Simultaneous Monitoring of 3 Antiviral Drugs in Serum Using Liquid Chromatography-Tandem Mass Spectrometry: Full Validation and Clinical Application

Abstract

Background: Patients undergoing solid organ and hematopoietic stem cell transplantation are at risk of opportunistic pathogenic infections that increase morbidity and mortality. Universal antiviral prophylaxis improves the outcomes in this context. Therapeutic drug monitoring of antiviral drugs is not universally recommended but may be necessary in certain complex or polymorbid patients. The authors aimed to develop and validate a high-performance liquid chromatography-tandem mass spectrometry method to simultaneously quantify ganciclovir, acyclovir, and letermovir in human serum.

Methods: A stable isotopically labeled internal standard was used for each antiviral drug. Compounds were extracted by protein precipitation, evaporation, and reconstitution in an aqueous mobile phase. Samples were analyzed using reverse-phase chromatography with subsequent detection by electrospray ionization in the positive ion mode on a triple quadrupole mass spectrometer (run time: 6.5 minutes).

Results: Analytical curves for ganciclovir and acyclovir exhibited linearity within 0.1-25 mg/L (R2 > 0.993), whereas for letermovir, the linear range was 0.01-2 mg/L (R2 = 0.999). Matrix effects were not observed. Intraday and interday precision and accuracy were within ±15%. A therapeutic drug monitoring-guided strategy was explored to optimize preemptive antiviral drug therapy in 3 cohorts of transplant recipients. Seventy-nine samples from 35 patients were quantified, revealing median trough concentrations of 0.2 mg/L for ganciclovir (n = 21), 0.28 mg/L for acyclovir (n = 26), and 0.29 mg/L for letermovir (n = 32).

Conclusions: This method has been successfully applied in clinical settings and allows reliable and accurate drug-level measurements.