionization-Mass Spectrometry: Application to a Parasitic Plant–Plant Interaction

An AccQ•Tag ultra performance liquid chromatography-photodiode array-electrospray ionization-mass spectrometry (AccQ•Tag-UPLC-PDA-ESI-MS) method is presented here for the fast, robust, and sensitive quantification of (15)N isotopologue enrichment of amino acids in biological samples, as for example in the special biotic interaction between the cultivated specie Brassica napus (rapeseed) and the parasitic weed Phelipanche ramosa (broomrape). This method was developed and validated using amino acid standard solutions containing (15)N amino acid isotopologues and/or biological unlabeled extracts. Apparatus optimization, limits of detection and quantification, quantification reproducibility, and calculation method of (15)N isotopologue enrichment are presented. Using this method, we could demonstrate that young parasite tubercles assimilate inorganic nitrogen as (15)N-ammonium when supplied directly through batch incubation but not when supplied by translocation from host root phloem, contrary to (15)N2-glutamine. (15)N2-glutamine mobility from host roots to parasite tubercles followed by its low metabolism in tubercles suggests that the host-derived glutamine acts as an important nitrogen containing storage compound in the young tubercle of Phelipanche ramosa.

Rapid and precise measurement of serum branched-chain and aromatic amino acids by isotope dilution liquid chromatography tandem mass spectrometry.

BackgroundSerum branched-chain and aromatic amino acids (BCAAs and AAAs) have emerged as predictors for the future development of diabetes and may aid in diabetes risk assessment. However, the current methods for the analysis of such amino acids in biological samples are time consuming.MethodsAn isotope dilution liquid chromatography tandem mass spectrometry (ID-LC/MS/MS) method for serum BCAAs and AAAs was developed. The serum was mixed with isotope-labeled BCAA and AAA internal standards and the amino acids were extracted with acetonitrile, followed by analysis using LC/MS/MS. The LC separation was performed on a reversed-phase C18 column, and the MS/MS detection was performed via the positive electronic spray ionization in multiple reaction monitoring mode.ResultsSpecific analysis of the amino acids was achieved within 2 min. Intra-run and total CVs for the amino acids were less than 2% and 4%, respectively, and the analytical recoveries ranged from 99.6 to 103.6%.ConclusionA rapid and precise method for the measurement of serum BCAAs and AAAs was developed and may serve as a quick tool for screening serum BCAAs and AAAs in studies assessing diabetes risk.

