A Validated Method for the Determination of Firocoxib in Equine Tissues

Research Article

Austin Chromatogr. 2015; 2(3): 1036.

A Validated Method for the Determination of Firocoxib in Equine Tissues

Cox S¹*, Hayes J¹, Gordon K¹, White M¹ and Macpherson M²

¹Department of Biomedical and Diagnostic Sciences, University of Tennessee, USA

²Large Animal Clinical Sciences, University of Florida, USA

*Corresponding author:Cox S, Department of Biomedical and Diagnostic Sciences, University of Tennessee, College of Veterinary Medicine 2407 River Drive, Knoxville, TN 37996, USA

Received: July 08, 2015; Accepted: September 14, 2015; Published: September 21, 2015

Abstract

A new reversed-phase High Performance Liquid Chromatography (HPLC) method was developed and validated for the determination of firocoxib in equine tissue. Firocoxib was detected by ultraviolet detection at 290nm after undergoing a liquid extraction using ethyl acetate: hexane (40:60) and separation on a Sunfire C18 column. The mobile phase consisted of water with 0.025% trifluoroacetic acid and acetonitrile (50:50), with a flow rate 1.1 mL/min. A concentration range of 5-1500ng/gm produced a linear curve with r²>0.99. The lower limit of quantification was 5ng/gm. The intra and inter assay variability was <10% and the average recovery was 97%.

Keywords: Firocoxib; HPLC; Tissue; UV detection

Introduction

Firocoxib, 3-(cyclopropylmethoxy)-4-(4-(methyl-sulfonyl) phenyl)-5, 5-dimethylfuranone, is a coxib-class Non-Steroidal Anti- Inflammatory Drug (NSAID) approved for use in horses and dogs [1]. It is a Cyclo-Oxygenase (COX) inhibitor that is highly specific for COX-2 and has little effect on COX-1 enzymes. Cyclo-oxygenase plays an important role in the production of prostaglandins and thromboxanes [1]. The inhibition of COX blocks prostaglandin synthesis and prostaglandin-mediated effects including inflammation.

Placentitis is well established as a significant cause of pregnancy loss in mares. In a study looking at 1800 mares during a 24-year period it was found that 64% of the pregnancy losses were due to placentitis [2]. Data in horses support the addition of an anti-inflammatory agent to the antimicrobial therapy for prevention of preterm delivery after placental infection [3]. The specificity of firocoxib for COX-2 inhibition, and its potential anti-inflammatory effects, has made it a desirable drug for the treatment of placentitis. To validate the use of firocoxib in mares with placentitis, it is critical to determine if the drug attains therapeutic concentrations in target tissues.

There are methods for detecting firocoxib in plasma, urine and milk [1,4-8]. However, a literature search revealed no published methods for the determination of firocoxib in tissue. A simple and reliable method to determine the concentration of firocoxib in equine tissue has been developed in the Pharmacology laboratory at the University of Tennessee using a liquid extraction technique, ultraviolet detection, and reverse phase HPLC.

Materials and Methods

Equipment

The chromatography system consisted of a 2695 separation module and a 2487 ultraviolet detector (Waters, Milford, MA). Empower software (Waters) was used for data acquisition and processing. Firocoxib separation was achieved on a Waters Sunfire C18 (4.6mm x 150 mm, 3.5 μm) column preceded by a 3.5μm Sunfire C18 guard column (20 mm x 3.9 mm).

Chromatographic conditions

The mobile phase was an isocratic mixture of water with 0.025% trifluoroacetic acid and acetonitrile (50:50, v/v). All solutions were filtered through a 0.22 μm filter and degassed before their use. The flow rate was 1.1 mL/min, and the column temperature was ambient (22°C).Ultraviolet detection occurred at 290 nm.

Preparation of standard solutions

Firocoxib was a gift from Merial (Duluth, GA). Deracoxib, the internal standard, was a gift from Novartis (East Hanover, NJ). All reagent grade chemicals and solvents were purchased from Fisher Scientific (Pittsburg, PA). Water was obtained from a Barnstead Nanopure Infinity (Dubuque, IA) ultrapure water system. Firocoxib and deracoxib (internal standard) were dissolved in methanol to produce stock concentrations of 100 μg/mL. Dilutions of firocoxib were prepared in methanol to produce 10, 1 and 0.1 μg/mL working stock solutions. Standards were aliquoted into 2-mL vials to prevent evaporation and cross contamination. All solutions were protected from light in bottles wrapped in aluminum foil and stored at 4°C. By comparing standard areas over time, it was determined that they were stable for a minimum of 6 months.

Sample preparation

Previously frozen equine tissue was thawed then 0.5 gm weighed and placed into Potter-Elvehjem glass homogenizers with a PTFE pestle. Fifteen microliters of internal standard was added (deracoxib, 100 μg/mL) followed by 5 mL of ethyl acetate: hexane (40:60). The tissue was ground by hand for 3 min. The tissue and extraction solution were transferred to a 15mL glass screw top tube. The homogenizer tube and probe were rinsed with 1mL of extraction solution and the rinse added to the screw top tube. The tubes were vortexed for 60 seconds then centrifuged for 20 minutes at 1000xg. The supernatant was removed to a clean 16x100 glass tube and evaporated under a stream of nitrogen. They were re-dissolved in 300 μL of mobile phase (50:50), loaded into HPLC vials, and 50 μL were injected into the HPLC system.

Preparation of calibration standards

For preparation of calibration standards and quality control samples, appropriate volumes of stock solutions were added to untreated tissue. The final concentrations for our tissue calibration standard curve were 5, 10, 25, 50, 100, 250, 500, 1000, and 1500 ng/ gm with quality control standards of 7.5, 175, 750, and 1250 ng/gm. Calibration standards and control samples were treated the same as test samples (Figure 1).