Dissolution Testing of Nutritional Supplements

C. Jeanne Taborsky(1) and Royal Hanson(2)*

*Email address for correspondence: rhanson@hansonresearch.com

Nutritional and dietary supplements such as vitamins, minerals, and botanicals have become more popular although they have not been subjected to the same quality assurance or labeling scrutiny as have pharmaceuticals or food additives. This article reviews new trends in the requirements for the testing of nutritional supplements including recent USP dissolution protocols as well as the problems inherent in various product packaging when exposed to the critical conditions of temperature and humidity.

An odd, incredible repository of parts and extracts of flora and fauna resides in a musty, museum-like courtyard next to the Temple of Heaven in Beijing, China. This collection -- under the aegis of the Chinese Ministry of Health and protected in Sichuan during the revolution -- showcases two millennia of Chinese pharmacology. Several rooms of floor-to-ceiling shelves house thousands of thick-glass jars of specimens -- everything from regional variations of ginseng to samples of deer and monkey organ (an ancient Oriental therapy for impotence).

Nutritional supplements -- not only essential vitamins and minerals, but also various herbal remedies --are now becoming vogue in the West (1). The Chinese have maintained a centuries-old tradition of using various nutritional supplements and herbs to treat the many maladies of the body and spirit. How will Western science and medicine qualify and validate these ingredients and practices?

During the past decade, through a rigorous process of proposal review and guideline selection, the United States Pharmacopeia (USP) established its first protocols for testing the vitamin and mineral contents of nutritional supplements (2,3). Additionally, FDA focused serious attention on product claims and labeling practices. Botanicals -- such as garlic, ginseng, ginkgo, and St. John's wort -- are now considered in new USP/National Formulary (NF) monographs (4,5). FDA is also amending its food-labeling regulations to establish requirements for the identification of dietary supplements and for their nutrition and ingredient labeling (6).

A primary concern is the quality, potency, and efficacy of nutritional supplements vis-à-vis their label claims. Under the 1994 Dietary Supplement Health and Education Act (DSHEA), FDA's required premarket review of nutritional supplements is less in-depth than that of other products the agency regulates, such as drugs and food additives. This means that consumers and manufacturers are responsible for checking the safety and truthfulness of label claims.

A recent study at Tufts University reviewed 10 vitamin brands -- five labeled "USP" and five not labeled "USP" (7). The five brands with USP label claims all passed dissolution test requirements, whereas two of the five without USP label claims failed to meet USP specifications. This result certainly suggests the need for a closer look at the process of qualifying and validating methods and standards for nutritional supplements.

Compounding these concerns are the problems of product packaging. How might the potency and biorelease rate of various ingredients vary over time, particularly when packaged samples or purchased products are subjected to the vagaries of uncontrolled temperature and humidity conditions that often exist in the real world? Specifically, what packaging types are superior in this regard?

Significant events in the establishment of test requirements for nutritional supplements
1991	USP holds an open conference on vitamins, minerals, and nutritional supplements. The first proposals are discussed and reviewed.
1993	Supplement 8 to USP XXII: The first standards are established, including requirements for initial disintegration testing and for dissolution testing if disintegration attempts fail. USP Pharmacopeial Forum publishes a proposal for establishing dissolution requirements for nutritional supplements (13).
1994	US Congress passes DSHEA.
1995	Supplement 1 to USP 23: Dissolution testing is established as a requirement for nutritional supplements, including folic acid, index vitamins, and index elements.
1995-1998	Subsequent USP 23 supplements: fine-tuning of requirements, test methods, and assays.
1998	Inclusion of botanicals in USP/NF monographs.

USP monographs provide methods for evaluating the acceptability and stability of pharmaceuticals in approved applications. FDA's Center for Drug Evaluation and Research (CDER) regulates both over-the-counter (OTC) and prescription drugs, and section 211 of 21 CFR provides standards for current good manufacturing practices (CGMPs) and testing (8). Although these standards for the manufacture and testing of pharmaceuticals are well established, the standards for dietary supplements are still being developed. Some of the concerns about the manufacture of pharmaceuticals also apply to the manufacture and testing of dietary supplements. The Center for Food Safety and Nutrition regulates foods, vitamins, minerals, dietary supplements, and cosmetics. However, until recently, dietary supplements had little or no standards for potency, dissolution, or stability (see sidebar, "Significant events in the establishment of test requirements for nutritional supplements").

