A Standard Addition Method Utilizing an Endogenous Substance as an Internal Standard for Quantitating Arabinofuranosylguanosine 5’-Triphosphate in Human Peripheral Blood Mononuclear Cells by Lc-Ms/MsYoshiyuki Minamide1*, Harue Igarashi2, Akira Wakamatsu2, Shinobu Kudoh1
- *Corresponding Author:
- Yoshiyuki Minamide, Ph.D.
2-13 Nishinokyo-Sanjyo Boucho, Nakagyo-ku
Kyoto 604-8435, Japan
E-mail: [email protected]
Received date: October 28, 2011; Accepted date: December 06, 2011; Published date: December 08, 2011
Citation: Minamide Y, Igarashi H, Wakamatsu A, Kudoh S (2011) A Standard Addition Method Utilizing an Endogenous Substance as an Internal Standard for Quantitating Arabinofuranosylguanosine 5’-Triphosphate in Human Peripheral Blood Mononuclear Cells by Lc-Ms/Ms. J Anal Bioanal Techniques S5:002. doi: 10.4172/2155-9872.S5-002
Copyright: © 2011 Minamide Y, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
A highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method was developed for quantitation of arabinofuranosylguanosine 5’-triphosphate (ara-GTP) in human peripheral blood mononuclear cells (PBMC). The determination of ara-GTP in PBMC was validated using a standard addition method with the human Tlymphoblastoid cell line as an alternative blank matrix. Ara-GTP was extracted with methanol/250 mmol/L ammonium carbonate solution (7/3, v/v) from the cells at a density of 106 cells per 0.5 mL. Extracts were subjected to LC-MS/ MS using a TurboIon spray interface and selected reaction monitoring with the transitions of m/z 524 to m/z 152 for quantitation. Endogenous guanosine triphosphate in the extract was used as an internal standard. Separation of the analytes was achieved on a porous graphitic carbon column (100 mm length × 2.1 mm i.d., 5 μm particle size) by isocratic elution with 250 mmol/L ammonium carbonate buffer (pH 9.5)/water/acetonitrile (40/51.5/8.5, v/v/v) at a flow rate of 0.2 mL/min. The method was validated in the range of 2–250 pg/mL. The pharmacokinetic profile of ara-GTP in PBMC in a Phase I clinical study of nelarabine in relapsed or refractory T-ALL/T-LBL patients was successfully determined using this method.