Quantification of several acidic drugs in equine serum using LC-MS-MS.

This research article discusses the development and validation of a method for simultaneous detection and quantification of various drugs commonly used in racehorses. The drugs can be determined using a specific volume of horse serum, and various liquid extraction methods followed by liquid chromatography tandem mass spectrometry.

Detection and Quantification Method

• The researchers have developed a method for the detection and quantification of drugs often administered to racehorses in therapeutic or illegal cases. These drugs include furosemide, flunixin, ketoprofen, phenylbutazone, and oxyphenbutazone.
• The detection and quantification method uses a specified volume (500 µL) of horse serum. The serum is subjected to liquid extraction using a specific mixture of chemical solvents: diethyl ether, hexanes, and dichloromethane.
• Following this extraction, the samples undergo liquid chromatography tandem mass spectrometry. This is a two-step process that first separates components in the serum sample (liquid chromatography) and then identifies and quantifies individual components (mass spectrometry).

Method Validation and Accuracy

• To test the validity and accuracy of this method, the researchers performed inter- and intraday precision and accuracy tests. These tests assess whether similar results can be reliably achieved on different days (interday) and during the same day (intraday).
• Additionally, the method was tested for stability. The researchers examined bench-top, freeze-thaw, processed, and long-term storage stability—looking at how the compounds in the serum samples were affected by the testing process and the duration of sample storage.
• According to the results, all compounds showed acceptable breakdown levels of less than 15% in all forms of stability testing.

Expression of Uncertainty and Confidence

• Uncertainty levels were calculated using a simplified version of the Guide to the Expression of Uncertainty in Measurement approach. This method allows the researchers to express how certain they are in their measurements of drug levels in the samples.
• The uncertainty level for all drugs was found to be less than 30% at a confidence level of 95%. This means that the researchers are 95% certain that their measurements are accurate within a margin of error of less than 30%.
• The method was found to be both robust and accurate for all tested drugs, indicating its potential application in future research or drug testing procedures.