Anal. Chem. 1993, 65,334R-363R
Food+ Sam K. C. Chang,'J*5 Patricia Rayas-Duarte,! Edna Holm,t and Clarence McDonald5 Department of Food Nutrition and Department of Cereal Science and Food Technology, North Dakota State University, Fargo, North Dakota 58105 Review Contents
Introduction Water Activity, Moisture, and Water Distribution Nitrogen and Proteins Carbohydrates Inorganics (Minerals) Enzymes Flavor Color
Lipids Vitamins
334R 334R
335R 337R 339R 341R 343R 348R
352R 354R -
INTRODUCTION This review was written based on the literature searches using Chemical Abstracts covering the dates primarily between 1988 (Vol. 109) and 1992 (Vol. 117). Thousands of papers were published during this period of time on the development and applications of methodologies for analyzing food components. We have selected nine major aspects of food analysis. The subtopics presented in each area were arbitrarily selected by the authors. The selected abstracts were predominantly those published in English. Only a few published in other languages were used. The journal reviews and book chapters published within each subject area are given in each respective section. There were not many new advances in methodologies for measuring water activity and moisture in foods. The nitrogen combustion method has been increasingly used to measure crude protein content. And the methods for identification and quantification of specific proteins in foods using electrophoresis and enzyme-linked immunosorbent assays have been developed. For the analysis of sugars, several methods using biosensors have been developed. Liquid chromatographic methods for the analysis of sugars and polysaccharides also have been developed. Flow injection flame atomic absorption, electrothermal atomic absorption, inductively coupled plasma atomic spectrometry, and neutron activation analysis have been widely used for the analysis of metallic elements. Ion chromatographic methods were used for inorganic anions in foods. Biosensors using enzymes have been widely used for the analysis of substrates in foods. In addition, several enzymes have been used in enzyme-linked immunosorbent assay for analyzing several food components. Flow injection spectrophotometric analysis using enzymes for analysis of food components has been used. Various instrumental methods, including GC/MS, chemical, and sensory methods are still being explored for analyzing flavors in foods. In addition to HPLC for food colorant analysis, several new instrumental techniques, including capillary isotachophoresis, near-IR FT/ Raman spectroscopy, fast-atom-bombardment mass spectrometry, flow injection analysis, and Curie point pyrolysis high-resolution GUMS, have increased detection sensitivity. New sensitive methods for vitamin analysis generally utilize HPLC with various types of detectors. + An abbreviated version of citations in Chemical Abstracts is given in the references. If expanded, the correct citation would be (for example, in ref A16) Chem. Abstr. 1990, 112(9),75571b. t Department of Food and Nutrition. I Department of Cereal Science and Food Technology. 334R
0003-2700/93/0365-0334R$12.00/0
WATER ACTIVITY, MOISTURE, AND WATER DISTRIBUTION A review on NMR uses for the determination of moisture in food and feed was reported by Schmidt ( A I ) . Schmidt and Lai (A2) reviewed the theory of NMR and pulsed-field gradient NMR and MRI techniques in the study of water relations in food systems. The use of DSC in the analysis of freezable water was reviewed by Schenz et al. (A3). The relation between chemical composition of cheese and its water activity predicted by linear re ression analysis was reviewed by Esteban and Marcos (A4). &ade and Levine (A5)reviewed the impact of water content on food quality and moisture management. The water sorption behavior of solute-polymer-water and the competition for water among the solutes were reviewed by Chinachoti and Schmidt (A6). Baianu et al. (A7)reviewed the theoretical and experimental multinuclear spin relaxation approaches in the study of the molecular dynamics of water in foods. A new method for the water activity analysis of biological samples based on the equilibrium moisture absorption of microcrystalline cellulose was reported by Alcaraz et al. (A8). A study on the influence of water activity and temperature in the kinetics of quality deterioration in dried onion and green beans allowed prediction of the shelf life of these products (A9). Prediction of water activity in aqueous nonelectrolyte solutions containing monosaccharides and disaccharides was reported by using a UNIFAC model (A10). The model showed an accuracy of 0.006 a, unit in the range of 0.85 C a, C 1,comparable to the accuracy obtained with a Thermoconstanter Novasina apparatus (AIO). Lilley and Sutton ( A l l ) proposed a method for the prediction of the water activity of multicomponent systems from the properties of solutions of one and two solutes. A semiempirical model of water activity changes in the presence of electrolytes and nonelectrolytes was reported using an extension of Raoult's law (A12). The predicted values had an accuracy of 0.001 a, unit up to 4-14 M (A12). An empirical three-parameter model was developed from published moisture sorption data of selected polysaccharides at water activities up to 0.98-0.99 a, (A13). Pollio et al. (A14)reported an average water activity (a,) of saturated KN03solution of 0.927 f 0.001and proposed to discontinue use of the value 0.936. An improved simple method for measuring higha, in cheese was reported by Esteban et al. (AI5). The average underestimation of a,,, > 0.990 was decreased to 0.001unit with the proposed method compared to 0.005 unit when a thermocouple psychrometer was used (A15). Gilbert (A16) described a kinetic method to determine sorption isotherms by inverse gas chromatography (IGC). Among the advantages of the method was the elimination of hysteresis errors (A16). IGC was later reported as an effective tool to characterize the hydrophilic and structural properties of food systems (A17). The moisture sorption isotherm of apple and citrus pectins (at 25-50 "C) were obtained by using a computerized IGC method with good agreement with the gravimetric static method ( A M ) . Water sorption isotherms of wheat flour were reported by using IGC and determined at ranges of 25-55 "C and 0 < a, < 0.8 (A19). Experimental data fitted the Brunauer, Emmitt, and Teller (BET), Freundlich, and Guggenheim, Anderson, and De Boer (GAB) equations (A19). Transverse water proton relaxation was proposed to be a potential sensitive robe in water distribution studies during freeze-thawing anzdehydration-rehydration in food systems (A20). The authors reported that chemical exchange and molecular diffusion information can be obtained from relaxation times of characteristic variations with Carr-PurcellMeiboom-Gill pulse spacing and morphology (A20). Proton 0 1993 American Chemical Society
FOOD
Dr. Sam K. C. Chang, Associate Professor of Department of Food and Nutrition with a joint appointment in the Department of Cereal Science and Food Technology, North Dakota State University, Fargo, NO (B.S. 1973, ChineseCuRure University; M.S. and Ph.D. 1977 and 1980, respectively, University of Nebraska-Lincoln), teaches food analysis and selected topics in food chemistry, food processing, and food engineering. His research interests include - .method development and/or applications A for the analysis of proteins, amino acids, carotenoids, pectin, and nonstarchy polysaccharides. He has authored many publications on the chemistry, analysis, and processing of foods, including dry edible beans, soybeans, carrots, rice, sugar beet fiber, and sunflower pectin. He is a member of the Institute of Food Technologists and is the President of The Chinese American Food Society. Patricia Rayas-Duarte, Ph.D. is assistant professor in the Department of Cereal Science and Food Technology, North Dakota State University(B.S. 1978, Universrty of Sonora, Mexico; M.S. 1985, University of Nebraska-Lincoln; Ph. D. 1988 University of Nebraska-Lincoln), teaches cereal carbohydrates and studies processes that will add value to alternate crops. Current focus of research includes the biochemicalcomposition of amaranth, buckwheat, and lupin grains, starch chemistry and processing, and development of food and nonfoodapplications of alternative crops. Prior research activities included starch irradiation, chromatographic analysis of protease inhibitors, and in vivo digestibility of sorghum. She is member of IFT and AACC.
NMR imaging spectroscopy was used to investigate lipid and water distribution of food samples. The noninvasive, dynamic measurement gives different relaxation values for each component (AZI). The relative amount of fat and water content in vitro and in vivo in pork and mice was reported by Mitchell et al. (A22). A report on the determination of water in coffee by drying methods and Karl Fischer titration was reported by Maier (A23). Modifications to the Karl Fischer titration were used in roasted coffee samples. A collaborative study involving nine laboratories gave exceptional repeatability and reproducibility (0.14 % and 0.2570, respectively) (A23). Apparently, better results were obtained when the paste Karl Fischer extraction method was used as the primary reference method in flour from cereal grain samples when correlated to near-IR spectra (A24). The water transport (diffusion) in starches and in starchsugar mixtures was reported by Leslie et al. (A25). Volume changes caused by sorption of water in starch samples were determined by dilatometry (A26). The contributions from strongly or weakly sorbed water molecules to volume changes were obtained from the parameters of the GAB equation (A26).
NITROGEN AND PROTEINS Introduction. In the past four years, many research papers have appeared in the four areas of determination of total nitrogen and the identification of proteins in foods. These include (1)the determination of total nitrogen using combustion followed by detection using a thermal conductivity sensor, (2) rapid and nondestructive determination of total proteins using near-infrared and infrared spectroscopic methods, (3) protein identification and quantification in various food systems using electrophoretic methods, and (4) determination of proteins using immunoassay, particularly the enzyme-linked immunosorbent assays. Analysis of Total Nitrogen Using the Combustion Met hods. Total nitrogen determination based on DUMAS and its modifications continues to be developed by many researchers for analyzing food protein content because of its rapidity and ease of operation. Theobald ( R I )measured total nitrogen by a modified DUMAS procedure, which is based on the conversion of total nitrogen to elemental nitrogen
Edna Holm, Ph.D., associateprofessor and chair of the Food and Nutrition Department and assistant graduate dean at North Dakota State University (M.S. 1960, North Dakota State University; Ph.D. 1987, University of Minnesota) teaches food sanitation, sensory evaluation, and food selection and preparation courses. Current research areas: natural food colorants, quality evaluation of potato products, and alternate uses for commodities. Memberships include IFT Microbiology and Sensory Evaluation Divisions.
Clarence E. McDonald, Ph.D. is a professor in the Department of Cereal Science and Food Technology, North Dakota State University (M.S. 1953, Kansas State University; Ph.D. 1957, Purdue University). He teaches cereal proteins and lipids and supervises an analytical laboratory. Research in areas of protein analysis, food protein modification, proteinase and lipoxygenase enzymes, and near-infrared analysis. Chair of American Association of Cereal Chemists technical committee for wheat and flour analysis for ten years.
