904
`
`J. Agrlc. Food Chem. 1986. 34, 904-907
`
`Hawks, S. NJ; 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, 1%.; Miller, C.; Roberts, Dr, Giles, J.; Dickerson, J.;oNelson,
`N.; Rix, C.; Ayers, P. Tobacco Sci. .1976, 20, 40.
`,
`Rodgers, J. D.; Mitchum‘, A. R. Rec. Adv. Tab. Sci; 1976, 2, 112.
`
`‘
`
`Se1tmann,_l-I. Proceedings of the 5th International Tobacco
`Science Congress, 1970, 77.
`Seltmann, H. Beitr. Takforsch. Int. 1980, 10, 120.
`
`’
`
`Received for review November 18, 1985. Accepted April 1, 1986, 5
`
`Supercritical Carbon Dioxide Extraction of Oils from Antarctic Krill
`Katsumi Yarnaguchi,* Masahiro Murakami, I-Iiroshi Nakano, Shoji Konosu, Tsunehiko Kokura,
`‘
`c
`Hiroshi Yamamoto, Masahiko Kosaka, and Kazuhiko Hata —
`V
`
`‘ S
`
`upercritical carbon ‘dioxide extraction of the Antarctic krill yielded oils that were composed solely of
`nonpolar lipids, largely triglycerides, without phospholipids. The extradted oils were fluid and of red
`color due to astaxanthin, whichtended to be decomposed at temperatures higher than 60 °C and a fixed
`pressure of 250 kg/cm”. Analyses of the fatty acids showed a comparatively.-high proportion of eico-
`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. A
`
`Organic solvent extraction of oils from raw materials is
`a well-developed technology. However, after the extraction
`process, further purification steps aregenerally required.
`to remove impurities and gum-forming‘ compounds from
`the extracted oil, especially "in foodstuffs intended for V
`human use.
`p
`.
`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-
`and waste-processing technologies.‘ Various kinds of su-
`percritical fluids have been studied (Wilke, 1978), but ‘most
`work done so far has used carbon dioxide as the extracftant.
`Carbon -dioxide’ has the advantages of nontoxicity, incom-
`bustibility, low critical temperature (31 °C) and pressure
`(75 kg/cm2), and low price, all of which meet the recent
`energy and health concerns.‘ -
`‘
`V
`e
`-
`' The application of supercritical carbon dioxide (SC-C0,)
`extraction to foods has-had limited success, as -exemplified
`by decaffeination of green coffee beans in large-scale in-
`dustrial plants (Zosel, -1974) and production ofhop extract
`(Hubert and Vitzthum, 1978)‘. _Further.,' SC-CO5 has been
`used to extract oils from soybean (Friedrich and List,
`1982), coconut p_alm- (Brannolte et al., 1983), butter
`(Kaufmann et al.,_1983), etc. Applications of "SC—CO2 ex-
`traction‘ to animal sources, "in particular seafoods, are ‘still
`more limited.
`'
`'
`Lipids of"aquatic organisms are generally rich inlhighly
`unsaturated fatty’ acids and phospholipids, which are
`readily deteriorated. The Antarctic krill, Euphausia su-
`perba,,possesses_an especially high proportion. of phos-
`pholipids (Mori and Hikichi,_ 1976), which hampers the
`
`5 Laboratory of Marine Biochemistry, Faculty of Agri-
`culture, The University of Tokyo, Bunkyo-ku, Tokyo 113,
`Japan (K-.Y., M.M., H.N., S.K.);-Iwatani & Company,
`Limited, Higashi-ku, Osaka 541, Japan ‘('I‘.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-CO2 ex-
`traction to krill samples and proved that the extracted oils
`were composed solely of nonpolar lipids without contam-
`ination by phospholipids and their deteriorated lipids.
`' The present paper deals with the extraction and char-
`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 werekept
`below "-25 °C_ until used. Standard ‘commercial prepara-
`tions of lipid reagent were used without further purifica-
`tion.
`«
`»
`
`'
`
`'Proximate composition of the samples was analyzed
`according to the AOAC procedures (No. 7.003, 7.009, 7.015;
`and 7.060; 1934).
`_
`'
`The extraction with SC-CO2 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/cmz
`and 100 °C, respectively; .
`_
`c
`.
`.
`'
`.
