Extension of AOAC official method 999.14 (choline in infant formula and milk) to the determination of choline in dietary supplements

AOAC Official Method 999.14 is applicable for the determination of choline in milk and infant formulas. To date, its use has not been extended beyond these matrixes. We modified Official Method 999.14 and applied it to the determination of choline in a range of choline-containing dietary supplements. Dietary supplement tablets, capsules, wafers, softgels, liquid products, and drink powders were included. We found that the standard curve could be extended to cover a wider range of choline concentrations and defined a procedure for the use of Norit for samples in which the vitamin C content was high enough to interfere with the analysis. Recoveries of choline added to infant formula powders and to representative dietary supplement tablets, capsules, powdered drink mix, and wafer products were 85-114%. The use of Norit during the procedure did not affect the recovery of choline added to infant formula powders or to dietary supplements. An alkaline digestion was included for use with a product containing lecithin as the sole source of choline. Ten of 11 dietary supplement products analyzed by the modified method contained amounts of choline at or above declarations found on the product labels. The remaining product contained about 40% of the label-declared amount of choline.     

The use of multi-element stable isotope analysis to monitor the origin of chondroitin sulfates

Continuous-flow isotope ratio mass spectrometry (CF-IRMS) of deuterium, carbon, nitrogen, oxygen and sulfur has been used to analyse samples of pure chondroitin sulfates from known animal sources (shark, squid, salmon, pig and bovine). There is a need to control the origin of this dietary supplement, which is extracted from several types of animals: for traditional, ethical, or economic reasons, a given source of natural products of animal origin can be preferred to another, and can therefore have a different price. Twenty-three samples collected in Europe and Asia were analysed by IRMS. The results, especially the isotopic deviations of sulphur, oxygen and deuterium, show a significant discrimination between marine and terrestrial origins of this compound which will provide a convenient and efficient way to control the declared sources in the market. The differences observed between origins are further discussed.     

Cattle breeding: A fast screening procedure to control the bovine fodder contamination

The European Commission has adopted a large number of emergency measures to prevent the spread of the Bovine spongiform encephalopathy (BSE) in the Member States, and among them, a European Regulation prohibits the feeding to ruminants of animal protein and animal feed containing such protein. Aim of this work has been to propose the thermal analysis as a new rapid and sensitive screening tool for a preliminary determination of possible contamination in products destined to bovine growth, since the possibility to screen the samples by a rapid and sensitive tool could reduce the time of analysis required by the actual methodology and could allow the systematic surveillance of bovine feeding, with the official EU methods applied only to positive samples.     

Determination of isoflavones in dietary supplements containing soy, Red Clover and kudzu: extraction followed by basic or acid hydrolysis

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to beta-estradiol, health benefits of isoflavones have been evaluated in age-related and hormone-dependent diseases. Dietary supplement preparations contain extracts from soy, Red Clover and kudzu. Soy products contain primarily genistein, daidzein, and glycitein, while Red Clover products contain primarily formononetin and biochanin A. Kudzu extracts contain puerarin and daidzein among other components. Previous methods of analysis focused on the determination of isoflavones from a single botanical source, while dietary supplements are often a blend of extracts from different plants. We developed a method for the analysis of isoflavones in dietary supplements regardless of their botanical composition, using HPLC-PDA because of its applicability to routine analysis. Isoflavones are found as free compounds, glucoside derivatives, 6'-O-malonyl-beta-d-glucoside and 6'-O-acetyl-beta-d-glucoside derivatives. In this study, the samples were extracted at room temperature with 50:50 (v/v) MeCN/water, and then analyzed before and after hydrolyzing the isoflavones by acid or basic digestion. 2'-Methoxy-flavone and 6-methoxy-flavone were used as internal standards and were added together to every sample. Daidzein, glycitein, genistein, puerarin, calycosin, pratensein, pseudobaptigenin, formononetin, biochanin A and prunetin were among the isoflavones determined.     

