Histamine quantification in human plasma using high resolution accurate mass LC–MS technology

• A Q Exactive mass spectrometer is suitable for routine use in medical laboratories.
• An LCHRMS method for histamine quantification has been developed for human plasma.
• The LCHRMS method shows a good correlation with the gold standard RIA assay.

BackgroundHistamine (HA) is a small amine playing an important role in anaphylactic reactions. In order to identify and quantify HA in plasma matrix, different methods have been developed but present several disadvantages. Here, we developed an alternative method using liquid chromatography coupled with an ultra-high resolution and accurate mass instrument, Q Exactive™ (Thermo Fisher) (LCHRMS).MethodsThe method includes a protein precipitation of plasma samples spiked with HA-d4 as internal standard (IS). LC separation was performed on a C18 Accucore column (100 ∗ 2.1 mm, 2.6 μm) using a mobile phase containing nonafluoropentanoic acid (3 nM) and acetonitrile with 0.1% (v/v) formic acid on gradient mode. Separation of analytes was obtained within 10 min. Analysis was performed from full scan mode and targeted MS2 mode using a 5 ppm mass window. Ion transitions monitored for targeted MS2 mode were 112.0869 > 95.0607 m/z for HA and 116.1120 > 99.0855 m/z for HA-d4. Calibration curves were obtained by adding standard calibration dilution at 1 to 180 nM in TrisBSA.ResultsElution of HA and IS occurred at 4.1 min. The method was validated over a range of concentrations from 1 nM to 100 nM. The intra- and inter-run precisions were < 15% for quality controls. Human plasma samples from 30 patients were analyzed by LCHRMS, and the results were highly correlated with those obtained using the gold standard radioimmunoassay (RIA) method.ConclusionOverall, we demonstrate here that LCHRMS is a sensitive method for histamine quantification in biological human plasmas, suitable for routine use in medical laboratories. In addition, LCHRMS is less time-consuming than RIA, avoids the use of radioactivity, and could then be considered as an alternative quantitative method.