DEVELOPMEMENT OF A HPLC-MS/MS METHOD FOR THE ANALYSIS OF HYDROZINOCURCUMIN IN PLASMA
.". In: 12 East and Central African Journal of Pharmaceutical Sciences
. FA Okalebol , L Wiesner, AN Guantai, K Chibale, P Smith; 2009. Abstract
The natural product curcumin has a wide range of useful biological effects. However its use in humans is limited due to its short half-life, rapid metabolism and poor oral bioavailability. It is metabolized by sequential reduction of carbon-carbon double two carbonyl groups. To overcome these limitations, a hydrazide derivative of curcumin was synthesized to improve its water solubility and reduce its rate of metabolism. The objective of study was, therefore, to develop a method for the analysis of hydrazinocureumin in murinc blood. This method will be eventually used to evaluate the pharmokinetic profile of hydrazinocurcumin in mice. Method: LC/MS/MS was selected as the analytic method because its high sensitivity allowed for sampling of small volumes of blood from mice. The optimal chromatographic and mass spectrometry conditions were determined by trial arid error. The column performance was monitored by measuring retention time, peak symmetry factor. A calibration curve was generated by using standard solutions with concentrations ranging from 0.78 - 10 rig/ml. The method was validated by determining the recovery, limit of detection, accuracy, linearity and intraday precision. The optimal method for extracting hydrazinocurcumin from biological fluids was determined by spiking human blood and plasma and extraction was done by solvent extraction from a spotted filter paper and liquid-liquid extraction. Instrumentation: The samples were assayed using an Agilent LC/MS/MS 3200Q Trap system (1100 series, USA) in the positive ionization mode. HPLC separation was done using an Agilent 1200 system (Agilent Technologies, Japan) interfaced with the MS/MS system. Ionization was done by electron spray ionization with a collision energy was 33eV. Chromatograms were integrated using Analyst version 1.4 software. Weighted linear regression was used to generate calibration curves from standard solutions. Results: The optimal conditions for HPLC separation was a mobile phase of 0.1% formic acid in acetonitrile: acetonitrile (1:1) with an isocratic flow rate of 0.3m1/minute and run time of 2 minutes. The analytical column was a 50 by 2.0 mm Pheromex C18 column with a particle size of 5 microns. The injection volume was 1 Out The hydrazinocurcumin formed molecular ion at M+H+ m/z 365.2 and two metastable ions at m/z 351 and m/z. 349.2. The transition monitored was m/z 365.2 to 349.2 at unit resolution and a dwelling time of 150 milliseconds. The retention time was 1.11 minutes. The optimal method of extraction was liquid-liquid extraction using 250 ul of ethyl acetate from 10 ul of whole blood in 50 ul of buffer at pH 10. The filter paper method of extraction was found to give erratic results. The calibration curve was linear over the concentration range of 10n/m1 to 0.5 ng/ml of hydrazinocurmin in whole human blood. Conclusion: The precision, accuracy, recovery and applicability were found to be adequate for pharmacokinetic studies in mice.