(690) Simultaneous Quantification of TRIKAFTA^© via Reverse Phase HPLC

Background: Ivacaftor/tezacaftor/elexacaftor compose a breakthrough cystic fibrosis (CF) drug combination therapy known as TRIKAFTA^©. These directly modulate the activity and trafficking of the defective CF transmembrane-conductance regulator protein (CFTR) responsible for the CF disease state. As of today, there are no therapeutic drug monitoring assays available for these expensive but life-saving drugs. A rapid and precise method for the quantification in human serum is in development at National Jewish Health, suitable for high-throughput applications in the clinical settings using high performance liquid chromatography (HPLC). The deployment of this rapid yet accurate assay will offer clinicians the resources to develop exposure−response relationships, aid with understanding of predictive biomarkers, and achieve the best possible clinical efficacy.

Methods: Observations were performed on a ThermoFisher UltiMate 3000 HPLC System using a C18 column (phenomenex Gemini 5mm 150 × 4.6mm) with a C18 guard column (phenomenex 2.1mm SecurityGuard ULTRA cartridges, 3/pk). Internal standard (ISTD) Inactive metabolite ivacaftor-M6- carboxylate are utilized to control for product loss and extraction efficiency. Aliquots of serum were obtained from National Jewish Health’s Immunology Department during method development. A mobile phase pump gradient was prepared containing 0.1% formic acid in DI water mixed 60:40 volume to volume (v/v) with ACN progressing to 90:10 v/v over 10 minutes at a flow rate of 1 mL/min. Temperature is maintained at 30°C with a Multichannel wavelength UV Detector set to 295nm.

Results: Retention times of ivacaftor/tezacaftor/elexacaftor are 4.88 min, 7.18 min and 8.88 min respectively. Good fit to these three calibration curves were observed as represented by the correlation coefficients with a linear range of 0.2-15 μg/mL (R² = 0.9883−0.9998). Linear least-squares regression analysis was performed using peak height relative response ratios plotted against an external calibration curve prepped in spiked serum. Linear fit with 1/C² weighting factor was employed for all three-target components. The linear equations were as follows: ivacaftor (y = 0.8573+ 0.0585; R² = 0.9998), tezacaftor (y = 0.1566x + 0.0558; R² = 0.9989), and elexacaftor (y = 0.5376x + 0.088; R² =0.9926). In all blank serum samples, no interference with the retention time of either of the analytes was observed, nor was interference detected from the combination of ivacaftor with tezacaftor with or without elexacaftor. The method used to determine the endogenous interferences was the percentage of the area of the lower limit of detection (LOD) for each analyte. The LOD and quantification (LOQ) were established: for ivacaftor, LOD 2.0 × 10^-2 μg/mL and LOQ 2.82 × 10^-2 μg/mL; for tezacaftor, LOD 1.80 × 10^-2 μg/mL and LOQ 6.01 × 10^-2 μg/mL; and for elexacaftor, LOD 7.40 × 10^-1 μg/mL and LOQ 2.64 × 10^-2 μg/mL.

Conclusions: A simple, rapid yet precise method will continue to clinical validation for the simultaneous estimation of Ivacaftor/tezacaftor/elexacaftor in serum drawn patients. This assay will allow for the high through put analysis using peak height HPLC analysis of serum samples from patient with CF receiving TRIKAFTA^© combination therapy. Our method allows for high throughput of patient samples, short turnaround time, and provide clinicians with an adaptive resource for dose managing CF patients.

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