Determination of plasma Ntau-methylhistamine in vivo by isotope dilution using benchtop gas chromatography-mass spectrometry.

A practical sensitive and specific method for determination of the stable metabolite of histamine, Ntau-methylhistamine, in human plasma using benchtop gas chromatography-stable isotope dilution mass spectrometry has been developed. Ntau-Methylhistamine, a principal metabolite of histamine in humans, was extracted and purified from human plasma using a two-step procedure with Sep-Pak silica cartridges. Quantitation of Ntau-methylhistamine was made possible by the synthesis of Ntau-[2H3]methylhistamine used as an internal standard. Derivatization with pentafluoropropionyl anhydride of extracts of human plasma yielded the bis-pentafluoropropionyl derivative of Ntau-methylhistamine for measurement using selected ion monitoring of the m/z 417/420 ion pair after electron impact on a benchtop gas chromatography-mass spectrometry (GC-MS). By improvements in the plasma extraction technique, inclusion of a synthetic internal standard and the development of a sensitive and stable derivative of the histamine metabolite, Ntau-methylhistamine was found to be significantly elevated in the plasma of patients with the dermal fibroproliferative disorder, hypertrophic scarring as compared to age-matched normal volunteers (98.5+/-29.5 pg/ml, n=9, versus 43.3+/-16.5 pg/ml, n=8, p<0.05). As such, this method affords a sensitive, specific and practical approach to measurement of histamine metabolites in plasma and other biological fluids.