Document jyvGjn8mapnxp9oRewyBJbnYy

VOLUME 19, KO. 1, JANUARY i 9 4 7 activity 8$ provitamin A, Of lesser importance are nco-S-caroi<n< B, neo-j3-cirotce lT (5n canned samples), y-earotene, and u-caro*ene. Lycopene and its isomers arc inactive and {'-carotene has recently been found inactive (9). Results of several biological assays (rat-growth tests), recently conducted go canned tomato samples of widely different ^-carotene cOTriems, i'ndicaio that the content of d-carotene is at present the best index of provitamin A activity, buj that other carotenoids may increase this activity of Lycopo'iifOH fruits. ACKNri'WJ-EBGMENT The authors wish lo thank R. M. Caldwell. 0. W, Kohler, and Wayne Silver of the Department of Botany and Plan; Pathology for supplying various L'j&opcnicon fruit types necessary for ilm extensive testing of these methods. UTERXTORE CITED (13 Beadle, B. W.. and 2acheiie. F. P.. J. Bio!. Chem.. 144.21 (i&42>>2} Cnry. PI. H., and Beckman. A, O.. J. Optical Sec Am.. 3). 6$2 U041-. 51 (3) Ellis. G. H.. end Hfvnacr. K. C., J. Xvifi/ion. 25. 539 (1943). (4) Lincoln. It. .. Zacbctlo. F. P., Porter. J. W.. Kohler. G. W,, aod Caldwell. R. M.. Boi. Gat.. 105. 113 0943). (3) Mnokinnev. G.. Aronoff. F.. and Born.urir., 3. T.. [.yd. Exo. Chei/.. A_val.Ed.. 34. 3SI (19421. {$) Miller, C. Crrtcl Chem., U, 310 U938), (7) MvlUr. F-. S., Plon> Phynal. 9. 081 (1534). (9) Nash. H. A., and ZscheiD, F. P.. ArzA. ?. 305 (1 P45). (9) Porter, J W.. NWn. H. A,. ZaehoUe, F. P.. and Quackoobush. f. w.,/,.u io. 26i <me>, (10) Porter, J W,, and Zscheik, F. P.. 2Ud. 10, 537 C1&46). (1U Went. F. W.. LcRosifen. A. L-, arid ZechmeUtor. L.. P/anl PAy- ' fo!.. 17, 01 0942). 0 2) ZtchmcLstar, L.. lA-Roctn. A. L., Scbrotder. W. A., Polgdr, h.. and PoulinR. L.. J. .4m. Chem. .Sac.. 65. 19-40 (1943). 13) Ztchir.eistcr. L., find Polgdr. A., Ibid.. 65, to22 0&431. M4) Zschejlc. F. P.. Beadle, B. W.. and KraybUl, H. R-. food Ra- ienrcA. 8. 239 (1943;. '15) ZschcHe. !' P.. and Comw, Q. L.. Bot. G<u-, 102, 463 (1041). (16) Zrcheite. T. V.. White. J. W., Jr.. Boodle, B. W.. tod Roach, J. R.. Plant Phyn*l. 17,331 (1642). JovhYAt. Paper 246. Purdue Cuiveraity AirieuiturM Espeomoot Suncm. TKii InvftsufMioD s siiftporud In part by > araoi }rorr> the Nutrition Foundation, !<- Colorimetric Determination of DDT and Fatty Materials MILTON S. SCHECHTER, MILTON A. POGORELSKIX, and H. L. HALLER U. 5. Department of Agriculture^ Agricultural Research Administration, Bureau of Entomology and Plant Quarantine, BefiJcf/fe, \Jd. A procedure bas been developed for the determination of DDT os such in food stuffs containing considerable smounls of fatty matter such as milk, butter, and ajtiimal fat. Although ibe method is not rapid.. It permits the detection und del ermina lion of DDT in milk in quantities ns low as 1 p.p.m. ECENTsnides (H, IS, 16, 17, BJ)have focused attention on Attempts to rJtr&u 0.5 gram or more of butterfat contuhring R the possible danger to the public health from contamination DDT led to violent nitrations, and even when the nitration was of such products as milk, butter, eggs, meat, and fata when farmcarefully carried out to prevent & violent reaction, the results animal? consume DDT-tmated feed, Phajvrmcologieui investiga obtained were low or negative. Neither Woodard claL (21) nor tions (21) have shown that ingested DDT accumulates ns such in 0/ncr and Calvary (?) mentioned this difficulty when they used the fatty tissues of experimental animals and cad be excreted tn the method in pharmacological investigations, probably because milk. Some of the DDT is metabolized to bis/p-chtoropheiiyj) there