through combustion of food samples at a high temperature. The nitrogen produced was separated using column chromatography and detected by a thermal conductivity detector. The apparatus for nitrogen determination has been automated. Compared to the Kjeldahl method, the automated method produces similar results and is safer and faster. A determination takes approximately 3 min. Total nitrogen in brewing grains was studied collaboratively by a group of laboratories using a combustion method with nicotine acid-p-toluenesulfonate as calibration standard (B2). The combustion method had a detection limit of 0.03% N. The relative standard deviations for repeatability and reproducibility were 1.3-5.57; and 4.2-6.7 %, respectively. Donhauser et al. (B3)used an automated nitrogen analyzer, which was based on the DUMAS combustion method, to analyze wheat, barley, and malt samples. The automated nitrogen analyzer generally gave a higher value than the value obtained by the Kjeldahl method. The results suggest that the analyzer is a good alternative to the standard Kjeldahl method if samples are homogenized and a sufficient number of samples are analyzed. A strong correlation existed between an automated nitrogen combustion method and the standard Kjeldahl method for determining nitrogen in soy sauce (B4). However, the coefficient of variation of the nitrogen combustion method was greater. Koenig (B5)used a fully automated nitrogen combustion method to determine food and feed nitrogen. The method took only 1.5 min for a single sample analysis and was applicable for nitrogen content of 0.2-500 mg. The nitrogen recovery rate was nearly 100% . An AOAC procedure (method 990.03) is available for measuring nitrogen based on the combustion method. A variation of the DUMASmethod for determiningnitrogen using ion chromatographyafter combustion has been reported by Wijesundera and Ackerman (R6). The method also could determine sulfur, halogens, and selenium simultaneously. Ion chromatography also has been used to determine ammonium after Kjeldahl digestion (R7). Rapid and Nondestructive Spectroscopic Methods for Total Protein Determination. The infrared (IR) and nearinfrared (near-IR) methods were the major methods for measuring proteins and other food components (B8-BIO). The nondestructive method has the advantages of being rapid and free from the use of potentially hazardous chemicals for analysis. An automated analyzer equipped with a flow cell for milk analysis takes approximately 20 s for a single sample analysis. ANALYTICAL CHEMISTRY. VOL. 65, NO. 12, JUNE 15, 1993
335R
FOOD
The near-IR method has been used by many researchers for the analysis of le ume proteins. The accuracy was found to be as good as the Ejeldahl method. Tsou and Hong ( B I I ) measured proteins in soybean plant and seed samples bynearIR. The R2 was greater than 0.95, and the standard error was less than 0.52 % . The procedure was good for a breeding program, in which a large number of samples are screened for the selection of beans for the processing industry. A near-IR method was used to determine proteins for soy sauce samples using 1114 nm for the reference wavelength and 2164 nm for detecting peaks (B12).Akiyama et al. (B13)used near-IR to determine commercial soy milk protein content, which was found to be in the range of 1.6-3.3% The moisture content of soy milk in the range of 86.4-93.7 % was also determined by the near-IR method. Chao et al. (B14) determined 7 s and 11sglobulins in ground whole soybean by near-IR spectroscopic analysis usin the second derivatives of the spectral data of the 7s a n c f l l s proteins. The standard errors of the difference between the values by ultracentrifugal analysis and near-IR prediction were 1.4%, 1.3%, and 0.1 % for 7S, 11S, and llS/7S ratio, respectively. This method could be used for screening beans for a desired functionality of the soybean food products. NearIR was proven to be good to determine the protein content of ground eas. However, the prediction for the whole pea was poor, $ut was improved after the spectral data were mathematically derived (B15). Near-IR methods continue to be used for predicting protein content and other components in cereal grains. Yoshikawa et al. (B16)used a near-IR method to determine the proteins in milled and brown rice. High correlation coefficients and low standard errors were obtained. McDonald and Bruns (B17)showed that both the K'eldahl and near-IR method were not able to distinguish adulterated fertilizer nitrogen from proteins in wheat. The nitrogen from the adulterations could be determined by chemical and enzymatic methods. Dauberte et al. (B18)determined chocolate proteins using near-IR spectroscop and showed satisfactory results as compared to the K'elchl method in terms of ease and ra idity of analysis. Near- R spectroscopyalso was used for pre icting proteins, oil, carbohydrates in corn (B19), crambe (B20), fish and millet (B22). muscle (B21), IR spectroscoy also has been used for a number of food products. Cereal flour proteins were determined with an IR reflectometer. The method used a rotating filter disk and PbS detector a t a reflectance angle of 45" to measure a range of wavelengths from 1050to 1400 nm (B23).IR spectroscopy was used for soybean-based beverages. For protein content in the range of 0.9-3.5 % ,the reproducibility was in the range of 0,008-0.28% (B24).Fourier transform IR spectroscopy was used to analyze wheat proteins and starch characteristics of various cultivars related to hardness and baking quality (B25). Fourier transform IR spectroscopy was used to analyze proteins, fat, lactose, and water. The precision and accuracy were similar to that obtained using an instrument equipped with an IR filter (B26). Protein Identification and Quantification Using Electrophoretic Methods. Polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing (IEF), two-dimensional (2D) electrophoresis, and electroimmunochemical assay have been used by many researchers to determine proteins in a variety of food systems, including milk, wheat, fish, meat, and legume, for the detection of adulteration and identification of animal species or plant cultivars, and for the detection of the changes of proteins in food processing. Milk and Cheese. Electrophoretic methods for analysis of milk (B27)and cheese (B28)have been reviewed. The genetic variants of caseins and whey proteins from cow's milk can be identified in 60 min. using a rapid electrophoresis system and a precast gel (B29).The gel contained 7 M urea, Triton X-100, and ampholytes in a narrow pH range. Approximately 150 samples could be screened daily. The method is fast and reproducible and has a high resolution of milk variants. A two-dimensional polyacrylamide gel electrophoresis (2DPAGE) combining SDS-PAGE and IEF was able to characterize major proteins in milk. The method was able to distinguish milk from different species of cows (B30).Another high-resolution two-dimensional electrophoresis usin is0 electric focusing on a capillary system, followed by 80sI
i
336R
B
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
PAGE in a minigel was faster than the traditional 2D method (B31).This method could differentiate easily the various isoforms of caseins and dephosporylated proteins in milk. This method also could detect the effect of bacterial proteinase on milk proteins (B31). An automated Phast System (an IEFsystem),usingalkaliie urea-PAGE and precast ultrathin gradient gels, was unable to separate caseins and whey proteins according to their charge-to-mass ratio (B32).The protein atterns of cheese samples varied among different types of cgeese. A capillary electrophoresis system was able to detect the adulteration of fresh milk with 25% or more of dry milk (B33).In dry milk, the whey protein a-lactalbumin showed extensivedegradation, which could be differentiated by the electrophoresis system. Rocket immunoelectrophoresis was able to detect approximately 30 ng of whey proteins (a-lactalbumin and &lactoglobulins A and B) with approximately 100%recovery. This method is better than olyacrylamide gel electrophoresis in terms of sensitivity anzrecovery. The method also could be used to detect the denaturation of whey proteins by varied pasteurization practices. Detectable a-lactalbumin decreased with increase in heating temperature. This method could be used to evaluate the extent of heatin Overheating could reduce the nutritional value of milk (%34 B35). Proteins and degradation of proteins in various cheese types and stages of ripening could be detected by SDS-PAGE (B36), high-resolution 2D electrophoresis (B37), and isoelectric focusing (B38,B39). Using SDS-PAGE, the advantages include minimal sample treatment, rapidity, simplicity, high resolution, and sensitivity. Fish and Shrimp. A number of studies have been done on identification of fish and shrimp species using electrophoresis. Sarcoplasmic proteins from fish muscle from 12 species of marine and 4 species of freshwater fish had species-specific patterns based on electrophoretic analysis. Patterns of marine fish were much different from that of freshwater species (B40). Processed fish species have been identified by electrophoresis and isoelectric focusing (B41-B44).Some fish proteins are relatively stable and the could be extracted for analysis. The fish species of seafoodlsuch as imitation crab and surimi could be identified by peptide ma ping of the myosin heavy chain (MHC) after separation by [DS-PAGE. The MHC was hydrolyzed with enzyme and separated by a second SDS-PAGE. The resulting peptide fragments were species-specific (B45). In addition to fish species identification, shrimp species can be identified by electrophoretic methods, including SDSPAGE (B46), urea gel IEF (B47), and electroimmunochemical methods (B48). Wheat. Bietz and Simpson (B49)gave a very comprehensive review of the use of electro horesis and chromato raphy for wheat protein analysis. 8DS-PAGE was suitabfe for analyzing wheat glutenins and was used to predict baking quality. Wheat proteins also have been separated by a highresolution 2D electrophoresis method (B50)using a capillary tube for nonequilibrium pH gradient electro horesis, followed by SDS-PAGE. The characteristics of suLur-rich proteins of durum and bread wheat were studied by electrophoresis. The sulfur-rich proteins bound to other gluten components during dough formation, probably through hydrophobic interactions. The sulfur-rich proteins may have important functional role in the formation of dough. Meat and Soy Proteins. Discrimination of beef from pork proteins in a raw meat mixture can be accomplished using isoelectric focusing (B51) and countercurrent immunoelectro horesis (B52).Countercurrent immunoelectrophoresis h a f a high sensitivity and could detect 0.4% pork in beef. Isoelectric focusing could detect 5% ork in beef. Use of SDS-PAGE to detect adulterationsof ifferent meat sources was effective a t the 10% level without risk (B53).A t the 5 7% level, care must be exercised in interpreting the data. Isoelectric focusing could be used to detect adulteration of raw and cooked meat in a binary mixture at 1-5 % according to the results of Matsuoka et al. (B54).A method based on PAGE, electroblotting on nitrocellulose, and immunoblotting was able to detect 25 ng of soybean protein in milk replacers. Proteins from other plant products also were detected (B55). Immunochemical Assays. A large volume of research papers and reviews (B56, B57) on applying immunochemical assays to qualitative and quantitative analysis of a wide range
FOOD
of food components, including proteins, has been published since 1988. Generally the reliability and sensitivity (at nanogram or picomole levels of antigen sensitivity) of the immunochemicalmethods are superior to traditional chemical methods. Because of the versatility of the immunochemical methods, many food applications are being developed. Generally, the princi le of the immunochemical assays is based on the noncov3ent interactions between antigen and antibody. Both antigen and antibody can be labeled with radioisotopes, enzymes, and other molecules to facilitate detection and quantification of the components of interest. Two of the most widely used methods are radioimmunoassays and enzyme-linked immunoassays. The enzymelinked assay has advantages in terms of the low cost of the required equipment and the freedom of risk associated with exposure to the radioisotopes. The enzyme-linked immunosorbent assay (ELISA) has become a popular method for detecting and analyzing several food protein components. For meat products derived from several animal sources it is difficult to detect and quantify the sources of meat using traditional means. Adulteration has been a concern in the food industry. Utilization of the meat extract, which contains sarcoplasmic proteins, to induce antibodies to develop an ELISA system for detecting meat sources has been reported. Myofibrillar proteins from various animal species have man similarities and are poor antigens for establishing a system for identification of a meat source. Commercialquality control diagnostic kits have been produced for identifying meat and non-meat proteins (B58). The presence of chicken meat in pork and beef meat mixtures was detected by monoclonal antibody sandwich ELISA. The antibody was raised using soluble chicken proteins (B59). An ELISA assay using purified lactate dehydro enase to raise antibodies for the assay has been developed y Wang et al. (B60)to determine the extent of cooking of the turkey rolls. Poultry products have been required to be cooked to an internal temperature of 71.1 "C. Currently, the USDA does not have a standard assay for verifying the processing temperature in poultr The lactate dehydrogenase content determined by the EEISA decreased as the internal temperature increased. The method could be used to indicate the adequacy of the heat treatment for cooking turkey rolls. The re lacement of expensive goat milk with cheaper cow's milk in t\e production of goat cheese products has been a problem. Immunoassay could be used to differentiate milk proteins, includingladoglobulin, lactalbumin and casein,from different species. Purified milk proteins have been used to induce antibodies for the precipitation immunoassays. Soy proteins have been used widely as a food additive in meat products. ELISA procedures have been proved to be the best for detecting soy proteins in the meat products. An AOAC method (method 988.10) using ELISA is available for detecting soy proteins. Medina (B61) developed an ELISA procedure to quantitate 23 commercial sausage samples using food-grade soy isolate as a standard. The results showed that experimental values were 94% in a reement with all levels of added soy isolate up to 5 % his method was suggested to be suitable for monitoring adherence to legal restrictions for the amount of protein additions to the meat products. Another ELISA method was developed by Yasumot0 et al. (B62)to quantitate soy proteins in pork sausages containing 0-20.8% soy protein isoIate. A peptide fragment, which was isolated from an enzyme-hydrolyzed peptide mixture of the soy protein, reacting to the antibody raised against 11s rotein was used as an indicator antigen. The method has Ettle interference from other food components. Mifek and Blawischnig (B63) showed an ELISA procedure was suitable to determine heated sausages containing soy proteins with a detection limit of 0.7% soy protein, while electroimmunodiffusion, radial double-diffusion, or histological examination could not detect soy proteins in cooked sausages. Brett et al. (B64)studied legume protein structure and functional properties using monoclonal antibodies raised against 11s stora e proteins of peas and soybean and found that some antiboiies were specific to the target protein and others could bind 11s proteins from other legume species. Some antibodies recognized surface epitopes (antigenic determinants), while others bound to the peptide chains buried within the protein. Soybean trypsin inhibitor in
ELISAY
g,
.5
processed foods and in heated rotein-carbohydrate mixtures can be analyzed by an EL1SA)procedure (B65).The use of ELISA procedures has two advantages com ared to the traditional enzymatic methods because it could3measurelow levels of the inhibitors in processed foods and it could differentiate between the two types of soy bean trypsin inhibitors, i.e., the Kunitz and Bowman-Birk inhibitors. A monoclonal antibody was raised against wheat gliadin and labeled with colloidal gold for identification of glutens with electron microscopy. Baking did not destroy the epitopes reco nized by the monoclonal antibody (B66).Monoclonal antigodies raised to gluten proteins from a number of related cereal species showed cross-reactions. Amino acid sequence homologieswere found to exist among proteins in cereal grains (B67). Antibody specificity or cross-reactivity is affected by the variations of sample extractant, solid absorbent phase, and assay format (B68). An inhibition ELISA was successfully developed using a purified polyclonal prolamin antibody. The antibody was specificto wheat prolamins and did not react with corn, millet, rice, or soybean prolamins. The assay was able to detect 1 ng of antigen. A monoclonal antibody raised against protein M in rock shrimp was used to develop an ELISA procedure for detecting the presence of rock shrimp in 26 seafood and meat mixture samples. The sensitivity of the ELISA was 4.3 ng and the monoclonal antibody correctly identified rock shrimp in all samples (B69). ELISA was successfully used to identify the addition of egg white in a sausage product (B70).Using a rapid amperometric electrochemical immunoassay s ecific for protein A-bearing Staphylococcus aureus, protein Lcould be detected as low as 10 pg/mL (B71). ELISA systems for detecting and quantifying other proteins suchas egg albumin, total wheat proteins (B72), gliadin (B73), amylases, proteinases, lipases, amyloglucosidase, bacterial enterotoxins, and corn zein in food products also have been summarized in the review article of Gama et al. (B57).
