Separation of plasma carotenoids and quantitation of beta-carotene using HPLC.

A method was developed for the extraction and separation of human plasma carotenoids and quantitation of beta-carotene. Carotenoids were extracted from plasma with ethanol: hexane and separated by C18 reversed phase HPLC using spherical 3 micron packing. beta-Carotene was identified and quantitated using an external standard. The within-run precision of three different plasma pools ranged from 3.53-5.72% relative standard deviation (RSD). The between-run precision was 7.34% RSD. The method was linear to 500 micrograms/l with a statistical detection limit of 3.80 micrograms/l. Recovery of added beta-carotene was from 90.41-100.37%. This method was compared to a spectrophotometric 'total carotene' method. The mean plasma concentrations of 25 male and 25 female human volunteers for the 'total carotene' were 1,549 micrograms/l for all samples, 1,487 micrograms/l for males and 1,611 micrograms/l for females. The corresponding true beta-carotene concentrations obtained by HPLC analysis were 134.8, 115.9 and 153.7 micrograms/l, respectively. The true beta-carotene concentrations were on the average only 8.76% (8.07% for males and 9.46% for females) of the concentrations obtained by the spectrophotometric 'total carotene' method. Correlation between the methods had an r = 0.6107. The poor correlation is due to the difference in the measured components. Total carotene methods measure all solvent extractable moieties having absorbance in the 430-460 nm region, while the HPLC method quantitates true beta-carotene after chromatographic separation from other carotenoids. Reference intervals were established for plasma beta-carotene using REFVAL, an IFCC computer program for determining statistical reference intervals. The reference interval for all samples is 40 to 344 micrograms/l.