Standards for vitamins, including disintegration and dissolution test requirements, first appeared in 1993 in the eighth supplement to USP XXII/NF XVII (2). The USP section describes monograph test methods for vitamins and calcium supplements. Testing is based on the product's end use and strength. For example, if a calcium product is a compendial article and will be used as an antacid, the test is a disintegration test. However, if the article will be used as a calcium supplement for the treatment of osteoporosis, the test is a dissolution method (9). Because manufacturers have no control over a product's end use, it becomes their responsibility to see that a tested product would pass both disintegration and dissolution tests throughout its shelf life. Additionally, if the product is labeled "USP," it must be packaged, stored, and distributed as required in the monographs and in conformance with USP's General Chapters.

When establishing the test requirements for vitamins, USP considered the degree of difficulty of the test and the number of active ingredients in the formulation. A list of index vitamins and minerals were chosen based on their solubility. It is presumed that if the least-soluble item in the formulation is dissolved, then the other components in the product are also in solution. Furthermore, USP provides for a less work intensive test by combining aliquots from six tests for a single determination.

Dissolution Test Requirements for Nutritional Supplements

Specifications are provided in USP 23, Supplement 9.

USP classifications. Table I lists the dissolution test requirements and references for the six USP classes of vitamins, minerals, and vitamins with minerals.

Table I. USP classifications with respective dissolution test requirements and references.

Class	Dissolution	Reference
I. Oil-soluble vitamins	Not required
II. Water-soluble vitamins	One index vitamin; folic acid if present	Tablet and capsule assays (10)
III. Water-soluble vitamins with minerals	One index vitamin and one index element; folic acid if present	Tablet assays (11) Capsule assays (12)
IV. Oil- and water-soluble vitamins	One index water-soluble vitamin; folic acid if present	Tablet and capsule assays (11)
V. Oil- and water-soluble vitamins with minerals	One index water-soluble vitamin and one index element; folic acid if present	Tablet assays (5) Capsule assays (11)
VI. Minerals	One index element	Tablet and capsule assays (11)

Index vitamins and index minerals. In general, the dissolution conditions for index vitamins and index minerals are

Formulations containing >25 mg of the index vitamin riboflavin. The dissolution conditions for this vitamin are

Folic acid. This test is also required if the product labeling includes a health claim concerning the relationship between folate deficiency in pregnant women and the risk of neural tube defects in the fetus. The dissolution conditions are

Note: Perform this test under subdued light.

Selection of an index vitamin. Test one vitamin present in the product formulation according to the following hierarchy.

Note: It has been proposed that folic acid become the first index vitamin.

Selection of an index element. Test one element present in the product formulation according to the following hierarchy.

Procedures. Combine equal volumes of the filtered solutions of the six individual specimens withdrawn and determine the amount of folic acid, index vitamin, or index element dissolved based on the average result of the six units tested. Methods for minerals usually involve atomic absorption, but vitamins are usually assayed by high-performance liquid chromatography with ultraviolet (UV) light detection. Refer to USP monographs on nutritional supplements for specific assay requirements for folic acid, vitamins, and minerals.

Tolerances. The requirements are met if at least 75% of the labeled content of folic acid and at least 75% of the assayed content of the index vitamin or index element from the units tested are dissolved in 1 hour.

Currently, there is an increased concern about folic acid and its importance in the treatment and prevention of disease and in the decreased risk of neural tube defects during fetal development. Additionally, patients are often titrated on medications based on their regular dietary intake of folic acid-containing products. Folic acid is a food additive to bread products, an ingredient in multiple vitamins, an OTC pharmaceutical product, and a prescription product in high doses. It is critical to disease treatment that the folic acid content of these products be as claimed on the label.

Standards for nutritional supplements were first proposed in Pharmacopeial Forum in 1993 and accepted in 1995 (13). The standards are provided in the Official Monographs and General Chapters-General Test and Assays sections of USP 23/NF 18 (14). These include chapters <2021> Microbial Limit Tests, <2040> Dissolution of Nutritional Supplements, <2091> Weight Variation, and <2750> Manufacturing Practices for Nutritional Supplements. These standards are very similar to those published as cGMPs for pharmaceuticals in 21 CFR 211 (8). The standards in USP 23/NF 18 apply only to articles that are labeled "USP". If tested, these materials must comply with USP monograph standards, including those for dissolution and potency.

