In 1994, the U.S. Congress enacted the Dietary Supplement Health and Education Act (DSHEA) to "protect the right of access of consumers to safe dietary supplements."1 One way in which this is expected to occur is through the implementation of Good Manufacturing Practices (GMPs), which went into effect August 24, 2007.2 The GMP Final Rule is intended to ensure product quality by establishing minimum practices related to manufacturing, packaging, labeling, or holding dietary supplements. As part of this requirement, manufacturers must establish specifications for identity, purity, strength, and composition, and set limits on contaminants and adulterants.
Figure 1 - Dietary supplement SRMs.
Dietary supplements must be tested using appropriate analytical methods to demonstrate that specifications are met. In support of this legislation, the National Institute of Standards and Technology (NIST), the National Institutes of Health-Office of Dietary Supplements (NIH-ODS), and the Food and Drug Administration-Center for Drug Evaluation and Research (FDA-CDER) are collaborating to develop Standard Reference Materials (SRMs) for the measurement of chemical constituents in dietary supplements. In general, well-characterized reference materials are needed to promote quality and consistency in manufacturing, and accuracy in product labeling. Reference materials may also be useful in the development of new analytical methods and validation of these methods, and provide a component in achieving traceability of measurements. The goal of this ongoing effort is to provide tools to the dietary supplement industry and measurement communities that will lead to improved quality of dietary supplements, and ultimately reduce public health risks that could potentially be associated with these products (Figure 1).
Design and production of dietary supplement SRMs
NIST offers a wide variety of complex-matrix SRMs that are characterized for chemical composition. Examples include natural-matrix environmental SRMs (e.g., soils, sediments, and animal tissues), clinical SRMs (biological fluids and tissues), nutritional SRMs (foods), fossil fuels, and forensic SRMs (energetic materials and solutions). Dietary supplements are frequently manufactured from botanical materials or their extracts. Because different analytical challenges are represented by different types of sample matrices, dietary supplement SRMs have been developed to include natural, extracted, and extensively processed forms of each botanical material. These SRM suites typically consist of dried, ground, and sieved plant parts; extract(s) of the plant that are spray dried or otherwise converted to a dry powder form; and blend(s) of commercially available finished products. The latter SRM type is not intended to be an archetype for product formulation, but since it is prepared from mixtures of commercially available products, the materials should be representative of measurement challenges that an analyst might encounter for individual dietary supplements. In some cases, the development of multiple finished product, dietary supplement SRMs may be warranted if the matrix types differ significantly.
Figure 2 - Packaging of dietary supplement SRMs.
The production of dietary supplement SRMs requires consideration of a variety of factors. During the acquisition of botanical materials, voucher specimens are obtained in cases for which proof of identity may be required. Corresponding herbarium sheets are available for inspection as described in the Certificates of Analysis for the botanical SRMs (note that this does not apply to extract or finished product SRMs unless they are directly linked to the plant material associated with the voucher specimen). Raw materials are ground and sieved (typically using 80-mesh screens that pass ≈180-µm particles). Special packaging has been developed to enhance the stability of these SRMs (Figure 2). Materials are placed in polyethylene bags and flushed with nitrogen prior to heat sealing. These packages are placed in nitrogen flushed aluminized mylar bags with two packets of silica gel drier, and are heat sealed. Following packaging, the materials are (typically) irradiated at a level of 3 kGy to 15 kGy, depending on prior assessment of material stability to various dosage levels.
The processes used in the value assignment of the chemical composition of SRMs have been described.3 Several modes of assignment are detailed, and in most cases multiple independent analytical methods are utilized. The rationale for this approach is based on the assumption that potential biases in measurements will differ among independent methods. Thus, if a bias is present in an analytical method (for example, from an incomplete extraction step), this bias will be apparent when data from other methods are compared. Measurements from collaborating laboratories are sometimes utilized in value assignment to supplement data generated by NIST. Data are reported as certified, reference, or information values. A NIST certified value is a value for which NIST has the highest confidence in its accuracy in that all known or suspected sources of bias have been investigated or accounted for. Reference values are noncertified values that are the best estimates of the true values based on available data; however, the values do not meet the NIST criteria for certification. For example, uncertainties may reflect only measurement reproducibility, may not include all sources of uncertainty, or may reflect a lack of sufficient statistical agreement among multiple analytical methods. Information values may be of interest to the SRM user, but insufficient information is available to provide an estimate of the uncertainty. Information values are not recommended for use in quantitation, but are provided to give additional general information about the material.
