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Indonesia has a lot of germplasm diversity, one of which is the banana plant. Tongka Langit banana plant (Musa troglodytarum L.) is a type of banana endemic to eastern Indonesia, namely Maluku and Papua, which is quite unique. Morphological characterization is an approach used to identify a plant. In banana plants, characterization was carried out in the vegetative and generative phases which included the characters of leaves, stems, flowers and fruit. The method used in this research is a survey and direct observation at the research site. Based on the results of observations, it can be concluded that there is variation in the leaf character, while the flower and fruit characters for the two research locations show no variation

Currently, biotherapeutic medicines are the most effective options for the treatment of many severe and chronic diseases. For faster market entry of biotherapeutic products and their cost reduction, the principles of "biosimilarity" have been developed. Development and licensing of biosimilars is allowed only after the end of patent exclusivity of the original preparation period.Characteristics of the main safety parameters of biosimilar hormone preparations licensed by EMA.This paper analyzes the results demonstrating the similarities and differences between biosimilar and reference hormone products indicated in the EPAR (public assessment report) for the examination of materials presented for the licensing of biosimilar products.During the development of biosimilar hormone medicines, differences in the glycosylation profile between biosimilar and reference preparations are revealed. As biotherapeutical preparations are produced by cells, the differences in glycosylation profile between biosimilar and referent preparation are predictable. While carrying out clinical studies, a high similarity of biosimilar and reference product effectiveness is shown, but some differences between them in the safety profile are revealed.The study of biosimilar product safety has shown the necessity of further improvement in safety and standard approaches for the assessment of the immunogenicity of biosimilar products.

This study aimed to determine the effect of different biostimulant applications (solid and liquid) on the growth and yield of mustard plants. This study used a single factor Randomized Block Design (RBD), consisting of solid or liquid biostimulant treatment, each has twelve levels, i.e. K0 = without biostimulant, K1 = NPK 1 g per plant, solid and liquid biostimulant each consisting of 10 treatments = B1, B2, B3, B4, B5, B6, B7, B8, B9, B10. Solid biostimulant was given at 2.5 g per plant and liquid biostimulant at 10 ml per plant. The results showed that liquid biostimulant gave a significant effect on plant height, crop fresh weight, and crop dry weight; whereas solid biostimulant gave a very significant effect on entire weight, i.e. fresh weight of mustard plant, fresh root weight, and crop dry weight. The best treatment for liquid biostimulant was B1 treatment; whereas for the solid biostimulant was B7 treatment. Keywords: biostimulant, mustard, growth, yield ABSTRAK Penelitian ini bertujuan untuk mengetahui pengaruh aplikasi biostimulan yang berbeda (biostimulan padat dan cair) terhadap pertumbuhan dan produksi tanaman sawi. Penelitian ini menggunakan rancangan acak kelompok faktor tunggal yang terdiri dari perlakuan Biostimulan (padat dan cair) yang masing-masing perlakuan terdapat dua belas taraf (P): K0 = tanpa biostimulan, K1 = NPK 1 g/tan, Biostimulan padat dan cair masing-masing terdiri dari 10 perlakuan = B1, B2, B3, B4, B5, B6, B7, B8, B9, B10. Biostimulan padat 2.5 g/tan dan biostimulan cair 10 ml/tan. Hasil penelitian menunjukkan bahwa biostimulan cair memberikan pengaruh yang nyata terhadap tinggi tanaman sawi, berat segar tanaman, dan berat kering tanaman, sedangkan biostimulan padat memberikan pengaruh yang sangat nyata terhadap semua bobot berat tanaman sawi yaitu berat segar tanaman, berat segar akar, dan berat kering tanaman. Perlakuan terbaik untuk biostimulan cair adalah perlakuan B1, sedangkan untuk biostimulan padat perlakuan yang terbaik adalah perlakuan B7. Kata kunci: biostimulan, sawi, pertumbuhan, produksi

Inceptisol is a young and newly developed soil so it needs to be optimized for crop cultivation. The aim of this research is to know the availability of soil P, P uptake and corn yield after application of granular composting of sago waste (KGES) together with phosphate fertilizer to Inceptisols soil. The experimental treatment was KGES which consisted of three levels: without KGES, 40 tons KGES ha-1, and 80 tons KGES ha-1, and the use of SP-36 (P) fertilizer consisting of three levels: without P fertilizer, 120 kg P ha-1, and 240 kg P ha-1. The experiment used a complete randomized design with 3 replications. The result of the experiment was analyzed variance and relationship test using interlaced analysis. The results showed that KGES together with phosphate fertilizers could increase soil pH causing phosphate to be available and plant roots could absorb nutrient phosphate well, so that the yield of corn kiln dry weight also increased. The highest yield of dry weight of grains (7.85 tons per ha) was obtained from a combination of 80 tons of KGES ha-1 and 240 kg P ha-1.Keyword: Keywords: Ella Sago, Inseptisols, maize, phosphate

Serum and erythrocyte (RBC) total folate are indicators of folate status. No nationally representative population data exist for folate forms. We measured the serum folate forms (5-methyltetrahydrofolate (5-methylTHF), unmetabolised folic acid (UMFA), non-methyl folate (sum of tetrahydrofolate (THF), 5-formyltetrahydrofolate (5-formylTHF), 5,10-methenyltetrahydrofolate (5,10-methenylTHF)) and MeFox (5-methylTHF oxidation product)) by HPLC–MS/MS and RBC total folate by microbiologic assay in US population ≥ 1 year (n approximately 7500) participating in the National Health and Nutrition Examination Survey 2011–2. Data analysis for serum total folate was conducted including and excluding MeFox. Concentrations (geometric mean; detection rate) of 5-methylTHF (37·5 nmol/l; 100 %), UMFA (1·21 nmol/l; 99·9 %), MeFox (1·53 nmol/l; 98·8 %), and THF (1·01 nmol/l; 85·2 %) were mostly detectable. 5-FormylTHF (3·6 %) and 5,10-methenylTHF (4·4 %) were rarely detected. The biggest contributor to serum total folate was 5-methylTHF (86·7 %); UMFA (4·0 %), non-methyl folate (4·7 %) and MeFox (4·5 %) contributed smaller amounts. Age was positively related to MeFox, but showed a U-shaped pattern for other folates. We generally noted sex and race/ethnic biomarker differences and weak (Spearman's rP

