An optimized HPLC/MS/MS method for quantification of excitatory amino acids in rat hippocampus and its application in brain ischemia/reperfusion research.

An optimized HPLC/MS/MS method was established to quantify glutamate (Glu) and aspartic acid (Asp) in rat hippocampus with glutamate-d5 (Glu-d5) as internal standard. The mass spectrometry was operated under the multiple reaction monitoring mode using electrospray ionization in the positive ion mode for Glu and negative ion mode for Asp. The retention times of Glu, Asp and Glu-d5 were 1.53, 2.07 and 1.52 min, respectively. The linearity of calibration curves was good, with r(2)  > 0.99 and a lower limit of quantitation of 10 ng/mL. The intra-day precisions (relative standard deviation, RSD) of Glu and Asp were in the range of 3.61-8.17 and 4.22-10.09%, respectively; the inter-day precisions (RSD) of Glu and Asp were in the range of 3.57-5.19 and 2.49-5.04%, respectively. The accuracies of Glu and Asp were in the range of -2.10-6.20 and -0.90-10.00%, respectively. The recovery rates of 10, 100 and 1000 ng/mL were found to be 0.89 ± 0.24, 1.01 ± 0.10 and 0.90 ± 0.12 for Glu and 0.99 ± 0.26, 0.93 ± 0.07 and 1.13 ± 0.13 for Asp, respectively. This optimized method was successfully applied to quantify the concentration of Glu and Asp in rat hippocampus in brain ischemia/reperfusion research.