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  • P-ISSN 2233-4203
  • E-ISSN 2093-8950

Direct Quantitation of Amino Acids in Human Serum Using a Stepwise- Dilution Strategy and a Mixed-Mode Liquid Chromatography-Tandem Mass Spectrometry Method

Mass Spectrometry Letters / Mass Spectrometry Letters, (P)2233-4203; (E)2093-8950
2018, v.9 no.1, pp.30-36
Lee Jaeick (Korea Institute of Science and Technology)
Lee Seunghwa (Korea Institute of Science and Technology)
Kim Byungjoo (Research Institute for Natural Sciences)
Lee Joonhee (Research Institute for Natural Sciences)
Kwon Oh-Seung (Korea Institute of Science and Technology)
Cha Eunju (Daegu-Gyeongbuk Medical Innovation Foundation)
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A quantitation method for free amino acids in human serum was developed using a stepwise-dilution method and a bimodal cation exchange (CEX)/hydrophilic interaction liquid chromatography (HILIC)-tandem mass spectrometry system equipped with an electrospray ionization source (ESI/MS/MS). This method, which was validated using quality control samples, was optimized for enhanced selectivity and sensitivity. Dithiothreitol (DTT) was used as a reducing agent to prevent the oxida- tion of a serum sample (50 µL), which was then subjected to stepwise dilution using 3, 30, and 90 volumes of acetonitrile con- taining 0.1% formic acid. Chromatographic separation was performed on an Imtakt Intrada Amino Acid column (50 mm × 3 mm, 3 µm) in mixed mode packed with CEX and HILIC ligands embedded in the stationary phase. Underivatized free amino acids were eluted and separated within 10 min. As a result of the validation, the precision and accuracy for the inter- and intraday assays were determined as 2.11-11.51% and 92.82-109.40%, respectively. The lowest limit of quantification (LLOQ) was 0.5- 4.0 µg/mL and the matrix effect was 80.22-115.93%. The proposed method was successfully applied to the quantitative analysis of free amino acids in human serum.

Amino acid, mixed-mode chromatography, quantitation, serum, LC-ESI/MS/MS



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