Amino Acids In Human Plasma Research Articles

血漿中の乳糜およびビリルビンがHPLC-ESI/MS による アミノ酸濃度分析に与える影響

血漿中の乳糜およびビリルビンがHPLC-ESI/MS による アミノ酸濃度分析に与える影響

Determination of Amino Acids in Plasma and Nutritional Supplements by Capillary Electrophoresis with Copper(II) Coordination

A capillary zone electrophoresis (CZE) method was developed using interactions between amino acids and copper(II) without derivatization. The optimized separation buffer included 10% isopropanol, 50 mmol L−1 CuSO4, 50 mmol L−1 acetic acid, and 50 mmol L−1 sodium acetate at pH = 3.98 at a detection wavelength of 233 nm. This method allowed baseline separation and determination of ten amino acids in less than 21 min. Quality parameters such as linearity (r > 0.99), precision (relative standard deviation <2.51%), limits of detection (0.05 µg mL−1), and recovery (82.25% to 117.6%) were measured. The separation of amino acids in high salt solutions was explored. The method was used to directly determine amino acids in human plasma and nutritional supplements.

A novel method for simultaneous measurement of concentration and enrichment of NO synthesis-specific amino acids in human plasma using stable isotopes and LC/MS ion trap analysis

Stable isotope studies offer the opportunity to study the in-depth metabolic pathway of glutamine, citrulline, and arginine amino acids involved in NO synthesis. The use of multiple stable isotopes can be used to elucidate the exact transformation of glutamine to citrulline and arginine de novo synthesis. This novel method provides a purification step using cation exchange resin in combination with a rapid and easy derivatization procedure for a precise and robust measurement of the concentration and isotopic enrichments of NO synthesis-specific amino acids using a liquid chromatography mass spectrometry (LC/MS) ion trap system with high sensitivity and selectivity. The ethyl chloroformate derivatization procedure is beneficial in terms of robustness, velocity, simplicity, and derivative stability. In addition, the ethyl chloroformate derivatization can be performed at room temperature in an aqueous environment without incubation and the isolation of the derivatives from the reaction mixture also serves as a purification step. The concentration and enrichment of NO synthesis-specific amino acids as well as phenylalanine and tyrosine to determine protein turnover, were measured with good inter-day precision for the concentration (<7.4%) and enrichment (<12.7%) in plasma samples at low and high levels. The low limit of quantification was 0.2μmol/L for most of the amino acids and the purification method showed to have good recoveries between 78% and 98%. No ion-suppression was observed by post-column infusion experiments. In order to develop new nutritional strategies, this novel method can be used to support the elucidation of the effect of administration of specific supplements on the glutamine-citrulline-arginine pathway by using stable isotope studies.

Analysis of 26 amino acids in human plasma by HPLC using AQC as derivatizing agent and its application in metabolic laboratory

The present study reports the simultaneous analysis of 26 physiological amino acids in plasma along with total cysteine and homocysteine by high-performance liquid chromatography (HPLC) employing 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) as precolumn derivatizing reagent. Separations were carried out using Lichrospher 100 RP-18e (5 μm) 250 × 4.0 mm column connected to 100 CN 4.0 × 4.0 mm guard column on a quaternary HPLC system and run time was 53 min. Linearity of the peak areas for different concentrations ranging from 2.5 to 100 pmol/μL of individual amino acids was determined. A good linearity (R (2) > 0.998) was achieved in the standard mixture for each amino acid. Recovery of amino acids incorporated at the time of derivatization ranged from 95 to 106 %. Using this method we have established the normative data of amino acids in plasma, the profile being comparable to the range reported in literature and identified cases of classical homocystinuria, cobalamin defect/deficiency, non-ketotic hyperglycinemia, hyperprolinemia, ketotic hyperglycinemia, urea cycle defect and maple syrup urine disease.

Direct quantitative analysis of aromatic amino acids in human plasma by four-way calibration using intrinsic fluorescence: Exploration of third-order advantages

A novel intrinsic fluorescence method for the direct determination of l-phenylalanine, l-tyrosine, and l-tryptophan in human plasma is presented. By using fluorescence excitation-emission-pH-sample data array in combination with four-way calibration method based on the quadrilinear component model, the proposed approach successfully achieved quantitative analysis of the aromatic amino acids in human plasma, even in the presence of an unknown, uncalibrated serious interferent. It needs little preparation, uses the "mathematical separation" instead of "analytical separation", what makes it fast and environmentally friendly. Satisfactory results have been achieved for calibration set, validation set, and prediction set. The ranges for phenylalanine, tyrosine, and tryptophan are 2.0 × 10(3)-20.0 × 10(3), 50.0-500.0, and 20.0-200.0 ng mL(-1) respectively. Average spike recoveries (mean ± standard deviation) are 93.3 ± 7.7%, 104.3 ± 6.6%, and 99.5 ± 9.0% respectively. The real concentrations in human plasma are 10.2 ± 0.3, 6.6 ± 0.1, and 5.3 ± 0.1 μg mL(-1) respectively, which are consistent with the results obtained by LC-MS/MS method and reference values. In addition, we explored the third-order advantages through the real four-way array; it has shown that higher resolving power is one of the main advantages of higher-order tensor calibration method. These results demonstrated that the proposed method is sensitive, accurate, and efficient for direct quantitative analysis of aromatic amino acids in human plasma.