The performance of a novel, transmission-mode, portable, Fourier transform infrared (FTIR) analyzer was evaluated and compared to that of a benchtop attenuated total reflection (ATR)-FTIR spectrometer. The total concentration of trans fatty acids in the fat extracted from 19 representative fast foods was rapidly (

The adverse effects of dietary trans fat on biomarkers of chronic disease are well documented. Regulatory authorities in many countries have enacted legislation aimed at reducing trans fat content of their food supplies, either by requiring trans fat labeling on pre-packaged foods or by limiting the amount of trans fat in oils used for food production. Increased use by the food industry of oils with a low trans fat content necessitates reevaluation of official methods used by the food industry and regulatory agencies for the determination of total trans fat. Attenuated total reflection-Fourier-transform infrared (ATR-FTIR) spectroscopy and gas chromatography with flame ionization detection (GC-FID) are two techniques used in official methods approved by method-endorsing organizations, for example AOAC International and the American Oil Chemists' Society. Here, we review current official ATR-FTIR and GC-FID methods for determination of trans fat, with a focus on factors affecting quantification of low levels of trans fat. We include new data on method performance that have only recently become available, and provide an overview of notable recent developments in lipid analysis (e.g. IR spectroscopy procedures, ionic-liquid GC columns, and multidimensional chromatographic techniques) that have the potential to substantially improve the accuracy, sensitivity, and/or speed of trans fat determination.

Declarations of the total content of trans fatty acids (FA) and saturated FA (SFA) are mandatory on food labels in the US and Canada. Gas chromatography (GC) has been the method of choice for the determination of FA composition. However, GC is time consuming and requires conversion of fats and oils to their FA methyl esters. In the present study, a recently published Fourier transform near-infrared (FT-NIR) spectroscopic procedure was applied to the rapid (

The separation of fatty acid methyl esters (FAME) provided by a 200 m × 0.25 mm SLB-IL111 capillary column is enhanced by adding a second dimension of separation ((2)D) in a GC × GC design. Rather than employing two GC columns of different polarities or using different elution temperatures, the separation in the two-dimensional space is achieved by altering the chemical structure of selected analytes between the two dimensions of separation. A capillary tube coated with palladium is added between the first dimension of separation ((1)D) column and the cryogenic modulator, providing the reduction of unsaturated FAMEs to their fully saturated forms. The (2)D separation is achieved using a 2.5 m × 0.10 mm SLB-IL111 capillary column and separates FAMEs based solely on their carbon skeleton. The two-dimensional separation can be easily interpreted based on the principle that all the saturated FAMEs lie on a straight diagonal line bisecting the separation plane, while the FAMEs with the same carbon skeleton but differing in the number, geometric configuration or position of double bonds lie on lines parallel to the (1)D time axis. This technique allows the separation of trans fatty acids (FAs) and polyunsaturated FAs (PUFAs) in a single experiment and eliminates the overlap between PUFAs with different chain lengths. To our knowledge, this the first example of GC × GC in which a chemical change is instituted between the two dimensions to alter the relative retentions of components and identify unsaturated FAMEs.

Simultaneous separation of steviol and steviol glycosides is challenging because of differences in their polarity and chemical structure. In this study, simultaneous analysis of steviol and steviol glycosides was achieved by LC with UV detection using a mixed-mode RP weak anion exchange chromatography column. Steviol and seven steviol glycosides were analyzed on an Acclaim Mixed-Mode Wax-1 (Dionex) column with a linear gradient of deionized water adjusted to pH 3.00 with phosphoric acid and acetonitrile. The extraction was performed by sonicating dry plant material at 40°C in acetonitrile–water (30 + 70, v/v). LOQ values (mg/g dry weight of plant material) were rebaudioside B, 0.50; steviol, 0.70, dulcoside A, 1.0; steviolbioside, 1.2; stevioside and rebaudioside C, 2.0; rebaudioside D, 3.3; and rebaudioside A, 5.0. The method demonstrated suitable performance for all analytes tested with respect to accuracy (mean recoveries 95–99%), intraday and interday precision for retention times (0.070–0.28% and 0.33–1.0% RSD, respectively), and linearity. The method was used to authenticate steviol glycosides in several samples of Stevia plant material as well as to quantitate steviol glycosides in dietary supplements containing Stevia.

Current interest by the food industry in exploring reformulation options that lower the content of trans fat in edible fats and oils requires methods to accurately measure low levels of trans fat. In the present study, the quantitation of trans fat in 25 edible fat and oil samples was evaluated using two current analytical approaches, attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and gas chromatography with flame ionization detection (GC-FID) according to Official Methods of the American Oil Chemists' Society. Significant differences between the ATR-FTIR and reference GC-FID quantitations were found for samples with a trans fat content

The NHANES has monitored folate status of the U.S. population from prefortification (1988-1994) to postfortification (1999-2010) by measuring serum and RBC folate concentrations. The Bio-Rad radioassay (BR) was used from 1988 to 2006, and the microbiologic assay (MBA) was used from 2007 to 2010. The MBA produces higher concentrations than the BR and is considered to be more accurate. Thus, to bridge assay differences and to examine folate trends over time, we adjusted the BR results to be comparable to the MBA results. Postfortification, assay-adjusted serum and RBC folate concentrations were 2.5 times and 1.5 times prefortification concentrations, respectively, and showed a significant linear trend (P < 0.001) to slightly lower concentrations during 1999-2010. The postfortification prevalence of low serum (

The content of trans fat in foods is most commonly determined by summing the levels of individual trans fatty acids (FAs), analyzed as FA methyl esters (FAME) by gas chromatography. Current Official Methods of the American Oil Chemists' Society (AOCS) enable quantitation of total trans fat in foods but were not designed for the determination of transFA isomeric compositions. In the present study, the content of trans fat in 32 representative fast food samples ranged from 0.1 to 3.1 g per serving, as determined according to AOCS Official Method Ce 1j-07. Further analysis of FAME using the 200 m SLB-IL111 ionic liquid column yielded quantitative results of total, trans, saturated, and cis unsaturated fat that were comparable to those of Method Ce 1j-07 and also allowed for the complementary determination of individual trans 18:1, trans 18:2, and trans 18:3 FA isomeric compositions under conditions suitable for routine sample analysis.