Studying variations in the PCDD/PCDF profile across various food products using multivariate statistical analysis

Polychlorinated dibenzo-p-dioxins (PCDD) and polychlorinated dibenzofurans (PCDF) are widely recognized by the scientific community as persistent organic pollutants due to their toxicity and adverse effects on wildlife and human health. The actual regulation dedicated to the monitoring of dioxins in food is based on the measurement of 17 congener concentrations. The final result is reported as a toxic equivalent value that takes into account the relative toxicity of each congener. This procedure can minimize the qualitative information available from the abundances of each PCDD/PCDF congener: the characteristic contamination profile of the sample. Multivariate statistical techniques, such as principal component analysis (PCA) or linear discriminant analysis (LDA), represent an interesting way to investigate this qualitative information. Nevertheless, they have only been applied to the analysis of contamination data from food products and biological matrices infrequently. The objective of the present study was to analyze a large data set from dioxin analyses performed on various food products of animal origin. The results demonstrate the existence of differences in congener-specific patterns between the analyzed samples. Variability was first demonstrated in terms of the food type (fish, meat, milk, fatty products). Then a variability was observed that was related to the specific animal species for meat and milk samples (bovine, ovine, porcine, caprine and poultry). Some practical applications of these results are discussed. The origin(s) of the observed differences, as well as their significance, now remain to be investigated, both in terms of environmental factors and transfer through living organisms. A better knowledge of the relation between a contamination profile and its specific source and/or food product should be of great interest to scientists working in the fields of contaminant analysis, toxicology and metabolism, as well as to regulatory bodies and risk assessors in charge of final decisions regarding the eventual hazards associated with theses substances.     

Development and validation of an HPLC-DAD analytical procedure for quality control of damiana (Turnera diffusa), using an antioxidant marker isolated from the plant

Damiana is a shrub used as a dietary supplement and as an additive in several foods. This report presents the first validated analytical method for QC of products made from damiana. Through bioassay-guided fractionation, the compound showing the best antioxidant activity, determined using 2,2-diphenyl-1-picryl-hydrazyl assay in TLC, was purified and identified as 8-C-beta-[6-deoxy-2-O-(alpha-1-rhamnopyranosyl)-xylo- hexopyranos-3-uloside] (Cl50 5.56 g/mL). This is the only natural source from which this compound has been reported, and has been found in all native damiana samples analyzed. Therefore, it was used as a biomarker to develop an HPLC-diode-array detection analytical procedure using the first-order derivative of the chromatograms. Separation was achieved using an AccQ Tag C18 150 x 3.9 mm (4 microm) column, with a gradient of methanol and 0.1% trifluoroacetic acid in water. The method was shown to be accurate, sensitive, and reproducible, providing a useful quantitative means to apply QC to damiana products.     

Choice of dietary protein of vegetarians and omnivores is reflected in their hair protein 13C and 15N abundance

Stable isotopic (15N, 13C) composition of tissues depends on isotopic pattern of food sources. We investigated whether the isotopic compositions of human hair protein and amino acids reflect the habitual dietary protein intake. Hair samples were analyzed from 100 omnivores (selected randomly out of the 1987-1988 German nutrition survey VERA), and from 15 ovo-lacto-vegetarians (OLV), and from 6 vegans recruited separately. Hair bulk and amino acid specific isotopic compositions were analyzed by isotope-ratio mass spectrometry (EA/IRMS and GC/C/IRMS, respectively) and the results were correlated with data of the 7 day dietary records. Hair bulk 15N and 13C abundances clearly reflect the particular eating habits. Vegans can be distinguished from OLV and both are significantly distinct from omnivores in both 15N and 13C abundances. 15N and 13C abundances rose with a higher proportion of animal to total protein intake (PAPI). Individual proportions of animal protein consumption (IPAP) were calculated using isotopic abundances and a linear regression model using animal protein consumption data of vegans (PAPI = 0) and omnivores (mean PAPI = 0.639). IPAP values positively correlated with the intake of protein, meat, meat products, and animal protein. Distinct patterns for hair amino acid specific 15N and 13C abundances were measured but with lower resolution between food preference groups compared with bulk values. In conclusion, hair 13C and 15N values both reflected the extent of animal protein consumption. Bulk isotopic abundance of hair can be tested for future use in the validation of dietary assessment methods.     

