Approaches by the US National Institutes of Health to support rigorous scientific research on dietary supplements and natural products.

Mechanistic, clinical, and epidemiological research relevant to dietary supplements (DS) is supported by the U.S. National Institutes of Health. The Office of Dietary Supplements and the National Center for Complementary and Integrative Health promote the development and appropriate use of rigorous and comprehensive DS analyses which are critical for research reproducibility, particularly when the investigational DS include chemically complex natural products with unclear mechanisms of action.

The development and implementation of quality assurance programs to support nutritional measurements.

The National Institute of Standards and Technology administers quality assurance programs devoted to improving measurements of nutrients and related metabolites in foods, dietary supplements, and serum and plasma samples. These programs have been developed in collaboration with the National Institutes of Health to assist measurement communities in their efforts to achieve accurate results that are comparable among different laboratories and over time. Targeted analytes include micronutrients, botanical markers, nutritional elements, contaminants, fatty acids, and vitamin D metabolites.

Development and certification of green tea-containing standard reference materials.

A suite of three green tea-containing Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST): SRM 3254 Camellia sinensis (Green Tea) Leaves, SRM 3255 Camellia sinensis (Green Tea) Extract, and SRM 3256 Green Tea-Containing Solid Oral Dosage Form. The materials are characterized for catechins, xanthine alkaloids, theanine, and toxic elements. As many as five methods were used in assigning certified and reference values to the constituents, with measurements carried out at NIST and at collaborating laboratories. The materials are intended for use in the development and validation of new analytical methods, and for use as control materials as a component in the support of claims of metrological traceability.

Certification of vitamins and carotenoids in SRM 3280 multivitamin/multielement tablets.

A new multivitamin/multielement dietary supplement Standard Reference Material (SRM) has been issued by the National Institute of Standards and Technology (NIST), with certified and reference concentration values for 13 vitamins, 24 elements, and 2 carotenoids. The constituents have been measured by multiple analytical methods with data contributed by NIST and by collaborating laboratories. This effort included the first use of isotope dilution mass spectrometry for value assignment of both fat-soluble vitamins (FSVs) and water-soluble vitamins (WSVs). Excellent agreement was obtained among the methods, with relative expanded uncertainties for the certified concentration values typically ranging from <2% to 15% for vitamins.

Certification of standard reference materials containing bitter orange.

A suite of three dietary supplement standard reference materials (SRMs) containing bitter orange has been developed, and the levels of five alkaloids and caffeine have been measured by multiple analytical methods. Synephrine, octopamine, tyramine, N-methyltyramine, hordenine, total alkaloids, and caffeine were determined by as many as six analytical methods, with measurements performed at the National Institute of Standards and Technology and at two collaborating laboratories. The methods offer substantial independence, with two types of extractions, two separation methods, and four detection methods. Excellent agreement was obtained among the measurements, with data reproducibility for most methods and analytes better than 5% relative standard deviation. The bitter-orange-containing dietary supplement SRMs are intended primarily for use as measurement controls and for use in the development and validation of analytical methods.

Detecting potential teratogenic alkaloids from blue cohosh rhizomes using an in vitro rat embryo culture.

The novel alkaloid thalictroidine (1), as well as the known alkaloids taspine (2), magnoflorine (3), anagyrine (4), baptifoline (5), 5,6-dehydro-alpha-isolupanine (6), alpha-isolupanine (7), lupanine (8), N-methylcytisine (9), and sparteine (10), were identified from an extract of Caulophyllum thalictroides rhizomes. N-Methylcytisine exhibited teratogenic activity in the rat embryo culture (REC), an in vitro method to detect potential teratogens. The structure of 1 was elucidated using various spectroscopic methods, primarily by NMR techniques. Thalictroidine, anagyrine, and alpha-isolupanine were not teratogenic in the REC at tested concentrations. Taspine (2) showed high embryotoxicity, but no teratogenic activity, in the REC.

Determination of salicin and related compounds in botanical dietary supplements by liquid chromatography with fluorescence detection.