`Ground freeze-dried krill (20 g) and krill meal (25 glwere
`put into the extraction vessel, gaseous C0, of which
`pressure was increased to a supercritical state by a pressufle
`pump was introduced, and extraction-was carriedout 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;‘
`100-20() mesh), and 50 mL of chloroform and then 50 ml»
`of methanol. The eluted oils were measured gravimetrr
`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-CO2 extraction. After evaporation of the
`solvent, the oilsand lipids weremeasured gravimetricalbb
`The oils and lipids extracted with SC-CO2 and by the
`method of Bligh and Dyer (1959) were analyzed by TLC
`on silica gel 60F25.,. (Merk, 0.25 mm thick) with petroleum
`' etherédiethyl ether-acetic acid (90:10:1) or chloroform’
`methanol—w.ater (65:25:4) as solvents and 50% aqueous
`sulfuric acid or Drag*end_orff'reagent as indicator.
`A
`
`.oo21-e561/as/14s+o9c4$o1.50/o © 1986 Amed‘can'ChemIca’| Society
`
`NEPN 2012)
`
`NEPN 2012
`
`

`
`I 50-062 Extraction of ous trom Antarctic Krm
`
`J. Agrlc. Food Chem, Vol. 34. No. 5, 1986
`
`905
`
`Table I. ‘Pr-oxim"ate Composition of Krill -Samples
`(as Percentage of Total)
`frozen
`‘krill
`
`freeze-dried
`krill
`
`'
`
`_
`
`krill
`mean
`
`.
`
`.
`
`moisture
`protein“
`fat
`ash
`total
`
`4
`
`.
`
`77.7
`14.0
`3.01
`2.74
`97.5
`
`7.83
`52.0
`16.7
`12.2
`88.7
`
`‘
`
`1.75
`80.7
`11.5
`13.4
`_ 87.4
`
`“Total nitrogen was multiplied by 6.25.
`
`Table 1;. 1’_ields_of oils Extracted from Krill Samples with
`
`Supercritical Carbon Dioxide
`.
`‘
`‘
`amt, g/100, 2 sample
`rec,‘ .
`.~extr
`resid 1
`total
`extractn
`
`
` sample condn‘ oil lipid” lipid” %
`
`
`
`freeze-dried
`250/40 ,
`. 11.2
`7.6
`95
`krill
`250/60
`11.7
`7.9
`98
`250/80
`11.5 I
`8.1
`l 99
`250/40
`4.5
`8.1
`34
`4.1
`9.0 r
`81
`' 4.0
`8.4
`77
`4.0
`9.0
`80
`
`i
`
`.
`krill meal
`
`‘250/80
`‘400/40
`
`19.7
`
`,
`
`i 16.2
`
`V
`
`‘Measured by the Bligh»
`‘Pressure, kg/cm’/temperature, ‘.’C.
`Dyer method.
`‘Recovery == [extr oil 4- resid lipid] /total lipid X
`100.
`
`0
`
`‘
`
`After saponification and" then esterification with boron
`trii‘luoride—-metha‘.nol complex, the fatty acid composition
`of the oils was determined by GLC with a_ Shimadzu QC-
`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 portwas 250 °C:
`The composition of carotenoids in the SC—CO2-extracted
`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-
`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-
`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
`T20 be 16.2% when measured by the method of Bligh and
`Dyer (1959) with chlorot_‘orm-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 frozenkrill.
`»
`_ Figure 1 presents the SC-CO2 extraction curves ‘with
`time of oils from the freeze—dried krill and krill meal with
`SC—C02 at 250 kg/cm? 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 CO2 at a flow rate of about 0.6 kg/h.
`Extraction of the krill meal oil, however, ceased after 2-43.
`h and the use of 1-2 kg of CO2. Reproducibility in the
`experiments was very high.
`‘Table II shows the yields of oils extracted with SC~CO2 A
`under‘ different conditions. In every sample the extracted
`oil was fluid and bright red from carotanoids. When the
`temperature was increased from 40. to6O and _80 °C at a
`fixed pressure of 250 kg/cm” and when .the pressure was 7
`increased from 250 to 400 kg/cm” at a fixed temperature
`of 40 °C, yields of extracted oils remained almost constant.