was enough DDT present so that very small samples could acetic acid (5, B0), which is excreted in urine (d, 7, 20). Telford's be u.vCrd. US) and Telford and GuthnVs {(7) DDT-feedmg experiments on In order to detect very small amounts of DDT, il h necessary goats And rats, using rather high dosages, indicate that such milk to find some method of removing extraneous material rmd concen may become toxic enough to kill other animals drinking it. In trating the DDT. Aucmptfi to concentrate the DDT by chromn- order to evaluate some of these factors, the Bureau of Entomology lography werts not successful. I/ow-tempenUun? precipitation of and Plant Quarantine started an investigation of the cottl&rnina* the fat by cooling a solution with solid carbon dioxide gave rise lion of f.hes* foods as a result of feeding DDT-treatcd crops lo to diffieul lies in, filtration. Saponification of the fni and nae of the farm animals. unssponlfiable portion would eliminate most of the (oily matter The problem of analyzing these foodstuffs is complicated con but would, at the .-amc time, convert DDT to as dohydroc.hlori- siderably by the presence 0/ large amounts of fatty matter which nated derivative. Such a prvrccdurs might bo useful ' one were accompany the DDT in organic solvent extracts---for example, not interested in whether the DDT was present n.- such or had about 4 gTarua of butterfst will be extracted with the DDT from decomposed to :k ilcliydrochlorinatcd clcrh'otivc. The method each 100 grams of milk. Any attempt to detect 0,1 mg. of DDT of S'.ifi' and Csv^tillo (/.{, >0) has this chaadvantnge In 100 grams of milk (equivalent to 3 p.p.m.) by &ny of the pub lished methods of analysis (J, 4, G, )2, IS, IS, Lk likely to en [n the authors* experiments ri w.-is highly desirable tn establish incomrovcrtihly the presence ol DDT a>; such and 10 estimate its counter difficulty because 0/ the large arrmuut of interfering fettv concfeuuiuimi. The oh-ervaiion ot the solubility of lots ami the matter. The Sehectuer-HaUer colorimetric method (10, 12). al insolubility hi DDT io concentrated sulfuric acid led to the de- though having the advantage of oxidizing a considerable amount velripmeu p? the procedure described in ihi* nrticlv for eliminating of extraneous matter during the nitration, cannot be uxed on all boi a small rcuduc from fatty motCN.-dv. Tin';- residue prob samples cootaining more than a few tenths 0: a gram cf futty mat ably consisix )nu-tly of hydrocarbon.s. A senreh of the literature ter because of the danger of an uncontrollably violent nitration. discl/jccd vjmilxr method? rar the determitinliun i>f oil dcpoijta on PLTEXP009315 WATER PCB-SD0000054570 52 Table I. Recovery of DDT Added to Milk DDT Added P p.n. 0 (Dlini"1 O (bUuk3 1.00 1.00 5.00 5.00 DDT Fouad t'ocorrecid Corrftf i ed lor blink Jcr bUnW P.p.pi. P.p.n. 0.35** 0.35'* 1.2? 1.30 5 50 i to 0.90 0.M 5.22 5 03 Refoveiy Corrected for Btaot `&0 03 105 101 a CxJcuUud 3 DDT; the charsrter.ine blue color um not produced. leaves {9) and of dipbcuyl in orange- (t IS). The relationship to the refining of petroleum with sulfuric ocfd is also interesting to note. apparatus and reagents Some o: the apparatus ond reagems ore described by Schecbu.-r el ol. (J2); it,is Advisable to use cert tubes 25 X 200 mm. or larger for theriitratioits. Scpatotory funnel.-;, 600-inl. capacity, should he prepared in iltt- same manner as the 125-rol. seprimtorv funoeb mentioned by Schechtor el ol. (12) and should be dry when used. Centrifuge and centrifuge bottles with rubber caps. Sodium stib'ate-fulfuric acid. Dissolve )00 grams of c.J>. anhy drous sodium sulfate (oven-dried) in ) liter of c.?. concentrated sulfuric acid (sp. gr. 1.