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Analytical and Bioanalytical Chemistry (v.389, #1)

Food and dietary supplements by Katherine E. Sharpless; Franz Ulberth (pp. 1-2).

is a research chemist in the Analytical Chemistry Division of the National Institute of Standards and Technology.is head of the Food Safety and Quality unit of the European Commission’s Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), located in Geel, Belgium.

“Doctrine of Signatures” challenge by Stephen G. Saupe; Katherine E. Sharpless (pp. 3-9).

Solution to quality assurance challenge 5 by Manfred Reichenbächer; Jürgen W. Einax (pp. 11-12).

Standard operating procedure for the collection and preparation of voucher plant specimens for use in the nutraceutical industry by Jana Hildreth; Eva Hrabeta-Robinson; Wendy Applequist; Joseph Betz; James Miller (pp. 13-17).

A vital part of the development of any standardized protocol for the extraction of plant-derived crude extracts to be used in herbal medicine or nutritional supplementation is proper documentation of the original botanical source of the extract via acquisition of a voucher specimen. The purpose of this document is to serve as an accepted protocol for voucher specimen collection, handling, and storage, with specific guidelines to address commercial and research uses.

Keywords: Dietary supplements; Nutraceuticals; Quality control; Vouchers

The NIH analytical methods and reference materials program for dietary supplements by Joseph M. Betz; Kenneth D. Fisher; Leila G. Saldanha; Paul M. Coates (pp. 19-25).

Quality of botanical products is a great uncertainty that consumers, clinicians, regulators, and researchers face. Definitions of quality abound, and include specifications for sanitation, adventitious agents (pesticides, metals, weeds), and content of natural chemicals. Because dietary supplements (DS) are often complex mixtures, they pose analytical challenges and method validation may be difficult. In response to product quality concerns and the need for validated and publicly available methods for DS analysis, the US Congress directed the Office of Dietary Supplements (ODS) at the National Institutes of Health (NIH) to accelerate an ongoing methods validation process, and the Dietary Supplements Methods and Reference Materials Program was created. The program was constructed from stakeholder input and incorporates several federal procurement and granting mechanisms in a coordinated and interlocking framework. The framework facilitates validation of analytical methods, analytical standards, and reference materials.

Keywords: Bioanalytical methods; Natural products; Quality assurance/control; Reference materials

Recent studies on selected botanical dietary supplement ingredients by Jeanne I. Rader; Pierluigi Delmonte; Mary W. Trucksess (pp. 27-35).

The market for botanical dietary supplements in the US has grown rapidly during the last 15 years. Use of newly introduced botanical ingredients has often outpaced an adequate scientific understanding of the ingredients themselves. This may lead to problems, including misidentification, mislabeling, adulteration, and toxicity related to the intended ingredient or one substituted for it. This article reviews recent work with several botanical ingredients (Ephedra, Citrus species, Hoodia gordonii, Teucrium, isoflavones) that illustrates the complexity of the current situation and approaches that contribute to ensuring the quality of botanical ingredients. Recent work with contamination of botanical products by mycotoxins is also reviewed. The need for tools for botanical authentication and methods for reproducible extraction of bioactive constituents is critical. Such tools, and improved analytical techniques for identifying potentially bioactive constituents in fresh plant material and in concentrated extracts and for detection of hazardous contaminants, are expected to improve the overall quality and safety of botanical dietary supplement ingredients.

Keywords: Botanical ingredients; Ephedra ; Hoodia ; Teucrium ; Isoflavones; Mycotoxins

Measuring vitamins and minerals in dietary supplements for nutrition studies in the USA by Johanna T. Dwyer; Joanne Holden; Karen Andrews; Janet Roseland; Cuiwei Zhao; Amy Schweitzer; Charles R. Perry; James Harnly; Wayne R. Wolf; Mary Frances Picciano; Kenneth D. Fisher; Leila G. Saldanha; Elizabeth A. Yetley; Joseph M. Betz; Paul M. Coates; John A. Milner; Jackie Whitted; Vicki Burt; Kathy Radimer; Jaime Wilger; Katherine E. Sharpless; Constance J. Hardy (pp. 37-46).

This article illustrates the importance of having analytical data on the vitamin and mineral contents of dietary supplements in nutrition studies, and describes efforts to develop an analytically validated dietary supplement ingredient database (DSID) by a consortium of federal agencies in the USA. Preliminary studies of multivitamin mineral supplements marketed in the USA that were analyzed as candidates for the DSID are summarized. Challenges are summarized, possible future directions are outlined, and some related programs at the Office of Dietary Supplements, National Institutes of Health are described. The DSID should be helpful to researchers in assessing relationships between intakes of vitamins and minerals and health outcomes.

Keywords: Dietary supplements; Dietary supplement ingredient database; Multivitamin mineral supplements; Analytical values

Profiling methods for the determination of phenolic compounds in foods and dietary supplements by James M. Harnly; Seema Bhagwat; Long-Ze Lin (pp. 47-61).

