Acetonitrile (high-performance liquid chromatography-grade) and high-purity water were obtained from Thermo Fisher (Waltham, MA, USA). 1-Butanol and acetyl chloride were commercially acquired from Sigma-Aldrich (St Louis, MO, USA). Isotope-labeled internal standards of 12 amino acids (NSK-A) and eight acylcarnitine (NSK-B) from Cambridge Isotope Laboratories (Tewksbury, MA, USA) were used for absolute quantification purpose. All the standards were mixed and dissolved in 2 mL pure methanol and stored at 4°C. Working solution was prepared through 100-fold dilution for metabolite extraction. Amino acids and carnitines quality control (QC) standards were provided by Chromsystems (Grafelfing, Germany). The QC samples were treated as real samples and processed according to the provided instructions to ensure the analysis stability.