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

Sabrina Forni, Xiaowei Fu, Susan E. Palmer, Lawrence Sweetman

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

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, 1 × 100 mm, 1.7 μm UPLC column, at 60 °C; run time is less than 10 min. 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.

Original languageEnglish (US)
Pages (from-to)25-32
Number of pages8
JournalMolecular genetics and metabolism
Volume101
Issue number1
DOIs
StatePublished - Sep 2010
Externally publishedYes

Keywords

  • 2-Methylbutyryl-CoA dehydrogenase deficiency
  • Isobutyryl-CoA dehydrogenase deficiency
  • Isovaleric acidemia
  • Pivaloylcarnitine
  • Short-chain acyl-CoA dehydrogenase deficiency
  • Short-chain acylcarnitines
  • Ultra performance liquid chromatography mass spectrometry

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Biochemistry
  • Molecular Biology
  • Genetics
  • Endocrinology

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