Liquid chromatography–electrospray ionization mass spectrometry for simultaneous detection of mtDNA length and nucleotide polymorphisms

We demonstrate the applicability of ion-pair reversed-phase high-performance liquid chromatography—electrospray ionization time-of-flight mass spectrometry (ICEMS) for the simultaneous characterization of length and nucleotide polymorphisms. Two sections within the first (HVS-I) and second (HVS-II) hypervariable segments of the mitochondrial (mt)DNA control region were selected as targets, both containing poly-cytosine (C) tracts, which display length heteroplasmy at a substantial frequency in the population. The two mtDNA sections were simultaneously amplified and analyzed by ICEMS in 90 maternally unrelated mother–offspring pairs from Austria. The findings were confirmed by direct sequencing of the polymerase chain reaction products. For the detailed characterization of present-length heteroplasmic variants, the results retrieved through ICEMS were more informative compared with those derived from direct sequencing. Hence, ICEMS represents an interesting option for successful application in forensic science.