Vancomycin hydrochloride (purity > 80%), teicoplanin (teicoplanins A2, purity ≥ 80%), methanol (HPLC grade) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Vancomycin hydrochloride (USP grade) for the rabbit experiments was from Hospira (Lake Forest, IL, USA). Formic acid (purity 98%) was from Acros Organics (Geel, Belgium), ammonium acetate (purity 97%) from Caledon Laboratories (Georgetown, ON, Canada). Pooled drug-free serum from female New Zealand white rabbits (protein content 63.0 ± 0.3 mg∙mL⁻¹ normalized to bovine serum albumin) was from the Centre for Comparative Medicine (Vancouver, BC, Canada). Ultra-pure water was prepared in our laboratory using a Milli-Q Synthesis system (Millipore, Billerica, MA, USA).

Animal experiments conducted in this study were approved by The University of British Columbia Animal Care Committee (Certificate number A10-0149) and adhered to the guide for the care and use of experimental animals [13] . New Zealand white rabbits (female, 1.75 - 5.5 kg) obtained from Charles River Laboratories (Wilmington, MA, USA) were used in the study. Rabbits were acclimatized for at least 7 days in group pens in a temperature-controlled room under a dark and light cycle of 12 h. They had access to typical diet (i.e., pellets, hay, and vegetables) and water ad libitum before and after the testing procedures. Five i.v. doses of VAN (vancomycin hydrochloride, diluted in sterile sodium chloride 0.9% solution) were administered to the animals (n = 5). One loading dose of 20 mg∙mL⁻¹ followed by four maintenance doses of 15 mg∙mL⁻¹ of VAN were administered with a dosing interval of 1.5 h. Blood samples were taken from the opposite ear using intraarterial catheters and transferred into serum collection tubes at the following time points: pre- dose at 0, 1.5, 3, 4.5, 6 h (trough concentration), and post-dose at 6.25, 6.5, 7, 7.5, 8, 8.5, 9 h. Clotted blood samples were centrifuged at 1000 × g for 10 min, serum kept at 4˚C until the end of the study period of 12 h, and then frozen at −20˚C until analysis.

The UHPLC-MS/MS system consisted of an Agilent 1290 Infinity Binary Pump, a 1290 Infinity Sampler, a 1290 Infinity Thermostat, and a 1290 Infinity Thermostatted Column Compartment (Agilent, Mississauga, ON, Canada) connected to an AB Sciex QTrap^® 5500 hybrid linear ion-trap triple quadrupole mass spectrometer equipped with a Turbo Spray source (AB Sciex, Concord, ON, Canada). The mass spectrometer was operated in positive ionization mode and data were acquired using the Analyst 1.5.2 software on a Microsoft Windows XP Professional operating platform. Chromatographic separation was achieved using a Waters Acquity UPLC BEH C₁₈ (1.7 µm, 2.1 × 100 mm) column that was protected by a Waters Acquity UPLC BEH C₁₈ VanGuard (1.7 µm, 2.1 × 5 mm) guard column. The columns were maintained at 35˚C and the autosampler tray temperature was maintained at 10˚C. Solvent A was water with 5 mM ammonium acetate (AA) and 0.1% formic acid (FA), and solvent B was methanol with 5 mM AA and 0.1% FA. The mobile phase initial conditions were solvent A (95%) and solvent B (5%) that was ramped to solvent A (5%) by 0.80 min held until 4.0 min and followed by an equilibration with solvent A (95%) and solvent B (5%) for 2 min. The flow rate was 0.2 mL∙min⁻¹, the injection volume was 5 µL with a total run time of 6.0 min. Samples were injected from a 384-well plate.

A stock solution of 1 mg∙mL⁻¹ VAN was prepared in 50% methanol/water (v/v) then further diluted to a second stock solution of 40 µg∙mL⁻¹ in rabbit serum, and stored on ice. From this solution, nine calibration standards were prepared to yield the concentrations of 0.1, 0.25, 0.5, 1, 5, 10, 20, 30 and 40 µg∙mL⁻¹ VAN. With each calibration curve, a zero sample (containing only IS) was prepared and the samples were stored at 4˚C. Quality control (QC) samples as QC-Low 0.3 µg∙mL⁻¹, QC-Mid 12 µg∙mL⁻¹, and QC-High 32 µg∙mL⁻¹ were also prepared and stored at 4˚C. TEI stock solution of 1 mg∙mL⁻¹ was prepared in 50% methanol/water (v/v) then further diluted to yield 600 ng∙mL⁻¹ solution in methanol. This solution was stored at −20˚C and used as the precipitation reagent. The final concentration of the IS in the analyzed samples was 250 ng∙mL⁻¹.

