By on-line addition of a central atom (for example, Ag , B , Pd , Li ) positively or negatively charged complexes of analytes can be formed for CIS-MS. This technique is applicable to both polar and nonpolar compounds-for example, for alcohols, ethers, and a large number of olefins, polyolefins, and arenes as well as steroids, vitamins of the D and E families, carotinoids, polystyrols, terpenes, and unsaturated fatty acids-and can be readily coupled with separation techniques.

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.

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.

No abstract

No abstract