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

Journal of Pharmaceutical Analysis

Wei Zhang¹  Hua Zhou²  Caiyun Wang³  Zheng Li³  Peng Zhu³  Huixia Zhang³  Christopher Wai-Kei Lam³  Yan Li³ 

[1] State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China;Corresponding author.;State Key Laboratory of Quality Research in Chinese Medicines, Macau Institute for Applied Research in Medicine and Health, Macau University of Science and Technology, Taipa, Macau, China;

关键词: Ribonucleotides;    Deoxyribonucleotides;    Derivatization;    N-(t-butyldimethylsilyl)-N- methyltrifluoroacetamide;    High-performance liquid chromatography tandem mass spectrometry;   

DOI  :  

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown