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 

Table 1.

Problems With Immunoassays: Data From the College of American Pathologists Proficiency Testing Program, 2002*

Problems With Immunoassays: Data From the College of American Pathologists Proficiency Testing Program, 2002*
Problems With Immunoassays: Data From the College of American Pathologists Proficiency Testing Program, 2002*

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 measurement of 9 steroids in a 760-μL sample of serum or plasma, without derivatization and with minimal sample workup—acetonitrile protein precipitation. The reliability of the method has been evaluated by correlation with currently used immunoassays, and assessment of within-day and between-day imprecision, recovery, and accuracy. Comparison of results obtained by tandem MS with the all method mean (CAP PT Program, 2002)14 has also been performed.

Chemicals

Androstenedione (4-androstene-3,17-dione), testosterone (4-androsten-17β-ol-3-one), DHEA (5-androsten-3β-ol-17-one), DHEAS (5-androsten-3β-ol-17-one sulfate, sodium salt), cortisol (4-pregnen-11β,17α,21-triol-3,20-dione), 11-deoxycortisol (4-pregnen-17α,21-diol-3,20-dione), progesterone (4-pregnen-3,20-dione), 17α-hydroxyprogesterone (4-pregnen-17α-ol-3,20-dione), 17β-estradiol (1,3,5[10]-estratriene-3,17β-diol), estriol (1,3,5[10]-estratriene-3,16α,17β-triol), ammonium acetate, and bovine albumin (96%) were purchased from Sigma-Aldrich (St Louis, Mo). Deuterium-labeled internal standards (testosterone-1,2-d2, cortisol-9,11,12,12-d4, and estradiol-2,4,16,16-d4) were from Cambridge Isotope Laboratory, Inc (Andover, Mass); 4-androstene-3,17-dione-2,2,4,6,6,16,6-d7, dehydroepiandrosterone-16,16-d2, 4-pregnen-17α-ol-3,20-dione-2,2,4,6,6,21,21,21-d8, 11-deoxycortisol-21,21-d2, estriol-2,4-d2, and progesterone-2,2,4,6,6,17α,21,21,21-d9 were from C/D/N Isotopes Inc (Pointe-Claire, Quebec). High-performance liquid chromatography–grade water and methanol were obtained from Burdick & Jackson (Muskegon, Mich). Optima-grade acetonitrile and toluene were from Fisher Scientific (Fair Lawn, NJ). All chemicals (except those noted otherwise) had a purity of at least 98%, as reported by the manufacturer.

Standard Solutions

Stock solutions of 1.0 mg/mL in methanol were prepared for each steroid of interest and stored at −20°C. Working profile standard solutions at various concentrations were prepared as follows: appropriate amounts of the stock solutions were mixed and diluted with methanol to obtain a solution containing 7.81 μg/mL of 11-deoxycortisol, 2.96 μg/mL of 17α-hydroxyprogesterone, 1.26 μg/mL of androstenedione, 0.344 μg/mL of estradiol, 2.73 μg/mL of DHEA, 2.09 μg/mL of testosterone, 3.31 μg/mL of progesterone, 68.1 μg/mL of cortisol, and 469.4 μg/mL of DHEAS. This mixed steroid standard solution was added to a 4% solution of albumin in water to provide steroid profile standards that were diluted 10-, 20-, 100-, 200-, 500-, and 1000-fold. These 7 solutions, including the blank albumin solution, were then used to prepare a calibration curve covering the clinically important range of concentration for each steroid. A solution of 80 ng/mL for each of 8 deuterium-labeled steroids in acetonitrile was used as internal standard and precipitant of proteins either for the albumin standards or serum/plasma samples. Quality control samples at 3 concentration levels were purchased from Bio-Rad Laboratories (Irvine, Calif) or prepared in-house and were used to evaluate the within-day and between-day precision, as well as the accuracy of the method.

Sample Preparation

Seven hundred sixty microliters of each profile standard or serum sample containing steroids of interest was placed into a 2.0-mL conical plastic centrifuge tube. One thousand one hundred forty microliters of internal standard solution in acetonitrile was added to the tube to precipitate the proteins in the sample. The tubes were capped, vortexed vigorously for at least 30 seconds, and centrifuged at 13 000g for 10 minutes. The supernatant in the tubes was transferred into autosampler vials for injection into the LC-MS-MS system. Sample preparation was performed at room temperature.