A sensitive and reliable method is described for quantitative determination of salicin (including salicyl alcohol) and salicylic acid in botanical dietary supplements by reversed-phase liquid chromatography (LC) with wavelength-programmed fluorescence detection. One gram sample material was extracted with 20 mL aqueous phosphate buffer (pH 5.0), which was heated in an 80 degrees C water bath for 30 min. After centrifugation and cooling of the extract to room temperature, the supernatant was diluted with additional buffer. A 1 mL portion of diluted extract was mixed with 1 mL beta-glucosidase solution (2 mg/mL) and incubated for 40 min in a 37 degrees C water bath. The extract was passed through a 0.45 micron syringe filter and analyzed by LC. Limits of quantitation for salicin and salicylic acid were 20 and 1 microgram/g, respectively. Recoveries from samples fortified with salicin at 20, 100, and 1000 micrograms/g and with salicylic acid at 5, 20, and 50 micrograms/g ranged from 85 to 110%, with standard deviations less than 7%.

Chiral gas chromatographic determination of ephedrine-type alkaloids in dietary supplements containing Má Huáng.

Má Huáng is a traditional Chinese medicine derived from the aerial parts of several Ephedra species (Ephedraceae). These plants produce (-)-ephedrine, (+)-pseudoephedrine, (-)-norephedrine, (+)-norpseudoephedrine, (-)-N-methylephedrine, and (+)-N-methylpseudoephedrine. Racemic and (-)-ephedrine, (+)-pseudoephedrine, and (+/-)-norephedrine (phenylpropanolamine) are used clinically in the United States and are largely synthetic in origin. Current interest in Má Huáng is spurred by reports describing a "thermogenic" (calorie burning) effect provided by mixtures of ephedrine, caffeine, and aspirin. Products providing the key thermogenic compounds from natural sources are available as dietary supplements in retail outlets. Reports of potentially unsafe levels of the alkaloids, as well as possible fortification of Má Huáng-containing products with synthetic Ephedra alkaloids, prompted the development of a chiral gas chromatographic (GC) method that allows determination of alkaloid patterns and identification of isomerically impure synthetic alkaloids. Nine products were analyzed on a gamma-cyclodextrin capillary GC column. Identity of the alkaloids was verified by GC/mass spectrometry (MS) and GC/matrix isolation/Fourier transform infrared spectroscopy. No synthetic isomers were found in the dietary supplements analyzed. Three products contained only one of the ephedrine-type alkaloids. One product that listed Má Huáng as an ingredient contained no detectable ephedrine-type alkaloid. In products containing measurable quantities of these compounds, total alkaloid levels ranged from 0.3 to 56 mg/g.

Analysis of aporphine and quinolizidine alkaloids from Caulophyllum thalictroides by densitometry and HPLC.

High-performance liquid chromatographic (HPLC) and densitometry procedures were developed to determine the principal alkaloids in the roots of C. thalictroides. In both techniques the alkaloids content was assessed using cytisine as an internal standard. The purity and identity of the peaks of the alkaloids was examined by diode array detection and by comparison with the standards. The content of individual alkaloids was found to be in the range 0.02-1.1% w/w.

Gas chromatographic determination of yohimbine in commercial yohimbe products.

The bark of Pausinystalia yohimbe [K. Schumann] Pierre (Rubiaceae), long valued as an aphrodisiac in West Africa, recently has been promoted in the United States as a dietary supplement alternative to anabolic steroids for enhancement of athletic performance. As the number of yohimbe products on the retail market increases, concerns about their safety are raised because of the reported toxicity of yohimbine (the major alkaloid of the plant). Although plant materials are usually identified microscopically, we were unable to identify them in many of the products, because as their labels indicated, the products were mixtures of various botanicals or were bark extracts and contained little or no plant material. A method for extraction and capillary gas chromatographic (GC) separation of the alkaloids of P. yohimbe was, therefore, developed and used to analyze a number of commercial yohimbe products. The method involved solvent extraction and partitioning in chloroform-water followed by separation on a methyl silicone capillary GC column (N-P detection). Comparisons of chromatograms of extracts of authentic bark with those of commercial products indicated that, although many products contained measurable quantities of the alkaloid yohimbine, they were largely devoid of the other alkaloids previously reported in this species. Concentrations of yohimbine in the commercial products ranged from < 0.1 to 489 ppm, compared with 7089 ppm in the authentic material. Authentic bark has been reported to contain up to 6% total alkaloids, 10-15% of which are yohimbine. The possible presence of undeclared diluents in the products was indicated by peaks in product chromatograms but not in those of authentic bark.

