OPTIMIZING EXTRACTION AND CONCENTRATION METHODS FOR LC-MS/MS ANALYSIS OF VETERINARY PHARMACEUTICALS IN EQUINE-ASSOCIATED ENVIRONMENTAL MATRICES.

Overview

• This research developed and validated an analytical method to simultaneously detect and measure six veterinary pharmaceuticals in various environmental samples related to horses, such as soils, sediments, faeces, and water.
• The goal was to optimize extraction and concentration techniques for accurate quantification using LC-MS/MS, enabling effective environmental monitoring of these potentially toxic compounds.

Introduction and Objective

• Veterinary pharmaceuticals used in equine treatments can enter the environment through manure, runoff, or waste, potentially posing ecological risks.
• Accurate measurement of these drugs in environmental matrices is necessary for assessing contamination and impact.
• This study focused on six drugs: benzylpenicillin (an antibiotic), acepromazine (a sedative), flunixin (an anti-inflammatory), dexamethasone (a corticosteroid), ketamine (an anesthetic), and romifidine (a sedative).
• The objective was to develop a reliable method for simultaneous quantification of these compounds in solid matrices (soil, sediment, equine feces) and natural waters.

Method Development

• Three extraction methods were tested to isolate the pharmaceuticals from solid matrices:
• Ultrasonic Extraction (USE): Uses ultrasound waves to enhance solvent penetration and compound release.
• QuEChERS: A quick, easy, cheap, effective, rugged, and safe extraction technique commonly used for pesticides and pharmaceuticals.
• Accelerated Solvent Extraction (ASE): Uses high temperature and pressure to improve extraction efficiency.
• ASE was further optimized by adjusting temperature and the number of extraction cycles to improve recovery rates.
• Liquid Chromatography coupled with Tandem Mass Spectrometry (LC-MS/MS) was used for quantification due to its high sensitivity and selectivity.
• Both chromatographic and mass spectrometric parameters were refined for optimal detection of all six compounds.

Validation and Calibration

• The developed method was validated according to the French Standard NF T 90-210, ensuring compliance with recognized quality requirements.
• Calibration curves were established over a concentration range of 1 to 100 µg/L, showing strong linearity with correlation coefficients (r²) ≥ 0.97.
• Accuracy of measurement was assessed at 5, 10, and 50 µg/L levels, with results typically within ±30%, except for flunixin at the lowest concentrations where accuracy decreased.
• The limits of quantification (LOQ) for the pharmaceuticals ranged between 0.1 and 2 µg/L, indicating the method’s sensitivity for environmental monitoring purposes.

Results and Findings

• USE and the optimized ASE methods achieved the best extraction recoveries, varying between 20.6% and 115.3%, depending on the type of matrix (soil, sediment, feces).
• QuEChERS method was less effective compared to USE and ASE under the tested conditions.
• Optimized ASE was beneficial due to its adjustable parameters (temperature, cycles), facilitating improved extraction efficiency.
• This comprehensive approach enabled reliable monitoring of pharmaceutical residues originating from equine sources in complex environmental samples.

Significance and Applications

• The method allows for simultaneous detection of multiple veterinary drugs, reducing time and resource expenditure versus analyzing each compound separately.
• Environmental samples relevant to equine activities—such as soils near stables, sediments in runoff areas, and natural water bodies—can be effectively assessed for contamination.
• This contributes to better environmental risk assessment and management of pharmaceutical pollution linked to horse farming and related practices.
• The validated approach can support regulators, environmental scientists, and veterinary health professionals in monitoring and mitigating potential ecological impacts.