Rapid high-performance liquid chromatographic method for the determination of ketamine and its metabolite dehydronorketamine in equine serum.

The research describes a quick, straightforward, and sensitive procedure for identifying ketamine and its metabolite, dehydronorketamine, in horse serum using high-performance liquid chromatography.

Methodology

• The process begins with sample preparation, which comprises of mixing equal volumes of serum and acetonitrile-phosphoric acid (85%)-water in the ratio of 20:2:78 by volume. The sample is then passed through ultrafiltration using a 10,000 molecular mass cut-off filter.
• Next step was the separation of ketamine and dehydronorketamine from the ultrafiltrate. They were separated on a reversed-phase phenyl column and eluted with a methanol-acetonitrile-phosphate buffer solution.

Detection Process

• Ketamine and dehydronorketamine were then detected by a variable photometric UV-Vis detector set at 215 nm, and their presence are double-checked by a photodiode array detector operating in the 200-320 nm range.
• The limit of detection for ketamine was determined to be 5-15 ng/ml in equine serum, providing a sense of the sensitivity of the method.

Additional Verification

• Furthermore, the research incorporated a thermospray liquid chromatography-mass spectrometry examination to tentatively confirm the identity of the dehydronorketamine peak. This served as an additional safeguard to ensure accurate identification of ketamine and its metabolite within the serum samples.
• Through this method, the research team was able to quickly and efficiently measure the presence and concentration of ketamine and its metabolite in equine serum, demonstrating the efficacy of their method.