Profiling methods are needed that separate and detect all the phenolic compounds in a single extract of a food material. These methods must be comprehensive, rapid, and rich in spectral information. Fourteen methods that meet, or have the potential to meet, these criteria have been selected from the recent literature for review. In general, the methods employ a single aqueous methanol extraction, separation on a reversed-phase C column, and detection by UV/vis spectroscopy and mass spectrometry. The variations in extraction, separation, and detection are discussed. An increasingly important aspect of these methods is the archiving of data to permit cross-comparison of samples and standards and retrospective analysis. This review shows that the necessary technology is available to achieve the desired analytical goals.

Keywords: Chromatographic profiling; Phenolic compounds; Flavonoids

Analytical procedures for water-soluble vitamins in foods and dietary supplements: a review by Christopher J. Blake (pp. 63-76).

Water-soluble vitamins include the B-group vitamins and vitamin C. In order to correctly monitor water-soluble vitamin content in fortified foods for compliance monitoring as well as to establish accurate data banks, an accurate and precise analytical method is a prerequisite. For many years microbiological assays have been used for analysis of B vitamins. However they are no longer considered to be the gold standard in vitamins analysis as many studies have shown up their deficiencies. This review describes the current status of analytical methods, including microbiological assays and spectrophotometric, biosensor and chromatographic techniques. In particular it describes the current status of the official methods and highlights some new developments in chromatographic procedures and detection methods. An overview is made of multivitamin extractions and analyses for foods and supplements.

Keywords: Water-soluble vitamins; Foods; Dietary supplements; Microbiological assay; Chromatography; Biosensor

Evaluation of gas chromatographic methods for the determination of trans fat by Pierluigi Delmonte; Jeanne I. Rader (pp. 77-85).

Consumption of trans fat has been associated with increased risk of coronary heart disease. For nutrition labeling purposes, the US Food and Drug Administration (FDA) defines trans fat as the sum of all the fatty acids with at least one nonconjugated double bond in the trans configuration. The FDA regulation states that label declarations of trans fat are not required for products that contain less than 0.5 g of trans fat per serving if no claims are made about fat, fatty acids or cholesterol. While attenuated total reflection Fourier-transformed infrared spectroscopy (ATR-FT-IR) provides reproducible measurements for samples containing more than 5% trans fat, methods based on gas chromatography (GC) are needed to measure lower trans fat levels. Trans fat quantitation by GC has recently been updated by considering more fatty acids, focusing more attention on fatty acids present in low amounts, and by using 100-m high-polarity capillary columns for optimal separation. The consistently high interlaboratory relative standard deviations (RSD, e.g., 21% at 1% trans fatty acids (TFA), 60% at 0.17% TFA), and intralaboratory RSD values (e.g., 10% at 1% TFA, 16% at 0.17% TFA) for trans fat at 1% or less of total fat reported in the collaborative study data for American Oil Chemists Society Official Method Ce 1h-05 suggest the need to carefully define the parameters associated with GC analysis of fatty acids.

Keywords: Trans fat; Fatty acids; Gas chromatography

Determination of total trans fats and oils by infrared spectroscopy for regulatory compliance by M. M. Mossoba; V. Milosevic; M. Milosevic; J. K. G. Kramer; H. Azizian (pp. 87-92).

The mandatory requirement in many countries to declare the amount of trans fat present in food products and dietary supplements has led to a need for sensitive and accurate methodologies for the rapid quantitation of total trans fats and oils. Capillary gas chromatography (GC) and infrared spectroscopy (IR) are the two methods most commonly used to identify and quantify trans fatty acids for food labeling purposes (see the article by Delmonte and Rader in this ABC issue for a detailed presentation of GC methodology). The present article provides a comprehensive review of the IR technique and the current attenuated total reflection (ATR) Fourier-transform (FT) IR methodologies for the rapid determination of total trans fats and oils. This review also addresses potential sources of interferences and inaccuracies in FTIR determinations, particularly those done at low trans levels. Recent observations have shown that the presence of saturated fats caused interferences in the FTIR spectra observed for trans triacylglycerols. The recognition and resolution of previously unresolved quantitative issues improved the accuracy and sensitivity of the FTIR methodology. Once validated, it is anticipated that the new negative second-derivative ATR-FTIR procedure will make IR spectroscopy more suitable than ever, and a rapid alternative and/or complementary method to GC, for the rapid determination of total trans fats for regulatory compliance. Figure Infrared light bouncing inside an internal reflection crystal

Keywords: Fourier transform infrared; Attenuated total reflection; Trans fats; Validated official methods

Analysis of bovine immunoglobulin G in milk, colostrum and dietary supplements: a review by Leyton W. Gapper; David E. J. Copestake; Don E. Otter; Harvey E. Indyk (pp. 93-109).

The immunoprotective properties of bovine milk immunoglobulin G (IgG) have led to a recent proliferation of nutritional products incorporating this protein. It has therefore become critical that reliable analytical techniques for the measurement of the IgG content in such products are available. This literature review surveys current methods of analysis for IgG, including separation-based or immuno-based concentration analysis. The review also discusses nutraceutical applications, regulatory issues, stability of IgG and the significance of primary reference material in IgG analysis.

Keywords: Immunoglobulin G; IgG; Bovine; Milk; Colostrum; Protein; Analysis; Dietary supplements

Food allergen detection methods and the challenge to protect food-allergic consumers by Arjon J. van Hengel (pp. 111-118).

