A Highly Sensitive Assay for Histamine Using Ion‐Pair HPLC Coupled with Postcolumn Fluorescent Derivatization: Its Application to Biological Specimens

Abstract

A simple and highly sensitive method for the determination of histamine (HA) was developed using ion-pair, reversed-phase HPLC coupled with postcolumn o-phthalaldehyde derivatization fluorometry, and it was applied to the unpurified extracts of human and rat plasma, and brains of rats and mice. The HA concentrations both in the plasma and brains determined by the present method were well consistent with the values obtained by cation-exchange HPLC with postcolumn fluorescent derivatization currently in use. The present method was more advantageous than the assay using cation-exchange HPLC: (1) it was three to four times more sensitive (the detection limit was 0.5 pg of HA), and (2) it enabled the measurement of HA in samples containing (R)alpha-methylhistamine, a potent and specific H3-receptor agonist, which could not be separated from HA by cation-exchange chromatography. Using the present method coupled with intracerebral microdialysis, we found in the rat hypothalamus that (R)alpha-methylhistamine (5 mg/kg i.p.) markedly decreased the extracellular concentration of HA with a maximal effect (83% reduction) during 30-60 min after injection, suggesting that most of HA in the microdialysate fraction is neuronal in origin.

Highly Sensitive Determination of Histamine by Narrow-Bore High-Performance Liquid Chromatography Using Postcolumn Fluorescence Detection

“…Therefore, we tried to develop a more sensitive method by changing the ion-exchange column to a reversedphase narrow-bore column. Compared with the prior method 13 , the sensitivity increased about 4-fold, and the detection limit was 5 fmol/injection, the same as that of Itoh et al 7 Measurements were possible even with a small volume of plasma (20 µl). However, histamine in guinea pig and monkey plasma, especially the latter, could not be determined because of its rapid disappearance.…”

“…Highly specific and sensitive highperformance liquid chromatography (HPLC) methods have also been reported. [5][6][7][8][9][10][11][12][13][14][15][16][17][18] Widely used today is postcolumn fluorescence detection; 5,7 this employs a twostep condensation reaction with o-phthalaldehyde (OPA) under alkaline conditions, followed by acidic conditions. 19 Although the specificity of this method for histamine is high, the sensitivity and reproducibility are still problematic in terms of reagent transfer and OPA stability in aqueous solutions.…”

Prophylactic effect of pemirolast, an antiallergic agent, against hypersensitivity reactions to paclitaxel in patients with ovarian cancer

“…The concentration of histamine in plasma was determined by ion-pair high-performance liquid chromatography (HPLC) coupled with postcolumn fluorescent derivatization method, as described previously. 16 Briefly, plasma protein was precipitated by adding perchloric acid (0.4 M in final concentration), and the mixture was centrifuged at 10,000g for 20 min. A 20-ll aliquot of the supernatant was directly injected into the HPLC system.…”

Enhancement by Morphine of Radiographic Contrast Media-Induced Histamine Release in Rat Peritoneal Mast Cells.

“…To the lower fraction, 0.75 ml of HBSS and 30 ul of 5.1 N perchloric acid were added, and the mixture was then ultrasonicated. The histamine contents of both fractions were determined with ion-pair HPLC coupled with postcolumn fluorescent derivatiza tion (10). None of the drugs used in the present study interfered with the fluorometric assay.…”