Determination of phosphorus using capillary electrophoresis and micro-high-performance liquid chromatography hyphenated with inductively coupled plasma mass spectrometry for the quantification of nucleotides

Abstract

We performed the quantification of phosphorus in deoxynucleotides using capillary electrophoresis (CE) and micro-HPLC (microHPLC) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). DNA and its component units have conventionally been determined by photometry; however, more selective and sensitive methods are needed for small biological samples. CE and microHPLC offer the advantages of good separation and small consumption of samples, and ICP-MS is a highly sensitive technique for the determination of a chemical element. Therefore, we have developed an interface device for combining CE and microHPLC with ICP-MS for quantifying nucleotides based on phosphorus content. The interface utilizes 4.5 microL/min for nebulizing and effective introduction of the sample into ICP. The samples of nucleotides and free phosphoric acid were well separated in the CE-ICP-MS measurement, and the calibration curves (1-100 microg/mL) of the nucleotides showed a linear (R2>0.999) increase in intensity. Similarly, the samples of nucleotides were baseline separated using microHPLC-ICP-MS, and the calibration curves of the nucleotides were linear (R2>0.998). The detection limits of these species and phosphorus in nucleotides using CE-ICP-MS and microHPLC-ICP-MS were 0.77-6.5 ng/mL and 4.0-6.5 ng/mL, respectively. These values were about one or two orders lower than those in a previous report. The sample volumes of these experiments were calculated to be about 10 nL and 50 nL per analysis. Therefore, these analytical methods have the potential to be useful for the determination of biological samples, such as DNA and RNA molecules.