title: Rapid quantification of metabolic intermediates in blood by liquid chromatography-tandem mass spectrometry to investigate congenital lactic acidosis.
abstract: A novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been established to quantify metabolic intermediates, including lactate (Lac), pyruvate (Pyr), acetoacetate (ACAC) and 3-hydroxybutyrate (3-HB) in blood. Samples were deproteinized with methanol-acetonitrile solution, and analytes were separated on an adamantyl group-bonded reverse phase column and detected in multiple reaction monitoring mode. Total analysis time was 4 min per sample. Method validation results displayed that limits of quantification were 10.0 μmol L-1for Lac and Pyr, and 5.0 μmol L-1for ACAC and 3-HB. The within- and between-run coefficients of variation were in the range of 1.2-6.4% for all analytes. The recoveries were ranged from 95.6 to 111.5%. The reference values of analytes were determined for the pediatric population. Duo to instability of Lac, Pyr and ACAC in vitro, a comprehensive stability assay was performed to determine optimal conditions for sample collection, pretreatment and storage. Results showed that precipitation of protein in blood at bedside combined with low storage temperature could effectively preserve the integrity of Lac, Pyr and 3-HB, but the precipitated protein accelerated degradation of ACAC. Isolation of supernatant fluid slowed degradation of ACAC. Supernatant samples could store at -20 °C for 10 days. The use of plasma or serum to determine these intermediates was not recommended. In this study, 450 samples from patients were analyzed, and 7 patients were diagnosed as congenital lactic acidosis. With the advantages of rapid, accurate and reliable, this method is very suitable for congenital lactic acidosis screening and researches related to energy metabolism.
authors.affiliation: Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, The Affiliated Hospital of Guangzhou Medical University, 9 Jinsui Road, Guangzhou, China.
authors.forename: Minzhi
authors.initials: M
authors.lastname: Peng
authors.affiliation: Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, The Affiliated Hospital of Guangzhou Medical University, 9 Jinsui Road, Guangzhou, China.
authors.forename: Yanna
authors.initials: Y
authors.lastname: Cai
authors.affiliation: Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA.
authors.forename: Xiefan
authors.initials: X
authors.lastname: Fang
authors.affiliation: Department of Genetics and Endocrinology, Guangzhou Women and Children's Medical Center, The Affiliated Hospital of Guangzhou Medical University, 9 Jinsui Road, Guangzhou, China. Electronic address: liliuxia2013@gmail.com.
authors.forename: Li
authors.initials: L
authors.lastname: Liu
mesh_terms: D000090:Acetoacetates / Q000097:blood; D000140:Acidosis, Lactic / Q000097:blood* / Q000151:congenital; D002853:Chromatography, Liquid; D006801:Humans; D006885:Hydroxybutyrates / Q000097:blood; D007773:Lactates / Q000097:blood; D011773:Pyruvates / Q000097:blood; D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D000090:Acetoacetates; D006885:Hydroxybutyrates; D007773:Lactates; D011773:Pyruvates; C016635:acetoacetic acid
keywords: 3-Hydroxybutyrate; Acetoacetate; Energy metabolism; Hyperlactacidemia; Lactate; Pyruvate