Liquid Chromatography–Tandem MS Analysis

We used a SCIEX (Applied Biosystems/MDS SCIEX, Foster City, Calif/Concord, Ontario, Canada) API-3000 triple quadrupole tandem MS equipped with an APPI (Applied Biosystems/MDS SCIEX) source. The photoionization lamp used was a 10-eV Cathodeon Ltd type number PKS100 krypton discharge lamp. Nitrogen produced by a high-purity nitrogen generator (PEAK Scientific Instruments Ltd, Chicago, Ill) was used as curtain, nebulizer, collision, and lamp gases. Unit mass resolution was set in both mass-resolving quadrupole Q1 and Q3. The high-performance liquid chromatography system consisted of 3 Shimadzu SCL-10Avp pumps, Shimadzu SIL-HTc autosampler, and Shimadzu DUG-14A degasser (Shimadzu Corp, Kyoto, Japan). Data were collected using a Dell Optiplex GX400 workstation and processed by Analyst 1.3.1 software package (MDS SCIEX).

Aliquots of 1700 μL were injected by the autosampler onto a Supelco LC-18-DB (3.3 cm × 3.0 mm, 3.0-mm internal diameter) chromatographic column equipped with Supelco Discovery C-18 (3.0 mm) guard column with identical packing material (Supelco, St Louis, Mo) at room temperature. The steroids adhered to the column, which was then washed with solvent C, a mixture of 15mM ammonium acetate and water-methanol (98:2 vol/vol, pH 5.5), at a rate of 1.0 mL/min. After 5.0 minutes of washing, the switching valve (VICI, Valco Instruments Co Inc, Houston, Tex) was activated, and the column was eluted with a gradient (Table 2) at a rate of 0.5 mL/min, and the sample was introduced into the MS. The column was flushed for 4 minutes with 100% solvent B (methanol) before the next injection. The dopant (Optima-grade toluene) was delivered into the source using a syringe pump (model 22, Harvard Apparatus Inc, Holliston, Mass) at a flow rate of 50 μL/min. Analytes were then quantified in MRM mode.

Table 2.

Gradient Timetable*

Gradient Timetable*
Gradient Timetable*

Calibration, using internal standardization, was done by linear regression analysis over various concentration ranges for the different steroids of interest. For each standard curve, a minimum of 6 different concentrations was used. Stable isotope dilution was used for 8 of the 9 steroids. For DHEAS, we used testosterone-d2 as the internal standard, because we could not find a sensitive and specific MRM transition for the deuterated DHEAS-d2 in positive-ion mode. Peak area ratios between target analytes and their respective internal standards were used for quantification.

Precision was evaluated by assaying Bio-Rad Liquichek Immunoassay Plus Control (lot 40620) in replicates (n = 10 for within-day, n = 20 for between-day) at 3 levels of concentration. Accuracy for cortisol, progesterone, and testosterone was evaluated by assaying Bio-Rad Lyphochek Immunoassay Plus Control (lot 40130), which provides target MS values for these steroids.

The Figure shows the chromatograms obtained for each of the steroids in a standard solution using the assay conditions described. The 9 steroids investigated in positive-ion mode and their respective deuterated internal standards were separated well in 18 minutes. Although both DHEA and 17α-hydroxyprogesterone have peaks at 14 minutes, no interference for these steroids was observed due to the specificity of the MRM mode in tandem MS. We used the same MRM transition, 271→213, for compound DHEA and DHEAS in positive-ion mode. Because of the huge difference in concentration in human plasma or in profile standard, the peak of DHEA at 14.1 minutes is almost invisible in the same panel of DHEAS, which has a peak at 7.5 minutes. Optimal MRM transition, collision energy, and declustering potential for each analyte were obtained by continuous infusion of each analyte solution (1.0 μg/mL in water-methanol [50:50 vol/vol]) separately into the tandem MS, as recommended by the manufacturer. Table 3 shows the optimum conditions chosen for the steroid profile assay, and Table 4 shows the main working parameters used. Linear regression analysis (GraphPad Prism version 3.02 for Windows, GraphPad Software, San Diego, Calif) gave the values shown in Table 5 for correlations of the tandem MS method with current immunoassays. Correlation coefficients obtained were between 0.886 and 0.988. Within-day and between-day imprecision at 3 concentration levels is shown in Table 6. Between-day results gave a coefficient of variation (CV) of 7.1% to 22% at the low concentration level and of 4.2% to 13.4% at the high concentration level. Poorest precision was obtained for androstenedione. Accuracy was evaluated using several approaches. Cortisol, progesterone, and testosterone were measured in replicates of 10 on the Bio-Rad Lyphochek Immunoassay Plus Control (lot 40130), and the mean result was obtained. This result compared well with the Bio-Rad MS values provided (Table 7). Accuracy was also assessed through the addition of known amounts of the steroids to a plasma pool (Table 8) and by method comparison (Table 5). In Table 8, the amount of steroid measured was very close to the target added, with the exception of DHEAS. As shown in Table 5, correlation coefficients were excellent. Samples used were either serum or heparinized plasma. The recovery of the 9 steroids investigated in positive-ion mode was determined at 2 concentration levels in replicates of 5. As shown in Table 8, the mean recoveries of the steroids under study were all higher than 82% at both concentration levels, except for DHEAS. (We were unable to use the deuterated internal standard for DHEAS. It is clear that the recovery of DHEAS is lower than that of the other steroids evaluated.) A comparison between the tandem MS results of the steroids studied and the all method mean from the CAP PT Program, 2002, is summarized in Table 9. Not surprisingly, tandem MS results were lower than the all method mean, ranging from 22.1% for 11-deoxycortisol to 88.7% for DHEAS.