&4.) with t Ue aid of hem, and cool Lo tooui temperature. Sodium hisulfoic is probably formed in the sohilion. Turning sulfuric acid-cohcemrated sulfuric acid. A mixture of equal volumes of fuming sulfuric acid (20 to 20% sulfur trioxide) and concentrated sulfuric acid (?p. gr. J.S4). Sodium bicarbonate solution, 6%. Tcchrucal acetone, technical chloroform, and Skellj'soive B {.*) petroleum fraction, boiling at 603 to 70a C.). These solvents should be redistilled before using. ft i3 advisable to Kayo all apparatus rinsed several times with redistilled technical acetone- and dried. The acetone may bv saved and recovered. PROCEDURE To 100 grams o( milk which has been thoroughly mixed before sampling add an equal volume of 95% ethanol Divide thesolu* lion equally between two 209-col. centrifuge buttles. When the concentration of DDT is liigher than 5 p.p.m., it is advantageous ui take a correspondingly smaller milk sample. Where rnTger bottles are available, the sample need not be divided. Add 50 ml. o{ Skellysolve B to each bottle, cover the bottles with rubber raps, shake\igoruusly, and centrifuge at 2000 r.p.m. for lo min utes; Pour the contents of both hollies into a 500-ml. separatory funnel. .After the layers have separated, drain the. lower layer inequo) portions directly into the same centrifuge bottles. Drain the upper Skellysolve B layer through a 6-ctn. lightly packed plug oJ cotton held in a glass G-oocb crucible holder into a d00-ml. Erfenrueycr flask with a standard ground-glass joint. Extract the solution in each centrifuge boirie in the same manner ba before with two successive 25*ml. portions of Skellysolve B and a fioal 50-roL portion centrifuging for about 10 minutes each time; re turn the lower layers from the separatory funnel to the centrifuge. boule3 nnd filter the upper layers ilirough the plug of cotton into the EricniDcycr flask. After the Inst extraction, rinse the separa tory funnel with 50 ml. of Sketlvsolve B, which is Also run through the plug nf cotton into the Erlenmeyer flask. Add a glass bead to the Erlenmever Bask end recover the Skellysolve B from the milk extract by*distillation on the stenm bath, using on nli-gjas.' apparatus, V?hib the flask is being heated, insert a tube con nected to a vacuum line to remove the last traces of solvent. Quantitatively wash the residue from the distillation into * 500-ml. separatory funnel with 150 ml. of chloroform. For tbe analysis of butter ot fat, substitute for this residue- a 6-grem sample or an cxtroct thereof from which the solvent has been re moved.. Place 100 ml. of chloroform in a second 500-ml. separa tory funnel, and extract the chloroform solution? successively with (1) 50 ml. of .sodium sulfate-sulfuric acid. (2) 50 ml. of so dium sulfate-sulfuric. acid, (3) 50 ml. of fuming sulfuric otj<)r concentrated sulfuric acid, and yt) 50 ml. of .sodium sul/otc-aolfunc add. Jf this last wash is not light in color, it is advisable to use ANALYTICAL CHEMISTRY >till another sodium .xulfoW'.-sulfurie acid wash. Drain each acid wash (lower layer) from the first funnel into Ihe second funnel anJ finally ioto a 250-ml. cylinder. The extraction in the second funnel Is used to minimize the loss of DDT by the slight emulsi fication of chloroform 5n the acid washings. Tbe funnels should W shaken vigorously each time and then allowed to stand for 10 u> 15 minutes before draining off the acid layer. In the rore ea>e where an emulsion forma and does not separate to 30 minutes, ihc mixture may be centrifuged and poured gemly back 'mid die