Untargeted UHPLC-MS deferential screening method and targeted UHPLC-MS/MS quantitative method for the determination of amino acids in mouse brain tissue

Abstract

An untargeted UHPLC-MS method was developed for the preliminary screening of differential amino acids between two mouse brain tissue samples, and a targeted UHPLC-MS/MS method was developed for the quantitative determination of amino acids in mouse brain tissue samples. The sample processing procedure, chromatographic conditions and MS parameters were optimized. The mouse brain tissue samples were homogenized with HCl solution (0.01 mol/L), protein precipitated with methanol and centrifugation. For the untargeted method, one sample was derivatized with butanol (D0)-HCl and another sample was derivatized with deuterated butanol (D9)-HCl, then combined together and analyzed with UHPLC-MS. Chromatographic peak area ratio for each amino acid was then used for differential analyses between two samples. Results showed good accuracy with standard spiked samples (83.4–113.0 %). For the targeted quantitative method, the sample supernatants were dried and residues were derivatized with butanol (D0)-HCl. Internal standards of amino acids derivatized with deuterated butanol (D9)-HCl were added before analyzed with UHPLC-MS/MS. The intra- and inter-day precisions were in the intervals of 0.5–8.1 % and 0.3–9.1 %. The LOD and the LOQ were ranged from 0.03 × 10−3 to 0.92 × 10−3 µg mL−1 and 0.08 × 10−3 to 3.08 × 10−3 µg mL−1, respectively. Spiked recoveries were from 96.8 % to 111.7 %. Both methods were applied to analyze amino acids in brain tissue of C57BL/6 mice treated and untreated with piperine and the result showed that the levels of glutamic acid, aspartic acid, histidine, asparagine, γ-aminobutyric acid, alanine, leucine and isoleucine significantly decreased in the piperine treated group comparing with the untreated group.