Highly sensitive and specific determination of pravastatin sodium in plasma by high-performance liquid chromatography with laser-induced fluorescence detection after immobilized antibody extraction.
A method for the determination of pravastatin sodium (PS), a cholesterol-lowering agent, in plasma was developed by using an immobilized antibody column extraction followed by high-performance liquid chromatography (HPLC). The analyte was monitored by a laser-induced fluorescence detector after fluorogenic derivatization. The PS antibody was coupled to Sepharose 4B and used as an extraction phase for sample cleanup and extraction of the drug. A plasma sample was applied to the column and washed with water, and the drug was eluted with methanol. N-Dansylethylenediamine (DNS-ED) was coupled to the carboxyl moiety of the drug in the presence of diethyl phosphorocyanidate (DEPC) and triethylamine (TEA) in dioxane. Derivatization was completed in 5 min at room temperature. A column-switching technique was utilized to remove excess reagents and byproducts. A He-Cd laser-induced fluorescence detector was applied to achieve an ultrasensitive determination. The detection limit was 2 pg/injection of PS, which was 20 times more sensitive than the conventional fluorescence detection. The limit of quantitation was 100 pg/mL when 1 mL of plasma sample was available. An average coefficient of variations of the overall method were less than 8% at the concentration range of 1-100 ng/mL. A single oral dose of PS in rats (20 mg/kg) and dogs (5 mg/kg) resulted in average maximum concentrations of 142 and 310 ng/mL, respectively.
Dumousseaux
C
C
Analytical and Metabolic Research Laboratories, Sankyo Co, Ltd., Tokyo, Japan.
Muramatsu
S
S
Takasaki
W
W
Takahagi
H
H
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000302:isolation & purification; D002851:Chromatography, High Pressure Liquid; D004285:Dogs; D004797:Enzyme-Linked Immunosorbent Assay; D007202:Indicators and Reagents; D007834:Lasers; D008297:Male; D010972:Platelet Activating Factor / Q000276:immunology / Q000302:isolation & purification; D017035:Pravastatin / Q000097:blood* / Q000276:immunology / Q000493:pharmacokinetics; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence
D000906:Antibodies; D007202:Indicators and Reagents; D010972:Platelet Activating Factor; D017035:Pravastatin