Validation and qualification

As with any equipment or analytical method, validation is a critical part of development. Validation is what demonstrates data accuracy and precision. The qualification process is divided among the facility, the equipment systems, the process, and the analytical method. The facility portion of testing should include temperature and humidity controls in the test area, demonstrated procedures that prevent contamination, a low-vibration workstation, and reliable utilities (e.g., electrical and water).

Equipment qualification procedures should include

Equipment validation protocols should

Processes and process controls should be qualified. If the results of a process cannot be fully verified by subsequent inspection and testing, the process should be qualified with a high degree of assurance and approved according to established pro- cedures. Essential processing parameters should be qualified for both procedures and equipment. For equipment, these parameters should include the use of automated systems and the verification of the stability of the dosage form while open to ambient conditions. As demonstrated in the graph of the dissolution rate of prednisone tablets when exposed for 3 days to high temperature and humidity, the tablets failed to dissolve completely (see Figure 1) (15). An automated delivery system should provide protection from light and excessive temperature and humidity.

Generally, qualification of any dissolution operating system includes

Method validation should include the testing of the active components, the product matrix or placebo, and the market product. The test method should provide instructions to protect the product from degradation, such as "protect from light" if the article is light sensitive. The product should meet the solubility requirements for the amount of dissolution media in the container. The method should be accurate, precise, and rugged. CDER's Guidance for Industry on Dissolution Testing of Immediate Release Solid Oral Dosage Forms provides information that can assist the nutritional products industry (16).

Unlike pharmaceuticals, nutritional products are not subject to limits on impurities and degradants. It is sometimes difficult to determine the article's active chemical component because most nutritional supplements are natural products or extracts thereof and the material can be a complex mixture of naturally occurring substances in amounts that vary depending on the season and source. Still, these products can have properties similar to those of pharmaceuticals, and manufacturers can benefit from the extensive history and knowledge of pharmaceutical chemists.

Packaging and storage conditions and their effects on dissolution

Many nutritional supplements are compounded into solid oral dosage forms using established tableting or capsule technology. These processes incorporate starch and other materials as binders and disintegrants. Like pharmaceuticals, these preparations can be sensitive to moisture, light, oxygen, and heat. The manufacture, packaging, storage, and distribution chain should be developed with these sensitivities in mind. The critical conditions of temperature and humidity must be determined for the product to be safely delivered to the consumer. Critical conditions are those parameters beyond which the product would suffer an irreversible change, would be unsuitable for use, or would not meet label claim. Critical test parameters for solid oral dosage forms should include potency and dissolution or disintegration limits.

For pharmaceutical products, the International Conference on Harmonization (ICH) and USP test conditions are:

The labeled storage conditions for controlled room temperature are 15-30 °C USP MKT 25 °C. Shipping conditions are yet to be determined. At this time, no specific FDA requirements exist for the storage and testing conditions instability studies of dietary supplements. Because the pharmaceutical test conditions for temperature and humidity are based on climatic conditions, it is scientifically sound to use these same conditions for the testing and storage of dietary supplements.

Figure 1: Dissolution profiles of prednisone tablets after storage in containers constructed of various materials.

Containers used to package dietary supplements typically include

Some bottles have foil seals to further protect the product. The rate of moisture permeation can be significantly higher without the foil seal depending on the type of liner used in the screw cap. CDER tested bottles with foil inner seals using USP <661> methods and bottles with screw caps lacking inner seals using ICH accelerated conditions (18,19). Especially in the case of the small (60 cc) bottles, water vapor permeation increased significantly (see Figure 2).

Figure 2: Comparison of bottles with foil inner seals tested according to USP <661> at 25 °C and 75% RH for 2 weeks and bottles with screw closures tested for 3 months at ICH accelerated conditions of 45 °C and 75% RH.

In the past, unit-dose containers -- with the exception of foil pouches -- did not protect the contents from water vapor permeation as well as bottles. With the use of Aclar (AlliedSignal, Inc., Morristown, NJ), poly(vinylidene chloride), and foil blisters, these containers now offer significant improvement in protection from water vapor permeation.

USP conducted a study in which the dissolution rates of prednisone tablets were measured after being packaged and stored in various multiple-unit and unit-dose containers. Figure 1 shows the results and illustrates the significant effect of packaging and storage on the dissolution rate and performance of a solid oral dosage form. Furthermore, one should note the degree of sensitivity of these tablets under increased temperature and humidity. It is obvious from these data that the prednisone calibrator should be well protected from increased temperature and humidity to obtain reliable calibration results.

