Elsevier

Clinica Chimica Acta

Volume 409, Issues 1–2, 3 November 2009, Pages 78-84
Clinica Chimica Acta

Development and validation of a sensitive liquid chromatography–tandem mass spectrometry assay to simultaneously measure androgens and estrogens in serum without derivatization

https://doi.org/10.1016/j.cca.2009.09.003Get rights and content

Abstract

Background

Immunoassays are widely used to quantify steroid hormones in biological samples. However, they lack specificity, especially at low levels. This study aimed to develop a sensitive LC–MS/MS method to measure serum androgens and estrogens without derivatization within a single run.

Methods

A stable-isotope dilution LC–MS/MS method was established using atmospheric pressure photoionization to quantify testosterone (T), dihydrotestosterone (DHT), estradiol (E2) and estrone (E1) from serum. Sample preparation involved liquid–liquid extraction (LLE) with hexane:ethyl acetate (3:2) containing deuterated internal standards. Accuracy was assessed by spiked recovery of serum pools, and imprecision by quality controls.

Results

Using 200 μL serum, limits of quantification were 0.3 pg (1.5 pg/mL) E1, 0.5 pg (2.5 pg/mL) E2, 2 pg (10 pg/mL) T and 10 pg (50 pg/mL) DHT. Accuracy (93–110%) and precision (median 4%, all <15%) were determined to be well within acceptable limits for bioanalytical method validation. An analysis time of less than 10 min allowed up to 150 samples (600 analytes) to be processed per day.

Conclusions

The method is sufficiently sensitive and precise to accurately quantify serum T levels in females and E2 in males, and is readily adapted to tissue and non-human samples.

Introduction

Testosterone (T) and 5α-dihydrotestosterone (DHT) are the two natural androgens responsible for the development and maintenance of the male phenotype. DHT is a 5α reduced metabolite of T with 3–10 times greater androgenic potency due to its higher binding affinity for the androgen receptor but circulating at ~10% of the concentrations of T [1]. Accurate measurement of these androgens in serum remains important to confirm the diagnosis of androgen deficiency states [2] and the therapeutic efficacy of DHT [3] or 5α reductase inhibitors [4]. Furthermore, there is growing interest in the primary DHT metabolites, 5α-androstane-3α,17ß-diol (3αDiol) and 5α-androstane-3β,17β-diol (3βDiol), which have androgenic and estrogenic properties by back-conversion to DHT or binding to the estrogen receptor β, respectively [5], [6], [7].

Similarly, estradiol (E2) and estrone (E1) are the major estrogens involved in the development and maintenance of the female phenotype and pregnancy, as well as having important indirect roles in men via aromatization of T within tissues. Accurate measurement is required for diagnosis and monitoring of female reproductive disorders including menstrual disorders, ovulation induction and IVF hyperstimulation. High sensitivity methods are required to accurately measure estrogen concentrations in serum as E2 levels are low (picomolar) in pre-menopausal women and very low in post-menopausal women and men [8].

Traditionally, steroid hormones have been measured using either gas chromatography/mass spectrometry (GC/MS) or immunoassay. Both methodologies have significant limitations. GC/MS analysis typically requires large sample volumes (>1 mL) and conversion to volatile derivatives [9], [10], [11], [12]. Although effective and often used as reference methods for steroid quantification [13], [14], sample preparation is cumbersome, and combined with long run times, limits throughput. Alternatively, steroid immunoassays are fast, simple and sensitive but specificity and accuracy are often compromised [15], [16] by cross-reactivity with structurally related components within samples, and by matrix effects arising from unextracted serum samples [17], [18]. Interference of immunoassays can lead to an overestimation of steroid levels by >60% [19], [20], [21]. For these reasons there has been growing interest in development of liquid chromatography–mass spectrometry (LC–MS) based methods to quantify steroid hormones from biological samples as affordable quantitative bench-top mass spectrometers now offer sensitivity to match immunoassays while maintaining reference level specificity.

Numerous liquid chromatography–tandem mass spectrometry (LC–MS/MS) methods have recently been described that quantify steroid hormones from biological fluids, mostly serum or plasma with a focus on T, and to a lesser extent E2 [22], [23], [24], [25], [26], [27], [28], [29], [30], [31], [32], [33], [34]. In many cases sensitivity has been improved by monitoring derivatized adducts. However, there are major drawbacks using steroid derivatives for LC–MS/MS analysis; substantial sample clean-up procedures are required, no one agent is available for both androgens and estrogens, and fragmentation patterns of derivatives are typically non-specific resulting in poor analytical specificity. It remains desirable to measure serum androgens and estrogens accurately using LC–MS/MS within a single run without derivatization. Of the published methods that do this [33], [35], [36], sensitivity remains insufficient for serum samples where circulating steroid concentrations are low.

Here we report the development and validation of a stable-isotope dilution LC–MS/MS method for the simultaneous quantification of six steroid hormones (including the DHT metabolites 3α and 3β androstanediols) from 200 μL of serum without derivatization. The method is sensitive enough to enable serum quantification of T from females and E2 from men. Six samples can be analyzed per hour with further potential for greater throughput by integration into a microtitre plate format and facile adaptation to tissue and non-human samples.

Section snippets

Reagents and equipment

T, DHT, 3αDiol and 3ßDiol were from the National Measurement Institute (NMI; Sydney, Australia). Deuterium labeled internal standards of these steroids were also from the NMI: testosterone-1,2,3-d3 (d3-T), dihydrotestosterone-16,16,17-d3 (d3-DHT), 5α-Androstane-3α,17β-diol-16,16,17-d3 (d3-3αDiol) and 5α-Androstane-3β,17β-diol-16,16,17-d3 (d3-3βDiol). E2 and E1 were from Steraloids (Newport, RI, USA). Deuterium labeled estradiol-2,4,16,16-d4 (d4-E2) was from Cambridge Isotope Laboratory

Sample preparation

Serum samples were prepared using either LLE or deproteination by acetonitrile precipitation prior to LC–MS/MS analysis (Fig. 1). LLE included an overnight drying step but resulted in a cleaner extract, and although samples were not able to be prepared for analysis on the same day, required minimal contact time. Acetonitrile treatment is a one-step sample preparation procedure that efficiently precipitates serum proteins. It is fast but results in a discolored extract containing impurities that

Discussion

We have developed a stable-isotope dilution LC–MS/MS method for the quantification of six steroids, including all the key bioactive sex steroids (T, DHT and E2) from 200 μL serum in a single 9 minute run without derivatization. The method is specific, accurate and precise for the target steroids over the specified ranges and matches the sensitivity of the best steroid immunoassay. Considering that multiple analytes can be quantified in a single run, it has equivalent or superior throughput to

Acknowledgement

This work was supported by an Australian Research Council LIEF grant.

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