Sensitive quantitative LC-MS/MS method of steroid hormones determination in adipose tissue as a helpful tool in obesity research

Rafał Szewczyk^1,2, Anna Lenartowicz¹, Alina Kuryłowicz³, Katarzyna Krupczyńska-Stopa^1,2, Maciej Stopa^1,2

1. LabExperts sp. z o. o., Gdańsk, Poland; 2. Bioanalytic sp. z o. o., Gdańsk, Poland; 3 Department of Human Epigenetics, Mossakowski Medical Research Centre, Polish Academy of Sciences, Warsaw, Poland

Introduction

Obesity is a growing problem that affects more than 1 billion people worldwide. Estrogens deficiency play an important role in the pathogenesis of obesity. Therefore, there is a need to characterize sex hormones profile in adipose tissues in normal-weight and obese individuals to predict the effectiveness of estrogen-oriented approach in prevention and treatment of obesity. Determination of steroid hormones in human adipose tissue is challenging due to the low concentration of hormones, low amount and complexity of matrix. LC-MS/MS is an attractive analytical technique for development of such challenging methods because of high specificity, sensitivity and short analysis time. In this work we present LC-MS/MS method for a quantitative determination of estradiol, estrone, androstenedione and testosterone in human adipose tissue.

Methods

Sample preparation method consists of two stages: 1) liquid–liquid extraction of previously homogenized and frozen tissue using ethanol:ethyl acetate (1:1) mixture with internal standards addition. Upper layer obtained after this extraction was collected and evaporated under nitrogen stream and reconstituted in 20% MetOH; 2) Solid-phase extraction (SPE) using Sep-Pak® C18 Cartridges; 2g sorbent (Waters). Elution fraction collected after SPE procedure was evaporated under nitrogen stream and reconstituted in 40% MetOH. Compounds were analyzed using reversed-phase chromatography on Nexera LC40 (Shimadzu) LC coupled with QTRAP 6500+ (SCIEX) operating in positive and negative electrospray ionization scheduled MRM mode. Analyte presence confirmation was based on 2 criteria: specific retention time and ion ratio. Data were processed in SCIEX OS software.

Bioanalytic: Figure 1. Schematic representation of the sample preparation procedure.Bioanalytic: Table 1. LC-MS/MS method parameters.

Preliminary data

Literature data regarding methods determining both estrogens and androgens in human adipose tissue are limited. Because of different physicochemical properties of these two groups of steroid hormones it was considered to prepare two distinct methods in the optimization stage. Finally, after several different approaches tested, it was possible to develop single sample preparation and analytical method suitable for all the tested analytes with sufficiently good sensitivity. The method was validated separately for visceral (VAT) and subcutaneous (SAT) adipose tissue and fulfilled the following criteria for each analyte: linearity (R ≥ 0.995), reproducibility (%CV ≤ 15%), accuracy in the range: 80 – 120%. Method’s working range depends on adipose tissue type and analyte (visceral: androstenedione 1-100 ng/g; testosterone 0.1-10 ng/g; estradiol and estrone 0.05-10 ng/g; subcutaneous: androstenedione 1-100 ng/g; testosterone, estradiol and estrone 0.1-10 ng/g). Developed method was successfully applied to analyze adipose tissue samples collected from normal-weight and obese patients using only 0.25 g of homogenized tissue sample, what is important in case of limited clinical material availability.  The analysis was performed on pairs of visceral and subcutaneous adipose tissue from 15 obese patients (10 women and five men) and five pairs of adipose tissues from normal weight subjects (40 tissues in total). It was found that the concentrations of androstenedione, testosterone and estrone were higher while estradiol lower in visceral tissues than in subcutaneous tissues, irrespective of body weight. The tissues of obese patients (both VAT and SAT) were characterized by higher concentrations of estrone, while normal weight subjects had significantly higher testosterone concentrations in visceral than in subcutaneous adipose tissue depot.

Bioanalytic: Figure 3. Chromatogram of LC-MS/MS analysis of extracted tissue sample with marked ionization parameters.

Bioanalytic: Table 2. Summary of method validation results depending on the adipose tissue type.

Bioanalytic: Figure 4. Charts representing results of analysis of adipose tissue samples collected from normal-weight and obese patients. VAT-O - visceral tissue from obese patients; SAT-O subcutaneous tissue from obese patients; VAT-N - visceral tissue from normal-weight patient; SAT-N - subcutaneous tissue from normal weight patient.

Novel aspect

Sensitive LC-MS/MS method allowing for simultaneous quantitation of estrogens and androgens in single run using low amount of adipose tissue.