MTBSTFA derivatization-LC-MS/MS approach for the quantitative analysis of endogenous nucleotides in human colorectal carcinoma cells
- Author:
Huixia ZHANG
1
;
Yan LI
;
Zheng LI
;
Wai-Kei-Christopher LAM
;
Peng ZHU
;
Caiyun WANG
;
Hua ZHOU
;
Wei ZHANG
Author Information
1. State Key Laboratory of Quality Research in Chinese Medicines
- Keywords:
Ribonucleotides;
Deoxyribonucleotides;
Derivatization;
N-(t-butyldimethylsilyl)-N-methyltrifluoroacetamide;
High-performance liquid chromatography tandem mass spectrometry
- From:
Journal of Pharmaceutical Analysis
2022;12(1):77-86
- CountryChina
- Language:Chinese
-
Abstract:
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-methyltri-fluoroacetamide(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 temper-ature contributed to shortened analysis time.The derivatives of 22 nucleoside mono-,di-and tri-phosphates were retained on the typical Cig 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 HCT116 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.