Optimization and validation of a capillary electrophoresis laser‐induced fluorescence method for amino acids determination in human plasma: Application to bipolar disorder study

Quantitative and qualitative analysis of amino acids in biofluids offers relevant information in diagnosis of diseases, evaluation of nutritional state, and in elucidating metabolic influences on physiology. A simple, rapid, and robust procedure in terms of sample treatment, separation, and quantitation based on CE-LIF has been optimized for use in human plasma samples. Time required for derivatization was 15 min and analysis time was 35 min. 4-Fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F) was the labeling agent used for obtaining fluorescent derivatives. Electrophoretic conditions were: 175 mM borate buffer at pH 10.25 prepared with 12.5 mM β-cyclodextrin. The voltage applied was +21 kV. Fourteen amino acids could be quantified: L-proline, L-phenylalanine, L-leucine, L-isoleucine, L-ornithine, D-ornithine, L-glutamine, L-alanine, L-threonine, glycine, L-serine, D-serine, taurine and L-glutamate. With this chiral CE-LIF method, L- and D-amino acids are adequately separated. The method was validated for a representative group of amino acids in human plasma: L-proline, L-isoleucine, L-ornithine, L-glutamine, L-alanine L-threonine, glycine, L-serine, D-serine, and glutamate. The method has been successfully applied to human plasma from patients with bipolar disorder, all of whom were taking lithium as a mood stabilizer. Eleven amino acids were quantified in plasma from nine patients, aged 24-55 years. The results were in accordance to published values for the bipolar patients. The method is useful particularly in studies where plasma amino acid levels can be used as biomarkers for diagnosis of diseases, evaluating the disease progression, and monitoring response to drug therapy.

Bioanalytical LC Separation Techniques for Quantitative Analysis of Free Amino Acids in Human Plasma

The quantitative analysis of free amino acids in human plasma has become an important and essential analysis parameter in different areas of life sciences. Free amino acid concentrations in human plasma samples are generally determined by means of GC or LC after chemical derivatization followed by UV, fluorescent or MS detection of the amino acid derivatives. Derivatization of free amino acids is done either pre- or post-column, and the amino acid derivatives obtained posess improved chromatographic behavior, increased detection sensitivity and selectivity compared with non-derivatized free amino acids. This work gives an overview of different chemical derivatization methods applied and their liquid separation techniques in bioanalytical assays for quantitative free amino acid analysis in human plasma samples. Important plasma preparation procedures, pre- and post-column derivatization, and different LC separation techniques are presented.

Surrogate Matrix And Surrogate Analyte Approaches For Definitive Quantitation of Endogenous Biomolecules

Quantitation of biomarkers by LC-MS/MS is complicated by the presence of endogenous analytes. This challenge is most commonly overcome by calibration using an authentic standard spiked into a surrogate matrix devoid of the target analyte. A second approach involves use of a stable-isotope-labeled standard as a surrogate analyte to allow calibration in the actual biological matrix. For both methods, parallelism between calibration standards and the target analyte in biological matrix must be demonstrated in order to ensure accurate quantitation. In this communication, the surrogate matrix and surrogate analyte approaches are compared for the analysis of five amino acids in human plasma: alanine, valine, methionine, leucine and isoleucine. In addition, methodology based on standard addition is introduced, which enables a robust examination of parallelism in both surrogate analyte and surrogate matrix methods prior to formal validation. Results from additional assays are presented to introduce the standard-addition methodology and to highlight the strengths and weaknesses of each approach. For the analysis of amino acids in human plasma, comparable precision and accuracy were obtained by the surrogate matrix and surrogate analyte methods. Both assays were well within tolerances prescribed by regulatory guidance for validation of xenobiotic assays. When stable-isotope-labeled standards are readily available, the surrogate analyte approach allows for facile method development. By comparison, the surrogate matrix method requires greater up-front method development; however, this deficit is offset by the long-term advantage of simplified sample analysis.

