Straightforward quantification of endogenous steroids with liquid chromatography-tandem mass spectrometry: Comparing calibration approaches

Gioele Visconti; Miguel de Figueiredo; Olivier Salamin; Julien Boccard; Nicolas Vuilleumier; Raul Nicoli; Tiia Kuuranne; Serge Rudaz

doi:10.1016/j.jchromb.2023.123778

Straightforward quantification of endogenous steroids with liquid chromatography-tandem mass spectrometry: Comparing calibration approaches

J Chromatogr B Analyt Technol Biomed Life Sci. 2023 Jul 15:1226:123778. doi: 10.1016/j.jchromb.2023.123778. Epub 2023 Jun 16.

Authors

Gioele Visconti¹, Miguel de Figueiredo¹, Olivier Salamin², Julien Boccard³, Nicolas Vuilleumier⁴, Raul Nicoli⁵, Tiia Kuuranne⁵, Serge Rudaz⁶

Affiliations

¹ School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland.
² Center of Research and Expertise in Anti-Doping Sciences - REDs, Institute of Sport Sciences, University of Lausanne, Lausanne, Switzerland; Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Lausanne University Hospital and University of Lausanne, Switzerland.
³ School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Basel, Switzerland.
⁴ Department of Genetic and Laboratory Medicine, Geneva University Hospitals (HUG), Geneva, Switzerland.
⁵ Swiss Laboratory for Doping Analyses, University Center of Legal Medicine, Lausanne and Geneva, Lausanne University Hospital and University of Lausanne, Switzerland.
⁶ School of Pharmaceutical Sciences, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel-Servet 1, Geneva, Switzerland; Swiss Centre for Applied Human Toxicology (SCAHT), Basel, Switzerland. Electronic address: serge.rudaz@unige.ch.

PMID: 37393882
DOI: 10.1016/j.jchromb.2023.123778

Abstract

Different calibration strategies are used in liquid chromatography hyphenated to mass spectrometry (LC-MS) bioanalysis. Currently, the surrogate matrix and surrogate analyte represent the most widely used approaches to compensate for the lack of analyte-free matrices in endogenous compounds quantification. In this context, there is a growing interest in rationalizing and simplifying quantitative analysis using a one-point concentration level of stable isotope-labeled (SIL) standards as surrogate calibrants. Accordingly, an internal calibration (IC) can be applied when the instrument response is translated into analyte concentration via the analyte-to-SIL ratio performed directly in the study sample. Since SILs are generally used as internal standards to normalize variability between authentic study sample matrix and surrogate matrix used for the calibration, IC can be calculated even if the calibration protocol was achieved for an external calibration (EC). In this study, a complete dataset of a published and fully validated method to quantify an extended steroid profile in serum was recomputed by adapting the role of SIL internal standards as surrogate calibrants. Using the validation samples, the quantitative performances for IC were comparable with the original method, showing acceptable trueness (79%-115%) and precision (0.8%-11.8%) for the 21 detected steroids. The IC methodology was then applied to human serum samples (n = 51) from healthy women and women diagnosed with mild hyperandrogenism, showing high agreement (R² > 0.98) with the concentrations obtained using the conventional quantification based on EC. For IC, Passing-Bablok regression showed proportional biases between -15.0% and 11.3% for all quantified steroids, with an average difference of -5.8% compared to EC. These results highlight the reliability and the advantages of implementing IC in clinical laboratories routine to simplify quantification in LC-MS bioanalysis, especially when a large panel of analytes is monitored.

Keywords: External calibration; Internal calibration; LC-MS; Quantification; Steroids.

MeSH terms

Calibration
Chromatography, Liquid / methods
Female
Humans
Reproducibility of Results
Steroids*
Tandem Mass Spectrometry* / methods

Substances

Steroids