Establishment and validation of basophil histamine release test using highly sensitive LC-MS/MS method for the determination of histamine

초록

Background: The basophil histamine release test (HRT) is an important in vitro diagnostic assay to evaluate IgE-mediated allergic responses. Among numerous methods to detect histamine including immunologic, fluorometric, and chromatographic methods, liquid chromatography-tandem mass spectrometry (LC-MS/MS) may be the best measurement method due to its excellent selectivity and high sensitivity. This study was conducted to develop a rapid and simple LC-MS/MS method for the determination of histamine, and performed to establish HRT and to confirm its clinical applicability. Method: LC-MS/MS method was developed to quantify histamine in the leukocyte suspension, and validated for specificity, calibration, accuracy, precision, matrix effect, recovery, and stability according to the acceptance criteria of the FDA guidance for bioanalytical method validation. Both HRT and basophil activation test (BAT) were simultaneously performed for patients with respiratory or cutaneous allergy in order to compare the results of these two assays. Results: The method used pre-column derivatization with phenylisothiocyanate and the resulting phenylthiocarbamyl histamine was analyzed by positive-ion electrospray ionization using the multiple reaction monitoring mode. This method had a lower limit of detection of 0.1 ng/mL of histamine, and the quantifiable range was 0.1?100 ng/mL. Preparation of an antigen dose-response curve required only 0.1 mL of the leukocyte suspension and histamine could be detected quickly (up to 20 samples every hour). The intra-day and inter-day precision and accuracy results were within the acceptable limits. It was confirmed that histamine quantification should be performed within 2 hours of preparing the leukocyte suspension, and freezing and thawing should be avoided. Both HRT and BAT showed almost the same results, and HRT showed a dose-dependent pattern of histamine release within the range of allergen concentrations.