Food James A. Yeransian, Katherine G. Sloman, and Arthur K. Foltz
General Foods Technical Center, White Plains, N. Y .
10602
The authors again have attempted in this review to pro,vide a survey of the advances, innovations, and improvements in food analysis since the last such survey (16P) was made. The time period covered is essentially that from October 1970 to October 1972. Citations are included which predate this period if they had not come to the attention of the authors in time for previous inclusion. As before, where similar work has appeared in both American and foreign journals, the domestic publications have generally been cited for reasons of convenience and availability to the authors. Texts which have been recently published which are of use to the food analyst are a new edition of the “Laboratory Handbook of Methods of Food Analysis” (24P) and “Modern Food Analysis” (19P)by Hart and Fisher.
ADDITIVES Interest continues in the dLtermination of all types of additives used in foods. A qualitative and quantitative procedure for butylated hydroxyanisole described by Alessandro et al. ( I A ) also includes color reactions for propyl gallate and nordehydroguairetic acid with iodic acid, and with strontium hydroxide. The fluorescent properties of the common antioxidants have been investigated by Hurtubise (34A), and methods for their determination in lard and cereals described. Column separation on polyamide powder has been used by Lehmann et al. (45A) to separate types of antioxidants. Separation of phenolic antioxidants from edible fats and oils followed by thin-layer chromatography on silica gel has been used by LemieszekChodorowska et al. (46A) to detect as little as 3 pg of these constituents. The phenolic antioxidants including the two isomers of butylated hydroxyanisole have been determined by Stoddard (64A) in vegetable oil a t the level of 0.05 pg using gas chromatography of the trimethylsilyl derivatives. Gallic acid esters have also been determined by Wachs et al. (71A) using silylation techniques. Gas chromatography has also been applied by Takemura (66A) to 15 preservatives and seven antioxidants and used to determine 4-hydroxybenzoates in soy sauce. Twenty-two preservatives used in beer have been determined by Silbereisen et al. (61A) after extraction and methylation using gas chromatography on a two-column system. Preservatives have been separated by Fujiwara et a!. (17A)on a strongly basic ion-exchange resin Dowex-2 X8, C1-form, and on Dowex-2 X8, acetate form (18A), using a gradient elution technique. Thin-layer chromatography on polyamide has been used by Clement et al. ( 6 A ) to separate preservatives using UV detection and by Lee (44A) for food preservatives using colorimetric detection. Sorbic acid in cheddar cheese has been determined by gas chromatography after column extraction of the cheese by LaCroix et al. (42A). A rapid spectrophotometric method for sorbic acid in fresh dairy products has been described by Maxstadt et al. (49A) using ultraviolet measurement after extraction. High-speed liquid chromatography has been employed by Krinitz (38A) for the simultaneous analysis of saccharin and sodium benzoate in beverages and other liquid foods. Thin-layer chromatography on polyamides has been
used by Wang et al. (72A) to separate 11 preservatives and three artificial sweeteners. Avicel SF plates have been used by Nagasawa et al. (55A) to separate and detect cyclamate, saccharin, and dulcin, using pinacryptol yellow. as detecting spray. The color reactions of sweeteners on thin-layer plates have been investigated by Matsumoto (48A). Aniline phthalate has been uscLd by Das et al. (13A) to detect saccharin, cyclamate, and sucrose on paper chromatograms. Sweeteners have been separated and detected a t levels of 10-100 ppm by Takeshita (67A) using column and thin-layer techniques. Dulcin, suosane, ultrasweet, and p-methoxy-o-benzoylbenzoic acid have been detected after ether extraction by Woidich et al. (74A) using thin-layer chromatography. A gas chromatographic method for cyclamate described by Conacher et al. (7A) converts the cyclamate to the methyl ester with diazomethane. Infrared spectrophotomctry has been used by Coppini e t al. (11A) to determine cyclamates in wine by obtaining the spectrum from 1800 to 1500 cm-1, after extraction and clarification. Cyclamate has been detected and estimated by Das et al. (14A) using thin-layer chromatography on silica gel after ether exxaction, and detection by bromination and fluorescein spraying. Dialysis has been used by Hayashi et al. (32A) to separate cyclamate from foods, and the sweetener is determined, after extraction, by thin-layer chromatography on silica gel, using otoluidine and hydrogen peroxide as spotting reagent. A colorimetric procedure for cyclamate has been described by Saturley (59A) in which the,cyciamate is converted into cyclohexylamine and then coupled with picryl chloride. Another colorimetric procedure, described by Shibat a e t al. (60A), uses the blue reaction product of cyclamate with methylene blue which is extracted into chloroform. Mixtures of cyclamate and saccharin salts have been analyzed by Amirjahed e t al. (24) using nonaqueous titration with sodium methoxide. Dulcin has been determined by Stclya (65A) by colorimetry after reaction with nitric acid. Saccharin has been determined by Kuroda et al. ( 4 I A ) by gas chromatography after conversion to thiophenol with hydrochloric acid and zinc. Gas chromatography for saccharin has been described by Gerstl et al. (21A) after purification with a liquid ion-exchanger and silylation, and by Conacher et al. (1OA) after conversion to the N-methyl derivative with diazomethane. A method for saccharin proposed by Corigliano (12A) is based on the ferroin-saccharin reaction. Methods for the chromatography of dihydrochalcone sweeteners, including paper and thin-layer chromatography, and paper electrophoresis, have been described by Gentili e t al. (20A). A method for i,he detection of cyclamate, dehydroacetic acid, saccharin, and salicylic acid has been proposed by Hayashi (31A) using nitrobenzene extraction with tris(1,lO-phenanthro1ine)-iron I1 chelate cation. A specific thin-layer chromatographic method for the detection of monochloro- and monobromacetic acid in wines has been developed by Hailer et al. (28A) using conversion to glycine with ammonium carbonate and ninhydrin detection on the plate. A qualitative and quantitative analysis for propionic acid in bread has been de-
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scribed by Takeshita et al. (68A) using separation by distillation and gas chromatography. Propionic, sorbic, and benzoic acids in rye bread and margarine have been determined by Graveland (26A) by gas chromatography after ether extraction. Acetic, propionic, and sorbic E cids in bakery products have been identified by Tjan et a!. (69A) by thin-layer chromatography after steam distillat on and concentration. A microdiffusion technique has been developed by Karasz et al. (35A) for the isolation of propionic acid in bread preliminary to gas chromatography. Ammonium glycyrrhizinate has been determined by Larry et al. (43A) by gas chromatography, after hfdrolysis, extraction, and preparation of the silyl ether :lerivative. Brominated vegetable oils in soft drinks have been determined by Conacher et al. (8A) by gas chromzttography after extraction and methylation; by Green et al. (27A) by extraction and combustion and colorimetr c bromide determination; and by Hayashi et al. (30A) :)y extraction, combustion, and fluorescein colorimetry. Kroeller (39A) has presented a method for calcium stear.il-lactate in bread utilizing acidification, extraction, and reverse phase paper chromatography. Diethyl pyrocarf. onate has been determined by Moncelsi (52A) by its reaction with the primary group of 4-aminophenazone. A s p x t r o photometric determination of EDTA has been desc fibed by Bhattacharyya et al. (4A) using the reaction with iron, and Mihara e t al. (50A) have used gas chromatoginphy after methylation. A method for the determination of diacetyl-tartaric acid esters of mono- and diglyceride:; has been proposed by Pentiti (56A) using colorimetric measurement of the tartaric acid obtained by hydrolysis. Circular thin-layer chromatography has been used by ‘Nurziger (75A) for the detection of emulsifiers in baked goods and confections. An extensive study of the composition of several commonly used nonionic emulsifiers has beeii reported by Srebrnik et al. (63A) including infrared :)hotometry, chromatographic separations, and a prop x e d analysis scheme. “False positives” for gums in mayonnaise have h e n traced by Krinitz (37A) to the presence of mustard fl:,ur, and infrared analysis is suggested as a means of detecting this interference. Several methods of determining gums in food have been investigated by Graham, including a colorimetric method for Keltrol (xanthan gum) (22A) usin(: a difference method, a study of the specificity of the fer,*icsulfuric acid reagent for alginates (23A), and two prtcedures for carboxymethylcellulose in food, one using the 2,7-naphthalenediol test (24A), and one using chromoliopic acid (25A). A method for lecithin, using thin-layer chromatograp -iy, has been used by Venturini (70A) for the analysis of baked products. Monosodium glutamate has been detixrmined by Gal et al. (19A) using gas chromatography of the N-trifluoroacetic butyl ester derivative, and by Bailey et al. (3A) using ascending paper chromatography. Nitrr le has been determined, colorimetrically by Krupowicz et 111. (40A) by the reaction of phenazone. An indirect method based on the selective interference of nitrate in the determination of rhenium with stannous chloride and biacetyl monoxime has been adapted by Holz et al. (33A) to automatic analysis. A rapid method for the reduction of nitrate to nitrite by shaking with cadmium has been proposed by Elliott et al. (15A) for the determination of nitrite and nitrate in bacon. A method for relatively lark\> amounts of nitrite has been described by Erben (16A ) using amperometric titration after reaction with sulfanil2 mide in hydrochloric acid. A method for the determinetion of 0.1 to 12 ppm nitrite has been developed by Weis:, 78
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et al. (73A), using antipyrine. Spectrophotometric methods have been used by Rammell et al. (57A) to determine low levels of nitrite in cheese and dairy products. Brucine sulfate has been suggested by Saito et al. (58A), for the spectrophotometric determination of nitrate and nitrite. A sodium salicylate method has been used by Kawana et al. (36A) to determine nitrate and nitrite in foods. Hassan (29A) has determined micro quantities of nitrates and nitrites by differential gasometric reactions. A method for the detection and determination of nitrous oxide in meat products has been described by Moehler et al. (51A) using gas chromatography. Reaction gas chromatography has been used by Lundquist et al. (47A) to determine polysorbates in food using a saponifying column. Sucrose diacetate hexaisobutyrate has been determined by Conacher et al. (9A) using direct gas chromatography. Procedures for the determination of anionic and cationic surfactants have been described by Clark et al. ( 5 A ) using titration techniques. Special separations have been employed by Mori et al. (53A) to determine alkylbenzenesulfonate in tomato juice. An infrared method using KBR disks for the determination of dimethylpolysiloxane in beer has been described by Sinclair et al. (62A). A microcolorimetric method for the determination of sulfur dioxide in fruits has been proposed by Nagaraja et al. (54;4),which uses the reaction of sulfur dioxide with a titanium sulfate salt for determination after distillation. ADULTERATION, CONTAMINATION, AND DECOMPOSITION Schemes of analysis to detect and quantify adulteration of food commodities continue to appear in the literature. Kato et al. (111B) calculated carbon number area ratios for triglyceride gas chromatograms in a method to estimate foreign fats in ice cream fat. Hahn et al. (87B) used methyl ester ratios to detect other fats in butter biscuits. A TLC separation of unsaponifiables and also the melting points of the mixed sterol acetates permitted the identification of esterified and trans-esterified fats in butter for Maglitto et al. (130B). De Ruig (41B) listed the detection of beef tallow in milk fat and lard as an application of his infrared spectroscopic method. Ivanov et al. (105B) found the lack of a maximum in the temperature curve of solidification to indicate adulteration in butterfat. Keeney et al. (112B) used detection of vitamin E from the adulterating fat to detect soybean or corn oil added to ice cream. Horse fat in beef fat was detected by Langner (124B) by calculating unsaturated CIS to other acid methyl ester ratios. Fincke (61B) used methyl ester ratios as well as sterol ratios to detect cacao butter substitutes. In a scheme to detect olive oil adulteration, Amati et al. (4B) first separated the unsaponifiables by TLC and then used GLC to ratio amounts of sterol T M S ethers after derivative formation of the sterol band. Losi et al. (1298) also used TLC followed by GLC of T M S ethers to separate and determine ratios of tocopherols in olive and peanut oil mixtures. Methyl ester adulterants in milk powder were detected by Valfre et al. (232%) by extraction, hydrolysis, and methanol measurement. A polyacrylamide-gel electrophoretic separation of cheese casein was used to detect cow milk added to goat milk by Pierre et al. (I59B) and they (160B) also found that storage of the cheese in 95% EtOH was necessary to prevent degradation of the casein fraction responsible for the differentiation. Kuschfeldt (122B) extracted casein with NaCl solution and developed a biuret color after heat-coagulating other proteins present in sausages in his
A N A L Y T I C A L CHEMISTRY, VOL. 45, N O . 5, APRIL 1973
method for milk protein. An enzymic method to identify skimmed milk powder in sausages based on lactose hydrolysis was reported by Bahl ( 7 8 ) . Polyacrylamide elec-8 trophoresis of characteristic protein bands to determine soya bean protein in sausages was described by Fischer (62%). This technique also allowed Hofmann et al. (98B) to differentiate soy and meat proteins. Soybean extracts in orange juice concentrates were found to be detectable by the presence of raffinose and stachyose by Benk (1IB) should such adulteration occur. Benk et al. (12B) advocated analysis for the ratio of total to ammonia F! as another indicator of orange juice adulteration. Koch (118B) listed a number of chemical procedures to detect adulterated orange juice. Lifshitz et al. (128B) presented statistical data they considered best for detecting adulteration of Israel lemon juice. GLC analysis for ricinoleic acid as the T M S 'ether-methyl ester was used by DiGiacomo et al. (45B) to detect traces of castor oil adulteration in lemon essence. Fitelson (64B) reported the suitability of sugar patterns by GLC analysis as a means of confirming adulteration in fruit juices. Lang (123B) determined isocitric acid enzymically and calculated citric to isocitric acid ratios as a means of detecting citrus juice adulteration. Synthetic acetic acid in vinegar was detected by carbon-14 scintillation counting of the distillate after concentration of the acid by Mecca et al. (137B). Mecca e t al. (138B), also used a carbon-14 measurement to determine nonbiogenic ethanol and acetic acid in wine and pickling liquids. Okamoto (154B) reported the discrimination between brewed and synthetic vinegar by several methods including formation of a precipitate with 12-KI and GLC of headspace vapor, Potter (167B) described a GLC method to detect adulteration of vanilla extract by nonvanillin volatiles. The ratio of ethyl acetate to isoamylacetate as determined by GLC of a distillate was applied to the detection of dark beer added to light beer by Hoellerer et al. (97B). Analysis of methanol content and infrared absorption characteristics enabled Nosko (1538) to correlate them with adulteration of cherry schnaps. The presence of salviginen, as determined by TLC, was used by Brieskorn et al. ( 2 4 8 ) to perceive addition of Saluia triloba to Salvia oficinalis (sage). Bromate and other oxidiiing agents in bread were separated and identified by paper chromatography by Pregnolatto et al. (168B). The problem of contamination of food with chemically harmful, as well as aesthetically unacceptable, material has continued to generate a large amount of research and analysis. Among those substances considered of prime importance are the mycotoxins. A review covering the methods of detection of mycotoxins in foods was written by Ayres et al. ( 2 B ) . Stoloff e t al. (207B) described a multiple screening TLC procedure for aflatoxins, ochratoxins, zearalenone, sterigmatocystin, and patulin. Eleven mycotoxins were separated and detected in a thin-layer method by Steyn (205B). Whitaker et al. (243B) published the methodology for a sampling plan for the analysis of peanuts for aflatoxin. The mechanics of preparing good lot samples of nut meats for mycotoxin assay were discussed by Stoloff et al. (208B). A qualitative two-dimensional TLC procedure for detecting aflatoxins BI and B2 in toasted cottonseed meal was reported by Yin et al. (252B). Velasco (238B) detected aflatoxin in cottonseed and its products by the fluorescence exhibited by a small florisil column containing a cleaned-up sample extract. Reddy et al. (170B) described their solvent system for one-dimensional TLC separation of aflatoxins B1, Bz, GI, and Gz in their work with wheat, rice, and cultures. A procedure for determining aflatoxins in moldy sugar-beet
pulp that used two stages of TLC separation was reported by Teng et al. (216B). McKinne$ (136B) recovered 0.3 ppb of aflatoxin MI from raw milk and concluded that a natural 0.2-ppb level could be quantitized. Jacobson et al. (107B) studied levels of aflatoxins B1 and MI in the milk of cows fed contaminated feeds. Afl3toxin B1 in cheese was measured by Kiermeier et al. (1.r3B) with their t,wodimensional TLC procedure. A screening method for corn and its products that was essentially a shortened AOAC procedure was reported by Dantzmart et al. (34B). Moor (114B) modified the AOAC peanut method for aflatoxins to make it applicable to products conkaining over 10% alcohol. A collaborative study of a procedure for aflatoxins in copra and coconut was reported by Baur et al. (9B). Scott et al. (191B) reported results of a collaborative study of cacao bean analysis. Three rrethods for aflatoxin determination in ground-nuts and their products were studied collaboratively by Waltking (241B). Reiss ( 171B) reported sharper separation bands foi. aflatoxins B1, B2, GI, and Gz when TLC separations we're carried out with circular and radial techniques. Schuller et al. (188B) used two-dimensional TLC with a fluoroilensitometric measurement to achieve good recovery of B1 spikes added to food in the low ppb range. Pohland c!t al. (164B) developed some reactions useful for the confirmation of aflatoxin Bl. Toth et al. (224B), quantitized aflatoxin spots after TLC with direct photofluorometry using 375 nm for excitation and 460 nm for emission. Muecke et al. (147B), visualized aflatoxins after TLC by spraying with NaOH, Fast blue salt B and then HC1 after dr5,ing. An adsorbosil5 column provided adequate separaticn of aflatoxins B1 and GI for Chu (29B) in his work wi;;h an infected rice concentrate. Masri (135B) studied exti,action systems for use with ground-nut meal and separated aflatoxins B1 and MI on silica gel columns with progriimmed elution. A rapid method for aflatoxin separation and detection involved a four-minute thin-layer separ3tion on a microscope slide in a paper by Jacquet et al (108B). Friedlander et al. (69B) advocated using polyamide TLC to separate B1 and GI from each other and fro:n some interferences. De Zeeuw et ai. ( 4 4 8 ) improved s?parations of aflatoxins B1, Bz, GI, and GP with vapor programmed TLC and controlled relative humidity. Child3 et al. (27B) performed fluorimetric measurements on iiflatoxin solutions and reported the precision attainable. Manabe et al. (132%) investigated liquid chromatographic separation of six aflatoxins and found best results weie given by Sephadex G-10 (CM). Haddon et a!. (86B) co.ifirmed aflatoxins after TLC separations by mass spectrometric measurements on material from zone scraping;. Hanssen et al. (88B) described their studies on aflaf.oxin B1 and its changes during food processing. An improvement of the AOAC kilogram sample method involving substituting an alumina column for silica gel was reported by Stack et al. (199B). Confirmation of aflatoxins by KBr pellet infrared spectroscopy in conjunction with TLC wiis investigated by Bencze et al. (1OB).Interfering materials were removed on a mixed basic alumina and oxalic acid dihydrate column by Steyn (204B) during his isolation of pure aflatoxins. Stefaniak (203B) differentiated ethoxyquin from B1 with different TLC solvent systems. Stack ct al. (200B) reported the formation of two derivatives of aflatoxin MI, useful for confirmatory work. Scoppa et c!l. (190B) studied the loss of aflatoxin B1 in contact with different plastics. Pons et al. (166B) investigated the acid catalyzed conversions of B1 to Bza and GI to GzR. Changes in the composition of aflatoxin standards stored dry and in solution were reported by Pons et al. (165B). Rodricks e t al. ( I 78B) ex-
