Figure 3: LC-MS/MS system used for validation analysis. Urine sample was subjected to an on-line sample clean-up with an extraction column (TSK guard gel ODS-100V, 5 μm, 3.2 x 15 mm) via a 10-port column switching valve. 5 mM ammonium formate buffer in 10 % acetonitrile was employed as the mobile phase at a flow rate of 1 mL/min for 2 minutes after the completion of the sample load. Then, the flow was changed so that the extraction column was connected in series with an analytical column (Discovery HS F5, 5 μm, 2.1 x 50 mm) and 5 mM ammonium formate buffer in 80% acetonitrile was pumped through the both columns at a flow rate of 0.4 mL/min. The column temperature
was maintained at 40°C.
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