`
`7
`
`10
`
`
`
`
`
`nerdsofoils(9/{O09sample)
`
`15
`
`
`
`Freeze—dr1ed
`krill
`
`Krill. meal
`
`.
`
`'°'
`
`1
`
`'
`"3
`27
`,(k<J)
`V
`-
`A Cozflux
`‘
`Figure 1. Extraction curves oikrill oils" with supercritical carbon ’
`dioxide at 250 kg/cm‘ and 80 °C.
`‘
`
`4
`
`5
`
`6
`
`‘
`
`_In this connection, Brogle (1982) reported that the solvent
`power of SC-CO2 for organic substances is highly de-
`pendent on its pressure and temperature; ‘at low pressures,
`around 100 kg/cm’, solvent power‘ drops with rising tem-
`perature?» and at pressures higher than approximately 150
`kg/cm’, its solvent power increases. For the krill samples,
`however, the amounts of extracted oils were almost con-
`stant regardless of pressures and temperatures examined.
`That is, even under the: lowest ’temperature/pressure
`combination (250 kg/cm’ and 40 °C), nearly all oils‘ex~
`tractable with SC-CO2 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-
`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-CO2.
`Judging from this result, we think that SC—CO2 extraction
`issuitahle method to obtain undenatured oilsfrom meals
`ofrnarine origin.
`,.
`’
`V
`’
`_
`_
`_
`. 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-
`proximately 100% of the oils from both krill samples was
`recovered in the fraction eluted‘ with chloroform, proving
`that theoils extractednwith SC-C02 were composed ex-
`clusively of nonpolar lipids with practically no polar lipids.
`* Figure 2 shows TLC patterns of the oils extracted with
`SC-CO5 and the residual lipids. ‘By co-TLC with standard
`reagents,‘ the main~_conip_onent of the extracted oils was
`found to be triglycerides (spot 3) and the minor compo-
`nents were .identified_ as hydrocarbon (1), cholesteryl ester
`(2), ‘free fatty acids (4), diglycerides (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-
`_NEPN 2012
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`NEPN 2012
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`

`
`Yamaguchi ei at.‘
`
`Table IV. Content and Composition of Carotenoids in Oils
`Extracted from Freeze-Dried Krill with Supercritical
`Carbon Dioxide
`
`
`
`'
`carotenoid
`
`temp of extractn,‘ °C
`40
`60
`-
`
`80
`
`total content, mg/100 g oil
`composition, %
`astaxanthin diester
`astaxanthin monoester
`astaxanthin
`unidentified
`
`‘
`
`43.5~5(_).4
`
`19.1-24.3
`
`7.0-8.7>
`
`48-58
`33-44
`5-'7
`0—2
`
`>
`
`78-83
`13-15.
`3-4
`1
`
`‘
`
`b
`b
`b
`b
`
`“Pressure: 250 kg/cm’.
`composition.
`
`‘Unable to be determined due to‘de~
`-
`
`freeze-dried krill at a fixed pressure of 250 kg/cm’ 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-
`clusively of astaxanthin and its esters and that their sta-
`bilities against heat and organic solvents are in the order
`of astaxanthin diester, astaxanthin monoester, and aste-
`xanthin. It is evident that the astaxantbin tends to be
`decomposed according to their instabilities during ex-
`traction with SC-CO2. Although Zosel (1978) pointed out
`that SC-CO is suitablefor the isolation of thermally labile
`substances
`ecause of its low critical temperature, the
`above finding indicates that some compounds like aste-
`xanthin could be‘ unstable under high pressures of SC-S-
`CO2 at a temperature, for example 80 ‘’C, that never in-
`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-CO2
`. is effective in obtaining nonpolar lipids from Antarctic krill
`by a simple, one-step extraction that excludes the phos-
`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 removalof
`polar lipids from seed oils with SC-CO2, but the reason why
`polar lipids can be removed with SC~CO2 has not yet been
`elucidated.
`.
`.
`t
`.
`As shown in Table III, the oils extracted from krill
`1, samples with SC—C02 contained fairly high proportions of
`eicosapentaenoic acid (EPA), which is known as a med!-_
`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-CO2, supercritical carbon
`dioxide; EPA, eicosapentaenoic acid.
`‘
`ACKNOWLEDGMENT
`.
