Liquid-chromatographic determination of progesterone in serum, with spectrophotometric detection

In this precise, accurate, and specific liquid-chromatographic procedure for determining progesterone in serum, the serum is diluted 10-fold with water/methanol (65/35 by vol), and the progesterone is extracted from the sample by passage through a column of graphitized carbon black (Carbopack B, Supelco). After washing the column, we elute the progesterone with chloroform/methanol (90/10 by vol), which is then evaporated. The progesterone is separated on a reversed-phase C18 column with a mobile phase of acetonitrile/water, (46/54 by vol) at a flow rate of 1.6 mL/min. The eluted compound is detected by absorbance at 242 nm. Analytical recoveries for progesterone varied from 96.0 to 97.8%. Within-day and day-to-day precision, determined by analyzing pooled serum, ranged from 3.4 to 6.4%, and 4.1 to 7.9%, respectively. The limit of detection was about 0.2 micrograms/L. Numerous drugs and steroids tested did not interfere. Results correlated (r = 0.997) well with those by radioimmunoassay.