2.3. Laboratory Analysis for Meloxicam Quantitation in Plasma

Processed plasma samples received at the diagnostic laboratory (Alberta Veterinary Laboratories, Calgary, AL, Canada) were rapidly transferred to frozen storage (−20 °C). Storage temperature was monitored by a National Institute of Standards in Technology (NIST)-certified temperature-recording device.

Plasma samples were analyzed with a validated high-performance liquid chromatography (HPLC; Agilent 1200 HPLC, Agilent, CA, USA) procedure that was conducted using UV detection. An internal standard (piroxicam) was added to the untreated plasma sample. Standard concentrations used were 0.05, 0.1, 0.5, 1, 2, 3, 4, and 6 µg/mL respectively. Meloxicam and the internal standard were then extracted from plasma by solid-phase extraction (SPE). The SPE cartridges were connected to a vacuum manifold and conditioned with 1 mL methanol followed by 1 mL of water. The samples were passed through sorbents at a flow rate of less than 1 mL/min. The cartridges were then rinsed with 1 mL of 5% methanol and dried under vacuum for 2 min. Analytes were eluted with 1.5 mL of methanol. The eluent was then dried under vacuum at 40 °C for 2 h and the dried residue was reconstituted in 100 µL of mobile phase. The reconstituted sample was vortexed for 15 s then centrifuged at 14,000× g for 10 min to remove any particulate from the sample. Following centrifugation, 10 µL of supernatant was injected into the HPLC system. HPLC apparatus consisted of a pump system equipped with an automatic injector and UV detector (360 nm). Separation was achieved using a reverse-phase column (C18, 3 mm, 125 × 3.0 mm) and a guard column (C18, 10 × 30 mm). The mobile phase consisted of a mixture of acetic acid: 1% methanol (40:60) at a flow rate of 0.4 mL/min. For these conditions, meloxicam and piroxicam were eluted at a retention time of 7.8 and 5.4 min, respectively. Results for the method were linear over the calibration range of 10 to 1250 ng/mL as determined by use of a weighted linear regression model. Within-day and day-day precision were <10%. Accuracy ranged from 96% to 99%. The validated limit of quantification (LOQ) was 10 ng/mL.