904
`
`J. Agric. Food Chem. 1986, 34, 904-907
`
`Hawks, S. N.; Collins, W. K. Principles of Flue-Cured Tobacco;
`North Carolina State University: Raleigh, NC, 1983; p 233.
`Leffingwell, J. C. Rec. Adv. Tob. Sci. 1976, 2, 1.
`Lloyd, R.; Miller, C.; Roberts, D.; Giles, J.; Dickerson, J.; Nelson,
`N.; Rix, C.; Ayers, P. Tobacco Sci. 1976, 20, 40.
`Rodgers, J. D.; Mitchum, A. R. Rec. Adv. Tob. Sci. 1976, 2, 112.
`
`Seltmann, H. Proceedings of the 5th International Tobacco
`Science Congress, 1970, 77.
`Seltmann, H. Beitr. Takforsch. Int. 1980, JO, 120.
`
`Received for review November 18, 1985. Accepted April 1, 1986.
`
`Supercritical Carbon Dioxide Extraction of Oils from Antarctic Krill
`
`Katsumi Yamaguchi,* Masahiro Murakami, Hiroshi Nakano, Shoji Konosu, Tsunehiko Kokura,
`Hiroshi Yamamoto, Masahiko Kosaka, and Kazuhiko Hata
`
`Supercritical carbon dioxide extraction of the Antarctic krill yielded oils that were composed solely of
`nonpolar lipids, largely triglycerides, without phospholipids. The extracted oils were fluid and of red
`color due to astaxanthin, which tended to be decomposed at temperatures higher than 60 °C and a fixed
`pressure of 250 kg/cm2• Analyses of the fatty acids showed a comparatively high proportion of eico(cid:173)
`sapentaenoic acid (11 % ). These results indicate that supercritical carbon dioxide is effective in obtaining
`nonpolar lipids from the krill by only one-step extraction and in excluding phospholipids that interfere
`with the utilization of krill oils.
`
`Organic solvent extraction of oils from raw materials is
`a well-developed technology. However, after the extraction
`process, further purification steps are generally required
`to remove impurities and gum-forming compounds from
`the extracted oil, especially in foodstuffs intended for
`human use.
`In recent years supercritical fluid extraction has received
`much attention, though its fundamental principles were
`known over 100 years ago (Hannay and Hogarth, 1879).
`The theory and practice of the supercritical fluid extraction
`process have been reviewed by Paul and Wise (1971), who
`predicted its application to foods, pharmaceuticals, fine
`chemicals, petrochemicals, mineral extraction, and fuel(cid:173)
`and waste-processing technologies. Various kinds of su(cid:173)
`percritical fluids have been studied (Wilke, 1978), but most
`work done so far has used carbon dioxide as the extractant.
`Carbon dioxide has the advantages of nontoxicity, incom(cid:173)
`bustibility, low critical temperature (31 °C) and pressure
`(75 kg/cm2), and low price, all of which meet the recent
`energy and health concerns.
`The application of supercritical carbon dioxide (SC-C02)
`extraction to foods has had limited success, as exemplified
`by decaffeination of green coffee beans in large-scale in(cid:173)
`dustrial plants (Zosel, 197 4) and production of hop extract
`(Hubert and Vitzthum, 1978). Further, SC-C02 has been
`used to extract oils from soybean (Friedrich and List,
`1982), coconut palm (Brannolte et al., 1983), butter
`(Kaufmann et al., 1983), etc. Applications of SC-C02 ex(cid:173)
`traction to animal sources, in particular seafoods, are still
`more limited.