Ten µL of ice-cold precipitation reagent containing the 600 ng∙mL⁻¹ IS was pipetted into 0.5 mL tubes (Eppendorf, lowBind, Mississauga, ON, Canada) on ice. After adding 2 µL of calibration standards, QC samples, or study serum sample, the tubes were vortexed (1 s, ~3000 rpm), sonicated for 5 min, again vortexed (1 s, ~3000 rpm) and subsequently centrifuged for 2 min at 9000 rpm (Eppendorf, Mississauga, ON, Canada). On ice, 9.7 µL of the supernatant was transferred to a 384 well plate containing 10 µL of Milli-Q water in each well and mixed by repeated aspiration. After a sonication of approx. 10 s, the plate was covered with a silicone mat. A maximum of 31 samples were prepared in one batch and analyzed with UHPLC-MS/MS.

The precursor ions and the product ions of VAN and TEI were determined by the direct infusion of their approx. 1 µg∙mL⁻¹ solutions in 50% methanol/water (v/v) into the mass spectrometer. Using Turbo Spray positive ionization mode, the doubly charged precursor ions of VAN m/z 725.5, and m/z TEI 940.6 were

observed. The precursor ions were fragmented by collisionally activated dissociation and the product ions were generated. The mass spectrometry parameters were optimized to achieve the most intense signals. MRM transitions were created and VAN and TEI were quantitated using their quantifier MRM transitions, and their identity confirmed by monitoring their qualifier MRM transitions. The Waters Acquity UPLC BEH C₁₈ 1.7 µm 2.1 × 100 mm column, the mobile phase composition, and gradient programming provided a fast analysis with a run time of 6.0 min. Using the current experimental conditions, the retention times were for VAN, 2.27 min, and for TEI, 2.68 min. A representative chromatogram of VAN (5 µg∙mL⁻¹) and TEI (0.25 µg∙mL⁻¹) in rabbit serum is presented in Figure 1.

The developed method was successfully applied to measure serum VAN levels after the i.v. administration of five doses of VAN to rabbits (n = 5). The concentrations of VAN over time are shown in Figure 2. A loading dose of 20 mg∙kg⁻¹ VAN was administered at time zero followed by four maintenance doses of 15 mg∙kg⁻¹ VAN administered in every 1.5 h. Serum samples were taken shortly before administering the next dose (i.e., at trough levels). After the last dose of VAN, six subsequent samples were taken to investigate the pharmacokinetic profile of VAN in rabbit serum. Trough levels of VAN showed a constant pattern with serum concentrations of 20.9, 21.0, 21.5, and 23.9 µg∙mL⁻¹. The highest concentration was 53.5 µg∙mL⁻¹ after the last maintenance dose. In a previously unpublished study, a 20 mg∙kg⁻¹ VAN i.v. dose was administered to rabbits and the serum samples analyzed using PETINIA (Siemens Dimension Vista^® System, Siemens Canada, Oakville, ON, Canada). The VAN serum concentrations obtained with PETINIA were superimposed over the data obtained with the current UHPLC-MS/MS method and the results were comparable (Figure 2). Using the serum elimination phase data obtained with UHPLC-MS/MS, a non-compartmental pharmacokinetic analysis was performed (Phoenix WinNonlin 6.3, Certara USA Inc., Princeton, NJ, USA) and the results are presented in Table 2.

AUC_0-∞: Area under the concentration time curve from time zero to infinity, C₀: Concentration at time zero, determined through back extrapolation of the log linear transform of the first two sampling points post dose, λ_Z: elimination rate constant, MRT_0-∞: Mean residence time from time zero to infinity, Vss: Volume of distribution at steady-state.

The present study reports a novel UHPLC- MS/MS method that is quick, sensitive, accurate and precise for the quantitation of VAN requiring only 2 µL of sample. The matrix used was rabbit serum, but the method may be easily transferable to other matrices, such as dried blood spots or ISF where only low sample volumes are available. The applicability of the method in ISF would be particularly useful, because once the method is transferred and fully validated in ISF, it can be implemented in studies, such as TDM of VAN in ISF [12] . One shortcoming of the present study may be noted as the method was qualified, not fully validated. However, a full validation was not the aim of the current pilot study, but to qualify the method and apply it for PK studies in rabbits. The significance of the current study is that the method qualification results obtained may provide useful information to assess the feasibility of expanding the process to larger studies in the future where the method is fully validated in appropriate matrices, such as ISF, and implement it in studies on TDM of VAN in ISF.