`-
`-
`_ We are indebted to Dr. Y. Miyake, Dir'ector»of the
`Central Research Institute of Iwatani 82 Co., Ltd., and DY-
`’_I‘. Fujita, Director of the Central Research Laboratory Of
`Nippon Suisan Keisha, Ltd., for their valuable advice;
`Thanks are also due to N. Ando and K. Kajiyama, Iwataffl
`& Co., Ltd., fdr their help in SC~CO2 extraction expel"
`rnents. We also thank Professor George J. Flick. D9‘
`_ pertinent of Food Science and Technology, Virginia P0‘-
`lytechnic Institute and State University, for his review ‘Pf
`this paper.
`Registry No. EPA, 1553-41.9; C0,, 124-38-9; astaxantliiflr
`. 4'/2-61-7.
`
`'
`
`_.
`_
`LITERATURE CITED ‘
`Association of Official Analytical Chemists, Inc. Official Methfldi
`of Analysis of the Association of_0fficial Analytical Chemists-
`14th ed.; AOAC: Washington, DC, 1984.
`-
`
`NEPN 2012
`
`
`
`111
`
`906
`'
`
`J. Agrlé. Food Chem, Vol. 34, No. 5. 1986
`A
`B
`A
`
`B “
`
`lD®~U\Lhfi1
`
`II
`
`Figure" 2. Thin-layer chromatograms of krill oils extracted with
`supercritical carbon dioxide (1) and the residual lipids (II and III);
`A, freeze-dried krill; B, krill meal. Stationary phase: silica gel
`601%, inchromatograms l—IIl. Mobile phase: petroleum eth-
`ervdiethyl ether~acetic acid (90:l0:1) in chromatograms I and II;
`chloroform—methano1—water (65:25:4) in chromatogram III.
`Identification: 50% H2804 in chromatograms I and II; Dragen-_
`_dorff reagent in chromatogram III.
`p
`.
`
`_
`
`Table 111. Fatty Acid Composition of Oils Extracted from
`Krill Samples with Supercritical Carbon Dioxide“
`freeze-
`‘
`.
`‘
`freeze-
`_
`V
`dried
`~
`dried
`,
`-
`
`fatty acid
`krill
`krill meal
`fatty acid _ krill
`krill meal
`_ 12:0
`0.25
`0.28
`18:4 A
`1.98
`3.21
`14:0
`17.42
`19.08
`20:1
`1.82
`1.86
`15:0
`0.28
`0.21
`20:3‘
`0.15
`0.09
`16:0
`22.05
`18.78 ,
`020:-40:6 +
`1.08
`0.55
`16:1
`11.78
`16.30
`22:1
`17:1
`1.09
`1.70
`2o:4...3
`18:0
`1.47 _
`1.33
`7 20:5
`18:1
`21.37
`22.40
`22:5
`18:2
`2.24 ~
`3.56
`22:6
`18:3
`0.30
`0.26 ‘
`24:1
`'
`unide_nti-
`fied
`V
`“Conditions of extraction: 250 kg/cm”, 60 °C.
`
`,
`
`_
`
`.
`
`~
`
`-
`
`0.32
`11.36
`0.20
`4.44
`0.32 »
`0.08
`
`0.30
`6.62
`. 0.18"
`2.71
`0.42
`0.16
`
`'
`
`'0
`
`veloped with petroleum ether—diethyl ethervacetic acid
`(90:10:1), indicating that the residual lipids werelcomposed
`almost exclusively of polar lipids together with denatured
`and polymerized lipids}; Furthermore, as shown in chro-
`matogram III, when the residual lipids were developed
`using.chloroform-methanol~water ‘(65:25:4) and visualized
`with Dragendorffreagent, atleast three orange-red spots
`(12—-14) of phospholipids‘ were detected. VThese results
`confirmed that the SC-CO2 extraction of. krill samples
`yielded only nonpolar undenatured oils, as mentioned
`above.
`‘
`‘
`A
`Table III shows the fatty acid compositions of the oils '
`extracted with SC-CO2 at a pressure of 250 kg/cm” 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 freezeédried
`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 showsthe content and composition of caro-
`tenoids in the oils extracted "with SC—C02 from the
`
`NEPN 2012
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`

`
`Bligh, E. G.; Dyer; W. J. Can. J. Biochem. Physiol. 1959, 37, 911.
`Brsnnolte, H. D.; Mangold, H. K.; Stahl, E. Chem. Phys. Lipids
`1983, 33, 297.
