Quantitative ionspray liquid chromatographic/tandem mass spectrometric determination of reserpine in equine plasma.

This research presents a method for determining the amount of reserpine (a drug used in racehorses) in equine plasma using a liquid chromatography/tandem mass spectrometry technique. The study compares the efficiency of liquid-liquid extraction and solid-phase extraction for reserpine isolation from plasma, with a preferable outcome for liquid-liquid extraction.

Methodology and Extraction Comparison

• The main purpose of the research was to develop a method for determining the concentration of reserpine in horse plasma using ionspray liquid chromatography/tandem mass spectrometry (LC/MS/MS), a powerful technique which combines the physical separation capabilities of liquid chromatography (LC) with the mass analysis capabilities of mass spectrometry (MS).
• To isolate reserpine from the equine plasma, two extraction methods were employed: liquid-liquid extraction and solid-phase extraction. The performance of these two methods was compared. These extraction techniques are commonly used in biochemistry for separating mixtures.
• Rescinnamine, a molecule structurally related to reserpine, was used as the internal standard, meaning it was used as a known quantity to which other quantities can be compared in the experiments.

Calibration and Quantitation

• Two sets of calibration graphs were created: one set for reserpine extracted using liquid-liquid extraction and another set for reserpine extracted using solid-phase extraction. Calibration graphs are used to understand how the detector’s response changes with varying concentrations of the analyte (in this case, reserpine).
• The calibration graph for reserpine using liquid-liquid extraction was linear from 10 to 5000 pg ml-1. Meanwhile, for solid-phase extraction, linearity was observed from 100 to 5000 pg ml-1. This indicates that liquid-liquid extraction had a higher sensitivity, as it was able to detect reserpine at lower concentrations than solid-phase extraction.
• The lower limit of quantitation (LLQ) represented the lowest concentration of reserpine that could be reliably measured by the LC/MS/MS method. These were 50 pg ml-1 and 200 pg ml-1 for liquid-liquid and solid-phase extraction respectively, further indicating a higher sensitivity for the former.

Accuracy and Recovery Rates

• The accuracy of the assay is reflected by the intra-assay accuracy, and results of this research suggested neither method exceeded 13%. This implies a generally high level of agreement between observed and accepted true values.
• An important measure of extraction efficiency is the recovery rate, referring to the percentage of the original sample that is retrieved after extraction. Here, recovery rates were observed to be 68% for liquid-liquid and 58% for solid-phase extraction methods – giving the advantage to liquid-liquid extraction.