Coupling of capillary electrochromatography to coordination ion spray mass spectrometry, a novel detection method.

Miniaturized separation techniques such as capillary electrochromatography (CEC), pressurized capillary electrochromatography (pCEC) and capillary high performance liquid chromatography (CHPLC) have been coupled to a new detection technique: coordination ion spray mass spectrometry (CIS-MS). Electrospray ionization (ESI) has found widespread applications in mass spectrometry (MS) for the analysis of polar compounds such as peptides or nucleotides. However, for weakly polar or nonpolar substances, ESI-MS yields poor sensitivity since, in the absence of basic or acidic groups, protonation or deprotonation is not possible. CIS is a universal ionization technique capable of detecting these compounds. Through the addition of a central complexing ion, charged coordination compounds are formed, enabling the detection with good sensitivity. Using the coaxial sheath flow interface commonly employed in CE-MS coupling, we were able to separate and detect various important natural compounds such as unsaturated fatty acid methyl esters, vitamins D2 and D3, and four different estrogens. A central ion solution of 100 microg/mL AgNO3 in water was used as sheath flow liquid, resulting in the formation of positively charged coordination compounds.

Niosomes as a novel peroral vaccine delivery system.

The feasibility to develop a peroral vaccine delivery system based on non-ionic surfactant vesicles (niosomes) was evaluated using BALB/c mice. Ovalbumin was encapsulated in various lyophilized niosome preparations consisting of sucrose esters, cholesterol and dicetyl phosphate. Two different formulations were compared in this study. The specific antibody titres within serum, saliva and intestinal washings were monitored by ELISA on days 7, 14, 21 and 28 after intragastric administration. Only encapsulation of ovalbumin into Wasag7 (70% stearate sucrose ester, 30% palmitate sucrose ester (40% mono-, 60% di/tri-ester)) niosomes resulted in a significant increase in antibody titres. Administration of ovalbumin and empty niosomes did not exert a similar effect, neither did administration of any control formulation. In contrast to ovalbumin loaded Wasag7 niosomes, application of the more hydrophilic Wasag15 (30% stearate sucrose ester, 70% palmitate sucrose ester (70% mono-, 30% di/tri-ester)) niosome preparations did not result in an increase in antibody titres.

Isolation, purity analysis and stability of hyperforin as a standard material from Hypericum perforatum L.

In 1996 131.5 million daily doses of preparations containing extracts of Hypericum perforatum L. were prescribed in Germany for treating mild to moderately severe depressive disorders. New pharmacological and clinical results focus on hyperforin as the main active ingredient of the drug. Hyperforin (C35H52O4) is one of the main components (2-4%) of the dried herb Hypericum perforatum L. It was isolated after six consecutive steps: extraction of deep-frozen blossoms (-20 degrees C) with n-hexane by means of an Ultra Turrax at room temperature; separation of lipophilic substances on a silica gel column; purification of the relevant fraction by preparative HPLC; evaporation of the mobile phase under reduced pressure; removal of the remaining water by freeze-drying; and storage of hyperforin at -20 degrees C under nitrogen. The identity and purity of the isolated substance were determined by high-performance thin-layer chromatography (HPTLC), high-performance liquid chromatography (HPLC) with diode-array and ultraviolet detection (DAD and UV), Fourier-transformed infrared (FTIR) and proton nuclear magnetic resonance (1H NMR) spectroscopy, and liquid chromatography coupled with positive-ion electrospray-ionization tandem mass spectrometry (LC-ESI(+)-MS-MS). By use of these methods the purity of hyperforin was shown to be >99.9%. Peroxides present at each step of the isolation were detected by titration and by means of Merckoquant analytical peroxide test-strips. Elimination of the peroxides and stabilization of hyperforin was achieved by consistent protection from oxidation-the mobile phases were protected by use of ascorbic acid; evaporation and freeze-drying were performed under nitrogen; and the mobile phase used for preparative HPLC was sparged with helium. Stability testing was performed by HPLC-the samples were stored at -30 degrees C in a normal atmosphere and at -20, 4, and 20 degrees C in a normal atmosphere or under nitrogen. Results were compared with those obtained after storage under liquid nitrogen (-196 degrees C). Because of its high sensitivity to oxidation, hyperforin was more stable under nitrogen under all test conditions. There was no statistically significant difference between results obtained after 8 months at -20 degrees C under nitrogen or at -30 degrees C under a normal atmosphere and those from the reference sample stored under liquid nitrogen (-196 degrees C). Despite this, because of the tendency of hyperforin to degrade, long-term storage at -70 degrees C under nitrogen is recommended.

Separation and Identification of Tocotrienol Isomers by HPLC-MS and HPLC-NMR Coupling.

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-, ß-, γ-, and δ-tocotrienol, α-tocoenol, and α-tocopherol.

Silver-plated vitamins: a method of detecting tocopherols and carotenoids in LC/ESI-MS coupling.

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.