Development and Validation of the Simultaneous Measurement of Estrone and 17β-Estradiol in Serum by LC-MS/MS for Clinical Laboratory Applications Neus Fabregat-Cabello (Presenter) University of Liège, CHU de Liège >> POSTER (PDF) Presenter Bio: Neus Fabregat Cabello is Post-Doc at the Department of Clinical Chemistry, University Hospital of Liège (CHU de Liège, Belgium) under the supervision of Prof. Etienne Cavalier since November 2015. At the moment she is responsible for the development of new mass spectrometry applications based on LC-MS/MS for endocrinology research dealing with small molecules, most of them related to steroids and vitamin D metabolites.

Authors: J. Pitarch-Motellón(1), E. Cavalier(2) C. Le Goff(2), S. Peeters(2), N. Fabregat-Cabello(2).
(1) Research Institute for Pesticides and Water, Universitat Jaume I, Castelló, Spain (2) Department of Clinical Chemistry, CIRM, University of Liège, CHU Sart-Tilman, Liège, Belgium

A straightforward analytical method for the determination of estrone (E1) and 17β-estradiol (E2) at ultra-low levels in serum samples by LC-MS/MS has been developed and validated. This method entails an extraction and derivatization with dansyl chloride followed by separation with a C18 column. A comparison of the developed LC-MS/MS method against our routine immunoassay shows a good correlation for E2 while an important negative bias is observed for E1 by RIA. The later comparison for E1 shows the need to switch from the current routine automated immunoassays to highly-sensitive LC-MS/MS quantifications in order to provide accurate and reliable clinical results, especially at very low levels.

Introduction

Estrogens are sexual steroidal hormones, derived from cholesterol, which can exert strong biological effects even at low pg/mL levels. There are two main active estrogens in non-pregnant humans, which are estrone (E1) and 17β-estradiol (E2).

Nowadays, there is an increasing interest in the simultaneous high-sensitivity measurement of these estrogens for both clinical research and routine analysis. The main samples of interest include pediatric, pre-pubertal, post-menopausal and male serum samples. Unfortunately, inmunoassays are not able to reach the very low levels needed in some cases and usually show poor correlation with LC-MS/MS methods, particularly for E1[1,2]. Besides, in the case of E1 determination, the vast majority of routine methods are based on competitive radioimmunoassay (RIA) attained to health and safety risks posed by the use of radiation.

Despite of the known advantages of estrogen measurement by LC-MS/MS, which include increased sensitivity and specificity, these compounds do not contain highly ionizable functional groups. In order to increase the ionization efficiency for these compounds, different derivatization agents have been proposed, including picolinoyl, dansyl chloride or N-methyl-nicotinic acid N-hydroxysuccinimide ester, among others[3].

Therefore, the aim of this work is to establish a rugged and easy-to-handle method for E1 and E2 analysis in serum samples suitable for routine laboratories dealing with low concentration levels and sample volumes.

Methods

Identification and quantification of analytes was carried out using an Sciex QTRAP® 6500 triple quadrupole MS/MS (Framingham, MA, USA) equipped with LC-30A Nexera ultra-performance liquid chromatography (UHPLC) system (Shimadzu Co., Kyoto, Japan). The mass spectrometer was operated in electrospray ionization in positive mode and selected reaction monitoring (SRM).

Analysis of estrone and 17β-estradiol in serum was performed as follows. Extraction of analytes was achieved by means of liquid-liquid extraction with 2 mL of methyl tert-butyl ether, on 250 µL of sample, calibration or validation standards spiked with a mix of labeled analogs acting as internal standards. Extracts were dried under vacuum and derivatization was carried out with dansyl chloride under basic conditions, incubating at 60ºC for 10 min. Extracts were diluted to 100 µL with water and 30 µL were injected in the LC-MS/MS system. Chromatographic separation was achieved in under 8 min with a H2O/acetonitrile gradient containing 0.01% of formic acid in both mobile phases in a C18 solid-core column.

Validation standards and calibrators were prepared in commercial steroid-free serum materials, spiking with mix solutions of natural analytes prepared gravimetrically. Calibration curves were prepared in the range of 5 to 1000 pg/mL while validation standards (or quality controls) were prepared at 5, 10, 200, 400 and 800 pg/mL, following the European Medicines Agency (EMEA) Guideline on bioanalytical method validation. In addition, 3 certified reference materials for 17β-estradiol (CRM) were analysed, as well as control solutions employed in commercial methods installed in the laboratory.

For comparison of the newly developed LC-MS/MS assay with our routine assays, 17β-estradiol was measured by electrochemiluminescense immunoassay “ECLIA” on modular E170 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany) while E1 was measured by RIA (DIAsource ImmunoAssays, Louvain-la-Neuve, Belgium).

Results

Method development

Based on our previous experience on analysis of steroids in serum, several extraction methods were tested, including solid phase extraction (SPE), supported liquid extraction (SLE) and liquid-liquid extraction (LLE) with different extracting solvents.

Following the bibliography available, different mobile phase additives were also tested, including formic acid, ammonium formate and ammonium fluoride. Besides, different columns were tested, including C18 and pentafluorophenyl stationary phases. The best conditions have been described in the previous section.

Method validation

Mean recoveries within 92–113% and 87-101% were achieved for E1 and E2, respectively, even for the lowest concentrations at 5 pg/mL. Coefficients of variation or CVs(%) for intra-assay (repeatability) and inter-assay (intermediate precision) were lower than 14% in all both metabolites, which are in line with the EMEA guidelines. LOQ was established as the lowest validated level.

Method comparison

The correlation based on Passing-Bablok regression between our newly developed LC-MS/MS was performed against our routine assays for each metabolite.

For E2, we obtained a good correlation, with a regression equation ECLIA=1.04•LC-MS -1.45, which stands for a proportional bias (slope) of 1.04 (95% CI 0.97 to 1.1) and a constant bias (Y-intercept) of -1.45 pg/mL (95% CI -7.0 to 1.9) (n=41). Contrary to E2, for E1 the regression equation was RIA=2.51•LC-MS-3.58, meaning a proportional bias of 2.51 (95%CI 2.3 to 2.9) and a constant bias of -3.58 (95% CI -19,1 to 7.5) (n=36).

Conclusions & Discussion

In this work, we have developed and validated an LC–MS/MS method for the simultaneous determination of E1 and E2 at low pg/mL levels in serum samples, according to EMEA validation guidelines. Despite including a derivatization step, the proposed sample preparation is still easy and fast. Our LOQ, established as the lowest fully validated level, is only 5 pg/mL.

Compared with LC-MS/MS, ECLIA showed a good correlation for E2. On the contrary, RIA showed a marked negative bias for E1 compared with the newly developed LC-MS/MS method. Regression comparisons showed that, on average, LC-MS/MS generated results 150% higher than RIA. The disparity between LC-MS/MS and RIA could be attained to an ineffective displacement of steroid hormone for its binding protein. However, the source of this difference remains uncertain even thought it have been already observed in previous works [1]. Finally we have also observed that several non-menopausical patients presented a E1 level under the LOQ of the RIA method, 18 pg/mL. As a consequence, new reference values for E1 must be established by LC-MS/MS.

In conclusion, the presented method permits to quantify simultaneously and accurately E1 and E2 at very low levels in serum samples.

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