Chemical studies of phytoestrogens and related compounds in dietary supplements: flax and chaparral.

High-performance liquid chromatographic (HPLC) and mass spectrometric (MS) procedures were developed to determine lignans in flaxseed (Linum usitatissimum) and chaparral (Larrea tridentata). Flaxseed contains high levels of phytoestrogens. Chaparral has been associated with acute nonviral toxic hepatitis and contains lignans that are structurally similar to known estrogenic compounds. Both flaxseed and chaparral products have been marketed as dietary supplements. A mild enzyme hydrolysis procedure to prevent the formation of artifacts in the isolation step was used in the determination of secoisolariciresinol in flaxseed products. HPLC with ultraviolet spectral (UV) or MS detection was used as the determinative steps. HPLC procedures with UV detection and mass spectrometry were developed to characterize the phenolic components, including lignans and flavonoids, of chaparral and to direct fractionation studies for the bioassays.

Determination of pyrrolizidine alkaloids in commercial comfrey products (Symphytum sp.).

The presence of hepatotoxic pyrrolizidine alkaloids in comfrey (Symphytum sp.) and the widespread use of decoctions of this plant as a beverage (herbal tea) are of increasing concern. A method for the extraction and solid-phase concentration and capillary gas chromatographic determination of these alkaloids and their N-oxides in botanical materials has been developed and was applied to eleven comfrey-containing products purchased from retail health-food outlets in the Washington, DC, area during May-June 1989. Nine of the 11 products were found to contain measurable quantities of one or more of the alkaloids, in ranges from 0.1 to 400.0 ppm. Products containing comfrey leaf in combination with one or more other ingredients were found to contain the lowest alkaloid levels. Highest levels were found in bulk comfrey root, followed by bulk comfrey leaf. The species of the bulk material was verified by thin-layer chromatography and other means.

Determination of ethanol in alcoholic beverages by liquid chromatography using the UV detector.

Ethanol in certain beverages and in similar solutions may be determined by reversed-phase liquid chromatography (LC) using the UV detector. The mobile phase in this indirect photometric detection technique contains a low concentration of a UV-absorbing compound, such as acetone, that coelutes with the ethanol peak. Several variables such as the choice and concentration of the UV-detection agent are examined regarding their effects on the retention time, magnitude and linearity of peak area, and other aspects of quantitation. Except for filtering to remove particulate matter, samples can be injected without pretreatment. The concentration of ethanol in several types of beverages can be determined with 2% relative standard deviation, calibration is linear to 40% ethanol, and the minimum detectable concentration is 0.1%.

Toxicity of Gonyaulax tamarensis var. excavata cells to the brine shrimp Artemia salina L.

Brine shrimp (Artemia salina L.) were exposed to concentrations of live Gonyaulax tamarensis var. excavata cells ranging from 0 to 13,400 cells/ml in unialgal culture. The shrimp ingested the dinoflagellate cells and an LD50 determination was made. The shrimp had a 72-hr LD50 of 1258 G. tamarensis cells/ml, which was calculated to correspond to 73,385 micrograms/kg of body weight. This figure can be compared with an oral LD50 value of 2100 micrograms/kg for the mouse. Since it requires one-tenth as much toxin to kill a mouse (LD50 approximately 0.04 mg) as to kill an Artemia (LD50 approximately 0.4 mg), the whole cell G. tamarensis bioassay is a poor alternative to the current mouse assay.