Single nucleotide polymorphism genotyping by on-line liquid chromatography-mass spectrometry in forensic science of the Y-chromosomal locus M9

J Chromatogr B Analyt Technol Biomed Life Sci. 2002 Dec 25;782(1-2):89-97. doi: 10.1016/s1570-0232(02)00694-3.


A method is described for genotyping alleles of the Y-chromosomal locus M9, incorporating DNA extraction, amplification by polymerase chain reaction (PCR), sample purification by ion-pair reversed-phase high-performance liquid chromatography (IP-RP-HPLC), and allele identification by on-line hyphenation to electrospray ionization mass spectrometry (ESI-MS). The alleles G and C were differentiated in 114 base pair amplicons on the basis of intact molecular mass measurements with a mass accuracy between 0.007 and 0.017%. The accuracy of mass determination was significantly reduced to less than 0.0036% upon amplification of a short, 61 bp fragment. The application of steep gradients of acetonitrile in 25 mM butyldimethylammonium bicarbonate not only enabled the efficient separation of non-target components from the PCR product in a monolithic, poly-(styrene-divinylbenzene)-based capillary column, but also allowed the high-throughput analysis of the PCR products with cycle times of 2 min. The new method was compared to a conventional restriction fragment length polymorphism assay with capillary gel electrophoretic analysis. In a blind study, 90 samples of unrelated individuals were genotyped. The high accuracy (<0.004%) and small relative standard deviation (<0.007%, n=20) of mass measurements, which enables even the differentiation of A and T alleles with a mass difference of 9 mass units, make IP-RP-HPLC-ESI-MS a potent tool for the routine characterization of SNPs in forensic science.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Chromatography, High Pressure Liquid / methods*
  • Chromosomes, Human, Y*
  • DNA Primers
  • Forensic Medicine*
  • Genotype*
  • Humans
  • Polymorphism, Single Nucleotide*
  • Spectrometry, Mass, Electrospray Ionization / methods*


  • DNA Primers