The detection of allergenic ingredients in food products has received increased attention from the food industry and legislative and regulatory agencies over recent years. This has resulted in the improvement of measures aimed at the protection of food-allergic consumers. The controlled production of food products and control activities executed by food inspection agencies rely on the availability of methods capable of detecting traces of allergenic ingredients. The development of such methods faces a multitude of analytical challenges. Those challenges will be identified and discussed in this review. Furthermore, future developments and trends in analytical methodology as applied to the detection of food allergens are reported.

Keywords: Bioanalytical methods; Foods; Beverages; Immunoassays; ELISA; PCR

Analysis of heat-induced contaminants (acrylamide, chloropropanols and furan) in carbohydrate-rich food by Thomas Wenzl; Dirk W. Lachenmeier; Vural Gökmen (pp. 119-137).

Heat-induced food contaminants have attracted attention of both the scientific community and the public in recent years. The presence of substances considered possibly or probably carcinogenic to humans has triggered an extensive debate on the healthiness of even staple foods. In that respect, acrylamide, furan and chloropropanols are the main substances of concern. Their widespread occurrence in processed food, which concomitantly causes considerable exposure to humans, led either to the setting of maximum limits (for some chloropropanols) or at least the initiation of monitoring programmes in order to put risk assessment on a solid data basis. Acrylamide, furan and chloropropanols are small molecules with physicochemical properties that make their analysis challenging. Their amount in food ranges typically from below the limit of detection to hundreds of micrograms per kilo or even milligrams per kilo. However, a number of recently published scientific reports deal with the analysis of these substances in different kinds of food. The aim of this publication is to give an overview of analytical approaches for the determination of acrylamide, furan and chloropropanols in foodstuffs.

Keywords: Foods; Beverages; GC; HPLC

Analysis of heterocyclic aromatic amines by M. Murkovic (pp. 139-146).

Heterocyclic aromatic amines are formed in protein and amino acid-rich foods at temperatures above 150 °C. Of more than twenty heterocyclic aromatic amines identified ten have been shown to have carcinogenic potential. As nutritional hazards, their reliable determination in prepared food, their uptake and elimination in living organisms, including humans, and assessment of associated risks are important food-safety issues. The concentration in foods is normally in the low ng g⁻¹ range, which poses a challenge to the analytical chemist. Because of the complex nature of food matrixes, clean-up and enrichment of the extracts are also complex, usually involving both cation-exchange (propylsulfonic acid silica gel, PRS) and reversed-phase purification. The application of novel solid-phase extraction cartridges with a wettable apolar phase combined with cation-exchange characteristics simplified this process—both the polar and apolar heterocyclic aromatic amines were recovered in one fraction. Copper phthalocyanine trisulfonate bonded to cotton (“blue cotton”) or rayon, and molecular imprinted polymers have also been successfully used for one-step sample clean-up. For analysis of the heterocyclic aromatic amines, liquid chromatography with base-deactivated reversed-phase columns has been used, and, recently, semi-micro and capillary columns have been introduced. The photometric, fluorimetric, or electrochemical detectors used previously have been replaced by mass spectrometers. Increased specificity and sub-ppb sensitivities have been achieved by the use of the selected-reaction-monitoring mode of detection of advanced MS instrumentation, for example the triple quadrupole and Q-TOF instrument combination. Gas chromatography, also with mass-selective detection, has been used for specific applications; the extra derivatization step needed for volatilization has been balanced by the higher chromatographic resolution.

Keywords: Heterocyclic aromatic amines; HPLC; Mass-selective detection; Electrochemical detection; Fluorescence detection; Clean-up

Regulations relating to mycotoxins in food by Hans P. van Egmond; Ronald C. Schothorst; Marco A. Jonker (pp. 147-157).

Regulations relating to mycotoxins have been established in many countries to protect the consumer from the harmful effects of these compounds. Different factors play a role in the decision-making process of setting limits for mycotoxins. These include scientific factors, for example the availability of toxicological data and occurrence data, detailed knowledge about possibilities for sampling and analysis, and socio-economic issues. By the end of 2003, approximately 100 countries (covering approximately 85% of the world’s inhabitants) had specific regulations or detailed guidelines for mycotoxins in food. The regulations were related to aflatoxins (B₁, B₂, G₁ and G₂), aflatoxin M₁, trichothecenes (deoxynivalenol, diacetoxyscirpenol, T-2 toxin and HT-2 toxin), fumonisins (B₁, B₂, and B₃), agaric acid, ergot alkaloids, ochratoxin A, patulin, phomopsins, sterigmatocystin, and zearalenone. In Europe, and in particular in the EU, regulatory and scientific interest in mycotoxins has undergone a development in the last decade from autonomous national activity towards more EU-driven activity with a structural and network character. Harmonized EU limits now exist for 40 mycotoxin–food combinations. It is expected this number will grow in 2007 to approximately 50. The direct or indirect influence of European organizations and programs on the EU mycotoxin regulatory developments is significant. They include the European Food Safety Authority, the Scientific Cooperation on Questions relating to Food, the Rapid Alert System for Food and Feed, the creation of an EU Community Reference Laboratory for Mycotoxins and a mandate of the EC to the European Standardization Committee in methods for analysis for mycotoxins in food. Large pan-European research and networking projects as “BioCop” and “MoniQA” are also important.