Stable isotope (13C, 15N and 34S) analysis of the hair of modern humans and their domestic animals

Relationships between dietary status and recent migration were examined by delta(13)C, delta(15)N and delta(34)S analysis of hair samples from 43 modern humans living in a rural community in SW England. The isotopic content of 38 'local' hair samples was compared with that of five recently arrived individuals (from Canada, Chile, Germany and the USA). Hair samples from domestic animals (i.e. mainly cats, dogs, cows and horses) were analysed to examine the difference in delta(13)C, delta(15)N and delta(34)S values between herbivores and carnivores. Generally, modern human hair data from the triple stable isotope (delta(13)C, delta(15)N and delta(34)S) provided enough information to confirm the dietary status and origin of the individual subjects. The dietary intake was generally reflected in the animal hair delta(15)N and delta(13)C values, i.e. highest in the carnivores (cats). However, a non-local origin of food sources given to domesticated omnivores (i.e. dogs) was suggested by their hair delta(34)S values.     

Potentiometric determination of saccharin in dietary products using mercurous nitrate as titrant

A rapid, precise and low cost method for saccharin determination in dietary products is proposed. Saccharin in several samples is potentiometrically titrated with mercurous nitrate solution using a silver wire coated with a metallic mercury film as the working electrode, and the end point was found using a Gran's plot. The detection limit of sodium saccharin was 0.5 mg/ml and the best pH range was from 2.0 to 3.5. Sucrose, glucose, aspartame, sodium cyclamate, sorbitol, fructose, benzoic acid, salicylic acid and lactose do not interfere even in significant amounts. The interference due to the presence of chloride and/or phosphate ions can be eliminated by previous solvent extraction of this sweetener. Recovery of saccharin from various dietary products gave from 95.2 to 103.2% of the label claim.     

Evidence of natural occurrence of the banned antibiotic chloramphenicol in herbs and grass

Chloramphenicol (CAP), a broad-spectrum antibiotic, was detected in several herb and grass samples from different geographic origins. Due to its suspected carcinogenicity and linkages with the development of aplastic anemia in humans, CAP is banned for use in food-producing animals in the European Union (EU) and many other countries. However, products of animal origin originating from Asian countries entering the European market are still found noncompliant (containing CAP) on a regular basis, even when there is no history of chloramphenicol use in these countries. A possible explanation for the continued detection of these residues is the natural occurrence of CAP in plant material which is used as animal feed, with the consequent transfer of the substance to the animal tissues. Approximately 110 samples were analyzed using liquid chromatography coupled with mass spectrometric detection. In 26 samples, the presence of CAP was confirmed using the criteria for banned substances defined by the EU. Among other plant materials, samples of the Artemisia family retrieved from Mongolia and from Utah, USA, and a therapeutic herb mixture obtained from local stores in the Netherlands proved to contain CAP at levels ranging from 0.1 to 450 μg/kg. These findings may have a major impact in relation to international trade and safety to the consumer. The results of this study demonstrate that noncompliant findings in animal-derived food products may in part be due to the natural occurrence of chloramphenicol in plant material. This has implications for the application of current EU, USA, and other legislation and the interpretation of analytical results with respect to the consideration of CAP as a xenobiotic veterinary drug residue and the regulatory actions taken upon its detection in food.     

Aquaculture Production of the Brown Seaweeds Laminaria digitata and Macrocystis pyrifera: Applications in Food and Pharmaceuticals

Seaweeds have a long history of use as food, as flavouring agents, and find use in traditional folk medicine. Seaweed products range from food, feed, and dietary supplements to pharmaceuticals, and from bioenergy intermediates to materials. At present, 98% of the seaweed required by the seaweed industry is provided by five genera and only ten species. The two brown kelp seaweeds Laminaria digitata, a native Irish species, and Macrocystis pyrifera, a native New Zealand species, are not included in these eleven species, although they have been used as dietary supplements and as animal and fish feed. The properties associated with the polysaccharides and proteins from these two species have resulted in increased interest in them, enabling their use as functional foods. Improvements and optimisations in aquaculture methods and bioproduct extractions are essential to realise the commercial potential of these seaweeds. Recent advances in optimising these processes are outlined in this review, as well as potential future applications of L. digitata and, to a greater extent, M. pyrifera which, to date, has been predominately only wild-harvested. These include bio-refinery processing to produce ingredients for nutricosmetics, functional foods, cosmeceuticals, and bioplastics. Areas that currently limit the commercial potential of these two species are highlighted.     

