Automated liquid chromatography-tandem mass spectrometry method for the analysis of firocoxib in urine and plasma from horse and dog.

The research paper is about the development and validation of an efficient, sensitive, and precise method for using liquid chromatography-tandem mass spectrometry for the quantitative analysis of a drug called firocoxib in the urine and plasma of horses and dogs.

Methodology:

• The method developed makes use of liquid chromatography, which is a technique used for separating a mixture into its individual components, and tandem mass spectrometry, which is an analytical technique used to determine the mass-to-charge ratio of ions.
• Sample preparation was conducted using 96-well solid phase extraction (SPE) plates with a BIOMEK 2000 Laboratory Automated Workstation. This utilized automation to reduce manual labor, and increased accuracy and speed.
• Chromatographic separation, which separates the mixture into individual components, was used to ensure firocoxib was distinct from any other materials in the matrix. This was achieved by using isocratic reversed phase chromatography on a PHENOMENEX LUNA Phenyl-Hexyl column.
• The mobile phase comprised 45% acetonitrile and 55% of a 2 mM ammonium formate buffer to help facilitate the separation.

Results:

• The results of the study indicated the method was accurate (with accuracy ranging between 88-107%) and precise (with a coefficient of variation less than 12.2%) both within and between sets.
• The extraction efficiencies achieved were also found to be high (more than 93%), indicating the method was very effective at extracting firocoxib.
• Ionization efficiencies were also found to be high (more than 72%), showing the method could effectively ionize the substance for the purposes of mass spectrometry.
• Stability and ruggedness testing was also performed to ensure the long-term usability and durability of the method.

Implications:

• The method is capable of being used for pharmacokinetic studies, which look into how a drug moves within a body, and also for drug monitoring and screening.
• The research also reports it as the first LC-MS/MS method for the quantitation of firocoxib in plasma with a LLOQ (Lower Limit of Quantitation) of 1 ng/mL, which is a 25-fold improvement in sensitivity over the HPLC-UV method.
• This is also the first quantitative method for firocoxib in urine with a LLOQ of 5 ng/mL.