Analysis of Chemical Contaminants

Although agricultural and industrial practices have drastically changed in recent decades, the threat of harmful residues in foods remains a major concern in the minds of consumers. In addition to pesticide residues, the presence of environmental contaminants such as dioxins, heavy metals and furans are of concern, as well as compounds created in processing, such as acrylamide and leachables from packaging. These concerns are compounded due to the explosion in the use of botanical ingredients that are often imported from countries where agrichemical monitoring is less stringent than in the United States. As a result, industry and regulatory agencies continue to be extremely vigilant in supplying accurate and timely data on residues in food. Because of the huge volume of testing needed to ensure the safety of both domestic and imported commodities, it is essential that fast, accurate and cost-effective methods are available to test samples.

The Food Quality Protection Act (FQPA) made several substantive changes to the manner in which pesticides are regulated. Primary among these are the standards for establishing tolerances and the impact on regulations pertaining to aggregate exposure to pesticides. Determining pesticide-residue levels that may be present in food products is a key part of a risk assessment process that must measure potential exposure from a variety of sources. Regulatory guidelines require that this testing include measuring levels of the compound present in food crops, products derived directly from the crops, and foods derived from the livestock fed treated crops or exposed to environmental sources (e.g., water). As a result of FQPA, the risk-assessment process must consider aggregate exposures from all potential routes of exposure, including dietary, residential and environmental. The data gathered from the studies determine which components of the total toxic residue (TTR) are present, and at what concentration secondary residues could appear. The sheer volume of testing required to monitor human exposure and evolving regulatory requirements have mandated the continuing development and adaptation of analytical methods.

Multiresidue methods

The need for efficient, high-quality testing led to the development of multiresidue screening methods that enable scientists to detect trace concentrations (parts per billion) of many pesticides using a single procedure. In addition to identifying compounds that are expected to be present, the multiresidue screen can alert researchers to the presence of compounds that are not expected or normally encountered in a specific matrix. Modern analytical technology now provides low-cost and high-throughput pesticide methods that can screen for hundreds of pesticides for less than $1 per compound.

Multiresidue screens allow cost-effective and timely samples analysis, produce reliable and verifiable data, and can identify many pesticides in a wide range of matrices at or below tolerance levels. These methods can monitor for parent compounds, metabolites, impurities, alteration products and other pesticide-associated chemicals. FDA’s Pesticide Analytical Manual (PAM) contains protocols and guidelines for these methods, and many have been adopted as official methods by AOAC International. If a pesticide is detected, confirmatory analysis by conducting additional chromatographic or mass spectrometry techniques can provide more comprehensive and sensitive data.

Although the specific parameters and techniques vary, traditional methods for the determination of chemical residues generally involves seven basic steps. Extraction removes the analyte from the matrix, usually by solvent extraction. The cleanup removes coextractives by such operations as column chromatography, liquid-liquid partitioning or volatilization. Some samples may undergo a modification process to convert the analyte to a readily analyzed derivative. The resolution step then separates the analyte from remaining interferences. Using a variety of detectors isolates a response related to the amount of analyte. This response is then measured and compared to that of a standard. A confirmation step provides assurance that the primary method gives correct results by use of a second method

Several multiresidue methods are listed in PAM for regulatory use. All of the methods routinely used are either GC- or HPLC-based. Although each of these employs similar techniques, they differ in the type of product and class of pesticide they are used to detect. The first of the multiresidue screens, the Mills method, was developed in 1959 for the measurement of nonionic organochlorine compounds in nonfatty foods. This method can be used for the determination of more than 150 chemicals, including several organophosphorus pesticides via gas chromatography (GC).

The PAM 302 assay, also known as the “Luke Method,” was originally designed to recover essentially all nonionic pesticides in the organochlorine, organophosphate, organonitrogen and hydrocarbon classes. Because of its ability to measure a wide range of pesticides, this method is used extensively. The Storherr method is applicable to nonfatty foods such as fruits, vegetables and grains for the detection of organophophorus pesticides.

Many of the traditional methods are being replaced by more efficient and accurate methodologies. For example, crises such as the recent incidences of alleged high pesticide levels in soft drinks in India accelerated the development of a new liquid chromatography/mass spectrometry (LC/MS) method that can detect minute levels (0.1 ppb) of pesticides. In addition to providing increased sensitivity, these methods reduce the complexity of the cleanup and extraction processes, thereby greatly enhancing the efficiency of the assay and accuracy of the data. These methods have successfully undergone validation and are being expanded to an increasing range of matrices.