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amined standards for concentration and purity. Robe] ison et al. (176B) also investigated the stability of standards during storage as solutions. Rodricks et al. (179B) discussed using molar extinction coefficients as a measure of purity of standards. The gas chromatographic analysis of patulin both directly and as its trimethylsilyl derivative was discussed by Suzuki et al. (209B). Reiss (I72B) detected patuliii in moldy bread and pastries with a thin-layer proceilure, using an o-dianisidine-acetic acid spray reagent. Gas chromatography of patulin as the silyl ether, acetate and chloroacetate was investigated by Pohland et al. (16313) in analyzing apple juice. Stability studies of patulin in foods and solvents were reported by Pohland et al. (162B and these authors (161B) also described a method of andysis by fluorescent plate TLC applicable to grains. S u z ~ l t iet al. (210B) reported the GLC separation and analysis of TNIS derivatives of penicillic acid and patulin applicable to rice. Per0 et al. (158B) also reported such an an3lysis with moldy corn samples. Chu (30B) measured ochri.toxin A in cereal products using TLC and direct spectrophotofluorometric densitometry. Trenk et al. (2258) improved the detection of ochratoxin A after TLC separation by ammoniating the plates before fluorescence measurement. Stack et al. (198B) presented a TLC method for analysis and confirmation of sterigmatocystin in grains. Uch yania et al. (2288) examined miso for the presence of griheofulvin with a TLC-fluorimetric technique. The GLC a r d y s i s of mycotoxins of the trichothecene group as natural esters and T M S derivatives was reported by Ikediobi et al. (102B). Wilson et al. (248B) described their technique for the preparative and analytical gas chromatography of ipomeamarone derived from damaged sweet potatoes, Considerable activity about the detection and idontification of nitrosamine compounds in food has been evident. Walters (239B) gave a review of some metho:lology toward this end. Eisenbrand et al. (49B) suggested heptane-acetonitrile partition as a preliminary cleanup stage. Walters e t al. (240B) examined steam distillation and charcoal adsorption as preliminary separation schemes before analysis by polarography or as NOz- after conversion. Sakai et al. (182B) modified Howard’s method to determine traces of dimethylnitrosamine in ham and also diethylnitrosamine in salted salmon roe. It0 et al. (104B) described a colorimetric method for secondary amines and nitrosamines. The extraction and identification of secondary amines in foods was also discussed by Ito et al. (103B). An automated screening procedure for foo:ls and beverages by Fan e t al. (55B) determined nitrosamines as NOS- colorimetrically after UV irradiation in a quartz coil. Eisenbrand et al. (53B) studied distillation recoveries of 16 nitrosamines a t neutral, alkaline, and acid conditions and a t reduced or atmospheric pressure. A spectrophotometric method utilizing Bratton-Marshall *eagent after N-nitroso group cleavage with HBr in acetic acid was described by Eisenbrand et al. (50B). A c o h m n of Sephadex LH-20 separated dialkylnitrosamines from a wheat flour extract which were subsequently detected by UV absorption in a study by Eisenbrand et al. (51J3). Van Ginkle (235B) investigated the formation of nitror,amines in cheese containing nitrates by TLC and spectroylhotometry but only found uncorrelated traces. Sen et al. (193B) published a method for determining nitrommines in alcoholic beverages using TLC after an alumina column cleanup, Various TLC systems were tried by Eisenbrand et al. (52B) to optimize cleanup separations before UV extinction measurements. Williams (247B) commented on interferences found in analyzing alcoholic bever *ges by
polarography and GLC for nitrosamines. Saxby (1858) removed pyrazine interferences in a GLC method by adsorption on a CuSCN precolumn a t the inlet. Pyrazine interferences were also discussed by Havre et al. (9OB) with respect to GLC, polarographic, and TLC methods. Kadar (11OB) heated D-glucose and glycine and generated pyrazines, which interfered with the polarographic procedure, and identified them with spectroscopic techniques. Heyns e t al. (958) also heated amino acids with saccharides and subsequent GLC mass spectrometry or polarography detected no nitrosamines. Hdward et al. (99B) published a method for N-nitrosodimethylamine in smoked fish consisting of saponification, distillation, extraction; column cleanup, and GLC with a thermionic detector. Foreman et al. (67B) analyzed for nitrosamines in corned beef by GLC with direct injection of an aqueous distillate into a Chromosorb 101 column. Hams were analyzed for traces of Nnitrosodimethylamine by Fiddler et ul. (60B) using a GLC procedure with thermionic detection after digestion, distillation, partition, and concentration. Althorpe ( 3 B ) claimed increased sensitivity by two to three orders of magnitude by using electron capture GLC after reaction with peroxytrifluoroacetic acid. Sen (192B) also achieved electron capture sensitivity by reaction of nitrosamines with peroxide and trifluoroacetic acid a t room temperature to generate the nitramines. Capillary column GLC followed by mass spectrometric identification of nitrosamines was discussed by Heyns et al. (96B). Telling e t al. (215B) determined traces of volatile nitrosamines in meat products by simultaneously monitoring GLC effluent by flame ionization and mass spectrometry a t the NO+ peak. Gough et al. (82B) discussed the use of a membrane separator for simultaneous GC-MS operation. The results of analysis of 51 various meat products were reported by Fazio et al. (59B) and 5 ppb was found to be the highest level. Fazio et al. (56B) determined traces of nitrosamines in smoked fish by GLC and confirmed them by MS. Crosby et al. (32B) also studied a series of foods for nitrosamines, utilizing combined GC-MS if preliminary GLC with nitrogen selective detection proved positive. Bryce et al. (26B) also screened meat proqucts for nitrosamines with GC-MS and discussed techniques for individual nitrosamines. Polycyclic aromatic hydrocarbons were extracted from smoked meat, separated on cellulose acetate-alumina TLC plates and identified by in situ fluorescence spectra by Toth (223B). White et al. (244B) developed methods for determining polycyclic aromatics in liquid smoke flavors. Fourteen polycyclic aromatic hydrocarbons in various food types were separated using solvent partition and column chromatography by Grimmer et al. ( 8 3 8 ) . Heffter (92B) analyzed for 3,4-benzopyrene in oils using a silica column cleanup and alumina TLC. Chiusa et al. ‘(28B)investigated the aromatic hydrocarbon content of olive oil. Levels of 3,4-benzopyrene in grains were established by Hertel et al. (93B) using a final TLC separation after initial Sephadex LG20 chromatography. Shiraishi e t al. (194B)determined 3,4-benzopyrene in Japanese tea. A review by Van Schothorst (2368) covered the detection and identification of antibiotic residues in animal meat. A bacteriological zone inhibition method for various antibiotics in food was described by Murakami et al. (148B). Forschner ( 6 8 8 ) reported a modified agar diffusion method for antibiotics in milk with the inhibition zones perceivable after a 4-5 hour incubation period. A technique involving zone inhibition after agar-gel electrophoresis for the analysis of antibiotics in food was demonstrated by Dubost et al. ( 4 7 B ) . Tysset e t al. (226B) pro-
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posed a nephalometric procedure using the Sarcina lutea organism to detect streptomycin residues in honey. Chloramphenicol in foods was separated and detected using thin-layer chromatography after an alumina column cleanup by Takeshita et al. (213B). Debackere et al. (35B) extracted tylosin from feeds and biological samples with chloroform-ethyl acetate and then did two-dimensional TLC for estimation.. A method was automated for sulfamethoxine in feeds that performed the Bratton-Marshall reaction on manually digested samples by Araujo et al. (5B). Variations in results for chlortetracycline, tetracycline, monomycin, and streptomycin in cow's and sheep's milk due to protein binding in a n agar diffusion method were reported by Danielova et al. ( 3 3 8 ) . The trifluoroacetyl derivative of 2-aminoquinoxaline was determined by electron capture GLC by Crisp (31B) in a sulfaquinoxaline procedure applicable to egg and poultry samples. Kroeller (12UB) reported a fast method for diethylstilbestrol detection in meat sensitive to less than 1 ppm. Smith e t al. (195B) measured diethylstilbestrol traces in meat a t the ppb level using a n electron capture GLC method after a solvent partitioning cleanup. Traces in feeds were also determined by electron capture GLC by Donoho et al. (46B) after ester formation by reaction with dichloroacetyl chloride. George et al. (BOB) reported the results of a collaborative study for the determination of roxarsone in animal feeds by spectrophotometry. Multiple residues of organic fumigants in cereals were determined by Malone (131B) using electron capture GLC after acid distillation and trapping in solvent. Gutsche et al. (85B) described a simple flame spectrometric method to detect bromide residues from methyl bromide fumigation of cocoa. Ionized and organic bromides in foods were determined by GLC, the inorganic having been converted first to 2-bromoethanol by ethylene oxide, in a paper by Heuser et al. (94B). Methyl iodide residues on grains were measured colorimetrically by Rangaswamy et al. (169B) after conversion to KI which catalyzed cerium(1V) oxidation of arsenic(II1). Wildbrett et al. (246B) presented a scheme for analyzing for traces of quarternary ammonium disinfectants in milk by spectrophotometry of the eosin complex. Van Gils (2348) detected residual chloramine T in dairy products with a color test. Kong Tse Lam et al. (119B) used TLC after conversion of chloramine T to toluene-p-sulfonamide to detect traces in ice cream. N Acetylhexosamines in milk were determined colorimetrically by Kumar et al. (121B) by reaction with p-dimethylaminobenzaldehyde and formic acid. Nitrous oxide formed from heating meat with nitrites was trapped on silica gel and subsequently desorbed for GLC estimation in a procedure by Moehler et al. (142B).Acetone residues in oilseed meals were measured by a headspace gas chromatography technique by Dupuy et al. (48B). Fore et al. (65B) released residual hexane from oilseed meals and flour samples directly in the inlet of a gas chromatograph. Fore e t al. (66B) also determined residual isopropanol in similar sample types. Dichloromethane traces in decaffeinated coffee were gas chromatographed by Gal et al. (72B) after distillation. Schilling et al. (187B) distilled dichloromethane from roasted decaffeinated coffee and used electron capture gas chromatography. The residual acetone in pepper oleoresin was measured by GLC by Orsi (156B) after H20 distillation and benzene partition. Uchiyama e t al. (227B) used an acetylacetone reagent to measure formaldehyde in foods by a fluorimetric method. Positive interferences in the determination of formaldehyde in maple syrup were investigated by Underwood (229B) using GLC separations, and he (230B) reported that some
compounds codistilled in the AOAC method were identified and were not the cause. Chlorobutanol in milk was measured by electron capture GLC by Wiskerchen et al. (250B) after steam distillation and solvent partition. Stijve (206B) chromatographed 2-ch~oroethanol formed from ethylene oxide fumigation using a Porapak Q column. Chlorohydrins in hydroxypropyl starch were determined after hydrolysis in a GLC method by Brobst e t al. (25B). Onley (155B) reported measuring ethylene thiourea residues in foods by TLC and also GLC of the l-bromobutane derivative. Berry et al. (14B) reported an automated method for cyclohexylamine in cyclamates that used a diazotization reaction. Erskine et al. (54B) described colorimetric and TLC methods for dicyclohexylamine in sodium cyclamate. Fazio et al. (57B) listed results of a collaborative study of a GLC method for cyclohexylamine in foods. Fazio et al. (58B) reported results of an analytical survey of foods for cyclohexylamine content. Gunner et al. (84B) were able to measure traces of cyclohexylamine in cyclamates by electron capture GLC after reaction with picryl chloride. Solomon et al. (196B) also used EC GLC after derivative formation with l-fluoro-2,4-dinitrobenzene.Howard et al. (IOUB) published a GLC method for ti,aces of dicyclohexylamine in cyclamates. Rohleder et al. (180B) determined various plasticizers in fatty foods by gas chromatography with direct injection onto a precolumn in work to investigate migration from plastics. Sampaolo et al. (183B) used gas chromatography to study migration of dioctyl phthalate to foods from rubber. Wildbrett ~t al. (245B) presented an analytical scheme to determine monomer plasticizer transfer to milk and milk fat to.plastics. Migration of organotin stabilizers into beer from vinyl bottles was studied by Koch et al. (117B),who measured tin with catechol violet after acid treatment. Fishbein (63B) reviewed chromatographic and ecological aspects of polychlorinated biphenyls. Mulhern e t al. (149B) measured PCB components in tissue samples using a Florisil cleanup and TLC. Hayashi et al. (91B) determined biphenyl in 5itrus fruit by thinlayer and gas chromatographic techniques. Free chlorophenols in fats were quantitized with electron capture gas chromatography on various liquid phases containing phosphoric acid in a method by Ress et trl. (173B). Mineral oil pollution of fish meat was found t c i be correlatable with headspace gas chromatography by Deshimaru (42B) and he (43B) also analyzed fish for oil dispersers. Argemone oil was detected down to 0.005% in otker oils with SbC13 reagent in a colorimetric method by EIose et al. (17B). Gossypol in cottonseed oil was measured by an adsorption method on a column packed with nylon by Avlyanova et al. (6B). A qualitative test for hydrogen peroxide in herring roe was discussed by Fukumi e t al. (70B). Fukumi e t al. (71B) also devised a method to measure it in the bleaching waste. A gas chromatogranhic method for traces of phosphine from zinc phosphide in sugarcane was described by Robison et al. (17773)in which they evolved the phosphine from acid, trapped it in toluene, and then did their instrumental analysis. Berck e,: al. (13B) also did gas chromatographic analysis of phosphine residues in foods, comparing microcoulometric, thermionic, and flame photometric systems. Small amounts of cyanide residues in wines and spirits were rapidly screened for using test papers in a method by Bates (8B). Johansson e t al. (109B) identified methylmercury compounds extracted from fish by combination gas chromatography-mass spectrometry. A rapid method for methyl mercury in fish involving serial solvent partitioning and GLC was described by Uthe et al. (231B). Kins (114B) reported modi'ications of the Higgin-
ANALYTICAL CHEMISTRY, VOL. 45, N O . 5, APRIL 1973
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botham method for chick oedema factor to shorten iinalysis time for fatty samples. Tafuri et al. (211B) iiivestigated a GLC procedure to determine chlorocholine chloride residues in tomato juice by reacting it with sodium benzenethiolate. Residual surfactant from rice wmhing was measured by Iimori (101B) using Abbot’s method after acid hydrolysis and solvent removal of oily material. A paper chromatographic procedure enabled Klochkova et al. (115B) to determine polyethylenepolyamines in syrups. Lee (126B) extracted 2-pyrrolidone-5-carboxylicacid from foods with ethyl acetate and then formed the trimetliylsilyl derivative for GLC analysis. Seeley et al. (189B) measured abcissic acid in apple juice by electron capture GLC after methylation with diazomethane. Opium in teii was detected by Bose (16B) using a microscope slide scale TLC separation. An alumina TLC separation enabled Abbasov ( I B ) to determine 2,4-dichlorophenoxyacetic acid residues in milk and meat. The impurity 6,6’-oxydi(iiaphthalene-2-sulfonic acid) in FD & C Yellow No. 6 food dye was separated on a cellulose column by Marmion c’t al. (134B) but Marmion (133B) reported insufficient se claration for quantitative analysis. Basic dyes, not legal food additives, were separated and detected on polyamide thin layer plates by Takeshita et al. (21223). Foreign pigments in spice extracts were identified by a TLC technique by Lehmann et al. (127B). A spot test on filter paper for urea in foods that yielded some separation from interferc ]ices was described by Roy et al. (181B). Thrasher (218B1detected wheat grains contaminated with urine by contacting the grain with the urease-bromothymol blue reagent in an agar medium. This author (221B) later suggcsted additional changes in the medium. He (222B) also described a two-dimensional TLC method for detection of urine on wheat. Analytical techniques for detecting extraneous mat xial in food have also evolved. Brickey et al. (23B) published a review on the subject. Jackson (106B) estimated shell in chocolate products. Leach (125B) detected isinglass finings in beer by hydrolysis and colorimetry witk 4dimethylaminobenzaldehyde reagent. Winter reported collaborative results for soil in frozen spinach and stiawberries. Light filth methods have been reported for many food types such as nutmeg by Vasquez (237B),ground coffee by Thrasher (22OB), ground chicory by Thrasher (219B), bread and doughnuts by Thrasher (217B), w h a t germ by Roaf et al. (175B), high bran breads by M ller (141B), white flour by Miller (140B),other grain flour: by Miller (139B), soya flour by Dent e t al. (39B), wheat ,;luten by Brickey et al. (22B), and starches by Gecan et al. (78B). The isolation of light filth from nutmeat has btlen accomplished by Gecan et al. (79B), from dehydrated potato products by Gecan (76B), from pork sausage by this same author ( 7 5 8 ) from spices, fruits, and candies by Hanssen (89B), from water insoluble candies by Ge :an (74B), from maize and rice cereals by Dent et al. (4G13), from ground pepper by Dent et al. (38B), from wkole wheat cereals by Dent et al. (37B), from canned fish by Dent (36B), from cocoa press cake by Brickey et al. (2113), from casein and sodium caseinate by Brickey (18B) rnd from crude and refined papain by this author (19U). Gecan et al. (77B) separated larvae from pecan pieces i 1 a rapid method. A bleaching and staining procedure for insect fragments and rodent hairs in cocoa products iias given by Brickey et al. (20B). Salwin (184B) gave a report on progress in determining decomposition and filth in foods. Vandercook (233B) s t l d ied UV spectral changes in lemon juice with respect to i dverse storage. A turbidity test was useful to Weip?rt 82
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(242B) in detecting heat damage in rye. Springer (197B) employed urea sedimentation values to indicate heat damaged wheat. Ritter (174B) discussed enzymic methodology to infer storage history. A collaborative study of a method for trimethylamine nitrogen in fish was reported by Boland et al. (15B). Gasco-Sanchez (73B) determined volatile amines from fish by GLC after sweeping them off with nitrogen and cold trapping to concentrate them. Wood et al. (251B) investigated lipid changes occurring in degraded fish tissue by column and gas chromatography. A simple method for fish freshness was described by Kobayashi et al. (116B) using column chromatography on Dowex-1 X4. Nakai et al. (151B) detected deterioration of whale meat by paper chromatographic determination of 1-methylhydantoin. Tanaka (214B) explored using TLC to separate nucleosides and nucleotides in a rapid method for meat freshness. A thin-paper chromatographic inosine and hypoxanthine separation was used to determine their amounts as muscle tissue breakdown products by Gissell et al. (81B). Staruszkiewicz et al. (201B)reported collaborative results for the GLC determination of B-hydroxybutyric acid in eggs to detect incubator rejects. This author et al. (202B) later investigated interfering GLC peaks in this method that might cause anomalous results. The formation of malonic dialdehyde during food irradiation was studied by Scherz (186B) using a colorimetric method. Nawar et al. (152B) reported using GLC techniques to de‘ tect hydrocarbons formed during pork irradiation. O’Sullivan (157B) correlated electrophoretic patterns of whey. protein with milk heat processing. A simple colorimetric test for detecting lipolytic milk rancidity was given by Nakai et al. (150B). Monikowski et al. (143B) assayed for lactoalbumin and lactoglobulin in milk as a sanitary index. Morr (146B) investigated starch and polyacrylamide gel electrophoresis as means of determining casein degradation in cheese. Moorhouse et al. (145B) applied a method for determining volatile reducing substances as decomposition indices to ground beef, shrimp, and peaches but found i t valid only for raw foods.
