Showing papers in "Journal of AOAC International in 2006"

The Horwitz ratio (HorRat): A useful index of method performance with respect to precision.

Abstract: The Horwitz ratio (HorRat) is a normalized performance parameter indicating the acceptability of methods of analysis with respect to among-laboratory precision (reproducibility). It is the ratio of the observed relative standard deviation among laboratories calculated from the actual performance data, RSDR (%), to the corresponding predicted relative standard deviation calculated from the Horwitz equation PRSDR (%) = 2C(-0.15), where C is the concentration found or added, expressed as a mass fraction. It is more or less independent of analyte, matrix, method, and time of publication (as a surrogate for the state of the art of analytical chemistry). It is now one of the acceptability criteria for many of the recently adopted chemical methods of analysis of AOAC INTERNATIONAL, the European Union, and other European organizations dealing with food analysis (e.g., European Committee for Standardization and Nordic Analytical Committee). The origin and applications of the formula are described. Consistent deviations from the ratio on the low side (values 2) may indicate inhomogeneity of the test samples, need for further method optimization or training, operating below the limit of determination, or an unsatisfactory method.

An overview of the health effects of isoflavones with an emphasis on prostate cancer risk and prostate-specific antigen levels.

Abstract: Soybean isoflavones possess hormonal and nonhormonal properties that may reduce the risk of coronary heart disease, osteoporosis, and certain cancers, and alleviate hot flashes in menopausal women. Among the various cancers whose risk may be reduced by isoflavones, there is particular interest in prostate cancer. Eleven trials have examined the effects of isoflavones on serum prostate-specific antigen (PSA) levels. The dose of isoflavones in these trials from supplements or soy protein ranged from 60 to 900 mg/day (typical Japanese intake is 30-50 mg/day), subject number/group ranged from 8 to 62, and study duration from 20 days to 1 year. Isoflavones did not affect serum PSA in healthy subjects. In contrast, in 4 of 8 trials involving prostate cancer patients, isoflavones significantly favorably affected PSA although in no studies was there an absolute decrease in PSA concentrations. The mechanism by which isoflavones affect PSA could not be determined from the existing research, although hormonal changes do not seem to be a factor. The clinical evidence is sufficiently encouraging to justify considering additional Phase II and III clinical trials investigating the efficacy of soy isoflavones in different populations of prostate cancer patients alone and in combination with other treatments.

Biosensors for the analysis of food- and waterborne pathogens and their toxins.

Abstract: Biosensors are devices which combine a biochemical recognition element with a physical transducer There are various types of biosensors, including electrochemical, acoustical, and optical sensors Biosensors are used for medical applications and for environmental testing Although biosensors are not commonly used for food microbial analysis, they have great potential for the detection of microbial pathogens and their toxins in food They enable fast or real-time detection, portability, and multipathogen detection for both field and laboratory analysis Several applications have been developed for microbial analysis of food pathogens, including E coli O157:H7, Staphylococcus aureus, Salmonella, and Listeria monocytogenes, as well as various microbial toxins such as staphylococcal enterotoxins and mycotoxins Biosensors have several potential advantages over other methods of analysis, including sensitivity in the range of ng/mL for microbial toxins and <100 colony-forming units/mL for bacteria Fast or real-time detection can provide almost immediate interactive information about the sample tested, enabling users to take corrective measures before consumption or further contamination can occur Miniaturization of biosensors enables biosensor integration into various food production equipment and machinery Potential uses of biosensors for food microbiology include online process microbial monitoring to provide real-time information in food production and analysis of microbial pathogens and their toxins in finished food Biosensors can also be integrated into Hazard Analysis and Critical Control Point programs, enabling critical microbial analysis of the entire food manufacturing process In this review, the main biosensor approaches, technologies, instrumentation, and applications for food microbial analysis are described

Immunoassay as an analytical tool in agricultural biotechnology.

Abstract: Immunoassays for biotechnology engineered proteins are used by AgBiotech companies at numerous points in product development and by feed and food suppliers for compliance and contractual purposes. Although AgBiotech companies use the technology during product development and seed production, other stakeholders from the food and feed supply chains, such as commodity, food, and feed companies, as well as third-party diagnostic testing companies, also rely on immunoassays for a number of purposes. The primary use of immunoassays is to verify the presence or absence of genetically modified (GM) material in a product or to quantify the amount of GM material present in a product. This article describes the fundamental elements of GM analysis using immunoassays and especially its application to the testing of grains. The 2 most commonly used formats are lateral flow devices (LFD) and plate-based enzyme-linked immunosorbent assays (ELISA). The main applications of both formats are discussed in general, and the benefits and drawbacks are discussed in detail. The document highlights the many areas to which attention must be paid in order to produce reliable test results. These include sample preparation, method validation, choice of appropriate reference materials, and biological and instrumental sources of error. The article also discusses issues related to the analysis of different matrixes and the effects they may have on the accuracy of the immunoassays.

