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High-performance liquid chromatographic method for the evaluation of possible interferences in basophil-histamine release measurements.

Siegel-PD; Lewis-DM; Olenchock-SA
Anal Biochem 1990 Aug; 188(2):416-421
A method for the measurement of histamine release by basophils using condensation to o-phthalaldehyde (OPT) and high performance liquid chromatography (HPLC) was described. Protein was precipitated from dextran sedimented and lysed peripheral leukocytes, and the histamine extracted into butanol, back into hydrochloric-acid, and then condensed to OPT. Fluorescence was measured on a filter fluorometer or by HPLC with a cyano-propyl column. A single fluorescent peak was seen with authentic OPT condensed histamine at 2.5 minutes. Chromatographic detection of histamine standards from 10 nanograms/milliliter to 1mg/ml was linear. Nonstimulated leukocyte supernatant extracts, perchloric-acid lysed leukocyte extracts and extracts from antibody stimulated leukocytes demonstrated single peaks that eluted at the same time as the authentic histamine. Unextracted lysed leukocyte preparations had decreased histamine peaks due to negative interferences that were removed by prior extraction of the histamine. Similar interference problems were seen using human urine extracts. The addition of interfering substances without extraction before derivatization resulted in interference of OPT/histamine fluorescence when measured with a fluorometer, but some positive interference could be filtered out by using a sharp cutoff filter.
NIOSH-Author; Analytical-methods; Fluorescence-spectrometry; Blood-cells; Histamine-liberation; Chromatographic-analysis; Laboratory-techniques
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Analytical Biochemistry