Simultaneous determination of curcumin diethyl disuccinate and its active metabolite curcumin in rat plasma by LC-MS/MS: Application of esterase inhibitors in the stabilization of an ester-containing prodrug

- An LC-MS/MS was developed and validated for simultaneous determination of curcumin (CUR) and its prodrug, curcumin diethyl disuccinate (CDD).
- Bis(4-nitrophenyl) phosphate (BNPP) was an effective esterase inhibitor for preventing ex vivo hydrolysis of CDD in rat plasma.
- The LLOQ of CUR and CDD was 1 ng/mL.
- The method was successfully applied to support the pharmacokinetic study of in rats.
- CDD is converted to CUR after i.v. administration in rats and a higher CUR plasma level at 5 min is achieved comparing to direct i.v. injection of CUR.

Four esterase inhibitors, ethylenediamine tetraacetic acid disodium (Na2EDTA), sodium fluoride (NaF), bis(4-nitrophenyl) phosphate (BNPP) and phenylmethanesulfonyl fluoride (PMSF), were evaluated for their inhibitory effects on enzymatic hydrolysis of labile phenolate esters in curcumin diethyl disuccinate (CDD), a prodrug of curcumin (CUR), in rat plasma. BNPP and PMSF at 10 mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10 mM was subsequently selected to prevent ex vivo metabolism of CDD throughout LC-MS/MS analysis of CDD and CUR in rat plasma. A simple protein precipitation technique using acetonitrile as a precipitating agent was used to extract CDD, CUR and dimethylcurcumin (DMC), an internal standard, from rat plasma. Chromatographic separation was performed on a Halo C8 column (4.6 × 50 mm, 2.7 μm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27 v/v) with a flow rate of 0.4 mL min−1. An AB SCIEX QTRAP® 6500 mass spectrometer was operated using a positive ion electrospray mode for ionization and detection of analytes and internal standard. Calibration curves for CDD and CUR were established using 50 μL of rat plasma over the concentration range of 1-500 ng mL−1. The developed method was fully validated according to US Food and Drug Administration (FDA) guidelines for selectivity, sensitivity, linearity, accuracy, precision, dilution integrity, recovery, matrix effect, and stability. The validated method was applied to evaluate the pharmacokinetics of CDD and CUR in rats after a single intravenous dose of 40 mg kg−1. The method using BNPP as an esterase inhibitor was successful in determining the remaining CDD in rat plasma. The pharmacokinetic results indicate that CDD in rats is converted instantaneously to CUR after intravenous administration and a higher CUR plasma concentration at 5 min is achieved in comparison with direct intravenous injection of CUR.