ScienceAsia 33 (2007): 405-410 |doi: 10.2306/scienceasia1513-1874.2007.33.405

High-Performance Liquid Chromatography with Amperometric Detection of Medroxyprogesterone Acetate in Human Plasma with 2,4-Dinitrophenylhydrazine as Derivatizing Agent and Solid-Phase Extraction for Sample Clean-up

Thanee Tessiri,^a Jinda Wangboonskul,^b Chalerm Ruangviriyachai^c and Saksit Chanthai^c*

ABSTRACT:    A specific and sensitive method is presented for the analysis of medroxyprogesterone acetate (MPA) in human plasma using reversed phase high-performance liquid chromatography (HPLC) with amperometric detection. The blood plasma spiked with trace amount of MPA was cleaned up to remove natural interfering matrices by solid-phase extraction (SPE). The MPA extract was then derivatized with 2,4- dinitrophenylhydrazine (DNPH) as an electroactive agent. The MPA-DNPH derivative was re-extracted using SPE prior to analysis by reversed phase HPLC. Quantitative analysis of the MPA-DNPH using prednisolone-DNPH as an internal standard were optimized on a Hypersil ODS column using acetonitrile: methanol:30 mM phosphate buffer, pH 3 (39:39:22; v:v:v) as mobile phase at a flow-rate of 1.0 ml min-1. It was found that the method was selective and gave linear calibration curve for a concentration range of 1.0 – 10.0 ng ml^-1 for 2 ml spiked plasma samples. The relative standard deviation (RSD) of inter-day precision for a period of three validation days was 11.5 ± 3.4 % for all concentrations used. The RSD of intra-day precision (n = 5) was 5.05 ± 2.7 % with accuracy (n = 5) of 102.3 ± 7.4 %. The average recovery was found to be 102.9 ± 4.4 %. The correlation coefficient of the calibration curve was 0.9985. The limits of detection and quantitation were found to be 0.2 and 1.0 ng ml-1, respectively. Using DNPH as a derivatizing agent can enhance both selectivity and sensitivity of MPA in plasma and is suitable for routine analysis.

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^a Academic and Research Services Unit, Khon Kaen University, Khon Kaen 40002, Thailand.
^b Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand.
^c Department of Chemistry, Faculty of Science, Khon Kaen University, Khon Kaen 40002, Thailand.

* Corresponding author, E-mail: sakcha2@kku.ac.th

Received 4 Oct 2006, Accepted 29 May 2007