Factors that influence radioimmunoassay of human plasma melatonin: a modified column procedure to eliminate...

We describe here the effect of various agents and conditions that can influence the assay of plasma melatonin and report a modified column method which minimizes these interferences. Melatonin was measured by a sensitive radioimmunoassay which can detect melatonin from 5 to 500 pg/ml; the assay was linear about 0-60 pg/ml range. The hemolysis of samples increases the apparent level of melatonin in plasma. When the hemolyzed samples were passed through the SEP-cartridge and washed with methanol, the normal basal level was restored. Elution from the column was highest at 100% methanol and proportionate with the "spiked" value of melatonin. The addition of hemoglobin standard and serum albumin decreases the assayed value of melatonin while the addition of red blood cells and heparin increases the level. EDTA, oxalate, and citrate do not influence the basal level of melatonin. Samples that have been affected by these various agents and conditions can be normalized by passing samples through the column. Up to three cycles of freezing and quick thawing of plasma had no effect on the assay of melatonin; however, beyond three cycles levels were reduced. Long-term storage (up to 4 years) has no influence on the assay.