Mass Spectrometry Letters

Korean Society for Mass Spectrometry (한국질량분석학회)
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Direct Quantitation of Amino Acids in Human Serum Using a Stepwise-Dilution Strategy and a Mixed-Mode Liquid Chromatography-Tandem Mass Spectrometry Method

  • Lee, Jaeick (Analytical Science and Technology Laboratory, Doping Control Center, Korea Institute of Science and Technology) ;
  • Lee, Seunghwa (Analytical Science and Technology Laboratory, Doping Control Center, Korea Institute of Science and Technology) ;
  • Kim, Byungjoo (Analytical Chemistry Center, Division of Metrology for Quality Life, Korea Research Institute of Standards and Science) ;
  • Lee, Joonhee (Analytical Chemistry Center, Division of Metrology for Quality Life, Korea Research Institute of Standards and Science) ;
  • Kwon, Oh-Seung (Doping Control Center, Korea Institute of Science and Technology) ;
  • Cha, Eunju (Analytical Science and Technology Laboratory, Doping Control Center, Korea Institute of Science and Technology)
  • Received : 2018.03.07
  • Accepted : 2018.03.13
  • Published : 2018.03.30
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Abstract

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 oxidation of a serum sample ($50{\mu}L$), which was then subjected to stepwise dilution using 3, 30, and 90 volumes of acetonitrile containing 0.1% formic acid. Chromatographic separation was performed on an Imtakt Intrada Amino Acid column ($50mm{\times}3mm$, $3{\mu}m$) 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 $0.5-4.0{\mu}g/mL$ 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.

Keywords

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Figure 1. Representative MRM chromatograms for 19 free amino acids and internal standards in normal human serum using LC？ESI/MS/MS with mixed-mode column and three-step dilutions.

Table 1. Dynamic ranges, linearities (r2), and LLOQs of 19 freeamino acids using LC？ESI/MS/MS.

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Table 2.Validation results for intra- and interday accuracy, precision, and matrix effect of 19 free amino acids using LC？ ESI/MS/MS.

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Table 2.Continued.

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Table 3. Results for the determination of the concentrations of 19 free amino acids in normal human serum samples.

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