Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
1998945 | Molecular Genetics and Metabolism | 2010 | 8 Pages |
BackgroundFlow-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.MethodsA 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.ResultsIntra-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%.ConclusionsTwo 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.