Keywords: Mycotoxin; International; Regulation; Analysis; Food safety

Analysis of food for toxic elements by Stephen G. Capar; William R. Mindak; John Cheng (pp. 159-169).

The levels of the toxic elements Al, As, Cd, Hg, Pb and Sn are routinely monitored in food to protect the consumer. Increasingly, the chemical forms of As and Hg are also monitored. Analyses are performed to enforce regulatory standards and to accumulate background levels for assessing long-term exposure. The analytical procedures used for these activities evolve as requirements to determine lower levels arise and as both the types and sheer number of different foods that need to be analyzed increase. This review highlights recent work addressing improvements in the analysis of toxic elements in food. The topics covered include contamination control, analytical sample treatment and the common analytical techniques used for food analysis.

Keywords: Review; Food; Analysis; Toxic elements

Standard reference materials for foods and dietary supplements by Katherine E. Sharpless; Jeanice Brown Thomas; Steven J. Christopher; Robert R. Greenberg; Lane C. Sander; Michele M. Schantz; Michael J. Welch; Stephen A. Wise (pp. 171-178).

Well-characterized certified reference materials are needed by laboratories in the food testing, dietary supplement, and nutrition communities to facilitate compliance with labeling laws and improve the accuracy of information provided on product labels, so that consumers can make good choices. As a result of the enactment of the Nutrition Labeling and Education Act of 1990 and the Infant Formula Act of 1980, the National Institute of Standards and Technology (NIST) worked to develop a series of food-matrix standard reference materials (SRMs) characterized for nutrient concentrations. These include SRM 1544 Fatty Acids and Cholesterol in a Frozen Diet Composite, SRM 1546 Meat Homogenate, SRM 1548a Typical Diet, SRM 1566b Oyster Tissue, SRM 1846 Infant Formula, SRM 1946 Lake Superior Fish Tissue, SRM 1947 Lake Michigan Fish Tissue, SRM 2383 Baby Food Composite, SRM 2384 Baking Chocolate, SRM 2385 Slurried Spinach, and SRM 2387 Peanut Butter. With the enactment of the Dietary Supplement Health and Education Act of 1994, NIST has been working to develop suites of dietary supplement SRMs characterized for active and marker compounds and for toxic elements and pesticides, where appropriate. An updated SRM 1588b Organics in Cod Liver Oil, a suite of ephedra-containing materials (SRMs 3240–3245), a carrot extract in oil (SRM 3276), and a suite of ginkgo-containing materials (SRMs 3246–3248) are available. Several other materials are currently in preparation. Dietary supplements are sometimes provided in forms that are food-like; for these, values may also be assigned for nutrients, for example SRM 3244 Ephedra-Containing Protein Powder. Both the food-matrix and dietary supplement reference materials are intended primarily for validation of analytical methods. They may also be used as “primary control materials” in assignment of values to in-house (secondary) control materials to confirm accuracy and to establish measurement traceability to NIST.

Keywords: Dietary supplement; Food; Reference material; Standard reference material

Characterization of a suite of ginkgo-containing standard reference materials by Catherine A. Rimmer; Samuel B. Howerton; Katherine E. Sharpless; Lane C. Sander; Stephen E. Long; Karen E. Murphy; Barbara J. Porter; Karsten Putzbach; Michael S. Rearick; Stephen A. Wise; Laura J. Wood; Rolf Zeisler; Diane K. Hancock; James H. Yen; Joseph M. Betz; Agnes NguyenPho; Lu Yang; Christine Scriver; Scott Willie; Ralph Sturgeon; Brian Schaneberg; Christina Nelson; Jules Skamarack; Meide Pan; Kerri Levanseler; Dean Gray; Edward H. Waysek; Anne Blatter; Eike Reich (pp. 179-196).

A suite of three ginkgo-containing dietary supplement Standard Reference Materials (SRMs) has been issued by the National Institute of Standards and Technology (NIST) with certified values for flavonoid aglycones, ginkgolides, bilobalide, and selected toxic trace elements. The materials represent a range of matrices (i.e., plant, extract, and finished product) that provide different analytical challenges. The constituents have been determined by at least two independent analytical methods with measurements performed by NIST and at least one collaborating laboratory. The methods utilized different extractions, chromatographic separations, modes of detection, and approaches to quantitation. The SRMs are primarily intended for method validation and for use as control materials to support the analysis of dietary supplements and related botanical materials.

Keywords: Liquid chromatography; Mass spectrometry; Ginkgo biloba ; Standard reference material; Dietary supplements

Determination of bitter orange alkaloids in dietary supplement Standard Reference Materials by liquid chromatography with atmospheric-pressure ionization mass spectrometry by Karsten Putzbach; Catherine A. Rimmer; Katherine E. Sharpless; Stephen A. Wise; Lane C. Sander (pp. 197-205).

A liquid chromatographic atmospheric-pressure ionization electrospray mass spectrometry (LC–API–ES–MS) method has been developed for the determination of five bitter orange alkaloids (synephrine, octopamine, n-methyltyramine, tyramine, and hordenine) in bitter orange-containing dietary supplement standard reference materials (SRMs). The materials represent a variety of natural, extracted, and processed sample matrices. Two extraction techniques were evaluated: pressurized-fluid extraction (PFE) and sonication extraction. The influence of different solvents, extraction temperatures, and pH were investigated for a plant material and a processed sample. The LC method uses a new approach for the separation of highly polar alkaloids. A fluorinated, silica-based stationary phase separated the five alkaloids and the internal standard terbutaline in less than 20 min. This method enabled the determination of the dominant alkaloid synephrine and other minor alkaloids in a variety of dietary supplement SRMs.

