A dilute and shoot method for urinary free cortisol analysis by LC-MS/MS

Area of Science:

• Clinical Chemistry
• Analytical Chemistry
• Mass Spectrometry

Background:

• 24-hour urinary free cortisol (UFC) is the primary screening test for Cushing's syndrome.
• Accurate UFC assays require reliable sample preparation and method-specific reference intervals for diagnosis.

Purpose of the Study:

• To develop and validate a simple, accurate liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for 24-hour UFC determination.
• To evaluate urine preservatives for optimal sample storage and analysis.

Main Methods:

• A "dilute and shoot" LC-MS/MS method using 50 µL urine and a methanol/water solution with an internal standard.
• Method validation including linearity, precision, and recovery assessments.
• Evaluation of urine preservatives and investigation of reference intervals.

Main Results:

• The LC-MS/MS method demonstrated a wide linear range (0.625–500 ng/ml) with high accuracy and precision.
• Excellent recovery (93.3–109%) and low coefficients of variation (CVs ≤ 5.70%).
• No preservatives were recommended for urine collection to ensure optimal peak detection.

Conclusions:

• The developed "dilute and shoot" LC-MS/MS assay is a simple and accurate method for routine clinical testing of 24-hour UFC.
• The method's validation and reference interval investigation support its potential for reliable Cushing's syndrome diagnosis.
• Optimized sample collection without preservatives is recommended for improved analytical performance.