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  3. A simple HPLC-UV method for monitoring therapeutic adherence in pulmonary arterial hypertension

A simple HPLC-UV method for monitoring therapeutic adherence in pulmonary arterial hypertension

  • J Chromatogr B Analyt Technol Biomed Life Sci. 2025 Feb 1:1252:124443. doi: 10.1016/j.jchromb.2024.124443.
Paweł K Kunicki 1,  Maciej T Grymm 2,  Tomasz Pawiński 2,  Daniel Szulczyk 3,  Marcin Waligóra 4,  Grzegorz Kopeć 5
Affiliations

Affiliations

  • 1 Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Warsaw, Poland. Electronic address: pawel.kunicki@wum.edu.pl.
  • 2 Department of Drug Chemistry, Pharmaceutical and Biomedical Analysis, Medical University of Warsaw, Warsaw, Poland.
  • 3 Chair and Department of Biochemistry, Medical University of Warsaw, Warsaw, Poland.
  • 4 Pulmonary Circulation Centre, Department of Cardiac and Vascular Diseases, Jagiellonian University Medical College, Krakow, Poland; Department of Cardiac and Vascular Diseases, St. John Paul II Hospital, Krakow, Poland; Center for Innovative Medical Education, Department of Medical Education, Faculty of Medicine, Jagiellonian University Medical College, Krakow, Poland.
  • 5 Pulmonary Circulation Centre, Department of Cardiac and Vascular Diseases, Jagiellonian University Medical College, Krakow, Poland; Department of Cardiac and Vascular Diseases, St. John Paul II Hospital, Krakow, Poland.
PMID: 39787725 DOI: 10.1016/j.jchromb.2024.124443
Abstract

A considerable percentage of ineffective treatment in pulmonary arterial hypertension (PAH) may be related to subtherapeutic dosage or non-adherence. The aim of the study was to develop a simple analytical method suitable for plasma determination of selected drugs: riociguat (RIO), bosentan (BOS) and macitentan (MAC) administered to PAH patients. An isocratic HPLC-UV system (Spectra Physics - Shimadzu) with a manual injector (50 μL loop) was applied. Chromatographic analysis was performed using a Suplecosil LC-CN column (150 × 4.6 mm, 5 μm) protected with a Supelguard precolumn at room temperature. The separation was carried out using the mobile phase: CH3CN:H2O:0.5 M KH2PO4:85 % H3PO4 (172:324.2:3.7:0.1, v/v) at a flow rate of 1.8 mL/min. Ethyl acetate (4 mL) was used for 10-min liquid-liquid extraction from 0.4 mL alkalized plasma sample. Detection was performed at λ = 245 nm chosen as a compromise between signal intensity and matrix interference. The analytes were eluted at retention times of 4.4 min (RIO), 5.4 min (BOS), 8.9 min (MAC) and 7.8 min for gallopamil (internal standard, GAL). The method was found linear and calibrated in the ranges: 5-1000 ng/mL for RIO, 10-2000 ng/mL for BOS and 20-2000 ng/mL for MAC, with r2 of 0.9991 for RIO, 0.9983 for BOS, and 0.9949 for MAC, respectively. Within the given ranges, the method ensured reliable results with the required precision and accuracy: ≤15 % (≤20 % for LLOQ). There was no significant carryover effect. The method has been successfully used in pilot study on adherence in patients treated for PAH, enabling monitoring of RIO, BOS and MAC. Drug concentrations were assessed in samples taken before (C0) and 3 h after drug administration (C3). For RIO, BOS and MAC, the developed method was suitable for both C0 and C3 samples, allowing steady-state drug determination if used. The presented method can be recommended to laboratories equipped with basic HPLC apparatus as an attractive analytical tool for both TDM and adherence studies.

Keywords

Adherence; Bosentan; HPLC-UV; Macitentan; Pulmonary arterial hypertension; Riociguat.

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