Methods for Absolute Quantification of Human Plasma Free Amino Acids by High-Performance Liquid Chromatography/Electrospray Ionization Mass Spectrometry Using Precolumn Derivatization.

Plasma free amino acid (PFAA) concentrations in humans are affected by various diseases. However, the variations caused are not dramatic, so a high accurate and precise method for analyzing PFAAs is required. The PFAA analysis protocol described in this chapter covers blood sampling, sample pretreatment, amino acid derivatization, and LC-MS analysis. Each procedure is important for accurate and precise quantification.In the protocol, a human blood sample is collected using an EDTA-2Na or 2K vacuum collection tube and then immediately cooled in water mixed with crushed ice. The sample is then centrifuged on cooling to allow a plasma sample to be removed. A stable-isotope-labeled internal standard solution is added to the plasma, and then the plasma is deproteinized with acetonitrile. The amino acids in the plasma are then derivatized using 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS) reagent which is designed for LC-MS analysis. The derivatized amino acids are separated by reverse-phase HPLC and detected by electrospray ionization mass spectrometry. Using this method, 21 amino acids in human plasma can be analyzed with a 12 min cycle. The accuracy and precision are both better than the required criteria given by the US Food and Drug Administration in guidance of Bioanalytical Method Validation.

Genetic basis for plasma amino acid concentrations based on absolute quantification: a genome-wide association study in the Japanese population.

To assess the use of plasma free amino acids (PFAAs) as biomarkers for metabolic disorders, it is essential to identify genetic factors that influence PFAA concentrations. PFAA concentrations were absolutely quantified by liquid chromatography-mass spectrometry using plasma samples from 1338 Japanese individuals, and genome-wide quantitative trait locus (QTL) analysis was performed for the concentrations of 21 PFAAs. We next conducted a conditional QTL analysis using the concentration of each PFAA adjusted by the other 20 PFAAs as covariates to elucidate genetic determinants that influence PFAA concentrations. We identified eight genes that showed a significant association with PFAA concentrations, of which two, SLC7A2 and PKD1L2, were identified. SLC7A2 was associated with the plasma levels of arginine and ornithine, and PKD1L2 with the level of glycine. The significant associations of these two genes were revealed in the conditional QTL analysis, but a significant association between serine and the CPS1 gene disappeared when glycine was used as a covariate. We demonstrated that conditional QTL analysis is useful for determining the metabolic pathways predominantly used for PFAA metabolism. Our findings will help elucidate the physiological roles of genetic components that control the metabolism of amino acids.

The effects of pre-analysis sample handling on human plasma amino acid concentrations.

BACKGROUND: The accurate and reliable quantification of amino acid concentrations in human plasma is important for the investigation of a number of diseases. However, few systematic studies investigating the changes in amino acid concentrations related to blood collection and storage conditions have been completed. METHODS: Blood samples were collected with EDTA-Na2 from 3 healthy volunteers and subjected to a number of different treatments; hemolysis, temperature after blood collection, time from blood collection to cooling, the influence of platelets, long term storage conditions, and repeated freeze-thaw cycles. Changes in the concentrations of 22 amino acids were determined using an Amino Acid Analyzer. RESULTS: Of the conditions influencing sample stability between blood collection and amino acid analysis, hemolysis, temperature after blood collection, and long-term storage at -20°C affected the concentrations of 11 amino acids. Time from blood collection to cooling, platelet contamination and repeated freeze-thaw cycles altered the levels of 4 amino acids. CONCLUSIONS: We observed changes in amino acid concentrations relating to blood collection and storage conditions. If attention is paid to 4 key factors (hemolysis, temperature immediately following blood collection, time from collection to cooling, and long-term storage temperature) 19 amino acids can be reliably quantified.

Reference intervals for plasma-free amino acid in a Japanese population.

BACKGROUND: Plasma amino acid concentrations vary with various diseases. Although reference intervals are useful in daily clinical practice, no reference intervals have been reported for plasma amino acids in a large Japanese population. METHODS: Reference individuals were selected from 7685 subjects examined with the Japanese Ningen Dock in 2008. A total of 1890 individuals were selected based on exclusion criteria, and the reference samples were selected after the outlier samples for each amino acid concentration were excluded. The lower limit of the reference intervals for the plasma amino acid concentrations was set at the 2.5th percentile and the upper limit at the 97.5th percentile. RESULTS: By use of the nested analysis of variance, we analysed a large dataset of plasma samples and the effects of background factors (sex, age and body mass index [BMI]) on the plasma amino acid concentrations. Most amino acid concentrations were related to sex, especially those of branched-chained amino acid. The citrulline, glutamine, ornithine and lysine concentrations were related to age. The glutamate concentration was related to body mass index. CONCLUSIONS: The concentrations of most amino acids are more strongly related to sex than to age or body mass index. Our results indicate that the reference intervals for plasma amino acid concentrations should be stratified by sex when the background factors of age and body mass index are considered.

