title: Measurement of succinyl-carnitine and methylmalonyl-carnitine on dried blood spot by liquid chromatography-tandem mass spectrometry.
abstract: Methylmalonic aciduria (MMA) is one of the most frequent organic acidurias, a class of diseases caused by enzymatic defects mainly involved in the catabolism of branched-chain amino acids. Recently, mild MMA and C4-dicarboxylyl-carnitine (C4DC-C) accumulation have been reported in patients carrying mutation in genes encoding the α-subunit (SUCLG1) and the β-subunit (SUCLA2) of the ADP-forming succinyl-CoA synthetase (SCS). We developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to quantify in dried blood spot the two isobaric compounds of C4DC-C, succinyl-carnitine and methylmalonyl-carnitine, to allow the differential diagnosis between classical MMA and SCS-related defects. This method, with an easy liquid-phase extraction and derivatization procedure, has been validated to demonstrate the specificity, linearity, recovery, lowest limit of quantification (LLOQ), accuracy and precision for quantitative determination of blood succinyl-carnitine and methylmalonyl-carnitine. The assay was linear over a concentration range of 0.025-10 μmol/L and achieved the LLOQ of 0.025 μmol/L for both metabolites. The average slope, intercept, and coefficient of linear regression (r(2)) were respectively: 0.3389 (95% confidence interval 0.2888-0.3889), 0.0113 (95% confidence interval -0.0157 to 0.0384), 0.9995 (95% confidence interval 0.9990-1.0000) for succinyl-carnitine and 0.5699 (95% confidence interval 0.5263-0.6134), 0.0319 (95% confidence interval -0.0038 to 0.0677), 0.9997 (95% confidence interval 0.9995-1.0000) for methylmalonyl-carnitine. Within-day and between-day coefficients of variation (CV) were 1.94% and 3.19% for succinyl-carnitine and 3.21%, and 2.56 for methylmalonyl-carnitine. This method is accurate and provides a new tool to differentiate patients with classical methylmalonic acidemia from those with SCS-related defects.
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Cristiano
authors.initials: C
authors.lastname: Rizzo
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy. Electronic address: sara.boenzi@opbg.net.
authors.forename: Sara
authors.initials: S
authors.lastname: Boenzi
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Rita
authors.initials: R
authors.lastname: Inglese
authors.affiliation: NeuroFarba Department, University of Florence, Florence, Italy; Newborn Screening Lab, Clinic of Pediatric Neurology, AOU Meyer, Florence, Italy.
authors.forename: Giancarlo
authors.initials: G
authors.lastname: la Marca
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Maurizio
authors.initials: M
authors.lastname: Muraca
authors.affiliation: Bioquimica Clinica y Istituto de Genetica Medica y Molecular, Hospital Universitario La Paz, Madrid, Spain.
authors.forename: Tegra Barreiro
authors.initials: TB
authors.lastname: Martinez
authors.affiliation: Department of Chemical Pathology, Women's and Children's Hospital, North Adelaide, South Australia, Australia.
authors.forename: David W
authors.initials: DW
authors.lastname: Johnson
authors.affiliation: Department of Laboratory Medicine, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Eleonora
authors.initials: E
authors.lastname: Zelli
authors.affiliation: Division of Metabolism and Research Unit of Metabolic Biochemistry, Bambino Gesù Children's Research Hospital, IRCCS, Rome, Italy.
authors.forename: Carlo
authors.initials: C
authors.lastname: Dionisi-Vici
mesh_terms: D000293:Adolescent; D002331:Carnitine / Q000031:analogs & derivatives* / Q000097:blood*; D002648:Child; D002675:Child, Preschool; D002853:Chromatography, Liquid; D059788:Dried Blood Spot Testing / Q000379:methods*; D005260:Female; D006801:Humans; D007223:Infant; D007231:Infant, Newborn; D057230:Limit of Detection; D016014:Linear Models; D008297:Male; D053719:Tandem Mass Spectrometry
chemical_list: D002331:Carnitine
keywords: C3-C; C3-C/C16-C; C3-C/C2-C; C4-dicarboxylyl-carnitine; C4DC-C; CE; CXP; DP; FOA; LLOQ; LOD; MMAs; MMC; MRM; MS/MS; Methylmalonic acidurias; S/N; SC; SCS; SD; SUCLA2; SUCLG1; TDHFA; collision cell exit potential; collision energy; declustering potential; formic acid; limit of detection; liquid chromatography-tandem mass spectrometry; lower limit of quantification; methylmalonic acidemia; methylmalonyl-carnitine; multiple reaction monitoring; propionyl-carnitine; propionyl-carnitine/acetyl-carnitine ratio; propionyl-carnitine/palmitoyl-carnitine ratio; signal-to-noise ratio; standard deviation; succinyl-CoA synthetase; succinyl-carnitine; tandem mass spectrometry; tridecafluoroheptanoic acid