Determination of 7alpha-hydroxycholesterol in dog plasma by high-performance liquid chromatography with fluorescence detection.
A new method was developed for the determination of 7alpha-hydroxycholesterol (7-HC) in dog plasma by means of high-performance liquid chromatography (HPLC) with fluorescence detection. 7-HC extracted with organic solvent from plasma was purified with Bond Elut 2OH and converted to a sensitive fluorescent derivative containing double coumarin groups at the C-3 and C-7 positions of the steroid nucleus with 7-methoxycoumarin-3-carbonyl azide. After removal of the excess reagent with Bond Elut NH2, the 7-HC derivative was separated by reverse-phase HPLC method. The detection limit of the authentic 7-HC-3,7-coumarin derivative was 4 pg (S/N = 5), approximately four times less than that of the 7-HC-3-anthroyl derivative yielded by reaction of 7-HC with 1-anthroylcyanide. The newly developed method was used to investigate the effects of consecutive oral administrations of cholestyramine (CA) on 7-HC levels in dog plasma. The plasma 7-HC levels of the CA-treated group were two times greater than those of the control group.
Saisho
Y
Y
Shionogi Research Laboratories, Shionogi & Company, Ltd., 12-4 Sagisu 5-Chome, Osaka, Fukushima-ku, 553-0002, Japan. yutaka.saisho@shionogi.co.jp
Shimada
C
C
Umeda
T
T
D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D002792:Cholestyramine Resin / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004285:Dogs; D006888:Hydroxycholesterols / Q000097:blood* / Q000737:chemistry / Q000302:isolation & purification; D007202:Indicators and Reagents; D008297:Male; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence
D000924:Anticholesteremic Agents; D006888:Hydroxycholesterols; D007202:Indicators and Reagents; D002792:Cholestyramine Resin; C011724:cholest-5-en-3 beta,7 alpha-diol