Scanning the Analytical Landscape

During an interview, legendary food industry attorney George Burditt once told me the food scientists of today have the talent to develop the methodologies required to solve the proverbial problem of fitting the square peg in a round hole. Although (with all due respect to George) this may be a bit of an exaggeration, there is no doubt the quality and complexity of the data being generated in the analytical laboratory are a primary factor in the unprecedented quality and safety of food products on the market.

The measurement of traditional nutrients for compliance with labeling regulations remains the primary job of the analytical laboratory, and these data are being generated much more efficiently and with greater sensitivity than ever before. In addition, new information concerning the beneficial and detrimental effects of food ingredients has resulted in scientists being asked to accurately quantify compounds such as trans fat and an expanding range of phytochemicals. In food safety, researchers are responding to the challenges posed by intentional contamination (e.g., bioterrorism), as well as a growing number of imported ingredients with potentially violative levels of chemical residues.

The science of sensitivity

Perhaps the most significant revolution in food analysis lies in the migration of many methods to mass spectrometry. Chromatography has long been the backbone of the analytical laboratory and the methodology of choice for a majority of food and botanical assays. Although chromatography can effectively separate the compounds of interest, the sensitivity and selectivity of the detection device are key to accurate quantification. The adoption of a combination of liquid chromatography (LC) for separation and mass spectrometry (MS) for quantification has provided researchers with more accurate and efficient tools to detect and measure trace levels of constituents in an expansive range of matrices.

A mass spectrometer measures the masses of individual molecules that have been converted into ions (i.e., electrically charged). The electrical signals, resulting from the detection of the ions, are converted into digital information that is used to indicate ion intensity and compound concentration. While LC/MS has long been an integral component of pharmaceutical bioanalysis, recent developments have expanded the applicability of these techniques to multifaceted food and botanical matrices that contain myriad components. These systems provide excellent limits of detection, reduce the need for many of the purification or clean-up steps, and allow the determination of multiple compounds at the same time.

Verifying vitamins

Traditionally, the determination of vitamin content using instrumental techniques presented special problems. For example, the concentration and recommended intake levels can be extremely low and, therefore, require highly sensitive tests. In addition, several vitamers and provitamins may be present, thus requiring the measurement of all appropriate substances. The form of certain vitamins is also important; the measurement of vitamins that occur naturally in foods is more difficult than analyzing dietary supplements, pharmaceutical products or fortified foods that contain vitamins in free (i.e., unbound) forms of known structure and biological activity.

The low concentration of some vitamins has made it difficult to measure chromatographically and, traditionally, a microbiological method was used. However, this method is extremely laborious and prone to poor reproducibility. Fortunately, the sensitivity and precision of LC/MS can distinguish these compounds without significant analytical issues. As a result, assays using LC/MS are increasingly utilized to measure vitamin content. These include assays for individual vitamins, as well as "profile" techniques for combinations of vitamins A, D and E, and all carotenoids. In one recent study, vitamins B₁, B₂, B₃, B₅, B₆ and PABA were measured by LC-MS/MS (tandem MS) simultaneously with a single injection. Assays have also been validated for quantification of "quasi-vitamins" such as inositol and choline. These LC/MS assays are rapidly becoming a mainstay of accurate, cost-effective vitamin analyses. Other new methods for vitamins are also being validated. For example, a reversed-phase HPLC assay for the determination of ascorbic acid has been developed, and methods using the enzyme-linked immunosorbent assay (ELISA), capillary electrophoresis, radioimmuno assay (RIA), and Biacore systems are also being validated.

Additional applications

MS is rapidly being adopted for use in other applications, including phytochemicals, minerals and chemical residues. The combination of inductively coupled plasma spectrometry (ICP) with MS provides the excellent limits of detection and multi-element determination capabilities required to quantify trace levels of minerals and heavy metals. This combination of instrumentation provides four modes of analysis, thereby allowing multielement screening with a single run, and it provides the sensitivity required to ensure levels fall within regulatory mandated or scientifically accepted tolerance levels. Multiresidue screening methods with LC/MS also allow the detection of trace concentrations (ppm) of pesticides using a single procedure.

A need for speed

Recent technological developments in chromatography have led to the introduction of a new technique and instrumentation known as ultra-high pressure liquid chromatography (uHPLC, UPLC). The theory of UPLC (i.e., rapid resolution chromatography) is based upon the Van Deemter equation, which theorizes that small HPLC particles give better chromatographic efficiency (i.e., more peaks per unit time) but require high pressure to maintain short run times. This sensitivity allows smaller sample volume to be used for analysis, and the speed helps to meet tight deadlines (true high-throughput analysis). The pharmaceutical industry employs uHPLC in the study of the metabolism of protein-based (i.e., genetically derived) drugs. The same principles are driving the increased use of the technique in the measurement of proteins and amino acids in food products.

