Contrast Agents: The study investigated two high relaxivity macrocyclic GBCAs: gadoquatrane (Phase 3 formulation, Bayer AG, Berlin), gadopiclenol (Vueway, Bracco Diagnostics Inc., USA). For relaxivity measurements gadobutrol (Gadovist, Bayer Vital GmbH, Leverkusen) was measured additionally as a reference.

Relaxivity measurements: Human plasma was sourced from three male and three female donors. Solutions of gadoquatrane, gadopiclenol and gadobutrol in aliquots of the individual donor plasmas at three different target concentrations of 250, 500 and 1000 μmol/L were produced. Additionally, one blank plasma sample per donor was prepared, leading to a total of 60 samples. The T1-relaxation rates of all samples were measured at 3 Tesla on a Stelar High Field relaxometer and at 1.41 Tesla on a Bruker MiniSpec relaxometer. Concentrations were corrected using ICP-OES measurements of the samples. The relaxivity was calculated with linear regression as the relaxation rate change per concentration change. NMRD profiles ranging from 50 mT to 3.0 T were measured using the highest concentration sample and the blank plasma from the six different donors.

Complex stability in human plasma: The kinetic profiles of Gd3+ dissociation of gadoquatrane and gadopiclenol were determined by incubation for 15 days in pooled human plasma (citrate) from healthy volunteers at a concentration of 1 mmol/L, pH 7.4, and 37°C. To simulate the situation in patients with end-stage renal disease who often have elevated blood phosphate levels, 10 mmol/L phosphate was supplemented. Additionally, 2 mmol/L of NaN3 was added to prevent microbial growth during the incubation. The initial rates of Gd3+ release and the amounts of Gd3+ released after 5, 8, 11 and 15 days were established by HPLC-ICP-MS analysis using a chelating Sepharose column. The lower limit of quantification (LLOQ) was estimated to be 0.1% of the total Gd. The integrity of each sample was additionally checked by ICP-OES.

Kinetic inertness in acidic medium: 50 mL solutions of Gadoquatrane and Gadopiclenol were prepared with a concentration of 50 μmol/L Gd each. The pH was adjusted to 1.2 and the solutions kept at 37°C. Concentrations were corrected using ICP-OES measurements. For the time points 0h, 2h, 4h, 24h, 48h, 120h, 168h, 216h, 288h, 336h, 384h and 552h (23d) the content of free Gd was determined by withdrawing an aliquot of 0.96 mL from the test solutions and mixing it with 0.12 mL of a 0.53 mmol/L Arsenazo III [2,2′-(1,8-dihydroxy-3,6-disulfonaphthylene-2,7-bisazo)bisbenzenearsonic acid,2,7-bis(2-arsonophenylazo) chromotropic acid] solution. Fifteen minutes after mixing, spectrometric measurement was performed at 654 nm in a 10-mm cuvette. This protocol was followed for each time point.