`Lipids of aquatic organisms are generally rich in highly
`unsaturated fatty acids and phospholipids, which are
`readily deteriorated. The Antarctic krill, Euphausia su(cid:173)
`perba, possesses an especially high proportion of phos(cid:173)
`pholipids (Mori and Hikichi, 1976), which hampers the
`
`Laboratory of Marine Biochemistry, Faculty of Agri(cid:173)
`culture, The University of Tokyo, Bunkyo-ku, Tokyo 113,
`Japan (K.Y., M.M., H.N., S.K.); lwatani & Company,
`Limited, Higashi-ku, Osaka 541, Japan (T.K., H.Y.); Osaka
`Hydrogen Industries, Limited, Amagasaki 660, Japan
`(M.K.); and Central Research Laboratory, Nippon Suisan
`Kaisha, Limited, Hachioji, Tokyo 192, Japan (K.H.).
`
`effective utilization of krill oils. We applied SC-C02 ex(cid:173)
`traction to krill samples and proved that the extracted oils
`were composed solely of nonpolar lipids without contam(cid:173)
`ination by phospholipids and their deteriorated lipids.
`The present paper deals with the extraction and char(cid:173)
`acterization of the extracted oils and residual lipids.
`Materials and Methods Commercial preparations by
`Nippon Suisan Kaisha, Ltd., of frozen Antarctic krill and
`its meal were sampled. A freeze-dried sample was prepared
`by lyophilization of frozen krill. The samples were kept
`below -25 °C until used. Standard commercial prepara(cid:173)
`tions of lipid reagent were used without further purifica(cid:173)
`tion.
`Proximate composition of the samples was analyzed
`according to the AOAC procedures (No. 7.003, 7.009, 7.015,
`and 7.060; 1984).
`The extraction with SC-C02 was carried out using a test
`plant instrument manufactured by Mitsubishi Kakoki, Co.,
`Ltd. This equipment has a 75-mL extraction vessel with
`upper limits of pressure and temperature of 500 kg/ cm2
`and 100 °C, respectively.
`Ground freeze-dried krill (20 g) and krill meal (25 g) were
`put into the extraction vessel, gaseous C02 of which
`pressure was increased to a supercritical state by a pressure
`pump was introduced, and extraction was carried out at
`different pressures and temperatures. Gaseous C02 of
`commercial purity (Iwatani & Co., Ltd.) was used. Average
`flow rate was 0.6 kg/h. The extracted oils were measured
`gravimetrically.
`The fractionation of the oils (150 mg) was carried out
`on a column (1 X 8.5 cm) using silica gel (Wakogel C-200,
`100-200 mesh) and 50 mL of chloroform and then 50 mL
`of methanol. The eluted oils were measured gravimetri(cid:173)
`cally after evaporation of the solvent.
`The method of Bligh and Dyer (1959) was applied to the
`extraction of whole lipids from the krill samples and the
`residues after SC-C02 extraction. After evaporation of the
`solvent, the oils artd lipids were measured gravimetrically.
`The oils and lipids extracted with SC-C02 and by the
`method of Bligh and Dyer (1959) were analyzed by TLC
`on silica gel 60F 254 (Merk, 0.25 mm thick) with petroleum
`ether-diethyl ether-acetic acid (90:10:1) or chloroform(cid:173)
`methanol-water (65:25:4) as solvents and 50% aqueous
`sulfuric acid or Dragendorff reagent as indicator.
`
`0021-8561/86/1434-0904$01.50/0
`
`© 1986 American Chemical Society
`RIMFROST EXHIBIT 1162 Page 0001
`
`

`

`SC-C02 Extraction of Oils from Antarctic Krill
`
`J. Agric. Food Chem., Vol. 34, No. 5, 1986 905
`
`Table I. Proximate Composition of Krill Samples
`(as Percentage of Total)
`frozen
`krill
`77.7
`14.0
`3.01
`2.74
`97.5
`
`freeze-dried
`krill
`7.83
`52.0
`16.7
`12.2
`88.7
`
`krill
`mean
`1.75
`60.7
`11.5
`13.4
`87.4
`
`moisture
`protein•
`fat
`ash
`total
`
`•Total nitrogen was multiplied by 6.25.
`
`15
`
`10
`
`"' 0
`0
`......
`"'
`"' "" ·~
`0
`"" 0
`"' ~ 5
`<ll
`·~ ><
`
`6
`(kg)
`
`Figure 1. Extraction curves of krill oils with supercritical carbon
`dioxide at 250 kg/cm2 and 80 •c.
