The aim of the investigation was to assess a stable isotope method for determining the relative bioavailability of food-derived lutein in humans. Subjects were administered a single dose of deuterium-labeled carotenoids from intrinsically labeled spinach or collard green; 10 mL blood samples were drawn at various time points over a 34 days period. The vegetables had been hydroponically grown using 25 atom-% deuterated water. Lutein molecules in the vegetables were partially deuterated with a highest abundance isotopomer at M0 + 8 (unlabeled molecular mass, M0, plus 8 additional mass units from 8 deuterium atoms in the molecules). This allowed labeled lutein to be distinguished from endogenous lutein in serum samples after consuming the labeled meal. The presence of labeled lutein in the circulation was determined by liquid chromatography-mass spectrometry (LC/MS) equipped with an atmospheric pressure chemical ionization (APCI) interface. The quantification of the labeled lutein in serum samples enabled the calculation of the enrichment for each time point after the dose; these values were plotted vs. time to generate absorption-clearance curves for each of the subjects. Area under the curve analyses of four different subjects (integrated over 29 days) yielded serum lutein responses of 128, 145, 149, and 262 μg-day/mg dietary lutein, following an acute dose of spinach containing 15.4, 18.8, 18.8 and 9.8 mg labeled lutein, respectively. This technique will facilitate the study of lutein bioavailability from different foods of diverse carotenoid composition and/or following various food preparation procedures.

The carotenoids lutein and zeaxanthin have been identified as the macular pigments of the human retina. Nutritional epidemiological reports indicate that high consumption of fruits and vegetables rich in these carotenoids is correlated with reduced risk of some illnesses, e.g. agerelated macular degeneration (AMD).

This chapter contains sections titled: Introduction Artifact-free Isolation of Geometrical Carotenoid Isomers Analysis of Lycopene Stereoisomers in Tomato Extracts and Human Serum Identification of Lycopene Stereoisomers in Tomato Extracts Employing LC–NMR Conclusions References

Gingival samples treated with toothpastes containing tocopherols (vitamin E) were investigated employing HPLC chromatography. The aim was to verify that vitamin E is actually enriched in the tissue, which could have beneficial effects on oral health. After determination of the tocopherols available in the toothpastes, control samples from healthy test persons and subjects suffering from gingivitis were analyzed. Subsequently, gingival tissues from diseased test persons who treated their teeth with the toothpastes containing tocopherols using various kinds of concentrations or applications were investigated. The first step of the analysis was a fast and careful extraction. employing matrix solid-phase dispersion (MSPD). Afterward, the separation of the different tocopherol homologues existing was performed by HPLC chromatography on highly selective C-30 RP phases. The identification of the tocopherol homologues was performed using the on-line coupling of HPLC with NMR spectroscopy and mass spectrometry.

The core-shell structure of lycopene micronizates can be verified by employing a combination of solid-state and suspended-state NMR spectroscopy. The type of molecular aggregation of carotenoid nanoparticles can be clearly determined from their characteristic fingerprint pattern in the solid-state NMR spectra.

The structural identity of two steroidal components in the low μg range of a pharmaceutical gel was determined by a rapid and efficient analytical method employing online HPLC-NMR coupling. The chromatographic separation conditions were optimised towards the requirements of on-line 1 H NMR detection, employing a tailored C 30 column together with a gradient elution of a methanol/D 2 O mixture. Stopped-flow 1D and 2D 1 H NMR spectra of the investigated chromatographic peaks unequivocally revealed the structure of the corresponding steroids norethisterone acetate and estradiol.

Amino acids lining the catalytic cavity of squalene–hopene cyclase of Alicyclobacillus acidocaldarius were mutated to investigate their catalytic functions. Mutagenesis of Leu607 to Lys in the central part of the cavity resulted in the production of the bicyclic γ-polypodatetraene (1) as main product, while the mutation of Phe605 to Lys near the deprotonation site of the cavity led mainly to the formation of tetracyclic 17-isodammara-20(21),24-diene (2).

