On-line procedures for the phase-transfer-catalyzed dansylation of phenolic steroids — Application to biological samples

Summary Commercially available equipment from two manufacturers served to set up an automated system for the precolumn phase-transfer-catalyzed dansylation of phenolic steroids, using ethynyl estradiol (EE) and estradiol (E) as model compounds. Using different mixing techniques, the on-line determination of EE and E in 200l untreated urine samples was achieved at a rate of 3–8 analyses per hour. Detection limits were calculated to be 3–5 ng/ml. Calibration curves in urine were linear over two orders of magnitude with r=0.999 (n=5) for EE and r=0.999 (n=6) for E. The repeatability of the determination of EE in urine (1g/ml) was 3.9% (RSD; n=20) and of E (1.5l/ml) 3.8% (RDS; n=10). The use of plasma instead of urine in the on-line procedures was not possible due to rapid formation of emulsions, but E and EE were determined in 100l plasma samples using a mild off-line mixing procedure in 10min. Detection limits were calculated to be ca 10ng/ml.A reaction detector, based on a solvent-segmented system, was developed for the on-line post-column dansylation of phenols and was coupled with a reversed-phase LC system. The highly selective system showed excellent linearity over at least two orders of magnitude with r=0.9999 (n=6) for both phenol and 2,5-dimethylphenol. The reproducibility was good with RSD values of around 2%. Detection limits for loop injections from standard solutions were calculated to be between 4 and 11ng.