Surface plasmon resonance biosensors for detection of pathogenic microorganisms: strategies to secure food and environmental safety.

Abstract: This review describes the exploitation of exclusively optical surface plasmon resonance (SPR) biosensors for the direct and indirect detection of pathogenic microorganisms in food chains and the environment. Direct detection is, in most cases, facilitated by the use of defined monoclonal or polyclonal antibodies raised against (a part of) the target pathogenic microorganisms. The antibodies were immobilized to a solid phase of the sensor to capture the microbe from the sample. Alternatively, antibodies were used in an inhibition-like assay involving incubation with the target organism prior to analysis of nonbound antibodies. The free immunoglobins were screened on a sensor surface coated with either purified antigens or with Fc or Fab binding antibodies. Discussed examples of these approaches are the determination of Escherichia coli O1 57:H7, Salmonella spp., and Listeria monocytogenes. Another direct detection strategy involved SPR analysis of polymerase chain reaction products of Shiga toxin-2 genes reporting the presence of E. coli O157:H7 in human stool. Metabolic products have been exploited as biomarkers for the presence of a microbial agent, such as enterotoxin B and a virulence factor for the occurrence of Staphylococcus aureus and Streptococcus suis, respectively. Indirect detection, on the other hand, is performed by analysis of a humoral immune response of the infected animal or human. By immobilization of specific antigenic structures, infections with Herpes simplex and human immunodeficiency viruses, Salmonella and Treponema pallidum bacteria, and Schistosoma spp. parasites were revealed using human, avian, and porcine sera and avian eggs. Bound antibodies were easily isotyped using an SPR biosensor to reveal the infection history of the individual. Discussed studies show the recent recognition of the suitability of this type of instrument for (rapid) detection of health-threatening microbes to food and environmental microbial safety.

Determination of aflatoxins and ochratoxin A in ginseng and other botanical roots by immunoaffinity column cleanup and liquid chromatography with fluorescence detection.

Abstract: Mycotoxins are toxic secondary metabolites produced by certain molds and are common contaminants of many important food crops, such as grains, nuts, and spices. Some mycotoxins are found in fruits, vegetables, and botanical roots. These contaminants have a broad range of toxic effects, including carcinogenicity, immunotoxicity, neurotoxicity, and reproductive and developmental toxicity. The public health concerns related to both acute and chronic effects of mycotoxins in animals have prompted more than 100 countries to establish regulatory limits for some of the well-known mycotoxins, such as the aflatoxins (AFL). Our research focused on method development for 2 of these toxins, AFL and ochratoxin A (OTA), in ginseng and other selected botanical roots. Methods using an immunoaffinity column (IAC) cleanup, liquid chromatographic separation, and fluorescence detection were modified and evaluated. Two types of IAC cleanup were evaluated: IAC for AFL, and IAC for both AFL and OTA. Three derivatization techniques to enhance the fluorescence of the AFL were compared: precolumn trifluoroacetic acid, postcolumn bromination, and postcolumn ultraviolet irradiation. No derivatization was needed for OTA. Results for AFL using the single analyte IAC cleanup and the 3 derivatization techniques were all comparable for ginseng and for other roots such as ginger, licorice, and kava-kava. Recoveries of added AFL for ginseng at levels from 2 to 16 ng/g were about 80%. Using IAC cleanup for both AFL and OTA recoveries of added AFL for ginseng at 4-16 ng/g were about 70%, and for ginger, licorice, and kava-kava were about 60%. Recoveries of added OTA for ginseng, ginger, and echinacea at 4 ng/g were about 55%.

Assessing exposure to lignans and their metabolites in humans.

Abstract: Phytoestrogens occur naturally in plants and are structurally similar to mammalian estrogens The lignans are a class of phytoestrogen and can be metabolized to the biologically active enterolignans, enterodiol, and enterolactone by a consortium of intestinal bacteria Secoisolariciresinol diglucoside (SDG), a plant lignan, is metabolized to enterodiol and, subsequently, enterolactone Matairesinol, another plant lignan, is metabolized to enterolactone Other dietary enterolignan precursors include lariciresinol, pinoresinol, syringaresinol, arctigenin, and sesamin Enterolignan exposure is determined in part by intake of these precursors, gut bacterial activity, and host conjugating enzyme activity A single SDG dose results in enterolignan appearance in plasma 8-10 h later--a timeframe associated with colonic bacterial metabolism and absorption Conjugation of enterolignans with sulfate and glucuronic acid occurs in the intestinal wall and liver, with the predominant conjugates being glucuronides Controlled feeding studies have demonstrated dose-dependent urinary lignan excretion in response to flaxseed consumption (a source of SDG); however, even in the context of controlled studies, there is substantial interindividual variation in plasma concentrations and urinary excretion of enterolignans The complex interaction between colonic environment and external and internal factors that modulate it likely contribute to this variation Knowledge of this field, to date, indicates that understanding the sources of variation and measuring the relevant panel of compounds are important in order to use these measures effectively in evaluating the impact of lignans on human health

Determination of residues of 446 pesticides in fruits and vegetables by three-cartridge solid-phase extraction-gas chromatography-mass spectrometry and liquid chromatography-tandem mass spectrometry.

