Stability of nonsteroidal anti-inflammatory drugs in urine and solution: effects of degradation on analytical assessment.

Aims: Knowledge of optimal storage conditions of drugs is crucial for properly interpreting analytical assessments. Materials & methods: The current study aimed to investigate the stability of some nonsteroidal anti-inflammatory drugs using a validated method by gas chromatography (GC)-MS. For this propose, long-term, short-term and solution stability were investigated. Results: The analytes remained stable in the sample, similar to the working solution. The most affected substance over time in both matrix and working solution was phenylbutazone. The freeze-thaw cycle affected flunixin and carprofen, but diclofenac and vedaprofen changed only in the third cycle. In short-term stability, high-temperature conditions changed carprofen. Conclusion: The present study is a comprehensive assay for nonsteroidal anti-inflammatory drug stability and can be used as a reference for results assessment.

Comprehensive stability study of benzimidazole drug residues in standard solution, muscle and milk.

The performance criteria of analytical methods and the necessity for stability analysis to provide the accuracy of the results of the analyzed samples are explained in European Commission Decision 2021/808/EC and the guidance document SANTE/2021/11312. Detection of time-dependent changes in drug concentrations during storage or transport and re-analysis of samples are crucial to obtain high-quality results and reliable data. In this way, it allows toxicologists to interpret the analytical results accurately in drug analyses. The aim of this study was comprehensively to investigate the stability of benzimidazoles (levamisole hydrochloride, albendazole, albendazole-sulfone, albendazole-2-amino sulfone, albendazole sulfoxide, oxfendazole, 5-hydroxythiabendazole, triclabendazole, ketotriclabendazole, thiabendazole, flubendazole, fenbendazole sulfone) in working solutions, muscle and milk samples. For this purpose, long-term stability was evaluated over 6 months and under four different storage conditions (4 °C, -20 °C, 20 °C light and 20 °C dark) in the matrix. The influences of three freeze-thaw cycles, autosampler stability, and 60 min storage at 40 °C were investigated for short-term stability. Simultaneously, the stability of the working solutions were established over 6 months and under five different conditions (4 °C, -20 °C, -80 °C, 20 °C light, and 20 °C dark). It was found that working solutions can be stored at -80 °C or -20 °C, and it is appropriate to prepare the standard working solution freshly once a month. Storage of milk at 4 °C is suitable for some analytes (ABZ-SO, FBZ-SO2, FLUBZ, ABZ, ABZ-NH2-SO2) whereas for the muscle almost all substances were stable only at -20 °C. Some freeze-thaw and short-term stability changes were detected.

Cocktail drug usage and etofenamate detection in post-race equine urine sample: A case report.

A recent trend in the use of high-resolution accurate mass screening (HRAMS) for doping control testing in both human and animal sports has emerged owing to significant improvement in high-resolution mass spectrometry in terms of sensitivity, mass accuracy, mass resolution and mass stability. Several HRAMS methods have been reported for the detection of multidrug residues in human or equine urine. These improved analytical technologies have led to changes in the use of prohibited substances, and the administration of more than one substance at low concentrations as a "cocktail" has become one of the methods used to alter performance in racehorses. In one of horse urine samples transferred to the analytical laboratory in Turkey for analysis, 5-hydroxymethyl meloxicam (2.96 ng/ml), etofenamate (2.15 ng/ml), flufenamic acid (108.92 ng/ml) and cobalt (200 ng/ml) were detected. These findings reveal that more than one prohibited substance was used together as a cocktail to alter the racing performance at low doses. In this case report, flufenamic acid was detected as a metabolite of etofenamate along with the parent drug. This case study also supports the advantages of metabolite analysis for anti-doping laboratories.

Simultaneous quantification of caffeine and its main metabolites by gas chromatography mass spectrometry in horse urine.

Caffeine is a naturally occurring alkaloid and it is metabolized to paraxanthine, theophylline and theobromine. Analysis of caffeine and its metabolites is challenging since the metabolites theophylline and paraxanthine generate similar product and precursor ions. In this study, a new method was developed for the simultaneous analysis of caffeine, paraxanthine, theobromine and theophylline in horse urine using gas chromatography-mass spectrometry (GC-MS). Urine samples were treated using solid-phase extraction followed by the elution with dichloromethane-isopropanol (90:10) after the pH was adjusted to 6, and then derivatization with N-methyl-N-trimethylsilyl-trifluoroacetamide-1% trimethylchlorosilane before analysis with GC-MS. Sample preparation and derivatization steps were optimized and the method permitted elution all of these analytes within 13 min. The method was fully validated according to Commission Decision, 2002/657/EC guidelines. The calibration curves were linear with a correlation coefficient of >0.99. Precision and accuracy were well within the 15% acceptance range and the method was robust. The validation results demonstrated that the method is highly reproducible, easily applicable and selective. The method was applied to urine samples collected from racehorses to demonstrate its applicability.

Quantification and validation of nine nonsteroidal anti-inflammatory drugs in equine urine using gas chromatography-mass spectrometry for doping control.

Nonsteroidal anti-inflammatory drugs (NSAIDs) are commonly used in therapeutic doses in human and veterinary medicine for the treatment of inflammation, pain, and fever. A method for the simultaneous determination of nine NSAIDs, known as therapeutic prohibited substances, in equine urine was developed and fully validated according to the European Commission Decision 2002/657/EC and Association of Official Racing Chemists criteria. The validation was performed for naproxen, flunixin, ketoprofen, diclofenac, eltenac, meclofenamic acid, phenylbutazone, vedaprofen, and carprofen in equine urine in accordance with the International Screening Limits (ISL) regulated by International Federation of Horseracing Authorities. After basic hydrolysis, samples were extracted with a C18 cartridge using automated solid-phase extraction. Several derivatization reagents were investigated, and trimethylphenylammonium hydroxide/methanol (20/80, v/v) was selected. Analyses were carried out using gas chromatography-mass spectrometry with selected ion monitoring mode, but the method can be applied to a large number of analytes. The within-laboratory reproducibility was not more than 12.8% (≤15%), and mean relative recoveries ranged from 91.1% to 104.1% for inter-day and intra-day precision. The decision limits (CCα) and detection capabilities (CCß) were evaluated at concentrations near the ISL for each therapeutic substance. The validation results demonstrated that the method is highly reproducible, easily applicable, and suitable for the analysis of some NSAIDs in equine urine that have not been previously published. Finally, the method was also applied to known positive samples.

A review of chromatographic methods for ketamine and its metabolites norketamine and dehydronorketamine.

Ketamine has a synthetic, sedative, nonbarbiturate and fast-acting anaesthetic properties and it is commonly used in both humans and veterinary surgery. There are many analytical methods available for the qualitative and quantitative determination of ketamine and its metabolites. We have focused on sample pre-treatment and chromatographic techniques used since the year 2000 for the determination of ketamine and its metabolites in biological samples. Liquid and gas chromatography coupled with various detection techniques (mass spectrometry, ultraviolet or fluorescence detection) have been used in these publications. This review gives information on the implementation of methods for studying ketamine and its metabolites in various research applications. It could be useful in forensic sciences including doping control and also in the therapeutic drug monitoring of ketamine and norketamine in human and animal clinical surgery.