The development of miniaturized hyphenated systems such as capillary high-performance liquid chromatography–and nuclear magnetic resonance spectroscopy (HPLC–NMR) remains challenging in the field of structure elucidation. In combination with a highly specific sample preparation technique, matrix solid-phase dispersion (MSPD), and a highly selective C30 reverse phase HPLC–NMR enables the identification of small amounts of natural compounds. Here, the investigation of five carotenoids in a standard solution and two carotenoids from a spinach sample demonstrate the potential of this new development. The separation of the carotenoids is performed with self-packed fused-silica capillaries with a binary solvent gradient consisting of acetone and water. The miniaturized system allows the use of fully deuterated solvents for on-line HPLC–NMR coupling. The 1H NMR spectra of the various carotenoids obtained in stopped-flow mode gave a high signal-to-noise ratio with a sample amount in the low nanogram range. All necessary parameters for structure elucidation such as multiplet structure, coupling constants and integration values can be detected unambiguously.

The protein kinase ZAP‐70 is involved in T‐cell activation and interacts with tyrosine‐phosphorylated peptide sequences known as immunoreceptor tyrosine activation motif s (ITAMs). We have studied the regulatory phosphorylation sites in the tryptic fragment containing amino acids 485–496 (ALGADDSYYTAR). The four possible peptides with phosphorylation at none, one, or both of the Y‐492 and Y‐493 tyrosines were specifically synthesized and analyzed by 1 H/ 13 C–NMR at 600 MHz using a capillary HPLC–NMR microprobe. Unambiguous discrimination of the peptides was possible via effect of chemical shifts of phosphorylation on the aromatic tyrosine protons. With the microprobe and the detection volume of 1.5 µl, it was possible to perform structure elucidation with the very small amounts available for the various peptides. For the syringe injection, 15 µg of the analyte were used (corresponding to ca 2 mg in classical 5‐mm tubes). Capillary HPLC–NMR spectra were recorded in the stopped‐flow mode from less than 400 ng of each peptide, using 1D and 2D techniques ( 1 H, 1 H–COSY‐90, 1 H/ 13 C–HSQC, and 1 H/ 13 C–HMBC). Copyright © 2005 John Wiley & Sons, Ltd.

Miniaturization and hyphenation of chromatographic separation techniques to nuclear magnetic resonance spectroscopy is being increasingly demanded in the field of biomedical, drug metabolite and natural product analysis. Herein, capillary liquid chromatography was coupled on-line to microcoil 1H nuclear magnetic resonance spectroscopy (capLC–NMR) equipped with a 1.5μL solenoidal probe for structure elucidation of isoflavones in Radix astragali. The extract was screened by HPLC–UV–MS as the preliminary step and four major peaks were identified tentatively by ion trap mass spectrometry molecular weights and characteristic fragments. Then, stopped-flow capLC–UV–NMR was performed using 33μg extract injected on-column. The four peaks were parked manually in the micro probe one by one and corresponding 1H NMR spectra were recorded with good resolutions under the applied capLC–NMR conditions (120 and 220ng injected on-column for peaks 2 and 4, respectively). All aromatic regions of 1H NMR spectra correlated well to the characteristic signals of isoflavone aglycone protons. And the signal corresponding to the anomeric proton of the glucopyranoside of isoflavone glycoside was also obtained for peak 1. Therefore, these four peaks are determined as calycosin-7-O-β-d-glucopyranoside (1), ononin (2), calycosin (3) and formononetin (4) unambiguously. The capLC–NMR results indicate that this hyphenated technique could be used for the determination of a great variety of natural products from small sample amounts, e.g., only 5g R. astragali in this study.

HPLC atmospheric pressure chemical ionization (APCI)/MS, GC MS, HPLC diode array detection (DAD), and NMR were used for the identification of astaxanthin and astaxanthin fatty acid esters in krill (Euphausia superba Dana). Matrix solid phase dispersion was applied for the extraction of the carotenoids. This gentle and expeditious extraction technique for solid and viscous samples leads to distinct higher enrichment rates than the conventional liquid-liquid extraction. The chromatographic separation was achieved employing a C30 RP column that allows the separation of shape-constrained geometrical isomers. A methanol/tert-butylmethyl ether/water gradient was applied. (all-E) Astaxanthin and the geometrical isomers were identified by HPLC APCI/MS, by coelution with isomerized authentical standard, by UV spectroscopy (DAD), and three isomers were unambiguously assigned by microcoil NMR spectroscopy. In this method, microcoils are transversally aligned to the magnetic field and have an increased sensitivity compared to the conventional double-saddle Helmholtz coils, thus enabling the measurement on small samples. The carotenol fatty acid esters were saponified enzymatically with Lipase type VII from Candida rugosa. The fatty acids were detected by GC MS after transesterification, but also without previous derivatization by HPLC APCI/MS. C14:0, C16:0, C16:1, C18:1, C20:0, C20:5, and C22:6 were found in astaxanthin monoesters and in astaxanthin diesters. (all-E) Astaxanthin was identified as the main isomer in six fatty acid ester fractions by NMR. Quantitation was carried out by the method of internal standard. (13-cis) Astaxanthin (70 microg/g), 542 microg/g (all-E) astaxanthin, 36 microg/g unidentified astaxanthin isomer, 62 microg/g (9-cis) astaxanthin, and 7842 microg/g astaxanthin fatty acid esters were found.

