Analyzing This and That:

November 2003

By Richard Crowley
Contributing Editor

Consumer demand for functional foods and nutraceuticals has accelerated the debate over the scientific and regulatory requirements necessary to substantiate claims for content, quality and consistency. As a result, the analytical evaluation of food and dietary products has evolved significantly since Casimir Funk first identified "vital amines" - his name for vitamins - in the early 1900s. Foods can no longer be evaluated only in terms of traditional nutrients. Scientists are now being asked to identify levels of phytochemicals and other physiologically active components unheard of or of little interest even 5 years ago.

The presence of components such as phospholipids and carotenoids becomes even more acute in light of FDA's July 2003 announcement proposing new regulations on the use of qualified health claims. These would allow different levels of health claims on labels of products containing certain ingredients for which scientific evidence exists purporting beneficial properties. By proposing this initiative, FDA recognizes that it "needs to adopt policies that would allow us to shift the competitive balance to these healthier products." In addition, some compounds, such as polyols and inulin, although not recognized by regulatory agencies for labeling purposes, are of increasing interest to manufacturers and consumers. As a result, the ability to identify and quantitatively measure levels of nutrients and other compounds has become even more essential as a tool to differentiate products in the marketplace.

Analytical evaluation of functional foods involves traditional methodologies as well as recently validated assays for the identification of new components of interest and unique sample matrices. Unfortunately, the lack of methods for many of these has generated challenges due to the absence of published references and compendial standards. As a result, researchers have focused on developing and validating assays to fill the gap. Although the pressure to adopt these methods is intense, they must meet the standards required to withstand scientific scrutiny and must be validated using established and well-designed criteria.

A method to the madness
The ability to efficiently develop, validate and implement a new method is an essential prerequisite for the successful completion of an analytical study. This process, known as method engineering, has many components that establish benchmarks for acceptance criteria and ultimately determine the validity of a specific method. The adoption of validation standards against which methods are evaluated assists in ensuring that the technique represents the practice of "good science" and is robust enough for a global scale. The process is composed of three phases: feasibility, development and validation. In addition, most of the primary methods used for regulatory compliance undergo a collaborative study process officiated by a recognized scientific organization, such as AOAC International, Gaithersburg, MD. FDA specifies its preference for AOAC methods, as noted in the Code of Federal Regulations (CFR). "Where the method of analysis is not prescribed in a regulation, it is the policy of the Food and Drug Administration in its enforcement programs to utilize the methods of analysis of the Association of Official Analytical Chemists (AOAC) ..." (21 CFR. 2.19, 2002).

Method feasibility allows a rapid determination of the best method-development approach and an assessment of the technical risks. The actual development follows; this involves determining the appropriate chemistry and optimum instrument parameters. The extent of validation required depends on the status of the method (i.e., newly developed or modification of existing procedure). A full validation requires a comprehensive evaluation of a variety of components that indicates not only the accuracy of the data, but the scientific limits and parameters as well. Validation uses both spiked solutions and subject samples. Existing methods that are being modified for a new matrix, analyte or instrument may undergo a less-extensive validation process.

Even though a thorough method-engineering process is a good indicator of an assay's acceptability, most food-analysis methods also undergo a stringent AOAC collaborative study process. "A single laboratory validation proves a method works in my hands, at my lab, on one day," says Jim Bradford, AOAC executive director. "A collaborative study ensures that when followed, a method will work in any lab, with any scientist, on any day."

At least eight labs analyze a set number of samples and standards following a detailed protocol. After the results are compiled and statistically evaluated, the methods committee and experts in the component or technique of interest review the data. If accepted, laboratories employ the method for a minimum of 2 years, during which time it is further evaluated and modified. After the trial period, the official-methods board votes to adopt a method as official. Darryl Sullivan, head of the official-methods board for AOAC, feels that the stringent nature of the study process promotes confidence in a method with both scientists and regulators. "By statistically evaluating data produced in different laboratories under different conditions, the strengths and weaknesses of a method are quickly recognized," he says. "Of the hundreds of methods developed, only a small percentage eventually attains 'official' status."

Analytical overview
By definition, analytical chemistry is the process of separating something into component parts or constituent elements. While measuring the traditional macronutrients - protein, minerals, calories and fat - relies on basic tried and true chemical techniques, identifying more-specific components, such as individual minerals and fatty acids, is a complex process involving intricate extraction, detection and measurement phases.

Extraction isolates and concentrates the compounds of interest for measurement. This phase of analysis has garnered much of the attention for functional foods due to the unique and diverse compounds measured. Extraction uses solvents in which the compounds are soluble, and it must isolate all of the analytes of interest as well as eliminate potential interferences. To determine an extraction procedure's accuracy, researchers must compare the results to known concentrations through standard addition, matrix fortification (spiking) or known reference standards. When analyzing raw materials, the standard addition and spiking methods are sometimes inadequate due to the dearth of data on the true concentrations of many of the ingredients. This lack of certified reference standards has, at times, hampered methods development.

Douglas Park, director of the FDA division of natural products, says the agency recognizes the challenges this poses to scientists. "Dependable and well-characterized reference standards are essential components of the method development and validation process and their limited availability has definitely hindered researchers in some cases," he notes. "As a result, the FDA is actively working with the National Institute of Standards and Technology as well as manufacturers, suppliers and academia to develop these reference standards and materials."

The majority of food-analysis methods employ chromatographic or spectrophotometric techniques. In chromatography, the purified sample is mixed with a mobile phase and then injected into the instrument. This mobile phase is designed to facilitate passage through a stationary phase, where samples are separated. By measuring the time required by individual components to pass through various sections of the stationary phase, the instrument can detect the components and generate a graph that allows precise measurement.