1. Toxicity of pyrrolizidine alkaloids (PAs) largely depends on their metabolic activation by hepatic enzymes, including cytochrome P450s, to become chemically reactive pyrrolic derivatives. These then spontaneously release the esterifying acids to generate carbonium ions that form covalent adducts with cellular nucleophiles to exhibit toxicity. 2. In our investigation, metabolism-mediated toxicity of monocrotaline, retrorsine, lycopsamine, echimidine (retronecine-type PAs), heliotrine (a heliotridine-type PA) and senkirkine (an otonecine-type PA) was studied using an in vitro co-incubation assay. 3. Human hepatocarcinoma (HepG2/C3A) cells were incubated with PAs in the presence and absence of rat liver S9 fraction and the toxicity was assessed as lowered mitochondrial activity. 4. Bioactivation potential was measured by incubating PAs with rat liver S9 fraction, NADPH and GSH in a cell free system. Pyrrolic metabolites generated were entrapped as glutathione conjugates (7-GSH-DHP and 7,9-di-GSHDHP) which were quantified using LC-MS-MS analysis. 5. Our results indicated that PAs were metabolized by rat liver S9 fraction into reactive pyrrolic derivatives which were toxic to HepG2/C3A cells. This approach can be used to determine and compare bioactivation potential and metabolism-mediated toxicity of various PAs.

Since trans fat labeling requirements became mandatory in the US and many other countries, there has been a need for rapid and accurate analytical methodologies that can facilitate compliance with the various regulations. The determination of total trans fatty acids by mid-infrared (IR) spectroscopy is a widely used procedure that was standardized and validated as AOCS Official Method Cd 14e-09 (negative second derivative infrared spectroscopic method for the rapid (5 min) determination of total isolated trans fat) in 2009. The C–H out-of-plane deformation mid-IR band observed at 966 cm−1 is uniquely characteristic of isolated (non-conjugated) double bonds with trans configuration. AOCS Official Method Cd 14e-09, the most recent attenuated total reflection-Fourier transform IR (ATR-FTIR) official method, entails the measurement of the height of the negative second derivative of the trans absorption band. In the present study, the performance of a novel, portable FTIR system equipped with a heated 9-bounce diamond ATR crystal was evaluated and compared to that of a conventional benchtop single-bounce ATR-FTIR spectrometer. The introduction of the 9-bounce diamond ATR crystal resulted in the lowering of the limit of quantification of trans fat, as a percentage of the total fat, from approximately 2 to 0.34%. The data collected from accurately weighed gravimetric standards and 28 unknown test samples ranging in trans fat content from about 0.5 to 54%, as a percentage of the total fat, indicated that this IR official method and the use of the new 9-bounce portable ATR-FTIR instrumentation could lead to a five-fold enhancement in sensitivity relative to single-bounce systems. Implementing these changes would facilitate regulatory compliance and verification of fat and oil samples for trans fat content in the US and other countries, since all of the published regulations (e.g., “0 g trans fat per serving”) have levels of trans fat, as percentage of total fat, that exceed 0.34%.

A roundtable to discuss the measurement of folate status biomarkers in NHANES took place in July 2010. NHANES has measured serum folate since 1974 and red blood cell (RBC) folate since 1978 with the use of several different measurement procedures. Data on serum 5-methyltetrahydrofolate (5MTHF) and folic acid (FA) concentrations in persons aged ≥60 y are available in NHANES 1999-2002. The roundtable reviewed data that showed that folate concentrations from the Bio-Rad Quantaphase II procedure (Bio-Rad Laboratories, Hercules, CA; used in NHANES 1991-1994 and NHANES 1999-2006) were, on average, 29% lower for serum and 45% lower for RBC than were those from the microbiological assay (MA), which was used in NHANES 2007-2010. Roundtable experts agreed that these differences required a data adjustment for time-trend analyses. The roundtable reviewed the possible use of an isotope-dilution liquid chromatography-tandem mass spectrometry (LC-MS/MS) measurement procedure for future NHANES and agreed that the close agreement between the MA and LC-MS/MS results for serum folate supported conversion to the LC-MS/MS procedure. However, for RBC folate, the MA gave 25% higher concentrations than did the LC-MS/MS procedure. The roundtable agreed that the use of the LC-MS/MS procedure to measure RBC folate is premature at this time. The roundtable reviewed the reference materials available or under development at the National Institute of Standards and Technology and recognized the challenges related to, and the scientific need for, these materials. They noted the need for a commutability study for the available reference materials for serum 5MTHF and FA.

A roundtable to discuss the measurement of vitamin B-12 (cobalamin) status biomarkers in NHANES took place in July 2010. NHANES stopped measuring vitamin B-12 - related biomarkers after 2006. The roundtable reviewed 3 biomarkers of vitamin B-12 status used in past NHANES - serum vitamin B-12, methylmalonic acid (MMA), and total homocysteine (tHcy) - and discussed the potential utility of measuring holotranscobalamin (holoTC) for future NHANES. Theroundtable focused on public health considerations and the quality of the measurement procedures and reference methods and materials that past NHANES used or that are available for future NHANES. Roundtable members supported reinstating vitamin B-12 status measures in NHANES. They noted evolving concerns and uncertainties regarding whether subclinical (mild, asymptomatic) vitamin B-12 deficiency is a public health concern. They identified the need for evidence from clinical trials to address causal relations between subclinical vitamin B-12 deficiency and adverse health outcomes as well as appropriate cutoffs for interpreting vitamin B-12 - related biomarkers. Theyagreed thatproblems with sensitivityandspecificity of individual biomarkers underscore the need for including at least one biomarker of circulating vitamin B-12 (serum vitamin B-12 or holoTC) and one functional biomarker (MMA or tHcy) in NHANES. The inclusion of both serum vitamin B-12 and plasma MMA, which have been associated with cognitive dysfunction and anemia in NHANES and in other population-based studies, was preferable to provide continuity with past NHANES. Reliable measurement procedures are available, and National Institute of Standards and Technology reference materials are available or in development for serum vitamin B-12 and MMA.

