Measurement of 19-nortestosterone and its esters in equine plasma by high-performance liquid chromatography with tandem mass spectrometry.

This study presents a method using high-performance liquid chromatography with tandem mass spectrometry (HPLC/MS/MS) to detect 19-nortestosterone and its esters in horse plasma, which can trace the use of this anabolic steroid for up to 23 days after injection.

Research Method

• The research employed a method involving high-performance liquid chromatographic-tandem mass spectrometric (HPLC/MS/MS). It is known for its precision and its ability to detect and quantify specific compounds in a sample.
• It used an atmospheric pressure chemical ionization (APCI) interface in selected reaction monitoring (SRM) mode. APCI is a method used in mass spectrometry allowing ionization to occur at atmospheric pressure. SRM is an extra stage of mass selection that serves to increase the sensitivity and specificity of the analysis.
• Two internal standards were used in the experiment: 16,16, 17-(2)H(3)-19-nortestosterone for 19-nortestosterone and methenolone acetate for its esters. Researchers use an internal standard to improve and correct analytical measurements, reducing variations and providing a comparison base.
• The steroids were extracted from equine plasma samples with a mixture of diethyl ether/n-hexane. This process is necessary to separate the specific compounds to be later analyzed with the HPLC/MS/MS.

Results

• Quantification limits for the various steroid esters used in the study were determined. This helps researchers establish the minimum amount of substance which can be measured with reliable results.
• The recovery rates of most steroids were between 71.6% and 101%, except the decanoate, which only had a recovery rate of about 39.8%. Recovery rate shows how well a compound can be extracted for a given procedure. The lower recovery rates indicate that a significant amount of the substance could not be effectively extracted from the plasma sample.
• The responses were linear, with correlation coefficients ranging from 0.9897 to 0.9999 in a concentration range of 0.1 to 50.0 ng/mL for the steroids studied. This demonstrates that within the particular range, as the concentration increases, the measurement also increases proportionately.
• The method also successfully detected the presence of 19-nortestosterone in mare plasma samples for up to 23 days post-injection. This shows that the method could have real-world applications, detecting the use of this steroid in horses long after it has been administered.