Acquity 1 class ultra performance liquid chromatography

Acquity I-Class ultra-performance liquid chromatography and XEVO TQS-micro triple quadrupole mass spectrometer (Waters Corp, Milford, MA, USA) were used. Masslynx 4.1 software was used for data acquisition and instrument control.
A CORTECS T3 column (2.1 mm × 50 mm, 1.6 μm) was used, and its temperature was set to 40°C. The mobile phase consisted of acetonitrile and 0.1% formic acid, with a gradient elution program: 0–0.2 min, acetonitrile 10%; 0.2–1.0 min, acetonitrile 10–70%; 1.0–2.5 min, acetonitrile 70–90%; 2.5–2.8 min, acetonitrile 90–10%; 2.8–4.0 min, and acetonitrile 10%. The flow rate was set to 0.4 mL/min, and the elution time was 4 min.
Nitrogen was used as the desolvation gas (900 L/h) and cone gas (50 L/h). The capillary voltage was set to 2.0 kV, the ion source temperature was 150°C, and the desolvation temperature was 500°C. ESI-MS in negative mode MRM was used for quantitative analysis: senegenin m/z 679.4 > 455.4 (cone voltage 40 V, collision voltage 26 V), tenuifolin m/z 535.3 > 481.3 (cone voltage 36 V, collision voltage 30 V), and internal standard ion m/z 825.4 > 617.5 (cone voltage 20 V, collision voltage 40 V).

The samples were analyzed using the Waters Xevo TQ-S triple quadrupole mass spectrometer (parameters: Polarity ESI, Capillary (kV) 2.6) coupled to Waters Acquity I-Class Ultra Performance Liquid Chromatography (UPLC). A 2 min High-Performance Liquid Chromatography (HPLC) gradient method was set up on the Waters UPLC BEH 1.7 μm 2.1 × 100 mm column with thermostatic control at 70°C. Mobile phase A consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v) in MQ water. Mobile phase B consisted of 2 mM ammonium acetate with 0.1% formic acid (v/v) in LC/MS grade ACN (acetonitrile). The injection volume was 15 μL and flow rate 400 μL/min. The gradient used for separation was changed in time (min): initial, 0.2, 1.0, 1.5 and mobile phases were respectively A (%) 90, 10, 2, 90 and B (%) 10, 90, 98, 10. Monitored transmissions were: 212.00 > 79.96 CE (Collision energy) 20, 212.00 >132.04 CE 20 –for indoxyl sulfate, and 216.11 > 135.76 CE 20 for indoxyl sulfate -D4.
The samples were blinded to the case-control status and the principles of Good Laboratory Practice were followed.