Analysis of Adrenal Steroids in Dried Blood Spots and Serum Using Agilent Ultivo LC/TQ

Importance of the Topic

Adrenal steroids play a central role in maintaining electrolyte and water balance, as well as regulating stress responses. Disorders of steroid biosynthesis lead to conditions such as congenital adrenal hyperplasia and Cushing’s syndrome. Rapid, multiplexed quantification of these hormones in minimally invasive samples like dried blood spots (DBS) and serum supports clinical research, newborn screening, and therapeutic monitoring.

Goals and Overview of the Study

This study describes the transfer of a validated LC/MS/MS method for quantifying eleven adrenal steroids from a non-Agilent platform onto the Agilent Ultivo LC/TQ system. Key aims include evaluating limits of detection (LOD), limits of quantification (LOQ), recovery, precision, and overall method robustness in both DBS and serum matrices within a seven-minute runtime.

Analytical Methodology

• Sample Preparation: Two 4.7 mm punches of DBS or 15.2 µL serum are spiked with a deuterated internal standard mix, precipitated with 200 µL of 50:50 acetone:acetonitrile, incubated, centrifuged, dried under nitrogen at 35 °C, and reconstituted in 50% methanol with formic acid.
• Chromatography: Separation on a 2.1 × 50 mm, 1.7 µm BEH C18 column at 23 °C. Mobile phases are water and methanol with 0.1% formic acid. Gradient from 47% to 95% B over 6 min, flow 350 µL/min, 10 µL injection.
• Mass Spectrometry: Agilent Ultivo TQ in dynamic MRM mode, positive and negative electrospray with JetStream source. Cycle time 500 ms, unit resolution MS2, transitions optimized for each analyte and internal standard.
• Data Analysis: Quantification via MassHunter QQQ software with 5-point external calibration, 1/x weighting, Agile2 algorithm for peak integration, Savitzky–Golay smoothing, and RMS noise thresholding.

Instrumentation Used

• Agilent Infinity II 1290 UPLC System: High Speed Pump (G7120A), Multisampler (G7167B), and Multicolumn Thermostat (G7116B).
• Agilent Ultivo Triple Quadrupole Mass Spectrometer with JetStream Ion Source (G6465A).

Main Results and Discussion

Method performance in DBS showed LODs between 0.05–0.95 nM and LOQs between 0.18–3.16 nM. Recoveries ranged 83–113% and intraday/interday CVs remained below 17% and 12%, respectively. In serum, LODs were 0.09–1.25 nM, LOQs 0.28–4.15 nM, recoveries 57–78%, with precision under 19% CV. The seven-minute gradient yielded clear separation of all eleven steroids, supporting reliable quantification in both matrices.

Benefits and Practical Applications of the Method

This fast, high-throughput approach enables simultaneous profiling of multiple adrenal steroids from minimal sample volumes. The robust precision and sensitivity are well suited for clinical laboratories, newborn screening programs, and research into endocrine disorders. Dried blood spots facilitate remote sampling and storage.

Future Trends and Opportunities

Future developments may include automation of DBS processing, expansion of analyte panels to cover additional steroid pathways, integration with high-resolution MS for untargeted profiling, and application of machine learning to interpret complex steroidogenic patterns. Point-of-care LC/MS solutions may further streamline clinical decision support.

Conclusion

The Agilent Ultivo LC/TQ system reliably transfers an established adrenal steroid panel for both dried blood spots and serum, delivering comparable sensitivity, accuracy, and precision within a seven-minute analysis. This platform supports efficient endocrine profiling in clinical and research settings.

Reference

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