Sensitive, high throughput HPLC-MS/MS method with on-line sample clean-up for everolimus measurement

Area of Science:

• Analytical Chemistry
• Pharmacology
• Clinical Chemistry

Background:

• Accurate quantification of immunosuppressants like everolimus is crucial for therapeutic drug monitoring.
• Existing methods may have limitations in terms of speed, cost, or sample volume.
• The selection of an appropriate internal standard (IS) is critical for reliable quantification.

Purpose of the Study:

• To develop and validate a novel, rapid, and cost-effective high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for everolimus determination.
• To evaluate the performance of two potential internal standards: SDZ RAD 223-756 and ascomycin.
• To compare the developed method with a reference HPLC-MS/MS method.

Main Methods:

• Development of a multiple reaction monitoring (MRM) positive ion HPLC-MS/MS method with on-line extraction and sample cleanup.
• Utilized an API 2000 triple quadrupole mass spectrometer and a Nova-Pak C18 analytical column.
• Assessed precision, recovery, and matrix effects using spiked blood samples with varying everolimus concentrations.

Main Results:

• The method demonstrated a lower limit of quantification (LLOQ) of 1.0 microg/L for everolimus with either IS.
• Between-day precision ranged from 3.1% to 8.6%, and absolute recoveries averaged 76.8-77.3%.
• The method using SDZ RAD 223-756 as IS showed equivalent results to the reference method, while ascomycin yielded statistically lower concentrations.

Conclusions:

• The developed HPLC-MS/MS method is suitable for accurate and efficient everolimus measurement in blood.
• SDZ RAD 223-756 is a reliable internal standard for this assay, ensuring comparability with reference methods.
• The method's features, including small sample volume and fast preparation, make it advantageous for clinical settings.