Establishing Plasma Concentration Methodology for Trimethoprim/Sulfamethoxazole: Application in Therapeutic Drug Monitoring of Critically Ill Patients

This study aimed to develop a high-throughput and high-sensitivity method for simultaneously determining trimethoprim (TMP), sulfamethoxazole (SMX), and its metabolite acetyl-N-sulfamethoxazole (NSMX) in critically ill patients' plasma. The samples were precipitated using methanol and then purified using the Oasis PRiME HLB solid-phase extraction column. The mobile phase consisted of a mixture of 40% 0.1% formic acid aqueous solution and 60% acetonitrile, with a flow rate of 0.35 mL/min. Analyses were conducted using UPLC-Q-Exactive Plus Orbitrap mass spectrometry in positive ion mode. With only 50 μL of plasma required for accurate quantification, this method was applicable for therapeutic drug monitoring (TDM). Validation demonstrated precision and accuracy within 15% of nominal values. The samples remained stable for 12 h at 4°C and -20°C and for 15 days at -20°C and -80°C, which ensured the reliability of the method during sample storage and handling under different environmental conditions. The method can be applied for concentration monitoring and pharmacokinetic studies, enabling TDM and dose adjustments for critically ill patients. This approach will enhance patient outcomes and reduce medical resource waste, offering reliable sensitivity and specificity to support precision medicine in critical care.