separatory funnel. It is well lo keep a small beaker under euel) funnel and to h&v* a wet doth hnndy to wipe ony.add which may drip. After the acid extractions are completed, filler the chloroform from the first funnel and then from the second funnel through a 5-cm. tightly packed plug of cotton in a glass Gooch cruciblv holder into a third 500-ml. separatory funnel. Pipei off any chloroform which has risen to the surface from the combined ftcii! washings in the cylinder and run it through the plug of cotton. Hinse theiwo funnels And the cotton *rith chloroform, using about 50 to 100 ml. Add enough 5%. sodium bicarbonate solution about 40 ml.) to the combined chloroform fikrata so that it will remain alkaline when tested with liunus paper after vigorous .-baking. After allowing about 10 minutes fora reasonably clear .-eparation, filter only the chloroformisyer through ao-cm. plugof tightly packed cotton in a glass Gooch crucible holder into a 500- mf. Erlenmever flask with a standard joint. YTasb the sodium bicarbonate solution remaining m the funnel wjch two successive 30-ml. portions of chloroform. which are also run through the cot ton inlo the Erlenmever flask. If the filtrate is nol clear, filicT again. ` t Add a glass bead to the Erlenmever flask and recover the chloro iorm on thcsvcatQ boih, usingan all-glass system, until only about 10 ml. of solution are left* wash this quantitatively .into'a large test tube (25 X 200 mm. or larger) with acetone, add o glass bead, nod cautiously evaporate tnc solvent on the steam bath, removing the last traces by inserting a tube connected^ to a vacuum Line. Xitraie the residue with 5 ml. of nitrating mixture and complete the snalvsiaas described by Scbechter el oT. [12). Since there will still be some interference from the small amount; of raffinate from tbe bultcrfst, it is advisable to make spoctrophoiometric measurements at 600, 620, and 640 milli microns (a Beckman quam spectrophotometer was used) and average the results. Below 600 millimicrons there may be enough interference to cause rather high results, so that it may be im possible to calculate the amounts of p,p'-DDT and. oyp'-DDT and add them to obtain the total DDT. Consequently, it is desirable to use ss a standard ft sample of tbe same DDTas was used in the feeding experiment. If it-is not available. DDT of fhes&rne type and grade may be used. wscussro.v The method of extracting the milk differs from a similar pro cedure described by Olson tf ol. (8) in that a centrifuge is used Do break emuhions rapidly and Skellysolve B js used father than a mixture of Skellysolve B with ether. In the present procedure it is not necessary to-wash the extract with water to remove ethanol, ?ince Skellysolve B alone docs Dot emact ethanol along -with the buUertat. Chloroform is used for the sulfuric acid troatmeht, becouse it gives the least trouble with emulsification in the presence of buiterUU The .addition of 5odium sulfate to the sulfuric arid also seems to nid In preventing emulsification. If solvents other ri;nn chloroform arc used or if sodium sulfate is not added to Ihe -ulfurie oeid, emulsion? of rbe consistency or mayonnaise may form- The mixture of fuming sulfuric acid-concentrated sul jmc acid removes material from the chloroform solution of huucrfat which is not removed by the sodium sulfate-sulfuric nitid washes. The use of straight fuming sulfuric, acid cont-afiu'ng 20 Lo 20% of sulfur trioxide hns been found to give low results, probably b3' sulfonetion and removal of the DDT. Phosphoric acid (35%) has been tested in this extraction, procedure in place o{ the sulfuric acid but it does not seem to he a&Sfltisfoctory- Wilh the amounts of re&gents described, persistent emulsions hich do nol break in 20 minutes wj)J be formed only rarely. If mi emulsion is formed, it mny be centrifuged or, preferably, the analyses repeated using a ^cr.nMcr sample. If terser aamples must be used and emulsions are as a consequence regularly encoun tered, one or two preliminary extractions oi tbe chloroform aolu- PLTEXP009316 WATER PCB-SD0000054571 VOLUME ) 9, NO- \. JANUARY 1947 tioa with, sodium suli'au.