Semi-automated serum steroid profiling with tandem mass spectrometry

Abstract

Objectives: Highly selective and sensitive multi-analyte methods for the analysis of steroids are attractive for the diagnosis of endocrine diseases. Commercially available kits are increasingly used for this purpose. These methods involve laborious solid phase extraction, and the respective panels of target analytes are incomplete. We wanted to investigate whether an improvement of kit solutions is possible by introducing automated on-line solid phase extraction (SPE) and combining originally separate analyte panels.

Methods: Sample preparation was performed using automated on-line SPE on a high-pressure stable extraction column. Chromatographic separation, including isobaric compounds, was achieved using a 0.25 mM ammonium fluoride-methanol gradient on a small particle size biphenyl column. Standard compounds and internal standard mixtures of two panels of a commercially available kit were combined to achieve an optimized and straightforward detection of 15 endogenous steroids. Validation was performed according to the European Medicines Agency (EMA) guidelines with slight modifications.

Results: Validation was successfully performed for all steroids over a clinically relevant calibration range. Deviations of intra- and inter-assay accuracy and precision results passed the criteria and no relevant matrix effects were detected due to highly effective sample preparation. External quality assessment samples showed the applicability as a routine diagnostic method, which was affirmed by the analyses of anonymized clinical samples.

Conclusions: It was found possible to complement a commercially available kit for quantitative serum steroid profiling based on isotope dilution LC-MS/MS by implementing automated on-line SPE, thereby improving the practicality and robustness of the measurement procedure.

Keywords: A4, Androstendione; ALDO, Aldosterone; APCI, Atmospheric pressure chemical ionization; CAH, Congenital adrenal hyperplasia; CE, Collision energy; CE-IVD, Certified in-vitro-diagnostic device; CV, Coefficient of variation; DHEA, Dehydro-epiandrosterone; DHEA-S, Dehydro-epiandrosterone sulfate; DHT, Dihydrotestosterone; DOC, 11-deoxycorticosterone; E, Cortisone; E2, Estradiol; EMA, European Medicines Agency; EQA, External quality assessment; ESI, Electrospray ionisation; F, Cortisol; IVD, In-vitro-diagnostic; IVDR, EU In vitro Diagnostic Regulation; LC, Liquid chromatography; LC–MS/MS, Liquid chromatography tandem mass spectrometry; LDT, Laboratory developed test; LLOQ, Lower limit of quantification; MRM, Multiple reaction monitoring; On-line solid phase extraction (SPE); P4, Progesterone; QC, Quality control; Robustness; S/N, Signal-to-noise ratio; SID, Stable-isotope dilution; SPE, Solid phase extraction; SST, System suitability test; Serum steroid profiling; Stable-isotope dilution liquid chromatography-tandem mass spectrometry (SID LC-MS/MS); UHPLC, Ultra high performance liquid chromatography; ULOQ, Upper limit of quantification.

Fig. 1

UHPLC settings for the 10-port switching valve and both binary pumps. A, on the left side, position 1 of the 10-port switching valve is shown. The extract is injected and loaded from the autosampler (ALS) to the extraction column, and afterwards washed (yellow line, time 0.0 – 1.5 min). On the right side, position 2 is visible. showing the elution of the analytes from the extraction column to the analytical column, where the chromatographic separation take place (blue line, time 1.5 – 11.0) and detected in the mass spectrometer (MS). B, UHPLC gradients for the autosampler (Pump ALS) and second pump (Pump 2), each with methanol as mobile Phase B, with the selected valve positions. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 2

Total ion current chromatogram for all 15 analytes of a UHPLC-MS/MS analysis of calibrator 4.

Fig. 3

Chromatograms of the post-column infusion experiment.