Use of high-performance liquid chromatography-tandem mass spectrometry to distinguish Panax ginseng C. A. Meyer (Asian ginseng) and Panax quinquefolius L. (North American ginseng).

A liquid chromatography-tandem mass spectrometry (LC-MS-MS) method was developed to distinguish Asian ginseng (Panax ginseng C. A. Meyer) and North American ginseng (Panax quinquefolius L.). The method is based on the baseline chromatographic separation of ginsenoside Rf and 24(R)-pseudoginsenoside F11, two potential chemical markers present in ginseng root methanolic extracts, and their unambiguous on-line identification using tandem mass spectrometry. Consistent with the literature, 24(R)-pseudoginsenoside F11 was detected in abundance in North American ginseng roots in excess of 0.1% (w/w) of the dried root. In contrast to some reports, 24(R)-pseudoginsenoside F11 was also identified in Asian ginseng roots at trace levels using LC-MS-MS but at less than 0.0001% (w/w). Besides showing identical tandem mass spectra to authentic 24(R)-pseudoginsenoside F11, the corresponding compound in Asian ginseng root coeluted with standard under different HPLC conditions, thus confirming this compound as 24(R)-pseudoginsenoside F11. Another ginsenoside often used to distinguish Asian and North American ginseng, ginsenoside Rf, was found in abundance in Asian ginseng roots at more than 0.021% (w/w). In Asian ginseng roots, the ratio of ginsenoside Rf to 24(R)-pseudoginsenoside F11 exceeded 700:1. The limit of detection of ginsenoside Rf or 24(R)-pseudoginsenoside F11 was 120 pg injected on-column, and the limit of quantification was 240 pg on-column. In summary, LC-MS-MS analysis of ginseng products for the presence and ratio of ginsenoside Rf and 24(R)-pseudoginsenoside F11 may be used for the unambiguous identification of Asian and North American ginsengs.

Stimulation of interleukin-1 and -6 production in alveolar macrophages by the neotropical liana, Uncaria tomentosa (uña de gato)

Two extracts of different collections of the traditional medicine uña de gato (Uncaria tomentosa) from Peru were characterized by High Pressure Liquid Chromatography as containing approximately 6 mg/g total oxindole content prior to studies with alveolar macrophages. The plant preparations greatly stimulated IL-1 and IL-6 production by rat macrophages in a dose dependent manner in the range of 0.025-0.1 mg/ml. They were also able to enhance IL-1 and -6 in lipopolysaccharide-stimulated macrophages. The results suggest a strong immunostimulant action of this plant.

Effect of temperature on stability of marker constituents in Echinacea purpurea root formulations.

Stability of an alkamide and a phenolic phytochemical marker in a hydro-alcoholic extract of Echinacea purpurea root and a dried powder prepared by evaporation of the extract was assessed in storage for 7 months at three temperature regimes: -20, 25 and 40 degrees Celsius. In the extract, the major alkamide, dodeca-2E, 4E, 8Z, 10E/Z-tetraenoic acid isobutyl amide, was not significantly affected by storage at any of the temperatures, but cichoric acid content declined as significantly (P = 0.05) at both 25 degrees C and 40 degrees C as compared to low-temperature storage. In the powder, the major alkamide showed a significantly reduced level at 25 degrees C and 40 degrees C while cichoric acid did not decline significantly. These results suggest that more attention should be given to the effect of formulation and temperature on storage of Echinacea products.

A bioassay for inhibition of serotonin release from bovine platelets.

A bioassay was developed to study agents capable of inhibiting the release of serotonin from bovine blood platelets. It is a simple, inexpensive, and reproducible high-throughput bioassay suitable for quality control of feverfew, Tanacetum parthenium, a crude drug with proven migraine prophylactic activity that is being considered for governmental registration and regulation. The bioassay, which requires no experimental animals or human subjects, was used to assess the in vitro activity of T. parthenium samples grown from seed obtained from 10 different regions of Europe. The activity was found to vary significantly within and between samples, with no geographical correlation. Serotonin release inhibition was shown to be significantly correlated with the content of the germacranolide sesquiterpene lactone, parthenolide, although other sesquiterpene lactones from this plant and other members of the Asteraceae were also shown to be active. The activities of six other species of Tanacetum, as well as of Artemisia absinthium (wormwood) and Zingiber officinale (ginger), and two commercial drugs for migraine prophylaxis, verapamil hydrochloride and propranolol hydrochloride, were also assessed. The relevance of the bovine platelet serotonin release inhibition bioassay to antimigraine research is discussed.