Unlike chromatography, which measures components by separation, spectrometry determines the level of a component, primarily minerals, by measuring the wavelength of emitted particles. The instruments are based on the basic principle that minerals emit colors when subjected to high temperatures. Using this principle, scientists have developed highly advanced excitation sources, such as plasmas, which produce the extremely high temperatures needed for emission of some elements. These techniques significantly reduce interference and provide extremely low levels of detection. Some compounds require other methods, such as atomic absorption, which quantifies compounds based on the amount of radiant energy absorbed.

Nutrient analysis
The components of interest in functional foods run the gamut from vitamins and carbohydrates to lycopene and flavonoids. As a result, most food samples undergo a barrage of diverse assays to meet labeling claims and marketing goals. For example, vitamin analysis includes a wide spectrum of analytical methodologies, such as chromatographic, spectrophotometric, fluorometric and microbiological procedures. Although there has been an increasing trend toward automated methods like chromatographic and fluorometric for some foods and certain vitamins, laboratories still use microbiological and biological methods.

In the same vein, the carbohydrate content of foods traditionally shown in most compositional tables is carbohydrate by difference (residual mass after subtracting water, protein, fat and ash). This value includes sugars, starches, fiber and other organic compounds. The amount of complex carbohydrate is generally recognized as the sum of available starch and total dietary fiber. As the classification of these compounds evolves, there has been increased need for specific methodologies to identify levels of individual fiber components.

Dietary-fiber analysis uses one of three methods: enzymatic-gravimetric, nonenzymatic-gravimetric or enzymatic-chemical (i.e., enzymatic-colorimetric, enzymatic-GLC and enzymatic-HPLC). Laboratories widely use the enzymatic-gravimetric method for routine analysis, labeling and quality control. Its popularity is based, in large part, upon its relative precision and cost-effectiveness. Nonenzymatic-gravimetric methods have been used for crude fiber, acid-detergent fiber and neutral-detergent fiber. In addition, a nonenzymatic-gravimetric method has been developed for total dietary fiber in low-starch products. However, these methods are generally not used to support nutrition-labeling claims. Enzymatic-chemical methods are based on chromatographic detection and measurement.

Phyto focus
With a few notable exceptions, such as omega-3 polyunsaturated fatty acids, the components of interest in functional foods are phytochemicals - organic components of plants that may promote human health. In many cases, scientific information about these components is relatively sparse and, as a result, requires the development or modification of analytical methods. Don Hughes, who has seen many advances during his more than 40 years as a scientist at Covance Laboratories, Princeton, NJ, says the current explosion in analytical methodology is not without precedent. "Over the decades, research has continually identified groups of compounds that posed beneficial or hazardous impact on health. Whether it was vitamins in the 1950s, agrochemicals in the 1960s or indirect additives in the 1980s, science has responded with new analytical techniques." Among the most recent successes are new and modified methods for phytochemicals such as carotenoids, phospholipids and polyphenols.

Until recently, the primary method used for measuring phospholipids dated back to 1984. Scientists today use high-performance liquid chromatography (HPLC) and evaporative light-scattering detection to determine the phospholipid content of soy lecithin products. The HPLC analysis includes qualitative identification by retention time and quantitative calculation from a calibration curve constructed from multiple-level standards. The phospholipids measured include phosphatidylcholine (PC), lysophosphatidylcholine (LPC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), phosphatidylserine (PS), and phosphatidic acid (PA). The results are expressed as percent (fresh-weight basis) for raw materials or the amount (mg) per serving size or capsule/tablet for finished products.

Carotenoids, such as alpha-carotene, beta- carotene, lycopene and zeaxanthin, comprise another class of compounds subject to increasing analytical demand. To determine carotenoid levels, food products are initially blended with alcohol, filtered and extracted with hexane. The extracts are analyzed on a reversed phase HPLC system and compared to recognized standards.

Catechins, a family of polyphenol compounds found predominately in green tea, have antioxidant properties and are linked to a wide range of health benefits. The industry needed an efficient, cost-effective method for quantifying their content in teas, tea extracts and fortified products to support product development and clinical studies. Six compounds, including catechin, epicatechin (EC), epicatechin gallate (ECG), epigallocatechin (EGC), epigallocatechin gallate (EGCG) and gallocatechin gallate (GCG) were isolated utilizing reversed-phase HPLC and quantified with ultraviolet (UV) detection. By modifying and optimizing chromatographic parameters, a method was established that enables simultaneous quantification of all six compounds within a single aliquot, thereby enabling catechin determination for multiple analyses over a short period of time. Validation of this HPLC method is a key factor in providing dependable data to support scientific and regulatory decisions.

Good science for a global community
The tremendous challenges posed by the burgeoning number of functional components can only be met through cooperation of all interested parties. Anita Mishra, AOAC principal scientific liaison for government and industry, envisions a new era of collaboration. "Through a coalition of industry, government and science, we can forge a global association of the analytical communities involved, thereby facilitating the efficient and thorough testing and adoption of accurate methods," she says. A commitment to the strict scientific standards and stringent method engineering will enable food researchers to continue to provide consumers with valuable product information and will provide impetus for the appropriate regulatory acknowledgement of a compound's physiological value.

Richard Crowley is a senior science writer with Covance Laboratories in Madison, WI. He is the editor of the Covance Food Science Newsletter and the author of numerous articles in the field of analytical chemistry. He has a B.S. in agricultural journalism from the University of Wisconsin-Madison and is a senior member of the Society for Technical Communication. For more information, e-mail rick.crowley@covance.com, or visit www.covance.com/analytical.