CARBOHYDRATES Mono- and Disaccharides. The HPLC methods for the analysis of low molecular weight su ars and olyhydric alcohols in foods were recently reviewefi by Ball (&I. In the period of 1988-1992, a large number of biosensor-based methods for mono- and disaccharides were reported. The amount and type of immobilized enzymes in the electrodes or reactors varied widely, so did the linear range, sensitivity, selectivity, and response time. Most of these methods showed a good correlation with the official AOAC method. Collaborative work of 20 laboratories studied the validation of liquid chromatography and refractive index detection for mono- and disaccharide analyses in foods. Among the six sources of variation identified, the method used for the calculation of concentration was reported to have the greatest impact on the results. Consideration of selection between peak area and height was recommended (C2).An HPLC pattern of sugars was reported successful in the classification of authentic apple juice by variety and origin. Detection of adulterants included the L-malicacid test and isotopic carbon with detection levels of 20% by volume (C3). An automatic su ar analysis of beet molasseswas developed using a reversed-piase HPLC with a Zorbax ODS column, water elution, and light-scattering detection (C4). The presence of undeclared material in coffee was found by identifying mannitol (0.3% ) in soluble coffee using a Cle SepPak cartridge, HPLC with a CHO-682 column using water as mobile phase, and derivatization detection (C5). The detection of honey adulteration with starch hydrolyzates was reported using a combination of medium-pressure liquid chromatogra hy (charcoal4elite column) to remove monosaccharides ant! HPLC to detect higher oligosaccharides. Reversed-phase HPLC (Ultrasphere ODS column) with refractometry detection was used (C6). Lactose analysis in homogenized and pasteurized milk using HPLC (Bio-Rad HPX-87P ion exchange column) and a refractive index detector was comparable to the official AOAC method (C7). A pretreatment to inactivate invertase from persimmon fruit prior to sugar analysis by GC was reported by Matsui and Kitagawa (C8). Steps included heat retreatment, homogenization with MeOH at 4 "C, neutraEzaANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
3371
FOOD
tion, and centrifugation (C8). Acceptable recoveries (>91%) of ma'or sugars, sorbitol, glycerol, and ethanol were obtained by HJLC with a cation exchange resin column in a Ca form, an aqueous solution with Ca EDTA as eluent, and a refractive index detector (C9). Two HPLC procedures with pulsed am erometric detection for the analysis of neutral sugars a n f hexosamines were described by Quigley and Englyst (C10). The authors reported a 36-min run for the neutral sugars and a 40-min run for the amino sugars and all neutral sugars, except rhamnose and arabinose, which coeluted in this procedure ((210). Simultaneous determination of dextrose, sucrose, maltose, and lactose in cooked and fresh sausage samples with recoveries of >80% and relative standard deviation of < l o % was obtained with a normal-phase HPLC amino column and a differential refractometer detector (C11). A report of an automated enzymic determination of starch by unsegmented flow injection analysis permitted the analysis of 32 samples/ h. The continuous method started with the injection of hydrolyzates using thermostable bacterial a-amylase followed by the traditional manual method reagent sequence of glucose oxidase, peroxidase, and chromogen detection (C12). The determination of fructose was reported b using immobilized D-fructose dehydrogenase on an electrodYe membrane and hexacyanoferrate(II1) as redox mediator, recording amperometric change in the presence of fructose. The authors reported a linear response in the range of 1.0 X 10-5-1.0 X 10-3 M fructose, a correlation coefficient of 0.999, and stability of the electrode for several weeks (C13). Detection of sucrose and fructose was reported using immobilized sorbitol dehydrogenase on controlled-pore glass and measurin the disappearance of NADH spectrofluorometrically. keported detection limits were 10pM, with a linear range of 50-500 pM for sucroseand 5pM with a 25-250 pM linear range for fructose
(C14).
Determination of saccharides, organic acids, and amino acids combining HPLC, postcolumn enzyme reactor, and electrochemical detection was reported (C15). The method showed detection limits in the nano- to picomolar range and a promising wide range of applications (C15). A rapid (1-3 s) and reproducible (0.448% relative standard deviation) glucose determination in food stuffs was based on an immobilized glucose oxidase cross-linked to bovine serum albumin and gelatin. A linear relationship was observed in the range of 3.6-62.9 mg/dL @-D-glucosein the presence of fructose (C16). A flow injection determination of D-mannitol using immobilized mannitol dehydrogenase on poly(viny1 alcohol) beads coupled with fluorometric detection of NADH was reported (CI7). Thirty samples per hour, a linear response between 5 X le7and 1 X lo-' M mannitol, and a detection limit of 1X le7 M was reported with celery and chewing gum samples (C17).A glucose-sensingenzyme electrode has been reported using immobilized invertase for the simultaneous determination of glucose and sucrose (C18). The potential use in the field of a H202 electrochemical sensor coupled with the enzymes @-galactosidase and glucose oxidase in the analysis of lactose and glucose in milk was reported (C19). An amperometric analysis of lactose in human and cow's milk using a flow injection system with immobilized @-palactosidase, glucose oxidase reactors, and a precolumn packed with glucose oxidase and catalase was reported. The method showed a linear correlation between lactose concentration (0.01-2.0 mM) and peak current (C20). A method for determining the lactose in milk using flow injection analysis was based on the carmine color of lactose with methylamine. Up to 40 samples/h were analyzed with excellent correlations when compared to liquid chromatogra hy ((221). A glucose method used direct reduction of green 8u(II)-2-2-bicinchoninate complex to a violet complex. The method had a linear range of 20-800 pM glucose and was tested in fermentations using Saccharomyces cereuisiae and colored corn-steep media (C22). A solid-phase extraction using a strong anion exchanger was used to analyzethe major carboxylicacids,su ars,glycerol, and ethanol in wine and grape must. Mafic acid was determined, eliminating the interference from sugars (C23). The determination of lactose, glucose, and galactose in cheddar cheese using HPLC, an Aminex HPX-87 column (H+form) with UV, and refractive index detectors in tandem offered a simple technique to monitor cheese manufacture 338R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
practices (C24). Reducing sugar analysis was reported (C25) applicable to soft drinks, wines, spirituous liquors, and other colored and turbid samples without retreatment. The method consisted of the reduction of eu(11) to CUZOand detection of the unreduced Cu in the supernatant or reduced Cu in the Cu20 by potentiometric strippin analysis (C25). A new method for spectrophotometric anafysis of sugars in foods used 3,4-dimethylphenol and sulfuric acid to yield a red chromogen that follows Beer's law in the range of 1-10 pg/mL of sugar solution ((226). The reaction of aniline and its alkyl derivatives in acetic acid with hexoses and pentoses was reported to yield colored products with absorption at 530,470, and 330 nm. The method measured quantitatively glucose, fructose, maltose, and sucrose (C27). An enthalpimetric method of reducing sugars without separation of the various matrix material, fruit peel, preservatives, etc., showed potential for automation (C28). The method oxidized sugars with copper(II1) ions in an alkaline medium and had a glucose determination range of 12.5-50 mg/L with average standard error of 1.5% (C28). Thermogravimetric analysis was used for sucrose determination in cariogenic diets. A linear correlation of mass loss in the range of 180-240 "C and sucrose content was reported (C29). Two enzymic methods adapted for glucose analyses from liquid expressed from potato tubers were developed. One method used only 5 pL of sample (potato liquid) in multiwells, and the second a blood glucose strip, to estimate lucose within 3 min. Glucose concentrations were correlatei to potato crisp darkening (C30). A rapid uantitative analysis of sugar mixtures suitable for the soft \rink and brewing industries was suggested by using the Fourier transform IR spectroscopy (FT-IR) in the mid-IR (4000-400 cm-l) (C31). The calibration of mixtures of sucrose, glucose, and fructose using matrix methods was applied to real and synthetic samples, and the results were compared to the traditional analyses (C31). FT-IR in the midregion was also applied to sugar and organic acid analyses in fruitjuices (C32). Dupuy et al. (C32).reported that sampling appeared more difficult in mid-IR than in near-IR spectroscopy. Few reports of sugar analysis in intact fruits and vegetables were found in the reviewed period. Linear correlation of sugars with peak intensity was reported using high-resolution proton magnetic resonance (C33). Fructose, glucose, and sucrose resonances were studied in intact fruit tissues using low-speed magic-angle-spinning carbon-13 NMR (C34). Oligosaccharides. A carbohydrate analysis review of anion exchange and ligand exchange separation modes of ion chromatography with different detectors was reported (C35). Several oligosaccharidespresent in beet medium invert sugar and absent or nonexistent in orange juice Sam les were identified using anion exchange chromatography (8arbo Pac PA1 columns) with pulsed amperometric detector. A detection level of 5% beet invert sugar added to orange juice was possible (C36). A modification of this method allowin analysis of a large number of samples was latter reporte! (C37). The columns used were an ATC-1 anion trap, a CarboPac PA1 guard column, and two Carbo-Pac PA 1analytical columns. The estimated detection limit was 5% beet medium invert sugar with a 0.1 9% coefficient of variation (C37). The oligosaccharide pattern in honey was analyzed with two Dionex Carbo Pac PA1 anion exchange columns in series with a pulsed amperometric detector. It was suggested that this technique provides information to determine honey source authenticity (C38). Hydrolysis with 2 M trifluoracetic acid in a pressure cooker (1h at 105 kPa) improved the analysis of galactomannan gum in guar using HPLC analysis (C39). In the analysis of polydextrose (polymer), a simple liquid chromatography method usin a cation exchange column, 0.001 N sulfuric acid mobile pfase, and a differential refractive index detector was reported. This method showed romise for adaptation in routine quality control analysis ( B O ) . The polysaccharidecompositionin fermented milk, isolated by sonication, centrifugation, and filtration,was analyzed by liquid chromatography using an AX 300 anion exchange column coupled to a Biosil TSK 300 gel permeation column (C41). The description by Lecacheux and Brigand (C42) of a preparative fractionation of olysaccharides using size exclusion chromatography may {e a plicable to industrial polysaccharides whose molecular weigEt fraction is important
-
FOOD
in their application. In a comparison stud on the analysis of oligosaccharides using GC versus HPL8 in protein-rich feedstuffs, HPLC gave better precision overall (C43). A capillary fused-silica cross-linked with methyl silicone, programmin temperature, and flame ionization detector used in the G 8 gave a relative standard deviation of 1.5-5.0%. HPLC gave a relative standard error of 0.6-1.3% using a Sep-Pack Cle and Aminex HPX-87N (Na form) columns with refractive index detector (C43). Carlsson et al. (C44)reported a modified extraction and derivatization for oligosaccharides (with two to seven sugar units) analyzed b high-temperature capillary GC and GC/MS with reproducigle values obtained with rapeseed meal, mung beans, soybeans, and chickpeas among others. The use of high-resolutiongas chromatography (HRGC) for the determination of raffinose, stachyose, and verbascose in pea flour extracts was reported (C45). Starch. The reader is referred to a recent review of modern qualitative analysis of starches (C46) and their hydrolysis products (C47). A new approach to starch release from whole cassava chips used a combination of pectinase I and cellulase enzymes (C48). A yield of 596% of starchand the elimination of mechanical pulverization made this method competitive with the traditional starch extraction (0%). Starch and its degradation products were studied by using an HPLC with Hypersil-APS column and mass (light scattering) detector. The analysis compared favorably with the traditional titrimetric method and has potential for an automated process (C49). During molecular weight distribution studies of starch using gel permeation chromatography, care is advised on the selection of the eluent (C50). The reproducibility of results, column stability, and starch modification are some of the factors to take into account (C50). Hydrolysis with 1 M HC1 at 106 "C for 40 min of starch residue from ethanol extraction prior to automatic analysis gave better results (102% recovery and 0.7% standard deviation) than the enzymic hydrolysis (C51). A pretreatment of sugar extraction with 80% hot ethanol in the starch determination of banana fruits using the direct assay with anthrone-sulfuric acid showed to be faster and more efficient than the acid hydrolysis and reducing sugar determination (C52). The use of reversed-phase (RP) HPLC to analyze damaged starch from wheat gave an alternative to the traditional colorimetric manual determination. The use of RP HPLC requires a larger initial investment and a trained operator but has a larger sample analysis capacity (C53). High correlations were obtained between the amount of damaged starch analyzed by enzymic and near-IR reflectance spectroscopy. Both hard and soft wheats were tested, and it was possible to predict the amount of damaged starch produced in other mill types (C54). A proposed method for starch analysis in biological samples that contain glucose, maltose, and higher oligosaccharides used NaBHd followed by evaporation with 2,2-dimethoxypropane to remove the mentioned sugars and oligosaccharides (C55). The method was suggested to be useful in the study of starch mobilization in plants, i.e., the endosperm of germinating cereal grains (C55). Pectins. An extensive review of recent analytical methods for pectic substances was reported by Walter (C56). An enzymic HPLC method (industrial enzyme Driselase from Irpex lacteus, Shodex SUGAR SH1821column) was proposed for routine pectin analysis of galacturonate and neutral sugars. The method was reported simple and accurate (C57). Similar results on the identification and quantitation of galacturonate in pectic substances were obtained with an anion exchange HPLC method coupled with pulsed amperometric detection and a standard colorimetric procedure (C58). Three colorimetric methods were compared in their precision and accuracy in determining ectin content in fruit jams. The method using m-hydroxytiphenyl showed good precision (coefficientof variation 0.9-6.4 5% ) and nearly 100% recovery (C59). More accurate results of the avera e charge density of pectin chains were obtained when the Pb(f1) bound to pectin in dilute aqueous solutions was quantitated (C60). A pectin aggregation number was interpreted by the ratio of weight average measured by light scattering and the average molecular weight determined by the copper-arsenomolybdate method (C61). The aggregation number was proposed to give a quantitation of pectin weight-average mass under the
normally used conditions (C61). Celluloseacetate membranes were used to analyze electrophoretically pectin and galacturonic acid (C62). Dietary Fiber. The nature and determination of nonstarch polysaccharides (dietary fiber) was reviewed in 1990 (C63). An improved method for the measurement of total and insolubledietary fiber as non-starch polysaccharide (NSP) was described by Englyst et al. (C64). Starch was removed enzymicallywithin 50 min and NSP precipitated with ethanol followed by sulfuric acid h drolysis for 2 h. The hydrolyzate was then analyzed with &, HPLC, or colorimetry (C64). Comparison of several dietary fiber methods for non-starch polysaccharide amounts and composition determined in four food products was done by Wolters et al. (C65). The inaccuracy of acid-detergent and neutral-detergent fiber methods was reported for cellulose, hemicellulose, and lignin determination. The neutral-detergent fiber and Hellendoorn methods were less appropriate for insoluble dietary fiber determination. Discrepancies were also reported between the quantities and non-starch polysaccharide composition, determined by the AOAC and the Englyst methods. However, Wolters et al. preferred the Englyst method due to the information gained on the composition of non-starch polysaccharide (C65). A distinctive high-resolution scanning absorption spectrum (230-350 nm) was obtained for several insoluble fibers used in baked goods and dietary products. The specific spectrum obtained for each insoluble fiber source is proposed to distinguish them (C66). Near-IR reflectance spectroscopy used for the analysis of pectin, arabinose, xylose, and glucose from forage cell walls of different botanical sources gave moderately high precision, but it was less recise than other reports with single forage species (C67). $he use of near-IR reflectance scanning spectrophotometer on the analyses of soluble, insoluble, and total dietary fiber and starch in oat bran has been reported to yield accuracy and recision of the predicted values. The method was shown to adequate for quality assurance (C68). The arabinoxylans from wheat dietary fiber were extracted in large scale with aqueous and alkaline solutions followed by acid hydrolysis, methylation analysis, and 1 and 2D NMR experiments. The structure of arabinoxylans was confirmed as fl-(1-4)-xylan substituted with a-L-arabinose (C69). The identification at the nanomol level of cell wall sugars of cherries using TLC silica gel analyses and spectrophotometric quantitation was reported; a single run separated the neutral and acidic sugars (C70).
