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

Abstract

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.

Abnormal levels of seven amino neurotransmitters in depressed rat brain and determination by HPLC‐FLD

“…Several methods have been developed and used for analysis of amino acids, such as high‐performance liquid chromatography (HPLC), gas chromatography (GC) (Wolfensberger, Amsler, Canzek, & Cuénod, ) and capillary electrophoresis (CE) (Lorenzo, Navarrete, Balderas, & Garcia, ). HPLC is widely used, and is commonly coupled with mass spectrometric (MS) detection (Xu, Qian, Zhao, & Fang, ; Zhang, Fang, & Smagin, ), electrochemical detection (Tcherkas et al, ) and ultraviolet detection (Wu et al, ) to resolve mixtures. However, these methods show various limitations.…”