UPLC-MS Analysis of Flunixin Meglumine Concentrations

Plasma concentrations of FM were determined using ultrahigh performance liquid chromatography (Q Exactive Focus Orbitrap, Thermo Scientific, San Jose, CA, USA) coupled with mass spectrometry (Dionex Ultimate 3000, Thermo Scientific, San Jose, CA, USA), as previously described (8). The standard curve for FM was prepared in blank porcine serum and ranged from 2 to 5,000 ng/mL with a correlation coefficient greater 0.995 for all analyses. Quality control (QC) samples were prepared at concentrations 30, 300, and 3,000 ng/mL. For concentrations above 3,000 ng/ml, dilution using blank swine serum was validated as a precise method for quantifying concentration. All dilutions in this study were <0.1. The QCs were ±15% of the nominal value. The lower limit of quantification (LLOQ) and lower limit of detection (LLOD) were 2 and 0.3 ng/mL, respectively. For Phase I, the accuracy and precision for the QC samples were 109 and 3.0% for the 30 ng/mL QC; 102 and 4.7% for the 300 ng/mL QC; and 105 and 4.2% for the 3,000 ng/mL QC. For Phase II, the accuracy and precision for the QC samples were 103 and 5.5% for the 30 ng/mL; 100 and 3.3% for the 300 ng/mL; and 104 and 4.8% for the 3,000 ng/mL samples.