Development of a HPLC-FL method to determine benzaldehyde after derivatization with N-acetylhydrazine acridone and its application for determination of semicarbazide-sensitive amine oxidase activity in human serum

Abstract

A novel fluorescence labeling reagent N-acetylhydrazine acridone (AHAD) was designed and synthesized. A highly sensitive high performance liquid chromatography (HPLC) method coupled with fluorescence detection to determine benzaldehyde after derivatization with AHAD was developed. Optimum derivatization was obtained at 40 °C for 30 min with trichloroacetic acid as catalyst. Benzaldehyde derivative was separated on a reversed-phase SB-C18 column in conjunction with a gradient elution and detected by fluorescence detection at excitation and emission wavelengths of 371 nm and 421 nm. The established method exhibited excellent linearity over the injected amount of benzaldehyde of 0.003 to 5 nmol mL^-1. The method was successfully applied to the determination of serum semicarbazide-sensitive amine oxidase (SSAO) activity in humans. SSAO is a significant biomarker because serum SSAO activity is elevated in patients with Alzheimer's disease, vascular disorders, heart disease and diabetes mellitus. It was demonstrated that the SSAO activity of the hyperglycemic group (60 ± 4 nmol mL^-1 h^-1) was significantly higher than that of normal blood sugar group (44 ± 4 nmol mL^-1 h^-1) with P < 0.05.

Fig. 1. Derivatization scheme of AHAD with benzaldehyde.

Fig. 2. Excitation and emission spectra of AHAD-benzaldehyde derivative in different mixed solvents. (1) 100% acetonitrile, (2) 90% acetonitrile, (3) 80% acetonitrile, (4) 70% acetonitrile, (5) 60% acetonitrile, (6) 50% acetonitrile, (7) 40% acetonitrile.

Fig. 3. Effect of different reaction conditions on peak area of the reaction product of AHAD with benzaldehyde. (a) Effect of catalyst species, (b) effect of amount of catalyst, (c) effect of reaction time, (d) effect of reaction temperature, (e) effect of amount of AHAD.

Fig. 4. HPLC chromatograms after applying the procedure described in Section 2.7 (a) acetonitrile blank; (b) standard benzaldehyde (40.0 nmol mL⁻¹); (c) blank serum sample; (d) serum sample.