Direct injection horse urine analysis for the quantification and identification of threshold substances for doping control. III. Determination of salicylic acid by liquid chromatography/quadrupole time-of-flight mass spectrometry.

The research study developed and tested a fast and simple analytical method for detecting and measuring the concentration of salicylic acid, a substance banned in equine sports, in horse urine. The method utilizes liquid chromatography and mass spectrometry techniques, and proved with high accuracy and selectivity.

Method Development

• In this study, salicylic acid was detected and quantified by liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOF-MS). This technique combines the separating power of liquid chromatography (LC) with the detection power of mass spectrometry (MS).
• The specificity of this method was increased by the use of an internal standard (4-methylsalicylic acid) which was added to the urine samples before they were introduced to the LC/QTOF-MS system.
• Another aspect of the method’s design was the need for sample dilution. To prepare for the LC/QTOF-MS analysis, the horse urine samples were diluted 900 times.
• The run time for this method is only 2.0 minutes, emphasizing its rapid utility.

Quantification and Identification

• The salicylic acid was detected using electrospray ionization (ESI) in negative mode with full scan acquisition mode and product ion scan mode for its quantification and identification, respectively.
• The tested linear range for salicylic acid was from 2.5 to 50 micrograms per mL following a 100-fold sample dilution. The linearity of an analytical method refers to the ability to obtain test results that are directly proportional to the concentration of the analyte in the sample.

Validation of the Method

• The relative standard deviations of deterministic (intra-assay) and random (inter-assay) variations of salicylic acid in horse urine were less than 2.5% and 2.8%, suggesting excellent repeatability and reproducibility of the method.
• The overall accuracy measured in relative percentage error was less than 3.3% indicating a high precision of the method.
• Finally, the method was applied to two real samples that were found to be positive for salicylic acid, further confirming the method’s accuracy and selectivity in a real-world scenario.