8,
INORGANICS (MINERALS) Introduction. Many techniques continued to be used for analyzing various minerals and heavy metals in foods. However, the most frequently used techniques include flame atomic absorption, electrothermal atomic absorption, inductively coupled plasma emission, and neutron activation analysis. The use of microwave and far-IR apparatus for the digestion of food materials has been re orted by several researchers. Flow injection systems, w ich are used to substitute the traditional dry ashing and aspiration flame atomic absorption, are frequently reported. The sensitivity of electrothermal analysis and the simultaneous multielement analysis capacity of the inductively coupled plasma atomic atomic emission and mass spectroscopy have made these techniques suitable for a wide variety of food samples in many analytical laboratories. Ion chromato aphy for determining inorganic anions, including sulfites, sufates, nitrites, nitrates, and other anions in foods has been reviewed by Fernandez Pereira @I). Compared to the traditional methods, ion chromatography can determine several ions simultaneously. In addition, ion chromatographic methods are rapid, sensitive, and precise. Sample Preparation for Atomic Absorption. The applications of microwave for digesting foods and other materials for elemental analysis have been compiled by Kuss (02). Digestion of fish samples for the analysis of mercury using microwave greatly improved the speed (a proximately 10 min versus hours for wet digestion on a hot pyate) and had no loss of Hg (03). A microwave digestion was used to di est liver paste samples in 1min at a power level of 700 W. !"he subsequent determinations of Cu, Fe, and Mg were accurate
R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
339R
FOOD
compared to a wet ashing method (04). A microwave digestion method and wet ashing were found to produce comparable results for the determination of heavy metals, Pb, Cd, Cr, Cu, and Zn in shellfish. Recoveries for the microwave method and the wet ashing method were much higher (80-92 % ) than those obtained by a dry ashing method (54-7296). Four ashing methods, including dry ashing using a crucible in a gas burner, wet ashing in a beaker on a hot plate, wet ashing using a far-IR apparatus, and wet ashing using a microwave, were com ared for preparing samples for the determination of metahc elements using atomic absorption. The IR method gave the best results ( 0 5 ) . An automated digestionsystem and an atomic absorption spectrophotometer with an autosampler were used to determine a number of elements. The same tube used for digestion was used for dilution and analysis, thereby reducing sample handling time
Table I. Electrothermal Atomic Absorption Spectrometric Analysis of Elements in Foods
(06).
Landi et al. (07)described a simple and rapid method for determining total Hg in vegetable samples by cold vapor atomic absorption spectrometry. The samples were oxidized by otassium dichromate in diluted sulfuric acid. This met\od gave better accuracy than the AOAC procedure for Hg using nitric and sulfuric acid and another method using nitric and perchloric acid. The method also gave good precision and full recovery of organomercury. Methylmercury in fish was extracted and cleaned using column chromatography and determined using flameless atomic absorption. The method gave a reproducibility (relative standard deviation) of 10.5% at 1ppm Hg and 18.2% at approximately 0.1 ppm Hg. The accuracy was higher than 94% (D8). Flow Injection System for Atomic Absorption. Total calcium and free calcium in milk were determined simultaneously by atomic absorption using a flow injection system (D9).A dialyzer eliminated the interference in the determination of free Ca. The effect of phosphate on calcium determination was reduced by using a dinitrogen oxideacetylene flame. The relative standard deviation was less than 1.3% for 1300-1500 mg/L (ppm) total Ca and less than 0.85 % for 120-170 mg/L free Ca. The system could determine 60 sampledh. Flow injection atomic absorption spectrometry was compared with the conventional aspiration atomic absorption spectrophotometric, colorimetric, and ring-colorimetric procedures for the determination of iron and copper in powdered infant formula. The results showed that the relative standard deviations of the flow injection method for both iron and copper were ap roximately 5 % ,which was consistent with other methods (L10). Reverse single line flow injection flame atomic absorption spectrometry for determining Cu and Fe in oil samples had relative standard deviations of 3 % for Cu and 2 % for Fe (011). Flow injection flame atomic absorption spectrometry was used for determining Cu and Fe in cocoa powder (012). A resistively heated oven for digestion was found to produce a clean solution, increased sample throughput, and reduced contamination and reagent consumption. A flow injection device coupled with a slurry nebulizer with the flame atomic absorption spectrophotometer was found to be able to inject 120 samples/h. The system was used to determine Fe and Zn in legumes, grains, and vegetables. The relative standard deviations were 2-3 % ,and detection limits were 0.3 ppm for Zn and 0.6 ppm for Fe (013). Electrothermal Atomic Absorption. Due to the low detection limits of the electrothermal or graphite furnace atomic absorption methods, many trace elements, particularly the heavy metals, in foods can be determined. Electrothermal atomic absorption has been applied for the determination of Cd, Co, and Pb in foods (014).Barbera and Farre (015) compared electrothermal atomic absor tion with flame atomic absorption spectrophotometry for the fetermination of cobalt. Electrothermal atomic absorption was more accurate than flame atomic absorption. However, flame atomic absorption was more precise than the electrothermal absorption. The detection limit was 1.8 ppb by the electrothermal method and 2.27 ppb by the flame method. The phosphorus of milk could be analyzed precisely and accurately by electrothermal atomic absorption spectrophotometry with a stabilized temperature plateform furnace and Zeeman background correction (016). Zinc in human milk (017) was determined by electrothermal atomic absorption. 340R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
element
food
P
oils and fats
Cd Cu, Fe, Ni Cu, Fe, Ni, Mn, Co, Cr, Cd, Sn, Pb Cu, Fe
shrimp oils and fats oils oils
Fe
oils
F
Cr, Pb, Cd
infant formula oils and fats plant foods milk and cheese
Zn
human milk
Al, Cu, Mn, Mo Cr, Mn, Fe, Ni, Cu, Cd, Pb
infant formulas pasteurized milk
Zn, Cu, Mo, Pb, Cs, Cr, Cd, Se Pb, Cd, Co, Ni Cu,. Fe,. Ni
milk powder
ref
Hendrikse and Dieffenbacher (021) Blust et al. (022) Hendrikse et al. (023) Calapaj et al. (024) Sun (025) Chen et al. (026) Wagley et al. (027) Debeka (028)
Capar (029) Gomoz et al. (030) Larsen and Rasmussen (031) Arnaud et al. (032), Arnaud and Favier (033)
Dabeka et al. (034) Favretto et al. (035)
The precision was 4.6 74 and accuracy was 96 % . Normal Zn value for colostrum and milk varied from 45 to 318 and 30 to 146 pmol/L, respectively. Selenium in foods was determined by the electrothermal method (018).The interference due to phosphate and Fe could be reduced by a modifier mixture of Pt(1V) and magnesium nitrate. A rapid and sensitive electrothermal atomic absorption spectrometric method, using a combination of microwave digestion and palladium as a stabilizer, was used for determining Cd and Pb at the sub-p m levels (019)in vegetables and protein foodstuffs. Pallachm was found to be a more useful stabilizer than ruthenium for electrothermal atomic absorption analysis of elements in foods. Palladium nitrate-magnesium nitrate was used as a matrix modifier for determining selenium in yeast using electrothermal atomic absorption spectrometry. The coefficient of variation for between-day random error was 4.2%. The analysis of eight commercial samples showed the results were consistent with declared values (020). Nickel nitrate was found to be a better modifier than a mixture of Ni + Cd + Cu or Cd and Cu alone for reliable determination of Se in a variety of foods. Additional applications of electrothermal or graphite furnace atomic absorption for element analysis are summarized in Table I. Inductively Coupled Plasma Atomic Analysis. Three types of inductively coupled plasma atomic analysis include the detection systems with atomic emission spectrometry, mass spectrometry, and atomic fluorescence spectrometry. The most frequently reported techniques are inductively coupled plasma atomic emission spectrometry (ICP-AES)and inductively coupled plasma mass s ectrometry (ICP-MS). The advanta es of the use of ICP-AEE or ICP-MS for analysis are their muftielemental analysis capacity and the speed of analysis. Spectral interference could occur in ICP-AES. The accuracy of ICP-AES for analyzing Pb, Cd, Cu, Cr, Zn, and Ni in shellfish was reported to be similar to flame atomic absorption spectrometry (036). ICP-AES was used to analyze Co, V, Mo, Ni, and Cd in 234 types of foods by the Food and Drug Administration (037). ICP-AES was reported not suitable for the determination of Co and V due to high sample concentrations of Ti, which interfered with the 228.6-nm line of Co and the 292.4-nm line of V. In addition, spectral interference of Fe on the 238.9-nm line of Co was observed. A device with tungsten coil electrothermal vaporization for introducing sample into ICP was used for analyzing trace elements in rice (038).Plant samples was digested withnitric acid in closed Teflon (PTFE) tubes at 170 OC for 2 h to prepare samples for ICP-AES (039).Microwave di estion in Teflon PFA tubes has been used by Scheikoph anfMi1ne (040)for ICP-AES. A powder sample introduction device was used for analyzing elements in cereals and tea leaves by ICP-AES (041).An ultrasonic nebulization system was coupled with
FOOD
Table 11. Applications of Inductively Coupled Plasma Atomic Emission Spectrometry for the Analysis of Elements in Foods
element
foods
Cu, Zn Na, Si, Sn
citrus juice citrus juice
Ca, Fe, Mn, P
vegetables four food groups
Ca, Cu, Fe, K, Mg, Mn, Na, P, Zn Ca, K, Mg, Na, P
milk
Ca, Co, Cr, Cu, Fe, jelly Mg,Mn,Mo,Na, Ni, Sn, V, Zn Al, B, Ca, Fe, Pb, Mg, foods P, K, Na, Sr, Sn, Zn et al. (31 elements)
ref Nikdel et al. (043) Rezaaiyan and Nikdel(044) Ikebe et al. (045) Sims et al. (046) Takahasi and Sutoh (047) Ou and Lin (048) White and Thomas (049) Krushevska et al.