The ionic liquid SLB-IL111 column, available from Supelco Inc., is a novel fused capillary gas chromatography (GC) column capable of providing enhanced separations of fatty acid methyl esters (FAMEs) compared to the highly polar cyanopropyl siloxane columns currently recommended for the separation of cis - and trans isomers of fatty acids (FAs), and marketed as SP-2560 and CP-Sil 88. The SLB-IL111 column was operated isothermal at 168 °C, with hydrogen as carrier gas at 1.0 mL/min, and the elution profile was characterized using authentic GC standards and synthetic mono-unsaturated fatty acids (MUFAs) and conjugated linoleic acid (CLA) isomers as test mixtures. The SLB-IL111 column provided an improved separation of cis - and trans -18:1 and cis / trans CLA isomers. This is the first direct GC separation of c 9, t 11- from t 7, c 9-CLA, and t 15-18:1 from c 9-18:1, both of which previously required complimentary techniques for their analysis using cyanopropyl siloxane columns. The SLB-IL111 column also provided partial resolution of t 13/ t 14-18:1, c 8- from c 6/ c 7-18:1, and for several t , t -CLA isomer pairs. This column also provided elution profiles of the geometric and positional isomers of the 16:1, 20:1 and 18:3 FAMEs that were complementary to those obtained using the cyanopropyl siloxane columns. However, on the SLB-IL111 column the saturated FAs eluted between the cis - and trans MUFAs unlike cyanopropyl siloxane columns that gave a clear separation of most saturated FAs. These differences in elution pattern can be exploited to obtain a more complete analysis of complex lipid mixtures present in ruminant fats.

The NIH sponsored a scientific workshop, "Soy Protein/Isoflavone Research: Challenges in Designing and Evaluating Intervention Studies," July 28-29, 2009. The workshop goal was to provide guidance for the next generation of soy protein/isoflavone human research. Session topics included population exposure to soy; the variability of the human response to soy; product composition; methods, tools, and resources available to estimate exposure and protocol adherence; and analytical methods to assess soy in foods and supplements and analytes in biologic fluids and other tissues. The intent of the workshop was to address the quality of soy studies, not the efficacy or safety of soy. Prior NIH workshops and an evidence-based review questioned the quality of data from human soy studies. If clinical studies are pursued, investigators need to ensure that the experimental designs are optimal and the studies properly executed. The workshop participants identified methodological issues that may confound study results and interpretation. Scientifically sound and useful options for dealing with these issues were discussed. The resulting guidance is presented in this document with a brief rationale. The guidance is specific to soy clinical research and does not address nonsoy-related factors that should also be considered in designing and reporting clinical studies. This guidance may be used by investigators, journal editors, study sponsors, and protocol reviewers for a variety of purposes, including designing and implementing trials, reporting results, and interpreting published epidemiological and clinical studies.

A method for the determination of total dietary fiber (TDF), as defined by the CODEX Alimentarius, was validated in foods. Based upon the principles of AOAC Official MethodsSM 985.29, 991.43, 2001.03, and 2002.02, the method quantitates high- and low-molecular-weight dietary fiber (HMWDF and LMWDF, respectively). In 2007, McCleary described a method of extended enzymatic digestion at 37C to simulate human intestinal digestion followed by gravimetric isolation and quantitation of HMWDF and the use of LC to quantitate low-molecular-weight soluble dietary fiber (LMWSDF). The method thus quantitates the complete range of dietary fiber components from resistant starch (by utilizing the digestion conditions of AOAC Method 2002.02) to digestion resistant oligosaccharides (by incorporating the deionization and LC procedures of AOAC Method 2001.03). The method was evaluated through an AOAC collaborative study. Eighteen laboratories participated with 16 laboratories returning valid assay data for 16 test portions (eight blind duplicates) consisting of samples with a range of traditional dietary fiber, resistant starch, and nondigestible oligosaccharides. The dietary fiber content of the eight test pairs ranged from 11.57 to 47.83. Digestion of samples under the conditions of AOAC Method 2002.02 followed by the isolation and gravimetric procedures of AOAC Methods 985.29 and 991.43 results in quantitation of HMWDF. The filtrate from the quantitation of HMWDF is concentrated, deionized, concentrated again, and analyzed by LC to determine the LMWSDF, i.e., all nondigestible oligosaccharides of degree of polymerization 3. TDF is calculated as the sum of HMWDF and LMWSDF. Repeatability standard deviations (sr) ranged from 0.41 to 1.43, and reproducibility standard deviations (sR) ranged from 1.18 to 5.44. These results are comparable to other official dietary fiber methods, and the method is recommended for adoption as Official First Action.

In recent years, several countries have implemented new regulations regarding limitations or labeling of the trans fatty acid ( t FA) content in foods. In order to comply with the new requirements, gas chromatographic methods for fatty acid (FA) analysis have been refined toward the quantitation of a larger number of FAs . Increased attention is also being paid to those present in lower quantities. This article describes a simple procedure for obtaining, pure or in mixtures, geometric and positional isomers of a commercially available monounsaturated FA . cis 10–17:1 Fatty acid methyl ester (FAME) was isomerized into its positional/geometrical isomers by repeated hydrobromination/dehydrobromination of its double bond. Reaction products were fractionated into cis and trans geometric isomers by silver ion HPLC. Pure cis -17:1 FAME positional isomers were obtained by reversed-phase HPLC fractionation and identified by gas chromatography – covalent adduct chemical ionization MS/MS using acetonitrile as the reacting gas. The isomerization with p -toluenesulfinic acid of the purified FAME yielded the corresponding trans isomers; these products were analyzed by GC with flame ionization detection using a Supelco 2560 capillary column in order to determine their elution order and retention times ( t R ). A novel procedure was developed to determine t r for 17:1 FAME positional/geometrical isomers relative to that of the commercially available cis 10–17:1 FAME.