Determination of St. John's wort components in dietary supplements and functional foods by liquid chromatography

St. John's wort (Hypericum perforatum L.) preparations, a top-selling botanical dietary supplement used primarily as an antidepressant, has recently been used as an ingredient in some food products sold as functional foods. A rapid extraction technique followed by a liquid chromatographic (LC) method was developed to determine 4 characteristic bioactive compounds (pseudohypericin, hypericin, hyperforin, and adhyperforin) from St. John's wort in dietary supplements and functional foods to which it was added. Solid samples, including dried leaf/flower mixture, dietary supplement capsules, tea bags, puff and snack bar, were extracted with methanol by sonication. Noncarbonated, fruit-flavored drinks were centrifuged and mixed with methanol. Compounds were then determined by isocratic, reversed-phase LC with UV detection at 2 wavelengths and further identified or confirmed by photodiode array spectra and LC/mass spectrometry. Within-laboratory method variations (% RSD) were satisfactory. Very low amounts, if any, of the 4 components were found in drink and puff samples, and none was found in the snack bar. The methods developed provide a useful means for the determination of St. John's wort components in dietary supplements and functional foods.     

Iodine determination in dietary supplement products by TXRF and ICP-AES spectrometry

About two-thirds of the Hungarian population is living in areas where drinking water contains less than 0.025 mg/L I₂. Iodine is an essential element required for the synthesis of thyroid hormones. The recommended daily allowance (RDA) is 0.150 mg for adults (WHO [17]). In the case of iodine deficiency mineral water containing iodine and seaweed products could serve as the natural source of this element. In the present study, the capability and limitations of total reflection X-ray fluorescence (TXRF) and inductively coupled plasma atomic emission (ICP-AES) spectrometry for the determination of iodine are discussed, in the case of commercially available dietary supplements and differently processed seaweed (kelp) products. Multi-mineral tablets and kelp samples were digested using concentrated nitric acid before analysis. Extraction using a 25% ammonia solution was proved to be effective as an alternative sample preparation procedure for seaweed and dietary supplement tablets containing iodine. Precipitation of silver iodide, as a sample preparation step of mineral water, was applied to prevent loss of volatile iodine during solvent evaporation. ICP-AES measurement was found to be seriously affected by spectral line coincidence between the prominent line of iodine and the adjacent phosphorus line. TXRF was proved to be line coincidence free and suitable for iodine analysis. TXRF determination of iodine in mineral water, in seaweed samples and in dietary supplement tablets has not been reported yet.     

Determination of total soy isoflavones in dietary supplements, supplement ingredients, and soy foods by high-performance liquid chromatography with ultraviolet detection: collaborative study

An interlaboratory study was conducted to evaluate a method for determining total soy isoflavones in dietary supplements, dietary supplement ingredients, and soy foods. Isoflavones were extracted using aqueous acetonitrile containing a small amount of dimethylsulfoxide (DMSO) and all 12 of the naturally occuring isoflavones in soy were determined by high-performance liquid chromatography (HPLC) with UV detection using apigenin as an internal standard. Fifteen samples (6 pairs of blind duplicates plus 3 additional samples) of soy isoflavone ingredients, soy isoflavone dietary supplements, soy flour, and soy protein products were successfully analyzed by 13 collaborating laboratories in 6 countries. For repeatability, the relative standard deviations (RSDr) ranged from 1.07 for samples containing over 400 mglg total isoflavones to 3.31 for samples containing 0.87 mg/g total isoflavones, and for reproducibility the RSDR values ranged from 2.29 for samples containing over 400 mg/g total isoflavones to 9.36 for samples containing 0.87 mg/g total isoflavones. HorRat values ranged from 1.00 to 1.62 for all samples containing at least 0.8 mg/g total isoflavones. One sample, containing very low total isoflavones (< 0.05 mg/g), gave RSDR values of 175 and a HorRat value of 17.6. This sample was deemed to be below the usable range of the method. The method provides accurate and precise results for analysis of soy isoflavones in dietary supplements and soy foods.     

Determination of total iodine in foods and dietary supplements using inductively coupled plasma-mass spectrometry

A method was developed and validated for the determination of total iodine in a wide variety of food products and dietary supplements. The method involves a unique sample digestion with a KOH solution in an oven or by using an open-vessel microwave system. After digestion, a stabilizer is added and the solution is taken to volume, then filtered and analyzed either directly or after dilution. The amount of iodine is determined with inductively coupled plasma-mass spectrometry (ICP-MS). The method was validated by experiments to determine its precision, accuracy, linearity, specificity, ruggedness, and robustness. The LOQ of this method is 25-50 microg/kg. The method demonstrated an average RSD of 2.27% during analysis of milk powder and 4.30% during analysis of a dietary supplement tablet reference material. The accuracy of the method as determined with these same reference materials was 100 and 94.2%, respectively. The method has been used successfully on commodity foods, processed foods, dairy products, pet food, infant formula, animal feed, mineral premixes, and a variety of dietary supplements.     

