Direct background subtraction LC-MS/MS assay for human plasma progesterone: Full validation and comparative...

Area of Science:

• Analytical Chemistry
• Biomarker Quantification
• Mass Spectrometry

Background:

• Accurate quantification of endogenous progesterone in human plasma is crucial for clinical diagnostics.
• Traditional methods often involve laborious matrix preparation, introducing potential variability and matrix effects.
• Development of simplified, robust, and accurate quantification methods is essential.

Purpose of the Study:

• To develop and validate a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method.
• To quantify endogenous progesterone in human plasma utilizing direct background subtraction.
• To establish a simplified and efficient alternative to classical quantification techniques.

Main Methods:

• Sample preparation involved protein precipitation with deuterated progesterone as an internal standard.
• Chromatographic separation was performed on an ACQUITY C18 column with gradient elution.
• Mass spectrometry analysis utilized positive electrospray ionization and multiple reaction monitoring, with direct use of authentic plasma for validation.

Main Results:

• The method demonstrated excellent linearity (R² ≥ 0.99) over the 0.1-100 ng/mL range.
• All validation parameters, including precision, accuracy, recovery, matrix effect, and stability, met regulatory acceptance criteria (FDA, ICH M10).
• Comparison with the classical method showed no significant difference in Cmax and AUC0-t (bias within ±15%).

Conclusions:

• The direct background subtraction method offers a simple, efficient, and low-cost approach for progesterone quantification.
• This technique eliminates the need for laborious blank matrix preparation and mitigates matrix effect discrepancies.
• The developed method serves as a generalizable strategy for the determination of endogenous substances.