Flow injection analysis−tandem mass spectrometry (FIA-TMS) has been applied in a first-tier test of newborn screening (NBS). Although
isovalerylcarnitine (i-C5), which is a diagnostic
indicator of
isovaleric acidemia (IVA), is isobaric with
pivaloylcarnitine (p-C5), 2-methylbutyrylcarnitine, and n-valerylcarnitine, these isomers cannot be distinguished by the FIA-TMS. There are many reports of false positives derived from p-C5 due to the use of pivalate-conjugated
antibiotics. In this study, we developed a new FIA-TMS method to distinguish i-C5 and p-C5. We found that the intensity ratio of product
ions for i-C5 and p-C5 was different in a certain range even under the same analytical conditions. The product
ions with the most distinct differences in ionic intensity between the isomers and the collision energies that produce them were determined to be m/z 246.2 > 187.1 and −15 V, respectively. In addition to the quantification ion, a reference ion was defined, and the similarity of the i-C5 and p-C5 reference ion ratios (i-C5 score and p-C5 score, respectively) were used to estimate which isomer (i-C5 and p-C5) was responsible for elevated C5
acylcarnitine in dried blood spots (DBSs). As a result of analyses of 11 DBS samples derived from pivalate-conjugated
antibiotics and four DBS samples from IVA patients using our method, it was found that our method was able to correctly determine the type of C5-acylcarnitine (i-C5 or p-C5) in the DBS samples. Implementation of this new FIA-TMS method into the current NBS protocol will allow for a reduction in false positives in IVA.