`”
`-
`V
`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, O. G. Angew. Chem, Int. Ed. Engl. 1978,
`17, 710.
`-
`'
`'
`Kaufmann, W.; Biernoth, G.; Frade, E.; Mark, W.; Precht, Dr,
`Timrnen, H. Milchwissenschaft 1982, 37, 92.
`_
`‘
`. Krukonis, V.JAOCS 1934, 61," 698.
`Miki, W.; Torin, 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.
`
`9979
`A
`_
`-
`.
`.1. Agrlc. Food Chem. 1933, 34, 907-910 '
`Ferrendelli, J. A.; Sprecher,
`V Needleman, P.; Raz, A.; Mikes, S.
`.
`I-I. 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.
`V
`.
`Zosel, K. Ger. Pat. 2005 243, 1974.
`Zosel, K. Angew. Chem., Int. Ed. Engl. 1978, 17, 701.
`
`A
`
`‘
`
`' Received for re’view9December 9,.1985. Revised manuscript re-
`ceived April 2, 1986. Accepted April 22_, 1986. ‘
`
`_ Isolation of Estrogens in Bovine Plasma and Tissue Extracts Using
`Alumina and Ion-Exchange Mic-rocolumns
`
`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 intandem. 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
`9 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 17,8-estradiol or estrone. This
`' technique offers the advantages of simplicity, rapidity, and accuracy over traditional methods employe
`routinely in the purification of estrogens.
`
`
`Table I. Column Conditions for Isolation of [’H]Estradiol
`A Added to Bovine Plasma and,Tissue Extracts Using the
`Alumina ,Ion—l.'*3xchange Columns
`extr appl
`\
`<
`resin
`to col,
`basic alumina,
`suspensn,
`«
`
`sample >mL mL ’ ' g -
`
`
`
`
`
`
`'
`
`a
`
`( INTRODUCTION
`' Partial purification of estrogens extracted from animal
`tissues and fluids is necessary prior to most methods of‘
`quantitation. The methodscurrently employed in routine
`analysis of estrogens such as paper chromatcgraphy.(Shu_tt,
`1969), Sephadex. LH-20 (Sjovall and Nystrom, 1968;
`Murphy, 1970; Mikhail et al., 1970; Murphy and Diez
`lJ’Aux, 1975), and Celite column cleanup (Korenman et
`111., 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 (Jarvenpafi et al.,
`1979) with reported recoveries of 50-90%. Covey and
`co—workers (1984) also used an anion-exchange resin for
`purification of diethylstilbéstrol 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- ‘
`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
`
`.
`
`A
`
`.
`
`.
`
`A. Bovine Plasma (mL)
`.
`
`2
`4
`8
`8”
`
`‘
`
`-
`
`1.0 (3)“
`1.0 (3)
`1.5 (5)
`1.5 (5)
`
`1.0 (2)?
`V 2.0 (3)
`3.0 (4)
`3.0 (4)
`
`,
`
`'
`
`-
`
`acetone extractn
`0.1
`0.5
`1.0
`1.0
`ether extractn.
`11.0
`
`1.0~(2)
`
`1.0 (3)
`4
`l
`I
`B. Bovine Tissues (1 g/8 ml. of Acetone)
`liver
`-
`2
`..
`1.0 (3l-
`2.0 (3)
`_ muscle
`2
`1.0 (3)
`2.0 (3)
`heart
`2
`1.0 (3)
`2.0 (3)
`kidney
`-
`'
`2
`1.0 (3)
`v
`2.0 (3)
`“Total volume 95% acetone (mL) to wash alumina column.
`‘Total volume 10% HOAc in acetone (mL) to elute [31-Ilestradiol.
`‘ °Plasma/acetone mixture added directly to column with glass wool
`on top of alumina bed.
`
`Eastern Regional Research Center, Agricultural Re-
`search Service, U.S. Department of Agriculture, Phila-
`delphia, Pennsylvania 19118.
`—
`’
`
`such as 17fl-estradiol and to ascertain their absence such
`that safe and” wholesome food can he delivered to con-
`sumers.
`'
`'
`~‘
`
`
`
`This article not subject to us. Copyright. Published 1986 by me American Chemical Society N EPN 2012
`
`NEPN 2012