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Development of an ion‐pair HPLC method for investigation of energy charge changes in cerebral ischemia of mice and hypoxia of Neuro‐2a cell line
Author(s) -
Chen Yunyun,
Xing Dongming,
Wang Wei,
Ding Yi,
Du Lijun
Publication year - 2007
Publication title -
biomedical chromatography
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.4
H-Index - 65
eISSN - 1099-0801
pISSN - 0269-3879
DOI - 10.1002/bmc.798
Subject(s) - chemistry , chromatography , detection limit , tetrabutylammonium hydroxide , high performance liquid chromatography , potassium hydroxide , trichloroacetic acid , acetic acid , calibration curve , coefficient of variation , analytical chemistry (journal) , acetonitrile , standard curve , biochemistry , organic chemistry
The determination of adenine nucleotides and energy charge (EC) has great importance in the characterization of cerebral ischemic injury and post‐ischemic recovery. An IP‐HPLC method was developed for the quantification of AMP, ADP, ATP and EC in cerebral ischemia and hypoxia of the Neuro‐2a cell line. The chromatographic conditions were: a Zorbax SB‐C 18 reversed‐phase column; mobile phase 100 m m KH 2 PO 4 , 1 m m tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 m ), filtered through a 0.45 µm Millipore filter and degassed prior to use. The flow‐rate was 1.0 mL/min. The injection volume was 20 µL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 ± 1°C). The method was validated by means of linearity, using calibration curves constructed with five concentration levels of each compound. The limit of detection was also determined. The system precision was calculated as the coefficient of variation for five injections for each compound tested. Cerebral tissue was homogenized (4°C) in 1 mL of an ice‐cold 6% trichloroacetic acid that contained ATPase inhibitor and obtained good recovery (>90%). The results show that the described method for the determination of adenine nucleotides by HPLC has good linearity, limit of detection, precision and specificity, and is simple and rapid to perform. Copyright © 2007 John Wiley & Sons, Ltd.