Scientific article
Open access

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

Published inJournal of chromatography. B, vol. 1226, 123778
Publication date2023-07-15
First online date2023-06-16

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.

  • External calibration
  • Internal calibration
  • LC-MS
  • Quantification
  • Steroids
Citation (ISO format)
VISCONTI, Gioele et al. Straightforward quantification of endogenous steroids with liquid chromatography–tandem mass spectrometry: Comparing calibration approaches. In: Journal of chromatography. B, 2023, vol. 1226, p. 123778. doi: 10.1016/j.jchromb.2023.123778
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Article (Published version)
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ISSN of the journal1570-0232

Technical informations

Creation06/30/2023 1:33:21 PM
First validation07/04/2023 12:41:15 PM
Update time07/04/2023 12:41:15 PM
Status update07/04/2023 12:41:15 PM
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