Keywords: Dietary supplement; Standard Reference Material (SRM); Bitter orange alkaloids; LC–API–ES–MS; Synephrine; Fluorinated stationary phase

Preparation and characterization of standard reference material 3276, carrot extract in oil by Katherine E. Sharpless; Jeanice Brown Thomas; David L. Duewer; Karsten Putzbach; Catherine A. Rimmer; Lane C. Sander; Michele M. Schantz; Stephen A. Wise; Takashi Yarita; James H. Yen (pp. 207-217).

The National Institute of Standards and Technology (NIST), the Food and Drug Administration (FDA) Center for Drug Evaluation and Research (CDER) and Center for Food Safety and Applied Nutrition (CFSAN), and the National Institutes of Health (NIH), Office of Dietary Supplements (ODS) are collaborating to produce a series of standard reference materials (SRMs) for dietary supplements. Standard reference material (SRM) 3276 Carrot Extract in Oil is one in this series, with values assigned for trans-α-carotene, trans- and total β-carotene, δ- and γ-tocopherol, and twelve fatty acids. Results for carotenoids and tocopherols were obtained by use of combinations of liquid chromatography (LC), on columns of differing selectivity, with absorbance and mass spectrometric (MS) detection. Fluorescence detection also was used for the tocopherols. Results for fatty acids were obtained by use of gas chromatography (GC) with both flame-ionization and mass-spectrometric detection. This material is intended for use as a primary control material when assigning values to in-house (secondary) control materials and for validation of analytical methods for measurement of these analytes in similar matrices. Figure Carrot Extract in Oil

Keywords: Carrot extract; Carotenoid; Fatty acid; Standard reference material; Tocopherol

Reference materials to evaluate measurement systems for the nutrient composition of foods: results from USDA’s National Food and Nutrient Analysis Program (NFNAP) by Katherine M. Phillips; Wayne R. Wolf; Kristine Y. Patterson; Katherine E. Sharpless; Joanne M. Holden (pp. 219-229).

Over a 6.5-year period a total of 2554 values were reported by nine laboratories for 259 certified or reference nutrient concentrations in 26 certified reference materials (CRM) submitted to contract laboratories, blinded, as part of the qualifying process for analytical contracts and in the routine sample stream as part of the National Food and Nutrient Analysis Program. Each value was converted to a Z′-score, reflecting the difference from the assigned value related to the combined expected analytical uncertainty plus the uncertainty in the CRM value. Z′-scores >|3.0| were considered unacceptable. For some nutrients (Na, folate, dietary fiber, pantothenic acid, thiamin, tocopherols, carotenoids, monounsaturated, and polyunsaturated fatty acids), >20% of Z′-scores were >|3.0|. For total fat, vitamin C, and niacin >25% of Z′-scores were >|2.0|. Components for which CRM data were best (more than 90% of Z′-scores <|2.0|) were Mg, P, Mn, Se, and vitamin B12. In some cases deviations from assigned values were not uniform across laboratories and materials. For Na almost all high Z′-scores were for low-Na matrices, suggesting analytical problems related to concentration. Figure Z′-scores for vitamins in certified reference materials

Keywords: Reference materials; Uncertainty; Accuracy; Food composition data

The caffeine contents of dietary supplements commonly purchased in the US: analysis of 53 products with caffeine-containing ingredients by Karen W. Andrews; Amy Schweitzer; Cuiwei Zhao; Joanne M. Holden; Janet M. Roseland; Mary Brandt; Johanna T. Dwyer; Mary Frances Picciano; Leila G. Saldanha; Kenneth D. Fisher; Elizabeth Yetley; Joseph M. Betz; Larry Douglass (pp. 231-239).

As part of a study initiating the development of an analytically validated Dietary Supplement Ingredient Database (DSID) in the United States (US), a selection of dietary supplement products were analyzed for their caffeine content. Products sold as tablets, caplets, or capsules and listing at least one caffeine-containing ingredient (including botanicals such as guarana, yerba mate, kola nut, and green tea extract) on the label were selected for analysis based on market share information. Two or three lots of each product were purchased and analyzed using high-pressure liquid chromatography (HPLC). Each analytical run included one or two National Institute of Standards and Technology (NIST) Standard Reference Materials (SRMs) and two products in duplicate. Caffeine intake per serving and per day was calculated using the maximum recommendations on each product label. Laboratory analysis for 53 products showed product means ranging from 1 to 829 mg caffeine/day. For products with a label amount for comparison (n = 28), 89% (n = 25) of the products had analytically based caffeine levels/day of between −16% and +16% of the claimed levels. Lot-to-lot variability (n = 2 or 3) for caffeine in most products (72%) was less than 10%.

