An easy-to-use liquid chromatography method with fluorescence detection for the simultaneous determination of five neuroactive amino acids in different regions of rat brain

作者：Fonseca Beatriz M; Cristovao Ana C; Alves Gilberto^*

来源：Journal of Pharmacological and Toxicological Methods, 2018, 91: 72-79.

DOI：10.1016/j.vascn.2018.02.002

• Amino acids
• Bioanalytical method validation
• Liquid chromatography
• o-Phthalaldehyde
• Derivatization
• Brain tissue

Introduction: To comprehend the normal function and pathological characteristics of certain neurological disorders it is important to evaluate the neuroactive amino acids levels in animal models.
Methods: This work describes a simple liquid chromatography-fluorescence detection (LC-FLD) method for the simultaneous determination of aspartic acid (Asp), glutamic acid (Glu), glutamine (Gln), taurine (Tau) and gamma-aminobutyric acid (GABA), using methyl-L-arginine as internal standard, in samples of rat brain tissue. The five target analytes (Asp, Glu, Gln, Tau and GABA) were determined in a single chromatographic run of less 11 min after a derivatization step with o-phthalaldehyde. The derivatives were separated on a reversed-phase C-18 column and detected by fluorescence at excitation and emission wavelengths of 340 and 448 nm, respectively.
Results: The method was validated in accordance with international guidelines on bioanalytical methods validation and it presented limits of quantification in the range of 25-50 ng mL(-1) and calibration curves with determination coefficients (r(2)) equal to or higher than 0.9920. In addition, the precision (coefficient variation, %) and accuracy (bias, %) of the method meet the established criteria, and the stability of the analytes at the sample handling and storage conditions was demonstrated.
Discussion: Unlike other similar bioanalytical assays, the current method was validated using diluted biological matrix, which is advantageous in order to ensure the derivatization process integrity. Moreover, this LC-FLD method was successfully applied for the determination of the compounds of interest in different rat brain tissue regions (frontal cortex, amygdala, hippocampus, cerebellum and striatum). Thus, this bioanalytical assay represents a useful tool to support multiple nonclinical studies in the field of neurosciences, requiring the quantitative profiling and pattern analysis of neuroactive amino acids.

• 出版日期2018-6