`In this connection, Brogle (1982) reported that the solvent
`power of SC-C02 for organic substances is highly de(cid:173)
`pendent on its pressure and temperature; at low pressures,
`around 100 kg/cm2, solvent power drops with rising tem(cid:173)
`peratures, and at pressures higher than approximately 150
`kg/cm2, its solvent power increases. For the krill samples,
`however, the amounts of extracted oils were almost con(cid:173)
`stant regardless of pressures and temperatures examined.
`That is, even under the lowest temperature/pressure
`combination (250 kg/cm2 and 40 °C), nearly all oils ex(cid:173)
`tractable with SC-C02 were recovered. Further, yields of
`krill meal oil were one-third of those of freeze-dried krill
`oil. The lower yields from meal oil are probably attrib(cid:173)
`utable to the fact that the oil of the krill meal was in part
`deteriorated by oxidation or polymerization to such an
`extent that only limited extraction occurred with SC-C02•
`Judging from this result, we think that SC-C02 extraction
`is suitable method to obtain undenatured oils from meals
`of marine origin.
`To determine the composition of the extracted oils, we
`partitioned them by chromatography on a silica gel column
`using a mobile phase of chloroform and methanol. Ap(cid:173)
`proximately 100% of the oils from both krill samples was
`recovered in the fraction eluted with chloroform, proving
`that the oils extracted with SC-C02 were composed ex(cid:173)
`clusively of non polar lipids with practically no polar lipids.
`Figure 2 shows TLC patterns of the oils extracted with
`SC-C02 and the residual lipids. By co-TLC with standard
`reagents, the main component of the extracted oils was
`found to be triglycerides (spot 3) and the minor compo(cid:173)
`nents were identified as hydrocarbon (1), cholesteryl ester
`(2), free fatty acids (4), diglycerldes (5), cholesterol (7),
`monoglycerides (8), and carotenoids (6 and 9), as illustrated
`in chromatogram I. In addition, a small amount of a
`nonpolar lipid (spot 10) was found to be present in the oil
`from the krill meal, but it remained unidentified. The free
`fatty acid content of the oil from the krill meal was lower
`than that of the freeze-dried krill. Because of their rapid
`deterioration, some of free fatty acids in the meal should
`have been denatured during manufacture and storage of
`the meal.
`As shown in chromatogram II, trace amounts of some
`nonpolar lipids (spots 3-9) were observed, but almost all
`the residual lipids (spot 11) were not affected when de-
`
`RIMFROST EXHIBIT 1162 Page 0002
`
`Table II. Yields of Oils Extracted from Krill Samples with
`Supercritical Carbon Dioxide
`amt, g/100 g sample
`total
`ex tr
`resid
`lipidb
`lipidb
`oil
`11.2
`7.6
`11.7
`7.9
`11.5
`8.1
`4.5
`8.1
`4.1
`9.0
`4.0
`8.4
`4.0
`9.0
`
`sample
`freeze-dried
`krill
`
`krill meal
`
`extractn
`condn•
`250/40
`250/60
`250/80
`250/40
`250/60
`250/80
`400/40
`
`rec,'
`%
`95
`98
`99
`84
`81
`77
`80
`
`19.7
`
`16.2
`
`•Pressure, kg/ cm2 /temperature, °C. b Measured by the Bligh(cid:173)
`'Recovery = [extr oil + resid lipid]/total lipid X
`Dyer method.
`100.
`
`After saponification and then esterification with boron
`trifluoride-methanol complex, the fatty acid composition
`of the oils was determined by G LC with a Shimadzu GC-
`5A gas chromatograph using a glass column (2 m X 3 mm)
`packed with 10% DEGS on Celite 545 (80-100 mesh) at
`a temperature of 200 °C, with nitrogen carrier gas at a flow
`rate of 25 mL/min. The temperature of the detector and
`injection port was 250 °C.