Abstract: A method was developed for determination of residues of 446 pesticides in fruits and vegetables through the use of cleanup by a 3-cartridge solid-phase extraction-gas chromatography/ mass spectrometry (GC/MS) and liquid chromatography/tandem mass spectrometry (LC/MS/MS). Fruit and vegetable samples (20 g) were extracted with 40 mL acetonitrile, salted out, and centrifuged. Half of the supernatant was passed into an Envi-18 cartridge, eluted with acetonitrile, and cleaned up with Envi-Carb and aminopropyl Sep-Pak cartridges in series after concentration of the eluates. Pesticides were eluted with acetonitrile-toluene (3 + 1, v/v), and eluates were concentrated to 0.5 mL and then added into internal standards after solvent exchange with 2 mL hexane and used for determination of 383 pesticides by GC/MS. The other half of the supernatant was concentrated to 1 mL and cleaned up with Envi-Carb and aminopropyl Sep-Pak cartridges in series. Pesticides were eluted with acetonitrile-toluene (3 + 1, v/v), and the eluates were concentrated to 0.5 mL, dried with nitrogen gas, diluted to 1.0 mL with acetonitrile-water (3 + 2, v/v), and used for determination of 63 pesticides by LC/MS/MS. The limit of detection for the method was 0.2-600 ng/g depending on the individual pesticide. In the method, fortification recovery tests at high, medium, and low levels were conducted on 6 varieties of fruits and vegetables, i.e., apples, oranges, grapes, cabbage, tomatoes, and celery, with average recoveries falling within the range of 55.0-133.8% for 446 pesticides, among which average recoveries between 60.0-120.0% accounted for 99% of the results. The relative standard deviation was between 2.1-39.1%, of which a relative standard deviation of 2.1-25.0% made up 96% of the results. Experiments proved that the method was applicable for determination of residues of 446 pesticides in fruit and vegetables.

Analysis of isoflavones in foods and dietary supplements.

Abstract: 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. Daidzein, genistein, and glycitein are present as free forms or derivatives in foods containing soy or soy protein extracts. The analysis of isoflavones has become more complex, because preparations contain isoflavones from multiple sources (e.g., red clover, kudzu). Red clover contains primarily formononetin and biochanin A, while kudzu extracts, which are becoming increasingly common in dietary supplements, contain puerarin and daidzein, among other components. Isoflavones are present in foods and dietary supplements as free compounds, glucoside derivatives, 6"-O-malonyl-7-O-beta-D-glucoside derivatives, and 6"-O-acetyl-7-O-beta-D-glucoside derivatives. High-performance liquid chromatography (HPLC)/tandem mass spectrometry has been applied to the identification of isoflavone derivatives based on the fragmentation pattern of the parent ion, providing high selectivity and sensitivity in the quantitation of isoflavones in complex mixtures. HPLC with ultraviolet detection is often chosen for routine analysis, but a preliminary acid or basic hydrolysis of isoflavone derivatives is often required for the investigation of samples containing extracts from multiple sources. Several internal standards have been used in the analysis of isoflavones from a single botanical source (e.g., soy, red clover), but the identification of a general internal standard remains a challenging process.

Immunobiosensor detection of domoic acid as a screening test in bivalve molluscs: Comparison with liquid chromatography-based analysis

Abstract: A rapid and sensitive immuno-based screening method was developed to detect domoic acid (DA) present in extracts of shellfish species using a surface plasmon resonance-based optical biosensor. A rabbit polyclonal antibody raised against DA was mixed with standard or sample extracts and allowed to interact with DA immobilized onto a sensor chip surface. The characterization of the antibody strongly suggested high cross-reactivity with DA and important isomers of the toxin. The binding of this antibody to the sensor chip surface was inhibited in the presence of DA in either standard solutions or sample extracts. The DA chip surface proved to be highly stable, achieving approximately 800 analyses per chip without any loss of surface activity. A single analytical cycle (sample injection, chip regeneration, and system wash) took 10 min to complete. Sample analysis (scallops, mussels, cockles, oysters) was achieved by simple extraction with methanol. These extracts were then filtered and diluted before analysis. Detection limits in the ng/g range were achieved by the assay; however, the assay parameters chosen allowed the test to be performed most accurately at the European Union's official action limit for DA of 20 microg/g. At this concentration, intra- and interassay variations were measured for a range of shellfish species and ranged from 4.5 to 7.4% and 2.3 to 9.7%, respectively.

Food allergen detection with biosensor immunoassays.

