Quantitative analysis of lycopene isomers in human plasma using high-performance liquid chromatography-tandem mass sectrometry

An analytical method for the determination of the concentrations of total lycopene and its cis and all-trans isomers in human plasma has been developed using high-performance liquid chromatography-tandem mass spectrometry (LC-MS-MS). This method was based on the observation that, during negative ion atmospheric pressure chemical ionization with collision-induced dissociation, a unique fragment of m/z 467 was formed from the molecular ion of m/z 536 by elimination of a terminal isoprene group. The use of multiple reaction monitoring facilitated the selective detection of lycopene isomers and an internal standard without interference from the isobaric carotenoids alpha-carotene and beta-carotene, which are also abundant in human plasma. Measurement of total lycopene was carried out using a C-18 high-performance liquid chromatography (HPLC) column and an isocratic mobile phase consisting of acetonitrile/methyl tert-butyl ether (95:5) so that all lycopene isomers eluted as a single chromatographic peak. all-trans-Lycopene was separated from its various cis isomers by using a C-30 carotenoid column and a gradient solvent system from methanol to methyl tert-butyl ether. The effects of sample preparation and handling parameters on the stability of lycopene were evaluated such as the stability of lycopene in the HPLC autosampler and the effect of saponification upon lycopene isomerization. For example, the half-life of all-trans-lycopene in the HPLC mobile phase in the autosampler at 4 degreesC was determined to be similar to16 h. Also, saponification of plasma samples was determined to cause lycopene degradation and isomerization so that lycopene recovery was reduced. The accuracy and interassay precision of this LC-MS-MS assay for lycopene showed a standard deviation of less than 10% over the range of 5-500 pmol injected on-column. The limit of detection was 11.2 fmol injected on-column, and the limit of quantitation was 22.8 fmol.