Conclusion

In 1994, the DSHEA established new regulations for dietary supplements, including vitamins, minerals, herbs, botanicals, and related products. The law allows FDA to establish cGMPs for these products and gives manufacturers more freedom to market products and to provide information about the product's benefits in the labeling. However, manufacturers must be careful that the claims do not imply that the product cures, mitigates, treats, or prevents disease. The requirements for these products are based on the labeling claims. Consumers are encouraged to purchase products that are labeled "USP" because the standards for those products have been established. If a product fails to meet USP requirements during its shelf life, it is considered to be mislabeled and misbranded, and FDA can take appropriate action against the manufacturer. Nutritional supplements and vitamins should meet label claims at expiration date. Products without an expiration date must meet label claims indefinitely. If they fail to meet label claims, they are considered mislabeled and misbranded, and the agency can take appropriate action.

Table II: Typical blister container materials.

Blister	Lidstock
Poly(vinyl chloride)	(PVC) Foil
PVC/polyethylene/poly(vinylidene chloride)	Foil/paper
PVC/Aclar	PVC
Metalized foil
Light resistant (UV inhibitor and amber)	Peel, push, peel/push

There is an increased need for an understanding of regulations and the establishment of realistic stability testing protocols and expiration data based on validated test methods and standards. Since 1995, standards for nine nutritional supplements have appeared in NF, including monographs for ginseng, garlic, gingko, and St. John's wort. Many monographs are still being developed, including those for Hawthorne leaf with flower, cranberry extract, milk thistle, and valerian (20). It is in the industry's best interest to take an active role in this process to influence the methods and standards that will affect the manufacture of these products (21).

References

1. "The Herbal Medicine Boom," Time, 57-69 (23 November 1998).

2. USP XXII/NF XVII, Supplement 8 (United States Pharmacopeial Convention, Rockville, MD, 1993).

3. USP 23/NF 18, Supplement 1 (United States Pharmacopeial Convention, Rockville, MD, 1995).

4. USP 23/NF 18, Supplement 8 (United States Pharmacopeial Convention, Rockville, MD, 1998).

5. USP 23/NF 18, Supplement 9 (United States Pharmacopeial Convention, Rockville, MD, 1998).

6. Federal Register (Office of the Federal Register, National Archives and Records Administration, Washington, DC, 23 September 1997) pp. 49825-49858.

7. Health and Nutrition Letter (Tufts University, Medford, MA, November 1997).

8. Code of Federal Regulations, Title 21, Food and Drugs (General Services Administration, Washington, DC, 1 April 1998), Part 211.

9. USP 23/NF 18 (United States Pharmacopeial Convention, Rockville, MD, 1995) p. 247.

10. USP 23/NF 18, Supplement 4 (United States Pharmacopeial Convention, Rockville, MD, 1996).

11. USP 23/NF 18, Supplement 5 (United States Pharmacopeial Convention, Rockville, MD, 1996).

12. USP 23/NF 18, Supplement 7 (United States Pharmacopeial Convention, Rockville, MD, 1997).

13. Pharm. Forum 19 (6) (1993).

14. "Nutritional Supplements," in USP 23/NF 18 (United States Pharmacopeial Convention, Rockville, MD, 1995), pp. 2129-2207.

15. C.J. Taborsky-Urdinola, V. Gray, and L.T. Grady, "Effects of Packaging and Storage on the Dissolution Rate of Model Prednisone Tablets," Am. J. Hosp. Pharm. 38, 1322-1327 (September 1981).

16. Guidance for Industry on Dissolution Testing of Immediate Release Solid Oral Dosage Forms (US Department of Health and Human Services, FDA Center for Drug Evaluation and Research, Rockville, MD, August 1997).

17. International Conference on Harmonization (ICH), "ICH Tripartite Guideline for Stability of New Drug Substances and Products," ICH Q1A (23 September 1994).

18. USP 23/NF 18 (United States Pharmacopeial Convention, Rockville, MD, 1995), General Chapters <661> and <671>.

19. C. Jeanne Taborsky et al.,"An Evaluation of High-Density Polyethylene Bottles Used to Package Solid Oral Dosage Forms and a Determination of the Impact of Using Regrind," Pharm. Technol. 21 (9), 116-131 (1997).

20. Pharm. Forum 24 (5) (1998).

21. The authors are currently conducting a study wherein a dietary supplement is being tested in containers with known permeation rates to measure the effect on the product's dissolution profile and potency. They plan to publish the results and make them available at the Web site (www.hansonresearch.com).