Biphenyl based stationary phases for improved selectivity in complex steroid assays

Area of Science:

• Endocrinology
• Analytical Chemistry
• Mass Spectrometry

Background:

• Steroid hormone and corticoid precursor measurement is crucial for diagnosing endocrine disorders.
• High-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) is the preferred analytical method.
• Existing C18 stationary phases often fail to adequately separate isobaric steroids.

Purpose of the Study:

• To develop an isotope dilution ultra-high-performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) assay for clinical research.
• To quantify a broad panel of steroid hormones in human serum.
• To evaluate the efficacy of biphenyl stationary phases for separating challenging isobaric steroids.

Main Methods:

• Developed a UHPLC-MS/MS assay using a Kinetex Biphenyl column.
• Employed a simple protein precipitation with zinc trifluoroacetate (ZnTFA) in methanol.
• Utilized a mobile phase of 0.2mM ammonium fluoride in water and methanol for separation.

Main Results:

• Achieved baseline separation of isobaric steroids including corticosterone, 11-deoxycortisol, and 21-deoxycortisol.
• Demonstrated excellent method validation results for accuracy, imprecision, recovery, matrix effect, and linearity (R² > 0.99).
• Quantified a broad steroid panel including aldosterone, cortisol, progesterone, and testosterone.

Conclusions:

• Biphenyl-based columns are effective for comprehensive, MS-based steroid assays, particularly for isobaric compounds.
• The developed UHPLC-MS/MS method offers a robust and efficient approach for clinical research.
• Zinc trifluoroacetate (ZnTFA) is a suitable precipitation agent for steroid analysis.