Jourmd of Citromalography, 572 ( 1991) 103-116 Biomedical Applications Elsevier Science Publishers B.V., A m s t e r d a m C H R O M B 1 0 . 6074
Simultaneous determination of retinol, -tocopherol and fl-carotene in serum by isocratic high-performance liquid chromatography J. A R N A U D * . I. F O R T I S , S. B L A C H I E R , D. K I A and A. F A V I E R
Laboratoire tit, Biochimie C. CHRUG. BP 217. 38043 Grenoblt, Ct, de.v 9 (France) (First received M a y 22nd, 1991: revised manuscript received July I I th, 1991) ABSTRACT A simultaneous determination o f rctinol, ct-tocopherol and fl-carotcne in serum by lligh-pcrform;mce liquid c h r o m a t o g r a p h y is dcsdribed. Total analysis time is 13 rain. A reverscd-phase (Ultrasphere ODS, 5 l~m) column is uscd with a mobile phase o f a c c t o n i t r i l e - m e t h a n o l - d i c h l o r o m e t h a n c (70:10:20, v/v/v) and a flow-rate o f 1.2 ml/min. Retinol is m o n i t o r e d at 325 nm, ~-tocophcrol at 292 nm and fl-carotene at 450 nm. Serum is deprotcinizcd with ethanol containing the internal standard (~t-tocophcrol acetate), then extracted with hexanc. T h e e v a p o r a t e d organic layer is reconstituted with the mobile phase and injccted. T h e choice o f the eluent is discussed, as well as the choice o f an internal st:mdard and the need for an antioxidant during the extraction step. Sixteen different eluents arc c o m p a r e d in terms o f analysis time and selectivity. The linear coitcentration ranges (rctinol 0.016-13.7 ll~l, ~t-tocopherol 0. ! 8-91.8 ItM, fl-earotene 0.05-5.75 ItM), within-run coefficients o f variation (retinol < 7%; ~-tocopherol < 8%, fl-carotene < 7%), betweenrun coefficients o f variation (retinol < 13%, ~-tocopherol < 9 % , fl-carotene < 8 % ) and recoveries (retinol > 9 5 % , ~t-tocopherol > 9 1 % , fl-carotene > 8 0 % ) are suitable for clinical investigations. Scram rcfercnce values were found to be 2 . 4 7 4 - 0.61 I~M (retinol), 30.5 4- 6.8 lllt¢ (~t-tocopherol) and 0.91 ± 0.55 I~M (fl-carotene). A signillcant difference (p
Simultaneous determination of retinol, -tocopherol and fl-carotene in serum by isocratic high-performance liquid chromatography J. A R N A U D * . I. F O R T I S , S. B L A C H I E R , D. K I A and A. F A V I E R
Laboratoire tit, Biochimie C. CHRUG. BP 217. 38043 Grenoblt, Ct, de.v 9 (France) (First received M a y 22nd, 1991: revised manuscript received July I I th, 1991) ABSTRACT A simultaneous determination o f rctinol, ct-tocopherol and fl-carotcne in serum by lligh-pcrform;mce liquid c h r o m a t o g r a p h y is dcsdribed. Total analysis time is 13 rain. A reverscd-phase (Ultrasphere ODS, 5 l~m) column is uscd with a mobile phase o f a c c t o n i t r i l e - m e t h a n o l - d i c h l o r o m e t h a n c (70:10:20, v/v/v) and a flow-rate o f 1.2 ml/min. Retinol is m o n i t o r e d at 325 nm, ~-tocophcrol at 292 nm and fl-carotene at 450 nm. Serum is deprotcinizcd with ethanol containing the internal standard (~t-tocophcrol acetate), then extracted with hexanc. T h e e v a p o r a t e d organic layer is reconstituted with the mobile phase and injccted. T h e choice o f the eluent is discussed, as well as the choice o f an internal st:mdard and the need for an antioxidant during the extraction step. Sixteen different eluents arc c o m p a r e d in terms o f analysis time and selectivity. The linear coitcentration ranges (rctinol 0.016-13.7 ll~l, ~t-tocopherol 0. ! 8-91.8 ItM, fl-earotene 0.05-5.75 ItM), within-run coefficients o f variation (retinol < 7%; ~-tocopherol < 8%, fl-carotene < 7%), betweenrun coefficients o f variation (retinol < 13%, ~-tocopherol < 9 % , fl-carotene < 8 % ) and recoveries (retinol > 9 5 % , ~t-tocopherol > 9 1 % , fl-carotene > 8 0 % ) are suitable for clinical investigations. Scram rcfercnce values were found to be 2 . 4 7 4 - 0.61 I~M (retinol), 30.5 4- 6.8 lllt¢ (~t-tocopherol) and 0.91 ± 0.55 I~M (fl-carotene). A signillcant difference (p
