Circulating estrogens decrease after the menopause from 60400 to 530 pg/ml in postmenopausal women and in men are below 30 pg/ml. These low physiological concentrations present an analytical challenge. Analysis of steroids by tandem mass spectrometry is attractive due to its high specificity compared with immunoassays. However, estrogens do not ionise efficiently and therefore chemical modifications are necessary to achieve the sensitivity required.
Two derivatising agents were evaluated; dansyl chloride and 2-fluoro-1-methylpyridinium (FMP). These react with the phenolic moiety of estrogens and introduce an easily ionisable and charged group respectively. Reaction conditions, short term stability, linearity of response and limit of quantitation (LOQ) were assessed.
Analysis was performed on a TQ2 quantum discovery triple quadrupole mass spectrometer in positive electrospray ionisation mode with a Surveyor LC, equipped with a Waters Atlantis T3 3 mm; 2.1×100 mm column.
Transitions for dansylated estrone (E1) and estradiol (E2) were m/z 504->171 and m/z 506−>171 respectively, and in both cases the product ion related to the dansyl moiety. The FMP derivative of E1 gave m/z 362−>252 and that of E2 gave m/z 364−>128; the product ions represented the steroid fragment. Fragmentation patterns were corroborated using 13C- and 2H-labelled estrogens.
Rapid reactions were achieved with dansyl chloride (60 °C, 10 min) or FMP (30 °C, 5 min), permitting high-throughput. Responses were linear between 2 and 500 pg/ml (r2>0.99). Short term stability was acceptable with no detectable degradation of derivatives over 30 h. The LOQ was 2 pg/ml for both estrogens and both derivatives. E1 and E2 were analysed simultaneously with run times of 10 min/sample.
Derivatisation using FMP and dansyl chloride may be used for quantitative analysis of estrogens in men and postmenopausal women. The sensitivities achieved using these agents was comparable but enhanced selectivity was achieved using FMP since the product ion relates to the steroid moiety. This approach could be extended to include other phenolic steroids.