Analysis of GBP on plasma by HPLC-UV
GBP was determined in plasma by HPLC-UV (Shimadzu Inc., Kyoto, Japan), as described previously [44,48]. In summary, the analytes were resolved on LiChrospher® C18 RP column (125 × 4.0 mm, 5 µm, Merck, Darmstadt, Germany). The mobile phase consisted of 0.05 M sodium monobasic phosphate solution (pH 3.9):methanol (27:73, v/v), in a flow rate of 1.2 mL/min. The detection by ultraviolet was performed at λ= 360 nm. Plasma samples were prepared by protein precipitation with acetonitrile (ACN), followed by derivatization with 1-fluoro-2,4-dinitrobenzene (FDNB). Precision and accuracy, evaluated as relative standard deviation and relative errors, were below 15%, as well as the stability (evaluated in different conditions: short-term, long-term, post-processing and freeze-thaw cycles).