Novel LC–MS/MS method enables high-throughput quantification of steroids for clinical applications

A novel high-throughput method for quantifying steroids in human plasma using liquid chromatography–tandem mass spectrometry (LC–MS/MS) has been developed, addressing long-standing challenges in the accurate measurement of endogenous and exogenous hormones at very low concentrations.

Conventional immunoassay-based diagnostics, often employed in clinical laboratories, are constrained by cross-reactivity – particularly when detecting hormones at low levels. This has prompted a shift towards the use of (ultra) high-performance liquid chromatography–tandem mass spectrometry techniques. However, the absence of a true blank biological matrix has hindered the accuracy of external calibration for endogenous hormone quantification.

The lead author of the study was Min Su, a doctoral candidate with the Institute of Pharmaceutical Sciences, Pharmaceutical (Bio-)Analysis, at the University of Tuebingen, in Germany.

To overcome this limitation, the researchers have established a surrogate calibration approach that uses 1,2-dimethylimidazole-5-sulfonyl chloride derivatisation specifically for oestrogens. This enhances both sensitivity and selectivity for quantifying oestrogens, while allowing simultaneous analysis of non-derivatised steroids. The method incorporates stable isotope-labelled (SIL) surrogate calibrants and deuterated internal standards, enabling accurate, matrix-matched quantification across a clinically relevant concentration range.

Systematic verification of parallelism between endogenous analytes and surrogate calibrants was achieved in plasma at various calibration levels. The method was further refined by employing narrow-bore UHPLC columns and optimised chromatographic conditions, which improved sensitivity and resolution across a broad panel of analytes.

In addition to analytical performance, the method integrates practical features for clinical scalability. It includes efficient protein precipitation and solid-phase extraction in a 96-well plate format, achieving quantification at the picogram-per-millilitre level with high precision and accuracy. Crucially, this workflow enables the quantification of both endogenous hormones and exogenous contraceptives in a single analytical run by combining surrogate calibration with external calibration and derivatisation-based enhancement of oestrogen detection.

The assay was validated in accordance with US Food and Drug Administration bioanalytical method validation guidelines and supplemented with additional criteria to accommodate the absence of formal regulatory frameworks for surrogate calibration approaches. Commercially certified quality control samples were used to confirm quantification accuracy, offering a robust reference model for future studies.

While the current vacuum evaporation step may limit its use in rapid clinical workflows, nitrogen-based evaporation alternatives were shown to improve throughput. Enhanced ionisation efficiency was achieved by employing low-flow (0.1 mL/min) chromatography using a narrow-diameter (1.0 mm) core–shell C18 column.

The steroid panel was expanded through the use of scheduled multiple reaction monitoring, supporting simultaneous quantification of derivatised oestrogens and non-derivatised steroids within a single injection. The use of 13C-labelled SIL surrogate calibrants, in combination with internal standards, effectively corrected for matrix effects and variability in extraction, contributing to the overall accuracy and reliability of the method.

This is the first reported demonstration of combining surrogate calibration with chemical derivatisation in a hybrid strategy capable of maintaining analytical integrity across both endogenous and exogenous steroid classes without the need for separate analyses.

Future improvements are anticipated through broader availability of 13C-labelled SIL standards, which would further enhance internal standardisation and quantification precision. Additionally, advances in chromatographic technology and next-generation mass spectrometry are expected to enable steroid quantification in smaller and more challenging sample matrices, such as murine plasma.

For further reading please visit: 10.1021/acs.analchem.5c01912