Abstract: An optical biosensor was used to develop both direct and sandwich immunoassays for the detection of proteins from milk, egg, hazelnut, peanut, shellfish, and sesame in food samples Affinity-purified polyclonal antibodies raised against the proteins were immobilized on the biosensor chip Food samples were injected and the proteins that bound to the antibodies on the surface were detected by a shift in the resonance angle By adding a second antibody in a sandwich assay, matrix effects could be overcome and the sensitivity and selectivity enhanced Detection of allergen levels down to 1-125 microg/g in food samples was demonstrated for the various assays Good agreement of results was also obtained from parallel analysis with alternative immunoassays, including rocket immunoelectrophoresis, enzyme immunoassay, and immunoblotting The present study demonstrates that the sensitivity of the described biosensor technique is comparable to the most sensitive enzymed-linked immunosorbent assays

Application of a fast high-performance liquid chromatography method for simultaneous determination of furanic compounds and glucosylisomaltol in breakfast cereals.

Abstract: The high-performance liquid chormatography method reported in the present paper provides a fast, low-cost, precise, and simple technique to analyze simultaneously 3 different indicators of nonenzymatic browning, i.e., hydroxymethylfurfural (HMF), furfural, and glucosylisomaltol (GIM), in breakfast cereals. These compounds were extracted in aqueous solution and separated on a reversed-phase C18 column with water-acetonitrile (95 + 5, v/v) mobile phase under isocratic conditions. Average recovery rates for HMF, furfural, and GIM were 99.1, 98.4, and 99.4%, respectively. The coefficients of variation were 3.67, 2.42, and 1.59% for HMF, furfural, and GIM, respectively. The detection limit was 0.01 mg/kg, and the quantitation limit was 0.05 mg/kg for the 3 studied compounds.

Cutting boards in Salmonella cross-contamination.

Abstract: Cutting boards are commonly perceived as important fomites in cross-contamination of foods with agents such as Salmonella spp., despite the lack of supporting epidemiological data. A variety of woods and plastics have been used to make work surfaces for cutting. In general, wood is said to dull knives less than plastic, and plastic is seen as less porous than wood. Research to model the hypothetical cross-contamination has been done in a variety of ways and has yielded a variety of results. At least some of the work with knife-scarred plastic indicates that the surface is very difficult to clean and disinfect, although this may vary among the polymers used. High-density polyethylene, which is most used in commercial applications, has been shown to delaminate in response to knife scarring. Wood is intrinsically porous, which allows food juices and bacteria to enter the body of the wood unless a highly hydrophobic residue covers the surface. The moisture is drawn in by capillary action until there is no more free fluid on the surface, at which point immigration ceases. Bacteria in the wood pores are not killed instantly, but neither do they return to the surface. Destructive sampling reveals infectious bacteria for hours, but resurrection of these bacteria via knife edges has not been demonstrated. Small plastic cutting boards can be cleaned in a dishwasher (as can some specially treated wooden boards), but the dishwasher may distribute the bacteria onto other food-contact surfaces. Most small wooden boards (i.e., those with no metal joiners in them) can be sterilized in a microwave oven, but this should be unnecessary if accumulation of food residues is prevented. However, 2 epidemiological studies seem to show that cutting board cleaning habits have little influence on the incidence of sporadic salmonellosis. Further, one of these studies indicated that use of plastic cutting boards in home kitchens is hazardous, whereas use of wooden cutting boards is not.

Flax lignans--analytical methods and how they influence our lunderstanding of biological activity.

Abstract: Flaxseed (Linum usitatissimum L.) is a major source of dietary intake of lignans by virtue of the high concentrations (0.7-1.5%) that are present in the seed. The principal lignan present in flaxseed is secoisolariciresinol diglucoside (SDG), which occurs as a component of a linear ester-linked complex in which the C6-OH of the glucose of SDG is esterified to the carboxylic acid of hydroxymethylglutaric acid. Also present in flaxseed and in resulting lignan extracts are significant quantities of 2 cinnamic acid glycosides. Our emerging understanding of the biological activity of flax lignans is based on studies using a variety of materials ranging from whole ground seed to pure SDG. The underlying assumption of most of these studies is that the biological activity of flax lignans results from their conversion to the mammalian lignans enterolactone (EL) and enterodiol (ED). There are, however, several intermediate compounds generated during the digestion and metabolism of flax lignans, including SDG and its aglycones and secoisolariciresinol (Seco), that are good candidates to be the principal bioactive molecule. This review will document the history of the development of lignan analytical methods and illustrate how analytical methods have influenced the interpretation of animal and human trials and our understanding of the biological activity of flax lignans.

Method development for the analysis of N-nitrosodimethylamine and other N-nitrosamines in drinking water at low nanogram/liter concentrations using solid-phase extraction and gas chromatography with chemical ionization tandem mass spectrometry.