The determination and unambiguous identification of carotenoid stereoisomers from biological tissues, avoiding isomerization and oxidation due to the extraction process, is still a major challenge. Particularly, the analysis of lutein and zeaxanthin stereoisomers is of great importance, as these are the main constituents of the macula lutea, the central part of the human retina, and act as possible agents in the prevention and treatment of age-related macular degeneration (AMD). By combining a mild and quick extraction technique such as matrix solid-phase dispersion together with high-performance liquid chromatography (HPLC), the extremely light and oxygen sensitive lutein and zeaxanthin stereoisomers are extracted, enriched, and separated directly from the solid plant or tissue samples, excluding preparation of artifacts. HPLC separations are performed with C30 phases due to their enhanced shape selectivity compared to C18 phases and on-line coupled to mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. By using HPLC-MS with atmospheric pressure chemical ionization, the lutein stereoisomers can be distinguished from the zeaxanthin stereoisomers within one chromatographic run in the upper picogram range, whereas HPLC-NMR coupling allows the unequivocal identification of each stereoisomer with a concentration in the upper nanogram range. This article provides an analytical method for the artifact-free determination of lutein and zeaxanthin stereoisomers directly from the solid biological tissue spinach as a source of carotenoids and retina as the sphere of activity for AMD. In addition, the structures of these stereoisomers were unambiguously elucidated by employing hyphenated analytical techniques.

The 2(3),9(10),16(17),23(24)-tetrasubstituted metalphthalocyanines 1-7 (M = In, Ni, Zn) were synthesized, as mixtures of four different structural isomers, from the corresponding 4-alkoxy-1,2-dicyanobenzenes and the appropriate metal salts. Separation of the four structural isomers was successfully achieved on a C30 alkyl phase by high-performance liquid chromatography (HPLC). The determination of the point groups of the structural isomers was carried out for 1 and 3, the composition of the structural isomers of 4-7 was accomplished by comparing their retention times and UV/Vis spectra with the data of 1 and 3. For the phthalocyanines 8-10 and the naphthalocyanines 11 and 12 only the C4h and D2h isomers could be separated.

The metabolites of the antipsychotic drug trifluoperazine (TFP) were investigated with on-line coupling of high-performance liquid chromatography (HPLC) and NMR spectroscopy. A chromatographic method was developed using a reversed-phase C-30 silica gel column. Rats were given a cumulative dose of 90 mg kg(-1) TFP dihydrochloride and samples of brain and muscle tissues were extracted after 29 h. The peaks obtained on chromatographic separation were assigned to trifluoperazine and its metabolites. With the help of chromatograms obtained from test mixtures, 1D and 2D HPLC-NMR were used to identify the peaks found for these mixtures. Copyright (C) 2000 John Wiley & Sons, Ltd.

Looking to the future, evolutionary development can be expected because the separating power of multidimension liquid chromatography is greatly increased over single-dimension liquid chromatography. The simultaneous improvements on both the software and the chromatographic apparatus will lead to a system capable of automatically developing analytical methods for a wide range of analytes in many different matrices. Other desirable aspects are a reasonably short analysis time and Sexible operating conditions. The thrust of multidimensional research will most probably be to improve the combination of separation methods, including coupling to alternative separation techniques.

The application of on-line C30-reversed-phase high-pressure liquid chromatography–nuclear magnetic resonance spectroscopy is described for the analysis of tetraglycosylated flavonoids in aqueous and hydroalcoholic extracts of the leaves of Maytenus aquifolium (Celastraceae). Triacontyl stationary phases showed adequate separation for on-line 1H-NMR measurements at 600 MHz and allowed the characterisation of these flavonoids by detection of both aromatic and anomeric proton signals. Copyright © 2000 John Wiley & Sons, Ltd.

The hyphenation of chromatographic separation techniques with NMR spectroscopy is one of the most powerful and time-saving methods for the separation and structural elucidation of unknown compounds and molecular compositions of mixtures. Most of the routinely used NMR flow-cells have detection volumes between 40–180 μL for conventional separations with analytical columns, and the newest designs employ detection volumes in the order of 200 nL for capillary separations. The low flow rates used in capillary chromatography permit the use of deuterated solvents. Unequivocal structural assignment of unknown chromatographic peaks is possible by two-dimensional stopped-flow capillary HPLC-NMR experiments.