Novel inhibition of cis/trans retinoic acid interconversion in biological fluids--an accurate method for determination of trans and 13-cis retinoic acid in biological fluids.
All-trans retinoic acid (tRA, or tretinoin) can be metabolized through stereoisomerization to 13-cis retinoic acid (13-cRA) in vivo. We have developed a simple, sensitive and accurate method for analyzing tRA and 13-cRA in plasma with the addition of N-ethylmaleimide (NEM) and Vitamin C (Vit. C) to prevent the interconversion of cis/trans retinoic acid. All-trans RA, 9-cRA, and 13-cRA were well separated from each other in plasma by using a C18 precolumn and a column with a gradient solvent system of mobile phases A and B at a flow rate of 1.0 ml/min. In addition, thermal stability of tRA and cRA in plasma during the sample preparation under the temperature of 0 and 25 degrees C were studied. Our results showed that (1) the interconversion ratios (%) (cRA/tRA and tRA/cRA) were decreased with the addition of NEM and Vit. C and the minimum concentrations of NEM and Vit. C to inhibit the interconversion were 50 and 150 microM, respectively, (2) higher concentrations of NEM and Vit. C were required to prevent the interconversion at higher temperature, (3) the precision and accuracy of calibration curve with various concentration of tRA (1-1000 ng/ml) and 13-cRA (5-800 ng/ml) in plasma showed good linearity (r(2)=0.9992 and 0.9994), and between-day errors expressed by coefficient of variation (CV, %) for tRA and 13-cRA which were both less than 5.6%, (4) the mean recovery of the analytes were 78-94% for tRA and 80-92% for 13-cRA at concentration range from 1 to 1000 ng/ml and 5 to 800 ng/ml, respectively, and (5) the limit of quantitation of tRA and 13-cRA were 1 and 5 ng/ml, respectively. In addition, the HPLC method had been successfully applied to the tRA pharmacokinetic study in two hepatoma patients after receiving 45 mg/m(2) per day orally. Thus, our results suggest that the HPLC method for analyzing tRA and 13-cRA in plasma with the addition of NEM and Vit. C is a simple, sensitive and accurate method.
Graduate Institute of Life Science, National Defense Medical Center, Taipei, Taiwan, ROC.
Chao-Jih
CJ
Wang
Li-Heng
LH
Pao
Cheng-Huei
CH
Hsiong
Chih-Yuan
CY
Wu
Jacqueline Jia-Kang
JJ
Whang-Peng
Oliver Yoa-Pu
OY
Hu
D006528:Carcinoma, Hepatocellular / Q000097:blood; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D008113:Liver Neoplasms / Q000097:blood; D010865:Pilot Projects; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013056:Spectrophotometry, Ultraviolet; D013237:Stereoisomerism; D014212:Tretinoin / Q000097:blood / Q000737:chemistry* / Q000493:pharmacokinetics
D014212:Tretinoin