Junko Yamazaki

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.

Determination and quantification of the kokumi peptide, γ-glutamyl-valyl-glycine, in commercial soy sauces

Recent studies have demonstrated that kokumi substances, such as glutathione, are perceived through the calcium-sensing receptor (CaSR), and screening by CaSR assay and sensory evaluation has shown that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) is a potent kokumi peptide. In this study, the contents of γ-Glu-Val-Gly in six commercial brands of dark-coloured soy sauces, two brands of light-coloured soy sauce, and one brand of white soy sauce, were investigated by high performance liquid chromatography-tandem mass spectrometry (LC/MS/MS), followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate (AQC). The analyses indicated that γ-Glu-Val-Gly was present in all investigated soy sauces at concentrations ranging from 0.15 to 0.61mg/dl, demonstrating that it is widely distributed in soy sauces.

Determination and Quantification of γ-Glutamyl-valyl-glycine in Commercial Fish Sauces

It was recently reported that kokumi substances such as glutathione are perceived through the calcium-sensing receptor (CaSR). In addition, screening by the CaSR assay and sensory evaluation revealed that γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) was a potent kokumi peptide. In this study, the quantities of γ-Glu-Val-Gly in various commercial fish sauces originating from Vietnam (Nuoc Mum), Thailand (Nampra), China (Yu-lu), Korea, Japan (Shottsuru and Ikanago-shoyu), and Italy (Garum) were investigated using a LC/MS/MS method followed by derivatization with 6-aminoquinoyl-N-hydroxysuccinimidyl-carbamate (AQC). The analyses revealed γ-Glu-Val-Gly at concentrations ranging from 0.04 to 1.26 mg/dL, indicating that γ-Glu-Val-Gly is widely distributed among various commercial fish sauces.

Determination of γ-glutamyl-valyl-glycine in raw scallop and processed scallop products using high pressure liquid chromatography-tandem mass spectrometry.

The determination of the kokumi peptide, γ-glutamyl-valyl-glycine (γ-Glu-Val-Gly) in raw scallop and processed scallop products was carried out using high pressure liquid chromatography-tandem mass spectrometry (LC/MS/MS). The detection of γ-Glu-Val-Gly was achieved using a multiple reaction monitoring (MRM) method. The optimised condition enabled the precise determination of γ-Glu-Val-Gly. Raw scallop contained 0.08 μg/g γ-Glu-Val-Gly, and the γ-Glu-Val-Gly levels in processed scallop products, such as dried-scallop and scallop extract, were measured to be 0.64 and 0.77 μg/g, respectively. This is the first report to confirm the existence of γ-Glu-Val-Gly in foodstuff.

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.