`The composition of carotenoids in the SC-COrextracted
`oils and in the residual lipids was analyzed by the method
`reported by Yamaguchi et al. (1983).
`RESULTS AND DISCUSSION
`Table I shows the proximate compositions of the krill
`samples. Total recoveries of components of the freeze(cid:173)
`dried krill and the krill meal were rather low, because of
`the application of the Soxhlet method with diethyl ether
`for the extraction of lipid. The lipid of krill, which contains
`high proportions of polyunsaturated fatty acids and
`phospholipids (Mori and Hikichi, 1976), deteriorates rap(cid:173)
`idly and diethyl ether insoluble lipid increases when krill
`is dehydrated or treated at high temperatures. In this
`connection, the lipid content of the krill meal was found
`to be 16.2% when measured by the method of Bligh and
`Dyer (1959) with chloroform-methanol. Furthermore, the
`low recoveries in the freeze-dried krill and the krill meal
`may be also accounted for by chitin, contents of which
`should be higher than that in the frozen krill.
`Figure 1 pr~sents the SC-C0 2 extraction curves with
`time of oils from the freeze-dried krill and krill meal with
`SC-C02 at 250 kg/ cm2 and 80 °C. For freeze-dried krill,
`extraction of oil practically terminated in 3-4 h and after
`the use of 2-3 kg of C02 at a flow rate of about 0.6 kg/h.
`Extraction of the krill meal oil, however, ceased after 2-3
`h and the use of 1-2 kg of C02• Reproducibility in the
`experiments was very high.
`Table II shows the yields of oils extracted with SC-C02
`under different conditions. In every sample the extracted
`oil was fluid and bright red from carotenoids. When the
`temperature was increased from 40 to 60 and 80 °C at a
`fixed pressure of 250 kg/ cm2 and when the pressure was
`increased from 250 to 400 kg/ cm2 at a fixed temperature
`of 40 °C, yields of extracted oils remained almost constant.
`
`

`

`906
`
`J. Agric. Food Chem., Vol. 34, No. 5, 1986
`
`Yamaguchi et al.
`
`A
`
`B
`
`A
`
`B
`
`A B
`
`Table IV. Content and Composition of Carotenoids in Oils
`Extracted from Freeze-Dried Krill with Supercritical
`Carbon Dioxide
`
`10•
`
`~ f I
`~ ! I
`
`13. •
`
`3 0
`
`0
`
`8
`
`~ 8
`~ §
`
`9 0
`11
`
`III
`II
`I
`Figure 2. Thin-layer chromatograms of krill oils extracted with
`supercritical carbon dioxide (I) and the residual lipids (II and III):
`A, freeze-dried krill; B, krill meal. Stationary phase: silica gel
`60F254 in chromatograms I-III. Mobile phase: petroleum eth(cid:173)
`er-diethyl ether-acetic acid (90:10:1) in chromatograms I and II;
`chloroform-methanol-water (65:25:4) in chromatogram III.
`Identification: 50% H 2804 in chromatograms I and II; Dragen(cid:173)
`dorff reagent in chromatogram III.
`
`Table III. Fatty Acid Composition of Oils Extracted from
`Krill Samples with Supercritical Carbon Dioxide•
`freeze-
`freeze-
`dried
`dried
`krill
`krill
`0.25
`1.98
`1.82
`17.42
`0.28
`0.15
`22.05
`1.08
`11.78
`1.09
`1.47
`21.37
`2.24
`0.30
`
`fatty acid
`12:0
`14:0
`15:0
`16:0
`16:1
`17:1
`18:0
`18:1
`18:2
`18:3
`
`krill meal
`0.28
`19.08
`0.21
`18.78
`16.30
`1.70
`1.33
`22.40
`3.56
`0.26
`
`fatty acid
`18:4
`20:1
`20:3
`20:4w6 +
`22:1
`20:4w3
`20:5
`22:5
`22:6
`24:1
`unidenti-
`fied
`
`krill meal
`3.21
`1.86
`0.09
`0.55
`
`0.32
`11.36
`0.20
`4.44
`0.32
`0.08
`
`0.30
`6.62
`0.18
`2.71
`0.42
`0.16
`
`•Conditions of extraction: 250 kg/cm2, 60 °C.
