Efficient determination of purine metabolites in brain tissue and serum by high-performance liquid chromatography with electrochemical and UV detection.
The purine metabolic pathway has been implicated in neurodegeneration and neuroprotection. High-performance liquid chromatography (HPLC) is widely used to determine purines and metabolites. However, methods for analysis of multiple purines in a single analysis have not been standardized, especially in brain tissue. We report the development and validation of a reversed-phase HPLC method combining electrochemical and UV detection after a short gradient run to measure seven purine metabolites (adenosine, guanosine, inosine, guanine, hypoxanthine, xanthine and urate) from the entire purine metabolic pathway. The limit of detection (LoD) for each analyte was determined. The LoD using UV absorption was 0.001 mg/dL for hypoxanthine (Hyp), inosine (Ino), guanosine (Guo) and adenosine (Ado), and those using coulometric electrodes were 0.001 mg/dL for guanine (Gua), 0.0001 mg/dL for urate (UA) and 0.0005 mg/dL for xanthine (Xan). The intra- and inter-day coefficient of variance was generally <8%. Using this method, we determined basal levels of these metabolites in mouse brain and serum, as well as in post-mortem human brain. Peak identities were confirmed by enzyme degradation. Spike recovery was performed to assess accuracy. All recoveries fell within 80-120%. Our HPLC method provides a sensitive, rapid, reproducible and low-cost method for determining multiple purine metabolites in a single analysis in serum and brain specimens.
MassGeneral Institute for Neurodegenerative Disease, Department of Neurology, Massachusetts General Hospital, Harvard Medical School, 114 16th, Charlestown, MA 02129, USA.
Thomas C
TC
Burdett
Cody A
CA
Desjardins
Robert
R
Logan
Nikolaus R
NR
McFarland
Xiqun
X
Chen
Michael A
MA
Schwarzschild
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D003342:Corpus Striatum / Q000737:chemistry*; D055664:Electrochemical Techniques; D006801:Humans; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D011687:Purines / Q000032:analysis* / Q000097:blood / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet
D011687:Purines