Determination of Hormones in Serum by LC/MS/MS Using Agilent Bond Elut Plexa SPE

Significance of the Topic

A streamlined and sensitive analytical workflow for steroid and related hormone quantification in serum addresses critical needs in clinical research, pharmaceutical development and quality control. Precise determination of low-level hormones such as corticosteroids and sex steroids supports diagnostic assays, therapeutic monitoring and biomarker discovery. By combining a polymeric solid phase extraction cleanup with high-performance liquid chromatography tandem mass spectrometry, this method enhances data reliability, lowers matrix interference and increases laboratory throughput.

Objectives and Study Overview

This study aimed to develop and validate an LC/MS/MS method for simultaneous quantification of 13 endogenous hormones in human serum. Key objectives included evaluation of Agilent Bond Elut Plexa solid phase extraction cartridges for sample cleanup, optimization of chromatographic separation on a Poroshell HPH-C8 column and improvement of ionization efficiency using ammonium fluoride. Method performance was assessed through recovery, precision and chromatographic resolution of isobaric pairs.

Methodology and Instrumentation

Sample preparation employed Agilent Bond Elut Plexa SPE cartridges (30 mg, 1 mL) with a wash solvent of 30 % methanol in 0.5 % formic acid and elution with 100 % methanol. After evaporation, extracts were reconstituted in 50:50 methanol/water. Chromatographic separation used an Agilent InfinityLab Poroshell HPH-C8 column (2.1 × 50 mm, 2.7 µm) with a binary gradient of 1 mM ammonium fluoride in water and acetonitrile at 0.4 mL/min, column temperature 40 °C and a 10 µL injection volume. Detection employed an Agilent 6460A triple quadrupole LC/MS/MS system with Jet Stream electrospray ionization in positive and negative modes, utilizing dynamic multiple reaction monitoring for each analyte and its stable isotope internal standard.

Key Results and Discussion

Recoveries for the 13 hormones ranged from 80 % to 105 % with relative standard deviations between 2.8 % and 5.8 %. Baseline separation of challenging isobaric pairs such as aldosterone versus cortisone was achieved within a 10-minute run time. The use of 1 mM ammonium fluoride mobile phase significantly enhanced both positive and negative mode responses compared to formic acid or high-pH ammonium hydroxide phases. SPE cleanup effectively reduced protein and lipid interferences, resulting in clean chromatograms and robust quantification across multiple hormone classes.

Benefits and Practical Applications

• High recovery and precision support reliable clinical and pharmacokinetic studies
• Rapid 10-minute analysis cycle increases sample throughput
• Polymeric SPE minimizes matrix effects and labor in routine laboratory workflows
• Stable isotope dilution ensures accurate quantification across diverse hormone targets

Future Trends and Opportunities

Emerging analytical trends include integration of automated online SPE for higher throughput, adoption of microflow LC/MS/MS to reduce solvent consumption and expansion of hormone panels to include novel biomarkers. Coupling this workflow with high-resolution mass spectrometry may enable untargeted discovery alongside targeted quantification. Machine learning algorithms for data processing promise to further enhance sensitivity and reduce analysis time in complex biological matrices.

Conclusion

The developed method combines Agilent Bond Elut Plexa SPE with an InfinityLab Poroshell HPH-C8 LC column and Jet Stream ESI MS/MS to achieve fast, sensitive and reproducible quantification of 13 serum hormones. Excellent recoveries, low variability and clear chromatographic separation of isobaric analytes make this approach well-suited for clinical research, pharmaceutical bioanalysis and routine QA/QC laboratories.

Reference

• Fu R Zhai A Determination of Hormones in Drinking Water by LC/MS/MS Using an Agilent InfinityLab Poroshell HPH Column EPA 539 Agilent Technologies Application Note 2016
• Hindle R Improved Analysis of Trace Hormones in Drinking Water by LC/MS/MS EPA 539 using the Agilent 6460 Triple Quadrupole LC/MS Agilent Technologies Application Note 2013
• Fiers T Casetta B Bernaert B Vandersypt E Debock M Kaufman J Development of a highly sensitive method for the quantification of estrone and estradiol in serum by liquid chromatography tandem mass spectrometry without derivatization Journal of Chromatography B 2012 893-894 57-62