`
`veloped with petroleum ether-diethyl ether-acetic acid
`(90:10:1), indicating that the residual lipids were composed
`almost exclusively of polar lipids together with denatured
`and polymerized lipids. Furthermore, as shown in chro(cid:173)
`matogram III, when the residual lipids were developed
`using chloroform-methanol-water (65:25:4) and visualized
`with Dragendorff reagent, at least three orange-red spots
`(12-14) of phospholipids were detected. These results
`confirmed that the SC-C02 extraction of krill samples
`yielded only nonpolar undenatured oils, as mentioned
`above.
`Table III shows the fatty acid compositions of the oils
`extracted with SC-C02 at a pressure of 250 kg/ cm2 and
`a temperature of 60 °C. Essentially no differences were
`noticed in the fatty acid compositions of the oils extracted
`under other conditions. Whereas the main fatty acids were
`14:0, 16:0, 16:1, 18:1, and 20:5 in oils of both freeze-dried
`krill and krill meal, significant differences were found
`between krill meal and freeze-dried krill oils in the 16:1,
`20:5, and 22:6 acids.
`Table IV shows the content and composition of caro(cid:173)
`tenoids in the oils extracted with SC-C02 from the
`
`temp of extractn,0 °C
`40
`carotenoid
`60
`80
`total content, mg/100 g oil 43.5-50.4
`19.1-24.3 7.0-8.7
`composition, %
`astaxanthin diester
`astaxanthin monoester
`astaxanthin
`unidentified
`
`78-83
`13-15
`3-4
`1
`
`b
`b
`b
`b
`
`48-58
`33-44
`5-7
`0-2
`
`•Pressure: 250 kg/cm2 • bUnable to be determined due to de(cid:173)
`composition.
`freeze-dried krill at a fixed pressure of 250 kg/cm2 and
`stepwise increase of temperatures. In previous papers
`(Yamaguchi et al., 1983; Miki et al., 1983), we reported that
`the carotenoids of the Antarctic krill consist almost ex(cid:173)
`clusively of astaxanthin and its esters and that their sta(cid:173)
`bilities against heat and organic solvents are in the order
`of astaxanthin diester, astaxanthin monoester, and asta(cid:173)
`xanthin. It is evident that the astaxanthin tends to be
`decomposed according to their instabilities during ex(cid:173)
`traction with SC-C02. Although Zosel (1978) pointed out
`that SC-C02 is suitable for the isolation of thermally labile
`substances because of its low critical temperature, the
`above finding indicates that some compounds like asta(cid:173)
`xanthin could be unstable under high pressures of SC-S(cid:173)
`C02 at a temperature, for example 80 °C, that never in(cid:173)
`duces the decomposition of astaxanthin under atmospheric
`pressure (Miki et al., 1983). Attention should be paid to
`this fact in the extraction of natural products with SC-C02.
`In spite of such a disadvantage, we proved that SC-C02
`is effective in obtaining nonpolar lipids from Antarctic krill
`by a simple, one-step extraction that excludes the phos(cid:173)
`pholipids that have hampered the utilization of krill oils.
`In this connection, several workers (Stahl et al., 1980;
`Friedrich and List, 1982) have reported the removal of
`polar lipids from seed oils with SC-C02, but the reason why
`polar lipids can be removed with SC-C02 has not yet been
`elucidated.
`As shown in Table III, the oils extracted from krill
`samples with SC-C02 contained fairly high proportions of
`eicosapentaenoic acid (EPA), which is known as a medi(cid:173)
`cally useful substance (Needleman et al., 1979). Recently,
`Krukonis (1984) reported that, by using SC-C02 for the
`fractionation of fish oils, EPA could be concentrated to
`15% from 8%. Used in this manner, SC-C02 extraction
`will produce useful substances from aquatic organisms.
