Twelve healthy, university-owned, treadmill-exercised Thoroughbred research horses (5 mares and 7 geldings; 4–7 years; weight: 457–576 kg) were included in the study. No medications for were administered for a minimum of two weeks prior to drug administration. Prior to inclusion, a physical examination, complete blood count (CBC) and a serum biochemistry panel were performed for each horse. The CBC and biochemistry panel were performed by the Clinical Pathology Laboratory of the William R. Pritchard Veterinary Medical Teaching Hospital of the University of California, Davis. The study was approved by the University of California at Davis’ Institutional Animal Care and Use Committee of the (IACUC #22,110).
Instrumentation and drug administration
For sample collection, a 14- gauge intravenous catheter was placed in one external jugular vein, using aseptic technique, prior to administration of drug. Horses received a single subcutaneous injection of 1 mL of 2% lidocaine HCl (20 mg/horse) with Epinephrine 1:100,000 (Med-vet International, Mettawa, IL) over the palmar digital nerve. The palmar digital neurovascular bundle was palpated immediately proximal to the ungular cartilages of the distal phalanx. The area was subsequently prepared for injection using gauze swab saturated with 70% isopropyl alcohol. A 25-gauge hypodermic needle was placed percutaneously in a proximal to distal direction axial to the neurovascular bundle, immediately adjacent to the palmar digital nerve. The syringe containing the lidocaine and epinephrine was then attached to the hub of the needle and the drug combination deposited subcutaneously.
Blood samples were collected at time 0 (prior to drug administration) and at 15, 30, and 45 min, and 1, 1.5, 2, 3, 4, 5, 6, 8, 12, 18, 24 and 30 h post administration for determination of lidocaine concentration. Samples were collected into blood tubes devoid of anti-coagulant (red top) and were allowed to sit at room temperature for approximately 20 min, before centrifugation at 3000 x g. Serum was immediately transferred to storage cryovials and stored at -20◦ C (4 weeks) until analysis for determination of lidocaine concentrations.
Urine samples were collected at 4, 24 and 48 h post drug administration by free catch. Samples were stored at -20◦ C (4 weeks) until analyzed for determination of lidocaine concentrations.
Determination of drug and metabolite concentrations
Lidocaine (Cerilliant, Round Rock, TX), 3-hydroxylidocaine (Frontier BioPharm; Richmond, KY), GX (Toronto Research Chemicals, Toronto, ON) and MEGX (Cerilliant, Round Rock, TX) were combined into one working solution. Serum calibrators (0.005 to 20 ng/mL) were prepared by dilution of the working standard solutions with drug free equine serum. Negative control and calibration curve samples were prepared fresh for each quantitative assay. Quality control samples (drug free equine serum containing analytes at three concentrations within the standard curve) were included with each sample set as an additional check of accuracy.
Prior to analysis, 0.25 mL of serum was diluted with 0.1 mL of water containing 25 ng/mL of d10-lidocaine internal standard (Toronto Research Chemicals, Toronto, ON) and 0.2 mL of β-glucuronidase enzyme, (Sigma Aldrich, St Louis, MO) at 10,000 Units/mL in pH 5, 1.6 M acetate buffer. The pH of the samples was adjusted to 5.0 ± 0.5 with 2 N NaOH or 2 N HCl, as necessary, and heated in a water bath at 37 °C for 2 h. After cooling to room temperature, 0.2 mL of 0.5 N NaOH was added to adjust the pH to 10.0 ± 0.5 with. Methyl tert-butyl ether (MTBE; 3mL) was added to each serum sample, and the samples were mixed by rotation for 20 min at 40 revolutions per minute. Samples were then centrifuged at 3300 rpm (2260 g) for 5 min at 4 °C. The top organic layer transferred to glass tubes and samples were dried under nitrogen and dissolved in 120 uL of 5% acetonitrile in water with 0.2% formic acid. The sample (30 uL ) was injected into the LC-MS/MS system.
Liquid chromatography-tandem mass spectrometry using positive heated electrospray ionization (HESI(+)) was used to measure lidocaine and metabolite concentrations. A TSQ Altis triple quadrupole mass spectrometer coupled with a Vanquish liquid chromatography system (Thermo Scientific, San Jose, CA) was used for quantitative analysis. The spray voltage was 3500 V, the vaporizer temperature 350ºC, and the sheath and auxiliary gas were 50 and 10 respectively (arbitrary units). To optimize product masses and collision energies of each analyte standards were infused into the TSQ Altis. An ACE 3 C18 10 cm x 2.1 mm 3 μm column (Mac-Mod Analytical, Chadds Ford, PA) and a linear gradient of ACN in water with a constant 0.2% formic acid at a flow rate of 0.35 ml/min was used for chromatography. Initially the ACN concentration was held at 3% for 0.5 min, then ramped to 90% over 6.0 min and held at that concentration for 0.2 min, before re-equilibrating at initial conditions for 4.6 min.
Selective reaction monitoring (SRM) of initial precursor ion for lidocaine (mass to charge ratio 235.2 (m/z)), 3-hydroxylidocaine (mass to charge ratio 251.1 (m/z)), GX (178.9 (m/z)), MEGX (207 (m/z)), and the internal standard d10-lidocaine (245.2 (m/z)). The response for the product ions for lidocaine (m/z 30.2, 58.1, 86.1), 3-hydroxylidocaine (m/z 30.3, 58.1, 86.1), GX (m/z 122), MEGX (m/z 58) and the internal standard d10-lidocaine (m/z 64.1, 96.2) were plotted and peaks at the proper retention time integrated using Quanbrowser software (Thermo Scientific). Generation of calibration curves and quantitation of analytes by linear regression analysis was conducted using Quanbrowser software. For all calibration curve, a weighting factor of 1/X was used.
Working solutions for urine analysis were the same as described above for serum. Calibrators (0.05 to 1,500 ng/mL) were prepared by dilution of the working standard solutions with drug free equine urine. Urine calibration curves and negative control samples were prepared fresh for each quantitative assay. As an additional check of accuracy, quality control samples were included with each sample. The extraction method for the urine was the same as the serum except the sample size for urine was 0.5 mL, samples were hydrolyzed at 65 °C with 99 min of sonication, and after hydrolysis the urine was adjusted to pH 9. Methyl tert-butyl ether (5 mL) was used for extraction, samples were redissolved in 150 uL of 5% acetonitrile in water, with 0.2% formic acid and 20 uL into the LC-MS/MS system. Detection and quantification in urine was the same as for the serum.
Pharmacokinetic analyses of lidocaine and metabolite serum concentration data were conducted using non-compartmental analysis (NCA) and a commercially available pharmacokinetic software program (Phoenix Winnonlin v8.3, Certara, Princeton, NJ). Maximum concentrations (Cmax) and the time of maximum concentration (Tmax) were determined directly from the concentration data.
After performing NCA, pharmacokinetic modeling using a nonlinear mixed effect modeling (NLME) approach with the Phoenix NLME software program and concentration data was conducted. Two and three-compartment models with saturable and linear absorption, with and without a lag time and with different error models were assessed using lidocaine concentration data. Following selection of the best fit model for lidocaine, the metabolites were added to attempt to generate a parent-metabolite pharmacokinetic model. The goodness of fit of the models was determined by visual analysis of observed compared with predicted concentration graphs and residual plots, as well as CV, Akaike Information Criterion, and % CV of parameter estimates.