Measurement of Unconjugated Estriol in Serum by Liquid Chromatography–Tandem Mass Spectrometry and Assessment of the Accuracy of Chemiluminescent Immunoassays

Abstract

BACKGROUND Unconjugated estriol (uE3) is routinely analyzed in clinical laboratories as risk assessment for Down syndrome. Immunoassays of various types are the most commonly used methods. The accuracies of RIAs and ELISAs for uE3 have been questioned, and to date there have been no independent studies investigating the accuracy of the relatively new chemiluminescent immunoassays. We developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for uE3 measurements in serum. METHODS Serum samples from patients in the second trimester of pregnancy were used, and uE3 concentrations were measured by LC-MS/MS and the Beckman Coulter Access® 2 and Siemens IMMULITE 2000 automatic chemiluminescent immunoassay analyzers. RESULTS The LC-MS/MS method was validated and showed limit of detection 0.05 ng/mL; limit of quantification 0.2 ng/mL; linearity of response to 32 ng/mL; total imprecision of 16.2%, 10.4%, and 8.2% for uE3 at 1.10, 4.18, and 8.32 ng/mL, respectively; and analytical recoveries of 95.9%–104.2%. ANOVA of the correlation for LC-MS/MS results vs chemiluminescent immunoassays results showed R2 = 0.9678 (Access 2 = 0.9305 LC-MS/MS + 0.2177, Sy|x = 0.1786, P < 0.0001), and R2 = 0.9663 (IMMULITE 2000 = 0.8849 LC-MS/MS − 0.0403, Sy|x = 0.1738, P < 0.0001). Bland–Altman plots of uE3 results revealed concentration-dependent immunoassay biases. Mock risk analysis for Down syndrome showed no apparent difference in the risk assessment outcomes if the adjusted method-specific multiples of the median were used, and the assay imprecision was <10% CV. CONCLUSIONS Standardization of immunoassay methods for uE3 analysis is needed to improve the accuracy of the measurements.

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).…”

Possibilities and limitations of signal summing for an immunosuppressant LC-MS/MS method

“…Liquid chromatography-tandem mass spectrometry (LC-MS/ MS) monitoring targeted precursor and fragment ions (multiple reaction monitoring (MRM)) is the method of choice for quantifying small molecules in research as well as clinical setting [1,2]. The method development is most often Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-8799-7) contains supplementary material, which is available to authorized users.…”