`Abbreviations Used: SC-C02, supercritical carbon
`dioxide; EPA, eicosapentaenoic acid.
`ACKNOWLEDGMENT
`We are indebted to Dr. Y. Miyake, Director of the
`Central Research Institute of Iwatani & Co., Ltd., and Dr.
`T. Fujita, Director of the Central Research Laboratory of
`Nippon Suisan Kaisha, Ltd., for their valuable advice.
`Thanks are also due to N. Ando and K. Kajiyama, lwatani
`& Co., Ltd., for their help in SC-C02 extraction experi(cid:173)
`ments. We also thank Professor George J. Flick, De(cid:173)
`partment of Food Science and Technology, Virginia Po(cid:173)
`lytechnic Institute and State University, for his review of
`this paper.
`Registry No. EPA, 1553-41-9; C02, 124-38-9; astaxanthin,
`472-61-7.
`LITERATURE CITED
`Association of Official Analytical Chemists, Inc. Official Methods
`of Analysis of the Association of Official Analytical Chemists,
`14th ed.; AOAC: Washington, DC, 1984.
`
`RIMFROST EXHIBIT 1162 Page 0003
`
`

`

`J. Agrlc. Food Chem. 1986, 34, 907-9 to
`
`907
`
`Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911.
`Brannolte, H. D.; Mangold, H. K.; Stahl, E. Chem. Phys. Lipids
`1983, 33, 297.
`Brogle, H. Chem. Ind. 1982, 19, 385.
`Friedrich, J.P.; List, G. R. J. Agric. Food Chem. 1982, 30, 192.
`Hannay, J. B.; Hogarth, J. Proc. R. Soc. London 1879, 29, 324.
`Hubert, P.; Vitzthum, 0. G. Angew. Chem., Int. Ed. Engl. 1978,
`17, 710.
`Kaufmann, W.; Biernoth, G.; Frade, E.; Merk, W.; Precht, D.;
`Timmen, H. Milchwissenschaft 1982, 37, 92.
`Krukonis, V. JAOCS 1984, 61, 698.
`Miki, W.; Toriu, N.; Kondo, Y.; Murakami, M.; Yamaguchi, K.;
`Konosu, S.; Satake, M.; Fujita, T. Bull. Jpn. Soc. Fish. 1983,
`49, 1417.
`Mori, M.; Hikichi, S. Rep. Cent. Res. Lab. Nippon Suisan Co.
`1976, No. 11, 12.
`
`Needleman, P.; Raz, A.; Mikes, S. M.; Ferrendelli, J. A.; Sprecher,
`H. Proc. Natl. Acad. Sci. U.S.A. 1979, 76, 944.
`Paul, P. F.; Wise, W. S. The Principles of Gas Extraction; Mills
`and Boon, Ltd.: London, England, 1971.
`Stahl, E.; Schutz, E.; Manglod, H.K. J. Agric. Food Chem. 1980,
`28, 1153.
`Wilke, G. Angew. Chem., Int. Ed. Engl. 1978, 17, 701.
`Yamaguchi, K.; Miki, W.; Toriu, N.; Kondo, Y.; Murakami, M.;
`Konosu, S.; Satake, M.; Fujita, T. Bull. Jpn. Soc. Sci. Fish.
`1983, 49, 1411.
`Zosel, K. Ger. Pat. 2 005 243, 1974.
`Zosel, K. Angew. Chem., Int. Ed. Engl. 1978, 17, 701.
`
`Received for review December 9, 1985. Revised manuscript re(cid:173)
`ceived April 2, 1986. Accepted April 22, 1986.