CARBOHYDRATES Continuing investigations in the field of carbohydrate analysis include both classical chemical and instrumental techniques. Recommendations for uniform methods of sugar analysis for the sugar industry were adopted a t the 15th session of ICUMSA (32C). Clarifying agents for the quantitative determination of sugars in tea were compared by Iwasa et al. (33C) and a combination lead acetate, lead hydroxide reagent is recommended. Vizhintaite et al. (87C) have studied five methods for the determination of protein precipitate volume during the polarimetric determination of lactose in milk. All were satisfactory; the simplest is the double dilution method. Copper I11 has been used as a titrant by Jaiswal (34C) for hyxose, ribose, allose, and other sugars. Copper-EDTA in sodium hydroxide has been suggested by Miyake (57C) as a replacement for Fehling solution. Enzyme methods for reducing sugars include the use of glucose and galactose oxidases by Hettinga et al. (28C), for the analysis of glucose, galactose, and lactose and the use of NAD galactose dehydrogenase by Moehler et al. (58C) to didtinguish between L-arabinose and D-galaCtOSe. The different sugars in fruit juices have been determined by Baumann et al. (3C) also using enzymatic techniques. Spectrometric techniques include the use of aromatic diazo compounds by Kachalova et al. (37C) for the quantitative colorimetry of reducing carbohydrates; the analy-
1973
sis of glucose, fructose, and sucrose mixtures by Garrett et al. (22C) by conversion of fructose into a chromophore. A new sensitive ultraviolet detection system for carbohydrates eluted from columns has been described by Katz et al. (42C) using the reaction of the carbohydrates with sulfuric acid. Spectrophotometric determination after reduction of copper sulfate, described by Lunder (50C) uses the reaction of reduced copper with ammonia. Tateo (79C) has determined lactose, sucrose, invert sugar, glucose, and fructose, basing the procedure on the color formed when picric acid is reduced by reducing sugars. A means of correcting for the interference from hydroxymethylfurfural has been proposed by Ershov (16C) using ultraviolet measurement of the interfering compound. Paper chromatography has been used by Sandke (70C) to determine glucose, fructose, and sorbitol using reflectance photometry for quantitation. Thin-layer chromatography of polyhydric alcohols obtained by reduction of monosaccharides has been described by Borisovich et al. (6C). Multiple development on cellulose thin-layer plates has been shown by Raadsveld et al. (65C) to be useful for the separation of reducing sugars. Sugars in cocoa have been identified by Luke (49C) using gas chromatography and thin-layer chromatography. Davies et al. ( 9 C ) have investigated the sugars in tomato juice using volumetric, colorimetric, and gas chromatographic methods. Gas chromatographic procedures have been described by Reid et al. (66C) using an iso-thermal procedure; by Sennello (73C) for fructose and glucose in syrups, and have been used by Stepak et al. (78C) to determine sugars in fruit juices. Other gas chromatography procedures include the determination of free carbohydrates in milk products described by Reineccius et al. (67C); a chloroform extraction procedure prepared by Partridge et al. (63C);and a method for the preparation of trimethylsilyl ethers in aqueous solution suggested by Weiss et al. (89C). A gas chromatographic method for monosaccharides described by Zanetta et al. (94C) uses the trifluoroacetate derivatives of the 0methyl glycerides. Improvements in ion-exchange chromatography of sugars include a method for the separation of mono-, di-, and trisaccharides using borate buffers a t high temperatures described by Floridi (19C); the use of finely sized resins, 88% ethanol as eluant, and automatic detection in the eluate described by Martinsson et al. (53C);and the separations of 28 sugars on Dowex 50W are reviewed by Walker et al. (88C). Liquid chromatography on a cation exchange resin has been used by Hobbs et al. (29C) to determine lactose in milk. Binkley et al. ( 4 C ) have recorded the IR spectra of those anhydrides of fructose likely to be present in molasses. Mono- and polysaccharides have been determined by Guilbault (23C) using fluorometric techniques. Lactose in milk has been determined by Desmaison et al. (IZC) using the o-toluidine reaction after hydrolysis, in meat products by Salzer (69C) using an enzymatic determination, and also in meat products by Karasz e t al. (38C) using a colorimetric procedure which has been adapted to automatic analysis (2OC). Enzymic methods for the determination of maltose, starch, and lactate have been described by Gutmann (24C). Methods for sucrose in milk products using polarimetry have been proposed by Vizintaite (86C), and using thin-layer chromatography by Valdehita et al. (84C). Sucrose in syrups and liquors has been determined by Mahoney et al. (51C) using gas chromatography. Thin-layer chromatography and spectrofluorimetric detection have been described by Saglietto et al. (68C) for the determination of raffinose and 6-kestose in
sucrose solutions. Enzymic methods for raffinose plus melibiose, and arabinose plus galactose have been described by Drawert et al. (14C). Sorbose has been determined by Igloy et al. (31C) in the presence of monosaccharides using thin-layer chromatography. Disaccharides have been converted into disaccharide alditols and determined by gas chromatography-mass (3pectrometry by Karkkainen (41C). Studies of pentosans in sorghum grains have been described by Karim et al. (39C, 40C). A colorimetric procedure for pentosans in cereals described by Schmieder (72C) involves the conversion of the pentosan to furfural and colorimetric determinaticln of the furfural. Birch et al. (5C) have published an investigation of the dextrose equivalents of maltodextrins and the mechanism of the Lane-Eynon titration. Sugars in cereal and baby foods have been determined by ion-exchange chromatography by Liljamaa et al. (48C) using refractometry for monitoring. A Bio-Gel P-2 column has teen used by Dellweg et al. ( I I C ) , to separate fermentakile sugars in wort and beer with automatic determination of the carbohydrates eluted. A similar column has b e m used by Trenel et al. (82C) to separate oligosaccharides up to 15 glucose units in wort. Oligosaccharides in soybean meal have been determined by Delente et al. (1OC) by gas chromatography. Techniques of thin-layer chromatography have been used by Damonte et al. (8C) for mono- and oligosaccharide separation on cellulose, by Mezzetti et al. (55C, 56C) for oligosaccharide separation on tungstic acid or molybdic acid impregnated silica gel, and by Mansfield et al. (52C) for densitometric measurement OF certain malto-oligosaccharides. Spitschan (77C) has suggested an improved technique using a preliminary pyridinc extraction to improve the separation of mono- and oligclsaccharides on cellulose thin layers. Chromatography on a cellulose column has been used by Otter et al. (61C) to determine oligosaccharides in wort, beer, and brewing syr ips. La Berge et al. (46C) have made a study of the factors affecting losses of sugars during acid hydrolysis of polysaccharides. Enzymic and chemical methods have been desciibed by Hashimoto et al. (27C) for investigations of the structure of coffee arabinogalactan. Amylose in milled rice has been determined by Juliano (36C) using an iodometric colorimetric: procedure, and by Sowbhagya et al. (76C) using a similar procedure. Amperometric titration with iodine has been described by Kopriva et al. (44C) for the determiration of amylose in potato starch. Polarimetric determina :ion after solubilization in dimethyl sulfoxide and precipitation with ethanol has been proposed by Wolf et al. (91C') for the determination of starch in maize; amylose is then determined colorimetrically with iodine. Hemicellulosw in rye grain have been determined by chromatography on DEAE-cellulose columns by Holas et al. (30C). Studies of the Evers polarimetric determination of starch in wheat, corn, and potatoes by Dudas (15C) show that dissolution time should be carefully controlled. Grinding in 90% dimethyl sulfoxide has been used by Garcia e t al. (21C) to determine starch in corn by polarimetric measurement. Methods for the determination of starch i i potatoes have been evaluated by Kiryukhim et al. (43C), and either an anthrone method or the Evers method with corrections appear to be most accurate. A method of counting starch grains microscopically has been proposed by Barbiroli (2C) as a means of determining the starch content in wheat flour. Saunders et al. (71C) have evaluated starch methods and their applicability to wheat milling fractions; iodine blue and anthrone metnods as well as methods based on starch-specific enzymes gave satisfactory re-
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sults. Proton magnetic resonance has been used t y Jaska (35C) to study starch gelatinization. Methods of assessing starch damage in flour have. been reviewed by Wil iams et al. (9OC); the Wooster test was recommended for reliability and convenience. Titration with iodine by di tference potentiometry has been suggested by Banks et al. ( I C ) as a tool in characterizing starch and determining amylme. Modifications of the IUPAC method for the determination of glycerol by the periodate method have b e m presented by Mormont (6OC). Trimethylsilation a !id gas chromatography have been used by Parker et al. (62C) for the determination of glycerol and sugars in beer. Thinlayer chromatography has been used by Tateo (UJC) to determine mannitol, glycerol, glucose, and maltose in food and wine, and by Coles et al. (7C) to determine sorbitol. Mannitol has been found by Thaler et al. (81C) tcl interfere with the determination of sorbitol by polariinetry. Ion-exchange columns have been used by Menger ( 5 X )to clean-up foods for sorbitol determinations. An improved procedure for the determination of galacturonic acid with carbazole has been suggeswd by Filippov (17C), who has also proposed (1%) a metliod for methoxyl in pectin substances using oxidation to form' aldehyde and reaction of the latter with chromotropic: acid. A rapid and simple scheme for the separation and iclentification of common hydrocolloid stabilizers has been proposed by Morley et al. (59C). Dische's method for pectin has been modified by Ponomareva (64C) using bortite to stabilize the color. Gel electrophoresis has been applied to pectin and pectic substances by Do et al. ( I 3 C ) . Glucosinolates have been analyzed by Underhill e t al. (83C) using gas chromatography. Silica gel columns have been used by Labourel et a / . (47C) to separate iiulin gluco-fructosans. Color reactions of eriodictyol and cmriocitrin have been described by Hasan e t al. (26C). Sarionin in refined beet sugar has been determined by Hanr;;is et al. (25C) using colorimetry with antimony pentachloride, and in soybeans by ion-exchange chromatography and thin-layer chromatogr,aphy by Wolf et al. (92C, 93C). Tomatine in tomato plants is determined, after extraction, by reaction with anthrone by Socic (75C). Gas chromtitography has been used by Sloneker (74C) to measure neutral aldoses and cellulose in whole and digested plant tissue. A micro method for the determination of cellulose in vezetable materials has been proposed by Vecher et a / . (135C) using anthrone colorimetric determination. A methoti for the determination of crude fibre in the by-products of the fermentation industry using acetic acid, nitric acid, trichloracetic acid digestion mixture has been proposed by Krueger et a / . (45C).
COLOR Analytical studies of food colors cover the determination and examination of both natural and synthetic pigments..Herrmann ( 1 7 0 ) has published a review on anthocyanins in foods including methods of analysis. A previously identified pigment in strawberries has been isolated by Wrolstad et al. ( 5 9 0 ) as a n anomer of pelargoniti-3glucoside using paper chromatography. Column chromatography on an insoluble poly (vinylpyrrolidone) has h e n used by Wrolstad e t al. ( 5 8 0 ) to separate the anthocpinins of strawberries, rhubarb, and raspberries for prep,irative work. Details of the separation and identification of the main pigments of several fruits have been given by Van Teeling e t al. ( 5 4 0 ) using the same type of columns. Anthocyanin pigments in red tart cherries have been ECParated by Dekazos ( 1 1 0 ) using paper chromatograpliy. 84 R
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Lees et al. ( 2 2 0 ) have described methods for flavanols and anthocyanins in cranberries using thin-layer chromatography and spectrophotometric measurement, and suggested ( 2 3 0 ) methods for the standardization of pigment assay in cranberries using acid-ethanol extraction and colorimetric measurement. The techniques of paper electrophoresis and paper chromatography have been used by Cansfield et al. (6D) to quantitate the measurement of cranberry anthocyanins. Purified pigments from grapes have been isolated by Hrazdina ( 2 0 0 ) using column chromatography on Polyclar AT. A color reaction for catechins with 4-dimethylaminocinnamaldehyde has been used by Thies et a / . ( 4 8 0 ) as a spray reagent for thin-layer chromatography and as a means of spectrophotometric determination. Studies of catechins in grapes, husks, vinegar-wines, and vinegars have been made by Berger et al. including separation and quantitation (3D), and identification of vinegar-wine made from repeated use of grape husks ( 4 0 ) . A spray of picric acid in methanol has beenaused by Tirimanna et al. ( 4 9 0 ) to detect tea catechins on paper chromatograms. A rapid method for the estimation of tea total catechols using cellulose thin-layers has been described by Ozhindzholiya et al. ( 3 6 0 ) . Carotene stereoisomers in vegetables have been separated by Sweeney et al. ( 4 6 0 ) ,using column chromatography. Carotenoids of citrus peel hagre been separated by liquid chromatography on Sea Sorb 43, and xanthophylls on zinc carbonate columns by Stewart'et al. ( 4 7 0 ) . An improved procedure for carotenoids in egg yolk presented by Vogtmann e t al. ( 5 6 0 ) uses special extraction procedures before column chromatography and spectrophotometric measurement. Foppen ( 1 3 0 ) has published tables for the identification of carotenoid pigments including spectral data, melting points, and other physical data. The various types of chromatographic procedures used for separation and identification of hop hydroxyflavones and flavans have been described by Vancraenenbroeck et al, ( 5 3 0 ) . Sephadex separation of tannins in wort and beer and their spectrophotometric measurement has been developed by Yadav (60D). Gas chromatography has been used by Coffin e t a / . ( 8 D ) for the analysis of the trimethylsilyl derivatives of flavanones. Separation of tannins from fruits and vegetables using precipitation with methyl cellulose has been proposed by Nakabayashi et al. ( 3 4 0 ) . Flavanols in tea have been measured by Collier et al. (IOD) using gas chromatography after extraction into ethyl acetate and silylation. Ullah ( 5 2 0 ) has proposed a simple spectrophotometric method for determining theaflavins and thearubigens in black tea liquors using spectrophotometric analysis, and Lea et al. ( 2 1 0 ) have described the separation of theaflavines on Sephadex LH-20. Theaflavines have also been determined by gas chromatography of their trimethylsilyl ethers by Collier et al. (9D). The pigments and polyphenolic compounds of Montmorency cherries have been described by Schaller ( 4 0 0 ) . A thinlayer procedure on Kieselgel-G has been used by Vinkler ( 5 5 0 ) to determine the pigment content in the pericarp'of paprika. A study of the interaction of the food colors erythrosine and rose bengal with protein and of the fluorescence produced has been made by Aizawa et al. ( I D ) . A scheme for the isolation and detection of natural coloring matters in mayonnaise has been proposed by Benk et al. ( 2 0 ) using liquid-liquid partition and column and thin-layer chromatography. A method for the determination of betanin in jellied meats has been described by Spell (43D), using thin-layer techniques after separation, as a means of dis-
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tinguishing between beet juice and red wine in the product. Methods for the determination of dyes in food include a rapid method described by Lehmann et al. ( 2 4 0 ) for the identification of synthetic water-soluble coloring matter in foods using polyamide powder columns and subsequent elution. The stability of FD&C Red No. 2 in baked goods has been investigated by Singh ( 4 1 0 ) using ion-exchange chromatography and 40% of the color was found to be decomposed. A paper chromatographic method has been used by Hartman et al. (150) to detect metanil yellow and vanaspati in butter. Baker’s yeast has been used as an adsorbent by Onozaki et al. ( 3 5 0 ) to detect coal-tar in candies. Lehmann et al. have published methods for the detection of synthetic dyes in liquid egg ( 2 6 0 ) , in milk products ( 2 7 0 ) ,in pastries and dough products (280), and in wines and fruit juices ( 2 5 0 ) , using polyamide powder separation and chromatography after extraction. A method for Azo Rubine in skim milk and skim-milk powder has been proposed by Hendrickx et al. ( 1 6 0 ) . Added color in milk and milk products has been determined by Dhar et al. ( 2 2 0 ) using direct extraction after protein coagulation. Dyes added to mustard have been determined by Lehmann et al. ( 3 0 0 ) , who have also determined foreign dyes in spice extracts ( 2 9 0 ) . Paper chromatography has been used by Woidich et al. ( 5 7 0 ) to detect artificial water-soluble dyes after isolation by the wool-dyeing technique, by Tobolina et al. ( 5 0 0 ) for the separation of color components in sugar caramelization products, and by Mathew et al. ( 3 3 0 ) after adsorption on alumina powder. Thin-layer chromatography has been used by Hoodless et al. (180) to separate and identify water soluble food colors, by Chiang et al. ( 7 0 ) to study yellow food dyes, and by Bose et al. ( 5 0 ) to analyze oil-soluble dyes from foods after adsorption with silica gel. I t has also been used by Hoodless et al. (190) for oil-soluble food colors, by Rai ( 3 7 0 ) for both water-soluble and fat-soluble food dyes, and by Turner et al. (510) for blue triphenylmethane dyes. A technique for the evaluation of color additives using a differential-scanning calorimeter has been developed by Marmion ( 3 1 0 ) . A study of the properties of bixin and norbixin and of annatto extracts has been made by Reith et al. (380). Studies of Citrus Red No. 2 have been made by Stein ( 4 4 0 ) , as well as studies of subsidiary colors in FD&C Green No. 3 (450). Other compounds have been determined by Singh ( 4 2 0 ) in FD&C Blue No. 2 by column chromatography, and Marmion ( 3 2 0 ) has presented a column chromatographic method for uncombined intermediates in FD&C Yellow No. 6. Color studies of means of measuring the color of orange juice and orange juice beverages have been described by Rummens ( 3 9 0 ) , using a special hollow illuminating sphere, and by Gullett et al. ( 1 4 0 ) who suggest the use of a specific colorimeter.
