An on-line SPE-LC-MS/MS method for quantification of nucleobases and nucleosides present in biological fluids.

Solid phase extraction (SPE) and liquid chromatographic (LC) separation of nucleobases and nucleosides are challenging due to the high hydrophilicity of these compounds. Herein we report a novel on-line SPE-LC-MS/MS method for their quantification after pre-column derivatization with chloroacetaldehyde (CAA). The method proposed is selective and sensitive with limits of detection at the nano-molar level. Analysis of urine and saliva samples by using this method is demonstrated. Adenine, guanine, cytosine, adenosine, guanosine, and cytidine were found in the range from 0.19 (guanosine) to 1.83 μM (cytidine) in urine and from 0.015 (guanosine) to 0.79 μM (adenine) in saliva. Interestingly, methylation of cytidine was found to be significantly different in urine from that in saliva. While 5-hydroxymethylcytidine was detected at a very low level (<0.05 μM) in saliva, it was found to be the most prominent methylated cytidine in urine at a high level of 3.33 μM. Since on-line SPE is deployed, the proposed LC-MS/MS quantitative assay is convenient to carry out and offers good assay accuracy and repeatability.