Botanical products are in great demand in health food markets. Botanicals are used for treatment of diseases as well as to promote health (1). Raw materials for medicinal use and herbal supplements frequently are contaminated with toxigenic fungi originating in the soil; the plants themselves are susceptible to fungal growth both pre- and post-havest under the right environmental conditions (2). Contamination with mycotoxins produced by these fungi could pose human health problems. In recent years, many incidences of natural occurrence of mycotoxins in botanicals have been reported (3). Aflatoxins (AF), the naturally occurring aflatoxins B1, B2, G1, and G2 (AFB1, AFB2, AFG1, AFG2), and ochratoxin A (OTA) have been found in ginseng, ginger, licorice, turmeric, and kava-kava in the United States (4, 5). Fumonisins have been found in medicinal wild plants in South Africa (6) and medicinal plants in Turkey (7) and Portugal (8). Zearalenone was identified in ginseng root (9). The presence of AF and OTA in botanicals may pose risks to human health. AF and OTA were found to co-occur in ginger (5). A multitoxin method for the determination of AF and OTA in botanical roots is needed. A method that is capable of determining AF and OTA simultaneously in powdered ginger and ginseng had been developed (5). The method uses a single immunoaffinity column for cleanup, high-performance liquid chromatography (LC) with post-column derivatization for AF fluorescence enhancement, and fluorescence determination for the toxins. The purpose of this collaborative study was to establish the accuracy, repeatability, and reproducibility parameters of the method by the analysis of spiked powdered ginseng and ginger and naturally contaminated powdered ginger.

Preparations of blue cohosh (Caulophyllum thalictroides) have been used traditionally by Native Americans for medicinal purposes. Dietary supplements containing dried roots or extracts of blue cohosh rhizomes are available as dietary supplements. The safety and efficacy of these preparations have not been systematically evaluated. Recent studies indicate that ingestion of specific alkaloids in blue cohosh preparations can produce birth defects and neonatal heart failure. Blue cohosh also contains saponins, which may be responsible for uterine-stimulating effects. We determined the amounts of major alkaloids and saponins in preparations of blue cohosh by high-performance liquid chromatography (HPLC). Alkaloids and saponins were monitored with a photodiode array detector and an evaporative light-scattering detector, respectively. Profiles were compared with those of authenticated blue cohosh root extracts. Identities of the alkaloids and saponins were confirmed by HPLC/mass spectrometry and nuclear magnetic resonance spectrometry. Calculations based on the results of analyses of dietary supplements showed that maximum daily intake of alkaloids and saponins will vary with the form (e.g., root, liquid extract) and doses recommended in product labeling. Intakes may vary from

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.

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.

Isoflavones are phytochemicals found in many plants. Because of their structural similarity to -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--d-glucoside derivatives, and 6-O-acetyl-7-O--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.

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 416 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%.

In their recent report, Williams et al1 examined data from 21 population-based birth defect surveillance systems for trends in prevalence of spina bifida and anencephaly for specific racial/ethnic groups from 1995 to 2002. Data were stratified into pre-, optional, and mandatory fortification intervals, coincident with the authorization of folate fortification of enriched cereal-grain products by the Food and Drug Administration (FDA) in 1996. Williams et al1 found prevalence values per 10000 births for spina bifida for Hispanic, non-Hispanic white, and non-Hispanic black women of 4.18, 3.37, and 2.90, respectively, for the mandatory fortification interval versus corresponding values for the same groups of 6.49, 5.13, and 3.57, respectively, for the prefortification interval. These results suggested that folate fortification was associated with significant decreases in the prevalence of spina bifida among non-Hispanic white and Hispanic births (34% and 36% decreases, respectively). The 19% decline in the prevalence of spina bifida among non-Hispanic black births was borderline statistically significant.1 This failure to find a statistically significant decline among non-Hispanic black births may have been attributable to smaller samples examined (total cases were 515 for non-Hispanic black births, 2672 for non-Hispanic white births, and 1281 for Hispanic births). In their editorial on the article by Williams et al,1 Brent and Oakley2 argued that folic acid fortification levels in the United States are too low. They have inappropriately generalized the data of Williams et al1 into a broad criticism of the current folic acid fortification program and a call for increased fortification. This warrants our response. Consideration of whether increases in fortification are warranted depends a number of complex issues. For example, are the residual neural tube defects (NTDs) in all racial/ethnic groups folate sensitive? If so, then would increasing the levels of folic acid in enriched cereal-grain products … Address correspondence to Jeanne I. Rader, PhD, Division of Research & Applied Technology, Office of Nutritional Products, Labeling and Dietary Supplements, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 5100 Paint Branch Pkwy, College Park, MD 20740. E-mail: Jeanne.Rader{at}cfsan.fda.gov

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.

Teucrium species, such as germander, are rich in neo-clerodane diterpenoids and have been used in traditional folk medicine for their stimulant, diuretic, antipyretic and antiseptic properties. However, the furano neo-clerodane diterpenoids present in germander have been implicated in the in vivo hepatotoxicity of this botanical. In this study, authenticated germander (Teucrium chamaedrys L. and Teucrium canadense L.) was used as the source material. Methanol extracts of powdered plant mate rial were prepared and analysed by HPLC using Synergi Max-RP columns with monitoring at 220 nm. Limited amounts of teucrin A and other diterpenoid standards were analysed on a Synergi Max-RP column in order to determine their retention times and to generate calibration curves. The same standards were subjected to concurrent mass spectral analysis. Teucrin A and diterpenoids such as dihydroteugin, teuflin, teuflidin and teucvidin were tentatively identified in the plant extracts by HPLC-MS and 1H-NMR experiments. For the isolation of teucrium diterpenoids on a semipreparative scale, a solid-phase extraction method was developed for the first time using styrene divinylbenzene and strata-X sorbents for teucrin A and teuflin, respectively. Semi-preparative HPLC of the methanol extract of the powdered aerial parts of Teucrium plants was carried out on a semipreparative Synergi Max-RP column with photodiode array detection in order to confirm the identities of some diterpenoids by HPLC-MS and NMR.