ENZYMES
A review by Reed and Thorn (24E) dealt extensively with data and analytical methods for the enzymes of wheat and wheat flour. Wismer-Pederson (32E) authored a review covering enzymes in the meat industry. Methods for enzymes pertinent to predicting baking properties were discussed and reviewed by Becker (4Ej. Baerwald et al. ( 3 E ) compared the ASBC and dyed-starch methods for a-amylase in malt. Dellweg et al. ( 5 E ) reported a new method for cu-amylase utilizing azo dye-bound starch.
Lauber (17E) described a kinetic methcld using an iodineformamide reagent. a- and @-amylase enzymes from barley malt were separated on carboxymethyl-cellulose by MacGregor et al. (19E)using a Na+ gradient. Trachman et al. (32Ej automated the ASBC method for malt a-amylase. Disk electrophoresis a t low rathei than high pH was recommended for separation of miilt a-amylases by MacGregor et al. (20E). Olered et al. (22E) postulated different a-amylase systems showing different electrophoretic patterns as responsible for sprouting and enzyme activity disparity in wheat. Patratii (22E) found a more rapid test for alkaline phosphatase in milk was possible using baaium rather than sodium p-nitrophenylphosphate. Sharma et ul. (30E) simplified the method of Kosikowsky by placing reagents and sample in a cellulose bag and measuiing p-nitrophenol in the diffusate directly. Kleyn (13E, 14E) used phenolphthalein monophosphate to measui e milk phosphatase activity. Kleyn et al. (16E) and Huang et al. (10E) reported on this technique using dialysis of the color, and collaborative results were also reporl ed by these authors (15E). Rammell et al. (23E) used this substrate to determine cheese alkaline phosphatase activity. An automated method for acid phosphatase in p h n t extracts was described by Schwerdtfeger (28E) utilizing sodium p-nitrophenylphosphate. Loane (18E) modified the Griess-11osvoy nitrate reductase activity test for milk to improve its efficiency. Ault ( 2 E ) recommended diastatic power as the measure of malt carbohydrase activity after collaborative testing. An assay for P-glucanase in malt flour utilizing dyed pglucan was devised by Zitting et al. (33E). Electrophoretic enzymograms were used by Gould e ! al. ( 7 E ) to test malic enzyme activity a s an index of whcther oysters were frozen and thawed. The lauric acid liberated from coconut oil was measured by GC in a method by Gross et al. (8E) for lipase activity in spices. Hide F’owder Azure was used by Savage e t al. (27E) as their dyed substrate to assay for proteolytic enzymes in beer. Leuc ne aminopepdidase in egg white was determined by Guenther et al. (9E) from the extinction of liberated p-nitroaniline from L-leucinep-nitroanilide reagent. Estimation of low levels of antitryptic activity in soybeans was iwestigated by Delobez et al. ( 6 E ) by following the degwe of trypsin digestion. Kaiser et al. ( I I E ) separated proteins from potatoes with polyacrylamide gel electrophoresis and identifed those with proteolytic enzyme inhibiting characteristics. Rapid screening of wheat grains for tyrosinase activity was described by Abrol et al. ( I E ) , using color development of wheat grain scrapings under a microscope when L-tyrosine was added. An electrochemical method for xanthine oxidase activity in milk was reporbed by Kiermeier et al. (12E) as suitable for routine use A rennet assay method based on turbidity measurement was investigated by Saberwal et ul. (26E). Papain in beer was determined by Scriban et al. (29E) employing Hide Power Azure, and measuring absorbance a t 595 nni. The polyphenoloxidase activity of coffee bean extract;; was investigated as a means of classifying bean types using an automatic colorimetric method by Rotenberg et a1 (25E).
FATS, OILS, AND I’ATTY ACIDS Wolff (120F) has reviewed the determination of fatty acids, unsaponifiable fats, and glycerides, giving special attention to the use of spectrophotometry and gas chromatography. A review on the 1,opic of separation of triglycerides by GC is also given by Kuksis (70F), and Kaufmann (63F) presents a review cn the use of column chroA N A L Y T I C A L C H E M I S T R Y , VOL. 45,
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matography for the analysis of lipids. Effects of simple storage time and potassium dichromate concentration on Milko-Tester results are reported by Kroger (69F), and Thomasow et al. (103F) compare the determination of fat in milk by means of both the Milko-Tester Automatic and the Milko Tester I11 with data obtained by the Roese-Gottlieb method. A procedure is presented for automating the determination of milk fat in fluid milk by forming a uniform suspension of the fat globules and then meas.iring absorbance a t 600 nm (101F) and Malanoski et al. I 74F) have made a comparison of a rapid instrumental fat determination (using the Digital Fat Controller) witt the official A.O.A.C. procedure. Wiggall et a / . (115F) describe the use of low resolution NMR as a means of rapidljr determining fat in chocolate and related products and videline NMR is also used by Shanbhag e t al. (89F)t o amlyze for oil in aqueous emulsions. A new milk butyrometer, which measures milk fat by the change in intensit,y of the secondary flourescence of fluorochrome dyes hound t c fat globules, is described by Konev et al. (68F), and a scminimicro method for determining lipids in fish meal which incorporates a compact extraction apparatus (66F, ;'7F) has been collaboratively studied and adopted as an A.O.A.C. official method (first action). Moreau and Idavoie have developed an emulsion method for the rapid tletermination of fat in raw meats (79F) wherein the en ulsion which is formed is treated with alkali to separate the fat which is then measured directly 'in a Babcock bot ;le, and Stahl (100F) describes a refractometric determination of crude fat in corn and corn processing products hy grinding directly with tu-bromonaphthalene prior to 1he refractometric measurement. Wray (121F) describes a n w technique named thermoreflectometric analysis by which setting and hardening of fats and couvertures is monitoIed by recording changes in surface reflectance measured with a photocell. Solov'eva et al. (98F) describe a rapid teclinique for estimating the fat content of preserved vegetiibles by extracting with dichloroethane in a microhomcgenizer and then applying a solvent aliquot to a tared f i l ter paper strip and drying to constant weight, and the conditions for a quick, repeatable procedure for analyzing fat in foodstuffs by use of a mixture of chloroform and methanol is provided by Southgate (99F). Samuelsson ct al. (88F) describe the use of NMR for the estimation of liquid fat in cream and butter, and Per1 et al. (83F) repoi,l on a rapid method for the analysis of fat in cocoa protlucts which incorporates a differential density techniquc . The amount of butter fat in chocolate has been examined in a comparison of t,he classical semimicrobutyric acid value and two gas chromatographic methods (47F), and Wilson (117F) describes a method for determining fat in soybean by an IR attenuated total reflectance technique, Harris (49F) provides a review of the applications of chro matographic and NMR procedures for determining fa1 and fatty acids in oilseeds and oilcakes. Applications of complexometric procedures wherein fat is measured by the amount of EDTA required to sequester the divalent cations precipitated by fatty acids resulting from the alkaline hydrolysis of fat have been made for cocoa beans, cocoa butter, and chocolate ( 3 1 F ) , eggs (YOE'), poppy and turnip seeds (33F), butter and cream (34F), and fat mixtures (326'). Methods are reported for examining the polymorphic behavior and thermal properties of cocoa butter by means of programmed temperature X-ray diffraction and differential scanning colorimetry (23F), and Bose (18F) describes modifications of the Gerber method to reduce interference by the presence of cane sugar in milk. High frequency conductivity is used to measure the 86 R
concentration of sunflower oil in hexane (8F). An accelerated refractometric method for determining fat in fish meal after a 5-minute extraction with a-monochloronaphthalene (37F) is described by Golovin et al., and Geyer and Red1 present a rapid method (36F) for determining fat in mayonnaise and salad cream. Conway reports (25F) on the use of wide line NMR to measure fat in moist samples of defatted corn germ and NMR has also been critically examined (43F) to determine sources of error when this tool is used for the determination of solid fat content. Goto et al. (39F), report on their work in using X-ray diffraction patterns for definition of the solids in vegetable fats a t various temperatures, and Bittenbender (16F) presents a new physical method for determining fat in foodstuffs by use of heptane solvent and especially constructed hydrometers, measuring the change of density of fat as the ratio of fat to solvent changes. Burns et al. (20F), report on a method for obtaining synergistic complexation effects for the TLC separation of certain triglycerides on Silica gel G plates impregnated with AgN03, and Hites (51F) describes a technique for obtaining qualitative mass spectra of triglycerides by placing the oil sample directly into the ion source. Argentation-TLC has been used for analyzing the triglyceride composition of milk fat (92F) in conjunction with GC, and the fatty acid composition of cod liver oil has been determined by urea fractionation and programmed temperature GC, both before and after hydrogenation (59F). Hadorn and Zuercher (44F) make use of GC to determine butyric acid in various fat mixtures as a measure of the milk fat present, using methyl valerate as an internal standard and Golovkin et al. (38F), present a GC method for analyzing the methyl esters of fatty acids in herring oil by first fractionating samples by means of silica gel TLC plates impregnated with AgN03, while Gedam et al., apply a similar method to the analysis of sardine oil (35F). A procedure for determining volatile fatty acids (C1-CI2) is described by Dhont et al. (2%') wherein the p-nitrobenzyl and p-bromophenacyl esters are separated on TLC plates, and Kat0 and Yamaura describe a method (61F) for the direct trans-esterification of soybean and castor oils in methanol-containing solvents by injecting them onto an NaOH-H20 precolumn equipped gas chromatograph. Shehata e t al. (91F) provide a technique for the preparation of milligram amounts of methyl esters of fats for gas chromatographic analysis. The formation of the trimethyl silyl derivatives of partially saponified olive, coconut, and palm kernel oils provided a means of obtaining gas chromatographic resolution (86F) of many components which would otherwise not have been resolved, and Tiscornia and Bertini have reported (105F) on the chemical composition of olive oil applying a variety of procedures to 150 olive oils. Walker ( 1 1 1 F ) describes a novel charring technique for detecting lipids on TLC plates (claiming the detection of as little as 0.01 wg of lipid), and a review is given (19F) of physical, chemicai, and enzymic methods used to determine the fatty acid composition for each position on the glycerol molecule. Zuercher (123F) reviews the occurrence of lipids in malt and beer and describes TLC methods for effecting the isolation of various lipids from these products, and Berner provides a method ( Z I F ) for TLC-enzymic determination of mono-, di-, and tri-glycerides in glyceride mixtures and soybean oil (12F). Bezard et al. report on the GC analysis of the triglycerides of coconut oil (13F),and TLC and GC are used by Delanghe (27F) to characterize hop wax. Hardy and coworkers (48F) report on a column chromatographic method for the fractionation of fish neutral lipids
A N A L Y T I C A L CHEMISTRY, VOL. 45, N O . 5, APRIL 1!)73
and MacMurray e t al. (72F) have developed a procedure for elucidating wheat flour lipids by sequential extractions and chromatographic procedures into individual components. Timmen and Dimick (104F) characterize the major hydroxy compounds in milk lipids by forming the pyruvyl chloride 2,6-dinitrophenylhydrazonederivatives and then separate these with TLC and LC methods. Quackenbush (87F) reports satisfactory results from a collaborative test of a method for analyzing total fatty acids plus unsaponifiable matter that employs a newly designed liquid-liquid extraction apparatus which permits removal of hexane soluble unsaponifiable matter as a separate fraction. Pokorny e t al. (84F) describe a paper chromatographic method for the analysis of fatty acids and their derivatives on urea impregnated paper using an ascending technique, and a GC method is given for determining (C, to CIS) free fatty acids in cheese ( 8 I F ) after first effecting separation of the FFA on a silica gel column. Moerk (78F) presents a method for determining linoleic and linolenic acids by isomerization a t low temperatures by use of the potassium alkaoxide of triethylene glycol monomethyl ether in 1,4-dioxane, and a GC method employing a terephthalic acid-Haloport column for direct determination of the higher fatty acids of Emmental cheese is also described (76F). Investigation (60F) showed hydrogen chloride to compare well with boron trifluoride as a catalyst for preparing methyl esters of fatty acids from margarines, rape, soybean, and sunflower oils, and a new method, based on reacting potassium soaps with ethyl iodide, is reported as a n esterification procedure for use in TLC and GC studies of butter fat (56F).A statistical examination (50F) was made of the results of a collaborative GC study of standard mixtures of methyl esters and selected oils t o provide a measure of the precision of each analysis, and Barnes e t al. ( I O F ) describe a simple technique for methylating the fatty acids of peanut oil directly in the ground peanuts prior to GC analysis. A method (52F) based on converting long chain (C14 to CIS) fatty acids into corresponding 60Co salt, and measuring the radioactivity in the salt, provides a simple, ultra-sensitive determination of free fatty acid, and Waltking (112F) reports on studies which confirm the reliability of the lipoxidase enzymic method for measuring essential fatty acids for supporting nutritional statements. Artman and Smith (6F) describe work on the systematic isolation and identification of minor components in heated and unheated fat (cottonseed oil) by use of silica gel column chromatography followed by GC (observing 136 components in 0.42% of the fat). Gracian e t al. (40F) present a TLC procedure for the identification of nonglyceride esters in edible oils ( L e . , such as those of ethylene glycol, 1,2-propylene glycol, and 1,3-propylene glycol dioleates) and Bier1 et al. (14F) describe a method for locating epoxide positions by reacting fat compounds with H I 0 4 and then analyzing the products formed by GC. Craske e t al. (26F), describe a technique employing column chromatography of the bromo-mercurimethoxy adducts of fatty acid methyl esters as a means of isolating polyenoic acids present in low concentrations and demonstrate its use by analysis of safflower oil, mutton tallow, and lipid extracts of beef perirenal and pericardial tissue. Coleman (24F) presents an evaluation of five methods for the quantitative determination of cyclopropenoid fatty acids, finding GC of the methyl mercaptan derivatives and titration with hydrobromic acid in toluene to be unsuitable and the Halphen test to be the best general method when properly calibrated. Katte e t al. (62F), provide a spectrophotometric method for determining stercu-
lic acid using the Halphen reaction m d Sheehan et al. (9OF) describe an improved Halphen method for measuring cyclopropenoid fatty acids. Sioufl’i (97F) reviews the characteristics of prooxygenic substances and methods for determining them in fats and oils. Methods for analysis of heated oils are compared ( I I 3 F ) for their general applicability and Witte et al. (118F)present a new extraction method for determining 2-thiobarbit uric acid values for pork and beef during storage. A met’iod is reported ( 7 F ) for the estimation of free malmaldehj.de in vegetable oils, including its application for following the changes in autoxidized fats in relation to peroxicle value, TBA. value, and rancidity development, and Holland (53F) describes the determination of malonaldehyde as an index of rancidity in meat products, wherein nut:; judged to be slightly rancid by a taste panel were found to contain approximately 40 gg of malonaldehyde/gram of nut meat, with no malonaldehyde being detected in fresh nut meats. The Shaal test was investigated (82F) as to its applicability for determination of the oxidative stability of fats and oils by comparing both taste and peroxide values a t suitable intervals and Valencia e t al. (107F), describe a technique for determining the peroxide value cf lean meat by colorimetry after extraction with chloroform in a homogenizer. For several vegetable oils, the average molecular weight, iodine number, average number of vinyl groups, and a “polyunsaturation factor” were determined by means of a high resolution nuclear magnetic resonance spectrometer which was controlled by a dedicated computer system (95F). Koman (67F) provides a GO procedure for determining unsaturated fatty acids by e ifecting hydrogenation on a catalyst-containing precolumn and comparing chromatograms obtained with and without the catalyst as a means of identification of acids in mixtures. Bitner et al. (15F) have developed a procedure for the simultaneous conjugation and formation of methbl esters of polyunsaturated triglycerides using methanolic tetramethylammonium hydroxide in a microreaction chamber preceding a gas chromatograph, relating the rimount of conjugation that occurs to the amount of linolcic acid in the sample. Bailey et al. ( 9 F ) , make use of Raman spectroscopy for analyzing the cis/trans isomer composition of edible vegetable oils, and Haeffner (42F) describes the separaLion of the CZOunsaturated fatty acids from rapeseed oil by counter-current distribution. A rapid method is presented ( 2 F ) for determining trans-unsaturation in fats and fat derivatives by use of IR absorption values a t two wave-lengths and IR is also used (5F) as a mean; of determining the degree of unsaturation of nonprocessed fats and oils. Differential UV spectra are used to deteiaiine the quality of purified cocoa butter (45F, 46F) by measuring the specific absorption regions for peroxides, dienes, and trienes; and Huang and Firestone (55F)have made use of differential IR spectroscopy to determine low levels of isolated trans isomers in vegetable oils and derived methyl esters. Arana Campos (4F)provides a new method of separating unsaponifiable matter from f i t , and Tykheeva et al. (106F) present their work on the spectrophotometric and TLC analysis of the nonsaponifialde fraction of wheat lipids. Unsaponifiables from butter have been analyzed by TLC (73F), and a micromethod fcr the quantitative determination of cholesterol, cholesteryl palmitate, palmitic acid, and mono-, di-, and tripalmitins by TLC is also given (I16F). The preparation anll separation of the unsaponifiable fraction of olive oil and subsequent determination of the sterols by GC is d e s c d ~ e d(75F), and LaCroix e t al. (71F) compare the Roese-Gottlieb and silicic acid extraction procedures for the determination of cholesterol A N A L Y T I C A L CHEMISTRY, VOL. 45, NO. 5,
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in dairy products and recommend either the former or a similar multiple-solvent extraction. Use is made of YLC t o separate phytosterol from vegetable oils and sterol concentrates prior to obtaining quantitation by use of cdorimetry (57F, 58F) and Alcaide et al. ( I F ) describe '3'LC and M S procedures for analysis of triterpenes and sterols of coffee oil. Homberg et al. (54F) have elucidated the more significant sources of error encountered in the analysis of sterols and describe a chromatographic mer.liod for their isolation from fats and oils. The composition of the bound lipids in caseins and in ripening cheeses have been analyzed by histochen ical means ( I 7%') including neutral lipids and phospholipids and a correlation has been established between beef niuscle total lipids and phospholipids whereby the latter can be estimated by use of a regression equation from the former (21F). Volatile fatty acids, alcohols, and carbonyls produced from soybean phospholipids by autoxidai ion were analyzed by GC and TLC by Shibasaki et al. (5;?F, 94F), and Singh et al. (96F) have used column chromatography and TLC for the analysis of the glycolipids m d phospholipids of immature soybeans. Vioque et al. (1113F) make use of TLC and densitometry to analyze cholestfryl esters, triglycerides, fatty acids, and cholesterol on silica gel plates, and TLC methods are also described for delermining phospholipids in meat (80F), butter (122F), slybeans (64F, 65F), wheat flour, rice, fish (3F) and cit-us juice (108F). Vaver et al. (109F) describe a procedure for the simultaneous gas chromatographic determination of diol esters and triglycerides, Chakrabarty and Kundu provide a photometric method for the microdetermination of hydroxy lipids (22F), and a gas chromatographic method for the determination of free and esterified polyalcohols in fats has also been developed (41F). TLC methods are presented for the determination of elaidic acid in food fat (119F), for the estimation of EUcrose esters of palmitic acid (114F), and for separattm and analysis of ferulates in rice bran oil (102F). Pongracz reports on a method for the direct titration of ascorbylpalmitate in edible oils and fats (85F), and a colunin chromatographic method has been developed by Fujisl ima et al. (29F) for the isolation of ceramide from cheese.