Liquid chromatography–tandem mass spectroscopy (multiple reaction monitoring) profiles of the steroids of interest obtained for the injection of a standard mixture. Liquid chromatography conditions as in Table 2. DHEAS indicates dehydroepiandrosterone 3-sulfate; DHEA, dehydroepiandrosterone

Liquid chromatography–tandem mass spectroscopy (multiple reaction monitoring) profiles of the steroids of interest obtained for the injection of a standard mixture. Liquid chromatography conditions as in Table 2. DHEAS indicates dehydroepiandrosterone 3-sulfate; DHEA, dehydroepiandrosterone

Close modal
Table 3.

Tandem Mass Spectrometry (Multiple Reaction Monitoring [MRM]) Conditions for the Steroids Analyzed in Positive-Ion Mode

Tandem Mass Spectrometry (Multiple Reaction Monitoring [MRM]) Conditions for the Steroids Analyzed in Positive-Ion Mode
Tandem Mass Spectrometry (Multiple Reaction Monitoring [MRM]) Conditions for the Steroids Analyzed in Positive-Ion Mode
Table 4.

Tandem Mass Spectrometer Main Working Parameters

Tandem Mass Spectrometer Main Working Parameters
Tandem Mass Spectrometer Main Working Parameters
Table 5.

Correlation Between Tandem Mass Spectrometry and Immunoassays*

Correlation Between Tandem Mass Spectrometry and Immunoassays*
Correlation Between Tandem Mass Spectrometry and Immunoassays*
Table 6.

Tandem Mass Spectrometry Within-Day and Between-Day Precision After Running Quality Control*

Tandem Mass Spectrometry Within-Day and Between-Day Precision After Running Quality Control*
Tandem Mass Spectrometry Within-Day and Between-Day Precision After Running Quality Control*
Table 7.

Tandem Mass Spectrometry (MS) Accuracy Running Against Bio-Rad Quality Control*

Tandem Mass Spectrometry (MS) Accuracy Running Against Bio-Rad Quality Control*
Tandem Mass Spectrometry (MS) Accuracy Running Against Bio-Rad Quality Control*
Table 8.

Recovery From Addition at 2 Concentration Levels*

Recovery From Addition at 2 Concentration Levels*
Recovery From Addition at 2 Concentration Levels*
Table 9.

Comparison Between the Results of Tandem Mass Spectrometry (MS) and the Results From the College of American Pathologists Proficiency Testing Program, 2002

Comparison Between the Results of Tandem Mass Spectrometry (MS) and the Results From the College of American Pathologists Proficiency Testing Program, 2002
Comparison Between the Results of Tandem Mass Spectrometry (MS) and the Results From the College of American Pathologists Proficiency Testing Program, 2002

The method described allows for the simultaneous quantitation of 9 steroids in positive-ion mode by tandem MS within 18 minutes. The method is based on isotope dilution and unlike immunoassays is very specific for the analytes of interest. The method possesses adequate sensitivity (due to use of the APPI source, with the lower level of sensitivity being 100 pg/mL for each steroid) and precision to be used in the routine clinical laboratory. The method has been used for the measurement of steroid concentrations in patient samples. Unfortunately, we cannot measure estradiol below 100 pg/mL using the API-3000. We are now evaluating the API-4000, which enables quantitation down to 10 pg/mL. Results have been compared with immunoassay techniques. Generally, tandem MS provides lower values, no doubt due to improved specificity. The correlation coefficients shown in Table 5 are good. Unlike immunoassays, in which each steroid has to be assayed separately, the current procedure allows for the simultaneous measurement of many steroids, thereby providing a steroid profile on each sample measured. We believe that the improved specificity and simultaneous quantitation features afforded by this method represent distinct advantages over current immunoassays. No drug interferences have been detected.

This work was supported by grant M01-RR13297 from the General Clinical Research Center Program of the National Center for Research Resources, National Institutes of Health, Department of Health and Human Services, Bethesda, Md.

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The authors have no relevant financial interest in the products or companies described in this article.

Author notes

Reprints: Steven J. Soldin, PhD, Department of Laboratory Medicine, Children's National Medical Center, 111 Michigan Ave NW, Washington, DC 20010-2970 ([email protected])