A review is presented describing the nature and evolving definition of dietary fiber. The historical development of the current definition is discussed as are the efforts to develop analytical methods to support food labeling regulations. Also considered are the characterization and quantitation of resistance starch, a dietary starch that does not digest in the small intestine, behaves like dietary fiber and therefore may have potential as a health-related ingredient in foods. The current status of AOAC methodology is discussed along with the possibility of updating the definition of dietary fiber. The potential impacts of changing the dietary fiber definition on analytical issues and on food composition databases are also considered.

In 1996, U.S. Food and Drug Administration regulations mandated the fortification of enriched cereal-grain products with folic acid, thereby emphasizing the need for validated methods for total folates in foods, particularly cereal products. The AOAC Official Methods (944.12, 960.46) currently used for the analysis of folate in foods for compliance purposes are microbiological methods. When the fortification regulations were finalized, no Official AOAC or Approved AACC methods for folate in cereal-grain products were in place. The AOAC Official Method (992.05) for folic acid in infant formula does not incorporate important improvements in the extraction procedure and was not considered suitable for the analysis of folates in foods in general. Amicrobiological assay protocol using a trienzyme extraction procedure was prepared and submitted for comments to 40 laboratories with recognized experience in folate analysis. On the basis of comments, the method was revised to have the conjugase (gamma-glutamyl-carboxy-peptidase) treatment follow a protease treatment, to include the use of cryoprotected inoculum, and to include the spectroscopic standardization of the standard and optional use of microtiter plates. Thirteen laboratories participated in a collaborative study of 10 required and 10 optional cereal-grain products, including flour, bread, cookies, baking mixes, and ready-to-eat breakfast cereals. The majority of the participating laboratories performed the assay by the standard test tube method; others used the microtiter plate modification for endpoint quantitation with equal success. For the required products, the relative standard deviation between laboratories (RSDR) ranged from 7.4 to 21.6% for 8 fortified (or enriched) products compared with expected (Horwitz equation-based) values of 11–20%. RSDR values were higher (22.7–52.9%) for 2 unfortified cereal-grain products. For the optional products, the RSDR ranged from 1.8 to 11.2% for 8 fortified products. RSDR values were higher (27.9–28.7%) for 2 unfortified cereal-grain products. Based on the results of the collaborative study, the microbiological assay with trienzyme extraction is recommended for adoption as Official First Action.

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.

Mandatory fortification of enriched cereal-grain products became effective in the United States on January 1, 1998. This fortification was undertaken to assist women of child-bearing age in increasing their intake of folic acid to reduce their risk of having a pregnancy affected by a neural tube birth defect. The process by which the Food and Drug Administration modeled the level of fortification with folic acid illustrates the complex issues and general principles that emerge when fortification of a nation's food supply is evaluated as a means of addressing a public health concern. The effectiveness of fortification for a target population and safety for the much larger general population impose conflicting challenges that must be considered concurrently when making decisions regarding fortification. Recent data show improved folate status and apparent decreases in risk of neural tube birth defects in the U.S. Much about the long-term effects of the fortification program remains unknown and careful monitoring over time will be necessary to ensure that the program functions as intended.

We investigated the ethanol precipitation step of the gravimetric method of dietary fiber analysis (AOAC Official Method 985.29). Four different solvents: ethanol, propanol, acetone and acetonitrile at four ratios: 1:1, 2:1, 3:1 and 4:1 (solvent : supernatant, v/v) were studied. Using inulins that contain components with a full range of degree of polymerization values (DP), we found that the percents of precipitation by these solvents were proportional to the average DP of the products. Use of ethanol and propanol produced similar results. In general, acetonitrile and acetone precipitated more of the inulins than did ethanol. Supernatant solutions were analyzed by high performance anion exchange chromatography in order to determine the size of the components that were precipitated. Our data showed that components of inulin with DP 1–10 remain in solution after the addition of ethanol at a ratio of 4:1. However, significant amounts of molecules of DP 11 and 12 and smaller amounts of molecules of DP 14–18 also remain in solution. The precipitation patterns of oligoglucoses with DP 1–DP 7 were also investigated. Our data suggest that the precipitation behavior of oligoglucoses follows a pattern similar to that of inulins.

In 1996, the U.S. Food and Drug Administration issued a regulation requiring that all enriched cereal-grain products be fortified with folic acid by January 1998. An average increase in folic acid intake of 100 micro g/d was projected as a result of this fortification. The objective of the present study was to estimate the effect of this fortification on the intake of folic acid and total folate, and on the prevalence of individuals with inadequate folate intake and with high folic acid intake. We used data on food and nutrient intake from 1480 individuals who participated in the 5th and 6th examinations of the Framingham Offspring Cohort Study. Fortification was instituted during the 6th examination so that 931 participants were examined before its implementation (nonexposed) and 549 after implementation (exposed). Published data on total folate in enriched cereal-grain products were used to correct folate content in these foods to reflect fortification. Among nonsupplement users, folic acid intake increased by a mean of 190 [95% confidence interval (CI): 176, 204] micro g/d (P < 0.001) and total folate intake increased by a mean of 323 (95% CI: 296-350) micro g dietary folate equivalents (DFE)/d (P < 0.001) in the exposed participants. Similar increases were seen among supplement users exposed to fortification. The prevalence of exposed individuals with total folate intake below the estimated average requirement (320 micro g DFE/d) decreased from 48.6% (95% CI: 44.2-53.1%) before fortification to 7.0% (95% CI: 3.1-10.9%) after fortification in individuals who did not use folic acid supplements. This prevalence was approximately 1% or less for users of supplements both before and after fortification. Prevalence of individuals with folic acid intake above the upper tolerable intake level (1000 micro g folic acid/d) increased only among supplement users exposed to fortification (from 1.3 to 11.3%, P < 0.001). No changes in folic acid intake were observed over time in the nonexposed participants. By these estimations, folic acid fortification resulted in a mean increase in folic acid intake that was approximately twice as large as previously projected.

