Measurement of human serum unconjugated estriol without derivatization using liquid chromatography-tandem mass spectrometry candidate reference method and compared with two immunoassays

Abstract

A candidate reference measurement procedure (RMP) for measurement of unconjugated estriol in human serum has been developed and validated. The proposed method is highly reliable and uses isotope dilution coupled with liquid chromatography-tandem mass spectrometry (ID-LC-MS/MS) and requires no derivatization. An appropriate amount of serum was accurately weighed and spiked with an isotopically labeled internal standard. Unconjugated estriol and its internal standard were extracted from serum matrix using liquid-liquid extraction prior to reversed-phase LC-MS/MS. Calibrator bracketing was used to give higher specificity and accuracy for assigning serum level. The accuracy of the candidate RMP was validated by split-sample comparison to established RMPs. The lowest limit of detection (LLoD) and lowest limit of quantification (LLoQ) for developed RMP was estimated to be 0.14 nmol/L and 0.35 nmol/L, respectively. Both intra- and inter-assay imprecisions were ≤2.19% at 1.39, 17.34 and 69.35 nmol/L, respectively. Recoveries were 98.54% to 100.34% and linear response ranged from 0.35 to 173.38 nmol/L. No interference was observed. Biases were 5.6% and 2.8% against the targets of RELA2015A (3.87 nmol/L) and RELA2015B (40.62 nmol/L), respectively. Moreover, the candidate RMP was successfully applied to measure level of unconjugated estriol in serum samples of pregnant women (n = 3) and compared with two immunoassays in clinical laboratory. Our developed method is simple, accurate, and can be used as a candidate RMP to determine total unconjugated estriol level in human serum. Further improvement of certain immunoassays in accuracy and precision is needed. Graphical abstract Selected ion chromatograms by LC-MS/MS using a C18 column for uE from a serum sample.

Development and validation of a liquid chromatography–tandem mass spectrometry method for the simultaneous analysis of androgens, estrogens, glucocorticoids and progestagens in human serum

“…Once developed and validated, the LC–MS/MS analytical method was applied to the analysis of five serum samples collected by five different male volunteers (age, 39.8 ± 11.3) and five serum samples collected by five different female healthy subjects (age, 43.8 ± 12.8). The obtained results (Table 4) are in accordance with the previously published reference ranges (Eisenhofer et al, 2017; Fanelli et al, 2011; Huang et al, 2014, 2018; Schiffer et al, 2019), demonstrating the applicability of the developed procedure for the identification and quantification of serum steroids both in male and in female individuals.…”

“…The LLOQ values (Figure 3) were in accordance with the concentration reference ranges reported in the literature (Eisenhofer et al, 2017; Fanelli et al, 2011; Huang et al, 2014, 2018; Schiffer et al, 2019) and could be useful in the case of the analysis of serum samples collected from individuals affected by metabolic disorders characterized by the reduction of concentration of endogenous steroids (i.e. hypogonadism for testosterone and other androgens or congenital adrenal hyperplasia for cortisol and other glucocorticoids).…”

“…Owing to the lack of a steroid‐free matrix, calibration samples were prepared in a surrogate matrix consisting of a solution of BSA in PBS 4% w/v. One blank, a zero‐point level and seven levels of calibrators were used for each compound (10 pg/ml to 10 ng/ml for A4, T, S, 21DF and 11DB; 40 pg/ml to 40 ng/ml for DHT, E1, E2, E3, B, P4, 17OHP4 and 17OHP5; 100 pg/ml to 100 ng/ml for F and E; and 400 pg/ml to 1,000 ng/ml for DHEA‐S), according to their reference concentration ranges (Eisenhofer et al, 2017; Fanelli et al, 2011; Huang et al, 2014; Huang et al, 2018; Schiffer et al, 2019).…”

“…To assess the linearity of the method, a lack‐of‐fit test was done for each steroid considered in the study (Magnusson, 2014). The reported linearity ranges were investigated according to the reference concentration ranges described for each compound under study (Eisenhofer et al, 2017; Fanelli et al, 2011; Huang et al, 2014, 2018; Schiffer et al, 2019).…”

“…The linearity was determined in the range 10 pg/ml to 10 ng/ml for A4, T, S, 21DF and 11DB; 40 pg/ml to 40 ng/ml for DHT, E1, E2, E3, B, P4, 17OHP4 and 17OHP5; 100 pg/ml to 100 ng/ml for F and E; and 400 pg/ml to 1,000 ng/ml for DHEA-S. To assess the linearity of the method, a lack-of-fit test was done for each steroid considered in the study (Magnusson, 2014). The reported linearity ranges were investigated according to the reference concentration ranges described for each compound under study (Eisenhofer et al, 2017;Fanelli et al, 2011;Huang et al, 2014Huang et al, , 2018Schiffer et al, 2019).…”