1 m naoh

New Zealand white rabbits were treated with Bupaq^® (0.01–0.05 mg/kg buprenorphine, Richter Pharma, Wells, Austria) and Metacam^® (0.3 mg/kg meloxicam, Boehringer Ingelheim, Ingelheim am Rhein, Germany), and then were intubated and ventilated with isoflurane 2% (Ecuphar Veterinaria, Barcelona, Cataluña, Spain). After topical administration of double anesthetic Colicursi^® (0.1% tetracaine and 0.4% oxybuprocaine, Alcon, Geneva, Switzerland), the alkali-burn lesion was performed by applying a gauze soaked in 1 M NaOH (VWR, Radnor, Pennsylvania, USA) on the right eye (left eye served as healthy control) for 10 s. After that, eyes were rinsed with balanced salt solution (Alcon); the central epithelium was removed with eye spears and Tobrex^® (3 mg/mL tobramycin, Alcon) and Cetraflux^® (3 mg/mL ciprofloxacin, Salvat, Barcelona, cataluña, Spain) eye drops were administered before performing a tarsorrhaphy with 4–0 silk suture (Ethicon, Bridgewater, NJ, USA). Once a day, rabbits received subdermal injections of Metacam^®, Bupaq^® and Alsir^® (5 mg/kg enrofloxacin, Esteve, Barcelona, Cataluña, Spain) until treatment.

The CIMmultus OH monolith column (1 mL, 2-µm pores; BIA separations, Ajdovščina, Slovenia) was used for HIC. The sample was diluted 2-fold with 3-M K₂HPO₄ (Merck, Darmstadt, Germany), 2% glucose (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), pH 7.0, and then filtrated over a 0.45-µm mPES membrane filter. After equilibration with 1.5-M K₂HPO₄ and 1% glucose, pH 7.0, the sample was loaded onto the column with a flow rate of 2 mL/min. For elution, the flow rate was set to 3 mL/min, and the gradient was run in 20 column volumes (CV) from 0% to 100% 50-mM K₂HPO₄ and 1% glucose, pH 7.0. The columns were washed in the cleaning in place (CIP) mode with 1-M NaOH (VWR, Geldenaaksenbaan, Belgium) for reuse [23 ].