ANALYSIS OF CORTICOSTERONE IN RAT PLASMA BY HIGH-PERFORMANCE LIQUID-CHROMATOGRAPHY

A sensitive and specific high-performance liquid chromatographic assay for the determination of corticosterone in rat plasma using dexametha sone as the internal standard is reported. Rat plasma (0.5 ml) is extr acted with methylene chloride, washed with 0.1 M sodium hydroxide and then with water. The extract is analyzed by HPLC on a C-18 column with ultraviolet absorbance detection at 254 nm. Pooled rat plasma was tre ated with activated decolorizing carbon to remove endogenous corticost erone, and was then used to prepare standards for the assay. Using 0.5 mi plasma for extraction, the detection limit of the assay is 10 ng/m l. The standard curve is linear over the concentration range 10-500 ng /ml. The recovery of corticosterone after extraction was independent o f concentration and ranged from 87 to 95%. The coefficient of variatio n for intra-day and inter-day precision ranged from 2.4 to 7.4% and 2. 1 to 8.7%, respectively. In addition, for concentrations ranging from 10 to 500 ng/ml the accuracy is within 5% of the spiked standards. The assay was utilized to examine the circadian rhythm of plasma corticos terone, and to examine the effect of immobilization stress on corticos terone levels in rats.