Chlorogenic acid derivatives are potent inhibitors of hepatic glucose production by inhibition of the glucose-6-phosphate translocase component of the hepatic glucose-6-phosphatase system. The pharmacological proof of concept was clearly demonstrated during i.v. infusion of potent derivatives (S 4048, S 3483) in rats. However, the blood glucose lowering effect of S 4048 after bolus i.v. injection lasted only 60-90 min. Plasma clearance of S 4048 was very high, and the parent compound was rapidly and efficiently excreted into the bile of Wistar and GY/TR(-) rats, indicating that mrp-2 was not involved in this hepatobiliary elimination process. About 72% of the total administered radioactivity appeared in the bile within 20 min after i.v. bolus injection of the radiolabeled analogue [(3)H]S 1743 in a Wistar rat. However, in GY/TR(-) rats the dicarboxylic analogue of S 4048, S 3025, was cleared from the plasma less rapidly than its parent compound and its biliary elimination was comparatively low. In contrast, S 3025 exhibited comparable pharmacokinetics and biliary elimination profile as S 4048 in Wistar rats, suggesting that biliary elimination of S 3025 is facilitated by mrp-2, functionally absent in GY/TR(-) rats. Targeting to mrp-2 resulted in a significantly prolonged reduction of blood glucose levels in GY/TR(-) rats after i.v. bolus administration of S 3025.

Retinoic acid-glucuronides are known as retinoids with activity in acne therapy, limited placental transfer and reduced retinoid adverse effects. We synthesized the glucuronide of a novel retinoid, CD271 (adapalene), used for the treatment of moderate acne. The synthesis product (“CD271 glucuronide”, CD271G) was purified by preparative HPLC. It undergoes in aqueous solution, like other glucuronides, rapid acyl-migration of the bound aglycone leading to position isomers. Thus characterization of purified CD271G could be only achieved by HPLC–NMR coupling. A subfraction (“CD271GB”) consisting essentially of 2′- and 3′-CD271G was used for pharmacokinetic studies. After a single subcutaneous injection at a dosage of 30 mg/kg the substance showed considerable uptake and metabolism to CD271 indicating that CD271GB could serve as a prodrug for CD271.

In this paper the application of on-line HPLC–UV–APCI (atmospheric pressure chemical ionization) mass spectrometry (MS) coupling for the separation and determination of different carotenoids as well as cis/trans isomers of β-carotene is reported. All HPLC separations were carried out under RP conditions on self-synthesized polymeric C30 phases. The analysis of a carotenoid mixture containing astaxanthin, canthaxanthin, zeaxanthin, echinenone and β-carotene by HPLC–APCI-MS was achieved by scanning the mass range from m/z 200 to 700. For the characterization of a sample containing cis/trans isomers of β-carotene as well as their oxidation products, a photodiode-array UV–visible absorbance detector was used in addition between the column and the mass spectrometer for structural elucidation of the geometrical isomers. The detection limit for β-carotene in positive-ion APCI–MS was determined to be 1 pmol. In addition, an extract of non-polar substances in vegetable juice has been analyzed by HPLC–APCI-MS. The included carotenoids could be identified by their masses and their retention times.

The hyphenation of HPLC together with NMR spectroscopy proves advantageous for the structure elucidation of oxidation- and UV-sensitive compounds such as β-carotene isomers. In the closed-loop HPLC–NMR system, degradation or isomerization of separated compounds is largely hindered. With the help of 3-μm C30 stationary phases a better separation efficiency towards the different β-carotene cis/trans isomers could be obtained in comparison to a 5-μm material, resulting in sharper peaks and a better resolution of all compounds. This effect greatly faciliated the structure determination of the isomers by HPLC–NMR coupling. Due to the introduction of a superior stationary phase, the structure of seven cis-isomers of β-carotene could thereby be determined employing the stopped-flow HPLC–1H NMR mode.

A crude palm-oil extract rich in vitamin E homologues was investigated by HPLC-MS and HPLC-NMR coupling. For mass spectrometry a newly introduced ionization technique called Coordination Ion Spray (CIS) was used. Through the addition of silver ions to the HPLC eluent, the ionization process of nonpolar substances is facilitated. Chromatography and all coupling experiments were conducted on a C(30) column which exhibited an extraordinary shape selectivity and overwhelming sample-loading capability. Experiments were performed with pure methanol as an eluent which proved to be ideal for NMR spectroscopy as well as mass spectrometry. All necessary information for unambiguous structural assignment was collected within 45 min of the LC-NMR experiment and 15 min of the LC-MS experiment. Six compounds were identified, i.e., α-, β-, γ-, and δ-tocotrienol, α-tocoenol, and α-tocopherol.