Folic acid fortification of enriched cereal-grain products (which became mandatory in the U.S. on January 1, 1998) was intended to increase folate intake among childbearing-aged women to reduce their risk of neural tube birth defect (NTD)-affected pregnancies. Interest now focuses on assessing the effects of fortification on risk of NTDs and on folate intake relative to homocysteine (Hcy) concentrations and risk of vascular disease, although a causal relationship between the latter two has not been demonstrated. Increased serum folate levels were first reported in 1999. Data from the Framingham Offspring Study cohort showed increased mean serum folate in middle-aged and older adults; additionally, the prevalence of high Hcy concentrations had decreased by approximately 50% in subjects examined before (1995-1996) and after (1997-1998) fortification. Another analyses of samples collected between 1994 and 1999 identified a trend of increasing serum folate values from 1996 onward with values in 1998 160% of those measured in 1996. Comparisons between 1988-1994 National Health and Nutrition Examination Survey (NHANES) III data and 1999 NHANES showed increased serum and erythrocyte folate concentrations among childbearing-aged women. While recent data show improved folate status in a short period of time, much about long-term effects of the fortification program remains unknown. Interest in the effects of increased folate intakes on risk of NTDs or vascular disease needs to be balanced against concerns about masking the anemia of vitamin B-12 deficiency and the general lack of data about safety of continuous high intakes. Careful monitoring over time is necessary to determine that the program functions as intended.

Twelve powdered and 13 liquid infant formulas were analyzed by using an extension of AOAC Official Method 996.01 for fat analysis in cereal products. Samples were hydrolyzed with 8N HCl and extracted with ethyl and petroleum ethers. Fatty acid methyl esters were prepared by refluxing the mixed ether extracts with methanolic sodium hydroxide in the presence of 14% boron trifluoride in methanol. The extracts were analyzed by gas chromatography. In powdered formulas, saturated fatty acid (SFA) content (mean ± SD; n = 12) was 41.05 ± 3.94%, monounsaturated fatty acid (MUFA) content was 36.97 ± 3.38% polyunsaturated fatty acid (PUFA) content was 20.07 ± 3.08%, and total trans fatty acid content was 1.30 ± 1.27%. In liquid formulas, SFA content (mean ± SD; n = 13) was 42.29 ± 2.98%, MUFA content was 36.05 ± 2.47%, PUFA content was 20.65 ± 2.40%, and total trans fatty acid content was 0.88 ± 0.54%. Total fat content in powdered formulas ranged from 4.4 to 5.5 g/100 kcal and linoleic acid content ranged from 868 to 1166 mg/100 kcal. In liquid formulas, total fat content ranged from 4.1 to 5.1 g/100 kcal and linoleic acid content ranged from 820 to 1100 mg/100 kcal. There were no significant differences between powdered and liquid infant formulas in concentrations of total fat, SFA, MUFA, PUFA, or trans fatty acids.

The increased availability and use of botanical dietary supplements and herbal remedies among consumers has been accompanied by an increased frequency of adulteration of these products with synthetic pharmaceuticals. Unscrupulous producers may add drugs and analogues of various classes, such as phosphodiesterase type 5 (PDE-5) inhibitors, weight loss, hypoglycemic, antihypertensive and anti-inflammatory agents, or anabolic steroids, to develop or intensify biological effects of dietary supplements or herbal remedies. The presence of such adulterated products in the marketplace is a worldwide problem and their consumption poses health risks to consumers. Analytical methods that allow rapid and reliable testing of dietary supplements for the presence of synthetic drugs are needed to address such fraudulent practices. Mass spectrometry (MS) and hyphenated techniques such as liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS) have become primary tools in this endeavor. The present review critically assesses the role and summarizes the applications of MS in the analysis of pharmaceutical adulterants in botanical dietary supplements and herbal remedies. The uses of MS techniques in detection, confirmation, and quantification of known pharmaceutical adulterants as well as in screening for and structure elucidation of unexpected adulterants and novel designer drugs are discussed.

Marine oil omega-3 supplements are among the most frequently consumed dietary supplements in the United States. However, few studies have evaluated the overall fatty acid composition of these products. We investigated the content and composition of fatty acids in 46 commercially available marine oil omega-3 supplements by gas chromatography with flame ionization detection using the 200 m SLB-IL111 ionic liquid column. Seventy-three fatty acid isomers were quantified, including n-6, n-4, n-3, and n-1 polyunsaturated fatty acids and trans isomers of eicosapentaenoic acid (EPA; C20:5n-3) and docosahexaenoic acid (DHA; C22:6n-3), the chromatographic separations of which we report for the first time on the 200 m SLB-IL111 column. Contents of EPA and DHA met their respective label declarations in more than 80% of the products examined. Eleven of the products (24%) carried the Food and Drug Administration's qualified health claim for EPA and DHA omega-3 fatty acids.

A rapid, selective and sensitive ultra-high-performance liquid chromatography-multistage fragmentation mass spectrometry (UHPLC-MS³) method was developed and evaluated for the determination of aristolochic acids I and II (AA I and II) in herbal dietary supplements. A hybrid triple quadrupole/linear ion-trap mass spectrometry was used to monitor MS³ ion transitions m/z 359.2 > 298.1 > 268.0 and m/z 329.2 > 268.2 > 238.0 to detect AA I and II, respectively. The extraction and clean-up of target analytes from dry powdered samples was performed using the quick, easy, cheap, effective, rugged and safe (QuEChERS) procedure. Herbal liquid extracts were analysed directly. Average recoveries ranged from 89% to 112%, with relative standard deviations (RSDs) ranging from 3% to 16%. Limits of quantification (LOQs) estimated for three selected matrices were as follows (AA I/II): 5/10 ng g⁻¹ (tablets); 25/50 ng g⁻¹ (capsules); and 2.5/5.0 ng ml⁻¹ (liquid herbal extract). The method was applied in a limited survey of 30 herbal products marketed in the United States via the Internet. AA I and II were detected in 20% and 7%, respectively, of tested samples.

Publisher Summary The U.S. Food and Drug Administration (FDA) is the federal agency charged with protecting public health by ensuring, among other things, the safety of foods. Specifically, the FDA is responsible for ensuring the public has accurate, science-based information that they need to plan a healthy diet. Trans fat labeling and other regulations in many countries prompted the food industry to accelerate food reformulation to reduce the amount of total trans fat in most products. It also increased interest in the development of rapid yet reliable methods for the accurate determination of the fatty acid composition and for monitoring the declarations of total trans and saturated fatty acid contents on food labels. Optimized and validated analytical methods are needed to ensure high rates of compliance with new food safety and labeling regulations. This chapter discusses the recent advances in three chromatographic and spectroscopic methodologies—capillary Gas Chromatography (GC), mid-Infrared (mid-IR), and Near-infrared (NIR)—needed to meet this challenge. It also provides a brief comparison of gas chromatographic and infrared spectroscopic approaches.