Parthenolide content and bioactivity of feverfew (Tanacetum parthenium (L.) Schultz-Bip.). Estimation of commercial and authenticated feverfew products.

Three physicochemical methods (HPLC, NMR spectroscopy, and HPLC of a derivative) have been used to measure parthenolide in authenticated Tanacetum parthenium (feverfew) and in several commercial purported feverfew products. A bioassay based on inhibition of the secretory activity of blood platelets by extracts of feverfew in comparison with parthenolide was also used. Similar results were obtained for all three physicochemical assays and also for the bioassay. Thus different methodologies yield consistent values for parthenolide content of feverfew preparations. Parthenolide appears to be mainly responsible for the antisecretory effects of extracts of feverfew. Authenticated Tanacetum parthenium grown in the UK contained a high level of parthenolide in leaves, flowering tops and seeds but a low level in stalks and roots. The level of parthenolide in powdered leaf material fell during storage. The purported feverfew products varied widely in their parthenolide content and in some products parthenolide was not detected. Possible reasons for the variation in parthenolide content are discussed. Since therapeutic efficacy has only been demonstrated for preparations of feverfew that contain parthenolide, it is suggested that manufacturers of feverfew products should use measurements of parthenolide as a means of standardization and quality control.

Aggregation of commercial heparin samples in storage.

The size distribution of heparin aggregates in commercial heparin preparations was examined with the technique of quasi-elastic light scattering. The size distributions were initially examined to determine if any relationship existed between the physical state of the heparin preparation, its age, and its biological activity. It was found that commercial heparin samples change their aggregation state in storage. The amount of aggregation appears to be related to the amount of time in storage and to the storage history. Storage of the samples under conditions of refrigeration and handling represents the storage history that most noticeably increases the aggregation state of the heparin preparations. These aggregates, once formed, appear to be stable. The biological activity of the heparin samples (as measured by the official test) was found to still fall within the accepted limits, independent of the aggregation state of the samples. It is not known what effect, if any, a change in the physical state of the commercial preparation should have on its biological activity.

Gas chromatographic and mass spectrometric analysis of the products of methylation of sulfonylurea drugs.

Gas chromatographic analysis of the products of reaction of diazomethane with tolbutamide and chlorpropamide indicates the formation of three compounds in both cases. As expected, N-methylation (at sulfonamide nitrogen) is the predominant reaction; minor amounts of O-methylated product are also observed. The third product in both cases is the N-methylsulfonamide formed by decomposition of the N-methylated sulfonylurea during gas chromatography. Electron impact and chemulfonylurea during gas chromatography. Electron impact and chemical ionization mass spectrometric analysis, as well as 1H nuclear magnetic resonance examination of samples collected from gas chromatography, confirm the structural assignments. Additionally, proton magnetic resonance analysis of the crude reaction products established that N-methylsulfonamides are not formed in the course of the diazomethane reaction and that the O-methylated derivatives are true products of the reaction. The use of a paramagnetic shift reagent allowed direct estimation of the ratios of N- to O-methylation, and the demonstration that these ratios are not vitiated during gas chromatographic analysis.

Mass spectral and pyrolytic behavior of the two main products of phenylbutazone degradation: simulation of unusual mass spectral fragmentation.

The two major routes of degradation of phenylbutazone (I) are oxidation and hydrolysis. Hydrolysis gives rise to n-butylmalonic acid mono(N,N'-diphenyl)hydrazide (II). Oxidation at the C-4 position yields 1,2-diphenyl-4-n-butyl-4-hydroxpyrazolidine-3,5-dione (III), which is readily hydrolyzed to n-butyltartronic acid mono-(N,N'-diphenyl)hydrazide (IV). Whereas the mass spectra of I, III, and the methyl esters of carboxylic acids II and IV all demonstrate major peaks corresponding to their respective molecular ions, the mass spectrum of II is essentially identical with that of I, suggesting facile dehydration. In the mass spectrum of IV, the peak of highest mass is found at m/e 205; no peak could be perceived corresponding to the molecular ion or to loss of water, carbon dioxide, or both of these elements from the molecular ion. Under normal pyrolytic conditions, II is decarboxylated to N-caproylhydrazobenzene (V) and IV is readily dehydrated to yield III. The mass spectral fragmentation of IV was successfully simulated in the laboratory to give an excellent yield of aniline and alpha-keto-N-caproylaniline (VI) (mol. wt. 205). The probable course of this unusual transformation was elucidated from studies of the accelerated decomposition of IV and derivatives considered as possible intermediates in the degradation process.