FLAVORS A N D VOLATILE COMPOUNDS Kolor describes the application of mass spectrometric techniques to the specific analysis of food flavors (52G), and Issenberg et al. (40G) review methods of analyzing volatile food flavors by gas chromatography, including spccia1 attention to those isolation and concentration techniques that have proved useful. Teranishi's book (118G') on principles and techniques used in flavor research also serves as a reference work for food analysts dealing with analysis of food flavors. Conditions are given for the folmation of various derivatives (32G) for the GC identification of alcohols, primary and secondary amines, and thiol3 in food aromas, and Sanders and Schubert (103G) provid? a method for colorimetrically determining carbonyls in the presence of carbohydrates using 2,4-DNP under conditions that suppress reaction of the reagent with the latter. A TLC method (134G) for simultaneously determining 5 hydroxymethyl-2-furaldehyde and 2-furaldehyde in thth presence of ?ach other is reported as is a colorimetric method (65G) for determining thiols and HzS in foods Vogh (123G) describes a general procedure for the extrac. tive isolation of carbonyl compounds as oximes prior t c GC analysis using glass equipment. Andersen provides a GC method for the determination of allyl isothiocyanate in mustard seed (3G) and compares 88 R
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recoveries obtained with this method to the A.O.A.C. official method, and Modzelewska (73G) describes the GC analysis of isothiocyanates in Cruciferae oil seeds. A volumetric determination of allyl isothiocyanate ( I 7G) in black mustard (Brassica nigra) is described which is based on its reaction with piperidine or pyrrolidine to form the corresponding monosubstituted thiourea, and Shankaranarayana et al. (11OG) also describe an oxidimetric method for determining allyl isothiocyanate in black mustard by use of chloramine T and then back titrating after reaction to determine the excess chloroamine T with KI and standardized thiosulfate solution. Titrimetric methods are also described for the determination of p hydroxybenzyl isothiocyanate in sinalbin (95G) and in white mustard (109G). A TLC method for the determination of capsaicin in hot pepper paste is described by Yum (139G) who makes use of a spectrophotometric azo dye method and reports the loss of 30% of capsaicin after 15minute cooking. Mathew et al. (70G) also describe a n improved method for determining capsaicin in Capsicum oleoresin by scraping the capsaicin spot from a TLC plate and determining it colorimetrically with Folin-Denis reagent, and Spanyar and Blazovich (115G) provide a TLC determination for capsaicin in ground paprika, locating the spots by spraying with FeC13-K3Fe(CN)e solution. The determination of capsaicin in chili is also effected (100G) by UV reflectance photometry after its TLC isolation, and a rapid GC method is presented by Hartman (35G) which incorporates use of an alkali flame detector after silanization of capsaicin directly in the oleoresin or spice extract, thus obtainihg improved sensitivity over a standard FID. Mueller-Stock et al. (80G) also describe a GC method for capsaicin, dihydrocapsaicin, nordihydrocapsaicin, and vanillylpelargonic amide after formation of the corresponding trimethylsilyl derivatives. A GC method is presented (50G) for analyzing the volatile components of horseradish and black mustard which can be used for calculating the mixing ratios of these in commercial powders, and De Cleyn and Verzele (26G) provide TLC and high-pressure liquid chromatography techniques for determining piperine and its isomers in black pepper. TLC and GC methods are presented and compared by Connell et al. (20G) for the analysis of gingerols, shogaols, paradols, and related compounds of the oleoresins from ginger and grains of paradise. Van Gils et al. (122G) provide a rapid spectrophotometric determination for quinine in tonic waters and Hey (38G) presents a method for isolating quinine from food by extraction and then quantitates it by either TLC, UV spectrophotometry, or fluorescence photometry. A method for the quantitative determination of ammonium glycyrrhizinate is reported (54G, 55G) wherein the analysis is achieved by GC of the silyl ether derivative of the aglycone. Porcaro et al. (92G) report on the use of liquid crystals as substrates in the GC separation of aromatic terpene isomers and derivatives known to be difficult to resolve by phases most frequently used in packed columns and Rasmussen et al. (97G) describe a technique for determining terpenes and rklated substances in sage leaves by GC by placing small amounts of sample directly into the inlet of the instrument. Potter (93G) reports a GC method of determining nonvanillin vanilla volatiles in vanilla extract, and GC-MS methods are provided in a study (56G) of vanillin related compounds after derivatization. GC-MS is also used by Kahn et al. (45G) to identify volatile components in vinegars. Markova et ai. (66G) describe GC methods used to determine the flavor components of bread and of the flavor intermediates formed dur-
ing its preparation and baking, and a method of determining monocarbonyls in wheat bread crumb and crust by GC (141G) is given which makes use of 2,4-DNP derivatives to achieve the isolation, regenerating the carbonyls prior to analysis. A similar GC method is described in a study of changes of the carbonyl compounds of bread through storage ( 7 G ) . Determinations of aroma components among several types of black tea were made and compared using GC (SSG, 102G), and Sato et al. (105G) report on the analysis of the intermediate and high boiling neutral components of black tea flavor as determined by use of distillation, silica-gel chromatography, and gas chromatography. Pypker and Brouwer (94G) describe a method by which enrichment samples of the lesser volatile components of coffee can be concentrated a hundred-fold and then be analyzed by gas chromatography in a manner producing good resolution. A combined programmed temperature GC-MS procedure is presented for analysis of fractions of green and roasted coffees in a study (71G) showing the relationship between volatile compounds in roasted coffee to their precursors, and Daniel et al. (23G) present polarographic and spectrophotometric procedures for the determination of pyrazines in coffee and cacao. Methods for isolating and identifying volatile compounds of cocoa are reported by Ketenes et al. (47G) with separations being achieved by GC and identifications made by various spectral means. An original method is presented by Mitjavila et al. (72G) for the separation and colorimetric titration of astringent polyphenolic fractions of various beverages. Coppini et al. (21G) propose a n infrared spectrophotometric method for determining low-molecular-weight monohydric alcohols after esterification with " 0 2 , using the -NO stretching band as the basis of the measurement. A rapid headspace GC technique (84G) is described for the estimation of certain amino acids as a means of indicating differences in the protoeolytic enzyme system of wort and Trachman et al. (120G) report on a simplified GC method for determining beer volatiles. A GC method for analyzing for whydroxy ketones in fermentation solutions and beverages (IOIG) is described by Ronkainen and Brummer wherein the 2,4-DNP derivatives are formed, separated on activated carbon, and chromatographed after conversion to the corresponding ketones using electron capture detection. Sinclair et al. (113G) present a GC method for determining dimethyl sulfide in beer and lager, and a descending paper chrqmatographic method (114G) is described for the analysis of volatile amines in wort, beer, barley, malt, and hops. Electron capture gas chromatography is used by Steffen (117G) for determining vicinal diketones in beer headspace, and Zenz et al. (140G) describe a GC method for determining the 2-methyl-2-butene content of beer aroma as a measure of typical light damage in bottled beer. A TLC method is given for analyzing methylglyoxal in beer after formation of its osazone (87G) and the amounts found are correlated to off-flavor development of beer, and Baerwald and Miglio (6G) describe the use of a flame photometric GC method for determining volatile sulfur compounds in hops. A colorimetric determination for analyzing volatile phenols (5G) is presented for use on distillates from beer; Rebelein (98G) reports a rapid and direct colorimetric method for determination of acetaldehyde and acetaldehyde-bound sulfur dioxide in wine, and an improved automated determination for ethanol in beer and wine is described (57G) by Lidzey e t at. Reinhard (99G) provides a GC procedure for determining higher esters and phenethyl alcohol in brandies by direct injection and Duro et al. (28G) describe the determination of meth-
yl alcohol in Sicilian wines making use of NMR. Techniques are provided for the GC deteimination of the higher esters in wine distillate, brandy, ilnd wine after extraction with CS2 (51G), and trichlorofluoromethane has been used (34G) as a means of concentrating the minor volatile constituents of a Riesling wine prior to analysis by GCMS. Liebich et al. (61G) report on the analysis of rum flavor by extraction and concentraticn of the volatiles in pentane and pentane-ether, followf d by GC separations and MS identification of approxim itely 200 compounds. The identification of 4-ethoxy-4-h~~droxybutyric acid ylactone (5-ethoxydihydro-2(3H) -fur anone) was accom plished ( 8 I G ) by use of MS, IR, arid GC retention times after its isolation from the aroma of Ruby Cabernet wine. A procedure for the separation and gas chromatographic determination of y- and &lactones in edible fats is described by Martin and Berner (68G), and Johnson et al. (44G) provide a sensitive method fcr the determination of carbonyl compounds in oxidized fats by separating them as their 2,4,6-trichlorophenylhydrazones,separating these on TLC plates and then determ.ning the 2,4,6-TCPH compounds eluted from the TLC plates by electron capture gas chromatography. Feenstra and Meijboom (29G) present their work in identifying '2-trans-nonenal as the hardened flavor present in hydrogmated peanut oil, and tandem GC-MS has been used (108G) by Selke et al. to analyze the volatiles from soybean oil through storage. A GC procedure for determining products from the aglucon moiety of the progoitrins in crambe and rapeseed meals is given (25G), and Shimizt et al. (112G) describe their work in the separation and analysis of furfuryl alcohol, acetylfuran, and 3-hydroxypy~idinefrom the neutral flavor fraction of roast barley, making use of liquid column chromatography, TLC, GC, m d MS. The 2,4-DNP derivatives of cooked rice carbonyl!; are analyzed by paper and column chromatography (4G) and Walradt et al. report on the isolation and mass sl3ectrometric identification of the volatile compounds of popcorn flavor (126G). The volatile components of roasted peanuts have been analyzed by GC-MS, including a number of compounds not previously reported, obtained from steam volatile fractions (127G) and also from methylene chloride extractions of a vacuum distillate (124G, 125G). Brown et al. (11G) describe methods for cornraring concentrations of aldehydes and ketones in raw and roasted peanuts by separating their 2,4-DNP derivatives into classes by column chromatography and then separating the various classes by TLC with the individual spots being dissolved in chloroform, and quantitation then achieved by spectrophotometry. Wang e t al. (128G) havs characterized volatile constituents from roasted pecans by fractionation into classes and then applying GC-MS and also using direct probe M S on isolated 2,4-DNP derivatives of selected carbonyls. Methods for determining the volatiles from roasted filberts are given (49G, 111G) using a variety of techniques for separation and identification. The volatile compounds of wl-ey powder subjected to accelerated browning were isolated and identified by GC-MS methods (30G), and use Lsmade of trifluoroacetyl derivatization prior to GC-MS fo* the analysis of isomeric cyclic amines in cheese and cho1:olate (16G). Dartney et al. (24G) have measured the rate of formation of methyl ketones during blue cheese ripening by 2,4-DNP isolation on columns of activated celite-Slea Sorb 43, followed by regeneration and final determination by GC. A review ( I G ) of methods for the extraction of cheese flavors is reported and various techniques applied to the isolation of Cheddar cheese flavor volatiles, prior to GC-MS analysis, A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 5, A P R I L 1973
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are also presented (58G, 59G). Use of GC headspace a i d ysis is described as a means of monitoring changes in ciirbonyls and fatty acids to determine quality of eggs ( 6 2 Y ) , and Staruszkiewicz et al. (116G) report a GC method lor determining P-hydroxybutyric acid in eggs after format on of its propyl ester. A colorimetric method is described (53G) for determining phenols in smoked fish after steam distillation and 1IV spectrophotometry is used (2G) to evaluate the quality of frozen fish as a means of measuring distilled carbonjls. Kim et al. (48G) have investigated GC, IR, MS, and NMR methods for determining the basic, neutral, aiid phenolic aroma compounds of dried bonito. A review is given of the literature on the isolation and analysis of 178 chicken flavor components (136G) including 11 not p‘eviously reported which were identified by GC-MS. The neutral volatiles from heated pork fat were determined by GC, MS, and spectroscopy (130G), and techniques are also described (41G ) for determining the phenolic components of smoked meat products. Piotrowski et al. (91G) describe TLC and GC procedures used to differentiate betwe :-n aromas of cured and uncured hams and methods are also described (14G) for the GC analysis of volatile fatty aci:ls and carbonyl compounds in fresh and stored hams. Metho :Is are given for the fractionation of shallow-fried beef flavor by silicic acid column chromatography (132G) prior to GC artd M S analysis. GC and MS techniques are also used to an3lyze beef fat for heated flavor compounds (131G) and :o analyze for alkyl substituted pyrazines and pyridines 11 fried beef (133G). Tonsbeek et al. (119G) describe a GC method for determining 2-acetyl-2-thiazoline in beef brot.i, and Halvarson (33G) reports a GC method for evaluatir,:: the low molecular weight monocarbonyls in meat product j. Using GC and MS, 17 alkylpyrazines were identified in ii pyrolyzed extract of fresh beef (31G), and Brinkman et a l . (8G) describe a method for the isolation of flavor volatiliv; of simmering beef broth. GC and M S were used to ani\lyze the volatiles from roast beef (60G), and a review 1‘3 given of thermally produced flavor components in tl-c aroma of meat and poultry (129G). Peri et al. (89G) present a scheme for separating phenolic compounds in vegetable extracts wherein tannins ale separated from nontannins by selective precipitation with cinchonine sulfate. Williams et nl. (135G) describe a GC method for determining dimethyl sulfide in processed veg etables, and Johnson et al. (42G, 43G) survey the volatilcb components identified in a number of vegetables. Simplta procedures are reported for the isolation, concentratior , and GC analysis of vegetable oil volatiles (27G, 36G), and MacLeod et al. (63G) have used GC to analyze flavor volatiles of cooked Brussel sprouts, cauliflower, beans, and runner beans. Brodnitz et al. (9G) spectrally analyzed1 thiopropanol S-oxide in raw onions, and GC was used (10G) to analyze flavor components of garlic extract. Buttery et al. describe the use of capillary and packed columii GC to determine the basic (13G) and nonbasic (12G) volatiles of potato chips, and Sapers et al. (104G) provide art objective GC method for the determination of aldehyde!; associated with the flavor quality of explosion puffed de hydrated potato. Cronin and Ward (22G) present GC-MS technique for characterization of mushroom vola tiles, and Heatherbell et al. (37G) have developed an on column entrainment procedure for the routine analysis 0,’ aqueous carrot extracts by GC-MS. Methods have beer developed for the analysis of volatiles in fresh watermelor by TLC and GC (46G) and GC procedures are described a:, used for the determination and comparison of fresh anc dehydrated cabbage volatiles (64G). Yokota et al. (138G) 90 f7
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present GC methods for determining the major volatile components of cane molasses, and a simple spectrophotometric method is given (137G) for the determination of methanol for use in measurement of pectin ester content and pectin esterase activity. The use of liquid COz as an effective extraction solvent for aroma constituents has been reported (96G, 107G) as used to extract fruit juices and essences, and distribution coefficients are given for various systems. Colorimetric methods are given for determining total aldehydes by use of N-hydroxybenzenesulfonamide (39G, 90G) and also for determining carbonyls with 2,4-DNP (88G) in citrus essences and concentrates. A GC procedure for determining flavanones in citrus juices as their trimethylsilyl derivatives is described (18G) by Coffin et al., and direct GC methods are given for aqueous orange juice essence (75G) and aqueous headspace aroma (106G) which incorporate precolumns to separate water from the remainder of the volatiles. GC and MS methods for analyzing the aroma compounds of grapefruit essences (19G, 77G) are given, and a procedure used for the separation and GC identification of 22 carbonyl volatiles from grapefruit oil is reported (74G) after use of vacuum and molecular-still distillation and column chromatography. Moshonas and coworkers provide GC methods for identifying components of tangerine (78G), Valencia orange essence (76G), and lemon and lime essences (79G) using methylene chloride and ether as extraction solvents and IR and M S to achieve identifications. Paillard et al. (86G) describe three methods for analyzing fruit aromas by GC and report on their findings in analyzing apples, bananas, and pears; and Chandler (15G) reports on a TLC method for determining limonin extracted from orange juice with chloroform. A method for identifying coumarin in citrus oils by IR and GC is given (67G) including separation from the oil by extraction and purification by TLC, and Nishimura et al. (83G) describe the analysis of volatile flavors of caucas by GC and MS. Gas chromatographic methods are also presented for determining black currant juice aromas using headspace techniques (69G), pineapple aromas extracted by hexane (82G), and banana volatiles concentrated with a 2: 1pentane-dichloromethane solution (121G). IDENTITY This section includes those articles in the literature which provide information on the composition of genuine foods, those which provide information on the detection of foods in foods, and those concerning the quality of foods. The Food and Agriculture Organization of the United Nations has published information on the amino acid content of foods (22H). A method for detecting the ripeness of sugar beet .has been described by Statiscescu et al. (72H) which measures the sucrose-sodium ratio. Comparative characterization of different types of cheese has been obtained by Liebich et al. (44H) using gas chromatography of the oil on open tubular columns. Polyacrylamide gel electrophoresis has been used by Portmann et al. (63H) to detect cow’s milk in goat’s cheese by examination of the casein fraction, Free sugars in cocoa beans have been identified by Reineccius et al. (67H) by gas chromatography of the silylated sugars. A simple test which differentiates between Robusta and Arabica coffee beans has been suggested by Wurziger et al. (78H). Polysaccharides in raw coffee beans have been analyzed by Hashimoto et nl. (32H) including both water soluble and alkali soluble polysaccharides. Gas-chromatographic studies by Rumpf et al. (68H) have shown that the soluble substances of sweet corn kernels may be used as a measure of the degree
