MTBSTFA derivatization-LC-MS/MS approach for the quantitative analysis of endogenous nucleotides in human colorectal...

Endogenous ribonucleotides (RNs) and deoxyribonucleotides (dRNs) are important metabolites related to the pathogenesis of many diseases. In light of their physiological and pathological significances, a novel and sensitive pre-column derivatization method with N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide (MTBSTFA) was developed to determine RNs and dRNs in human cells using high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). A one-step extraction of cells with 85% methanol followed by a simple derivatization reaction within 5 min at room temperature contributed to shortened analysis time. The derivatives of 22 nucleoside mono-, di- and triphosphates were retained on the typical C₁₈ column and eluted by ammonium acetate and acetonitrile in 9 min. Under these optimal conditions, good linearity was achieved in the tested calibration ranges. The lower limit of quantitation (LLOQ) was determined to be 0.1-0.4 μM for the tested RNs and 0.001-0.1 μM for dRNs. In addition, the precision (CV) was <15% and the RSD of stability was lower than 10.4%. Furthermore, this method was applied to quantify the endogenous nucleotides in human colorectal carcinoma cell lines HCT 116 exposed to 10-hydroxycamptothecin. In conclusion, our method has proven to be simple, rapid, sensitive, and reliable. It may be used for specific expanded studies on intracellular pharmacology in vitro.