Efficient determination of purine metabolites in brain tissue and serum by high-performance liquid chromatography with electrochemical and UV detection

Abstract

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.

Figure 1.

Purine degradation pathway. Adonosine is converted to inosine through the removal of the amine moiety by adenosine deaminase, and inosine is degraded to hypoxanthine through the removal of phospho-1-ribose by purine nucleoside phophorylase. Guanosine is converted to guanine via the action of purine nucleoside phosphorylase, and guanine is then degraded to xanthine through the action of guanine deaminase. In the presence of xanthine oxidase, hypoxanthine and xanthine are converted to urate. Urate constitutes the end product of purine catabolism in humans owing to lack of urate oxidase activity.

Figure 2.

Mobile phase gradient paradigm. Mobile phase A: 0.2 m KH₂PO₄ monobasic, 0.52 mm sodium 1-pentanesulfonate, pH 3.5. Mobile phase B: 0.2 m KH₂PO₄ monobasic, 0.52 mm sodium 1-pentanesulfonate, 10% acetonitrile, pH 3.5. MP: Mobile phase.

Figure 3.

Hydrodynamic voltammogram curves for urate, guanine, and xanthine. Measurements were taken using a model 5011A coulometric cell. An analytical potential of +0.15 V (P₁) was selected for urate, and an analytical potential of +0.45 V (P₂) was selected for guanine and xanthine. A conditioning potential of −0.1 V (P₀) was chosen to minimize contaminant peaks.

Figure 4.

Chromatograms of 1 mg/dL standards mixture (a), mouse serum (b), mouse striatum (c) and human striatum (d). Detection of analytes was performed either by ECD at +0.15 V (P1), +0.45 V (P2), or UV–vis at 254 nm. Gua, Guanine; UA, urate; Hyp, hypoxanthine; Xan, xanthine; Ino: inosine; Guo: guanosine; Ado: adenosine. Internal Standards are 3,4-dihydroxybenzylamine (DHBA, 1 μm) and methyl-DOPA (MD, 50 μm).