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 5, A P R I L 1973
of maturity. A study of the fatty acid composition of corn endosperm and germ oils has been made by Jellum (36H) to determine the influence of extraction procedures, and he concludes that specific extraction procedures must be recognized as a factor when studying oil from endosperm. Egg lipids have been fractionated by Holopainen (34H) and data on fatty acids, phospholipids and sterols obtained. Methods for the detection of eggs in food products include gas chromatographic determination of cholesterol in baked goods described by Johansen et al. (37H), the quantitative determination of egg protein in egg pastes by disk electrophoresis described by Silano et al. (70H); a similar technique has been suggested by Kobrehel et al. (41H), and gas chromatography has been used for the determination of fatty acid distribution for the estimation of egg content in food pastes by Hadorn et al. (30H). Studies have been continued by Brammell ( 7 H ) on a colorimetric method for the determination of sterol content of egg noodles as a measure of egg solids. Triglyceride composition of butter fats has been compiled by Parodi (58H) and found to serve as a base for the detection of synthetic and adulterated butterfat. Methods of determining the authenticity of modified milk fats have also been proposed by Parodi (59H), using sterol analysis, gas chromatographic analysis, and softening point data. Cocoa butter and fats from chocolate have been examined by Garcia-Olmedo et al. (24H) using gas chromatography, and data obtained for genuine cocoa butter and fat from milk chocolate. The C16/C18 ratio and the sterol fraction data have been suggested by Fincke (19H) a s a means of detecting cocoa butter substitutes. A means of detecting the mixture of coconut oil in olive oil has been described by Garcia-Villanova et al. (25H), using the determination of the “c9mplexometric index” which is the number of milligrams of disodium EDTA necessary to sequester the bivalent cations precipitated by the fatty acids produced by alkaline hydrolysis of one gram of fat. The components of fat mixtures may be identified by gas chromatography of the methyl esters and of the sterol acetates according to a method described by Guyot (29H). A general method for the gas chromatographic study of edible fats and oils has been described by Hadorn et al. (31H), and applied to the determination of milk fat in mixed fats. A modification of the A.O.A.C. procedure for the determination of fish oil in vegetable oil has been proposed by Lauro (42H). A new color test for pongam seed oil in edible oils has been suggested by Rao et al. (65H),using the reaction with SbC13. Procedures for the identification of fish species include vertical gel electrophoresis described by Coduri ( 1 3 H ) ,the use of authentic flesh standards instead of photographs of electrophoretic patterns proposed by Learson (43H), and the application of polyacrylamide-disk electrophoresis to heat-sterilized canned fish described by Mackie et al. (47H). Another method for the identification of canned fish has been suggested by McLay et al. (49H), using thin-layer chromatograms of the 2,4-dinitrophenylhydrazine derivatives of the carbonyl compounds. Tests for determining whether fish have been frozen or thawed have been proposed by Gould (27H) which measure changes in soluble malic enzyme activity. Another test has been applied to shucked oysters by Gould e t al. (28H) which used the differing electrophoretic properties of soluble forms of L-malate: NADP oxide-reductase on polyacrylamide gel columns. Carotenoid index has been suggested by Kelley et al. (38H) as a measure of quality and an indicator of changes during storage in shrimp. Gas chromatographic determination of the free amino acids in fresh fish has been shown by Viviani et al. (76H) to provide a useful indi-
cator of sardine freshness on the basis of lysine, leucine, and phenylalanine levels. Hypoxanthine has been used as an index of freshness in Cape hake by Eurt et al. (9H). Paper chromatography and gas chromatography have been used to determine ketoheptoses in some fruits by Ogata ( 5 5 H ) ; mannoheptulose was found in avocado in appreciable amounts. Free amino-acids in 22 fruit varieties have been determined by Fer nandez-Flores et al. (18F) by gas chromatography after ion-exchange clarification. Sugars in 28 authentic fruit sainples have been determined by Kline et al. (40H) by means of gas chromatography of t h e ‘ T M S derivatives. Amino acids in citrus juices have been determined by Vandercook et al. (74H) and correlated with other juice contkituents; these relations appear to offer a means for assessing juice content or authenticity of a sample. Vesicular li:?ids from orange and tangor varieties have been analyzed by Nordby e t al. (54H), who determined the methyl esters in triglycerides, monogalactosyl diglycerides, steryl esters, and esterified steryl glucosides. Each variety gave a series of four profiles which could be distinguished from the others. The determined amounts of the major phospholipids in orange, lemon, and grapefruit juices have beon tabulated by Vandercook et al. (75H). The deuterium content of directly pressed orange juice has been found by Bricout (8H) to be significantly higher than in redilut Bd orange juice and thus provides a method of distingulshing between these two types of juices. The nitrate concentration has been suggested by Benk et al. ( 6 H ) as a ml?ans of distinguishing between natural and imitation 0rang.e juice since most of the imitation juices contain much less nitrate than freshly squeezed juice. Orange juice has been determined in fruit drinks by Pregnolatto et al. (64H) by the precipitation of the quarternary ammonium bases as reineckates; this determination was specific for citrus juices. Fifty-eight samples of natural lemon juice have been tested by Lifsliitz et al. (45H), for a number of analytical parameters including a detailed free amino acid analysis. The sugars in prune juice have been determined by Flynii et al. (21H), by gas chromatography of the silyl ethers. Vertical gel electrophoresis of frer8h meat sarcoplasmic proteins has been shown by Coduri t t al. ( 1 2 H ) , to distinguish between single species and mixed species protein. The migration of myoglobin levels on polyacrylamide gels has been used by Hoeyem et al. (.33H), to identify the source of animal meat but is not ap13licable to heat-treated meat. Horse meat and kangaroo meat lipids have been. examined by Payne (60H), using thin-layer chromatography, and’ this process can be used to identify these meats in meat mixtures. The chemical composition of extracts of whale meat has been described by Suyma et al. (73H). The suitability of differential therm a1 analysis for establishing the identity of meat produc1,s has been shown by Lorant (46H). A method for distinguishing between fresh and thawed frozen minced meat using the difference in extractibility of isozymes of glutamate-oxalacetate transaminase has been tested by Masic et al. (48H), and found not suitable. Electrophoretic! patterns have been used by Mihaita et al. (50H) and Moreno-Calvo e t al. (52”) to differentiate between frozen and chilled meat. A colorimetric method in which meat juice is treated with malachite green has been recommended by Otel et al. (57H), as a means of distinguishing frozen meat from refrozen meat. A simple and rapid niethod for the extraction of foreign proteins from meat products has been described by Gerigk et al. (26H) who ‘detect the foreign protein by immuno-diffusion techniques. Casein and soybean proteins have been detected in meat products by
A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 5, A P R I L 1973
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Olsman (56H) by vertical electrophoresis on urea-st irch gel, and by Hoog et al. (35H), using chemical and physical tests on the separated protein. Fischer et al. (:OH) have also described an electrophoretic method for the detection of soybean products in sausage. Yeast extract in meat extract has been detected by Baltes ( 4 H ) by dtmlermination of the individual purines in the yeast extract compared to the purines in meats. An enzymic methoc for the determination of skimmed milk powder in raw wusages has been developed by Bahl ( 1 H )using the estimation of free lactose by its hydrolysis with B-galactosidase. An electrophoretic method for the detection of milk solids in foods has been reported by Rout (66H) by detecting the distinct pattern of casein in polyacrylamide gel electrophoretograms. Both electrophoresis and polarography knve been tested by Doguchi (16H) as methods for the identification of soybean casein in milk casein; neither proceciure was useful for this purpose. Soy milk protein has teen compared with cow’s milk protein by Kim et al. (31H) using polyacrylamide gel electrophoresis, and some differences in patterns were observed. The fatty acid composition of 82 peanut genotypes has been determined by Worthington et al. (77H), who kave also determined the stability of the oils, the iodine numbers, and the per cent of fatty acids. Bahl (2H)has presented an enzymic method for dried skim milk in p o ~ a t o powders using enzyme determination of the lactose, ar tl a similar method ( 3 H ) has been applied to the determination of skim milk powder in soup and sauce mixes. The dipeptides, anserine, balenine, and carnosine in r~ cat cubes in prepared meat products and soup mixtures hilve been determined by Baltes ( 5 H ) and used to establish the content of whale-meat extract in the product. Gas chromatographic analysis has been used by Stahl et al. (71H) t o determine the geographical origin of oregrino using the carvacrol-to-thymol ratio. Paper chromiitographic separation has been applied by Pertot et al. (61H), to the identification of native and modified maize starches. Studies of tea amino acids and amides have been made by Popov et al. (62H), showing the changes that occur with aging. The sugar isomers in tomato juice have been determined by Miladi et al. (51H), using gas chromatography. A review of the current knowledge of carbohydrates in wheat has been compiled by D’Appdonia et al. ( 1 4 H ) . Among methods for distinguishing hiird and soft wheat, Fabriani et ai. (17H), have investigated infrared spectra of fatty acids and found some quant lative differences; D’Errico et al. (15H), have detected 1070 of soft wheat in mixtures by evaluating the amount of specific soft wheat albumen in extracts after gel eleciiophoresis; and column chromatographic separations of the lipids have been used by Cavallaro ( I I H ) to detect soft wheat. An immunochemical method for the identification of soft wheat in flours and pastes has been developed by Cantagalli et al. (IOH). Starch-gel electrophoresis ias been used by Garcia-Faure et al. (23H), to detect flm from triticum aestivun in macaroni. Studies of microscopical determination of rye flour in wheat flour have been made by Seidemann (69H) who found that, for best results, a t least two different methods must be used. A method for the electrophoretic detection of added gelatin in yogurt has been described by Ney et al. (53H), usulg gel electrophoresis.
INORGANIC The large volume of work published dealing with inorganic food constituents necessitated that many referen :es go unmentioned because they dealt with established rou92 R
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A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 5, APRIL
tine procedures. Therefore, an effort has been made to select work that seemed to present useful modifications of existing methodology or new techniques that extended limits of detection or offered unique specificity. Minerals and trace elements as determined in foods by the atomic absorption technique was the subject of a review by Ooghe ( 5 1 4 . Tanner ( 7 2 4 described the neutron-activation analysis program of the U S . Food and Drug Administration, citing some examples. The use of this technique for determining a multitude of elements in tea and coffee was reported by Shah et al. ( 5 9 4 . The results of a collaborative study of methods of ashing cocoa were presented by Iverson ( 3 1 4 . Separation of heavy metals by TLC with their subsequent visualization by urease inhibition was demonstrated by Geike (194. Handa et al. (204 used a ring oven technique to separate and measure trace metals in milk. Fetisou et al. (14J) read the diethyldithiocarbamate complexes of Au, Fe, Ni, Mn, and Zn extracted from milk ash a t five wavelengths and used correction factors to calculate amounts. Bergner et al. (4J) described his procedure for determining Fe, Au, Zn, Mn, and Br in wine by X-ray fluorescence. Tejam ( 7 3 4 extracted antimony with N-phenylbenzohydroxamic acid as part of a neutron-activation method. Reyrnont et al. ( 5 6 4 precipitated arsenic with added molybdenum as sulfides before determination by X-ray fluorescence. Tin, arsenic, and germanium were extracted into solvent as their iodides and measured in the UV in a method by Tanaka e t al. ( 7 1 4 . Total arsenic in composite foods was measured as the diethyldithiocarbamate complex after dry ashing and arsine evolution by Hundley et al. (30-4). Boron in foods was chelated with 2-ethyl-1,3hexanediol and measured by atomic absorption in an adaptation of a fertilizer method by Holak (24J). Low temperature ashing of rice prevented cadmium losses before atomic absorption spectrophotometry for Tanaka et al. ( 7 0 4 . Hundley et al. (295) extracted metals from acid digests as their dithizonates as an intermediate stage before anodic stripping cadmium quantification. Cesium-137 in food ash and water was separated by extraction and precipitation before counting by Flynn ( 1 6 J ) . Chromium in saccharin down to 0.2 pg was polarographically analyzed after ashing by Tvaroha ( 7 8 4 . Thirteen methods for copper not requiring digestion were studied for application to spirits by Szobolotzky (SM) and one employing 2,2’-biquinolyl was chosen as satisfactory. Schilt e t al. ( 5 7 4 reported a simultaneous iron and copper method utilizing either 5,6-diphenyl-3-(2-pyridyl)-,2,4-triazine or 5,g-diphenyl-3-(4-phenyl-2-pyridyl)-1,2,4-triazine reagents and visible spectrophotometry. Yoshida et al. (86s)demonstrated the direct ac polarographic determination of iron and copper in oils after adding supporting electrolytes. Evans (12T) advocated char-ashing of oils to attain higher atomic absorption sensitivity for copper and iron than by direct aspiration. Holak (25.5) used a background matrix similar to his samples to calibrate an atomic absorption method for iron and aluminum in baking powder. Meredith et al. (4444 found atomic absorption to be the technique preferred over colorimetry for iron in alcoholic beverages since wet oxidation of some sample types was required for the latter method. Procedures for determining cobalt in beer--i.e., spectrographic, complexometric, and atomic absorption-were reviewed by Schultze-Berndt ( 5 8 4 . Ssekaalo (624 described the use of 2-nitroso-l-naphthol as extractant and spectrophotometric reagent for cobalt in plant materials. Dewey et al. ( 1 1 4 substituted 1-nitroso-2-napthol for dithizone as cobalt’extractant in a modification of a spec-
1973