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.

In LC/MS, nonpolar substances in the majority of cases cannot be ionized by standard electrospray ionization (ESI) because they obviously lack a site for protonation or deprotonation. The ionization of carotenoids and tocopherols can be greatly enhanced by the addition of silver ions. The Ag(+)-carotenoid and Ag(+)-tocopherol adducts thus formed render these substances amenable to MS. alpha-, beta-, gamma-, and delta-tocopherol, alpha-tocopherol acetate, and the various isomers of lycopene and beta-carotene were separated by C30 RP-HPLC and could be identified by online ESI-MS. A mixture of six different carotenoids was analyzed by scanning the mass range from m/z 500 to 800. The mass spectra of the peaks revealed that all carotenoids and most tocopherols were partially oxidized to radical cations. The detection limit for canthaxanthin was approximately 500 fmol while that of beta-carotene was below 300 fmol. An increase in sensitivity in the MRM mode can be attained by monitoring ions formed by loss of elemental silver from the adducts in the CID cell. Dichloromethane extracts of tomato, carrot, and vegetable juices, a vitamin drink, and a commercial infant food product were analyzed by LC/MS. After postcolumn argentation, from the mass-selective extracts of the TIC, the carotenoids and tocopherols present could be identified by their masses and their retention times. For all studies, a silver perchlorate solution with an overall concentration of 50 micrograms/mL was used.

Vitamin E (tocopherol) acts in various organisms as the main free radical scavenger. This capacity, which is enhanced by the synergetic effect of vitamin C and carotenes, points to a possible application as anti-tumor agent in chemotherapy. There are several isomeric forms, namely alpha-, beta-, gamma-, and delta-tocopherol, having different antioxidative abilities, with alpha-tocopherol being the most biologically active. Using methanol as eluent and a C30 stationary phase, we achieved complete separation of alpha-, beta-, gamma-, and delta-tocopherol and alpha-tocopherol acetate by RP-HPLC within 14 min. Detection was performed by UV and 1H NMR spectroscopy. The advantage of NMR is the possibility of structural identification of chromatographic peaks. Also, coeluting peaks are easily recognized. The enhanced shape recognition of the C30 phase has been attributed to the high order of the alkyl chains of the stationary phase. This has, so far, been proven by solid-state NMR spectroscopy. We now introduce the technique of 13C MAS NMR spectroscopy of suspended stationary phases. The resulting NMR spectra reveal that, in the presence of weak eluents, like methanol, the overall high order of the C30 chains is slightly altered, whereas in stronger eluents, like MTBE, the alkyl chains possess a higher mobility.

The separation of cis/trans isomers of β-carotene has been performed with a C30 stationary phase employing 1H NMR spectroscopy as an on-line detection technique. 1D as well as 2D NMR spectra have been recorded in the stopped-flow mode for the predominant chromatographic peaks. Structural assignment of the five identified isomers was performed via comparison of simulated 1D 1H NMR spectra on the basis of the structures of β-carotene cis/trans isomers with the experimental data, and also by the analysis of the proton-proton connectivities in the 2D NMR spectra of three isomers with the highest concentration. The chromatographic retention behaviour of the isomers agreed well with previously reported data. The advantage of the applied hyphenated coupling technique compared to conventional off-line techniques lies in the fact that chromatographic separation and NMR detection are performed in a closed system, so that reisomerization of the separated compounds is inhibited.

Unlike HPLC-NMR coupling, the combination of supercritical fluid chromatography (SFC) with NMR spectroscopy is not yet a routine method, but the feasibility of this coupling technique has already been demonstrated with synthetic mixtures. In this paper the first real life application is presented and the potential of this new method is shown. A mixture of five cis/trans isomers of vitamin A acetate is separated by SFC using supercritical CO2 as eluent. Therefore no solvent signal suppression is necessary and the unrestricted observation of the whole spectral range is possible, contrary to the HPLC separation in n-heptane, where in the aliphatic region almost 2 ppm of the 1H NMR spectrum are affected by the suppression technique.

