Drug analysis

Deionized water was purified with a Barnstead™ NANOpure™ water system (Thermo Fisher Scientific, Waltham, MA) which provided 18.2 MΩ-cm water. Methanol and acetonitrile were OmniSolv^® from MilliporeSigma (Burlington, MA). Formic acid was Optima^Ⓡ LC/MS (Fisher Scientific, Fair Lawn, NJ). Lidocaine was from Sigma Aldrich (Milwaukee, WI) and deuterium-labeled lidocaine (lidocaine-d₁₀) was from Cayman Chemical (Ann Arbor, MI). EZ Flow^® nylon membranes (0.22 μm pores) used to filter mobile phases were from VWR (Avantor, Radnor, PA). Blank horse plasma and urine for calibration standards were collected from one of the study horses before the study began.

Plasma and BALF samples were analyzed using a high-performance liquid chromatography (HPLC) system consisting of an HP1100 HPLC (Agilent, Santa Clara, CA) coupled to an AB/SCIEX API 4000 triple quadrupole mass spectrometer (AB/SCIEX, Framingham, MA). The column used was a Kinetex^Ⓡ PFP (100 x 2.1 mm, 2.6 μm particles, 100 Å pores, Phenomenex, Torrance, CA) and the mobile phase consisted of water/acetonitrile (ACN), 85:15 (v/v) with 0.1% formic acid added to each solvent. The flow rate was 150 μL/min and lidocaine and the internal standard, lidocaine-d₁₀ eluted at ~3.4 min.

The mass spectrometer was used with TurboIonSpray™ positive ionization, in multiple reaction monitoring mode. Parameters optimized for lidocaine and lidocaine-d₁₀ were curtain gas 20, ion spray voltage (IS) 4000V, probe temperature 550°C, nebulizer gas (GS1) 50, auxiliary gas (GS2) 50, entrance potential 10V and collision energy 27V. The declustering and collision cell exit potentials were 60V and 13.7V for lidocaine and 52V and 15.3V for lidocaine-d₁₀. Transitions monitored were 235.3/86.3 for lidocaine and 245.3/96.3 for lidocaine-d₁₀. Control of the HPLC system and mass spectrometer and analysis of data were with Analyst 1.6.2 software.

Due to the high concentration of the condensate, samples were analyzed by an HPLC/UV method with a Waters e2695 separations module and a 2489 UV/VIS detector (Waters, Milford, MA). The column used was a Phenomenex Luna C₁₈(2) (150 x 4.6 mm, 5 μm particles, 100Å pores) with a mobile phase of 20 mM KH₂PO₄ (pH 5.5)/ACN, 65:35 (v/v), with a flow rate of 1.2 mL/min and detection at 230 nm. The retention time of lidocaine with this method was ~7.7 min.

A stock solution of lidocaine was prepared in methanol at 1 mg/mL. A 10 μL aliquot of this solution was added to 90 μL of blank horse plasma or urine which had been analyzed to ensure that no lidocaine was present. From this, dilutions were made in this plasma and urine to appropriate ranges of concentration for calibration. Standards for analysis of BALF samples were prepared in blank BALF.

Aliquots of plasma, urine, and BALF were diluted appropriately with ACN containing lidocaine-d₁₀ as an internal standard. These mixtures were centrifuged at 16,162-g for 10 min. Aliquots of the supernatants were diluted with H₂O/ACN in proportions to produce a final solvent ratio of 1:1 H₂O/ACN in samples to be injected. These mixtures were centrifuged at 16,162-g for 10 min and 2 μL aliquots of the supernatant were injected into the HPLC system. Standards in the appropriate matrix were prepared at an appropriate range of concentrations to analyze samples collected at different time points and were prepared identically to samples. The lower limit of quantification (LLOQ) was established at 2 ng/mL and the limit of detection (LOD) was 0.5 ng/mL for both lidocaine and MEGX.