Agilent 1290 series liquid chromatograph

Quantitative analysis of UAMC-3203 was performed with the LC-MS/MS system that consisted of an Agilent 1290 series liquid chromatograph connected to an Agilent 6495 triple-quadruple mass spectrometer (Agilent Technologies, Inc., USA). The mobile phase consisted of 0.1% formic acid (Fisher chemical, Geel, Belgium), 1 mM ammonium formate (Honeywell, Fluka, Seelze, Germany) in Milli-Q-H2O (eluent A), and acetonitrile (LC-MS Chromasolv, Honeywell, Riedel-de Haen, Seelze, Germany) (eluent B). Gradient elution was used: 0.0 to 3.0 min: 25%→95% B, 3.0 to 3.5 min: 95% B, 3.5 to 3.6 min: 95%→25% B, 3.6 to 5.0 min 25% B. Solvent flow was 0.3 mL/min, and Poroshell 120 SB-C18 column (2.1 mm × 50 mm, 2.7 µm, Agilent Technologies, Inc., USA) was maintained at 60 °C. The injection volume was 2 µL. Nitrogen was used as a drying, nebulizer, and collision gas. The following conditions were used in the electrospray ion source: positive ion mode, sheath gas flow rate 11 L/min at 350 °C, drying gas flow rate 16 L/min at 200 °C, nebulizer gas pressure adjusted to 25 psi, capillary voltage 3500 V (ESI+), fragmentor voltage 380 V, dwell time 150 ms and cell accelerator voltage 5 V. The data were analyzed with Agilent Mass Hunter Quantitative Analyzed software (vB.09.00, build 9.0.647.0, Agilent Technologies, CA, USA). The precursor and fragment ions are presented in Supplementary Table S1.

Samples were analyzed using an Agilent^® 1290 series liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA), coupled with an ABSCIEX^® API 5500 mass spectrometer (SCIEX, Framingham, MA, USA), and a SunfireTM C18 chromatographic column (Waters Corp., Milford, MA, USA) of 3.5 μm and 150 × 2.1 mm was used. The analytes were separated chromatographically using a mobile gradient of 0.1% formic acid in water (Phase A) and 0.1% formic acid in methanol (Phase B). The flow rate was adjusted to 0.2 mL/min, the injection volume was 20 µL, the duration was 25.423 min, and the column temperature was set at 35 °C ± 1 °C. The liquid chromatographic pump gradient is shown in Table 1. The scans per peak are shown in Table S1.
The criteria to identify the different AMs and their active metabolites was the monitoring of the masses of the precursor and fragment ions (Table S2). In addition, different parameters were used for the operation of the mass detector (Table 2). The chromatographic integration of the samples was performed using Analyst^® software version 1.6.2 (SCIEX, Framingham, MA, USA).
Samples were processed and analyzed at the Veterinary Pharmacology Laboratory (FARMAVET, as per initials in Spanish) of the Faculty of Veterinary and Animal Sciences at the University of Chile, which is accredited under ISO 17,025 standards.