Ultrapure solvents

The determination of the IM plasma concentrations followed the protocol as described in Ubhayasekhera et al. [25 (link)]. IM standard was kindly provided by Novartis (Basel, Switzerland). All chemicals including internal standard (Trazodone) and ultrapure solvents were purchased from Sigma Aldrich (Stockholm, Sweden), unless otherwise stated. The stock solutions of IM and internal standard were prepared by dissolving methanol to obtain a final concentration of 1 mg/mL. Protein precipitation was applied as a sample pretreatment. Twenty-five microliter of methanol containing 1 µg/mL internal standard and 0.5 mL of methanol were added to 100 µL of plasma, shaken in 10 min and centrifuged for 10 min at 4 °C at 14,000g. The supernatant was dried by vacuum centrifugation and the residue was reconstituted in 100 µL of 5% acetonitrile containing 0.1% formic acid. Aliquouts of 10 µL were injected into the LC–MS system. Chromatography and mass spectrometry was performed as previously described [25 (link), 26 (link)].

The ATR spectra for the surface layer of the analysed samples and the solutions were registered with the use of an ATR attachment with a diamond crystal. The ART spectra were registered on an FTIR Thermo Nicolet 8700 spectrometer with a Smart Orbit™ diamond ATR attachment and DTGS (Deuterated Triglycine Sulphate) detector. The spectra underwent ATR correction, baseline correction, and ATR scale normalisation to achieve transmission spectra equivalence.
The quantitative measure of the quality of fit between the measured spectrum and the benchmark is the correlation index or HQI (hit quality index), where HQI = 100 indicates a perfect spectral fit. Values of HQI > 90 indicate very high levels of spectral similarity. The results of the FTIR-ATR analyses are presented in a graphic format as sets of comparative spectra. The attachment crystal was always thoroughly cleaned using ultrapure solvents purchased from Sigma-Aldrich (Saint Louis, MO, USA). The spectra were measured within the spectral range from 450 to 3700 cm⁻¹ at a resolution of 2 cm⁻¹. The results were processed and prepared for publication using Grams AI software from ThermoGalactic Industries (Waltham, MA, USA) and OriginPro 2021 software from OriginLab Corporation (Northampton, MA, USA).