Torasemide

Amlodipine besylate was purchased from Pfizer (Karlsruhe, Germany); canrenone from the European Directorate for the Quality of Medicines & HealthCare (Strasbourg, France); bisoprolol fumarate, metoprolol tartrate, carvedilol, candesartan, valsartan, olmesartan, torasemide, and a diazepam-d5 solution (1 mg/mL in methanol) from LGC Standards (Wesel, Germany); atorvastatin-d5 calcium salt from Alsachim (Shimadzu Group, Illkirch Graffenstaden, France); and methanol, acetonitrile (ACN), diethyl ether, ethyl acetate, and natrium sulfate from VWR International GmbH (Darmstadt, Germany), as well as ammonium formate and formic acid from Merck KGaA (Darmstadt, Germany). All chemicals were of analytical grade. Water was purified with a Millipore filtration unit (18.2 Ω × cm water resistance) from Merck (Darmstadt, Germany). Drug-free pooled human blank plasma was obtained from a local blood bank. The analysis of human samples was approved by the local ethics committee to assess medication adherence using toxicological analyses objectively and complied with the Declaration of Helsinki. All patients provided written informed consent.

The internal standards (IS) canrenone-d₄, hydrochlorothiazide (HCT)-¹³C₆, lisinopril-d₅, torasemide-d₆, and ramiprilat-d₅ were purchased from Alsachim (Illkirch Graffenstaden, France), canrenone from European Pharmacopoeia (Strasbourg, France), HCT, furosemide, and torasemide from LGC (Luckenwalde, Germany), lisinopril from Merck (Darmstadt, Germany), ramiprilat from Aventis Pharma (Strasbourg, France), and enalaprilat from Sigma Aldrich (St. Louis, USA). All other chemicals (LC-MS grade or analytical grade) were from VWR (Darmstadt, Germany). Mitra VAMS with a 10 μL absorbing tip were purchased from Neoteryx (Torrance, USA). Blank blood, stabilized with ethylenediaminetetraacetic acid (EDTA), used for development and validation of the procedure was submitted to the authors’ laboratory for regular toxicological analysis and handled in accordance with the institutional protocol and regulations concerning data privacy and sample handling. FPB loaded VAMS, matching EDTA-stabilized blood samples, and medication plans for applicability studies were collected from volunteers with hypertension as part of their regular consultation at Saarland University Hospital, Homburg, Germany. All volunteers provided written informed consent and the local ethic committees approved the study (Homburg Hypertonie Register, 142/10).