QUANTIFICATION OF AMINO ACIDS IN RAT URINE BY SOLID-PHASE EXTRACTION AND LIQUID CHROMATOGRAPHY/ELECTROSPRAY TANDEM MASS SPECTROMETRY: APPLICATION TO RADIATION INJURY RAT MODEL

Abstract

A robust and sensitive liquid chromatography/electrospray tandem mass spectrometry (HPLC–ESI–MS/MS) method for the determination of urinary amino acids was developed. Silica-based cationic exchange (SCX) solid-phase extraction (SPE) cartridges were used to extract amino acids and minimize the matrix effect. The SPE cleanup protocol was optimized to get satisfactory recoveries and reproducible data. The phenomenon of retention time shift between the standards and samples was eliminated using SCX cleanup. For most amino acids, extraction efficiency recoveries were between 53% and 100%, the recoveries induced by matrix effect were between 70% and 120% and the total recoveries were between 50.4% and 125.2%. The other analytical characteristics such as intra-day precision (CV < 8%), inter-day precision (CV < 10%), and accuracy (88.2–117.0%) were also satisfactory for most of the amino acids. Only a few of isotope-labeled internal standards cover the LC–MS/MS analysis with satisfactory results, which is cost effective. The method was successfully applied to early dynamic changes in amino acid concentration of urine samples from rats after exposure to ionizing radiation.

Keywords:

• amino acids
• ionizing radiation
• liquid chromatography/electrospray tandem mass spectrometry
• matrix effect
• solid-phase extraction
• urine

ACKNOWLEDGMENT

The study has been supported by the foundation (No. 31000385) from the National Natural Science Foundation of China, Jiangsu University Natural Science Research Project (No.10KJB310012), Jiangsu University Natural Science major projects (No.09KJA310004), postdoctoral research funding of Jiangsu Province (No.0901052C), and a project funded by the Priority Academic Program Development (PAPD) of Jiangsu Higher Education Institutions.

Notes

Recovery 1: comparison of extracted standards to non-extracted standards (extraction efficiency); recovery 2: comparison of standards from extracted spiked urine samples to extracted standards (matrix effects); recovery 3: comparison of extracted urine samples to non-extracted standards (extraction efficiency plus matrix effects).

Recovery 1: comparison of extracted standards to non-extracted standards (extraction efficiency); recovery 2: comparison of standards from extracted spiked urine samples to extracted standards (matrix effects); recovery 3: comparison of extracted urine samples to non-extracted standards (extraction efficiency plus matrix effects).

^a With stable isotope dilution method, the other with external standard method.

Results are mean ± SD, n = 10.

^*Significantly different at P < 0.05 compared to control.

^**Significantly different at P < 0.01 compared to control.

Authors Xinxing Tang and Yuan Gu contributed equally to this manuscript.

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