Validation of an analytical method for human plasma free amino acids by high-performance liquid chromatography ionization mass spectrometry using automated precolumn derivatization.

The analysis of human plasma free amino acids is important for diagnosing the health of individuals, because their concentrations are known to vary with various diseases. The development of valid, reliable, and high-throughput analytical methods for amino acids analysis is an essential requirement in clinical applications. In the present study, we have developed an automated precolumn derivatization amino acid analytical method based on high-performance liquid chromatography/electrospray ionization mass spectrometry (so-called UF-Amino Station). This method enabled the separation of at least 38 types of physiological amino acids within 8min, and the interval time between injections was 12min. We also validated this method for 21 major types of free amino acids in human plasma samples. The results of the specificity, linearity, accuracy, repeatability, intermediate precision, reproducibility, limits of detections, lower limits of quantification, carry over, and sample solution stability were sufficient to allow for the measurement of amino acids in human plasma samples. Our developed method should be suitable for use in clinical fields.

Positional effects of phosphorylation on the stability and morphology of tau-related amyloid fibrils.

Hyperphosphorylated forms of tau protein are the main component of paired helical filaments (PHFs) of neurofibrillary tangles in the brain of Alzheimer's disease patients. To understand the effect of phosphorylation on the fibrillation of tau, we utilized tau-derived phosphorylated peptides. The V(306)QIVYK(311) sequence (PHF6) in the microtubule-binding domain is known to play a key role in the fibrillation of tau, and the short peptide corresponding to the PHF6 sequence forms amyloid-type fibrils similar to those generated by full-length tau. We focused on the amino acid residue located at the N-terminus of the PHF6 sequence, serine or lysine in the native isoform of tau, and synthesized the PHF6 derivative peptides with serine or lysine at the N-terminus of PHF6. Peptides phosphorylated at serine and/or tyrosine were synthesized to mimic the possible phosphorylation at these positions. The critical concentrations of the fibrillation of peptides were determined to quantitatively assess fibril stability. The peptide with the net charge of near zero tended to form stable fibrils. Interestingly, the peptide phosphorylated at the N-terminal serine residue exhibited remarkably low fibrillation propensity as compared to the peptide possessing the same net charge. Transmission electron microscopy measurements of the fibrils visualized the paired helical or straight fibers and segregated masses of the fibers or heterogeneous rodlike fibers depending on the phosphorylation status. Further analyses of the fibrils by the X-ray fiber diffraction method and Fourier transform infrared spectroscopic measurements indicated that all the peptides shared a common cross-ß structure. In addition, the phosphoserine-containing peptides showed the characteristics of ß-sandwiches that could interact with both faces of the ß-sheet. On the basis of these observations, possible protofilament models with four ß-sheets were constructed to consider the positional effects of the serine and/or tyrosine phosphorylations. The electrostatic intersheet interaction between phosphate groups and the amino group of lysine enhanced the lateral association between ß-sheets to compensate for the excess charge. In addition to the previously postulated net charge of the peptide, the position of the charged residue plays a critical role in the amyloid fibrillation of tau.

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

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

Advantage of LC-MS metabolomics methodology targeting hydrophilic compounds in the studies of fermented food samples.

The utility of a liquid chromatography mass spectrometry (LC-MS) method, using a pentafluorophenylpropyl (PFPP) bonded silica, was demonstrated in a metabolomics study of fermented food samples. Our LC-MS method was applied to Japanese fermented food (miso) of different stages of ripeness. The data acquired were evaluated by principal component analysis (PCA). The score plots indicated that the miso samples could be approximately classified into three groups, based on the stage of miso ripeness. The loading plots indicated that the ions responsible for group separation included not only amino acids and citric acid but also Amadori compounds. On the other hand, the miso samples were also analyzed by a conventional LC-MS method using an octadecyl (C(18)) column for comparison. The group separation of score plots from the conventional method was less clear than that from our method. The advantage of our LC-MS method is due to the different retention properties of the PFPP column and the C(18) column with hydrophilic compounds. Our LC-MS method will be useful for the metabolic profiling of fermented food samples.

Comprehensive analytical method for the determination of hydrophilic metabolites by high-performance liquid chromatography and mass spectrometry.

