Plasma samples were analyzed for fentanyl concentration using liquid chromatography and tandem mass spectrometry (LC-MS/MS). For the plasma samples collected from the transdermal fentanyl solution portion of the study, a 1 mg/mL stock solution of fentanyl (Cerilliant®, Round Rock, TX, USA) dissolved in methanol was diluted in 50:50 methanol (Honeywell Burdick & Jackson®, Morristown, NJ, USA)/water (Milli-Q; Millipore Corp., Billerica, MA, USA) to a 25 μg/mL working solution. Control dog plasma (Bioreclamation Inc., Hicksville, NY, USA) was then serially diluted with the fentanyl working solution to create standard curve samples ranging from 0.1 to 100 ng/mL (10 calibration standards in duplicate) and quality control (QC) samples at concentrations of 0.1, 0.3, 3.5, 40, and 85 ng/mL. Additionally, 100 μg/mL stock solution of the internal standard (IS) fentanyl-d5 (Cerilliant®) dissolved in methanol was diluted in 50:50 methanol/water to a 200 ng/mL working solution. One hundred microliters each of sample, standard, QC, or control blank was aliquoted directly into a 96-well block, and 20 μL of the IS working solution was added to all wells except for the control blanks. Twenty microliters of 50:50 methanol/water were added to the control blanks instead of the IS working solution, and all samples were vortexed for 30 s. Four hundred microliters of 5% acetic acid (Mallinckrodt Baker, Phillipsburg, NJ, USA) in water was then added to each well, and the samples were vortexed again followed by centrifugation at 2000 g and 4 °C. Solid-phase extraction (SPE) was then proceeded using Bond Elut® 96 Certify, 50 mg sample extraction blocks (Varian Corp., Palo Alto, CA, USA), and a Tomtec Quadra-96 Model 320 (Tomtech, Hamden, CT, USA). Sample blocks were preconditioned with 1 mL methanol followed by 1 mL water. Samples were then transferred to the extraction blocks and gently pulled through the SPE plate with low vacuum, followed by a wash with 1 mL of 5% acetic acid in water and then by 1 mL of methanol. Samples were eluted through the extraction blocks with 600 μL of 2% ammonium hydroxide (EMD Biosciences, Darmstadt, Germany) in acetonitrile (Honeywell Burdick & Jackson®), into a new collection plate. Eluted samples were evaporated under nitrogen at 40–45 °C to dryness with about 35 F3/h flow and reconstituted with 200 μL of 1% formic acid (EMD Biosciences) in acetonitrile.
Reconstituted samples were quantified using an API 3000 triple quadrupole mass spectrometer equipped with TurboIonSpray™ interface (Applied BioSystems/MDS SCIEX, Foster City, CA, USA) with peak area integration conducted using Analyst Software v 1.4 (Applied BioSystems/MDS SCIEX) data acquisition system. High-performance liquid chromatography separation was achieved using LC-10AD pumps (Shimadzu Co., Kyoto, Japan) and a Thermo Betasil Silica-100 column (50 × 3 mm, 5 μm) (Thermo Fisher Scientific, Waltham, MA, USA) with the flow rate set at 0.5 mL/min. Mobile phase A consisted of 1% formic acid in water and mobile phase B consisted of 1% formic acid in acetonitrile. The mobile phase gradient started at 90% mobile phase B from 0.0 to 1.0 min, switched from 90 to 70% mobile phase B from 1.0 to 1.5 min, and switched back from 70 to 90% mobile phase B at 2.5 min. The total run time per sample was 2.7 min. The injection volume was 1 μL, and mass spectrometer detection was conducted using positive ionization mode and monitoring of the transitions 337.2 m/z188.3 m/z for fentanyl and 342.2 m/z188.3 m/z for the IS fentanyl-d5. Both analytes were typically retained on the column for 1.93 min. Standard curves were determined using linear regression with 1/x2 weighting using Watson v7.0.0.01 (Thermo Fisher Scientific), where x is the nominal sample concentration and had typical squared correlation coefficient (R2) values of 0.9987 (range of 0.9968–0.9996). All concentration calculations were based on the peak area ratios of fentanyl to the IS. The intra- and interassay precision was ≤7.3%, and the accuracy ranged from −13.5 to 5.9%. The lower limit of quantification (LLOQ) of the method was 0.100 ng/mL.