Zn
milk
Cr Ba, Cr, Co, Cu, Sr, Ni, Pb, Cd, Mn, Mo, Zn
spinach, beef kidney Baucells et al. (051) vinegar Zhang et al. (052)
Table IV. Applications of Neutron Activation Methods for the Analysis of Elements in Foods and Reference Samples ref element food
Na, K, Br, As, Rb, Zn, Co, Fe, Sc Na, Mg, P, C1, K, I, Ca. Zn. Se. Br. Rb Zn, F;, cb, ~ dMo, , W, Th Se
Maihara and Vasconcellos (068) milk, milk powder Al-Jobori et al. (069) rice
certified references Woittiez (070)
wheat, rye, cocoa butter Br, Ca, C1, Cu, Fe, I, corn K, Mg, Mn, Na, Rb, S, V, Zn 15elements infant milk foods 30 elements brown rice 19 elements rice, wheat, barley 16 elements vegetables 18 elements spices
Langenauer and Kraehenbuehl(071) Armelin et al. (072) Durrant et al. (073) Suzuki et al. (074) Al-Jobori et al. (075) Al-Jobori et al. (076) Zaidi et al. (077)
(050)
Table 111. Applications of Inductively Coupled Plasma Mass Spectrometry for the Analysis of Elements in Foods element food ref I
Ba, Mo, Mi, Co, Cd, Cs, Ti, Pb, Bi, Th, U Na, Mg, Al, P, K, Ca, V, Cr, Mn, Fe, Co, Ni. Cu. Zn.. As,. Mo, Hg, Pb Fe, Sn, Pb Mn, Co, Ni, Cu, Zn, As, Mo, Sr
milk diet
Baumann (056) Shiraishi et al. (057)
human milk
Durrant and Ward (058)
fruit juice vegetable
Al-Swaidan (059) Jin and Cheung (060)
ICP-AES for analyzing elements ( 0 4 2 ) . Other applications of ICP-AES for analyzing elements in foods are summarized in Table 11. ICP-MS does not have problems with spectral interferences compared to ICP-AES. A flow in’ection system was used to introduce samples into the ICP-bS to eliminate problems associated with high salt content, viscosity, and high acid concentration ( 0 5 3 ) . The enhancement and su pression effects of major nutrients in foods (Na, Mg, P, K, Ea) on the response of the trace elements (Al, Cr, Zn, Mo, Cd, Pb) in ICP-MS were found to be less than 10% ( 0 5 4 ) . In a comparison of neutron activation to ICP-MS for the analysis of 50 different foods and diet for trace elements, (D55), ICP-MS had superior sensitivity for a wider range of trace elements. However, neutron activation analysis does not have the blank problems for low concentrations of trace elements in solid samples. In many applications, ICP-MS was more convenient and more rapid than the neutron activation analysis. The a plications of ICP-MS for the analysis of elements in f o o i are summarized in Table 111. Neutron Activation Analysis. Elements can be activated by nuclear bombardment with high-energy neutrons to generate radioactivity. Generally, the radioactive element to be determined is separated chemically from other induced radioactivities of other elements before measuring. In certain cases, trace elements in food samples can be determined without chemical separation of elements. The neutron activation analysis is sensitive (detection limits of 0.001-1 ppm) and has been used widely for determining approximately 20-30 elements in food and agricultural products (061-063). Na, C1, Mn, Br, Fe, Zn, Rb, Sb, Ca, Al, and Mg in bread and milk powder were determined by instrumental neutron analysis (064). The amount of the phosphorus-based additives that migrated from food packaging materials into foodsimulating solvent was determined by neutron activation and
that Cherenkov counting method (065). The method could detect as low as 1ng/mL and did not require elaborate sampleprocessing rocedures. Iodine values of oils and fats can be determinecfby saturation with bromine followed by neutron activation analysis of the reacted bromine (066).Compared to an official method (Huebl method) for determining the iodine value, neutron activation analysis gave similar results for several vegetable oils, including almond oil, sunflower oil, peanut oil, soybean oil, sesame oil, sunflower oil, eanut oil, soybean oil, sesame oil, corn oil, and olive oil. TEe neutron activation method is faster than the official method, and the reacted bromine can be determined within 150 days without a decrease in iodine value. Al, Ca, C1, Mn, Na, and Sr in chewinggums were determined using an instrumental neutron activation method (067). Compared to graphite furnace atomic absorption, the neutron activation method did not require sample treatment and took only a few minutes for each sample. The concentrations of these elements contributed only a very small fraction of the total adult dietary intake. Other applications of neutron activation methods for analysis of elements in foods are summarized in Table IV.
ENZYMES Enzyme Biosenors (Electrodes). Although the first paper on an enzyme electrode was published in 1967 ( E l ) , continued interest over the past two years in enzyme biosenors was indicated by the number of papers published. Chaplin and Buche (E2) have published a recent review on enzyme biosenors that is not an overly technical one. Chaplin and Buche’s (E21 definition of a biosensor is “an analytical device which converts a biological response into an electrical signal”. The continued interest, evidently, is due to the potential of an enzyme biosensor being very useful as a rapid, simple, and relatively low cost method for the determination of the concentration of an enzyme substrate in an often complex food mixture. For determining lactose and glucose in milk, dairy products, and foodstuffs and for postcolumn detection of these sugars after separation by HPLC, several pa ers reported using sensors with coimmobilized glucose oxifase and 0-galactosidase and monitoring hydrogen peroxide electrochemically (E3, E4). A number of researchers determined glucose in different foods or in postcolumn HPLC detection with their developed biosenors (E3, E5-E8). The biosensors used immobilized glucose oxidase with measurement of hydrogen peroxide amperometrically or dissolved oxy en by an oxygen electrode. A sucrose electrode used coimmogbilized invertase and glucose oxidase (E6)while another one used coimmobilized invertase and permeabilized cells containing glucose oxidase and gluconolactonasewith detection by a pH electrode (E9).For sucrose, Barlikova et al. (EIO)used a biosensor of coimmobilized yeast cell walls as a source of invertase and glucose oxidase with phosphate ions for mutarotation of aand @-glucose. A biosensor for D-glucono-&lactone in the presence of glucose first eliminated interfering glucose by an outer antiinterference layer containing hexokinase ( E l l ) . ImmoANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
841R
FOOD
bilized glucose dehydrogenase converted the gluconolactone to glucose, and the lucose was oxidized by an immobilized glucose oxidase wit oxygen consumption measured by an electrode. Enzyme biosensors were developed for postcolumn determination of dextrins from beer and wort (E12) and maltooligoaccharides from corn syrups (E13). The dextrin sensor used coimmobilized glucoamylase, mutarotase, and glucose dehydrogenase and the maltooligosaccharide sensor used an immobilized glucoamylase. Enzyme biosensors were developed for determinin amino acids. L-Glutamic acid and L-lysine biosensors use immobilized L-glutamic acid and L-lysine oxidases, respectively, with oxygen or hydrogen peroxide electrodes (E3, E8, E14, E15) or 1,l-dimethylferrocene as an electron mediator (E16). Other sensors for amino acids used NADH electrochemically generated by coimmobilized diaphorase and L-amino acid dehydrogenases with 2-ferrocenylethanol and vitamin K3 as a mediator (E17). Researchers also developed enzyme biosensors for cholesterol, henols, short-chain fatty acids, and free fatty acids. The cRolestero1 biosensor for butter and margarine used immobilized cholesterol oxidase, and the cholesterol was determined in a chloroform-hexane (1:lv/v) solution (E18). Wang et al. (E19)determined phenols in achloroform solution of olive oil with tyrosinase immobilizedon an electrode. Ukeda et al. (E20)measured total saturated even-numbered C&2 fatty acids in milk with a biosensor based on the immobilized Arthrobacter nicotiana and a oxygen electrode. A free fatty acid biosensor used coimmobilized acyl coenzyme A synthetase and acyl coenzymeA oxidasewith an oxygen electrode, but this electrode was stable for only 3 days (E21). Sulfite oxidase was immobilized for the determination of sulfite in foods by biosensors and the hydrogen peroxide produced was determined by an electrode (E22,E23). With a microsensor consisting of an oxygen electrode and an S-oxidizingautotrophic bacterium (Thiobacillus thiooxidants JCM7814), Kurosawa et al. (E24) measured free sulfite in wine and total sulfite after freeing bound sulfite with alkaline or acid treatment. For freshness in fish, Okuma et al. (E%)determined inosine monophosphate, inosine, and hypoxanthine with a coimmobilized nucleoside phosphorylase-xanthine oxidase reactor or a coimmobilized 5-nucleotidase-xanthine oxidase reactor. Chemnitius et al. (E26) determined fish freshness by measuring the reaction of an immobilized putrescine oxidase reactor with putrescine and other compounds produced during fish decomposition. Flow Injection Analysis. Researchers published a number of papers on the determination of food constituents by flow injection analysis (FIA). In FIA, one or more enzymes are immobilized in the flow path(s) of the substrate; then some physical change from the enzyme reaction, such as light absorption or fluorescence, is measured in the determination of the substrate concentration. In some FIA systems the enzyme is not immobilized. Luque de Castro and FernandezRomero (E27) reviewed the outstanding configurations used in FIA. Several papers report the determination of lactose in milk by FIA using &galactosidase and glucose oxidase reactors (E28-E30). Abdul Hamid et al. (E31) investigated different combinations of lactase, glucose oxidase, mutarotase, and galactose oxidase reactors in a FIA system for determining lactose. A number of FIA systems determined glucose. Most of them used immobilized glucose oxidase with monitorin of H202(E29, E32, E33). One system used a glucose dehyfirogenase reactor (E34). Tzowara-Karayanni and Crouch (E351 determined glucose, sucrose, and maltose in foods with a system that used a combination of immobilized and soluble enzymes. Matsumoto (E32)determined sucrose with a /3-fructosidase, mutarotase, and glucose oxidase reactor system with HzO2 monitoring while fructose was determined with a fructose5-dehydrogenase reactor with monitoring of hexacyanoferrate(I1). Sucrose in the presence of glucose was also determined by coimmobilized glucose dehydrogenase, invertase, and mutarotase with fluorometic measurement of produced NADH (E36). Glucose and fructose were also determined in fruit juices, yogurt, and dessert powders using immobilized
fl
f
342R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
hexokinase and glucose-6-phosphate dehydrogenase with fluorometric measurement of NADH (E37). For the determination of L-glutamic acid, FIA systems employed a glutamate oxidase reactor with H202 monitoring (E38, E391 or used glutamate dehydrogenasewith fluorescence monitoring of NADH (E40). In comparison to the enzyme electrode, the glutamic oxidase FIA system surpassed the enzyme electrode system in sensitivit and analysis speed. Dremel et al. (E41) investigated two figer-optic L-glutamate biosensors for FIA determination of L-glutamate in foods. Cheese amino acids were determined by FIA using L-amino acid oxidase with HzOz measured by a chemilumescence reaction (E42). In determining L-lysine by FIA, an L-lysine oxidase reactor was coupled with peroxidase, and the peroxide produced in the first reaction was converted by peroxidase with phenol and 4-aminoantipyrine to a dye with light absorption at 500 nm (E43). For detecting freshness in fish by FIA, Hungerford et al. (E44)used the inhibition of the reaction of histidine with the diamine oxidase reactor by inhibitors occurrin in decayin fish. Also, for fish freshness by FIA, Yao et a f (E45) use! 5’-adenylic acid deaminase, alkaline phosphatase reactors, and a nucleoside phosphorylase-xanthine oxidase coimmobilized reactor. The system measured hypoxanthine, inosine, inosine-5’-monophosphate, and adenosine-5’-monophosphate. For meat freshness Yao et al. (E46) determined polyamines (putrescine, cadaverine, spermidine) and hypoxanthine with putrescine oxidase and xanthine oxidase reactors at fixed positions in the flow system. For measurement of neutral fat in milk, one FIA method employed coimmobilized lipase, esterase reactor, and a glycerol dehydrogenasereactor with amperometric monitoring of the NADH produced (E47). For measuring free fatty acids used a butyrate kinase reactor. in butter, Schooner et al. (E48) The determination was highly specific for butyric, propionic, and valeric acids. For determining isocitrate in orange, grapefruit, and raspberry juices by FIA, Koch and List (E49)used a isocitrate dehydrogenase reactor. Yoshiaka et al. (E50) and Ukeda et al. (E51)determined L-malate and L-lactate in wine by using a malate dehydrogenase-diaphorase reactor and a lactate oxidase reactor, respectively. Ethanol in several beverages was determined by a FIA system that used a stopped-flow step for measuring activity of the alcohol dehydro enase in solution actin on the alcohol in the flow system ($2). Xie et al. (E53) fetermined ethanol using an alcohol oxidase electrode and measurement of hydro en peroxide with a chemiluminescence reaction that use potassium hexacyanoferrate(II1) as a catalysis. Canale-Gutierrez et al. (E54) determined ammonia in foods by a FIA system using immobilized glutamate dehydrogenase and measurement of NAD by decreasing light absorption. Enzyme Immunoassays. A number of researchers published methods that used the highly s ecific and sensitive enzyme-linked immunosorbent assay (ELISA) technique or other enzyme immunoassays. These methods can determine in foods high molecular compounds (antigens) that form antibodies when injected in the body of man or animals. The antigen and the antibody have a high affinity for each other. In ELISA techniques, antigen in the sam le and the same antigen linked to an enzyme (often peroxiise) are adsorbed to the antigen’s antibody that is immobilized. The activity of the adsorbed or unadsorbed enzyme-linked antigen is used to measure the concentration of the antigen in the sample. Introductory descriptions of the ELISA are found in books by Hartmeier (E55),Chaplin and Bucke (E56),and Stewart and Whitaker (E57). Some examples of the determination of antigens in foods are covered in this review of recent literature. Researchers used ELISA extensively for the determination of specific roteins in food products. Yasumoto et al. (E58) determine$ a peptide fragment of a soybean protein from a try sin digest of autoclaved samples as a way to measure soyiean protein in meats. McNeal (E591 reported on a collaborative study on a published method for determining soy protein in meat products. The method could be made to be quantitative with information on the specific soy protein and a sample of it for calibration. In cooked meat roducts, Andrews et al. (E60)successfully detected mutton, eef, horse meat, and venison except for a