Abstract: N-nitrosodimethylamine (NDMA) is a probable human carcinogen of concern that has been identified as a drinking water contaminant. U.S. Environmental Protection Agency Method 521 has been developed for the analysis of NDMA and 6 additional N-nitrosamines in drinking water at low ng/L concentrations. The method uses solid-phase extraction with coconut charcoal as the sorbent and dichloromethane as the eluent to concentrate 0.50 L water samples to 1 mL. The extracts are analyzed by gas chromatography-chemical ionization tandem mass spectrometry using large-volume injection. Method performance was evaluated in 2 laboratories. Typical analyte recoveries of 87-104% were demonstrated for fortified reagent water samples, and recoveries of 77-106% were demonstrated for fortified drinking water samples. All relative standard deviations on replicate analyses were < 11%.

Interlaboratory evaluation of two enzyme-linked immunosorbent assay kits for the detection of egg, milk, wheat, buckwheat, and peanut in foods.

Abstract: The labeling of 5 major allergenic ingredients (egg, milk, wheat, buckwheat, and peanut) is mandatory in Japan, and 2 series of enzyme-linked immunosorbent assay (ELISA) kits have been established as official screening methods. However, these official methods have not provided the necessary sensitivity, due in part to poor extraction efficiency. To address this need, 2 novel ELISA kits have been developed: the FASTKIT ELISA Ver. II Series and the FASPEK Allergenic Substances Detection Kit. The new kit systems use an improved extraction buffer that can extract insoluble proteins produced by processing and feature new antibodies that bind to the denatured proteins extracted with the new extraction buffer. The analytical performances of the 2 new ELISA kit series were evaluated in an interlaboratory study. Ten laboratories participated in the study and determined the major allergenic ingredients contained in 5 types of model processed food. The 2 ELISAs displayed fairly good reproducibility and sufficient recovery.

Factors affecting the bioavailability of soy isoflavones in humans.

Abstract: To fully evaluate the potential risks and benefits of isoflavones to human health, an understanding of the physiological behavior of these compounds following ingestion is required. Numerous researchers have investigated the kinetics and extent of polyphenol absorption by measuring plasma concentrations and/or urinary excretion among adults after the ingestion of a single dose of polyphenol, provided as either a pure compound, plant extract, or whole food/beverage. Available data suggest isoflavones are more bioavailable than other flavonoid subclasses. This review will focus on our current understanding of factors affecting isoflavone absorption and metabolism in humans.

High-performance thin-layer chromatography densitometric method for simultaneous quantitation of phyllanthin, hypophyllanthin, gallic acid, and ellagic acid in Phyllanthus amarus.

Abstract: Whole plant of Phyllanthus amarus Linn. is a reputed drug of the Indian systems of medicine that is used as hepatoprotective agent. A simple high-performance thin-layer chromatography (HPTLC) densitometric method has been developed for the simultaneous quantitation of phyllanthin, hypophyllanthin, gallic acid, and ellagic acid in the whole plant of P. amarus. They were found at levels of 0.37, 1.16, 0.36, and 0.17% (w/w), respectively. The method was validated for precision, repeatability, and accuracy. Instrumental precision was found to be 0.54, 0.93, 0.08, and 0.78% (coefficient of variation, CV); repeatability of the method was 1.01, 0.79, 0.98, and 1.06% (CV) for phyllanthin, hypophyllanthin, gallic acid, and ellagic acid, respectively. Accuracy of the method was determined by a recovery study conducted at 3 different levels, and the average recovery was found to be 99.09% for phyllanthin, 99.27% for hypophyllanthin, 98.69% for gallic acid, and 100.49% for ellagic acid. The proposed HPTLC method was found to be simple, precise, specific, sensitive, and accurate and can be used for routine quality control of raw material of P. amarus and formulations containing P. amarus. It also has the applicability in quantitating any of these marker compounds in other drugs.

Liquid chromatographic analysis of aflatoxin using post-column photochemical derivatization: collaborative study.

Abstract: Aflatoxin analysis, with post-column derivatization using a photochemical reactor for enhanced detection (PHRED) system for derivatization, has been compared to the officially recognized iodine and Kobra cell derivatization systems. This photochemical system has been extensively used for screening peanuts by some U.S. Department of Agriculture laboratories for many years. From their periodic method checks, using standard spiked samples, an 80 sample series with each of the 3 derivatization methods was statistically analyzed. Paired comparisons, using the same sample extract, were also made between the PHRED and one of the other 2 methods, among laboratories in 4 different countries, on a variety of naturally contaminated commodity products. The differences between the techniques were not significant for peanuts, but for corn the photochemical system consistently gave slightly higher values for aflatoxins B1 and B2 than the Kobra cell method. However, a comparison of all sample results showed no significant differences between methods. The Pearson correlation coefficients for aflatoxin B1 in 102 test samples and aflatoxin B2 in 94 test samples were 0.9994 and 0.9874, respectively. The probability factor was P < 0.0001, and the t-tests were not significantly different except for the corn. These indicated that the PHRED system is equivalent to the iodine and Kobra cell methods for peanuts relative to the current official procedures, but the PHRED system has a slightly high bias for corn compared to the iodine and Kobra cell systems.

