. UHPLC–MS/MS analysis
The chromatographic method for the determination of CRBs
was previously developed in our laboratory [19]. UHPLC separations
were performed on a C18 column using a mobile phase
consisting of 0.01% aqueous formic acid solution (solvent A) and
MeOH with the same percentage of acid (solvent B) at a flow rate
of 0.5 mL min1
. The gradient profile was 0% B at the beginning;
20% B from 0.7 to 1.2 min; 50% B from 2.5 to 3 min; 95% B from
6.5 to 7.0 min; and finally in order to come back to the initial
conditions, 0% B at 7.5 min, equilibrating for 3 min. Under optimum
conditions, all the analytes were eluted for 6 min, while the
run time for each injection was 9.5 min. The temperature of the
column was 25 1C and the injection volume was 10 mL. The massspectrometer
was working with ESI in positive mode under the
multiple reaction monitoring (MRM) conditions shown in supplementary
data (Table SD1). The ionization source parameters were:
source temperature 400 1C; curtain gas (nitrogen) 30 psi; ion spray
voltage 5000 V; and GAS 1 and GAS 2 (both of them nitrogen)
were set to 50 psi.