Efforts are made to assign values for constituents that will be most useful to the dietary supplement user community. Known and suspected active constituents (e.g., alkaloids in ephedra and bitter orange SRM suites, catechins in green tea SRM suites, ginkgolides in ginkgo-containing SRMs) are of high priority. Other analytes of interest such as constituents that are reported on product labels are also characterized. Botanical materials are screened for pesticides; however, these data are rarely reported since levels are typically very low. Levels of four toxic elements (cadmium, mercury, arsenic, and lead) are also screened in most of the dietary supplement SRMs.
Examples of dietary supplement SRMs
Table 1 - Dietary supplement Standard Reference Materials (current)
Table 2 - Dietary supplement Standard Reference Materials (in progress)
Table 2 - Dietary supplement Standard Reference Materials (in progress) continued
A listing of currently available dietary supplement SRMs is provided in Table 1; planned and in-progress dietary supplement SRMs are shown in Table 2. NIST’s efforts to provide dietary supplement SRMs began with the development of a suite of ephedra-containing reference materials. Prior to April 2004, ephedra-containing dietary supplements were widely marketed in the United States to promote weight loss and to increase energy (i.e., as a stimulant stimulant). A few products were formulated by adding ephedra to high-protein milk powders; these products were marketed to bodybuilders to increase muscle mass while reducing body fat. Five ephedra-containing SRMs have been developed and are currently available: SRM 3240 Ephedra sinica Stapf Aerial Parts, SRM 3241 Ephedra sinica Stapf Native Extract, SRM 3242 Ephedra sinica Stapf Commercial Extract, SRM 3243 Ephedra-Containing Solid Oral Dosage Form, and SRM 3244 Ephedra-Containing Protein Powder. SRM 3245 Ephedra Dietary Supplement Suite contains two bottles each of the five ephedra-containing SRMs. Levels of ephedrine alkaloids and selected trace elements are reported as certified or reference values. In addition, the level of synephrine (an ephedrine-like alkaloid present in many ephedra-free dietary supplements) is certified in SRM 3243, and levels of caffeine are certified in SRM 3243 and SRM 3244. Because SRM 3244 contains a high-protein milk powder base, a number of food-matrix constituents are also characterized. In addition to the ephedrine alkaloids, levels of fatty acids, amino acids, vitamins, nutrient elements, and proximates (i.e., moisture, solids, ash, protein, carbohydrates, and fat) are reported for SRM 3244.
The accuracy and precision of the data reported for the ephedra SRM suite are representative of many of the other dietary supplement SRMs. Data are reported for constituents present over a broad range, at percent levels to trace levels (i.e., ≈10% mass fraction to ≈10 mg/kg). Expanded uncertainties (calculated as U = kuc, where uc is intended to represent, at the level of one standard deviation, the combined contributions to uncertainty, and the coverage factor k corresponds to an approximate 95% confidence interval) range from about 2% for high-level analytes to about 15% for low-level analytes. Analytical data from four to nine data sets of (mostly) independent methods were combined to yield the certified and reference values.
After the FDA ruling in 2004 that restricted the sale of ephedra-containing dietary supplements, 4 many manufacturers of dietary supplements reformulated weight-loss products with ingredients considered to be safer alternatives to ephedra. Dietary supplements have been developed from extracts derived from the fruits of Citrus aurantium L. (Rutaceae). Mixtures of subspecies are often blended to adjust alkaloid content; these materials are commonly referred to as “bitter orange.”5 Active constituents in bitter orange are similar in structure and function to ephedrine alkaloids; however, alternate analytical methods are required than are used for the analysis of ephedra. For these reasons, a suite of bitter orange-containing SRMs has been developed to support measurements in related dietary supplements. Three SRMs are available: SRM 3258 Bitter Orange (Fruit), SRM 3259 Bitter Orange Extract, and SRM 3260 Bitter Orange-Containing Solid Oral Dosage Form. SRM 3261 Bitter Orange Dietary Supplement Suite contains two packets of each of the three bitter orange-containing SRMs. Levels of five citrus alkaloids and total citrus alkaloids are reported as certified, reference, or information values. Caffeine is often present in bitter orange finished products, and the level of this constituent is certified in SRM 3260. SRMs were screened for several elements, and the trace levels are provided as information values.
