Quantification of steroids in human serum for clinical research by TurboFlow using the new Transcend VTLX-1 UHPLC system

Abstract

Objective

To implement an analytical method for quantifying eight steroids in human serum using TurboFlow online sample cleanup on the new Thermo Scientific™ Transcend™ VTLX-1 UHPLC system, coupled to a Thermo Scientific™ TSQ Altis triple quadrupole mass spectrometer. The study aims to compare the analytical performance with the same method run on a Thermo Scientific™ Transcend™ TLX-1 system in terms of chromatography, limits of quantification, accuracy, and intra-assay precision.

Introduction

Liquid chromatography (LC) coupled with triple quadrupole mass spectrometry (MS/MS) is the preferred method for quantifying steroid hormones in biological matrices due to its superior specificity and sensitivity with low sample volumes. However, lab space and budget constraints often hinder its adoption.

To address these issues, we developed the new Thermo Scientific™ Transcend™ VTLX-1, an evolution of the Transcend TLX-1 UHPLC system. The VTLX-1 maintains the same functionalities but has a smaller footprint and a more affordable price. It supports online sample cleanup via TurboFlow or online SPE and UHPLC applications without online purification.

In this technical note, we compared the performance of the Transcend VTLX-1 and TLX-1 systems using a Thermo Scientific™ TSQ Altis triple quadrupole mass spectrometer. Serum samples, prepared by offline protein precipitation with internal standard addition, were injected into both systems. Detection was performed in SRM mode using the TSQ Altis with a HESI-II source in positive ionization mode. Method performance was evaluated based on chromatography, linearity, accuracy, and intra-assay precision using calibrators, controls, and internal standards from the ClinMass® Complete Kit for Steroids in Serum/Plasma from RECIPE Chemicals + Instruments GmbH.

Method

200 μL aliquots of redissolved calibrators and controls were protein precipitated using 200 μL of methanol containing six isotopically labelled internal standards, vortex-mixed and centrifuged. The supernatant was transferred to clean vials pending the injection. The same analytical method, including TurboFlow online sample cleanup, was used on the two UHPLC systems. The total runtime was 5.0 minutes. Analytes and internal standards were detected in SRM acquisition mode on a TSQ Altis triple quadrupole mass spectrometer with heated electrospray ionization operated in positive ion mode.

Results and Discussion

Comparison of peak symmetry and intensity obtained on both front ends for the highest calibrator showed a superior peak symmetry for all analytes using the Transcend VTLX-1, thanks to the optimized fluidics. A linear response with 1/ × weighting was obtained for all the analytes on both Transcend systems, with a correlation factor (R2) always above 0.9995. Experimental data demonstrate the outstanding equivalent performance of this method when applied to the two different Transcend systems, both in terms of accuracy and intra-assay precision.

Conclusion

The obtained results demonstrate the ability of the new Transcend VTLX-1 UHPLC system to provide higher quality analytical results when compared to the TLX-1 version in the quantification of steroids in human serum, meeting research laboratory requirements in terms of linearity of response, accuracy, and precision.