Uremic toxins and Indigo were analyzed using LCMS 8040 Triple Quadrupole LC/MS/MS (Shimadzu), as previously described [55 (link)].
Electrospray (ESI, negative mode at 4000 V) was performed for each analysis in LC-MS/MS. The mobile phase was composed of MeCN mixed with 2 mM of ammonium acetate. Nitrogen was employed for the nebulizer and desolvation gas and finally utilized as collision gas for molecule dissociation [28 (link)]. Notably, for Indigo determination, 10% MeCN was used for equilibrating the column (Luna Omega, size 150 × 4.6 mm, Phenomenex). Next, MeCN was raised up to 50% and finally scaled at 10% of MeCN [56 (link)]. The fragmentation voltages and collision energies of analytes for the first and second methods were optimized, as reported inTable 1 . The chromatograms and MRM (multiple reaction monitoring) spectrum of IS, pCS, DHTC, and Indigo are shown in Figure 5 .
Electrospray (ESI, negative mode at 4000 V) was performed for each analysis in LC-MS/MS. The mobile phase was composed of MeCN mixed with 2 mM of ammonium acetate. Nitrogen was employed for the nebulizer and desolvation gas and finally utilized as collision gas for molecule dissociation [28 (link)]. Notably, for Indigo determination, 10% MeCN was used for equilibrating the column (Luna Omega, size 150 × 4.6 mm, Phenomenex). Next, MeCN was raised up to 50% and finally scaled at 10% of MeCN [56 (link)]. The fragmentation voltages and collision energies of analytes for the first and second methods were optimized, as reported in