The deproteination method using methanol was utilized to extract CUR from plasma/brain homogenates. Brain homogenates were obtained by homogenization of brain samples in PBS: Acetonitrile (ACN) (50:50) followed by centrifugation at 10 000×g (10 min) to obtain supernatants. The supernatant (200 μL) was mixed with methanol 300 μL in 1.5-mL eppendorf tube and vortexed, followed by addition of 500 μL of 2% acetic acid (pH 3): ACN (50:50) and vortexing for 3 min. The supernatant obtained by centrifugation at 10 000×g (10 min) was quantified for CUR by a validated HPLC method.
Reverse-phase high-performance liquid chromatography (RP HPLC) method used to distinguish between CUR, its analogs and its degradation products to enable quantification of CUR was developed. The analysis of CUR was carried out using a HPLC system (Jasco PDA), having 100 μL injection loop with C18 column Waters Spherisorb® 5 μm ODS2 (4.6 × 250 mm). Analytical column equipped with solvent delivery pump and Jasco MD-2010 multiwavelength detector operated at 425 nm. The mobile phase composed of 2% glacial acetic acid (pH3) and ACN in a ratio of 50:50, pumped at a flow rate of 0.9 mL/min with retention time of 11.55 ± 0.28, 12.95 ± 0.36 and 13.28 ± 0.25 min, respectively, for bisdemethoxycurcumin, demethoxycurcumin and curcumin.
Reverse-phase high-performance liquid chromatography (RP HPLC) method used to distinguish between CUR, its analogs and its degradation products to enable quantification of CUR was developed. The analysis of CUR was carried out using a HPLC system (Jasco PDA), having 100 μL injection loop with C18 column Waters Spherisorb® 5 μm ODS2 (4.6 × 250 mm). Analytical column equipped with solvent delivery pump and Jasco MD-2010 multiwavelength detector operated at 425 nm. The mobile phase composed of 2% glacial acetic acid (pH3) and ACN in a ratio of 50:50, pumped at a flow rate of 0.9 mL/min with retention time of 11.55 ± 0.28, 12.95 ± 0.36 and 13.28 ± 0.25 min, respectively, for bisdemethoxycurcumin, demethoxycurcumin and curcumin.