II ELSEVIER Journal of Chromatography B, 678 (1996) 187-195 JOURNALOF CHROMATOGRAPHY B: BIOMEDICAL APPLICATIONS Liquid chromatographic determination of carotenoids in human serum using an engineered C30 and a C18 stationary phase Katherine E. Sharpless*, Jeanice Brown Thomas, Lane C. Sander, Stephen A. Wise Analytical Chemistry Division, Chemical Science and Technology Laboratory, National Institute of Standards and Technology, Gaithersburg, MD 20899, USA Received 21 August 1995; revised 31 October 1995; accepted 1 November 1995 Abstract A C3o stationary phase was specifically engineered for carotenoid separations, and carotenoid measurements using this column are compared with those obtained using a somewhat more conventional C~8 column. Both methods were used to contribute measurements for the certification of carotenoids in Standard Reference Material 968b, Fat-Soluble Vitamins and Cholesterol in Human Serum. Analytes were extracted from the serum into hexane. Measurements on the C~8 column were made using a gradient of acetonitrile, methanol, and ethyl acetate, which is described in detail elsewhere. Measurements on the C3o column were made using a gradient of water, methanol, and methyl tert.-butyl ether. Keywords : Carotenoids 1. Introduction Carotenoids constitute a group of compounds that continue to be studied for their possible ability to reduce the risk of developing certain types of cancer, and B-carotene has received the most attention in this regard [1 ]. '/3-Carotene', however, can occur as a variable mixture of trans-fl-carotene and several mono- and di-cis isomers. These cis isomers have less provitamin A activity than trans-fl-carotene, therefore accurate determination of the trans form in the presence of cis isomers is nutritionally important. AOAC International continues to recognize the need for reliable methods for the measurement of cis and trans isomers of/3-carotene [2]. *Corresponding author. Cis isomers may have cancer-preventive activity; however, lack of good measurement technology hinders the acquisition of epidemiological data for these compounds. Workers have separated some /3- carotene isomers in serum samples; these methods frequently separate cis and trans isomers, but may not resolve all cis isomers from each other [3-8]. Wide-pore polymeric C ~8 columns have been shown to separate geometric isomers of/3-carotene [9] and of lutein, lycopene, c~-carotene, and/3-carotene [10]. Calcium hydroxide and alumina columns are also able to separate the/3-carotene isomers [11,12], but these columns are not commercially available and do not separate other carotenoids present in serum [7]. In comparisons of carotenoid selectivity involving dozens of columns, polymerically modified station- ary phases [13] have been shown to provide better SSDI 0378-4347(95)00494-7