Keywords: Caffeine; HPLC; UV/VIS; Dietary supplement; Reference material

Biosynthetic production of universally ¹³C-labelled polyunsaturated fatty acids as reference materials for natural health product research by Phuong Mai Le; Catherine Fraser; Graeme Gardner; Wei-Wan Liang; Jaroslav A. Kralovec; Stephen C. Cunnane; Anthony J. Windust (pp. 241-249).

Long-chain polyunsaturated fatty acids (LCPUFA) including eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have become important natural health products with numerous proven benefits related to brain function and cardiovascular health. Not only are omega-3 fatty acids available in a plethora of dietary supplements, but they are also increasingly being incorporated as triglycerides into conventional foods, including bread, milk, yoghurt and confectionaries. Recently, transgenic oil seed crops and livestock have been developed that enhance omega-3 fatty acid content. This diverse array of matrices presents a difficult analytical challenge and is compounded further by samples generated through clinical research. Stable isotope ¹³C-labelled LCPUFA standards offer many advantages as research tools because they may be distinguished from their naturally abundant counterparts by mass spectrometry and directly incorporated as internal standards into analytical procedures. Further, ¹³C-labelled LCPUFAs are safe to use as metabolic tracers to study uptake and metabolism in humans. Currently, ¹³C-labelled LCPUFAs are expensive, available in limited supply and not in triglyceride form. To resolve these issues, marine heterotrophic microorganisms are being isolated and screened for LCPUFA production with a view to the efficient biosynthetic production of U-¹³C-labelled fatty acids using U-¹³C glucose as a carbon source. Of 37 isolates obtained, most were thraustochytrids, and either DHA or omega-6 docosapentaenoic acid (22:5n-6) were produced as the major LCPUFA. The marine protist Hyalochlorella marina was identified as a novel source of EPA and omega-3 docosapentaenoic acid (22:5n-3). As proof of principle, gram-level production of ¹³C-labelled DHA has been achieved with high chemical purity ( >99%) and high ¹³C incorporation levels (>90%), as confirmed by NMR and MS analyses. Finally, U-¹³C-DHA was enzymatically re-esterified to glycerol to yield a ¹³C-labelled tridocosahexaenoin.

Keywords: Polyunsaturated; Omega-3; Stable isotope; Thraustochytrid; Hyalochlorella

Chromatographic fingerprint analysis for evaluation of Ginkgo biloba products by Pei Chen; Mustafa Ozcan; James Harnly (pp. 251-261).

The flavonoids and the terpene lactones are regarded as the two main active components of Ginkgo biloba that affect human health. In the work discussed in this paper, two analytical methods for the characterization of G. biloba authentic materials and commercial products, an LC–UV chromatographic fingerprinting method and a traditional flavonol quantification method, were compared. The traditional method was used to determine the total flavonol content (as glycosides) after acid hydrolysis. The fingerprinting method examined the chromatographic profiles of methanol–water extracts using chemometric methods. The traditional method showed that all the commercial products met the current voluntary standard of 24% flavonols by weight. The chromatographic fingerprinting method revealed significant variations in the commercial products with regard to the relative concentration of individual flavonols.

Keywords: Ginkgo biloba ; HPLC; UV; Dietary supplement; Fingerprint

Enforcement of the ban on aristolochic acids in Chinese traditional herbal preparations on the Dutch market by Martijn J. Martena; Jacqueline C. A. van der Wielen; Leo F. J. van de Laak; Erik J. M. Konings; Henk N. de Groot; Ivonne M. C. M. Rietjens (pp. 263-275).

In traditional chinese medicine several Aristolochia species are used. Aristolochia spp. contain a mixture of aristolochic acids (AAs), mainly AA I and AA II which are nephrotoxicants and carcinogens. After AA-related nephropathy (AAN) and urothelial cancer were described in female patients in Belgium following intake of AA-contaminated herbal preparations, herbs with AAs were prohibited worldwide. Confusing nomenclature can cause AA contamination of certain Chinese traditional herbal preparations (THPs). Here we report the results of investigations by the Dutch Food and Consumer Product Safety Authority (VWA) into the presence of AAs in THPs sampled on the Dutch market using a liquid-chromatography–-mass spectrometry method. Between 2002 and 2006 we sampled 190 Chinese THPs using recent information on Chinese THPs potentially containing AAs. AA I was found in 25 samples up to a concentration of 1,676 mg/kg. AA II was also found in 13 of these samples up to 444 mg/kg. All 25 positive samples including Mu Tong, Fang Ji, Tian Xian Teng and Xi Xin were part of a group of 68 THPs identified as possibly containing AAs. In a worst-case scenario, use of a sample of Mu Tong with the highest AA content over a 7-day period would result in the same intake levels of AAs which significantly raised the cancer risk in the Belgian AAN cases. Our results show that contaminated THPs still can be found on the market following worldwide publicity. Therefore, it can be concluded that testing of possibly AA-contaminated THPs is still essential. Figure Various Chinese Herbs

Keywords: Chinese herb nephropathy; Aristolochic acid; Mu Tong; Traditional chinese medicine; Herbal remedies

Lack of effect of oral supplementation with antioxidants on cholesterol oxidation product concentration of human plasma, as revealed by an improved gas chromatography method by Francesc Guardiola; Alba Tres; Rafael Codony; Paul B. Addis; Scott D. Bergmann; James H. Zavoral (pp. 277-289).