The comparison of two different types of RP stationary phases in their ability to separate cis/trans isomers of retinoic acid (tretinoin) has been investigated by LC-NMR coupling. Only by recording of 1H NMR spectra the structural identification of the separated compounds was possible, since their absorption coefficients are very similar and their mass is identical, and therefore identification by UV-Vis is not unambiguous and identification with LC-MS fails due to identical fragmentation patterns. A commonly used C18 phase and a recently developed C30 phase have been used for the separation of a mixture of thermal isomerized retinoic acids. Three isomers could be separated and identified with the separation on a C18 column, whereas five cis/trans isomers could be identified by the use of a C30 column. It could be shown that even under optimized chromatographic conditions the separation efficiency of the two phases varies and that these differences are probably due to differences in the alkyl chain organization of the stationary phases.

Abstract The p-nitrosophenolate anion, PNPA (3), represents an excellent example of variable π-electron transfer demanded by a network of different hydrogen bonds. The limiting structures of mono-protonation in the solid state and in solution are either the p-nitrosophenol (1) or the p-quinonoxime (2) molecules. For the systems 1 to 3 and the related compounds 4 to 7 are results of ab initio 6-31G HF MO optimizations of molecular structures presented which are calculated additionally in variously rotated conformations. Solid state 13C-CP/MAS NMR spectra of two PNPA salts 8 and 9 have been recorded at 7.0 Tesla. The protonation of 8 and 9 was studied in solution using a 400 MHz NMR spectrometer recording the variations in 1H and 13C NMR chemical shifts on changing the solvent from DMSO-d6 to D2O in steps of 25%. The 2D 1H-13C correlated spectrum leads to unambigous assignment of NMR signals. From the coalescence temperature of 1H signals of 9 the experimental barrier of rotation in D2O was determined as 16.9 ± 0.1 kcal mol-1. The calculated gas phase rotational barrier for free PNPA is with 26.9 kcal mol-1 much higher, but is lowered by calculations for the Li-salt 6 c to 14.5 kcal mol-1 and to 11.8 kcal mol-1 for the protonated PNP If. The in-plane inversion of PNPA through 3c is calculated to be extremely high with 52.4 kcal mol-1. The effects of rotation of substituents to orthogonal conformations on calculated energies and on intramolecular distances were studied thoroughly.

Temperature-dependent C-13 CP/MAS NMR investigations of C-30 bonded interphases show two different signals for the (CH2)(n) chain, with a chemical shift difference of 2.6 ppm. They are characterized by selective population inversion (SPI) measurements and contact time variation experiments. One signal belongs to more rigid chains with trans conformations, the other one to more mobile chains with trans/gauche conformations. With decreasing temperature, more trans conformations are formed. HPLC separations of vitamin A acetate isomers have been performed from 50 degrees C to 2 degrees C. The on-line coupling of HPLC with H-1 NMR spectroscopy yields information necessary for determination of the structures of the separated isomers. The conformational change of the C-30 interphases with the temperature can also be seen in the retention order of the vitamin A acetate isomers. At low temperatures, the most angled 9-cis isomer is the most strongly retained compound, whereas at high temperatures, the linear all-trans isomer exhibits the strongest interactions with the C-30 phase.

Coupling HPLC and NMR is one of the most powerful techniques for simultaneous separation and structural elucidation of unknown compounds in mixtures. To date, however, minimizing the detection volume, as is required when coupling NMR with miniaturized separation techniques, has been accompanied by a dramatic loss in resolution of the NMR spectra. Here, we report on the coupling of gradient capillary HPLC with on-column, high-resolution NMR detection. On-line stopped-flow and static (1)H NMR spectra were acquired with capillary columns of 75-315 μm i.d. With detection over a length of 1.2 cm, cell volumes cover a range of 50-900 nL. An on-line-detected NMR separation of dansylated amino acids was carried out in a 315 μm i.d. fused silica capillary packed to a length of 12 cm with C(18) stationary phase. The low solvent consumption makes the use of fully deuterated solvents economically feasible. NMR spectra with resolution on the order of 3 Hz were obtained using a 50 nL detection cell to measure 1.1 nmol of dansylated γ-aminobutyric acid under static conditions in a 75 μm i.d. capillary.