Synchronous analysis method for detection of citrinin and the lactone and acid forms of monacolin K in red mold rice.

Abstract: The Monascus product known as red mold rice (RMR) has been found to contain the cholesterol-lowering agent monacolin K (MK), including the lactone form (MKL) and the acid form (MKA) and mycotoxin citrinin (CT). In current studies, CT and MK are usually detected by different analysis methods, which have a high level of error, and are inconvenient, expensive, and time-consuming. The goal of this study is to establish a rapid synchronous analysis method for the detection of CT, MKL, and MKA levels in RMR. In this study, CT, MKL, and MKA are extracted by the same extraction method and are then separated in a reversed-phase high-performance liquid chromatography (HPLC) C18 column. The elution from the C18 column is then passed through an ultraviolet detector and introduced directly into the fluorescence detector. The results show that higher recovery rates of CT, MKL, and MLK are yielded from RMR powder by extracting with 95% ethanol (10 mL) at 60 degrees C for 30 min. Regarding the optimal conditions of HPLC, the peaks of CT, MKL, and MKA can be clearly separated from any noise peaks by isocratic elution with optimum mobile phase, acetonitrile-water-trifluoroacetate (55 + 45 + 0.05, v/v).

Determination of the appetite suppressant P57 in Hoodia gordonii plant extracts and dietary supplements by liquid chromatography/electrospray ionization mass spectrometry (LC-MSD-TOF) and LC-UV methods.

Abstract: Hoodia gordonii is traditionally used in South Africa for its appetite suppressant properties. P57AS3 (P57), an oxypregnane steroidal glycoside, is the only reported active constituent from this plant as an appetite suppressant. Effective quality control of these extracts or products requires rapid methods to determine P57 content. New methods of liquid chromatography/mass spectrometry (LC/MS) and LC-UV for analysis of P57 from H. gordonii have been developed. The quantitative determination of P57 was achieved with a Phenomenex Gemini (Torrance, CA) reversed-phase column using gradient mobile phase of water and acetonitrile, both containing 0.1% acetic acid. The method was validated for linearity, repeatability, and limits of detection and quantification. Good results were obtained in terms of repeatability (relative standard deviation <5.0%) and recovery (98.5-103.5%). The developed methods were applied to the determination of P57 for H. gordonii plant samples, one related genus (Opuntia ficus-indica), and dietary supplements that claim to contain H. gordonii.

Determination of aflatoxin B1 in medical herbs: interlaboratory study.

Abstract: A method was developed for the determination of aflatoxin B 1 in medical herbs (senna pods, botanical name Cassia angustifolia; devil's claw, botanical name Harpagophytum procumbens; and ginger roots, botanical name Zingiber officinale) The method, which was tested in a minicollaborative study by 4 laboratories, is based on an immunoaffinity cleanup followed by reversed-phase high-performance liquid chromatography separation and fluorescence detection after post-column derivatization It allows the quantitation of aflatoxin B 1 at levels lower than 2 ng/g A second extractant (acetone-water) was tested and compared to the proposed methanol-water extractant Several post-column derivatization options (electrochemically generated bromine, photochemical reaction, and chemical bromination) as well as different integration modes (height versus area) were also investigated No differences were found depending on the choice of derivatization system or the signal integration mode used The method was tested for 3 different matrixes: senna pods, ginger root, and devil's claw Performance characteristics were established from the results of the study and resulted in HorRat values ranging from 012 to 075 with mean recoveries from 78 to 91% for the extraction with methanol-water and HorRat values ranging from 010-103 with mean recoveries from 98 to 103% for the extraction with acetone-water As a result, the method, with all tested variations, was found to be fit-for-purpose for the determination of aflatoxin B 1 in medical herbs at levels of 1 μg/kg and above

Quantification of eugenol, luteolin, ursolic acid, and oleanolic acid in black (Krishna Tulasi) and green (Sri Tulasi) varieties of Ocimum sanctum Linn. using high-performance thin-layer chromatography.

