Simultaneous quantitation of seven endogenous C-21 adrenal steroids by liquid chromatography tandem mass spectrometry in human serum

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Abstract

Quantitation of endogenous steroids is important in the diagnosis of several endocrine disorders. In this study we present a new method for simultaneous quantitation of cortisol, cortisone, 11-desoxycortisol, 21-desoxycortisol, corticosterone, 17-hydroxyprogesterone and 11-desoxycorticosterone in human serum by on-line extraction and LC–MS/MS. Analytes extraction was performed on-line using a 2-position and 6-port valve equipped with a monolithic silica cartridge. After chromatographic separation of all analytes, detection was performed in the multiple reaction monitoring mode using positive atmospheric pressure chemical ionization mode. Total imprecision of the assay ranged from 5.5 to 15.5%. Comparison with immunoassays yielded coefficients of 0.893 for cortisol, 0.848 for 11-desoxycortisol and 0.924 for 17-hydroxyprogesterone. The sensitivity of this method provides meaningful data for patients within normal and elevated range and it may be useful for the diagnosis of a variety of adrenal dysfunctions.

Introduction

Due to their low levels and their chemical similarity, the analysis of endogenous steroids is a bioanalytical challenge. Most methods for routine endogenous steroid quantitation are based on radioimmunoassay (RIA) that is simple and accessible to most of clinical laboratories and hospitals. However, this methodology is susceptible to interference from other structurally related endogenous and exogenous steroids [1]. It has been demonstrated that RIA, especially those based on direct assays often overestimate true steroid values [2], [3]. Although RIA with previous extraction steps to eliminate interfering compounds improves specificity, especially when some chromatographic step such as HPLC is used [4], [5], lengthy sample pretreatment procedures required limits their throughput and applicability in most of laboratories. Furthermore, even the use of analyte extraction techniques does not eliminate all interferences from some steroids [6].

Gas chromatography coupled to mass spectrometry (GC–MS) is considered the gold standard for steroids quantitation. In fact, it is highly specific and has been applied to quantify a large number of steroids [7], [8]. However, this technique requires complicated and time consuming sample preparation procedures limiting its usage to few specialized laboratories.

Liquid chromatography coupled to tandem mass spectrometry (LC–MS/MS) is being rapidly introduced as a better alternative to quantify steroids in the clinical context. Methods have been described for the determination of cortisol and cortisone [9], [10], 17-hydroxyprogesterone [3], 11-desoxycortisol [11], [12], 21-desoxycortisol [13] and corticosterone [14]. Some of them still demand manual sample preparation procedures such as liquid–liquid extraction [13], [14] or off-line solid-phase extraction [3], [12]. Derivatization, which is often necessary to achieve the required sensitivity to quantify endogenous levels of these analytes requires several steps [12], reducing the throughput.

One of the most important features of LC–MS/MS for analysis of steroids is the possibility to measure several analytes simultaneously without all workup required by GC–MS [11], [12], [15], [16]. This is especially interesting because it allows the discrimination among related disorders. For example, in congenital adrenal hyperplasia (CAH), the use of a steroid profile allows the acquisition of clinically more useful data than can be obtained through the measurement of a single steroid.

In this study, we report the development and clinical validation of a new method for simultaneous measurement of 7 related C-21 adrenal steroids: cortisol, cortisone, 11-desoxycortisol, 21-desoxycortisol, corticosterone, 17-hydroxyprogesterone and 11-desoxycorticosterone (Fig. 1). We aimed to achieve a simple, rapid, and sensitive assay using a reduced sample preparation procedure based on on-line extraction and LC–MS/MS.

Section snippets

Materials

17-Hydroxyprogesterone-d8 and 11-desoxycortisol-d2 were from CDN Isotopes (Pointe-Claire, Canada). Cortisol-d4, 21-desoxycortisol-d8, corticosterone-d8, and 11-desoxycorticosterone-d8 were from Medical Isotopes (Pelham, NH). All other steroids were purchased from Steraloids (Newport, RI). Ultrapure water was prepared from MilliQ system (Millipore, Billerica, MA). HPLC grade solvents were from Tedia (Rio de Janeiro, Brazil). Fetal bovine serum was obtained from Invitrogen (São Paulo, Brazil).

Mass spectrometry detection

Positive mode electrospray, APCI and atmospheric pressure photoionization (using toluene as dopant) were evaluated on the Quattro Premier tandem mass spectrometry in order to detect the seven steroids (data not shown). We found higher signal to ratio intensities detecting protonated forms [M + H]+ in positive mode APCI. Suitable precursor and product ions for each steroid and parameters optimization were performed by direct infusion of single-analyte solutions at 10 μmol/L into the mass

Discussion

LC–MS/MS is becoming the method of choice for steroid quantitation in clinical samples. Despite the high selectivity and sensitivity achieved by LC–MS/MS, sample preparation procedures are still necessary and they often bottleneck the throughput of this technique. Here we present a simple strategy for on-line extraction of seven endogenous steroids from serum samples. A single silica monolithic guard cartridge connected to a 6-port 2-position diverter valve is used as an extraction column. Due

Acknowledgments

We thank Fleury Medicine and Health for financial support.

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