Agilent 1200 sl binary pumps

The instrument parameters were same as Assay 2 (see Section 2.5.2) with a few modifications as described here. The HPLC system comprised of two Agilent 1200 SL binary pumps (Agilent, Palo Alto, CA, USA) and a CTC Analytics LCPAL (CTC Analytics AG, Zwingen, Switzerland) autosampler. Analytical column pump gradient conditions were as follows: 0.0–2.50 min, isocratic 40% B; 2.50–2.60 min, linear from 40% to 90% B; 2.60–4.50 min, isocratic 90% B. The analytical column flow rate was 0.150 mL/ min except for 3.60–4.50 mins when it was increased to 0.350 mL/min. The injection volume was 50 µL.
Mass spectrometric detection was carried out using ABI SCIEX API 5000 triple quadrupole mass spectrometer with positive electrospray ionization in MRM mode (see Table 1 and Table 2 for mass spectrometric parameters).

High performance liquid chromatography (HPLC) system comprised of two Agilent 1200 SL binary pumps (Agilent, Palo Alto, CA, USA) and a CTC Analytics LCPAL (CTC Analytics AG, Zwingen, Switzerland) autosampler. Chromatographic separation was achieved on a Waters Acquity BEH C18, 2.1 mmx 50 mm, 1.7 µm column using 5 mM ammonium bicarbonate as mobile phase A and acetonitrile as mobile phase B. The analytical column was maintained at 50 °C. The analytical pump gradient conditions were as follows: 0.0–0.25 min, isocratic 10% B; 0.25–4.00 min, linear from 10% to 22% B; 4.00–4.50 min, linear from 22% to 90% B; 4.50–4.70 min, linear from 90% to 95%; 4.70–7.60 min, isocratic 95% B; 7.60–7.70 min, linear from 95% to 10% B; 7.70–9.00 min, isocratic 10% B. The flow rate was 0.300 mL/ min except for 5.60–7.60 mins when it was increased to 0.450 mL/min. A CTC Analytics LCPAL autosampler was used. The autosampler temperature was 2–8 °C. The injection volume was 25 µL.
Mass spectrometric detection was carried out using an AB Sciex API 5000^TM triple quadrupole mass spectrometer (AB Sciex, Framingham, MA, USA) with positive electrospray ionization in multiple reaction monitoring (MRM) mode (see Table 1 and Table 2 for mass spectrometric parameters). The data were acquired using Analyst Version 1.6.2 (AB Sciex, Framingham, MA, USA).