High throughput screening of sub-ppb levels of basic drugs in equine plasma by liquid chromatography-tandem mass spectrometry.

The study reports on a highly efficient method using liquid chromatography-tandem mass spectrometry (LC-MS-MS) for detecting low levels of various drugs in horse plasma. A broad spectrum of drugs can be detected within an impressively short time frame, with some limits of detection reaching as low as 0.5 parts per billion.

Methodology and Results

• The researchers’ main task was to develop a method to detect various drugs at very low levels in horse plasma. To do this, they used a method involving liquid chromatography-tandem mass spectrometry (LC-MS-MS).
• They tested for a total of 75 different drugs across a range of classes, including various blockers, agonists, antihypertensives, analgesics, antidepressants, and many more.
• To begin the process, the researchers first removed proteins from the plasma sample using trichloroacetic acid. They then extracted basic drugs through solid-phase extraction, using a Bond Elut Certify cartridge.
• The samples were analysed using LC-MS-MS, specifically in positive electrospray ionization (+ESI) and multiple reaction monitoring (MRM) mode.
• To accelerate the analysis time, they performed the liquid chromatography with a short C(8) column. This led to the entire process taking only 8 minutes, including the time for post-run and equilibration.
• The team found no interference from the matrices at the expected retention times of the targeted masses, meaning there was no distortion of the drug analysis due to other substances in the samples.
• The limits of detection (LoD) for over 60% of the drugs studied were at or below 25 parts per billion (pg/mL), and some even reached down to 0.5 pg/mL.
• The relative retention times (the time it takes a substance to go from the injection point to the detection point in chromatography) had an inter-day precision ranging from 0.01 to 0.54%.
• The relative peak area ratios (relative to the internal standard), a measure used to quantify the amount of a substance within a sample, had an inter-day precision ranging from 4 to 37%.
• Overall, the study demonstrated that the method was adequately precise to be used on a daily basis for qualitative identification.

Significance of the Research

• This method with its fast turnaround time and low limit of detection can help with more efficient drug screening in the equine industry.
• It can be used to test for a broad range of drug types, improving the comprehensive nature of drug monitoring.
• From a veterinary perspective, this method may help rule out the presence of certain drugs when diagnosing equine conditions.
• The precision of this method suggests it could be used for frequent, regular testing and would yield consistent results.