When pesticide residues are detected in raw agricultural commodities for the production of livestock feed, data on the transfer of residue to meat, milk and eggs are required. These studies are also required if a pesticide is to be applied directly to animals or used in the agricultural premises in which the animals will be housed or feed is stored. The results of these studies are used to establish and enforce tolerances or maximum residue levels, and thereby reduce health hazards for humans and animals.

Botanical ingredients are also routinely tested to determine levels of pesticide residues. This is critical, because, in some cases, the use of these pesticides has not been approved and tolerances have not been established. These tests are performed using standard residue methods. Because there are no established tolerances for most of these products, any trace of an agrichemical discovered would result in the product being classified as adulterated. FDA is enforcing interim tolerance of 10 parts per billion (ppb) for some pesticides. As a part of the GMP process, testing is also conducted for solvent residues from the concentration and clean-up of the active ingredients.

Modifications in the techniques and instrumentation used with these methods continue to improve their value. As we look to the future, the use of LC/MS/MS, high-resolution GC/MS and supercritical fluid chromatography will play an increasing role is residue analysis. In addition, initiatives are underway to accelerate the worldwide harmonization of residue assays.

Other chemical hazards

Determining human exposure to possible carcinogens is not limited to pesticide residues. Other chemical substances of concern include toxic minerals (mercury, lead), compounds produced as a result of processing (acrylamide, PNA), environmental contaminants (dioxins, furans, benzene, heavy metals), and indirect additives that may migrate from packaging. In Europe, illegal dyes such as Sudan I, II, III and IV have been found in recent studies and prompted quick action from the European Union.

As the use of innovative packaging methods increases, it produces a corresponding effect on the regulatory and experimental guidelines for determining the impact of packaging components on safety. The primary concern is the migration of indirect additives from packaging that can cause health concerns and adversely affect the taste, color and aroma of foods. In the case of botanicals, migration could potentially have a negative effect on the efficacy of the active ingredient. Migration data are the quantitative description of the substances that transfer from the contact material into the product. Testing requires identifying potential classes of migrating substances and defining a method for determining human dietary exposure. Determining migration has proven difficult because the food products themselves, due to their heterogeneous nature, can interfere with the accuracy of the data. As a result, testing is done using solvents that simulate the leaching action of the foods.

In 2002, the release of data from the Swedish National Food Administration determining that carbohydrate-rich foods processed at high temperatures may contain significant levels of acrylamide resulted in a firestorm of debate. A liquid chromatography-mass spectrophometric/mass spectrophotometric (LC-MS/MS) method for the determination of acrylamide was developed and is being used for a variety of matrices. This instrumentation facilitates optimum chromatographic parameters and a mode of detection that provides improved precision, accuracy, selectivity and specificity. This method has a typical limit of quantitation of 10 ppb. Before analysis, fat is removed and an aqueous extraction is done to remove the acrylamide. The acrylamide is quantified using a triple quadrupole mass spectrometer.

As the list of elements thought to cause toxic reactions expands, the need for the efficient analysis of minerals will become more acute. The adoption of techniques, such as inductively coupled plasma spectrometry (ICP), that provide excellent limits of detection as well as multi-element determination provides researchers more sophisticated and efficient tools to detect and quantify trace levels of inorganic constituents in an ever-growing range of matrices. For analysis of heavy metals, ICP in conjunction with a MS detection system can screen for the presence of metals at minute levels.

A continuing effort

Global agricultural practices cannot change overnight, and despite the rise in the popularity of “organic” products, consumers, by nature, will continue to be concerned about the potential for chemical residues in food and dietary supplements. As a result, the efficient and accurate monitoring of these products for all potential chemical residues is essential to retain consumer confidence and assure regulatory compliance.

Table 1. Residue Assays Commonly Conducted

• FDA Residue Screens

• Chlorinated Pesticides

• Organophosphate Pesticides

• Chlorinated and Organophosphate Pesticides

• Triazine and Chloracetamide Herbicides

• Chlorphenoxy Acid Herbicides

• N-Methyl Carbamates

• Pheyl Urea Herbicides

• USP-NF Pesticides

• PCNB (Quintozene) and Degradants

• Aflatoxins

• Heavy Metals

• Arsenic

• Cadmium

• Lead

• Mercury

• Indirect additives: leachables

• Acrylamide

• Polynuclear aromatic hydrocarbons (PNA/PAH)

• Solvents

• Illegal dyes