Abstract: Ocimum sanctum (family Lamiaceae) is a reputed drug of Ayurveda, commonly known as Tulasi. In the present work, we quantified 4 marker compounds, viz., eugenol, luteolin, ursolic acid, and oleanolic acid, from the leaf of green and black varieties of O. sanctum using high-performance thin-layer chromatography (HPTLC) with densitometry. The methods were found to be precise, with relative standard deviation (RSD) values for intraday analyses in the range of 0.52 to 0.91%, 0.77 to 1.29%, 0.11 to 0.16%, and 0.34 to 0.42% and for interday analyses in the range of 0.73 to 0.96%, 1.02 to 2.08%, 0.11 to 0.12%, and 0.39 to 0.64% for different concentrations of eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Instrumental RSD values were 0.24, 0.39, 0.21, and 0.18% for eugenol, luteolin, ursolic acid, and oleanolic acid, respectively. Accuracy of the methods was checked by conducting a recovery study at 3 different levels for the 4 compounds, and the average recoveries were found to be 99.73, 99.3, 100.58, and 100.57%, respectively. Eugenol content ranged from 0.175 to 0.362% (w/w) and luteolin from 0.019 to 0.046% (w/w) in the samples analyzed. Green variety was found to contain higher amounts of ursolic acid [0.478 and 0.348% (w/w), from Sources 1 and 2, respectively] than the black variety [0.252 and 0.264% (w/w) from Sources 1 and 2, respectively]. Black variety had 0.174 and 0.218% (w/w) of oleanolic acid from Sources 1 and 2, respectively, while it was not detected in the green variety. Ursolic acid and oleanolic acid ran at the same Rf value and could not be resolved in several solvent systems tried. However, we observed that only ursolic acid gave yellow fluorescence under 366 nm ultraviolet light after derivatization with anisaldehyde-sulfuric acid reagent. The HPTLC-densitometry methods for the quantification of the 4 markers in O. sanctum leaf will have the applicability in quality control.

Chemical fingerprinting of valeriana species: simultaneous determination of valerenic acids, flavonoids, and phenylpropanoids using liquid chromatography with ultraviolet detection.

Abstract: The roots and rhizomes of various valeriana species are currently used as a sleeping aid or mild sedative. A liquid chromatography method has been developed that permits the analysis of chlorogenic acid, lignans, flavonoids, valerenic acids, and valpotrates in various valerian samples. The best results were obtained with a Phenomenex Luna C18(2) column using gradient elution with a mobile phase consisting of water and 0.05% phosphoric acid and 2-100% acetonitrile-methanol (1 + 1) with 0.05% phosphoric acid. The flow rate was 0.8 mL/min and ultraviolet detection was at 207, 225, 254, 280, and 325 nm. Different valerian species and commercial products showed remarkable quantitative variations. Chlorogenic acid (0.2-1.2%), 3 lignans, linarin (0.002-0.24%), and valepotriates were detected in all the valeriana species analyzed. Highest amounts of valerenic acids were detected in V. officinalis L., trace amounts in V. sitchensis, and none in the other species analyzed.

Spectrophotometric analysis of selective serotonin reuptake inhibitors based on formation of charge-transfer complexes with tetracyanoquinodimethane and chloranilic acid.

Abstract: A simple, accurate, and sensitive spectrophotometric method for analysis of selective serotonin reuptake inhibitors (SSRIs) has been developed and validated. The analysis was based on the formation of colored charge-transfer complexes between the intact molecule of SSRI drug as an n-electron donor and each of tetracyanoquinodimethane (TCNQ) or p-chloranilic acid (pCA) as electron acceptors. The formed complexes were measured spectrophotometrically at 842 and 520 nm for TCNQ and pCA, respectively. Different variables and parameters affecting the reactions were studied and optimized. Under the optimum reaction conditions, linear relationships with good correlation coefficients (0.9975-0.9996) were found between the absorbances and the concentrations of the investigated drugs in the concentration ranges of 4-50 and 20-400 microg/mL with TCNQ and pCA, respectively. With all the investigated drugs, TCNQ gave more sensitive assays than pCA; the limits of assay detection were 2.5-4.8 and 20-40 microg/mL with TCNQ and pCA, respectively. The intra- and interassay precisions were satisfactory; the relative standard deviations did not exceed 2%. The proposed procedures were successfully applied to the analysis of the studied drugs in pure form and pharmaceutical formulations with good accuracy; the recovery values were 98.4-102.8 +/- 1.24-1.81%. The results obtained from the proposed method were statistically comparable with those obtained from the previously reported methods.

A review of the animal models used to investigate the health benefits of soy isoflavones.

Abstract: This review considers the recent literature in which animal models were used to investigate the purported health benefits of soy isoflavones. The main conclusions are that our animal models demonstrate minimal effects in breast, prostate, and colon cancer prevention, and that, while some cancers may respond to isoflavones, it would appear that isoflavones do not prevent further development once cancer has become established. Regarding cardiovascular health, the lipid-lowering effects of isoflavones have been established, but their efficacy may be less than original research purported. However, it may be considered a bonus of habitual soy consumption that blood cholesterol levels would be reduced somewhat. With respect to osteoporosis and menopausal symptoms, animal models do not show any consistent benefit of isoflavones in preventing osteoporosis, and calcium fortification or the use of prescribed medications are likely much better approaches to combat bone loss. However, our animal models of osteoporosis and menopausal symptoms may not be entirely representative of the human situation. Perhaps the benefit of isoflavones in cognitive skills and in delaying Alzheimer's disease is an area where they can be of some advantage. However, this field is very recent and requires much more research in both humans and animal models before any definitive benefit can be propounded. On the other hand, isoflavones in moderation are probably not dangerous, as few studies have indicated adverse effects. However, large doses have been shown to increase apoptosis and cell degeneration, and in some cancer regimes, once the cancer has progressed beyond the hormone-dependent stage, high doses of isoflavones may be contraindicated. The prospect of mega-dosing from isoflavone supplements opens a new chapter in the risk assessment of isoflavone consumption.