A gas chromatographic method was successfully applied to determine cholesterol oxidation products (COPs) in human plasma. The linearity, precision, recovery and sensitivity of the method were determined. Oral supplementation with a combination of vitamin E (800 IU), C (1 g) and β-carotene (24 mg), given for 21 days to 21 patients, did not significantly decrease plasma COP content. No correlations (n = 26) were found between initial plasma COP content and the following parameters: age, body mass index, plasma content of α-tocopherol, cholesterol, high-density lipoprotein cholesterol and triglycerides, and fat, natural antioxidant and oxidized lipid intake. Differences in plasma COP content between type 2 diabetic (n = 6) and nondiabetic (n = 20) patients were not statistically significant. The results from this study lead us to hypothesize that the nonenzymatic oxidation of cholesterol in plasma is negligible compared to COPs originating from the diet. This article also includes a comprehensive review of the drawbacks of the analytical methods of COP determination in plasma and serum.

Keywords: Cholesterol oxidation product analysis; Human plasma; Vitamin E, vitamin C and β-carotene supplementation; Diet; Diabetes

An integrated procedure for the measurement of total dietary fibre (including resistant starch), non-digestible oligosaccharides and available carbohydrates by Barry V. McCleary (pp. 291-308).

A method is described for the measurement of dietary fibre, including resistant starch (RS), non-digestible oligosaccharides (NDO) and available carbohydrates. Basically, the sample is incubated with pancreatic α-amylase and amyloglucosidase under conditions very similar to those described in AOAC Official Method 2002.02 (RS). Reaction is terminated and high molecular weight resistant polysaccharides are precipitated from solution with alcohol and recovered by filtration. Recovery of RS (for most RS sources) is in line with published data from ileostomy studies. The aqueous ethanol extract is concentrated, desalted and analysed for NDO by high-performance liquid chromatography by a method similar to that described by Okuma (AOAC Method 2001.03), except that for logistical reasons, d-sorbitol is used as the internal standard in place of glycerol. Available carbohydrates, defined as d-glucose, d-fructose, sucrose, the d-glucose component of lactose, maltodextrins and non-resistant starch, are measured as d-glucose plus d-fructose in the sample after hydrolysis of oligosaccharides with a mixture of sucrase/maltase plus β-galactosidase.

Keywords: Total dietary fibre; Resistant starch; Non-digestible oligosaccharides; Available carbohydrates; Integrated method

Multi-element (H,C,N,S) stable isotope characteristics of lamb meat from different European regions by F. Camin; L. Bontempo; K. Heinrich; M. Horacek; S. D. Kelly; C. Schlicht; F. Thomas; F. J. Monahan; J. Hoogewerff; A. Rossmann (pp. 309-320).

Multi-element (H,C,N,S) stable isotope ratio analysis was tested for its suitability as a means for geographical provenance assignment of lamb meat from several European regions. The defatted dry matter (crude protein fraction) from lamb meat was found to be a suitable probe for “light” element stable isotope ratio analysis. Significant differences were observed between the multi-element isotope ratios of lamb samples from different regions. The mean hydrogen isotopic ratios of the defatted dry matter from lamb were found to be significantly correlated with the mean hydrogen isotopic ratios of precipitation and groundwater in the production regions. Carbon and nitrogen isotopic ratios were influenced by feeding practices and climate. Sulfur isotopic ratios were influenced by geographical location and surface geology of the production region. The results permitted differentiation of lamb meat, from most production regions, by inspection. However, more sophisticated evaluation of the data using multivariate methods, such as linear discriminant analysis, achieved 78% correct classification.

Keywords: Lamb; Geographic origin; Stable isotopes; Ratios; Authenticity

Polychlorinated dibenzo-p-dioxins, dibenzofurans and biphenyls in fish from the Netherlands: concentrations, profiles and comparison with DR CALUX® bioassay results by S. P. J. van Leeuwen; P. E. G. Leonards; W. A. Traag; L. A. P. Hoogenboom; J. de Boer (pp. 321-333).

Fish from Dutch markets were analysed for concentrations of polychlorinated dibenzo-p-dioxins/dibenzofurans (PCDD/Fs) and dioxin-like polychlorinated biphenyls (DL-PCBs) and compared with the new European maximum residue levels (MRLs), set in 2006. In a first study on 11 different fish and shellfish from various locations, concentrations of PCDD/Fs were nearly all below the MRL for PCDD/Fs [4 pg toxic equivalents (TEQ) per gram wet weight (ww)] and nearly all below 8 pg total TEQ/g ww, the new MRL for the sum of PCDD/Fs and DL-PCBs. Some samples exceeded the total TEQ MRL, such as anchovy, tuna and sea bass. Furthermore, 20 (out of 39) wild eel samples exceeded the specific MRL for eel (12 pg total TEQ/g ww), as the study revealed PCDD/F TEQ levels of 0.2–7.9 pg TEQ/g ww and total TEQ values of 0.9 to 52 pg/g ww. TEQ levels in farmed and imported eel were lower and complied with the MRLs. Smoking eel, a popular tradition in the Netherlands, only had marginal effects on PCDD/F and DL-PCB concentrations. Owing to volatilization, concentrations of lower-chlorinated PCBs were reduced to below the limit of quantification after smoking. DL-PCBs contributed 61–97% to the total TEQ in all eel samples. This also holds for other fish and shellfish (except shrimps): DL-PCB contributed (on average) from 53 (herring) to 83% (tuna) to the total TEQ. Principal-component analysis revealed distinctive congener profiles for PCDD/Fs and non-ortho PCBs for mussels, pikeperch, herring and various Mediterranean fish. The application of new TCDD toxic equivalency factors (TEFs) set by the World Health Organization in 2006 (to replace the 1997 TEFs) resulted in lower TEQ values, mainly owing to a decreased mono-ortho PCB contribution. This decrease is most pronounced for eel, owing to the relative high mono-ortho PCB concentrations in eel. Consequently, a larger number of samples would comply with the MRLs when the new TEFs are applied. The DR CALUX® assay may be used for screening total TEQ levels in eel, in combination with gas chromatography–high resolution mass spectrometry confirmation of suspected samples. An almost 1:1 correlation was found when the 1997 TEFs were applied, but, surprisingly, a 1.4-fold overestimation occurred with application of the 2006 TEFs.