Study of disulfide reduction and alkyl chloroformate derivatization of plasma sulfur amino acids using gas chromatography–mass spectrometry

Four disulfide-reducing agents, dithiothreitol (DTT), 2,3-dimercaptopropanesulfonate (DMPS), and the newly tested 2-mercaptoethanesulfonate (MESNA) and Tris(hydroxypropyl)phosphine (THP), were investigated in detail for release of sulfur amino acids in human plasma. After protein precipitation with trichloroacetic acid (TCA), the plasma supernatant was treated with methyl, ethyl, or propyl chloroformate via the well-proven derivatization-extraction technique and the products were subjected to gas chromatographic-mass spectrometric (GC-MS) analysis. All the tested agents proved to be rapid and effective reducing agents for the assay of plasma thiols. When compared with DTT, the novel reducing agents DMPS, MESNA, and THP provided much cleaner extracts and improved analytical performance. Quantification of homocysteine, cysteine, and methionine was performed using their deuterated analogues, whereas other analytes were quantified by means of 4-chlorophenylalanine. Precise and reliable assay of all examined analytes was achieved, irrespective of the chloroformate reagent used. Average relative standard deviations at each analyte level were ≤6%, quantification limits were 0.1-0.2 μmol L(-1), recoveries were 94-121%, and linearity was over three orders of magnitude (r(2) equal to 0.997-0.998). Validation performed with the THP agent and propyl chloroformate derivatization demonstrated the robustness and reliability of this simple sample-preparation methodology.

Analysis of 25 underivatized amino acids in human plasma using ion‐pairing reversed‐phase liquid chromatography/time‐of‐flight mass spectrometry

Amino acids in biological fluids have previously been shown to be detectable using liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) with perfluorinated acids as ion-pairing agents. To date, these studies have used precursor mass, retention time and tandem mass spectrometry (MS/MS) to identify and quantify amino acids. While this is a potentially powerful technique, we sought to adapt the method to time-of-flight (TOF)MS. A new application of a recently described liquid chromatographic separation method was coupled with TOFMS to employ accurate mass for qualitative identification; resulting in additional qualitative data not available with standard single quadrupole data. In the current study, we evaluated 25 physiological amino acids and one dipeptide that are routinely quantified in human plasma. Accuracy and precision of the method was evaluated by spiking human plasma with a mix of the 25 amino acids; in addition, the inclusion of a cation-exchange cleanup step was evaluated. The calibration curves were linear over a range from 1.56 to 400 microM. The dynamic range was found to be within physiological levels for all amino acids analyzed. Accuracy and precision for most of the amino acids was between 80-120% spike recovery and <10% relative standard deviation (RSD). The LC/MS technique described in this study relies on mass accuracy and is suitable for the quantitation of free amino acids in plasma.

Determination of d-serine and related neuroactive amino acids in human plasma by high-performance liquid chromatography with fluorimetric detection

A simple and versatile methodology using high-performance liquid chromatography (HPLC) with fluorimetric detection was developed to simultaneously determine d-serine along with other metabolically related neuroactive amino acids in the glutamatergic system: l-serine, l-glutamate, l-glutamine, and glycine. On-column sensitivity was in the lower picomole range. Of two chiral thiol reagents investigated, amino acid derivatives of o-phthaldialdehyde (OPA) in combination with N-isobutyryl- l-cysteine were found to have consistently higher responses than their corresponding N- tert-butyloxycarbonyl- l-cysteine derivatives. This methodology was applied to the quantitative detection of amino acids in human plasma and lays the foundation for further investigations of the role of neuroactive amino acids in the pathophysiology and treatment of neurological and psychiatric disorders.

On‐capillary derivatization and analysis of amino acids in human plasma by capillary electrophoresis with laser‐induced fluorescence detection: Application to diagnosis of aminoacidopathies

A capillary electrophoresis with laser-induced fluorescence detection method for the analysis of free amino acids (AA) in human plasma was developed. A mixture of 16 AA was on-capillary derivatized with 3-(2-furoyl)quinoline-2-carboxaldehyde (FQ) and separated inside the capillary in less than 30 min using 70 mM borax-3.5 mM SDS pH 9.3 as running buffer. Four plasma samples from a healthy donor and patients suffering from phenylketonuria, propionic acidemia, and tyrosinemia type II were studied. Repeatabilities calculated as intra-day RSD (n = 3) values for the AA involved in these aminoacidopathies (glycine, phenylalanine, and tyrosine) were in the range of 0.3 to 1.2% for migration time and 3.7 to 8.2% for peak height. Reproducibilities calculated as inter-day RSD (n = 4) values for the same AA were between 0.7 and 1.4% for migration time and 4.7 and 9.1% for peak height. A fast qualitative analysis allowed the identification of the corresponding disease by comparing the electrophoretic profiles from the patient and the healthy donor and noting the increased level of the specific AA accompanying each individual disease. The results of the quantitative analysis for glycine, phenylalanine, and tyrosine in the plasma samples studied using the developed method showed a good agreement with those provided by the Center of Diagnosis of Molecular Diseases using a standard method for AA analysis.

