Simultaneous Quantification of Filgotinib and Its Active Metabolite in Human Plasma Using Liquid Chromatography-Tandem Mass Spectrometry: Validation and Clinical Application

Filgotinib (FLG) is a selective inhibitor of Janus kinase 1 (JAK1) and undergoes metabolism to produce an active metabolite known as GS-829845. While FLG is an important therapeutic agent, no previously published method exists for the simultaneous quantification of FLG and GS-829845 in clinical samples. To address this gap, we developed an advanced and reliable liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the concurrent determination of FLG and GS-829845 levels in human plasma.

The analytical procedure begins with the extraction of FLG and GS-829845 from a 50 μL plasma sample. This extraction is achieved through a straightforward deproteinization process using methanol, ensuring the elimination of plasma proteins that could interfere with accurate quantification. For chromatographic separation, we utilized a Shim-pack Scepter C18-120 column, which was optimized with a mobile phase consisting of a mixture of water and methanol, both containing 0.1% formic acid. The separation was carried out under gradient elution conditions with a constant flow rate of 0.2 mL/min to achieve optimal separation of both compounds.

Detection of FLG and GS-829845 was performed using positive electrospray ionization (ESI) coupled with a QTRAP 4500 mass spectrometer, providing the necessary sensitivity and specificity for accurate measurement. The method was rigorously validated over a concentration range of 2.5–50 ng/mL for FLG and 250–5000 ng/mL for GS-829845, covering the typical therapeutic and clinical ranges of both compounds. Validation studies demonstrated that intra- and inter-day assay accuracy and precision were well within acceptable limits, with deviations of less than 11.4% for accuracy and 13.9% for precision.

Further, recovery rates and matrix effects were found to be consistent and reproducible, ensuring that the method is both reliable and robust for routine clinical use. This fully validated LC-MS/MS method is not only simple and rapid but also cost-effective, making it ideal for therapeutic drug monitoring (TDM). We successfully applied this method to monitor drug levels in a patient undergoing treatment for rheumatoid arthritis, demonstrating its practical utility in a clinical setting.

This newly developed technique represents a significant advancement in the clinical pharmacokinetics of FLG and GS-829845, offering an essential tool for optimizing therapeutic outcomes and ensuring safe and effective use of filgotinib in patients. GLPG0634