Analyzing antioxidants

The growing interest in the physiological benefits of antioxidants has been matched by acceleration in the development of both analytical and biological methodologies of the levels and quality of these compounds. These phytochemicals include a broad range of chemically diverse compounds, such as vitamins E and C, carotenoids and polyphenols (e.g., catechins). As the potential therapeutic value of antioxidants becomes further elucidated, approaches to determine the safety and efficacy of these substances are being developed. To determine total polyphenolic levels, a spectrophotometric method is employed. The samples are analyzed in a spectrophotometer and the absorbance is compared to a gallic acid reference standard. Results are presented as gallic acid equivalents (GAE). Levels of individual compounds are quantified by utilizing reversed-phase HPLC for isolation and quantification with ultraviolet (UV) detection.

The goal of these methods is to provide efficient techniques for the determination of multiple compounds with a single mode of analysis. For example, by modifying and optimizing chromatographic parameters, a gradient method was established that would enable simultaneous quantitation of all six catechin compounds within a single aliquot, thereby enabling multiple analyses over a short period of time. Through the use of modified extractions and multiple detection systems, these and a variety of phenolic compounds (e.g., trans-resveratrol, caffeine and gallic acid) can also be analyzed. The ability to utilize this unified platform for a series of assays increases efficiency and lowers costs.

Testing trans fat

As a result of labeling mandates, the methods of analysis for trans fat in food products have become increasingly important, and have also come under intensive scrutiny by the scientific community, manufacturers and consumers. Currently, a method (996.06) for the analysis of trans fat from AOAC International, Gaithersburg, MD, is specified as one of the preferred assays by FDA. Specifically, it is the method of preference for foods that contain low levels of trans fat. This technique, originally developed in 1996, involves the extraction of fat and fatty acids from food by hydrolysis, after which the fats are extracted and converted into fatty acid methyl esters (FAMEs). The FAMEs are a mixture of all of the cis and trans isomers derived from the food. A profile of the isomers is obtained using gas chromatography (GC), and individual reference standards are used to identify and quantify the individual isomers. When standards are not available, the quantitation is performed using the response factors of isomers of similar chemical structure. Identification is then established using peak retention times and comparison of the fatty acid profile. Using this FAME profile, trans fats are identified and summed to generate a total trans fat value.

After the development and validation of the AOAC method, the American Oil Chemists’ Society (AOCS), Urbana, IL, performed additional research to optimize trans fatty acid isomer separation and identification. This resulted in the release of method Ce 1h-05 in 2005. This method allowed the separation, identification and quantitation of at least 15 trans isomers. Because the AOCS method was written specifically for the analysis of pure oils and fats, it does not include the sample preparation procedures documented in the AOAC assay (i.e., the separation of the fats from food matrices). Recently, researchers developed a hybrid method with the sample extraction techniques described in the AOAC method, but including the improved analytical separation of the GC conditions as documented in the AOCS method.

This method uses different GC conditions (e.g., column temperature), as well as modifications in the interpretation and quantitation of the chromatography. Over the last two years this method has been used to analyze hundreds of food and dietary supplement matrices, and the data show a more accurate determination of trans fat in all food samples may be possible using this technique. The data and discussion from the first validation of this method are currently pending publication by the AOAC. It is expected that the results of this initiative will facilitate a collaborative effort to revise the current official methods.

Fighting for food safety

The ability to quickly develop and validate a method is essential to combat the potential for intentional product tampering, and researchers are working on protocols to accelerate method validation in these incidences. For example, AOAC International is working with the U.S. Departments of Homeland Security and Defense on the development of a validation protocol for bioterror (i.e., public health actionable assay) that would provide valid data in the decision to evacuate a building. In addition, several new bills are pending in Congress (e.g., HR 3610, "Food and Drug Import Safety Act") that would further strengthen food-safety regulations and require more testing. However, because most programs place the testing burden on the already stretched resources of FDA, many experts believe the addition of provisions allowing private laboratories to be involved in the process are essential.

Continuing quest for quality

As more information about specific nutrients and botanical ingredients becomes available, consumer attitudes and regulatory expectations change, requiring manufacturers and scientists to seek analyses for compounds at trace levels. The validity and accuracy of the methods used are integral to the success of any product. As a result, there must be a balance in the need for rapid method development and cost vs. the quality of the data generated. From a scientific and regulatory standpoint, the precision, accuracy and generation of a true value are imperative. However, it is essential that industry, regulators and the scientific community take a collaborative approach to ensure that technology, science and law can adequately support consumer needs.

Richard Crowley is the editor of Covance Food Science Newsletter and the author of numerous articles on food, dietary supplement and pharmaceutical analysis. He has a B.S. in Agricultural Journalism from the University of Wisconsin–Madison and is an elected member of the National Association of Science Writers. He can be contacted at rick.crowley@covance.com.