Determination of Proteinogenic Amino Acids in Human Plasma by Capillary Electrophoresis with Contactless Conductivity Detection

Capillary electrophoresis (CE) with contactless conductivity detection (CCD) represents a viable alternative to liquid chromatography in the analysis of amino acids (AA) in human plasma. After optimizing the composition of the background electrolyte (BGE), and introducing simple plasma pretreatment to remove spurious protein components, the CE/CCD methods allows determination of 18 from 20 proteinogenic AAs, three nonproteinogenic AAs and creatinine in 60 minutes. AAs are separated in their cationic forms in BGE containing 1.7 M acetic acid and 0.1% hydroxyethylcellulose, pH 2.2. LODs for individual AAs vary in an acceptable range with minimum of 4.3 μM for Arg and maximum of 42.9 μM for Cys. Mean concentrations and concentrations ranges for AAs in human plasma samples from 9 healthy individuals are found to agree well with those determined by an LC method in other two laboratories.

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.

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.

Separation of free amino acids in human plasma by capillary electrophoresis with laser induced fluorescence: potential for emergency diagnosis of inborn errors of metabolism

Free amino acids (AAs) in human plasma are derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) and analyzed by capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection. The labeling procedure is significantly improved over results reported previously. Derivatization can be completed in 40 min, with concentrations as low as 4×10 −8 M successfully labeled in favourable cases. Twenty-nine AAs (including 2 internal standards) are identified and can be reproducibly separated in 70 min. Migration time RSD values for 23 of these AAs were calculated and found in the range from 0.5 to 4%. The rapid derivatization procedure and the resolution obtained in the separation are sufficient for a semi-quantitative, emergency diagnosis of several inborn errors of metabolism (IEM). Amino acid profiles for both normal donor plasma samples and plasma samples of patients suffering from phenylketonuria, tyrosinemia, maple syrup urinary disease, hyperornithinemia, and citrullinemia are studied.

Amino acid quantitation in aqueous matrices via trap and release membrane introduction mass spectrometry: homocysteine in human plasma.

Trap and release membrane introduction mass spectrometry (T&R-MIMS) using a removable direct insertion membrane probe (DIMP) is employed to determine the total homocysteine concentration (tHcy) directly from human plasma after derivatization with ethyl chloroformate. The method uses no chromatographic separation, is linear, reproducible, and displays limit of quantitation (2 pM) sufficiently below the threshold concentration of tHcy in plasma. It also combines chemical, membrane, and mass spectrometric discrimination, and can be used to determine selected amino acids in human plasma simultaneously. After derivatization with ethyl chloroformate, many amino acids in aqueous solution are observed to be efficiently detected; hence T&R-MIMS is promising as a simple and sensitive technique for simultaneous quantitation of selected amino acids in plasma and urine, and in other aqueous matrices.

Precolumn Deproteinization Method of Human Plasma Using a Hydroxyapatite Cartridge for High-Performance Liquid Chromatographic Amino Acid Analysis

Determination of amino acids in human plasma was performed by postcolumn ninhydrin derivatization liquid chromatography using a hydroxyapatite cartridge for precolumn deproteinization. This method was suitable for the determination of amino acids in human plasma. The proposed method was simple, rapid (deproteinization time: within 1min) and reproducible (relative standard deviation: ca. 0.4 to 5.4% (n=3)). Average recovery of 22 amino acids was over 80%. It took about 2h to elute the amino acids in human plasma on the chromatogram completely. Protein-binding tryptophan was also determined by the proposed method.

Systematic approach to the development of plasma amino acid analysis by high-performance liquid chromatography with ultraviolet detection with precolumn derivatization using phenyl isothiocyanate

A reversed-phase liquid chromatographic method for the separation of 26 phenylthiocarbamyl derivatives of amino acids in human plasma in ca. 35 min. is described. The method used a C 18 column (150 × 4.6 mm I.D., 3 μm) thermostatted at 41°C, and a simple multistep linear gradient of two solvents. Solvent A was 0.05 M sodium acetate (pH 5.1)—acetonitrile (98:2, v/v), and solvent B was water—acetonitrile (40:60, v/v). A simple and successful approach to the optimization of the conditions for the separation of the 26 amino acid derivatives was realized. In the initial phase of development, the composition of the gradient, its timings, the column temperature, the flow-rate and the mobile phase compositions were optimized. At the end the influence of pH was studied, and this approach led to a clear resolution of the 26 amino acids. The method was validated by accuracy, precision, and recovery studies, by analyzing patient samples, and by comparing the quality control sample results with the classical ion-exchange method.

More pitfalls in human plasma amino acid analysis

More pitfalls in human plasma amino acid analysis