ti
E
FOOD
canned bab food meat and another canned meat product. Mifek and lawjschnig (E611 also detected beef, pork, and poultry proteins in unknown cooked meats and meat products Wang et al. (E62) investigated an ELISA method for determining lactate dehydrogenase in turkey breast rolls, and they concluded that the assay might serve as a simple and rapid method for determining the end cooking temperature. For cellulase added to animal feed to improve nutrient conversion, Burianova et al. (E63) developed an ELISA method that could be used up to a cellulase level of 24.0 pg/g of feed mixture. Makinen-Kuljunen and Palosuo (E64) developed a highly sensitive ELISA method for cow milk 8-lactoglobulin that can be used for measurements on infant feeding formulas and on human milk. Berankova et al. (E65) investigated the effect of temperature and pH on an immunochemical method for determinin milk-clotting rennet enzymes (proteinases) and concludef the method could not be used in place of measuring milk-clotting activity where denatured enzyme may be present. Clements et al. (E66) developed an inhibition ELISA method for detectinglow levels of proteinase from Pseudomonas fluorescens as a way to quantify spoilage proteases in dairy products. With a sandwich enzyme immunoassay, Rauch et al. (E67) determined Mucor miehei proteinase (Fromase) in cheese to a detection limit of 50 ng/mL. Researchers also determined compounds in food products that have the potential of being toxic. With the ELISA technique, using an antibody of w- liadin, Hill and Skerritt (E68) determined, in cereal pro ucts, meats, and soup, prolamine proteins of wheat, rye, and barley that are toxic to individuals with luten intolerance (coeliac disease). The AOAC after a cobaborative study adopted an enzyme immunoassay method for determinin gluten (E69). Ayob et al. (E70)published details on a simpfe, sensitive, and rapid ELISA for determining gliadin in a variety of food products. Brandon et al. (E71) used an ELISA to measure soybean trypsin inhibitor content in soybean protein concentrates, isolates, and flours (heated and unheated); soybean infant formula; and bean protein. With solanidine protein conjugates that were synthesized by a newly developed method, Plhak and Sporns (E72) developed enzyme immunoassays that detected and quantified the major solanidine glycoalkaloids in potatoes. Researchers published on the detection and/or quantification of antibodies, drugs, and pesticides with ELISA techniques. The antibodies for small molecules are prepared after attaching a large macromolecule to them. DixonHolland (E73)reviewed methods for determining antibiotics and drugs in products of animal origin including milk, and Jackman (E74) reviewed methods for determining antimicrobial substances in meats and milk. For tert-butylo-agonist drugs in serum and urine of animals for meat, Oriundi et al. (E75) developed and tested a simple, rapid, and sensitive solid-phase enzyme immunoassay. Skerritt et al. (E76) developed specific ELISA methods for quantitation of residues of three major organophosphate insecticides ( fenitrothion, chlorpyrifos-methyl and pirimihos-methyl) and tested them on wheat grain and milling fractions of wheat. Researchers also published on enzyme immunoassays for detection and/or quantitation of food microorganisms and for toxins from microorganisms. June et al. (E77) detected Salmonella spp. in low-moisture foods, and Kitagawa et al. (E78) detected and quantified Vibrio cholerae 01 species in foods. For tomato paste, Pate1 et al. (E79) developed an enzyme immunoassay for total mold content and concluded the assay has considerable potential to replace the traditional Howard mold count. Robison (E8O) reviewed rapid ELISA methods for detecting, in foods, the foodborne patho ens Salmonella, Listeria, and hemorrhagic Escherichia cok With enterotoxins produced by 52 biot ed and phagetyped s.aureus strains isolated from foozMathieu et al. (E81) found an ELISA ’kit” for detectin simultaneously five enterotoxins gave reliable and repeatab e results. A “kit” is a complete set of materials needed for an ELISA analysis that is sold commercially. Park et al. (E82)tested another kit (TECRA) for detecting staphylococcal enterotoxin types A-E on ham, cheese,and mushrooms and found the sensitivity to be similar to those of other kits and a nonenzyme nonimmunoassay; however, the TECRA kit gave nonspecific reactions with food samples contaminated by microorganisms
8
d
f
other than S. aureus. Patey et al. (E83) reported on a collaborative study with an ELISA method for total aflatoxin in peanut butter; the method was adapted by the AOAC International.
FLAVOR Techniques applied to study flavor are becoming more complex and sophisticated. The goal of being able to characterize odors instrumentally and chemically to reflect the sensory perception is still being pursued. The topic of flavor has been subdivided into areas that will help the reader to pinpoint particular interest areas. The literature reviewed was published primarily during the years 1987-1991.
General Methodology. Reineccius ( F I )compiled a review
(29 references) on the research and quality control of flavor by GC/MS. Cronin and Ca lan (F2) compiled a review (170
references) discussing comgined GC/MS in flavor analysis with em hasis on headspace methods. Grundschober (F3) re ortetresults from a collaborative trial to determine the reEability of identifying compounds in flavorings by GC/ MS. Imhof and Bosset (F4) compared two commercially available systems using dynamic headspace techniques for collection, enrichment, and injection. There were significant differences in repeatability. Aishima (F5) concentrated headspace volatiles on a porous polymer, passed them through a system containing six different semiconductor gas sensors, and analyzed the results by cluster analysis. Data are given for coffee, citrus oils, and alcoholic beverages. Barcarolo et al. (F6)developed a technique for performing headspace GC/ MS analysis of volatile components of food in which the carrier gas flow is reversed during sampling in order to overcome problems caused by the diffusion of substances not retained by the cold trap. The technique can be used for liquid and solid matrices and, with the use of internal standards, can be used quantitatively. A novel device for trapping headspace volatiles was described by Ishihara and Honma (F7).Risch and Reineccius (F8)compiled a review (27 references) on the analysis of thermally generated aromas from food or model systems. The challenges that remain in quantitative and qualitative isolation of aromas are discussed. Schreier (F9) compiled a review (54 references) describing analysis of flavors by high-resolution GC with MS or Fourier-transform IR spectroscopy and multidimensional GC. Fischboeck et al. (F10)compiled a library of vapor-phase Fourier-transform IR spectra from 500 contaminant-free flavor-relevant compounds Nitz et al. (F11)adapted a valveless switching system to facilitate transfer of enriched components from thermal adsorption traps onto a multidimensional GC operation. Hartman et al. (F12) compiled a review (21 references) on the use of desorption ionization techniques, FAB with LCIMS, tandem MS, and various hybrid techniques to determine nonvolatile or thermally labile flavor compounds in a food system. Feeney and Jennings (F13) compiled a review (11 references) concerning the importance of column stationary-phase selection in analyzing complex volatile samples. Hawthorne et al. (F14, F15) developed a method for extraction and analysis of flavor and fragrance compounds by supercritical fluid extraction with COZ combined with capillary GC and tested it on a variety of samples includin spices, chewing gum, orange peel, spruce needles, cedar woot dust, coal, nicotine, and river sediment. Shaath and Griffin (F16)compiled a review (52 references) on the use of analysis techniques in the development of new products in the flavor industry. Vercellotti et al. (F17)detected ppb of volatiles with minimal thermal decomposition by removing them from the product at 30-60 “C under vacuum and desorbing them from the Tenax-GC trap directly onto a cold capillary GC column with resolution by temperature programming. Nitz et al. (F18) combined cryogenic headspace enrichment and multidimensional GC to analyze leaves and oil of parsley and mar’oram, garlic, and coffee off-flavor. Adamczyk et al. (F19) used dynamic headspace analysis with a GC/photoionization MS system to produce fingerprints of onion, garlic, orange and lemon peel, peppermint leaves, and cloves. Bohnenstengel et al. (F20)manually injected large headspace volumes on a GC system to detect aroma changes in relation to parameters associated with processing or storage. N
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
343R
FOOD
Kenney (F2l)compiled a review (10 references) on the use of HPLC in flavor research and testing for adulteration of flavors. Hagedorn (3'22)studied the differentiation of natural and synthetic benzaldehydes by deuterium nuclear magnetic resonance. It was possible to differentiate petrochemical and botanical sources and to detect adulteration of bitter almond oil. Butzenlechner et al. (F23) used stable isotope ratio analysis to identify the source of benzaldehyde. Gillyon (F24) described the apparatus for GC/isotope ratio MS to detect adulteration of food flavors by differences in 13C/l*C ratio. Synthetic counterparts of vanilla and lemongrass oil were shown to have higher 13C amounts. Grosch et al. (F25) described quantitation of aroma and flavor compounds by using a stable isotope dilution assay method. The detection limit of flavor compounds was 0.05-0.5 pg/kg in the systems tested. Nitz et al. (F26)described a system which combines multidimensional GC with isotope ratio mass spectrometry. Potentials and limitations of the technique are discussed. Maignial et al. (F27) described an improvement to the simultaneous distillation-extraction system developed by Likens and Nickerson. The improvement allowed isolation of aroma compounds at 20-40 OC by operating under vacuum in a closed system. There was no artifact formation. Leffingwell and Leffingwell (F28) compiled a review (126 references) on detection thresholds of GRAS flavor chemicals. Zervos and Albert (F29)compiled a review (67 references) on statistical analyses which can be used to analyze, characterize, and classify flavors. Chirality. Mosandi (F30) compiled a review (76 references) on flavor chemistry with special em hasis on synthetic and analytical methods. Mosandi a n x co-workers have published several articles on separation, detection, and measurement of chiral flavor compounds (F31-F43). Tress1 et al. (F44) published a review (43 references) on analysis techniques and metabolic sequence investigation of chiral flavor compounds, especially in fruit. Bernreuther et al. published two articles (F45, F46) on separation of enantiomeric compounds from natural sources. Guentert and coworkers at the Research Department of Haarmann and Reimer GmbH published four articles (F47-F50) on chiral analysis in flavor and essential oil chemistry. Wines. Barros and Tubino (F51)developed a flow injection conductimetric method and a flow in'ection spectrophotometric method for determining the volatile acidity of wines quickly. Miranda-Lopez et al. (F52) compared the odor of Pinot noir wines from grapes of different maturities during 1987 and 1988. Wines from grapes harvested at the end of the ripening period had more odor-active peaks than did grapes harvested earlier in the season. The 1988 wines had more odor-active peaks than the 1987 wines. Gunata et al. (F53) used enzymes to liberate components of aromas from nonaromatic glycosides in wine and fruit juice. Chisholm and Samuels (F54) used gas chromatography-olfactometry to study the effect on odor of a bacterially spoiled red wine treated with potassium sorbate. Beer. A method relying on headspace samplin and GC reported by Buckee (F5.5) was approved by t%e Anal. Committee of the Institute of Brewing for inclusion in Recommended Methods of Analysis. This method should be viewed as a guideline, not an ideal method. Walker (F56) used capillary GC to quantify low levels (0.05-0.1 fig/L) of flavor-active volatiles in fermenting wort and beer. Barker et al. (F57) developed a capillary column GC technique to quantitate flavor volatiles, including ethyl acetate and acetaldehyde, by direct injection of wort or beer. The effects of temperature,oxygenation, and yeast pitching were studied. Dercksen et al. (F58) combined an in-bottle urge-and-trap sampling technique coupled to a capillary G8system with a sulfur chemiluminescence detector to elucidate sulfury notes detected by a sensory panel. Burmeister et al. (F59) found the sulfur chemiluminescence detector to have better selectivity, sensitivity, and linearity than flame photometric detectors commonly used. The contribution of P-damascenone to beer aroma was hi h, accordin to Sen et al. (F60), who extracted the compouncf using a staile isotope dilution assay. The compound was also found in coffee,tea, and honey. Xu et al. (F61) used capillary GC thermal energy analysis to study volatile N-nitrosamines in Chinese beers and reported a correlation between concentrations and mortality from digestive cancer. 344R * ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
