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.

Abstract

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-C18 reversed-phase column; mobile phase 100 mM KH(2)PO(4), 1 mM tetrabutylammonium hydroxide, and 2.5% acetonitrile, brought to pH 7.0 with potassium hydroxide (4 M), filtered through a 0.45 microm Millipore filter and degassed prior to use. The flow-rate was 1.0 mL/min. The injection volume was 20 microL. Detection was performed at a wavelength of 254 nm under a constant temperature (27 +/- 1 degrees 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 degrees 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.

• Authors: Yunyun Chen, Dongming Xing, Wei Wang, Yi Ding, Lijun Du
• Journal: Biomedical chromatography : BMC (Biomed Chromatogr) Vol. 21 Issue 6 Pg. 628-34 (Jun 2007) ISSN: 0269-3879 [Print] England
• PMID: 17385810 (Publication Type: Evaluation Study, Journal Article, Research Support, Non-U.S. Gov't, Validation Study)
• Copyright: Copyright 2007 John Wiley & Sons, Ltd.

Chemical References

• Adenine Nucleotides
• Ions

Topics

• Adenine Nucleotides (analysis, chemistry)
• Algorithms
• Animals
• Area Under Curve
• Brain (metabolism)
• Brain Chemistry
• Brain Ischemia (metabolism)
• Cell Line
• Chromatography, High Pressure Liquid (classification, methods)
• Energy Metabolism
• Hypoxia (metabolism)
• Ions (chemistry)
• Male
• Mice
• Mice, Inbred ICR
• Reference Standards
• Reproducibility of Results
• Sensitivity and Specificity
• Spectrophotometry, Ultraviolet