Validation of a liquid chromatography-tandem mass spectrometry method for quantification of glycopyrrolate in horse plasma.

The research paper describes the development and validation of a new method for detecting and quantifying glycopyrrolate – a drug used to treat various medical conditions such as chronic obstructive pulmonary disease – in horse plasma. The method utilizes ultra-high-performance liquid chromatography with heated electrospray ionization-tandem mass spectrometry.

Research Methods and Process

• The researchers developed a method using ultra-high performance liquid chromatography with heated electrospray ionization tandem mass spectrometry (UHPLC-HESI-MS-MS) to detect and quantify the presence of glycopyrrolate in horse plasma.
• They administered glycopyrrolate to Thoroughbred horses via oral and intravenous methods and collected plasma samples. The doses administered to the horses were clinically relevant.
• They utilized a deuterated analogue of the drug as an internal standard (IS) to calibrate the process.

Treatment of Samples

• They isolated glycopyrrolate and the deuterated analogue (GLY and GLY-d(3) respectively) from the plasma samples using a weak cation exchange and a simple solid-phase extraction technique.
• The analysis of the extracts was made on a reversed-phase UHPLC, which allows for rapid and efficient separation of compounds, on an Acquity™ column.
• The detection and quantification of precursor and product ions were performed using a type of mass spectrometer known as a triple-stage quadrupole (TSQ) instrument.

Results

• The method developed by the researchers exhibited a linear range from 0.125-25pg/mL, a lower limit of quantification of 0.125 pg/mL and a lower detection limit of 0.025 pg/mL, indicating the high sensitivity of this method for detecting and quantifying glycopyrrolate in horse plasma.
• The recovery rate of GLY ranged from 78% to 96%, suggesting a high extraction efficiency. In addition, the precision of the method showed a coefficient of variation (CV) ranging from 3.3-14.4% within batches and between batches, which suggests that the method is reliable.
• The study also revealed that glycopyrrolate is stable in horse plasma for up to 170 days when kept in -80°C conditions, through three freeze-thaw cycles, and for up to 48 hours after extraction in autosampler conditions of 20°C, providing evidence of sample stability under these conditions.

It should be stated that this new UHPLC-HESI-MS-MS method for detecting and quantifying glycopyrrolate in horse plasma has the rpotential to greatly improve our understanding of how this drug functions in equine care. However, subsequent studies with larger sample sizes and in different conditions should be subsequently performed to further substantiate this method.