Vinegar. Enzymic techniques were used by Plessi et al. (F62)to quantify organic acids and their derivatives in several t es of vinegar. Tubino and Barros (F63) described conE t o m e t r i c and colorimetric flow injection rocedures for determination of volatile acidity of vinegars. $he procedures are reported to be quick (-2 min) and accurate (SD 1% 1. Blanch et al. (F64) used GC/MS to identify 61 compounds from vinegar. Cocoa. Abeygunasekera et al. (F65),using GC/MS, found that increased fermentation time and maturation increased the level of volatiles of cocoa and improved the flavor. The results were corroborated by sensory panel analysis. Silwar (F66)used GC/MS to identify 99 components of roasted cocoa beans. Pino (F67) used a dynamic headspace method with thermal desorption and GC analysis to study the volatile components of cocoa butter. Arnoldi et al. (F68) quantitatively analyzed the pyrazines resulting from the reaction of eight amino acids and fructose in a deodorized cocoa butter-water model of the Maillard reaction in roastedcocoa beans. New pyrazines were reported. Meat. Cerny and Grosch (F69) identified 22 of 25 odor compounds isolated from roasted beef. Spanier and Edwards (F70)isolated and purified low molecular mass peptides from cooked and cooked-stored-recooked meat. Cooked meat appeared to have equal amounts of hydrophilic (associated with sweet taste) and hydro hobic (associated with bitter and often sour taste) peptiles while the cooked-storedrecooked samples appeared to contain predominantly hydrophobic peptides. Seven novel heterocyclic flavor components were identified in the reaction products of a meat flavor model system developed by Werkhoff et al. (F71). Possible pathways for the formation of these novel S compounds were postulated. An equation was develo ed by King and Nassos-Stalder (F72)relating odor acceptagility by a sensory panel and lactic acid content of coarsely ground beef after regrinding and storage under aerobic conditions. Nolan et al. (F73) stored precooked pork and turkey at 4 and -20 "C in vacuum and atmospheres of air, COZ, and N. Sensory panel scores correlated well with TBA values for warmed-over and rancid flavor and aroma but less well with fluorescence method values. Baloga et al. (F74) were able to identify more than 60 flavor compounds in ham. Atomic emission was useful in selective detection of N-, 0-,and S-containing compounds Ihekoronye (F75) used GC/MS to quantitate hexanal levels in nitrite and non-nitrite cured bacon stored at 4 OC for 2 weeks. Hexanal increased during stora e and paralleled subjective undesirable flavor estimates. exanal increased faster than four other warmed-over flavor markers tested by Lamikanra and Dupuy (F76) in cooked chevon. Pensabene et al. (F77) modified a solid-phase extraction procedure to determine volatileN-nitrosamines in hams processed in elastic rubber nettings. Artifactual nitrosamine formation was found to be a problem in detection by the mineral oil distillation and low-temperature vacuum distillation rocedures, two currently used methods. Pensabene a n t Fiddler (F78) developed a method which is free of artifact formation for quantifying volatile N-nitrosamines in cured minced fish or surimi meat frankfurters. Artifactual nitrosamine formation in cheese and cured meat products was a factor in both the mineral oil technique and the lycerol isolation technique as reported by Groenen et al. (479). A hazard to consumers does not appear to exist since the artifact formation occurs mostly in situations peculiar to laboratory analyses and not in normal household preparation. Grinwich et al. (F80)determined that RIA of 16-androstenes in the saliva is not a reliable method of estimatin boar taint levels in fat. However, Squires et al. (F81) founi good correlation between the levels of off-aroma and offflavor sensory scores and the androst-16-ene steroid levels measured by a colorimetric assay in fat and salivary glands. Results of a fry test for taint, which used four experienced testers, showed no significant correlations with the off-aroma and off-flavor sensory scores. Sausage. Prusa et al. (F82) found that summer sausage formulated from pork to which porcine somatotropin had been administered received greater acceptability scores and was preferred by 62 % of consumers when compared to porkbeef blended sausage or sausage from pork without soma-
-
l5
FOOD
totropin treatment. Baardseth et al. (8'83)used principal component analysis to reveal the effects on sensory properties and instrumental texture and color values of adding dairy ingredients to saus e. Frankfurtersprepared with corn germ protein were teste? for sensory properties and amino acid profiles by Zayas and Lin (F84). Weak, acceptable corny aroma and taste was detected at 2.4% and 3.5% levels of added corn germ protein. The amino acid composition was not greatly altered by the corn germ addition. Nagy et al. (F85)found that salami prepared with reduced NOz- or no NOS- increased in acid number, but the fats did not become rancid. Salami stored in cartons lost color, whereas those hung did not. Reduced nitrite-nitrate contents did not affect chemical or sensory characteristics. Wittkowski et al. (F86,F87) compared the headspace volatile components of liquid smoke flavoring and smoked meat. A series of monoterpene hydrocarbons were found in the smoked meat products but not in the liquid smoke. Chicken. Croasmun and McGorrin (F88)compiled a review (25 references) which describes an integrated GC/ matrix isolation IR/mass spectrometry instrument as a powerful tool for rapid identification of thermally generated aroma compounds. The technique is illustrated with analysis of charbroiled chicken flavor components. Viehweg et al. fF89)tested direct headspace analysis, lowtemperature, high-vacuum distillation and adsorption for detection of volatile metabolic products formed during the spoilage of chilled poultry carcasses. Each method provided useful, different information. Fish. Perez et al. (F90)described a GC method for quanitation of trimethylamine and dimethylamine in seafoods. St. An elo et al. (F91) used capillary GUMS to separate and i entify volatile compounds that can be used as markers for quality assessment. McCarthy et al. (F92) tested decomposition of fish usin volatile acid number, acetic acid, total volatile bases, and !'BA analyses. Acetic acid determinations closely paralleled volatile acid number values; total volatile bases was useful in evaluating lean seafood quality while TBA was better for evaluatin fatty fish quality. Antonacopoulosand Vyncke (F93)reporte on a collaborative study by the West European Fish Technologist Association to standardize methods for detection of total volatile basic N. A direct test is recommended for routine QC, but not for research or confirmation purposes. Murray and Lockhart (F94) tested fish tissue for the presence of water-soluble components of crude oils and petroleum products which may be associated with tainting in fish tissues. Heil and Lindsay (F95) tested for flavortainting alkylphenols and aromatic thiols in rainbow trout tissue. Davis et al. (F96)tried several sensory evaluation methods to detect taint from trout deliberately contaminated by diesel fuel. A triangle test appeared to be the best test. Jardine and Hrudey (F97) used a sensory panel to determine detection threshold values for chemical compounds associated with oil sands wastewaters. Flament (F98)identified 2 new alcohols amon 19 which were identified in crabmeat. Hollingworth et al. ( j 9 9 )tested several chemical, microbiological, and sensory analyses of imitation crabmeat to assess spoilage and concluded that sensory analysis was the only method which could assess product acceptability after prolonged storage at reduced temperatures. Matiella and Hsieh (F100)identified 53 volatile compounds from boiled and pasteurized blue crab samples. Tanchotikul and Hsieh (F101)analyzed volatile flavor components in steamed rangia clam after extraction by dynamic headspace sampling and simultaneous distillation and extraction (SDE). SDE gave more information on characteristic flavors. Tanchotikul and Hsieh (F102)reported a method for quantification of geosmin in rangia clam and measured levels of the compound in clam samples tested in different seasons of the year. Girard and Nakai (F103)studied factors which could improve a static headspace sampling method a plied to analyze volatiles in canned salmon. Optimizerfconditions enabled the detection of 80 volatiles, 34 of which were identified. Strachan et al. (F104) studied several methods as possible indexes of days in ice and extent of spoilage of cod. Nunes et al. (F105)employed physical, chemical, and sensory analyses to assess the shelf life of iced sardines. Sensory evaluation and Torrymeter readings gave the most
tf
6i
highly significant de ree of correlation. Run e and Steinhart (F206)determined CfC detection limits and i entifiedvolatile S-containing flavor compounds of carp meat. Mandeville et al. (F207,F208) explored the extraction of useful components such as flavor-active compounds from crustacean waste generated by the fishing industry. Milk and Cheese. Christensen and Reineccius (F209) tested the use of static headspace sampling with GC and a S-specific detector to determine heated milk flavor uality. The results were compared with the intensity of heatel flavor as determined by a sensory panel. Correlations were moderatelystrong for skim milk but weak for whole milk. Duncan and Christen (F110)evaluated the relationship between acid degree value and sensory detection when milk samples were sup lemented with varying concentrations of selected fatty aci8s. Acid degree value does not measure short-chain fatty acids, which contribute to the threshold sensory detection of rancid off-flavor. Coulibaly and Jean (F111 )developed a rapid and sensitive solid-phase extraction method to isolate relatively nonvolatile flavor compounds from ultra-high-temperature milk. GC and GUMS were used to analyze the compounds. Bouzas et al. (F122)compared proteolysis assessment, total acidity, pH, and detection of lactose and organic acids by HPLC with sensory evaluation by an expert judge as means of assessing Cheddar cheese uality. The objective measurements complemented the su$jectivetesting and facilitated the detection of quality problems. Sanz et al. (FI 13)designed and evaluated a mixed-phase capillary column for GC separation of volatile components of cheese. Klein (FI 24) compared four extraction, concentration, and injection techniques to study the volatile flavor of Swiss cheese. GUMS was employed to identify 185 volatile compounds. Gallois and Langlois (FI15) used high-vacuum distillation with GUMS analysis to identify and quantify 129 volatile odorous compounds in five French blue cheeses. De Frutos et al. (F116)used simultaneous distillationextraction with capillary GC and GUMS to characterize the aroma compounds of six artisanal cheeses. The mold-ripened varieties had the most aroma compounds. Horwood (F117) identified 28 volatile compounds of chain length IC, in the headspace of cheese by GC and GUMS. De Frutos et al. (F118)used a simultaneous distillation-extraction approach with GC and GUMS to qualitatively and quantitatively analyze volatile components of cheese. The technique was described as a fast method for characterization of cheeses. Ulberth (F119) used a headspace GC method to analyze yogurt volatiles. The volatiles were baseline separated in less than 5 min. Kang et al. (F220)monitored changes in flavor compounds of unflavored yogurts for up to 10 days by GC methods. Fewer artifacts were produced than occur with either extract injection method commonly used. Cormier et al. (F121)grew Pseudomonasfragi in skim milk, producing a pleasant strawberry-like odor. Volatiles were extracted and analyzed by GC-FID. Of approximately 90 compounds detected, 26 were odor-active with ethyl butyrate, 3-methylbutanoate, and ethyl hexanoate being major contributors. Fats and Fatty Acids. Ackman (F122)compiled a review (80 references) on lipid analysis with emphasis on fatty acid detection by GC. Boyd et al. (F123) determined conditions under which GC-headspace analysis for propanal and pentane-hexanal can be used to follow the oxidation of selected w-3 fatty acids and linoleic acid. Gandemer et al. (F124)fixed free fatty acids (FFA) from total lipid extracts from meat products onto an anionic exchange resin. The fixed FFA were esterified on the resin before quantification by GLC, resulting in minimal contamination by other lipid fractions. Ukeda et al. (F125) have previously reported the use of a biosensor for determining fatty acid composition in milk; this report indicated the potential for a rapid selective detection of off-flavor fatty acids. Park and Goins (F126)reported using a dynamic headspace capillary GC analytical technique with cryofocusing to quantitate volatile lipid oxidation compounds in milk-based nutritional products. Hexanal was present at sub-ppm levels. Ceccon (FI27)used a Nucol fusedsilica capillary column in a GC system to separate free volatile fatty acids from dairy products. Kim Ha and Lindsay (F228) obtained mass spectra by GC/MS of butyl esters of volatile fatty acids in milk fat and meat lipids. Methyl-branched,
d
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
345R
FOOD
Table V. Vegetable Flavors
vegetable tomatoes
ref Stern et al. (F151) Baldwin et al. (F152) Baldwin et al. (F153) Petro-Turza et al. (F154)
potatoes
Quilletteet al. (F155) Carlin et al. (F156) Przybylski and Hougen (F157) Kuo et al. (F158)
black truffles
Talou et al. (F159-F164)
cauliflower
Baardseth and Naesset (F165)
cucumbers (and melons)
Schieberle et al. (F166)
daikon
Ishii et al. (F167)