Determination of patulin in apple juice by liquid chromatography.

Abstract: A method was developed and validated in-house for the determination of patulin (PAT), a toxic mold metabolite, in apple juice. The sample was extracted with ethyl acetate–hexane and analyzed by liquid chromatography equipped with a C18 column and diode array detector. The mobile phase used for the quantification was water–ethanol, at a flow rate of 0.5 mL/min. The method showed a mean recovery of 84.8%, the relative standard deviation obtained in the precision study was <7.7%, the quantification and detection limits were 7a nd 3g/L, respectively, and the linear range for PAT in apple juice was 2.6–650 g/L. The ruggedness was evaluated by an intralaboratory experiment, in which 5 factors were studied, and only one was found to influence the observed results. The developed method is fast, practical, and simple; the solvents (except hexane) and reagents used were nontoxic. The results of the validation confirmed the efficiency of the method, which is sensitive enough to be used in studies required to quantify PAT in apple juice.

Determination of abamectin, doramectin, emamectin, eprinomectin, ivermectin, and moxidectin in milk by liquid chromatography electrospray tandem mass specrometry.

Abstract: The avermectin and milbemycin families of compounds are derived from naturally occurring yeasts. They have proven to be potent preventatives against a variety of pests such as insects and parasites. Only eprinomectin and moxidectin are currently approved for use on lactating cattle with tolerances in milk of 12 microg/kg for eprinomectin and 40 microg/kg for moxidectin. Detection of misuse or inadvertent contamination in milk requires a sensitive and definitive analytical method. A method has been developed for the determination of 5 avermectins and 1 milbemycin in milk using a simple liquid-liquid extraction and liquid chromatography/tandem mass spectrometry (LC/MS/MS) analysis. Ivermectin (IVR), doramectin (DOR), abamectin (ABA), eprinomectin (EPR), emamectin (EMA), and moxidectin (MOX) were extracted from whole milk by partitioning into acetonitrile with a subsequent solvent exchange into methanol-water. Simultaneous confirmation and quantification were achieved with LC separation, positive electrospray ionization (ESI+), and MS/MS. The limits of detection ranged from 16 pg/g (ppt) for EMA to 1.7 microg/g (ppb) for MOX.

Biosensor screening for veterinary drug residues in foodstuffs.

Abstract: The advent of the surface plasmon resonance (SPR) biosensor has led to many applications in diverse fields from the pharmaceutical industry to the life sciences and other areas within biotechnology. One area that has seen a significant increase in applications is the testing for veterinary drug residues in foodstuffs. These include tests for antibiotics, beta-agonists, and antiparasitic drugs. The introduction of the Biacore Q in the late 1990s, an SPR biosensor dedicated to the food industry, and the complementary development of kits to test for these residues mean that end users have a viable alternative screening test to the established enzyme-linked immunosorbent assay (ELISA) techniques. This paper reviews many SPR biosensor veterinary drug tests that have been developed, with particular emphasis placed on kit-based assays.

Biosensor Analysis of beta-Lactams in Milk Using the Carboxypeptidase Activity of a Bacterial Penicillin Binding Protein

Abstract: The applicability of a beta-lactam receptor protein for detection of beta-lactam antibiotics in milk using surface plasmon resonance (SPR) biosensor technology was investigated. The advantage of using a receptor protein instead of antibodies for detection of beta-lactams is that a generic assay, specific for the active form of the beta-lactam structure, is obtained. Two assays based on the enzymatic activity of the DD-carboxypeptidase from Actinomadura R39 were developed, using a Biacore SPR biosensor. The carboxypeptidase converts a tri-peptide into a di-peptide, a reaction which is inhibited in the presence of beta-lactams. Polyclonal antibodies against the 2 peptides were developed and used to measure the amount of enzymatic product formed (di-peptide assay) or the amount of remaining enzymatic substrate (tri-peptide assay), respectively. The 2 assays showed similar performances with respect to detection limits (1.2 and 1.5 microg/kg, respectively) and precision (coefficient of variation <5%) for penicillin G in milk. Several other beta-lactams were detected at or near their respective maximum residue limit. Furthermore, the 2 peptide assays were evaluated against 5 commercial kit tests in the screening of 195 producer milk samples. The biosensor assays showed 0% false-negative and 27% false-positive results, whereas the figures were 0% false-negative and 27-53% false-positive results for other screening tests investigated.