Determination of Histamine by High-Performance Liquid Chromatography After Precolumn Derivatization with o-Phthalaldehyde-Sulfite

Abstract

A fast and sensitive method was developed for in vivo determination of histamine in the brain microdialysate by reverse ion pair chromatography with electrochemical detection. The microdialysates were derivatized with o-phthalaldehyde and sodium sulfite, and separation was achieved using isocratic elution within 10 min. The separation was performed in an Agilent Eclipse Plus C18 column (3.0 × 150 mm, particle size 3.5 μm), and the mobile phase consisted of 100 mM monosodium phosphate (pH 6.0), 500 mg L(-1) OSA and 20% methanol (v/v). The linearity (R(2)) was found to be >0.999, with a range from 2 to 50 nM and excellent repeatability (relative standard deviation, 2.29-6.04%), and the limit of detection was 0.4 nM. This method was successfully applied to analyze the extracellular concentration of histamine in the hypothalamus of rats, with probe recovery calculated in vivo.

Figure 1.

Optimization of derivatization conditions. (A) Effect of OPA concentrations on the responses of OPA-derivative of HA. Different concentrations of OPA and sodium sulfite (OPA: sulfite = 1:1, c/c) were tested with microdialysate. (B) Effect of OPA-reaction time under in vitro conditions on response of the OPA-derivatives of 10 nM HA. Derivation agents: 5 mM OPA and sulfite in 0.1 M borax, pH 10.4. (C) Stability of the derivative with different ratios between OPA and sulfite, concentration of OPA fixed at 5 mM. Peak areas were normalized at the reaction time of 1.5 min. During all of the analysis, the HA standard solutions or samples were mixed with the derivatization reagents at a ratio of 10:1 (20:2 µL). The derivatization reagents were in the buffer of 0.1 M borax (pH 10.4).

Figure 2.

Optimization of separation conditions. (A) Effect of pH on the capacity factor of the HA and some hydrophobic amino acids. Mobile phase: 100 mM phosphate and 400 mg L⁻¹ OSA in 15% methanol. (B) Effect of OSA concentration on the capacity factor of the HA. Different concentrations of OSA were investigated with 100 mM phosphate (pH 6.0) in 20% of methanol.

Figure 3.

Optimization of detection conditions. (A) Hydrodynamic voltammograms of OPA-sulfite derivatives. Mobile phase: 0.1 M phosphate, 500 mg L⁻¹ OSA, pH 6.0, containing 20% (v/v) methanol. (B) Chromatograms (HA standard) from coulometric cell and amperometric cell, signals 1 and 2 were from the amperometric cell and coulometric cell, respectively.

Figure 4.

Elution profiles of HA standard solution and the microdialysate after derivatization with OPA-sulfite. Chromatograph 1: standard amino acids solution (10 nM). Chromatograph 2: microdialysis sample from HYP.

Figure 5.

Zero-net flux of four rats. HYP HA levels corrected for in vivo probe recovery are represented by the x-intercepts of the plotted linear regression lines.