Other dietary supplement SRMs currently available include a suite of three ginkgocontaining SRMs: SRM 3246 Ginkgo biloba (Leaves), SRM 3247 Ginkgo biloba Extract, and SRM 3248 Ginkgo-Containing Tablets. SRM 3249 Ginkgo Dietary Supplement Suite consists of two bottles each of the three ginkgo-related SRMs. Two families of organic constituents and four elements are characterized in these SRMs: terpene lactones (i.e., ginkgolides and bilobalide), flavonoids, and cadmium, mercury, arsenic, and lead. Two saw palmetto-containing reference materials are available: SRM 3250 Serenoa repens (Fruit) and SRM 3251 Serenoa repens Extract. These SRMs are characterized for phytosterol and fatty acid content; in addition, β-carotene and γ-tocopherol have been determined in SRM 3251. Fatty acids in saw palmetto are present as triglycerides and as free fatty acids, and both species are characterized in these materials. Nearly 30 other SRMs are characterized for fatty acid content, and these materials are discussed in a separate publication.6 Several of these SRMs can be categorized as dietary supplements. They include SRM 1588b Organics in Cod Liver Oil, SRM 3274 Botanical Oils Containing Omega-3 and -6 Fatty Acids, and SRM 3276 Carrot Extract in Oil. The oils differ significantly in fatty acid profiles, particularly in the levels of specific unsaturated fatty acids. SRM 3276 consists of an extract of carrots that is dispersed in a vegetable oil of unspecified origin. This SRM was developed to support measurements of tocopherols and carotenoids; however, levels of fatty acids have also been determined.
Most recently, a “multivitamin/multimineral” SRM has been issued. SRM 3280 Multivitamin/Multielement Tablets is provided with certified and reference concentration values for 13 vitamins, 24 elements, and two carotenoids. In the design of the SRM, tablets were utilized to enhance long-term stability and to provide a material that is similar in form to commercial dietary supplements.
SRMs for other dietary supplements are at various stages of development (see Table 2). These SRMs include a suite of green tea-containing SRMs that will be characterized for catechins, xanthines, theanine, and elements; several suites of Vaccinium berry SRMs (representing cranberries, blueberries, and bilberries) that will be characterized for organic acids, anthocyanidins, and procyanidins; suites of soy, red clover, and kudzu-containing SRMs that will be characterized for isoflavones; St. John’s Wort SRMs with values for naphthodianthrones and flavonoids; various black cohosh-containing SRMs characterized for triterpene glycosides; and fish oil SRMs characterized for fatty acids and vitamins.
Conclusion
Several SRMs are available or are in production to support analysis of dietary supplements. The constituents of these materials can be grouped into two broad categories: compounds that are specific to a particular botanical material (such as ginkgolides in Ginkgo biloba or citrus alkaloids in bitter orange) and compounds that are more widely distributed among botanicals (e.g., fatty acids, flavonoids, and isoflavones). The selection of an appropriate SRM depends on the intended use of the SRM as well as the nature of the samples to be analyzed. In general, the most useful results are obtained for reference materials that are similar in composition to the unknown samples. Complete information on each of the SRMs described is provided on the Certificates of Analysis, available at http://www.nist.gov/srm.
References
- Dietary Supplement Health and Education Act. Public Law 103-417 [S.784]; Oct 25, 1994.
- FDA. Current Good Manufacturing Practice in Manufacturing, Packaging, Labeling, or Holding Operations for Dietary Supplements; docket no. 1996N-0417 (formerly no. 96N-0417), 34752-34958 (2007); 21 CFR Part 111.
- May, W.; Parris, R.; Beck, C.; Fassett, J., Greenberg, R.; Guenther, F.; Kramer, G.; Wise, S.; Gills, T.; Colbert, J.; Gettings, R.; MacDonald, B. Definitions of terms and modes used at NIST for value-assignment of reference materials for chemical measurements; National Institute of Standards and Technology, ed. NIST Special Publication 260-136, U.S. Government Printing Office, Gaithersburg, MD, 2000.
- Food and Drug Administration. 21 CFR Part 119; Final Rule Declaring Dietary Supplements Containing Ephedrine Alkaloids Adulterated Because They Present an Unreasonable Risk; Final Rule; Fed. Reg.2004, 69, 6787-6854.
- Mattoli, L.; Cangi, F.; Maidecchi, A.; Ghiara, C.; Tubaro, M.; Traldi, P. A rapid liquid chromatography electrospray ionization mass spectrometry(n) method for evaluation of synephrine in Citrus aurantium L. samples. J. Agric. Food Chem.2005, 53, 9860-6.
- Sander, L.C.; Schantz, M.M.; Sharpless, K.E.; Wise, S.A. Standard Reference Materials to support measurement of fatty acids. Lipid Tech. 2009, 21, 7-12.
The authors are with the Analytical Chemistry Div., National Institute of Standards and Technology (NIST), Gaithersburg, MD 20899-8392, U.S.A.; tel.: 301-975-3315; e-mail: [email protected].