A Method for Analysis of Free and Total Ropivacaine in Dog Plasma Using UHPLC-MS/MS

Area of Science:

• Veterinary Pharmacology
• Analytical Chemistry
• Mass Spectrometry

Background:

• Ropivacaine is a widely used local anesthetic in veterinary anesthesia.
• Accurate quantification of free and total ropivacaine in plasma is essential for pharmacokinetic and safety studies.

Purpose of the Study:

• To develop and validate a sensitive liquid chromatography-mass spectrometry (LC-MS) method for quantifying free and total ropivacaine in dog plasma.
• To assess the method's performance according to International Conference on Harmonization M10 guidelines.

Main Methods:

• Development of a liquid chromatography-mass spectrometry (LC-MS) method incorporating rapid equilibrium dialysis.
• Ultra-high performance liquid chromatography (UHPLC) separation coupled with triple quadrupole tandem mass spectrometry (MS/MS) detection.
• Method validation for selectivity, specificity, matrix effect, calibration, accuracy, precision, stability, and reproducibility.

Main Results:

• The validated UHPLC-MS/MS method demonstrated high sensitivity, with lower limits of quantification of 0.05 ng/mL for free and 0.30 ng/mL for protein-bound ropivacaine.
• Accuracy ranged from 85.50% to 113.30%, and precision ranged from 0.40% to 5.30% across validated concentrations.
• All validation parameters met the acceptance criteria outlined by ICH M10 guidelines.

Conclusions:

• A robust and validated UHPLC-MS/MS method for quantifying free and total ropivacaine in dog plasma has been established.
• This method is suitable for use in pharmacokinetic studies and clinical applications in veterinary medicine.
• The method was successfully applied in a clinical study involving intraperitoneal ropivacaine instillation in dogs.