Current Initiatives in Analytical Method Development

The tremendous influx of phytochemical ingredients in functional foods and dietary supplements has resulted in increased scrutiny from consumers, regulatory agencies and health professionals about the quality and levels of active ingredients in these products. As a result, questions have been raised about the level of testing and certifications necessary to ensure an ingredient is what it purports to be, is not adulterated, and is not contaminated with pesticide residues or other impurities. This need for dependable analytical methods has resulted in an unprecedented initiative from industry, regulators and scientists to develop, validate and adopt standard assays for botanical products.

This is an enormous undertaking due to the hundreds of botanical ingredients used in thousands of products. In addition, although some of these compounds have been consumed by humans for thousands of years, many have not been subject to comprehensive analysis. To accelerate the process, AOAC International created a task group to evaluate and prioritize ingredients for which validated methods are most urgently needed and to facilitate the development of these assays. This proactive initiative brings together representatives from governmental agencies, industry associations and academia to address the issues that have hampered researchers such as the identification of specific active ingredients, lack of validated reference standards and a dearth of collaborative data.

Analysis of these products poses challenges that are not encountered with traditional matrices. For example, in many cases, the active constituents are not known, and appropriate marker substances must be selected. In addition, the natural variability of many of these products complicates method development. When the instability of many of the active components and marker compounds is factored in, the need for a single, rugged, thoroughly validated analytical method is magnified. Other issues include the appropriateness of the marker compounds and the applicability of the method for both raw materials and finished products. In some instances, matrix interferences result in the methods used for raw materials that are not applicable for finished products. Another factor is the lack of analytical standards resulting in additional method development costs.

The development of methods is based on identification of the known active ingredients and determination of the amount available in the raw material. In addition, methods must allow a determination of how much of the active ingredients should be included in the final product and analysis for quality control. As a result, the identification of the active compounds of interest was an essential first step. While much work still needs to be done, researchers have now isolated the known active ingredients in many of these products.

Although the specific techniques may vary, analysis of most botanical products involves essentially the same steps (i.e., extraction, separation, detection). Before extraction, the product is ground to a powder and stored in airtight, light-resistant containers. Then, using an appropriate solvent (e.g., methanol), the components of interest are extracted, and a chromatographic process is used for separation and detection. High performance liquid chromatography (HPLC) is the technique of choice for most products; to determine the accuracy of an extraction procedure, researchers must compare the results to known concentrations (e.g., reference standards).

Until recently, researchers have been hampered by the lack of known pure standards to use for comparison both in validation of extraction process and in the separation and detection steps. The availability of such standards has accelerated method development for these products and now several U.S. Pharmacopeia (USP) certified reference standards are available. Although a considerable amount of progress has been made, additional research including collaborative studies to validate methodologies remains.

Method Development and Validation

Although the issues associated with successful analysis are numerous, no single component of the process is more important than the development of validated methods using established and well-designed criteria. Method engineering is a stringent process designed to establish benchmarks for acceptance criteria and ultimately determine the validity of a specific method. The adoption of validation standards ensures that methods represent good scientific practice and are robust enough to be used on a global scale. The method engineering process is composed of feasibility, development and validation. Only after these phases are complete can the method be used with confidence.

Method feasibility allows for a rapid determination of the best method development approach, and an assessment of the technical risks. Development of a method involves determining the optimum instrument settings, appropriate method parameters, and isolation and concentration of the compound of interest. All methods used to generate data for regulatory compliance must be validated.

A partial validation is conducted when a method undergoes a minor modification such as a change in technique, site or instrumentation. When a method is developed for routine analysis, a full single-laboratory validation is conducted. This includes an evaluation of several critical parameters. Although a single-lab validation is a good indicator of the acceptability of an assay, the majority of methods also undergo a stringent AOAC collaborative study process, which involves a team of scientific and statistical experts responsible for study direction, data review and analysis.

A minimum of eight qualified laboratories is required, each of which must successfully analyze at least five sets of samples and standards; results are then compiled and statistically evaluated. By statistically evaluating data produced in different laboratories under different conditions, the strengths and weaknesses of a method can be readily identified. If accepted, the method is used and further evaluated in laboratories for a minimum of two years. After the trial period, adoption of a method as Official Final Action is determined by a vote of the official methods board. Methods designated as official are viewed as the gold standard in analytical chemistry by scientists, regulators and litigators worldwide. The Code of Federal Regulations (21CFR2.19) specifies a preference for AOAC methods for compliance purposes.

The ability to identify the primary active ingredients in botanical sources and quantify their content is a key factor in gaining consumer confidence and regulatory compliance. The work of the AOAC and others has resulted in significant progress toward providing the scientific methodologies required for this dynamic marketplace. 

Darryl Sullivan is senior manager of food and drug analysis at Covance Laboratories. He is a director on the AOAC International Board, and served as chair of the AOAC Presidential Task Force on Dietary Supplements. He can be contacted at [email protected]. For more information, visit www.covance.com/analytical.