trophotometric method. Jag0 et al. (324 used this same the mercury carrier by electrodeposition in their neutron activation analysis scheme. Anothm neutron activation reagent to complex cobalt in an atomic absorption methmethod by Filby et al. (15J) counted the irradiated samod. Davidek et al. ( 1 0 also used this reagent, but in an ples directly after allowing time for decay and then comindirect polarographic procedure measuring the NN deputer processed the y-ray spectrum. Montoloy et al. ( 4 5 4 crease after reaction. The rate of peroxide oxidation of described their activation analysis method employing an catechol activated by p-phenetidine was used to deteriodinated resin to separate interfering radionuclides. mine cobalt traces after a n initial paper chromatographic Wenger et al. (824 reviewed methods for molybdenum separation in a procedure applied to milk by Prik et al. and presented a universal spectrophotometric method ( 5 3 4 . Germanium in foods was extracted from a n ash sowith two stages of chelation-extraction, benzoin a-oxime lution into CCll and the absorbance a t 508 nm read after and then toluene-3,4-dithiol. Ssekaalo et al. (644 adapted reaction with phenylfluorone in a procedure described by the molybdenum method of Kirkbright and Yoe for appliHashinaga et al. (21s). The analysis of milk for lead was cation to rolled oats and milk. Toluene-3,4-dithiol chelaaccomplished by anodic stripping voltammetry in a n HCl tion permitted direct determination of molybdenum in ash solution by Stelte (654. Gajan et al. (184 reported acid digests of plant material without prior separation in the results of a collaborative study on lead in fish with a method by Ssekaalo (634. An autcmated method based atomic absorption and polarography as the methods under on molybdenum catalysis of I- oxide tion and spectrophostudy. Wald et al. (814 described his activation analysis tometric measurement of 13- formed was used by Fuge scheme for magnesium and manganese traces in rice. (174 for milk, among other sample ;ypes. A rapid nickel White (834 attributed higher recovery of manganese from determination for fats was published by Price et al. (524, ash after hydroxylammonium chloride treatment to chemsensitive to 0.1 ppm. Szy et al. ( 6 7 4 measured small ical reduction and improved oxide solubilization in his amounts of radium and radon with a cylindrical scintillaatomic absorption method. tion counter. The silica content of beer was measured by Variations in trace mercury methodology for foods have the molybdenum blue method after filtration of precipicontinued to appear since the problem became public tated tannins and proteins and reduction with 4-amino-3news: A procedure for determining mercury in canned fish hydroxynapthalene-1-sulfonicacid in a method reported with a dithizone extraction and photometric measurement by Hoover (265). Black ( 7 4 used direct flame photometry after wet ashing was presented by Nagy ( 4 9 4 . Woidich et of soybean oil in a solvent, calibrating in an oil matrix al. (844 used TLC and absorption chromatography to with Na oleate. Yamamoto (854 investigated a new colorseparate the mercury dithizonate from others before imetric method for the determination of tin in canned absorbimetry. A rapid digestion of oysters followed by foods using extraction of the 1,lO-phenanthroline complex dithizone colorimetry was reported suitable for both inorinto nitrobenzene and colorimetry. In a method for deterganic and organic mercury residues by Mayer ( 4 2 4 . mining tin in canned vegetables by Biston et al. (64, Hauser et al. ( 2 2 4 analyzed fish canned in oil in a rapid exprocedure utilizing alkaline permanganate digestion. traction with cold 10M HCl was done instead of mineraliThorpe (75J) reported a modified cold vapor mercury prozation, and then direct oscillographic polarography on the cedure that was derived from the Fisheries Research solution was performed. Inorganic arid organic tin comBoard of Canada method. Okuno et al. (505) also used pounds were separated from foods using TLC by Akagi et cold vapor photometry but collected mercury on silver al. ( 1 4 after ether extraction. Zima et ~ l (884 . examined wire after oxygen combustion and liberated the vapor by precipitation methods for removing laiage calcium excesses resistance heating. Thomas et ~ l .( 7 4 4 pyrolized fish before strontium analysis and concluded that most insamples in an air stream and measured the vapor absorbvolved large losses. These authors (893) also advocated ion ance after appropriate scrubbing to remove interferences. exchange and elution with 0.005M 1,2-diaminocyclohexA digestion system with a solid COZ cooled condenser was ane-N,N,N',N'-tetraacetic acid to efficiently separate excess calcium before measuring strontium by flame phoused by Malaiyandi et al. (395) before an AutoAnalyzer tometry. SnClz reduction and cold vapor measurement ( 4 0 4 . Bailey et al. (3J)automated the Hatch and Ott procedure, analyzA collaborative study of a fast pot,?ntiometric method for chloride to determine salt in canned vegetables was ing 22 samples per hour. Skare (614 extended the application of Pn automated cold vapor method previously rereported by Brammell (ar).Vander M'erf ( 8 0 examined ported. A technique by Ukita et al. (795) used either deAgzCr207 indicator strips to estimate chloride in food aqueous extracts and found their results comparable to composition of the dithizonate or stannous chloride reducthe Volhard method. A Conway dish diffusion method for tion to liberate mercury vapor after an initial oxygen fluorine with La-alizarin spectrophotometric measurement bomb combustion. Hoover et al. (285) described their syswas found comparable to a distillation procedure by Tusl tem for digestion and flameless atomic absorption using (774. Hoover et al. ( 2 7 4 employed the iodide specific multiple cycling of the vapor. Collaborative study results of a flameless A.A. method for fish were reported by electrode to directly measure traces in feeds and plants in Munns et al. ( 4 6 4 . Magos ( 3 7 4 was able to selectively a 10% phosphate solution to stabilize ionic strength and release total or only inorganic mercury to the vapor phase pH. Zhukov et al. ( 8 7 4 scaled down and modified a modepending on whether he used SnC12-CdC12 as reductant lybdenum blue phosphorus method by Black and Hamor SnC12 alone, respectively. Tong ( 7 6 4 , using a final mond to permit its application to oil:; containing low P measurement by spark source mass spectrometry, was levels. An automated phosphorus mcthod reported by able to determine mercury residues in apples after McNeal et al. ( 4 3 4 was designed to work in conjunction Schoniger combustion and dithizone extraction. Collecwith his automated nitrogen method for meat. Linden et tion on silver wire and subsequent release into a fluoresal. (365) determined phosphorus by forming the molybdocence cell by electrical heating was used by Muscat et ~ l . phospate complex, extracting it into niethyl isobutyl ke( 4 7 4 in their atomic-fluorescence method for mercury. tone and measuring molybdenum by atomic absorption. A Takeshita et al. (684 employed reversed phase TLC to collaborative study of a phosphorus method for gelatin separate and detect mercury and alkylmercury comadapted from an AOAC method for fertilizer was reported and recommended by Burkepile (9J). pounds in food or sewage. Heitzman et al. (23J) isolated ANALYTICAL C H E M I S T R Y , VOL. 45,
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Babuchowski et al. (w) analyzed rape oil for traces of tion of alkoxide, determined by titration with ethanolic sulfur by heating the oil with treated Raney nickel and HCI, gives a measure of the water. The method has also acid, sweeping with hydrogen, and trapping the H i S gcnbeen applied t o determining water in butter ( 6 K ) , meat erated for analysis. Beswick e t al. (54 also determined products ( 5 K ) , and fermented feeds (8K). An automated sulfur in foods, but by first reducing sulfate from an inisystem for monitoring the water in butter during continutial Schoniger combustion to HzS, and then sweeping into ous manufacture is described by Koenen ( 9 K ) and operdithizone and titrating with mercuric acetate. Sinclair et ates on the principle of measurement of dielectric conal. ( 6 0 4 in analyzing beer for sulfur, eliminated nitrfite stant (at a frequency of 13 MHz) which is relatable to interference by forming the zinc salts after oxygen coinmoisture levels. bustion, selectively decomposing the nitrate salt by igiiiA rapid technique for estimating the total solids of fluid tion, and then reducing to sulfide and determining it by milk is given by Lunder ( I I K ) who makes use of ceriomethe methylene blue method. Sulfur in phosphoproteins try of sugars and the constancy of the relationship of milk was determined by Kim et al (34J) utilizing a sealed tribe sugar to the total solids present to obtain an effective, indigestion, phosphate removal with silver oxide, ion exdirect procedure. change cleanup, and finally EDTA titration of excess The use of anhydrous 2-propanol as a means of extracting water from cheese is reported by Strange (14K, 1510 BaZ+ after B a s 0 4 precipitation. Traces of hydrogen sulfide in beer headspace were measured by Jansen et al. (;,?a prior to determining it either by gas chromatography or using GLC with flame photometric detection. Sulfur d oxby Karl Fischer titrimetry. An indirect gas chromatoide in fruit products caused a loss of color of peroxodiwlgraphic method for determining water in hops and hop exfatotitanic acid, which was quantified colorimetrically by tracts is also reported (IOK) wherein use is made of the Nagaraja et al. (4m.A gas chromatographic method for reaction of water with 2,2-dimethoxypropane to form aceoxygen in wort and beer by Kreuger et al. (355) swelk a tone. Swift (16K, 1 7 K ) describes the use of infrared reflectance measurements for assessing moisture in instant sample with helium, trapped COz and water vapor, and separated the oxygen peak on a 5A molecular sieve colcoffee, confections, fish meal, soup powders, and potato umn. Gasses dissolved in milk were measured by ditect granules. Vomhof and Thomas (19K) describe infrared injection into a gas chromatograph operated with its Poramethods for determining water in starch hydrolysates pak Q column subambiently programmed in a method by after extraction with either dimethyl formamide or dimethylsulfoxide while Vornheder and Brabbs (ZOIC) have Kreula et al. ( 3 w . Mass spectrometric headspace gas analysis of salad oils was accomplished by designirig a applied a similar approach to the determination of moisleakproof sampling system by Evans et al. (135).M i i t i n ture in potato chips, dough, instant coffee, and vegetable ( 4 1 4 reviewed methods for determining carbon dioxic e in oils. Electrical conductivity is reported as a suitable pabeer. Rees (554 automated a method for COz in beet by rameter for rapidly measuring and controlling the concenacidifying, air sweeping the gasses into a buffered ph( 1101tration of Espresso coffee decoctions (13K), and methods phthalein reagent, and measuring the color decrease with are described for making use of NMR for the on-line meaAutoAnalyzer apparatus. Quast e t a1 ( 5 4 4 constructed surement and control of moisture in starch streams (12K). their version of an oxygen probe to measure this gas iticiide packages. Two fluorescent compounds, 3-[1-(2-hydioxyORGANIC ACIDS propyl)-2-piperidyl] pyridine and its 1-(2-hydroxypropyl) analog have been proposed as pH indicators for wine by The volatile acidity of wine was made an automated Talipou et al. ( 6 9 4 . determination by Sarris et al. (37L) who used a continuous steam distillation in an AutoAnalyzer setup. Mass MOISTURE spectral values of 24 carboxylic acids obtained by Shaw et a!. (43L) have been used to identify volatiIe acids from A study was made by Hart (7K) as to the identitj and stored citrus powders. Ney et al. (27L) reported 13 impact of nonaqueous volatiles and also of chemical ieacmonocarboxylic and 8 keto acids among other components tions producing water during use of the USDA oven found in cheddar cheese. TLC after steam distillation into drying method for determining moisture in corn. Emriions NaOH allowed Tjan et al. (5I.L) to identify acetic, propiet al. (3K) report that improved precision can be effxted onic, and sorbic acids in bakery products. Organic volatile for determining total solids in heterogenous heat-sensitive acids in cocoa beans were determined by Kuznetsova et foods by freeze-drying samples prior to drying in a vacual. (20L) to explain unpleasant aromas. Staruszkiewicz um oven, and Thieme (18K) describes methods for deter(4615) reported additional .collaborative results on a GLC mining the equilibrium relative humidity and explains its and the AOAC methods for lactic and succinic acids in relationship to stability of a product. eggs. Starling et al. (45L) modified AOAC methods for volA comparison is made of 27 solvents which form ilzeoatile acids in eggs to improve speed and also allow detertropes with water for their effectiveness in shortening the mination of lactic and succinic acids in the same solution. time required for determining the moisture contcnt of meat and meat products by distillation methods ( 2 K ) , Lactic acid was rapidly estimated in a procedure by Lawrence (21L) applicable to skim milk powder although and Wyler (21K) also describes a rapid distillation methhigher results than the ion exchange method resulted. od for meat products which incorporates tetrachlorcethylKiely e t al. (17L) described a procedure for molasses in ene as the solvent in conjunction with specialized lippawhich volatile acids were determined directly by GLC and ratus. A technique for determining the water holding calactic acid after methylation. Oldfield et al. (31L) oxipacity of meat is given (110 wherein the water from a hodized lactic acid with HI04 directly upon injection into a mogenized sample is pressed into filter paper between gas chromatograph and measured the resultant acetaldeglass plates and estimated by the water stain arcu obhyde in a rapid method applied to beet molasses and protained. Glass reports ( 4 K ) on a method for deterrriining cess juices. Oldfield et al. (30L) also developed a routine water in vegetable oils by reaction with a solution of sodihigh precision method using spectrophotometry after oxium methoxide in anhydrous methanol-ethyl acetate, dation, acetaldehyde diffusion, and semicarbazide reacthereby making use of a saponification reaction to rtbmove tion in a Conway apparatus. An enzymic determination of the resulting NaOH formed. The decrease in conccntra94 R
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lactic acid by Bourzutschky et al. ( 4 L ) gave reproducible results for beet molasses but wide variability for cane molasses. A method of interpreting data from an AOAC-GLC method for vanilla organic acids was presented by Schoen (41L). Harvey et al. (12L) used methylation and GLC of the esters to determine malic, citric, and oxalic acids in plants and food products. GLC of Me esters after separation by P b precipitation allowed Weisberger et al. (57L) to identify and quantify cocoa bean nonvolatile acids. Boland (3L) chromatographed T M S derivatives of organic acids initially separated from fruit products by ion exchange. Martin et a / . (23L) precipitated fixed acids from wine with P b and separated their T M S products by GLC. A method for dehydroacetic acid in margarine and butter using spectrophotometry was described by Ohsaki et al. (2%). Sasaki ( 3 9 i ) studied the salicylaldehyde color reaction with dehydroacetic acid with respect to related compounds that would give interference. A headspace GLC electron capture method for determining 2-acetolactic and 2-hydroxybutyric acids in a fermented solution after conversion to diacetyl and 2,3-pentanedione was reported by Ronkainen et al. (36L).Methylation and GLC followed an initial TLC separation of foodstuff acids in a method by Stoll (47L). Bengtsson e t al. ( 2 L ) scaled quantities of dicarboxylic acids for an automated method down to micrograms to reduce tailing in anion exchange chromatography. The nonvolatile acids of passion fruit juice were identified by Chan et al. (5L) using GLC of their Me esters. Wasa et al. (56L) reported the applicability of a polarographic procedure for 2-oxo-acids as their o-phenylenediamine condensates to pyruvic and lactic acid in milk. A conductimetric titration with triethanolamine for fruit acid determination was reported as advantageous by Sarudi (38L). Pires et al. (32L) utilized periodate to oxidize angliceric and tartaric acids in wine to pyruvic and glyoxylic acids which were then determined enzymically. Olschimke et al. (29L) compared the enzymic and Rebelein methods for malic acid in wine. Succinic acid in wine was reacted with succinic dehydrogenase and KsFe(CN),j and absorbance a t 420 nm then measured in a method by Pires et al. (33L). Kiermeier et al. (18L) analyzed for cheese pyruvate content enzymically and by TLC of its rhodanine reaction product. A colorimetric method for aconitic acid in sugar cane juice by Fournier et al. (1OL) employed the Furth-Herrmann reaction. Mehltretter et al. (24L) precipitated aconitic acid from molasses as the lead salt and chromatographed its T M S reaction product. Poe e t al. (3415) extended use of his colorimetric aconitic acid method to oats. Paper chromatography was the separation technique employed by Jacorzynski (15L) to evaluate tomato juices for pyrrolidone-carboxylic acid content. Bean phytate was measured by Fe3+ precipitation of an extract and measurement of Fez+ after reduction of iron from the redissolved precipitate in a method by Makower (22L). Rombouts et al. (35L) worked out a scheme using absorption a t 232 nm to determine the number average degree of pectin polymerization. Verzele et al. (55L) evaluated methods for (Y acids in hops and extracts and suggested changes to improve some of them. Trolle (52L) reported that the BIRF conductimetric method gave truest acid values for hop extracts. De Ceuster et al. ( 6 L ) also described a conductimetric N acid method. GLC separation of TMS ethers and NMR spectroscopy were used to analyze for N acids, p acids, and 4deoxyhumulones in hops in a paper by Shannon et al. (42L). Kokubo e t al. (19L) used UV absorbance to deter-
mine iso-a-acids in wort and beer after ion exchange. The Institute of Brewing Analysis Committee (14L) reported collaborative results on eight methods for a- and iso-aacids and made recommendation;. Fantozzi (9L) used TLC and GLC of methyl esters to separate citramalic acid in beer. Acetate in wort and beer was measured with an acetate kinase reagent by Drawert et al. ( S L ) . These authors (7L)also enzymically determined L- and D-lactate. A polyamide column adsorption followed by elution, TLC separation, and UV spectrophotometry was the procedure followed by Heimann et al. (13L) to study hydroxycinnamic acids in vegetables. Olice total polyhydric phenols were reacted with Folin-Den,s reagent and read a t 725 nm in a method of Vazquez Roncero et al. (54L). Densitometric measurements of fluorescent TLC spots provided Van Deventer-Schriemei et al. (53L) with a method for chlorogenic acid in a p d e juice. An AlC13 reagent and UV absorbance changes permitted Tissut et al. (50L) to quantitatively determine chlorogenic acid and flavonols in vegetables. Diffusion e rtraction of chlorogenic acid from sunflower kernels was studied by Sosulski et al. (44L). A rigid GLC method for caffeic acid and quercetin type compounds using electron capture detection of their volatile silyl derivatives was given by Andersen et al. (1L). Schaller et a / . (4015)separated and identified a number of polyphenols from Montmorency cherries. U1traviolet extinction differences after Polyclar A T removal of polyhydric phenols allowed their determination in hops by Jerumanis (16L). Miskov et a / . (25L) described the TLC isolation and measurement of grape, must, and wine phenolic acids and catechins. Polyamide and Sephadex column separations enabled Gryuner et al. (11L) to identify crystalline catechol fractions from cacao beans. Tirimanna et al. (49L) described a new spray reagent for detection of catechols on paper chromatograms consisting of 2,4,6-trinitrophenol followed by KOH. Gallic acid and its esters were separated by polyam de TLC and detected in work done with 3,5-dichloro-p-benzoquinonechlorimine by Takeshita e t al. (48L). Nakabayashi (26L) studied color reactions between Fe salts and tannins of fruits and vegetables. Salicylic acid in vinegar was determined spectrophotometrically after extractioii using tris(1,lO-phenathroline)iron(II) chelate in a prxedure by Yamarnoto (58L). NITROGEN Analysis of nitrogen and nitrogen -containing compounds is of great interest, both from the point of view of nutrition and for the purpose of obtaining information on the composition and characteristics of the nitrogen components of food. Modifications of the final determination in the Kjeldahl digestion include conductimetric determination of the ammonia in boric acid described by Sarudi et al. (63M); use of oxidation by bromate, permanganate or dichromate after Kjeldahl digesticn in situ described by Siddiqui et al. ( 6 S M ) ;and Urban (7711rl) has suggested the use of p-hydroxybenzoic acid as t t e receiving solution for Kjeldahl distillates. Spectrophotonietric methods for protein determination include a methcd proposed by Toma et al. (74M) which uses ultraviolet measurement after dissolution in urea-sulfuric acid, or urc a-sodium hydroxide, a quick biuret method described by Craney ( I I M ) and applied to protein in wheat, a modified biuret method for protein in fish flour and fish meal suggested by Misra et al. (53M), and a dye binding method for dairy products which uses the Udy method has been recommended by Sherbon (67M) for adoption official final action in the AOAC methods.
