LC-MS/MS method for determination of curcumin in serum

The concentrations of curcumin in serum were determined using a high-performance liquid chromatographic method with mass spectrometric detection (LC-MS/MS). To 100 μl of plasma, 200 μl methanol containing 2% formic acid was added, and the mixture was vortex-mixed for 1 minutes, centrifuged at 14000 RPM for 5 minutes. Zileuton was used as internal standard. The supernatant was collected, and a volume of 10 μl was injected into the HPLC.

Chromatographic analysis was performed using a Waters Model 2695 separations system (Milford, MA, USA). Separation of the analytes from potentially interfering material was achieved using Waters Xterra MS C18 column (50 × 2.1 mm i.d., 3.5 μm) protected by a Waters Xterra MS C18 guard column (3.5 μm, 10 × 2.1 mm i.d ). The mobile phase used for the chromatographic separation was composed of methanol/0.45% formic acid in water (70:30, v/v), and was delivered isocratically at a flow rate of 0.2 mL/min. The column effluent was monitored using a Waters Quattro Micro^™ triple quadrupole mass-spectrometric detector (Milford, MA, USA). The instrument was equipped with an electrospray ionization source, and controlled by the Masslynx version 4.1 software. The samples were analyzed using an electrospray probe in the negative ionization mode operating at a cone voltage of 24 V for curcumin and in the positive ionization mode at a cone voltage of 13 V for internal standard zileuton. Samples were introduced into the ionization source through a heated nebulized probe (350°C). The spectrometer was programmed to allow the [MH]⁺ ion of curcumin at m/z 367.0 and zileuton at m/z 237.0 to pass through the first quadrupole (Q1) and into the collision cell (Q2). The collision energy was set at 22 and 19 eV for curcumin and zileuton, respectively. The product ions for curcumin (m/z 149.0) and zileuton (m/z 161.0) were monitored through the third quadrupole (Q3). Argon was used as collision gas, and the dwell time per channel was 0.2 sec for data collection. The linear calibration curve was set at curcumin serum concentrations ranging from 10 to 10,000 ng/ml. The intra- and inter-day accuracy and precision for QC samples were within 15%.