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

作　　者：Huixia Zhang Yan Li Zheng Li Christopher Wai-Kei Lam Peng Zhu Caiyun Wang Hua Zhou Wei Zhang 

机构地区：[1]State Key Laboratory of Quality Research in Chinese Medicines,Macao Institute for Applied Research in Medicine and Health,Macao University of Science and Technology,Taipa,Macao,China

出　　处：《Journal of Pharmaceutical Analysis》2022年第1期77-86,共10页药物分析学报（英文版）

基　　金：supported by Science and Technology Development Fund, Macao SAR (File Nos.: 0052/2018/A2 and 0077/2019/ A2);the National Natural Science Foundation of China (Grant No.: 61827819)

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 tri-phosphates were retained on the typical C;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 0.1-0.4μM for the tested RNs and 0.001-0.1μM 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.

关 键 词：RIBONUCLEOTIDES Deoxyribonucleotides DERIVATIZATION N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide High-performance liquid chromatography tandem mass spectrometry 

分 类 号：R735.34[医药卫生—肿瘤]