A novel and rapid (5 min) attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopic method AOCS Cd 14d-99 for the determination of total isolated trans fatty afids, which absorb at 966 cm‐, was recently developed, collaboratively studied, and applied to food products containing 1–50% rans fat (as percentage of total fat). Attempts to apply the ATR-FTIR method to biological matrices of low trans fat and/or low total fat content, and to dairy and other products were not satisfactory due to interfering IR absorptions in the trans region. One group of interfering compounds with absorption bands near 985 and 948 cm−1 was the cis/trans positional isomes of conjugated linoleic acid (CLA) found in dairy and meat products from ruminants at levels of

Fortification of cereal-grain products was introduced in 1941 when iron and three vitamins were added to flour and bread. Ready-to-eat cereals were fortified at about the same time. These fortifications have contributed to increased dietary iron intake and reductions in iron deficiency anemia in the US. In 1996, FDA finalized rules for fortification of specific enriched cereal-grain products with folic acid. This measure was instituted to increase the folate intakes of women of child-bearing age and thereby reduce the risk of having a pregnancy affected with a neural tube birth defect. However, with recent increases in fortification, public health officials in the US are concemed that excess intake of specific nutrients such as iron and folic acid may result in toxic manifestations.Our objective was to measure iron and total folate content in breakfast cereals and compare assay to label values for % Daily Value. We also determined by weight the amount of a ready-to-eat breakfast cereal adults would eat and compared this to the labeled serving size, for which the reference amount for this cereal per eating occasion was 1 cup or 30 g.Twenty-nine breakfast cereals were analyzed for iron content using the bathophenanthroline reaction. Twenty-eight cereals were analyzed for total folate, utilizing a microbiological assay with tri-enzyme digestion. Serving size quantities were estimated in seventy-two adults who regularly ate breakfast cereal and were asked to fill a 16 or 22 cm round bowl with the amount of cereal that they would consume for breakfast.When the labeled value was compared to the assayed value for iron content 21 of the 29 breakfast cereals were 120% or more of the label value and 8 cereals were 150% or more of the label value. Overall, analyzed values for iron ranged from 80% to 190% of label values. Analyzed values for folate ranged from 98% to 320% of label values. For 14 of 28 cereals, analyzed values exceeded label declarations by more than 150%. Bran-containing cereals contained the highest amounts of folate relative to their label declarations. The median analyzed serving size for the breakfast cereal was 47 g for females, 61 g for males with a combined median of 56 g as compared to the label value of 30 g.Analyzed values of iron and folic acid in breakfast cereals were considerably higher than labeled values. For adults, the amount of cereal actually consumed was approximately 200% of the labeled serving size. When the quantity of cereal consumed is more than the labeled serving size and when the levels of iron and folate are higher than declared, the intake of both will be significantly greater than the labeled values. It will be important to continue monitoring serum ferritin and folate levels in NHANES IV, since daily consumption of breakfast cereals may contribute to excessive intakes of iron and folate.

There is currently no official method for the analysis of fatty acids (including trans fatty acids) in infant formulas. AOAC Official Method 996.01 for Fat Analysis in Cereal Products was extended to the analysis of milk-based infant formula Standard Reference Material (SRM)1846 to determine its applicability for use with infant formulas. Following the analysis of SRM 1846, 2 infant formulas, one milk-based liquid and one soy-based powdered infant formula, were analyzed for total fatty acid composition. Fatty acid methyl esters were prepared and analyzed by gas chromatography. The results of the analysis of SRM 1846 show that the mean analyzed values were highly reproducible as indicated by low coefficients of variation (CV). The CVs were

The full compliance date for mandatory folic acid fortification of enriched cereal-grain products in the United States was 1 January, 1998. There is currently a great interest in determining the effectiveness of this measure, which was instituted to increase the folate intakes of women of child-bearing age to reduce their risk of having a pregnancy affected by a neural tube birth defect. We surveyed 83 enriched cereal-grain products that are required to be fortified with folic acid under the new regulations and an additional 79 foods that contain enriched cereal-grain ingredients or that are currently fortified with folic acid. Products were collected and analyzed between February 1998 and April 1999. Total folate was determined by microbiological assay using a tri-enzyme digestion. We compared analyzed values for total folate with amounts required by Federal regulations and/or with label declarations of folate content. For many enriched cereal-grain products, there were significant differences between amounts of folate found on analysis and amounts required by Federal regulations. In part because of this, label declarations of folate content were also in error. The high values found in some enriched cereal-grain products may represent manufacturers' averages as well as the presence of higher-than-expected levels of endogenous folates. These results indicate that reliable food composition databases cannot be developed without extensive new data on the actual concentrations of folate in recently fortified enriched cereal-grain products as well as in products containing enriched cereal-grain ingredients. Reliance on older data bases or on compositional information that has a weak analytical underpinning will lead to unsound estimates of folate intake, and hence, of the potential impact of the new fortification program.

We report the synthesis of fluorescently labeled ubiquitin (Ub) and its use for following ubiquitin transfer to various proteins. Using Oregon green (Og) succinimidyl ester, we prepared a population of Ub mainly labeled by a single Og molecule; greater than 95% of the Og label is associated with Lys 6 of Ub. We demonstrate that Og-Ub is efficiently accepted by Ub-utilizing enzymes, such as the human ubiquitin-activating enzyme (E1). We used this fluorescent substrate to follow the steady-state kinetics of human E1-catalyzed Ub-transfer to the ubiquitin-carrier enzyme Ubc4. In this reaction, E1 uses three substrates: ATP, Ubc4, and Ub. The steady-state kinetics of Og-Ub utilization by E1 is presented. We have also used analytical ultracentrifugation methods to establish that E1 is monomeric under our assay condition (low salt) as well as under physiological condition (150 mM NaCl).