Direct injection LC/ESI-MS horse urine analysis for the quantification and identification of threshold substances for doping control. I. Determination of hydrocortisone.

The research developed and tested two methods for identifying and measuring levels of the drug hydrocortisone in horse urine, as a way of detecting doping in equine sports. The methods used liquid chromatography and mass spectrometry, with minimal sample preparation.

Methodology

• The researchers introduced two distinct techniques to identify and quantify hydrocortisone in horse urine, leveraging the capabilities of ion trap mass spectrometry (IT-MS) and time-of-flight mass spectrometry (TOF-MS).
• A key benefit of these proposed methods is the minimal requirement of sample preparation. The researchers were able to directly inject diluted urine samples that are particle-free into both systems.
• Desonide was employed as the internal standard (IS), which helps in quantifying the amount of hydrocortisone.

Range and Matrix Effects

• The linear range, referring to the concentration at which the hydrocortisone could be accurately quantified, was established at 0.25-2.5 micrograms per milliliter for both methodologies.
• The scientists also assessed the effect of different matrices on the signal perception for hydrocortisone and the internal standard. Notably, the signal fluctuated significantly across various matrices.

Dealing with Matrix Effects

• To manage the matrix effects, absence of a blank matrix, and the high cost of the isotopically labeled internal standard, the researchers applied the standard additions calibration method.
• This research primarily explores the effectiveness of the standard additions approach in a method where neither nonisotopic internal standards nor extensive sample preparation is utilized and no blank matrix is available.

Results and Accuracy

• The variations of intra-and interday analysis of hydrocortisone in horse urine were found to be less than 10.2% and 5.4% respectively for the LC/IT-MS technique, and less than 8.4% and 4.4% for the LC/TOF-MS approach.
• The accuracy of the methods, depicted as bias percentage, was less than 9.7% for both methods, suggesting a high level of reliability.