--concenvtited sulfuric acid using gentte shaking is advisable. Biolpgical (.issues may be (rented with sodium sulfate o? de scribed by Smith and Stobl.Tian (Id) or O/ney and Calvery (>). A nor evaporation of Urn solvent used for c-stroction, the residua may bo dissolved in chloroform and submitted to the sulfuric acid eKlruc.tion procedure described above. p.p'-DDA, or bb.(p-cMoropbeT>yl)ocetic acid, when putthrough the sulfuric ncid treatment, is removed mid ooos not interfere in the ujiv.\y5te for DDT. If p.p'-DDA w to he deter mined in biological tissues, It- should first- be separated from the .'ample by uliludng its acidic properties. It cun be extracted ;rom a.u ether solution of the sample (or extract thereof) with sodium bicarbonate solution, which should be separated, acidified, and extracted with fresh ether, livoporation oi the ether will give .a residue containing the p,p'-DDA. This can usually bo nitrated directly and determined by comparison with p.p'-DDA Mandards run by the Schechter-IlaUcr colorimetric procedure (IB). It traces niT to be rlcrrrriiined and interference* ore en countered, the residue, innceod of being niLmted directly can be Mihnijtied to a modified sulfuric acid Ircmmem. The inacuficn* lion con-isU in omiltmg. the fuming sulfuric ncid-concenirMe.d 'ulfuric ncid and the sodium bicarbonate washes and using only our sodium sulXate-sulfunc acid wn.-jh&s and one wash with -10 ml. of water. Duplicate dctermiiuih&us on J.OO mg. of p.p`DDA using thh modification Have given recoveries of 05 and 01^. 53 dehydrocblorinated derivative likewise would be expected to he unaffected by the sulfuric acid treatment. Any p,p'-DDA|bta(pchlorophenyl)aeetiD acid | which is not removed by the sulfuric acid washes will be removed in the sodium bicarbonate wash of the chloroform solution and hence will not interfere in this de termination. The complete procedure, starting wilb the extraction of the milk sample, was toiled by adding known amounts of technical DDT to the milk. To 100 grants of milk 0.1 mg. of technics) DDT In acetone was added to give 1 u.yun., and to 100 grams of milk O.oO mg. of technical DDT in acetone was added to give 5 p.p.m. The results of these analyses together with the analyses of the original railk without any DDT added (blank Analyses) are given in Table I. The procedure when applied to milk con taining as low as 1 p.p.rn. of DDT gave ai> easily discernible, characteristic blue coldr. The blank milk, to which no DDT'ivas added, gave only a yellow color. Table II shows (.be results of analyses of milk, butter, and. fatsamples for DDT content. These sample? came from cows which were given DDT-treated feed, except ID 10,107, which came from a control cow that did not- receive nay DDT. 'fhe results are in duplicate or triplicate and have been rounded oft* to the nearest whole Dumber except for the blanks. Some of (he devia tions may have been due to inadequate mixing and sampling of the milk, which in many instance* Had separated on standing. The blue color, characteristic <if DDT, was developed in ail eases except for the blank milk, ID 10,197. Table 11. Sarr.plr, ID No i ilk IQ.OtK 10.0'iB 10,106 io.no 10.141 I0.H2 10,151 10. IAS \nalysics of Milk, Du Her, am) Fat for DDT DDT P.p.m. 3.0 4.4 >5. 