Keywords: Dioxins; PCBs; Fish; Eel; Consumption; DR CALUX®

Enzymatic hydrolysis of esterified diarrhetic shellfish poisoning toxins and pectenotoxins by Erin Doucet; Neil N. Ross; Michael A. Quilliam (pp. 335-342).

Okadaic acid (OA) and dinophysistoxins-1 and -2 (DTX1, DTX2), the toxins responsible for incidents of diarrhetic shellfish poisoning (DSP), can occur as complex mixtures of ester derivatives in both plankton and shellfish. Alkaline hydrolysis is usually employed to release parent OA/DTX toxins, and analyses are conducted before and after hydrolysis to determine the concentrations of nonesterified and esterified toxins. Recent research has shown that other toxins, including pectenotoxins and spirolides, can also exist as esters in shellfish, but these toxins cannot survive alkaline hydrolysis. A promising alternative approach is enzymatic hydrolysis. In this study, two enzymatic methods were developed for the hydrolysis of 7-O-acyl esters, “DTX3,” and the carboxylate esters of OA, “diol-esters.” Porcine pancreatic lipase induced complete conversion of DTX3 to OA and DTXs within one hour for reference solutions. The presence of mussel tissue matrix reduced the rate of hydrolysis, but an optimized lipase concentration resulted in greater than 95% conversion within four hours. OA-diol-ester was hydrolyzed by porcine liver esterase and was completely converted to OA in less than 30 min, even in the presence of mussel tissue matrix. Esters and OA/DTX toxins were all monitored by LC–MS. Further experiments with pectenotoxin esters indicated that enzymatic hydrolysis could also be applied to esters of other toxins. Enzymatic hydrolysis has excellent potential as an alternative to the conventional alkaline hydrolysis procedure used in the preparation of shellfish samples for the analysis of toxins.

Keywords: Diarrhetic shellfish poisoning (DSP); Okadaic acid; Dinophysistoxins; Pectenotoxins; Esters; Liquid chromatography–mass spectrometry; Enzymatic hydrolysis

Contents of selected B vitamins in NIST SRM 3280 multivitamin/multielement tablets by liquid chromatography isotope dilution mass spectrometry by Pei Chen; Mustafa Ozcan; Wayne R. Wolf (pp. 343-347).

There is increased interest in accurately assessing the total dietary intake of vitamins from all sources, including foods and dietary supplements. Consequently, a Dietary Supplement Ingredient Database (DSID), based upon analytical values, is being established by USDA with support of the Office of Dietary Supplements (ODS), NIH. The DSID necessitated the development of a new SRM, 3280 — Multivitamin/Multimineral Tablets, by the National Institute of Standards and Technology (NIST), with support from the ODS. As a continuation of a long-term project to develop and validate new methods of determining water-soluble B vitamins in foods and dietary supplements, and as part of a collaborative effort with NIST to characterize SRM 3280, values for the vitamin contents of SRM 3280 have been generated by a liquid chromatographic isotope dilution mass spectrometric (LC/IDMS) method. Isotope-labeled (¹³C and/or ²H) B vitamins (B1-thiamine, B6-pyridoxine, B3-nicotinamide, and B5-pantothenic acid) were obtained from commercial sources, with the support of the ODS/NIH. Our LC/IDMS method uses a C18 reversed phase column, an Agilent 1100 HPLC system, and a Quattro Micro triple-quad mass spectrometer (MS). B vitamin determination was achieved using a gradient LC profile combined with MS/MS detection in multiple reaction monitoring mode. Stock solutions of the isotope-labeled vitamins were calibrated against USP standard solutions. The SRM tablets, with added amounts of the four isotope-labeled B vitamins, were extracted and the vitamins simultaneously determined in a single LC run, in contrast with the single-component determinations performed via IDMS. Unknown vitamin concentrations were calculated by comparing the ratios of the integrated LC peaks at the different masses of the unlabeled and labeled vitamins.

Keywords: Vitamin B; Thiamine; Nicotinamide; Pantothenic acid; Pyridoxine; Isotope dilution mass spectrometry; Liquid chromatography

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Analytical and Bioanalytical Chemistry (v.389, #1)