title: Quantifying MMA by SLE LC-MS/MS: Unexpected challenges in assay development.
abstract: OBJECTIVE
  Analysis of serum/plasma methylmalonic acid (MMA) is important for the diagnosis and management of methylmalonic acidemia in pediatric populations. This work focuses on developing and validating a liquid chromatography tandem mass spectrometry (LC-MS/MS) method to monitor methylmalonic acidemia using a simple method preparation.
  
  
  METHODS
  MMA and stable isotope labeled d3-MMA was extracted using supported liquid extraction (SLE). Assay imprecision, bias, linearity, recovery and carryover were determined. The relationship between MMA and propionyl acylcarnitine (C3-acylcarnitine) was also evaluated using historical paired results from 51 unique individuals.
  
  
  RESULTS
  Baseline separation between MMA and succinic acid was completed in 7min. The assay was linear from 0.1 to 500μM. The intra-day and inter-day imprecision CV ranged from 4.1 to 13.2% (0.3 to 526μM) and 5.0 to 15.7% (0.3 to 233μM), respectively. Recovery ranged from 93 to 125%. The correlation with a national reference laboratory LC-MS/MS assay showed a Deming regression of 1.026 and intercept of -1.335. Carryover was determined to be <0.04%. Patient-specific correlation was observed between MMA and C3-acylcarnitine.
  
  
  CONCLUSIONS
  This report describes the first LC-MS/MS method using SLE for MMA extraction. In addition, we illustrate the challenges encountered during this method development that should be assessed and resolved by any laboratory implementing a SLE LC-MS/MS assay designed to quantify analytes across several orders of magnitude.
authors.affiliation: Department of Laboratory Medicine, Chemistry Division, University of Washington, Seattle, WA, United States.
authors.forename: Sheng-Ying
authors.initials: SY
authors.lastname: Lo
authors.affiliation: Department of Laboratories, Seattle Children's Hospital, Seattle, WA, United States.
authors.forename: Cindy
authors.initials: C
authors.lastname: Gordon
authors.affiliation: Department of Laboratories, Seattle Children's Hospital, Seattle, WA, United States.
authors.forename: Katerina
authors.initials: K
authors.lastname: Sadilkova
authors.affiliation: Department of Laboratories, Seattle Children's Hospital, Seattle, WA, United States.
authors.forename: Rhona M
authors.initials: RM
authors.lastname: Jack
authors.affiliation: Department of Laboratories, Seattle Children's Hospital, Seattle, WA, United States. Electronic address: jane.dickerson@seattlechildrens.org.
authors.forename: Jane A
authors.initials: JA
authors.lastname: Dickerson
mesh_terms: D002853:Chromatography, Liquid / Q000379:methods*; D006801:Humans; D008764:Methylmalonic Acid / Q000097:blood*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: D008764:Methylmalonic Acid
keywords: Inborn errors of metabolism; LC-MS/MS; Methylmalonic acid; Methylmalonic acidemia; Supported-liquid extraction