A versatile method for analysis of saliva, plasma and urine for total thiols using HPLC with UV detection

• Simultaneous HPLC determination of six biothiols in biological samples.
• Versatile chromatographic conditions are applicable to three different matrices.
• Assay uses less toxic ethanol instead of acetonitrile.
• Saliva appears to be a valuable diagnostic fluid alternative to plasma.
• The method exploit cheap and user friendly UV detection.

A simple and rapid HPLC method using 2-chloro-1-methyllepidinium tetrafluoroborate (CMLT) as a derivatization reagent was developed for simultaneous determination of homocysteine (Hcy), glutathione (GSH), γ-glutamylcysteine (γ-GluCys), cysteinylglycine (CysGly), N-acetylcysteine (NACys) and cysteine (Cys) in human saliva, plasma and urine. Separation of the analytes was achieved in just 7 min using an HPLC, followed by UV detection at 355 nm. Chromatographic separation was accomplished on Aeris PEPTIDE XB-C18 (150 mm×4.6 mm, 3.6 µm) column from Phenomenex with a gradient elution: 0–4.0 min, 7–30% B; 4.0–5.5 min, 30–7% B; 5.5–7.5 min, 7% B; (A: B, v/v); (A) 0.5% CH3COOH and (B) EtOH. Mobile phase was delivered at a flow rate 1.0 mL min−1. Linearity in detector response for total thiols was observed over the range of 0.1–20 μmol L−1 for Hcy, GSH and γ-GluCys, 0.25–50 μmol L−1 for NACys and CysGly and 5–300 for Cys. The LOQ values for Hcy, GSH, γ-GluCys, NACys, CysGly and Cys were 0.05, 0.05, 0.10, 0.06, 0.12 and 0.08 μmol L−1, respectively. The method was successfully implemented to analysis of the samples donated by 15 apparently healthy volunteers and 10 patients.

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