An overview of recent advances in nuclear magnetic resonance (NMR) coupled with separation technologies and their application in natural product analysis is given and discussed. The different modes of LC-NMR operation are described, as well as how technical improvements assist in establishing LC-NMR as an important tool in the analysis of plant-derived compounds. On-flow, stopped-flow and loop-storage procedures are mentioned, together with the new LC-SPE-NMR configuration. The implementation of mass spectrometry in LC-NMR is also useful on account of the molecular weight and fragmentation information that it provides, especially when new plant species are studied. Cryogenic technology and capillary LC-NMR are the other important recent developments. Since the plant kingdom is endless in producing potential drug candidates, development and optimization of LC-NMR techniques convert the study of natural products to a less-time-consuming task, speeding up identification.

The structures of biosynthetic deuterated carotenoids in labeled vegetables were investigated: (all-E)-lutein and (all-E)-beta-carotene from spinach, and (all-E)-beta-carotene and (all-E)-alpha-carotene from carrots. The vegetables were grown hydroponically using a nutrient solution enriched with deuterium oxide (D(2)O) and were extracted using matrix solid-phase dispersion (MSPD). Deuterium enrichment in the carotenoid molecules was determined by liquid chromatography-mass spectrometry (LC-MS). (all-E)-Lutein and (all-E)-beta-carotene in spinach showed partial deuteration from (2)H(1) to (2)H(12), with the abundance maximum at (2)H(5). (all-E)-beta-Carotene and (all-E)-alpha-carotene from carrots showed partial deuteration from (2)H(1) to (2)H(17), with the abundance maximum at (2)H(11). The (1)H NMR spectra of the four deuterated carotenoids showed additional signals for all methyl groups and decreased signal intensity for the olefinic protons and the methylene protons in the ring. These differences are due to isotopic effects and are based on the substitution of protons by deuterium atoms. The deuteration was distributed randomly throughout the carotenoid molecules.

Highly selective reversed phases (C-30 phases) are self-packed in 250,mum inner diameter fused-silica capillaries and employed for capillary HPLC separation of shape-constrained natural compounds (tocopherol homologues, vitamin E). Miniaturized hyphenated systems such as capillary HPLC-ESI-MS (positive ionization mode) and, with special emphasis, continuous-flow capillary HPLC-NMR are used for structural determination of the separated compounds. Despite the small amount of sample available (1.33 mug of each tocopherol), the authors have been able to monitor the capillary HPLC separation under continuous-flow H-1 NMR conditions, thus allowing an immediate peak identification. Further structural assignment was carried out in the stopped-flow NMR mode as shown, for example, by a 2D H-1,H-1 COSY NMR spectrum of cc-tocopherol. We demonstrate in this paper the considerable potential of hyphenated capillary separations coupled to MS and NMR for the investigation of restricted amounts of sample.

The isoflavonoids in Radix astragali were determined and identified by HPLC–photodiode array detection–MS after extraction employing matrix solid-phase dispersion (MSPD). As a new sample preparation method for R. astragali, the MSPD procedure was optimized, validated and compared with conventional methods including ultrasonic and Soxhlet extraction. The amounts of two major components in this herb, formononetin (6) and ononin (2), were determined based on their authentic standards. Four major isoflavonoids, formononetin (6), ononin (2), calycosin (5) and its glycoside (1), and three minor isoflavonoids, (6aR,11aR)-3-hydroxy-9,10-dimethoxypterocarpan (7), its glycoside (3), and (3R)-7,2′-dihydroxy-3′,4′-dimethoxyisoflavone-7-O-β-d-glycoside (4), were identified based on their characteristic two-band UV spectra and [M+H]+, [aglycone+H]+ and [A1+H]+ ions, etc. The combined MSPD and HPLC–DAD–MS method was suitable for quantitative and qualitative determination of the isoflavonoids in R. astragali.

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).

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.