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a The spectra correspond to the solvent shown in parenthesis: LP = light petroleum, E = ethanol, C =

chloroform, B = benzene, A = acetone, H = hexane. b A[%m an arbitrary value of 2500 is often taken when no experimentally determined value has been reported for an unknown compound or to give an estimate of the total carotenoid content of an extract.

the measurement of the bioavailability of P-carotene and retinol. Commonly, bioavailability is determined after the administration of high doses of P-carotene (12 to 30 mg/day) or deuteriated compounds; however, none of these conditions is physiological, and absorption or bioconversion could be affected. Another approach is the use of gas chromatography (GC)-MS, but carotenoids are very sensitive and sample preparation is tedious. Instead of these methodologies, LC-MS using 13C-labeled P-carotene and an atmospheric pressure chemical ionization (APCI) mass detector has been implemented. This equipment shows a linear response in the range of P-carotene concentrations of 0.4968 to 99.36 pmol/pl; the lower limit of quantification is 560 fmol and the limits of detection are similar to retinol and retinyl palmitate. In addition, it has been observed that labeled and unlabeled P-carotene show similar retention times resulting in a method suitable for evaluating the absorption and bioavailability of P-carotene.104 An APCI detector in combination with a C30 column has been used to differentiate stereoisomers of carotenoids, and it has been established that the limit of detection is in the range of mass-to-charge ratio (m/z) of 200 to 800 and 1 pmol concentration. On the other hand, the HPLC-NMR coupling is still not sensitive enough to identify samples in the picomole range, but it remains the only technique allowing an unequivocal structural elucidation of unstable substances such as carotenoid stereoisomers, excluding the influence of light and oxygen.105

To date, the criteria for carotenoid identification are minimally co-chromatog-raphy with authentic samples, UV-visible, and mass spectra. Interestingly, new methodologies have been introduced to study carotenoids in vivo (Table 7.10), and photoacoustic spectroscopy has been used to evaluate paprika carotenoids; it is possible to use it as a tool to make a semiquantitative evaluation. The main peaks associated with paprika photoacoustic peaks are in the near infrared region (800 to 1000 nm).106

Another important approach in carotenoid characterization is the use of chemical tests (Table 7.10). Besides such tests, reflectance measurements have resulted as good descriptors of color; Hunter-Lab and Minolta equipment are among the most often used.107,108

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