Buccal pH was measured by placing a strip of paper pH (Hydrion 5–9, Micro Essential Laboratory, Brooklyn, NY, USA) in the cheek pouch prior to drug administration. A 22 gauge, 2.5 cm catheter was placed in a cephalic vein, and used for intravenous drug administration. For buccal administration, drug was deposited in a cheek pouch, and the cat's mouth was held closed for 30 sec to 1 min. Morphine sulfate (15 mg/mL; Morphine Sulfate, Baxter Healthcare, Deerfield, IL, USA), 0.2 mg/kg IV1 or 0.5 mg/kg buccal (mean ± SD total volume 0.14 ± 0.01 mL); methadone hydrochloride (10 mg/mL; Dolophine Hydrochloride, aaiPharma, Wilmington, NC, USA), 0.3 mg/kg IV or 0.75 mg/kg buccal (mean ± SD total volume 0.34 ± 0.02 mL); hydromorphone hydrochloride (2 mg/mL; Hydromorphone Hydrochloride, Hospira, Lake Forest, IL, USA), 0.1 mg/kg IV or 0.25 mg/kg buccal (mean ± SD total volume 0.59 ± .05 mL); or oxymorphone hydrochloride (1 mg/mL; Opana, Endo Pharmaceuticals, Malvern, PA, USA), 0.1 mg/kg IV or 0.25 mg/kg buccal (mean ± SD total volume 1.21 ± 0.13 mL) were administered. Commercially injectable drug solutions were used and were not altered prior to administration. The order of the route was randomized according to a computer-generated list, while the order of drug was always as above. This order had been randomly selected for the entire group of cats, but the drug order was not selected randomly within cats for logistical reasons. All cats received all treatments, with at least 2 weeks between successive treatments.
Blood samples (2 mL) were collected from the vascular access port prior to drug administration, and 1, 2, 4, 8, 15, 30, 60, 120, 240, and 480 min following intravenous drug administration, or 5, 10, 15, 20, 30, 45, 60, 120, 240, and 480 min following buccal drug administration. Blood was transferred to tubes containing EDTA, immediately placed on ice, and then centrifuged for 10 min at 3901 g at 4 °C within 10 min of collection. The plasma was separated and frozen at −20 °C until analysis for drug concentration.
Because the vascular access port had lost patency in one cat for both oxymorphone studies, a 20-gauge, 5-cm catheter was placed in a medial saphenous vein in that cat and used to sample blood. In addition, due to technical problems, data from 1 cat in the hydromorphone IV group were discarded and are therefore not included in the analysis.
Morphine, methadone, hydromorphone, and oxymorphone were quantitated in feline plasma by LC-MS2 analysis of protein-precipitated samples. The calibration standards were prepared as follows: stock solutions were made by dissolving 10.0 mg of morphine, methadone, hydromorphone, or oxymorphone standard in 10.0 mL of methanol. Working solutions were prepared by dilution of the stock solution with methanol to drug concentrations of 1000, 100, 10, and 1 nanogram per milliliter (ng/mL). Plasma calibrators were prepared by dilution of the working solution with feline drug-free plasma following evaporation of the methanol to concentrations of 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 150, 200, 300, 400, and 500 ng/ml (morphine, methadone, hydromorphone), or 0.05, 0.1, 0.25, 0.5, 1, 5, 10, 25, 50, 100, 150, and 200 ng/mL (oxymorphone). In addition, quality control samples (plasma fortified with analytes) at concentrations of 0.3, 35, and 160 ng/mL (morphine, methadone, hydromorphone), or 0.35, 35 and 120 ng/mL (oxymorphone) were routinely included as an additional check of accuracy. The concentration of morphine, methadone, hydromorphone, or oxymorphone in each sample was determined by the internal standard (morphine-D3, methadone-D3, hydromorphone-D3, or oxymorphone-D3, respectively) method using the peak area ratio and linear regression analysis. The response was linear within the calibration range and gave correlation coefficients (R2) of 0.99 or better.
Prior to analysis, the plasma samples, controls, and calibrators were fortified with the appropriate internal standard to a final concentration of 100 ng/mL and submitted to solid phase extraction (morphine, methadone, hydromorphone: C18 200 mg/3 mL, UCT, Bristol, PA, USA; oxymorphone: Cerex Polychrom ClinII SPE 3 cc 35 mg, SPEware, Baldwin Park, CA, USA). Quantitative analyses were performed on a mass spectrometer (TSQ Quantum Ultra triple quadrupole mass spectrometer, Thermo Scientific, San Jose, CA, USA) equipped with a heated electrospray ionization probe that was kept at 355 °C. All analyses were performed in the positive ionization mode with a spray voltage set at 5000 V. The sheath and auxiliary gas used was nitrogen at 45 and 10 arbitrary units, respectively. The system was operated in the selected reaction monitoring mode with argon as the collision gas at a pressure of 1.5 mTorr. The ion transfer tube was kept at 300 °C, while the scan time and width were 0.25 s and 0.1 m/z, respectively. Data were processed using LCQuan software version 2.6 (Thermo Scientific, San Jose, CA,USA). The mass spectrometer was coupled with liquid chromatography (1100 Agilent LC system; Agilent, Santa Clara, CA, USA). Chromatographic separation employed a column (ACE C18, 100 × 2.1 mm, 3 μm, column; Mac Mod, Chadds Ford, PA) and a linear gradient of acetonitrile in water with a constant 0.2% formic acid at a flow rate of 0.35 ml/min. The acetonitrile concentration was held at 1% for 0.5 min, ramped up to 90% over 8.5 min. The injection volumes were 10.0 μl.
Detection and quantitation employed full scan LC-MS/MS transitions of initial product ions for morphine, methadone, hydromorphone, and oxymorphone (mass to charge ratio (m/z) 286.2, 310.3, 286.2, and 302.1, respectively). The response for the major product ions for morphine (m/z, 128.0), methadone (m/z, 265.1, 105.0, 77.1, 91.1, and 219.0), hydromorphone (m/z, 185.0, 157.0, 128.0, and 152.0), and oxymorphone (m/z, 284.1, 227.0, 198.0, and 181.0) was plotted and peaks at the proper retention time integrated using LCQuan. The software was also used to generate calibration curves and quantitate these analytes in all samples.
The lower limit of quantitation was 0.1 ng/mL, 0.25 ng/mL, 0.1 ng/mL, and 0.35 ng/mL, and the upper limit of quantitation was 500 ng/mL, 500 ng/mL, 500 ng/mL, and 200 ng/mL for morphine, methadone, hydromorphone, and oxymorphone, respectively. Accuracy (% nominal concentration) and precision (% relative standard deviation) at 0.3, 35 and 160 ng/mL were 104 and 9%, 110 and 6%, and 97 and 3%, respectively, for morphine; 91 and 3%, 111 and 6%, and 101 and 2%, respectively, for methadone; 92 and 12%, 95 and 10%, and 103 and 6%, respectively, for hydromorphone; and at 0.35, 35, and 120 ng/mL, they were 109 and 3%, 102 and 2%, and 93 and 8%, respectively, for oxymorphone. Accuracy and precision were considered acceptable based on FDA guidelines for bioanalytical method validation.