title: Rapid determination of C4-acylcarnitine and C5-acylcarnitine isomers in plasma and dried blood spots by UPLC-MS/MS as a second tier test following flow-injection MS/MS acylcarnitine profile analysis.
abstract: BACKGROUND
  Flow-injection MS/MS methods for elevated acylcarnitines are routinely performed in most newborn screening and biochemical genetics laboratories; however this technique cannot distinguish between isobaric compounds; therefore, chromatographic separation is required to quantitate isomers for differential diagnosis of some inborn errors of metabolism.
  
  
  METHODS
  A UPLC-MS/MS method has been developed for the simultaneous quantitation of isobutyrylcarnitine and butyrylcarnitine, and a second UPLC-MS/MS method for the quantitation of isovalerylcarnitine, (S) and (R) 2-methylbutyrylcarnitine, pivaloylcarnitine and valerylcarnitine. Plasma and dried blood spots samples are extracted with methanol and derivatized with butanolic HCl. Deuterium labeled internal standards are used for quantitation. Separation is obtained using a methanol/water gradient with a C18 BEH, 1x100mm, 1.7microm UPLC column, at 60 degrees C; run time is less than 10min. The isomers are detected with a Quattro Premier triple quadrupole, with electrospray ionization in positive ion mode.
  
  
  RESULTS
  Intra-day precision in plasma and dried blood spots ranged from 1.4% to 14% and accuracy from 88% to 114% respectively for butyrylcarnitine and isobutyrylcarnitine. Precision for the isomers of C5-acylcarnitine ranged from 1.3% to 15% and accuracy 87% to 119%, respectively in plasma or dried blood spots. Inter-day precision was within 20% at each concentration of isobutyrylcarnitine and butyrylcarnitine. Precision for 2-methylbutyrylcarnitine and isovalerylcarnitine at concentrations above the normal range was within 24%.
  
  
  CONCLUSIONS
  Two diagnostic tests based on the separation of C4-acylcarnitine and C5-acylcarnitine isomers by UPLC-MS/MS provide fast differential diagnosis of SCAD deficiency versus IBCD deficiency and IVA versus 2-MBCD deficiency. The separation of C5-acylcarnitines can reveal false elevation due to pivalic acid-containing antibiotics. Abnormal newborn screen results due to pivalate-generating prodrug antibiotics of maternal origin were confirmed. This separation of isomers can resolve multiple diagnostic challenges in both newborn screening and in cases with ambiguous metabolic test results.
authors.affiliation: Institute of Metabolic Disease, Baylor Research Institute, Dallas, TX 75226, USA. sabrinaf@baylorhealth.edu
authors.forename: Sabrina
authors.initials: S
authors.lastname: Forni
authors.forename: Xiaowei
authors.initials: X
authors.lastname: Fu
authors.forename: Susan E
authors.initials: SE
authors.lastname: Palmer
authors.forename: Lawrence
authors.initials: L
authors.lastname: Sweetman
mesh_terms: D002331:Carnitine / Q000031:analogs & derivatives* / Q000097:blood / Q000737:chemistry; D002853:Chromatography, Liquid / Q000379:methods; D006801:Humans; D007231:Infant, Newborn; D007536:Isomerism; D015997:Neonatal Screening / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C116917:acylcarnitine; C020381:isobutyryl-1-carnitine; C427065:butyrylcarnitine; C054040:pivaloylcarnitine; D002331:Carnitine