Background: Ethinylestradiol (EE) is the active component in most birth control products. It is especially difficult to analyze due to the presence of many closely related endogenous steroids. Endogenous components can coelute with EE making selective extraction and chromatographic separation challenging. Current MS systems are more sensitive to background, contamination and the overall cleanliness of samples and solvents, placing additional emphasis on sample preparation methodology.
Method: UPLC was combined with a sensitive triple quadrupole MS and a three-step sample preparation method to highlight and resolve method development challenges.
Results: EE was adequately resolved using an unendcapped high-strength silica C(18) column. The average matrix factor in six sources of plasma was 1.14 with a %CV of 4.48. Standard curves were linear with 1/x weighting and r(2) value of 0.999 over three orders of magnitude. Average accuracy for standard curves and quality control samples was 96%. LOD of 0.001 ng/ml was achieved.
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