ethyl-branched, and dimethyl-branched fatty acids were found. Ha and Lindsay (F129)extracted volatile FFA (CC12) from Parmesan cheese and butter and found, in addition to the usual even-numbered carbon FFA, substantial reservoirs of volatile branched-chain, odd-numbered carbon, and unsaturated fatty acids in cow milk fat. Elliott et al. (F130) improved an HPLC method for quantitative detection of saturated and unsaturated Cpls FFA. Madaan (F131)developed and standardized an oxidative method for measurin saturated mono- and diunsaturated fatty acids (CIS and higher). Uhler and Miller (F132)reported development of a multiple headspace extraction GC method that could rapidly quantitate volatile compounds from a wide variety of matrices. The use of the procedure in determining the volatile halocarbons in butter was described. Dobarganes and PerezCamino (F133)described a procedure for analysis of heated or used frying fats to determine oxidative, thermal, and hydrolytic degradation. Yasuhara and Shibamoto (F134) purged the volatile chemicals in the headspace above heated pork fat into aqueous solutions of cysteamine, NaHS03, or methylhydrazine to trap aldeh des and ketones. This is the first time that malonaldehydre has been detected in the vapor phase. Skala et al. (F135)used thermogravimetric analysis to measure the rate of volatile compound formation from lipids extracted from hog and cattle tissues. Hog tissues yielded about 20% more volatile compounds compared to correspondingsamples from cattle tissues. Activation energies for thermal degradation were correlated with the phospholipid content of the sample. Um et al. (F136)fractionated beef fat heated at 100 "C with supercritical CO1, analyzed the fractions by GC and GC/MS, and identified 66 volatile compounds. Oils. Comptonand Stout (F137)used headspace GC/FTIR to analyze flavor oils. Jirovetz et al. (F138)used GC/MS and GC/FTIR to analyze volatiles from the seed oil of Hibiscus sabdariffa. Rosales et al. (F139)determined the intensity of the bitter taste in virgin olive oil by extracting the oil with MeOH-H20, (1:l)and measuring the absorbance of the extract at 225 nm. This simple test was significantly correlated with the intensity as evaluated by a taste panel. Idris et al. (F140)compared sensory evaluation of alm oils of various qualities with chemical analyses incluing free fatty acid detection, peroxide value, and p-anisidine value. Guth and Grosch (F141) detected three furanoid fatty acids in soybean, wheat germ, rapeseed, and corn oils and measured the effect of light exposure in developing off-flavors. Coffee and Tea. Flament and Chevallier (F142)compiled a review (67 references) on the volatile constituents of coffee. 346R
ANALYTICAL CHEMISTRY, VOL. 65, NO. 12, JUNE 15, 1993
objective/technique/result
GC with spread sheet analysis used to develop tomato flavor profiles levels of color, flavor volatiles, sugars and org. acids in 2
varieties were compared to climacteric ethylene production six cultivars were tested for flavor parameters including sugar, acid, and volatiles two cultivars were tested for N compounds in fresh and pureed preparations volatiles from normal and disease-inducedpotatoes analyzed by GUMS showed differences two new compounds were identified in french fries a simple method was devised for detection and quantitation of volatile carbonyl compoundsin oil and chips at 0.1-0.5 ppm detection level a simple, selective method was described to detect pyrazines in a model product and potato chips-less laborious and timeconsuming than traditionalmethods dynamic headspace GC analysis provided profiles of 50 black truffle flavorings, with information on detection of imitation product blanched and unblanched cauliflower (3 varieties) frozen for up to 8 months was sensorily evaluated. Unblanched samples were unacceptable after 4 weeks extract dilution analysis was used to determine the odorants having the highest odor units sections of fresh daikon roots were analyzed for 4-(methylthio)3-butenyl glucosinolate to determine areas of concentration Since 1837,670 compounds have been identified in roasted coffee, including furans, pyrazines, pyrroles, ketones, and 90 S-containing compounds. Shimoda and Shibamoto (F143) isolated and identified headspace volatiles from brewed coffee with an on-column GC/MS method. Sixty-two compounds were identified, but some well-known volatiles were not found. Holscher and Steinhart (F144)studied the pleasant odor from freshly roasted coffee beans by an improved headspace technique with intermediate cryofocusing and simultaneous sniffing analysis. The loss of aroma freshness corresponded to a decrease in low-boiling volatiles, especially methanethiol. The method also differentiated between Arabica and Robusta coffees. Headspace GC/Fourier transform IR spectrometry was used to analyze instant and regular-grind coffee. The effects of roasting and decaffeination procedures could be detected. Spadone and Liardon (F145)analyzed the aroma compounds in coffee during storage by means of headspace GC and sensory panels. Headspace GC/FTIR was used by Compton and Compton (F146)for monitoring of volatiles in four instant and one ground coffee. Caffeine was detected in the decaffeinated coffees, but no peak was detected for chlorinated hydrocarbon solvents. The re ular-grind coffee had more peaks than the instant coffees. hshima (F147)applied a semiconductor gas sensor array to the discrimination of coffee aromas, essential oils, and volatile compounds with different functional groups. Two ground coffees and freeze-dried and spray-dried instant coffees were separated by cluster analysis and linear discriminant analysis. Aishima and Ozawa (F148)used R P HPLC to concentrate volatiles in aqueous systems at levels from ppm to ppb. It was tested in a model system and with coffee and juices. Spadone et al. (F149)used simultaneous distillationextraction with capillary GC, GC-sniffing, and FC/MS to identify 2,4,6-trichloroanisoleas the most likely key compound in the Rio off-flavor defect. Julkunen-Titto et al. (F150)used capillary GC to screen 26 contributory components in six Finish herbal plants used for tea. The aroma profile was markedly affected by withering and rolling followed by fermentation. The concentration of benzaldehyde and some undesirable aroma compounds decreased while higher boiling point aromatics and intermediates accumulated during fermentation. Vegetables. See Table V. Fruits. See Table VI. Spices, Herbs, and Flavorings. See Table VII. Irradiation Effects on Flavor. Nawar (F238)reviewed 12 articles on types of volatile products produced on irra-
FOOD
diation of foods and the possible usefulness in detecting the use of irradiation on foods. Speigelberg et al. (F239) found severalradiation-induced volatiles in irradiated frozen chicken that were not found in control samples. Meier et al. (F240) found that irradiation of chicken meat can be identified simply and quickly by determination of o-tyrosine by HPLC fluorescence detection. The same author (3'241) tested three chemical methods for the detection of irradiated foods. Sjovall et al. (F242) subjected several pure aroma comounds in spices to y-irradiation and analyzed the changes gy GUMS. Only (&)-linalool and a-terpineol changed substantially. Farkas et al. (F243) and Sharif et al. (F244) studied several possible indexes of irradiation in spices with a change in the apparent viscosity of a gelatinized suspension showing the most dramatic change. Sjoberg et al. (F245) tested three methods to evaluate irradiated spices: amicrobial method, a chemiluminescence procedure, and GC and GC/ MS. The microbial procedures combined with chemiluminescence were the best methods. No differenceswere detected between irradiated and nonirradiated samples by chemical analysis. Kolbak (3'246) tested whether UV light affects thermoluminescence signals in spices. Rosemar was more sensitivethan ground black pepper. Plnniojaand lutio (F247, F248) proposed that thermoluminescence of irradiated food appears to originate more from mineral contamination than from the food material itself. Giamarchi et al. (F249) showed no significant effects on the fatty acid composition of sunflower and peanut oils subjected to high-dose irradiation, but new C1~1,alkanes and alkenes appeared in proportion to irradiation dose. Singh et al. (F250)found differences in the volatile compounds resent in irradiated and unirradiated, cooked and uncookef bacon as detected by GC/MS analysis. Troup et al. (F25I) found that y-ray irradiation roasted coffee beans or powder, tea leaves or powder, and various foods irradiated by y-rays gave free-radical EPR signals. Packaging. Hodges (F252) reviewed two articles on packaging material interactions with foods that produce offodors, methods for collecting and analyzing these odors, and predicting the odor and taste performance in packaging materials. Booker and Friese (F253) studied volatile compounds formed by microwave or conventional heating of packaging for microwaveable foods. Rose (F254) developed a method for determining volatileextractives from microwave susceptorcontaining food packaging. Risch et al. (F255) analyzed volatile compounds generated by the ackage and/or food in microwave cooking. Letourneur and 8harlet (F256)analyzed volatile releases from commercial food-grade coated paperboards and identified nine compounds. Durst and Laperle (F257) studied the migration of styrene monomers from packaging into food by purge-and-trapGC and sensory panel analysis. Deionized water stored in styrene containers showed that styrene pickup was proportional to the square root of time. Nielsen et al. (F258)used supercritical fluid extraction with GC for analysis of aroma compounds absorbed by lowdensity polyethylene. Compton et al. (3'259) used GC/IR to analyze the type of compound absorbed by beverage container coatings after the cans had been stored at 100 "C (such as left in a transport truck during hot summer days). Several compounds were found, including flavor volatiles which would affect taste perception. Roland and Hotchkiss (F260)studied sorption of aroma compounds by food-contact polymers by using a microgravimetric balance in a vacuum chamber. Hunziker (F261) studied the stability of flavor compounds during storage of candies and found that wrapping the box increased retention of flavors. Miscellaneous. Clark (F262) used instrumental and sensorypanel analyses to determine that gels with a low degree of rupture strength released flavor properties more quickly than harder gels. Culp and Noakes (F263) determined that sufficiently different deuterium/hydrogen ratios existed in 11synthetic flavoring materials compared to their natural or fossil fuelderived counterparts so the D content could be used in determining adulteration. Fish (F264)reported a procedure for quantifying 5'mononucleotides in food and food ingredients.
Wellman et al. (F265)used a saccharin aversion paradigm to evaluate the potential aversive action of T-2 mycotoxin which induces emesis and weight loss in rats. Pons et al. (F266) developed a device which continuously trapped volatile compounds from aromatic caramel on adsorbent while heating. Following thermal desorption and analysis, 57 compounds were detected including some unexpected intermediates. Groesz and Johas (F267) developed a rapid, quantitative TLC method for determining aspartame and alitame. Haeusler and Monta (F268)used RP HPLC to isolate the norisoprenoid (8)-(+)-cfehydrovomifoliolin honey, which was identified by conventional methods. Heather-derived honey contained 56-264 mg/kg whereas other floral-derived honeys contained 0.03-6.02 mg/kg. Pattee et al. (F269)compared the color of roasted peanuts to the roasted peanut flavor response. In analysis of data over a 3-year period, an L value of 51-52 was determined to be the color to which peanuts should be roasted to obtain optimum roasted peanut attribute response.
COLOR Researchers continue to use normal-phase and reversedphase high-performance liquid chromatography (HPLC) as the ma'or technique to separate and identify colorants in food. h e of photodiode array detection, which has been particularly useful in characterizing anthocyanins, is now also applied to carotenoid study. New techniques that are reported include capillary isotachophoresis, near-IR FT Raman spectroscopy, fast-atom-bombardment mass spectrometry (combined with HPLC), use of flow injection analysis, and Curie point pyrolysis high resolution gas chromatograph (GC)/ mass spectroscopy (MS). These techniques have enailed the detection of smaller quantities, new pigmented com ounds, more efficient analyses of color compounds, a n t better detection of adulteration of colorants in foods. This section addresses selected colorants and selected research studies on those colorants. The studies were reported during the period of 1987-1991 primarily. Synthetic Colorants. Youn ( G I )reported a collaborative study by nine laboratories to cfetect and identify the seven FD and C color additives in five commercial food products and two dye mixtures. The methods used (C18cartridge and spectrophotometry at 750-350 nm or TLC on silica gel G-precoated plates) have been given official first action. Reynolds et al. (G2)reported on a collaborative study in the U.K. and Denmark using HPLC to determine synthetic coloringmaterials in six samples of soft drinks and two samples of sponge cakes. The method produced variable results with several cases of misidentification. Milovanocic and Veljkovic (G3)used TLC to separate 10 dyes in mixtures and extracts from foods, beverages, and drugs. Chaytor (G4) reported a procedure for rapid extraction and HPLC analysis of synthetic colors in fruit juices. Recoveries of tartrazine, Quinoline Yellow, and Sunset Yellow were 74-93 % . Karovicova et al. (G5, G6) used capillary isotachophoresis to detect synthetic colorants in liquid and solid foods. Greenway et al. (G7) extracted s thetic dyes from foods with water. After dialysis using an A G E D system, the extract was analyzed by HPLC. Ren and Hong (G8)used a reversed-phase HPLC method to determine preservatives, sweeteners, artifical colors, and caffeine in beverage systems. Roy et al. (G9) used TLC to separate chlorophyll and carotenoid bands from leafy green vegetables. This method was reported to detect both artificial and natural colorants in foods. Chakravarti (GIO)reported a simple test for butter yellow (and metanil yellow) in edible and cosmetic materials: the carcinogenic dye gave a red color when the substrate was mixed with 10% aqueous HC1. Naga and Koike ((211)used HPLC with methylguanidine as an ion-pair reagent to determine azo, xanthene, triphenylmethane, and indigo-type food dyes. Lancaster and Lawrence ((712, G13) extracted free and bound nonsulfonated aromatic amines in tartrazine, Sunset Yellow FCF, allura red, and amaranth. Most of the amines are bound to the coupling compound or R salt during manufacturing with