Amino acid analysis: current topics and trends

The history of amino acids begins with the discovery of aspartic acid in asparagus sprouts in 1806. Thereafter, various naturally occurring amino acids of more than 360 species were detected from living organisms. Amino acid analysis was first performed by a postcolumn derivatization method using ninhydrin, which was originally developed by Moore and Stein [1]. They analyzed 124 amino acids and the ribonuclease structure using an automatic and systematic analyzer, and were awarded the Nobel Prize in Chemistry in 1972. Amino acid analysis is an integral part of analytical biochemistry. Amino acid analysis generally includes derivatization, i.e., precolumn, on-column, and postcolumn, coupled with chromatographic separation. A wide variety of separation techniques, including ion-exchange chromatography, reversed-phase chromatography, hydrophilic interaction chromatography, gas chromatography, capillary electrophoresis, and microchip electrophoresis, have been used. Various detection techniques, such as ultraviolet–visible, fluorescence, chemiluminescence, and electrochemical detection, have also been developed for amino acid analysis. On the basis of recent advances in the software and hardware used in mass spectrometry (MS), the variety of amino acid analysis methods has dramatically evolved and use has shifted to various types of MS detection techniques. In the meantime, a number of relatively old techniques, based on derivatization and conventional separation, have acquired a new look. On the basis of these observations, The Japan Society of Amino Acid Analysis (JSA3) was established in the autumn of 2011 to promote and follow the bioanalytical and/or technological advances relating to analysis of amino acids and related molecules. Various labeling techniques using ultraviolet–visible and fluorescent reagents are still used for the trace analysis of amino acids by the combination of chromatographic separation and MS detection. Of course, electrospray ionization MS allows the analysis of underivatized amino acids. Another new aspect is high-throughput analysis, such as the use of a high-pressure liquid-chromatographic system such as ultraperformance liquid chromatography using a sub-2-μm particle column, and miniaturization analysis such as the use of a microfluidic system. However, the most important issue in amino acid analysis today seems to be in the context of metabolomics studies as part of systems biology. Besides, naturally occurring D-amino acids, such as D-serine and D-aspartic acid, were found in awide variety of living organisms, both in their free form and as isomeric residues in many peptides. Their detection is due to the tremendous advances in the analytical techniques in recent years. Stereoselective metabolism of amino acids has also been gradually unveiled, and D-amino acids are now considered as novel physiologically active substances and biomarkers even in mammals. In view of the growing importance of the enantioselective analysis of amino acids, articles related to D-amino acid analysis are provided in this issue. Several articles regarding the enantioseparation of DL-amino acids by direct methods using a chiral stationary phase column are also provided. This topical collection covers a broad range of amino acid analysis, with contributions from the most prominent researchers in the field. The review articles are focused on the analytical methods applied to amino acids and related molecules using liquid chromatography, capillary electrophoresis, and microchip electrophoresis. The topics of the original research articles are expanded to the research field of peptides and proteins. Clarification of both the quantity and the distribution of amino acids in biological samples, including physiological fluids and foods, is also presented. Several research articles describe new methods of amino acid analysis, such as the use of a pillar column and a novel immunometric assay. This topical collection is focused on analysis of amino acids, including peptides, and provides a glimpse of the current research activity in the field. As a guest editor, it is my pleasure to present this collection of research articles and critical reviews which address the many challenging areas of amino acid analysis, including chiral recognition. Thanks are due to all of the authors for their excellent and timely contributions as well as the editorial staff of Published in the topical collection Amino Acid Analysis with guest editor Toshimasa Toyo’oka.

Fast and quantitative analysis of branched-chain amino acids in biological samples using a pillar array column

In this study, a fast and quantitative determination method for branched-chain amino acids (BCAAs), namely leucine, isoleucine, and valine, was developed using a pillar array column. A pillar array column with low-dispersion turns was fabricated on a 20 × 20-mm(2) microchip using multistep ultraviolet photolithography and deep reactive ion etching. The BCAAs were fluorescently labeled with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), followed by reversed-phase separation on the pillar array column. The NBD derivatives of the three BCAAs and an internal standard (6-aminocaproic acid) were separated in 100 s. The calibration curves for the NBD-BCAAs had good linearity in the range of 0.4-20 μM, using an internal standard. The intra- and interday precisions were found to be in the ranges of 1.42-3.80 and 2.74-6.97%, respectively. The accuracies for the NBD-BCAA were from 90.2 to 99.1%. The method was used for the analysis of sports drink and human plasma samples. The concentrations of BCAAs determined by the developed method showed good agreements with those determined using a conventional high-performance liquid chromatography system. As BCAAs are important biomarkers of some diseases, these results showed that the developed method could be a potential diagnostic tool in clinical research.

New Chemodosimetric Reagents as Ratiometric Probes for Cysteine and Homocysteine and Possible Detection in Living Cells and in Blood Plasma

In this work, we have rationally designed and synthesized two new reagents (L(1) and L(2)), each bearing a pendant aldehyde functionality. This aldehyde group can take part in cyclization reactions with β- or γ-amino thiols to yield the corresponding thiazolidine and thiazinane derivatives, respectively. The intramolecular charge-transfer (ICT) bands of these thiazolidine and thiazinane derivatives are distinctly different from those of the molecular probes (L(1) and L(2)). Such changes could serve as a potential platform for using L(1) and L(2) as new colorimetric/fluorogenic as well as ratiometric sensors for cysteine (Cys) and homocysteine (Hcy) under physiological conditions. Both reagents proved to be specific towards Cys and Hcy even in the presence of various amino acids, glucose, and DNA. Importantly, these two chemodosimetric reagents could be used for the quantitative detection of Cys present in blood plasma by using a pre-column HPLC technique. Such examples are not common in contemporary literature. MTT assay studies have revealed that these probes have low cytotoxicity. Confocal laser scanning micrographs of cells demonstrated that these probes could penetrate cell membranes and could be used to detect intracellular Cys/Hcy present within living cells. Thus, the results presented in this article not only demonstrate the efficiency and specificity of two ratiometric chemodosimeter molecules for the quantitative detection of Cys and Hcy, but also provide a strategy for developing reagents for analysis of these vital amino acids in biological samples.

