Validation of HPLC Method for Determination of Histamine in Human Immunoglobulin Formulations.

ABSTRACT

BACKGROUND: Histamine fixed-immunoglobulin formulations, which consisted of 0.15 µg of histamine dihydrochloride and 12 mg of human immunoglobulin in a vial, are used for anti-allergic treatments, and controlling the amounts of histamine in the formulations is essential to avoid histamine intoxication. OBJECTIVE: A high-performance liquid chromatography (HPLC) method for determination of histamine contents of the formulations was established and validated. METHODS: Histamine extracted from the formulation was labeled with 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate and was analyzed by gradient elution HPLC with UV detection at 260 nm. RESULTS: The method showed linearity in the range 0.8-2.4 µM (R > 0.999), accuracy (100.1-105.8% recovery), and precision (relative standard deviation ≤ 1.93%). The validated method was applied for five lots of the pharmaceutical, and their histamine contents were determined to be 0.149-0.155 µg/vial. CONCLUSIONS: These results indicated that the validated method is useful to control amounts of histamine in biopharmaceutical products. HIGHLIGHTS The HPLC method was developed for quantitative determination of histamine content of the histamine fixed-immunoglobulin formulations.