`
`Isolation of Estrogens in Bovine Plasma and Tissue Extracts Using
`Alumina and Ion-Exchange Microcolumns
`
`Marjorie B. Medina* and Daniel P. Schwartz
`
`Estrogens (estradiol, estrone) in picogram quantities can be isolated quantitatively from bovine plasma
`and tissue extracts by a simple procedure. Bovine plasma (0.1-1.0 mL) was extracted with either acetone
`or ether while tissues (1 g) were extracted with acetone. Extracts were passed through two disposable
`plastic tubes vertically arranged in tandem. The top column (5-mL pipet tip) contained 1-1.5 g of dry
`basic alumina and removed interfering substances. The bottom column (transfer pipet) contained 0.3-1.0
`g of wet anion-exchange resin in the phosphate form and trapped the estrogens through their phenolic
`hydroxyl group. The estrogens were then eluted with acetic acid in acetone following a thorough washing
`of the columns. Recoveries greater than 95% were obtained when extracts of bovine plasma and tissue
`extracts of liver, kidney, and heart were spiked with either tritiated 17/j-estradiol or estrone. This
`technique offers the advantages of simplicity, rapidity, and accuracy over traditional methods employed
`routinely in the purification of estrogens.
`
`Table I. Column Conditions for Isolation of [3H]Estradiol
`Added to Bovine Plasma and Tissue Extracts Using the
`Alumina Ion-Exchange Columns
`extr appl
`to col,
`mL
`
`basic alumina,
`g
`
`resin
`suspensn,
`mL
`
`sample
`
`INTRODUCTION
`Partial purification of estrogens extracted from animal
`tissues and fluids is necessary prior to most methods of
`quantitation. The methods currently employed in routine
`analysis of estrogens such as paper chromatography (Shutt,
`1969), Sephadex LH-20 (Sjovall and Nystrom, 1968;
`Murphy, 1970; Mikhail et al., 1970; Murphy and Diez
`D'Aux, 1975), and Celite column cleanup (Korenman et
`al., 1969; Abraham et al., 1970) are tedious with reported
`recoveries of only 65-85%. Aqueous solutions of estrogens
`had also been purified by ion exchange (Jarvenpaa et al.,
`1979) with reported recoveries of 50-90%. Covey and
`co-workers (1984) also used an anion-exchange resin for
`purification of diethylstilbestrol and dienestrol.
`This paper describes a relatively simple and rapid
`technique that can quantitatively isolate the estrogens
`(estradiol, estrone) from acetone extracts of bovine blood
`plasma and tissues for subsequent chromatographic
`analysis. This study is a preliminary report on the de(cid:173)
`velopment of screening methods to detect and measure
`residues of estrogens in the blood and edible tissues of
`food-producing animals given growth-promoting hormones
`
`acetone extractn
`0.1
`0.5
`1.0
`1.0
`ether extractn
`1.0
`
`A. Bovine Plasma (mL)
`
`2
`4
`8
`sc
`
`4
`
`1.0 (3) 0
`1.0 (3)
`1.5 (5)
`1.5 (5)
`
`1.0 (3)
`
`1.0 (2)b
`2.0 (3)
`3.0 (4)
`3.0 (4)
`
`1.0 (2)
`
`liver
`muscle
`heart
`kidney
`
`B. Bovine Tissues (1 g/8 mL of Acetone)
`2
`1.0 (3)
`2.0 (3)
`2
`1.0 (3)
`2.0 (3)
`2
`1.0 (3)
`2.0 (3)
`2
`1.0 (3)
`2.0 (3)
`
`0 Total volume 95% acetone (mL) to wash alumina column.
`bTotal volume 10% HOAc in acetone (mL) to elute [3H]estradiol.
`'Plasma/acetone mixture added directly to column with glass wool
`on top of alumina bed.
`
`Easteri} Regional Research Center, Agricultural Re(cid:173)
`search Service, U.S. Department of Agriculture, Phila(cid:173)
`delphia, Pennsylvania 19118.
`
`such as 17 /j-estradiol and to ascertain their absence such
`that safe and wholesome food can be delivered to con(cid:173)
`sumers.
`
`This article not subject to U.S. Copyright. Published 1986 by the American Chemical Society
`
`RIMFROST EXHIBIT 1162 Page 0004
`
`