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Immunoassays are among the most sensitive and precise analytical methods. However, recent studies1-14 have shown that many immunoassays lack specificity owing to cross-reactivity. Furthermore, results from the College of American Pathologists Proficiency Testing Program (CAP PT Program) for the year 2002 (Y-Survey)14 clearly showed that the antibodies used in the commercially available immunoassays lack specificity. Table 1 presents the mean low and mean high values for each steroid using the different immunoassays currently available, and the data strongly illustrate their lack of specificity. In the past, steroids were analyzed individually using gas chromatography-mass spectrometry (MS) or immunoassay. Gas chromatography-MS is both sensitive and specific, but requires tedious and time-consuming sample preparation. Liquid chromatography-MS (LC-MS) and liquid chromatography-tandem MS are specific and offer simpler approaches to sample preparation without sample derivatization steps. Recently, a number of LC-MS-based methods using different ion sources have been reported for the determination of the following steroid hormones: testosterone,15-17 cortisol,18-22 11-deoxycortisol,23 androstenedione,16-17 dehydroepiandrosterone (DHEA),24 dehydroepiandrosterone 3-sulfate (DHEAS),16-24 progesterone,25 17-hydroxyprogesterone,26 estriol,27 and estradiol."16-27
Prior to the advent of the atmospheric pressure photoionization (APPI) ion source,28 the LC-MS-based methods mentioned above used either an atmospheric pressure chemical ionization (APCI) or electrospray ionization source. Without multistep sample preparation procedures, the APCI source usually cannot provide adequate sensitivity for some steroids, such as estradiol and DHEA in human serum. The electrospray ionization source is considered more sensitive than the APCI source for polar compounds. However, for the nonpolar or low-polar compounds, such as most steroid molecules, the sensitivity provided by the electrospray ionization source is less satisfactory22,25 than the APCI source. The more recently introduced APPI source has been demonstrated to be significantly more sensitive than APCI for certain compounds.28 Alary29 used APPI-tandem MS for the detection of steroids in biological matrices, and reported that in both selected ion monitoring mode and multiple reaction monitoring (MRM) mode, the signal obtained by photoionization was more intense by a factor of 3 to 10 when compared to the APCI source.
Because of the high sensitivity provided by the APPI source for steroids, we hypothesized that stable isotope dilution tandem MS in the MRM mode would allow for the rapid simultaneous quantitation of numerous steroids in a single sample. This article describes a method that permits the simultaneous...