Validated LC-MS-MS Method for Simultaneous Analysis of 17 Barbiturates in Horse Plasma for Doping Control.

This study presents a quick, sensitive and accurate method to screen and confirm the presence of 17 different types of barbiturates in horse blood, which can be used for doping control in horse racing.

Research Method

• The research introduces a method to simultaneously screen, quantify, and confirm 17 different types of barbiturates (types of drugs) in horse plasma (a component of blood).
• This method utilizes liquid chromatography-tandem mass spectrometry (LC-MS-MS), a technique that identifies and quantifies the amount of a substance in a sample. In this context, it’s being used to identify and quantify the barbiturates in horse plasma.
• The barbiturate substances were first extracted from the blood plasma using a process of liquid-liquid extraction. Methyl tert-butyl ether was used in this extraction process.
• Subsequently, the analytes were distinguished on a C18 column. This is a common type of column used in liquid chromatography, a technique that separates a mixture into its individual components.

Measurement and Analysis

• The researchers used negative electrospray ionization mode during the analysis. This is a technique that helps detect and measure compounds with high molecular weights.
• Utilizing multiple-reaction monitoring, the researchers performed screening and quantifying of the analytes.
• They confirmed the presence of the analytes by comparing the ion intensity ratio.

Efficiency and Accuracy

• The detection, quantification and confirmation limits were very low – 0.003-ng/mL to 1 ng/mL; 0.01-ng/mL to 2.5 ng/mL and 0.02-ng/mL to 5 ng/mL, respectively. This makes the method highly sensitive, able to detect even minute amounts of barbiturate in the plasma.
• The method demonstrated a linear dynamic range of 0.1-ng/mL to 100 ng/mL, meaning it remained accurate across this range of concentrations.
• The precision and accuracy of the method proved to be high, both for intra-day (1.6%-8.6% precision and 96%-106% accuracy) and inter-day (2.6%-8.9% precision and 96%-106% accuracy) assays. This consistency makes it reliable for multiple tests done within and across different days.
• Furthermore, the entire analysis of the 17 analytes was completed within 7 minutes, indicating the method is both rapid and efficient.

This robust technique provides a fast, simple, and reliable method for doping control in horse racing, with the capability to reliably detect very low levels of barbiturates in horse plasma.