Amino acid analysis using core–shell particle column

In this study, the separation efficiency of a core-shell particle column was compared with particle-packed and monolithic silica columns, which showed that the core-shell particle column had a smaller theoretical plate height and that its separation efficiency was not affected significantly by the increase in flow rate. A fast HPLC method using a core-shell particle column was developed for the determination of amino acids. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was used as a fluorescence derivatization reagent for amino acids, followed by separation on a core-shell Kinetex C18 column. The analysis time for 21 NBD-amino acids was within 7min, which was faster than that in our previous studies with conventional particle-packed columns or monolithic silica columns. The linearities of the calibration curves for all the amino acids were found to be good over a range of injection amounts from 40fmol to 40pmol. The accuracies for the amino acid determinations were 90.9-107%. The method was proved to have potential for the fast determination of amino acids in biological samples.

New approach for amino acid profiling in human plasma by selective fluorescence derivatization

A new approach for the separation of 6-aminoquinolyl-carbamyl (AQC)-derivatized amino acids has been proposed. The chromatography used ion-pairing mechanism to increase the method selectivity. Mobile phase was based on triethylamine buffer containing N,N-dimethyloctylamine as a modifier. A number of factors, buffer composition and pH, counterion concentration, temperature and acetonitrile gradient profile, were optimized to achieve final chromatographic conditions. With the presented analytical method, the separation and identification of 34 AQC-amino acids and amino compounds present in human plasma is possible. The results of validation proved the applicability of the method for quantification of 27 amino acids in biological samples. The ultrafiltration proposed as deproteinization procedure gave repeatable and reliable results for the amino acids under investigation. This method introduced in routine testing can be a suitable tool for amino acid profiling in plasma including all aspects of clinical application.

Metabolic profiling of fifteen amino acids in serum of chemical-induced liver injured rats by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

Amino acids (AA) play a crucial role in the metabolic process of animals, plants and microbial cells, which are useful for the diagnosis, follow-up and prognostics of liver disorders affecting AA metabolism. A rapid, simple and sensitive analytical method is in urgent need to investigate the intact metabolic profile and to simultaneously determine individual AA in biological samples. Here, a hydrophilic interaction liquid chromatography (HILIC) coupled with tandem mass spectrometry (MS/MS) analytical method was developed and validated for simultaneous quantification of 15 AA in rat serum using isotope stable-labeled phenylalanine and alanine as internal standards. The 15 AA without derivatization were separated on a hydrophilic interaction silica column (TSK-GEL AMIDE-80), the total analytical time was within 8 min, and the concentrations of the 15 AA were determined using a multiple reaction monitoring (MRM) mode. Limits of detection (LOD) ranged from 0.01 to 0.05 μg/ml, and the calibration curve was linear in the range of 0.05–10 μg/ml (r > 0.99). The HILIC-MS method was employed to the analysis of AA in serum samples obtained from N-acetyl-p-amino-phenol (APAP)- and chloropromazine hydrochloride (CH)- induced liver injured rats. The concentrations of each AA ranged from the low quantification value up to 10 μg/ml. Based on metabolic profile of AA, multivariate statistics using principal component analysis (PCA) and partial least squares discriminate analysis (PLS-DA) could differentiate two distinct groups corresponding to APAP-induced and CH-induced rats. This novel metabolic profile study of AA based on the HILIC-MS analysis and chemometric analysis provided not only an accurate quantitative assay of the serum concentrations of biomarkers, but also a promising methodology for evaluation of chemical-induced hepatotoxicity in reflecting AA metabolic pathway.