Relevant Financial Disclosures
(within past 24 months, reported on Jul 13, 2026)
No relevant financial relationship(s) to disclose.
Abstract
1. Problem
During routine measurements using a commercially available kit for steroid quantification by LC-MS/MS in human serum, a uniform shift of all measured parameters towards higher retention time (RT) was observed. The deviation appeared consistently in this specific method and on a single instrument, while other methods on the same instrument and the same method on other instruments remained unaffected.
2. Method Information
• Commercial kit for steroid quantification in human serum or plasma via LC-MS/MS from Chromsystems
• HPLC: Agilent Technologies – 1290 Infinity II
• MS: Sciex QTRAP 6500+
• 12 min gradient LC program, 0.8 – 0.4 mL/min changing flow rate
• Column oven: 32°C
• Injection volume: 20µL
• Quantitative MRM acquisition
3. Troubleshooting Steps
Chromatographic parameters, including mobile phases, column, gradient profile and consumables such as inline-filter, solvent filter, heat-exchanger within the column module, connection capillaries and diverter valves were systematically replaced to identify possible causes. The HPLC Pump and autosampler module were replaced. Injection vessels were changed and diluted internal-standard mix was used instead of prepared samples. The solvent channels were swapped.
4. Outcome
None of the interventions resolved the problem. Chromatographic parameters, the specified consumables, malfunction of the pump or parts of the autosampler and sample preparation could be essentially precluded as source of the RT shifts. Swapping the solvent channels did not change the direction of the RT shift.
The persistence of the method specific RT shift after replacing all essential parts of the chromatographic procedure suggests that subtle, method-specific interactions between mobile phase and HPLC hardware could be the cause of the RT shifts, which highlights the complexity of retention mechanisms in LC-MS/MS assays. Ongoing investigations in collaboration with the kit and HPLC manufacturer aim to further elucidate the underlying factors and ensure long-term method robustness and reproducibility of the commercial kit to prevent future issues for other users.
= Discovery stage. (57.21%, 2026)
= Translation stage. (23.38%, 2026)
= Clinically available. (19.40%, 2026)
