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.
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 10mM exhibited stabilization by preventing degradation of CDD. BNPP at a final concentration of 10mM 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 (4.6×50mm, 2.7μm) using an isocratic mobile phase containing acetonitrile-0.2% formic acid in water (73:27v/v) with a flow rate of 0.4mLmin(-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 50μL of rat plasma over the concentration range of 1-500ngmL(-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 40mgkg(-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 5min is achieved in comparison with direct intravenous injection of CUR.
Biomedicinal Chemistry Program, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand.
Pahweenvaj
P
Ratnatilaka Na Bhuket
Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand; School of Pharmacy, Eastern Asia University, 200 Rangsit-Nakhon Nayok Road (Klong5), Thanyaburi, Pathum Thani, 12110, Thailand.
Nuansri
N
Niwattisaiwong
Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand.
Patanachai
P
Limpikirati
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand.
Phisit
P
Khemawoot
Department of Pharmacology and Physiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand.
Pasarapa
P
Towiwat
Department of Biochemistry and Microbiology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand; Chulalongkorn University Drug and Health Products Innovation & Promotion Center, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand.
Boonsri
B
Ongpipattanakul
Department of Food and Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Chulalongkorn University, 254 Phayathai Road, Patumwan, Bangkok, 10330, Thailand. Electronic address: pornchai.r@chula.ac.th.
Pornchai
P
Rojsitthisak
D000818:Animals; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D003474:Curcumin / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism / Q000493:pharmacokinetics; D004355:Drug Stability; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D004950:Esterases / Q000037:antagonists & inhibitors*; D004952:Esters / Q000737:chemistry; D007275:Injections, Intravenous; D008297:Male; D011355:Prodrugs / Q000737:chemistry*; D017208:Rats, Wistar; D012015:Reference Standards; D015203:Reproducibility of Results; D013386:Succinates / Q000097:blood* / Q000737:chemistry / Q000378:metabolism* / Q000493:pharmacokinetics; D053719:Tandem Mass Spectrometry / Q000379:methods*
D004791:Enzyme Inhibitors; D004952:Esters; D011355:Prodrugs; D013386:Succinates; C000614237:curcumin diethyl disuccinate; D004950:Esterases; D003474:Curcumin
Curcumin; Curcumin diethyl disuccinate; Esterase inhibitors; LC–MS/MS; Rat plasma; Validation