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Bioanalytics - Chemical

(T1330-01-03) LC/MS/MS Assays for Three Novel Oxazolidinones with Potential Anticonvulsant Effects and Their Application in Pharmacokinetic Studies in Rats

Tuesday, November 11, 2025
1:30 PM - 2:30 PM CT
Purpose: Oxazolidinone heterocycle is an identifiable scaffold that is clinically useful as antimicrobial, psychotropic and anticoagulant drugs. We identified a novel series of triazolyloxazolidinones (PH066, PH162, PH166) with anticonvulsant activities, showing protection on in vivo rat model of electrically-induced seizure (1). The objectives of these studies were to develop LC/MS/MS assays for these compounds, to investigate their stability and analyze the samples obtained from pharmacokinetic studies.

Methods: PH066, PH162, PH166 and the internal standard (IS), figure 1, were synthesized and characterized using previously published methodology (2-4). Waters Acquity UPLC H-Class Xevo TQD system equipped with an electrospray ionization probe operated in the positive ionization mode (+ESI), was utilized in these studies. The mobile phase consisted of 0.1% formic acid in water and 0.1% formic acid in acetonitrile in ratios of 60:40, 40:60, and 50:50, the flow rates were 0.2 ml/min, 0.2 ml/min and 0.25 ml/min for PH066, PH162, and PH166, respectively. Chromatographic separation was achieved using Acquity UPLC BEH C18 (50mmx2.1mm, 1.7 um) column and the column temperature was kept at 35°C, 22°C, and 26°C for PH066, PH162, and PH166, respectively. The source dependent parameters maintained for the analytes and IS were: cone gas flow, 0 l/h for the three compounds and 50 l/hr for the IS and the desolvation gas flow, 1000 l/h for PH066, 650 l/hr for PH162 and PH166, and 800 l/hr for the IS. The optimum values for compound dependent parameters like source desolvation temperature, capillary source voltage, cone voltage and collision energy were set at 500°C, 0.50kV, 46V and 80V for PH066; 200°C, 3.4kV, 64V and 42V for PH162; 450°C, 3.5kV, 50V and 30V for PH166; and 300°C, 3.5kV, 42V and 25V for the IS. Quantitative determination was performed using multiple reaction monitoring (MRM) scanning mode of the parent to daughter mass ions of m/z 441.2700→95.0300 (PH066), 510.2027→150.0203 (PH162), 508.200→347.1300 (PH166), and 297.2200 → 163.1700 (IS). Mass Lynx software was used for data processing and reporting. The calibration standards were prepared by spiking 200 µl blank rat plasma or brain homogenates with 100 µl of the standard solutions to produce the calibration standards in the range 5- 30 µg/ml in plasma and 100-5000 ng/ml in brain homogenates. The standard samples were mixed with 30 µl of 10 ng/ml IS solution, vortex, then extracted with 600 µl of diethyl ether. After centrifugation, the ether layer was transferred into another tube and evaporated under nitrogen. The residue was reconstituted in 300 µl mobile phase and 10 µl of the solution was injected onto the LC/MS/MS. When unknown plasma or brain homogenates samples were analyzed 200 µl of sample was mixed with 100 µl of blank solvent and 30 µl of the IS solution and the solution was extracted and processed similar to the standards. The developed methods were validated for linearity, accuracy, precision, and extraction efficiency. Also, the methods were utilized for determining the concentration of the compounds in plasma and brain tissue samples obtained at different time points after IP administration of 100 mg/kg of each of the compounds to male SD rats (120-150 g; age 21-25 days), obtained from Kuwait University Animal Resource Center.

Results: The developed methods were linear in the concentration range of 5-30 µg/ml for plasma and 100-5000 ng/ml for brain tissue homogenates. The assay precision was determined from the coefficient of variation (CV%) in the assay response for 3 sets of calibration standards. The range of the intra-day precision were 1.0-10.2%, 0.4-7.8%, and 5.9-20.3%, while that for the inter-day precision were 2.3-6.0%, 2.3-8.6%, and 1.0-17.1% for PH066, PH162 and PH166, respectively. The accuracy was determined from the percent of nominal concentration of 3 sets of standards. The range of the intra-day accuracy were 96.4-108.3%, 95.3-108%, and 95.8-107%, while that for the inter-day accuracy were 97.1-106%, 95.5-109%, and 96.9-118% for PH066, PH162, and PH166, respectively. The average extraction efficiency were 89.8%, 76.0% and 40.9, for PH066, PH162, and PH166, respectively. The average maximum plasma and brain tissue concentrations for PH066, PH162, and PH166, were 14.6, 15.0, and 18.1 µg/ml and 2.7, 2.0, and 1.99 µg/gm, respectively. The average plasma and brain tissue concentrations for PH066, PH162, and PH166, declined to 2.72, 7.70, and 12.8 µg/ml, and 0.90, 1.51, and 1.59 µg/gm, respectively after 180 min, as in figure 2. A rough estimate for the average brain to plasma distribution ratios for PH066, PH162, and PH166 were 0.186, 0.156, and 0.131, respectively.

Conclusion: Sensitive, specific, and accurate LC/MS/MS assays were developed for the determination of the novel oxazolidinone compounds; PH066, PH162 and PH166 in rat plasma and brain tissue homogenates. The validation results prove that the developed methods are suitable for quantitative determination of the concentrations of these compounds which can therefore be applied to investigate their stability and analyze the plasma and brain tissue samples obtained for pharmacokinetic studies.

References: 1- Qaddoumi MG, Phillips OA, and Kombian SB (2019). A novel oxazolidinone derivative PH192 demonstrates anticonvulsant activity in vivo in rats and mice. Eur. J. Pharm. Sci., 130 21-26 (2019).
2- Phillips OA, Udo EE, Ali AAM and Al-Hassawi N, 2003. Synthesis and antibacterial activity of 5-substituted oxazolidinones. Bioorg. Med. Chem. 11(1), 35-41.
3- Phillips OA, Udo EE, Ali AAM and Samuel S, 2005. Synthesis and antibacterial activity of new N-linked 5- triazolylmethyl oxazolidinones. Bioorg. Med. Chem. 13(12), 4113-4123.
4- Phillips OA, Udo EE, Abdel-Hamid ME and Varghese R, 2013. Synthesis and antibacterial activities of N-substituted-glycinyl 1H-1,2,3-triazolyl oxazolidinones. Europ. J. Med. Chem. 66:246-257.

Acknowledgements: This research is funded by research grant PP02/19 and the General Facilities Science (GF-S) grants GS01/03, GS01/05, and GS02/10 from Research Sector, Kuwait University.


Figure 1: Chemical structures of oxazolidinones and the internal standard


Figure 2: Plasma and brain concentration-time profiles for PH066, PH162 and PH166 after IP administration of 100 mg/kg.