A sensitive liquid chromatographic procedure for the analysis of camphor in equine urine and plasma.

This research article discusses a sensitive method for analyzing small amounts of camphor in horse urine and plasma. The method involves the use of liquid chromatography and UV detection, yielding detectable limits as low as 10ng/mL and 20ng/mL in urine and plasma respectively. Camphorated oil was given to standardbred mares topically and intratracheally and its presence and disappearance in the biological samples were measured over time.

Research Methodology

• To begin their research, the scientists applied camphorated oil to standardbred mares in two ways: topically (6g) and intratracheally (1g).
• This oil was made up of 20% camphor by weight.
• Following its administration, urine and plasma samples were collected from the horses at different points in time.
• The scientists then used diethyl ether to extract the camphor from these samples and made it detectable by converting it into a 2,4-dinitrophenylhydrazone derivative.
• They then implemented a liquid chromatographic procedure for the analysis. Specifically, they used a reverse phase high-performance liquid chromatography (HPLC) process, coupled with UV detection. The UV detector was set at 368.5 nm.
• The samples were eluted from a C18 column with the help of 82% acetonitrile in water.

Findings from the Research

• The method was highly sensitive, achieving detection limits of roughly 10 ng/mL for urine and about 20 ng/mL for plasma.
• The scientists found only trace amounts of camphor in urine, while measurable amounts appeared in plasma from 20 minutes to 12 hours post-administration for topically applied camphor.
• With intratracheal administration, camphor appeared immediately in both urine and plasma, and at higher concentrations. The concentrations of camphor ranged between 11.4 and 21.6 ng/mL in urine over the first two hours, and between 46.7 and 1650 ng/mL in plasma during the first hour.
• All traces of camphor disappeared from urine after about 4 hours and from plasma after 12 hours.
• A gas chromatography/mass spectrometry (GC/MS) analysis of hydrolyzed urine extracts indicated two metabolites of camphor: one was trans-isoketopinic acid, while the second was an unrecognized type of hydroxycamphor.