Simultaneous determination of 12 steroids by isotope dilution liquid chromatography-photospray ionization tandem mass spectrometry

https://doi.org/10.1016/j.cca.2006.03.034Get rights and content

Abstract

Background

Serum steroid assays play an important role in the clinical evaluation of a number of common endocrine disorders. Among various assays, tandem mass spectrometry (MS/MS) has being increasingly applied in clinical laboratories for its high sensitivity, specificity, and simultaneous multi-analyte quantitation capability. Our first generation isotope dilution steroid profile assay by HPLC-tandem MS/MS with a C-18 column allowed for the measurement of 9 steroids in 18 min employing a sample volume of 760 ul serum. We describe our second generation steroid profile assay which allows for the quantitation of 12 steroids simultaneously employing HPLC-MS/MS and isotope dilution tandem MS in 11 min. This method requires a sample volume of 200 μl.

Methods

An API-5000 triple-quadrupole mass spectrometer (Sciex, Concord, Canada) coupled with the PhotoSpray source and Shimadzu HPLC system (Shimadzu Scientific Instruments, Columbia, MD) was used employing isotope dilution with deuterium labeled internal standard (IS) for each analyte. Two hundred microliters of serum were deproteinized by adding 300 μl of acetonitrile containing internal standards. After centrifugation, 450 μl of supernatant were diluted with 900 μl of water and 1000 μl aliquot were injected onto a C-8 column. After a 3 min wash the valve was activated to initiate the gradient elution program which eluted the steroids. Quantitation by MRM analysis was performed both in positive ion mode for 11 analytes and in negative ion mode for aldosterone. Within-day and between-day precision, reliability and accuracy of this method were assessed by correlation with other MS/MS and immunoassay methods and by recovery study.

Results

Within-day CVs were < 11.5% for all analytes tested and between-day CVs ranged from 3.5% to 12.2%. The results of the comparison study yield r values ranging between 0.908 and 0.999. Recovery ranged from 90% to 110%.

Conclusions

This method can simultaneously measure 12 steroids in serum within 11 min with minimal sample preparation. It can be routinely employed in a clinical environment and is attractive because of its simplicity in sample processing and high throughput.

Introduction

The steroid hormones, derived from cholesterol and synthesized in the adrenal cortex, the gonads, and the placenta, are of great clinical importance and act as biological messengers [1], [2]. Secreted into and transported by blood to their target cells, the steroids are in equilibrium between their protein-bound and free forms. 25-hydroxyvitamin D3 is the most stable and abundant metabolite of vitamin D and an important regulating factor of calcium metabolism. Serum concentration of 25-hydroxyvitamin D3 is considered to be a good marker for Vitamin D deficiency.

Clinically important endogenous steroids can be measured by numerous techniques which include HPLC with UV detection [3], [4], and various immunoassays (IAs) [5], [6], [7], [8]. HPLC–UV based methods have difficulties in separating multiple analytes of interest from contaminants in plasma or serum and generally lack sensitivity. IAs have been widely used in many laboratories and hospitals because of their simplicity, speed, and sensitivity. Major drawbacks of immunoassays for the steroids include their lack of specificity [9], [10], [11] and the fact that you need a different assay for each steroid.

A number of tandem-mass spectrometry based methods for the measurement of steroids and Vitamin D have been reported in the literature [12], [13], [14], [15], [16], [17], [18], [19]. However, most of these assays measure only one or a couple of the analytes of interest. We previously reported a nine-steroid profile approach employing tandem mass spectrometry utilizing the API-3000 with atmospheric pressure photoionization source [11]. Steroid profiles for example in congenital adrenal hyperplasia (CAH) allow for the acquisition of more clinically useful data than can be obtained through the measurement of a single steroid alone.

Section snippets

Chemicals

Androstenedione, aldosterone, testosterone, DHEA, DHEAS, cortisol, corticosterone, 11-deoxycortisol, progesterone, 17α-hydroxyprogesterone, 17β-estradiol, 25-hydroxyvitamin D3, ammonium acetate, and bovine albumin (96%) were from Sigma-Aldrich (St. Louis, MO). Deuterium labeled internal standard: testosterone-1,2-d2 (testosterone-d2), cortisol-9,11,12,12-d4 (cortisol-d4), and estradiol-2,4,16,16-d4 (estradiol-d4) were from Cambridge Isotope Laboratory, Inc. (Andover, MA);

Results and discussion

The HPLC separation was performed on a C-8 column which greatly shortened the analysis time. The gradient was optimized to provide the maximum separation possible in a minimum time period. The eluant and gradient conditions used are detailed in Table 3. As aldosterone and cortisol are both avidly bound to cortisol binding globulin it was found necessary to add an aliquot of cortisol equivalent to 30 μg/dl to the serum to prevent precipitation of the low amounts of aldosterone with the proteins.

Conclusions

We have developed a 2nd generation steroid profile assay employing an isotope dilution LC-APPI-MS/MS-based method for the analysis of 12 steroids in serum samples with an 11-min run time. The method requires 200 μl serum. This method correlates very well with the other MS/MS-based methods employed at the Mayo Clinic. The combined attributes of organic solvent protein precipitation coupled with LC-APPI-MS/MS techniques under MRM mode provide a unique combination of analytical capability which

Acknowledgements

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, USA. It was also supported in part by grant 1 U10HD45993-02 of the National Institute of Child Health and Development, Bethesda, MD.

References (20)

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