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Neutron activation has been tested by Tsen e t al. (75hI) after extraction of the protein from a column of flour. and found suitable for the determination of protein CO:I- Wheat flour proteins have been analyzed by Lawrence et tent in wheat flours and flour streams. An empirical d:!al. (41M) using polyacrylamide gel electrophoresis. Dyetermined half-life for interfering elements has been u s d binding capacity has been correlated by Lawrence et al. by Blake (6M) to improve neutron activation analysis for (42M)with amino acid content of gel electrophoresis nitrogen in gluten. Analysis of small samples based on tlic bands. Aniline blue-black has been found by Silano et al. determination of nitrogen by the 10.82-MeV gamma ra:p (69M) to give specific color reactions with albumin, globproduced by the 14N(n,y)15N reaction has been describd ulin, and gliadin after gel electrophoresis separation. Enby Tiwari (73M). Other methods of determining nitrogm zymic precipitation in agar gel has been proposed by include a modified Conway diffusion method described 11y Nordal et al. (58M) as a method for the detection of sodiKoval’shuk (38M) for nitrogen in meat, and a rapid metlrum caseinate in meat products. Proteins have been separated by King (37M) using reverse ammonium sulfate preod for sausage-like products and ham proposed by Heircipitation. A method for the removal of sodium chloride drik (27M) uses formaldehyde titration after decomposifrom C.I. Acid Black 1 for use in protein dye-binding has tion. to decrease variability in been described by Lakin (40M) Automatic methods for the determination by nitrogcmii results. Soy-bean trypsin inhibitor has been suggested by by the automatic Kjeldahl (AutoAnalyzer) have been d 3scribed by Uhl e t ai. (76M)for grain, by McNeal et ~ l . Eldridge et al. (19M) as a reference protein in calculating (48M)for meat and meat products, by Mitcheson et ~ l . the relative mobilities of other soy-bean proteins in gel electrophoresis studies. (54M)for barley and malt, and by Lento et al. (44M) for Enzyme electrode probes suitable for the assay of Dfoods. An automated trinitrobenzene sulfonic acid methcltl amino acids and asparagine have been described by Guilhas been applied by Gehrke et al. (21M) to 6N hydrolybault et al. (25M). An improvement in the AutoAnalyzer sates of grain. Two methods for the automatic determinitechnique for ion-exchange analysis of amino acids has tion of the protein content of flour described by Desbeen suggested by Heathcote et al. (26M)which uses chreider et al. (I7M)use either indophenol co1orimeti.y thin-layer chromatography to detect small peptides. A after digestion, or assay by the Lowry method for phenolic comparative study of amino acid analyses of five laboratogroups. A very sensitive method for ammonium ion 11 ries has been published by Cavins et al. (1OM) who note Kjeldahl digests which uses sodium salicylate and sodiu 71 that between-lab-variations were significant for most dichloroisocyanurate has been adapted to automatic ana 1amino acids. Gas chromatography has been used by Baerysis by Crooke et al. (12M). A new automatic unit for the wald e t al. (4M) for the determination of amino acids in determination of nitrogen has been described by Me r z wort and beer using a nitroge‘n selective thermionic detec(51M) using combustion in oxygen and gasometric me,ttor, and by Zumwalt e t al. (85M) for the analysis of nanosurement of the nitrogen. An evaluation of the Pro-Mi.Ir gram amounts of amino acids. A method for the preparaAutomatic instrument has been published by Thomasow tion of N-trifluoroacetyl methyl esters of amino acids has et al. (72M). An improved method for pepsin digestibiliiy been developed by Shearer et ai. (66M) and used for the of animal proteins has been described by Gehrt (22M). separation of 20 amino acids; the results were compared The use of cellulose to extract free protein from chick peas has been suggested by Ghose et al. (23M). to the ion-exchange method. Qualitative chromatographic A procsmethods have been used by Johnson et al. (34M)to isodure for the rapid analytical gel chromatography of p r i teins and peptides has been described by Catsimpoolas #?t late and identify the free amino acids in table sirups. al. ( 9 M ) using Sephadex microbore columns. A review (111 Improvements in the chloramine T method for hydroxythe chromatographic separation of milk proteins has bees11 proline have been described by Arneth et al. (3M). An auwritten by Yaguchi et a!. (84M). The displacement of tfl- tomatic amino acid analyzer has been used by Mack luidine blue absorption color in milk casein solution ti:! (46M)to determine hydroxyproline in meat products. the addition of sodium phosphates has been studied 11y Methods for the spectrophotometric determination of free lysine and methionine in amino acid fortified wheat have Abdulina et al. ( I M ) . The Pro-Milk MK I1 has been usc,cl been described by Ferrel et al. (20M) to estimate casein in milk arc1 using ninhydrin for by McGann et al. (47M) both amino acids and the sodium nitroprusside reaction protein in whey. Chromatography on Sephadex G-100 hiis for methionine. Anomalous results for the determination been described by Nakai et al. (56M) for fractionation if of available lysine in casein using 2,4,6-trinitrobenzenesulcaseins directly from skim milk using elution with phosfonic acid have been ascribed by Holsinger et al. (31M) to phate buffers. A modified ninhydrin reaction has becmii the presence of hexosamines. A method of screening for proposed by DeKoning et al. (16M) for the estimation ,If high lysine content in wheat using an amino acid analyzer whey proteins in casein coprecipitate or in milk powder. Dye binding has been used by Sanderson (62M) programmed so that only the lysine peak is recorded has to determine undenatured whey protein in skim milk powder. S ?been described by Mattern et al. (50M). The reaction of lective precipitation of whey proteins has been achieve (1 lysine in milk and cheese with Remazol Brilliant Blue R has been used by Ney et al. (57M) to determine available by Hidalgo et al. (28M), using carboxymethylcellulose I I lysine in these products. Hupf et al. (33M)have found selected conditions of p H and ionic strength. An0thi.r lanthionine in the proteins of wheat gluten and egg whites whey protein precipitant, “ferric-polyphosphate” has be6 n after heat treatment. found by Jones et al. (35M) to precipitate all the prote:ii in commercial acid whey. Casein and whey proteins h a w An accurate method for the determination of tryptophan in proteins has been developed by Hugli et al. (32M) been fractionated by Sedmerova et al. (64M)by colurii chromatography on DEAE-cellulose. using ion-exchange after alkaline hydrolysis. A study of Water soluble proteins from barley and malt have becii the optimal conditions for the extraction and determinaseparated by Radola et al. (60M) using isoelectric focusirq: tion of tryptophan in food proteins has been made by in a sucrose density gradient. Peptides, from cheddar Robin et al. ( 6 I M ) who suggest the use of alkaline hydrolcheese have been isolated by Polzhofer et al. ( 5 9 M ) on S’?- ysis in an inert atmosphere. Alkaline extraction and enzyme hydrolysis of the proteins in food products has been phadex QAE-A 25. Disk electrophoresis has been used tly used by Skibinska e t al. (70M) Williams et al. (80M) to characterize wheat flour protein:; for tryptophan determina96 R
ANALYTICAL CHEMISTRY, VOL. 45, N O . 5, APRIL. 1973
tion, and Monari et al. (55M) have compared enzyme hydrolysis methods for liberating tryptophan from food products. Different hydrolysis methods for tryptophan have also been compared by Vangala et al. (78M) who recommend hydrolysis with papain for legume products. A convenient method for the detection of N H containing compounds on thin-layer chromatograms has been described by Ariyoshi et al. ( 2 M ) using bleaching powder and starch-iodide solution. A study of the hydroxytryptamides of green and roasted coffee beans has been made by Wurziger et al. (83M) who have used chromatographic and spectrophotometric techniques to identify the compounds. The same authors (82M) have applied these techniques to the identification of carboxylic acid-5-hydroxytryptamides in cocoa. A special indicator, thymol blue and phenolpthalein, has been used by Sedov (65M) to improve the determination of amine-ammonia in colored extracts of meat. Thaler et al. (71M) have studied the behavior of amino acids and amides under the conditions used for the determination of ammonia in foods; only cysteine and cystine were found to release ammonia under these conditions. Volatile amines in dilute aqueous solution have been separated and determined by Keay et al. (36M) using gas chromatography on a n alkaline Dowfax 9N9 column. Volatile amines from fish have also been analyzed by Gruger (24M) using gas chromatography and thin-layer chromatography of the 5-dimethylamino-1-naphthalene sulfonamides. Betaine in sugar juice has been determined by Devillers et al. (18M) by the Kjeldahl method after ionexchange separation. An automatic apparatus for the determination of creatinine in soups has been described by Carisano et al. ( 8 M ) using cation exchange chromatography and ultraviolet detection. A simplified method for the analysis of glutamine has been proposed by Lin et al. (45M) using an automatic organic acid analyzer. Inosinic acid in muscle tissue has been determined by Davidek et ai. (14M) using chromatography on Dowex and spectrophotometric detection. Baltes ( 5 M ) has used reflectionspectrophotometry after thin-layer chromatography for the quantitative detection of inosinate in meat extracts. Another method for inosinic acid, inosine, and hypoxanthine in meat has been described by Velisek et al. (79M) using perchloric extraction and ion-exchange chromatography on Dowex-1 X 4. Gas chromatography, after silylation, has been used by Lee (43M) to determine 5-oxoproline. Methods for the determination of purine mononucleotides in foods have been described by Cuzzoni e t al. (13M) using densitometric assay after thin-layer chromatography. A complete method for the assay of nucleotides, nucleosides, and nucleic acid bases has been described by Davidek et al. (15M) using Dowex-1 X 8 (Cl-) column with a formic acid-sodium elution system. Investigation of a method for ribonucleic acid in milk by Majumder e t al. (49M) has indicated that high values, due to the presence of hexose, are obtained by the orcinol method. Cysteine and methionine have been determined by Wronski et al. ( 8 I M ) by a mercurimetric determination using Raney nickel. Amperometric titrations have been used by Bol’shakov, et al. ( 7 M ) for the determination of sulfhydryl groups in meat using a back-titration technique, and by Hofmann (30M) by indirect titration of excess silver after reaction with silver nitrate. Hofmann (29M) has also studied the effect of inorganic salts on the amperometric titration of mercapto groups with silver nitrate and noted decreases caused by the presence of copper sulfate and manganous sulfate. A potentiometric procedure has been used by Kuenbauch et al. (39M) to deter-
mine sulfhydryl and disulfide grours in wheat by means of silver nitrate titrant and a silvert hiol electrode. Urea in animal tissue (milk) has been determined by Miller (52M) by gas chromatography of the trifluoroacetyl derivative.
VITAMINS Newer trends in vitamin met iodology involve high speed liquid chromatography and automation of wet methods. The third volume of “The Vitamins” by Sebrell (43N) appeared and contains methodology for the D and K groups, among others. Deutsch 112N) reported the status of a variety of vitamin methods in a paper. An AutoAnalyzer method for ascorbic acid in orange and grapefruit juice utilized 2,6-dichlorophenolindophenolreagent and dialysis as reported by Hoffman et al. (19N). Aeschbacher et ai. ( I N ) automated a method for Vitamin C in tissue using 2,4-dinitrophenylliydrazinereaction after Chloramine-T oxidation. Bancher e t al. ( 3 N ) studied the effect of various sugars on the Roe-Kuether method for Vitamin C. Microamounts of ascorbic acid in citrus fruits were measured by reduction of ferric ion and subsequent ferrozine chelate color absorbance by Jaselskis et al. (23N). Toothill et al. (48N) studied interferences involved in analyzing milks by the 2,6-di1:hlorophenolindophenol method and the 2,4-dinitrophenylliydrazinemethod with and without subsequent derivative chromatography. Dehydroascorbic acid in meat was polarographically measured after condensation with o-phenylenediamine by Davidek et al. ( 9 N ) . The ascorbic u’as also included when 2,6-dichlorophenolindophenoloxidation preceded condensation in a later paper by Davidek et al. (ION).Kajita et al. (25N) used Br oxidation before o-phenylenediamine condensation and polarography to determine L-ascorbic acid and triose reductone simultaneously in foods. These same authors (24N) also described the behavior of L-ascorbic and erythrobic acids in canned fruit when directly polarographed. Potentiometric titration methods for ascorbic acid in colored food solutions anc ascorbyl palmitate in oils, respectively, were reported by Pongracz (37N, 38N). TLC of its osazone was used by Kclvacs e t al. (27N) to determine Vitamin C in some foods Ascorbic acid oxidase with subsequent manometric or spectrophotometric measurement enabled Marchesini et d.(31N) to determine ascorbic acid in vegetables. Marchesini et al. (32N) also reported on using a Clark electrodcm to measure the oxygen consumed in the presence of ascorbil: oxidase. The fat-soluble vitamins have been separated using high speed liquid chromatography by Williams et al. (51N) using a reversed phase modc. The tocopherols have been separated on hydroxyalkoxypropyl sephadex and monitored by their natural fluorescence in a paper by Thompson et al. ( 4 6 N ) . Linow et al. (29N) investigated the spectrophotometric method for tocopherol using 2,2diphenyl-1-picrylhydrazyl and applied it to margarine. The potassium ferricyanade oxidation product of cu-tocopherol in oils during storage was separated from interference by TLC in a technique reported by Schmandke et al. (42N). Fedeli et al. (15N) used TLC and GLC to identify individual tocopherols in vegetable oil unsaponifiables. Small amounts of Vitamin I) in milk that had been UV irradiated were measured by gas chromatography after purification and silylation by Br:ndel et al. (5N, 6 N ) . Janecke et al. (21N) also described this work. Panalaks (35N) determined Dz and Ds in dried milk by gas chromatography after extraction with ascorbic acid and BHA oxidation protection and column chIomatography. A colorimetric method for use with fortificd liquid milks was also described by Panalaks (36N). Low et al. (30N) separated A N A L Y T I C A L C H E M I S T R Y , VOL. 45, NO. 5, A P R I L 1973
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interferences by column chromatography of tocopherols from milk before bathophenanthroline spectrophotometi ic measurement. Touw ( 4 9 N ) used TLC for a final cleaniip before GLC detection for Vitamins Dz and D3 in infant formulas. Nelson e t al. (34N) claimed improvemerts through derivatization before the GLC determination of tocopherols and sterols in soybean sludges and residucat;. Rao et al. (40N) described a one-step method to identify and estimate tocopherols and tocotrieneols in vegetable o .Is using GLC-MS. A quantitative TLC method for total CYtocopherol in feeds and foods was reported by MuelltrMulot (33N). Ranfft ( 3 9 N ) determined Vitamin E in feeds and food by GLC after an alumina column cleanup. Collaborative data on a Vitamin E method was presented by Ames ( 2 N ) . Dean ( I I N ) used dry column chromatograpliy to separate tocopherol dimers that interfered with a spectrophotometric method. Gutfinger ( I 7N) studied tocop tierol dimers from soybean oil by means of reaction gtis chromatography. A simple method for isolating riboflavine from whey on a neutral ion exchange resin was reported by Brewingtiln et al. ( 7 N ) . Janicki et al. (22N) studied f1uorescen::e methods for flavines with respect to interferences aiid made recommendations. Gel filtration, paper chromatagraphy, and bioautography enabled Dougherty et al. (13iv') to characterize thiamine forms in raw peanuts. Ito et (11. (20N) improved recoveries of thiamine from milk powder by enzyme pretreatment. Hart et al. (18N) reduced losses of thiamine in flour samples with a preliminary extraction. Dialysis removed fluorescent contaminants and (11lowed Dougherty et ai. ( I 4 N ) to extend the limit of detection for the thiochrome assay. Brunink e t al. (8N) coriverted nicotinic acid to a fluorescent compound on TLIC by UV irradiation in a rapid method applied to met.(. Gorin et al. (16N) compared microbiological and spectrophotometric methods for nictonic acid. B,j constituents were analyzed in each others presence in plant juiws wi:h separate reagents in a method by Richter (41N). Pangarnic acid contents of cereals were determined by TLC b y Telegdy-Kovats et al. ( 4 5 N ) .Weil et al. (SON) automated the Sobel and Werbin method for Vitamin A in feed mirieral compounds. Vitamin A as well as D and E were separated on sephadex LH20 with isooctane mobile phase aiid the method was applied to margarine and eggs by Bi!!1 ( 4 N ) . Thompson et al. (47N) reported a rapid retinol method for dairy products using fluorescence of unsaporiifiable petroleum ether extracts. Knowles ( 2 6 N ) ana1yzi:d alfalfa and corn meal extracts by two dimensional TLC for carotenoids. The separation of carotene stereoisomers in vegetables was accomplished by a high pressure liquid chromatographic method in less than an hour as reported by Sweeney et al. (44N). Analytical schemes for the I< ~ i tamins and other bioquinones in foods were given I):! Kraszner-Berndorfer et al. (28N).
MISCELLANEOUS For those interested in obtaining a clearer picture of tlia roles and interactions of international organizations 011 development of methods of analysis, attention is directid to the articles by Chapman and Smith ( 6 P ) , who discuss cooperation between such organizations; by Miklovim (28P), who reviews the work of IS0 (the Int,ernational Standards Organization); and by Davies (IOP), who presents an assessment of the Codex Alimentarius. Egan (12P) provides a review of problems and progress in analytical methods for food toxicology and an extensiTia
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a
review is also presented on the occurrence and analysis of polychlorinated biphenyls in foods by Edwards ( I IP). Still another review describes methods for determining naturally occuring toxic chemicals in foods (32P). Baltes describes the application of reflectance spectrophotometry to food analysis ( I P ) and also provides an assessment of the application of the newer instrumental methods to foods (2P). Kohn (22P) discusses the use of dielectric methods for foods and additives, and reviews of chromatographic methods are also provided for various foods and natural products (5P, 23P, 3IP). A discussion of automat. ed gas chromatographic systems is presented (36P) which applies particularly to fatty acid methyl esters and wines or other ethanol-water systems. Cronin describes a simple quantitative method (9P) for trapping and transfeiing low concentration GC fractions from glass open tubular columns to other glass columns for additional separation, and Fleet et al. (15P) evaluate a range of commercially available fluidic devices for application to analytical sampling processes. Bhattacharya et al. (4P) have developed a colorimetric method for determining the degree of milling of rice after extraction of the bran pigment in aqueous, alkaline propanol, and a complete procedure is given for analyzing and comparing ready prepared cake and pastry doughs (1"). James ( 2 I P ) has outlined a general procedure for microscopically examining and geographically classifying honey based on the appearance of mounted pollen grains, and Lindner (27P) has evaluated methods for the determination of crude fiber. Smit (35P) has investigated the influence of extracting agents during the colorimetric determination of pectic substances and a method is presented for the automated analysis of hop resins (30P) by ion-exchange chromatography. Collier et al. provide methods for the determination of theaflavins (7P) and flavanols (8P) in tea by gas chromatography of their trimethylsilyl derivatives and Yamazaki et al. (39P) describe a column chromatographic-fluorometric procedure for determining Beclotiamine, a new coccidiostat, in chicken tissues and egg yolks. Feigl et al. (14P) have described spot tests for detecting theophylline and theobromine by reactions with mercuric cyanide, and an automated chemical method (33P) is described wherein coffee beans are evaluated for their beverage quality by extracting them with phosphate buffer and then measuring the o-diphenol oxidase activity of the extract using AutoAnalyzer equipment. Wisniewski et al. (38P) provide a method for determining theobromine and caffeine in cacao shells by nonaqueous titration with perchloric acid, and a new TLC method is also described (37P) for the determination of caffeine in roasted coffee beans. Sachse et al. (34P) describe their work in the extraction, isolation, and TLC identification of alkaloids in potato tubers, shoots, or leaves; and spectrophotometric methods are also described for the determination of caffeine in tea (26P, 29P) after column clean-up. Methods for caffeine in coffee are described which involve, respectively, comparison of periodate precipitates in coffee infusions ( I 7 P ) ,use of UV reflection spectrophotometry after TLC separation (3P) and also rapid automated spectrophotometry wherein trichloroethylene is used as the extraction solvent (13P). Heftmann and Schwimmer (2OP) describe a new TLC procedure for the separation and identification of caffeine and related methylxanthines, and a UV procedure is presented for determining purine alkaloids in cocoa after first purifying extracts through a polyamide column (25P).
ANALYTICAL CHEMISTRY, VOL. 45, N O . 5, APRIL. 1973
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(198) Ibid., 54, 565 (1971).
(688) Forschner, E., Arch. Lebensmitteihyg., 23, 101 (1972); Chem. Absfr., 77, 112554e (1972). (698) Friedlander, A., Gonen, M., lsrael J. Chem., 8, 87 (1970); Anal. Abstr., 20, 2117 (1971).
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