Determination of phosphorus in phosphorylated deoxyribonucleotides using capillary electrophoresis and high performance liquid chromatography hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell

Abstract

The determination of phosphorus in a biologically relevant sample matrix such as DNA is described. The analytical methodology used is based on a robust on-line coupling of capillary electrophoresis (CE) or high performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometry, equipped with an octopole reaction system (ORS-ICP-MS). Polyatomic ions formed in the plasma and the interface region of the ICP-MS that interfere with the determination of P at mass 31 were minimised by the addition of helium to the collision cell. Instrumental detection limits of 125 ng L⁻¹ for ³¹P, and in the range of 18 to 49 ng L⁻¹ for the other trace elements, were achieved under the conditions described. In order to demonstrate the high separation and detection efficiency of the system, the method developed has been applied to the element specific detection of phosphorus in monophosphorylated deoxynucleotides and to enzymatically digested calf thymus DNA after CE and HPLC separation, respectively. Both hyphenated techniques provide baseline separation of the four deoxynucleotide monophosphates (dNMP), which are present in the DNA chain. With CE-ICP-MS, detection limits for phosphorus down to 53 µg L⁻¹ (2′-deoxyguanosin-5′-monophosphate, dAMP) (corresponding to 0.6 pg P absolute) have been achieved. RSDs of the migration times were about 5%. Using HPLC-ICP-MS, detection limits down to 3 µg L⁻¹ (2′-deoxythymidine-5′-monophosphate, dTMP) (corresponding to 0.03 ng P absolute) were obtained. The migration/elution order of the deoxynucleotides investigated was obtained by comparison with the migration/retention times of commercially available dNMP's. Furthermore it was checked if HPLC-ESI-MS can be operated under the same chromatographic conditions for the confirmation of the elution order and for the characterisation of unknown peaks. Finally, flow injection analysis (FIA) was used for the quantification of the phosphorus content in the dNMP samples. Using FIA, detection limits for phosphorus of 2.5 µg L⁻¹ corresponding to 25 pg phosphorus absolute were achieved.