Challenges encountered in the analysis of phthalate esters in foodstuffs and other biological matrices

Phthalate esters are ubiquitous environmental pollutants and are recognized as environmental endocrine disruptors because of their potential to elicit reproductive and developmental toxicity. Several phthalate esters have been listed by the US Environmental Protection Agency (EPA) as chemicals of concern. Determination of concentrations of phthalate esters in foodstuffs, typically present at sub to low nanogram-per-gram concentrations (between 0.1 and 100?ng?g^?1), is essential for assessment of human dietary exposure. However, phthalate esters are commonly present as contaminants in several laboratory products, including organic solvents, that are used in sample preparation and analysis. Therefore, accurate analysis of phthalates in food samples is a challenging task. In this review, we summarize the methods available for the determination of phthalate esters in foodstuffs and report on concentrations of phthalates in foodstuffs and potential sources of contamination by phthalates in the analysis of foodstuffs. We offer suggestions to eliminate and/or reduce background levels of contamination by phthalates in the analysis of food and other biological samples. We also introduce methods that are suitable for trace analysis of phthalates in a variety of liquid and solid food samples, in particular, a liquid–liquid extraction method for removal of lipids from food samples, because these can substantially reduce background levels of phthalates in the analytical procedure.     

On-line microwave-assisted digestion of solid samples for their flame atomic spectrometric analysis

A new procedure has been developed for the on-line digestion of solids in a microwave oven. The direct injection in a water carrier flow of dispersions of solid samples in concentrated nitric acid, the merging of these slurries with 30% (v/v) H(2)O(2) and the microwave-assisted digestion in a Teflon coil of 100 cm permit a fast and quantitative extraction of Cu and Mn from different solid matrices, such as vegetables, powdered dietary products and sewage sludges. The development of an appropriate interphase, in which digested samples are cooled and degassified, previous to their introduction into the nebulizer of a flame atomic absorption spectrometer, makes possible the full automatization of the digestion and measurement steps of the elemental analysis of solids and it provides a sample frequency of 180 injections per hour. The developed procedure has also been applied for Pb and Zn determination in certified sewage sludge samples, with accurate results obtained for Pb but low results found for Zn.     

Two alternative derivations of Bridgman's theorem

One of the fundamental results of Dimensional Analysis is the so‐called Bridgman' s theorem. This theorem states that the only functions that may have dimensional arguments are products of powers of the base quantities of a given system of units. In this work, Bridgman's theorem is discussed and rederived in two different ways, one not involving calculus, and a second one based on a Taylor series expansion analysis. This revised version was published online in July 2006 with corrections to the Cover Date.     

Targeted and nontargeted screening and identification of 50 antihypertensive adulterants in dietary supplements and herbal medicines using quadrupole–orbitrap high resolution mass spectrometry with compound database

In the present work, a novel database of drug compounds and a rapid screening method based on ultra‐high performance liquid chromatography coupled to high resolution orbitrap mass spectrometry were developed and applied in the screening and identification of targeted and nontargeted antihypertensive adulterants in dietary supplements and herbal medicines. The established screening database includes retention time, exact mass, fragments, isotopic pattern, and MS² spectra library of the target compounds and thus provides automated search and identification of the targets with a single injection. The nontargeted compounds in the samples are identified through the full MS scan and MS² data by using the Chemspider database and the data analysis in XCalibur, MassFrontier and TraceFinder software. In addition, this method possesses excellent quantitative capacity. The novel approach was applied to 65 batches of samples that are claimed as “all‐natural” products having the antihypertensive function, among which nine batches were found to be positive. Multiple targeted and nontargeted antihypertensive adulterants were detected at levels ranging from 2.8 to 27.9 mg/g. The novel database and screening method demonstrated herein will be promising and powerful tools for rapid screening of antihypertensive adulterants in dietary supplements and herbal medicines.