A method for the comprehensive analysis of hydrophilic metabolites, based on a combination of high-performance liquid chromatography and mass spectrometry, is described. We evaluated three types of stationary phases to achieve the separation of highly hydrophilic metabolites. Good chromatographic retention and separation of these metabolites were achieved on a pentafluorophenylpropyl-bonded silica column with gradient elution, using 0.1% aqueous formic acid and acetonitrile as the mobile phase. The optimized conditions allowed the comprehensive determination of the standard 49 kinds of amino acids, 6 kinds of amines, 45 kinds of organic acids, 18 kinds of nucleic bases, 5 kinds of nucleosides, and 14 kinds of nucleotides, and then the linearity, dynamic range, detection limit, and precision of the retention time and the peak area were validated. We applied this method for the targeted analysis of the components in soy sauce. The results from the quantitative determination of amino acids were compared to those obtained with an amino acid analyzer, and the accuracy was in the range between 85 and 119%. The accuracy of other detected components was confirmed to be 105-133% by the recovery rate after the addition of standard compounds. We also applied the method for the nontargeted metabolic profiling of the components in several kinds of soy sauces with the principal component analysis. They were classified by the manufacturing methods, and the components that corresponded to the differences were identified. This method could be useful for the targeted analysis of hydrophilic metabolites as well as their nontargeted metabolic profiling.

On-line desalting-mass spectrometry system for the structural determination of hydrophilic metabolites, using a column switching technique and a volatile ion-pairing reagent.

A novel desalting method, using a column switching technique and a volatile ion-pairing reagent, pentadecafluorooctanoic acid, was developed. This system allows hydrophilic and cationic compounds in a nonvolatile buffer to be directly introduced into a mass spectrometer for structural elucidation. The desalting procedure consists of four steps: (1) the fractionation of a target compound from a separation column, (2) the removal of salts with pentadecafluorooctanoic acid on the trap column, (3) the desorption of the compound from the trap column, and (4) the re-equilibration of the trap column with a pentadecafluorooctanoic acid solution. In this procedure, we investigated the methods for optimizing the desalting and re-equilibration steps. Various amino acids, including branched chain amino acids, aromatic amino acids, basic amino acids and methionine, after separation with phosphate buffer on a cation-exchange column, were successively desalted by this method, and were observed as protonated ions by mass spectrometry. This desalting system could be useful for the structural elucidation of unknown hydrophilic compounds eluted by conventional high-performance liquid chromatography methods, such as ion-exchange chromatography, with mobile phases containing nonvolatile salts. As an example, we present the structural elucidation of unknown metabolites in bovine serum.

A further study on the combined use of internal standard and isotope-labeled derivatization reagent for expansion of linear dynamic ranges in liquid chromatography-electrospray mass spectrometry.

The combined use of a so-called internal standard and the isotope-labeled derivatization reagent for the quantification of analytes for liquid chromatography-mass spectrometry (LC/MS) was further studied. The sample solution (containing the analytes and an internal standard) was derivatized with the light form of the derivatization reagent, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (DBD-PZ-NH(2)) or 7-(N,N-dimethylaminosulfonyl)-4-piperazino-2,1,3-benzoxadiazole (DBD-PZ). A standard solution of the analytes (containing an internal standard) was derivatized with the isotope (d(6))-labeled derivatization reagent, DBD-PZ-NH(2) (D) or DBD-PZ (D), and served as the isotope-labeled internal standards. The peak heights of the targeted analytes derivatives in the sample solution were corrected using those of the internal standard and the heavy form derivatives of the standards, and the calibration curves were constructed. The curve bending of the calibration curves caused by the ion suppression at the ion source was suppressed and the linear dynamic ranges of the calibration curves were expanded. The derivatives of DBD-PZ-NH(2) were about 10 times more sensitively detected than those of DBD-PZ derivatives and, therefore, DBD-PZ-NH(2) might be suitable for sensitive detection.

Synthesis of the isotope-labeled derivatization reagent for carboxylic acids, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (d6) [DBD-PZ-NH2 (D)], and its application to the quantification and the determination of relative amount of fatty acids in rat plasma samples by high-performance liquid chromatography/mass spectrometry.

The isotope-labeled benzofurazan derivatization reagent for carboxylic acids, 7-(N,N-dimethylaminosulfonyl)-4-(aminoethyl)piperazino-2,1,3-benzoxadiazole (d6) [DBD-PZ-NH2 (D)] was synthesized. DBD-PZ-NH2 (D) was used for the accurate quantification of fatty acids by liquid chromatography/mass spectrometry (LC/MS). The standard fatty acids were derivatized with DBD-PZ-NH2 (D) to the stable isotope-labeled compounds for the fatty acids derivatives of DBD-PZ-NH2 and used for the internal standards. The obtained calibration curves for fatty acids were linear over the range 0.1-200 microM (r2 > 0.999). Fatty acids in plasma samples were determined after derivatization with DBD-PZ-NH2 and analyzed by LC/MS using standard fatty acid DBD-PZ-NH2 (D) derivatives as internal standards. Furthermore, the relative amounts of fatty acids in two plasma samples were determined after derivatization with DBD-PZ-NH2 and DBD-PZ-NH2) (D). The isotope-labeled derivatization reagent was useful for accurate quantification and the determination of relative amounts of the metabolites in biological samples having the target functional group.