title: Quantitation of oligonucleotides by phosphodiesterase digestion followed by isotope dilution mass spectrometry: proof of concept.
abstract: The importance of DNA as a regulatory analyte is well-known. Recent years have seen an increased interest in the quantitation of this analyte. Accurate quantitative measurements have been hampered by the lack of well-characterized standards and pure materials for this large-molecular-weight analyte. Outlined here is an approach for the accurate and reproducible quantitation of an oligonucleotide that is solely reliant on the availability of pure, well-characterized deoxynucleotides and not a sequence-specific pure DNA standard. The proposed procedure is intended to provide an accurate and definitive method for the quantitation of DNA for reference measurements as an improved alternative to the more conventional UV absorbance-based methods. For proof of concept, a gravimetrically prepared oligonucleotide solution was enzymatically digested to its constituent monomer-deoxynucleotide monophosphates (dNMPs), of which there are four different types. Qualitative mass spectrometry was used to confirm the 100% successful completion of the enzymatic digestion step. The dNMPs were then separated by liquid chromatography (LC) before being detected by electrospray ionization (ESI) mass spectrometry (MS). The method of quantitation was based on isotope dilution mass spectrometry (IDMS) analysis of the four different monomer units. The concentrations of the four dNMP residues were then summed to obtain the original concentration of the oligonucleotide. The concentrations determined by liquid chromatography/mass spectrometry (LC/MS) and also by liquid chromatography-tandem mass spectrometry (LC/MS/MS) differed by <2.5 and 1%, respectively, from the gravimetrically assigned value. These differences were well within the uncertainty of the gravimetrically assigned value. This highly accurate method, suitable for the definitive quantitation of oligonucleotides, should be ideal for characterizing primary calibration standards and certified reference materials that can then be used to underpin the more conventional quantitative techniques of UV and fluorescence spectroscopy.
authors.affiliation: LGC Limited, Queens Road, Teddington, Middlesex, UK. goc@lgc.co.uk
authors.forename: Gavin
authors.initials: G
authors.lastname: O'Connor
authors.forename: Carol
authors.initials: C
authors.lastname: Dawson
authors.forename: Alison
authors.initials: A
authors.lastname: Woolford
authors.forename: Kenneth S
authors.initials: KS
authors.lastname: Webb
authors.forename: Tim
authors.initials: T
authors.lastname: Catterick
mesh_terms: D002851:Chromatography, High Pressure Liquid; D007554:Isotopes; D009841:Oligonucleotides / Q000032:analysis* / Q000378:metabolism; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D007554:Isotopes; D009841:Oligonucleotides; D010727:Phosphoric Diester Hydrolases