Appendix. Supporting Data

Appendix. Supporting Data

Mass spectrometry

HPLC mass spectrometry (HPLC-MS) was conducted to identify the chemical structure of the three peaks observed in standard HPLC sample analysis. HPLC-MS was performed with an Agilent 1260 series (Agilent Technologies, Germany) HPLC with a binary pump (G1313B), column compartment (G1316C), DAD detector (G4212B), and HIP sampler (G1367E) coupled to a Bruker AmaZon x ion trap mass spectrometer (Brucker, USA). The amaZon x was controlled by ESI Compass 1.7 trapControl software Version 7.2, data were collected using HyStar software, version 3.2, and data were processed using Bruker Compass DataAnalysis Version 4.2 (Bruker Daltonik GmbH, Bremen, Germany).

The chromatographic separation was performed using the same column as standard HPLC-UV and with column temperature of 25 °C. The mobile phase was water with 0.1 %v/v trifluoroacetic acid (85%) (mobile phase A) and acetonitrile with 0.05 %v/v trifluoroacetic acid (85%) (mobile phase B). Chromatographic separation was conducted using gradient elution: 0 min, B 5%; 5 min, B 10%; 15 min, B 40%; 17 min, B 80%; 18 min, B 5%; 25 min, B 5%. The flow rate was 0.4 mL/min and the injection volume was 5 µL for both LC-DAD and LC-MS analysis. UV-detection was performed at 249 nm and 279 nm.

LC-MS analysis utilized electrospray ionization (ESI) operating in positive ionization mode. The following MS parameters, optimized for the corresponding analysis, were applied: flow rate drying gas (N2) = 8.0 mL/min, nebulizer gas pressure = 20 psig, temperature drying gas (N2) = 220 °C, capillary voltage = 4500 V, collision gas = Helium. The mass spectrometer was operated in full scan mode in the range from 100 to 1000 m/z. Tuning of the ion trap mass spectrometer was performed with Agilent ESI tune mix (G2431A). The tune solution was infused with a syringe pump at a flow rate of 180 µL/h. A torasemide standard solution at a concentration of 500 µg/mL was used as control standard to verify the collision energy. The target mass was 350 m/z and the compound stability 100% for SPS tune.

Mass spectrometry data for the three peaks observed in torasemide physical mixture and extrudate samples, along with a corresponding UV chromatogram, are shown in Figure A1. Due to the slight difference in the gradients used for HPLC-UV analysis and HPLC-MS analysis, the retention times for the three species are slightly shifted. The peaks in the HPLC-MS chromatogram elute somewhat later than in the HPLC-UV chromatogram because the gradient was less steep. For clarification, the peak at 2.8 min in the HPLC-UV chromatogram corresponds to the peak at 5.7 min in HPLC-MS chromatogram, the peak at 6.5 min to the peak at 10.3 min and the peak at 11.6 min to the peak at 12.7 min.

Figure A12. HPLC chromatogram (a) and mass spectra for the 3 peaks found in samples: thermal degradant (b), hydrolysis degradant (c) and torasemide (d).