15, Is t4. is. ie re,*ic 20.20 22, 23 20, 2C Saiupl*. ID Xo. Milk 10.157 10.I5S 10.1EE 10.1S9 10,107 (blank'* Butitr 10.200 10.20) KfcV from sle^k. 1O.3B0 Leas meat from susk. 10.350 DDT r.p.* 21.21 20. 20 21.24 35. 26 O 4, 0.4 456,456 530. 534 176. 170 4. i J CaliulaitJ a* DDT: :be charac'exvilic tlutf color char*<leri*u< ** aol tiCodued. The Application oi the sulfuric acid treatment prior to the ni tration of ihe sample may eliminate interfering substances if they are soluble In sulfuric acid or easily sulfonntabje. Tor example, a 0,0^ solution of DDT in Vetsico) XR-70 (chiefly tetra methylUApht-halane) when analyzed by direct nitration gave values of 10.5 nnd 10.7^, whereas when the sample was put through the sulfuric iWid treatment described under Procedure, values of* 5.2 and 3. D7# uere obtained. RESULTS The procedure was first tested on technical DDT and ctehydrochlorinated p.p'-DDTf1,t-dichloro-2-,2-his(p-clilorophenyl)$ihylenel to determine the percentage recovery of these materials in the absence of buiterfal. tVfcen t.00 mg. of technical DDT was dissolved in 150 ml. of chloroform end carried through the de scribed treiitme.rij, 100*^ recovery was obtained in duplicate runs. When 1.00 rng. of dcbydrochlorinated p,p'-DPT was treated in the same manner, it was recovered to the extent of 97 n.nd 99(~} in duplicolc runs. Since dehydroohlorinated p,p'))DT is evidently ttel removed by the sulfuric acid treatment, its presence to any appreciable extent in a sample may be detected by ita effect on the absorption spectrum of the developed color. Dehydiochlorinaled p.p'^DDT gives a red color in the Schec.hterHaller colorimetric Lest (18), with an absorption spectrum dif ferent from thnt of the blue color of DDT. o,p'-DDT and its ACKNOWLEDGMENT The authors wish to acknowledge the cooperation of J. C, Grime?, of the Alabama Polytechnic Institute, and W* C. Cowsert, of Mississippi State College, for the collection of samples used in. Chip study. LtTEJUTUB e cited (l; Bailes. E. L,, arid Pavnc, M. G.. Ivd. Exg, Chem.. A.val. E., 17.4.JSO&45). (2) Cos. H. E., Aml'jit. 70, 373 (1945). (.3) Grunimiu, O., Buck. A., and Stearns, I., J. Am. Cheui.Soc,, 67, loOU-Ala). C-4) Gunthuc. F. A., Ixa. Ej?g. Cuxh-i Ax.a. Ed.,17, 14.9 (1945), (5) Neal, P. A., Sweeney, T. 11., Spicer. S. S., and Von OeUingon. W. F., U, S. Pub. Health Serrice, Pub. Health RepU.. 6t, 403 (1946). (6) NcdI, P. A.. Von Oetimfien. W, F., and co-workera. Ibid,, Suppi. 177. 2 09441. (7) Ofner. R. R.. and Cnlvcry, H. O.. J. PKsnno&L, 65, 3W/.1945). (8) Olson, K. It.. Hegsted. D. .\J., and Peterson. W. IT.. J. fruity Ret., 22, 63 (1939}. (91 Redd. J. B.. IXD, E.*ro. CrmM,. Ax^t. Eo., 17, H21 (19451(10) SchccluGr, M, S.. and Haller, 31. L..,1m. Chtm. See., 66, 2129 (1044). (t\) Schechter. M. S.. Pogorolslcm, M. A., and Halier. H. L.. Ao?icuUuru! CheTyiicoli, l. (6). 27 (194$). (12) Scheelucr. \f. S.. Soloway, $. B., Hayes. It. A., and Halfer, H". ' L.. J-vd, Eno. Crem.. A.v.u.. Ed., 17,7W (1945). 03) Smith. M- I., and Stohlrna;i, E. T., C. S. Pub. Health Service, Pah. HtaUK Htpli., 59. 9S4 (\!M). (14) Stj/T, H. A., end CasiDIo, J. C.. J Bid. Chtm,. 159. 545 (1946). (15) Stiff, H. A., and Castillo. S. C-, Science, 101, 4d0 (1945). <m TeUorrf, K. S.. Saap S-mit. Chtm., 2t (12), J$1 (1^5). (17) Telford. H. S.. and Guthrie, J. E.. Scfrntt, 102, 647 (1945). (1$) Torr.kine. K. G., and laherwood. F. A., Anulyrt. 70, 330 (19451. ()9) Umhocfer. R. R.. Ixd. Exo. Cnru., Akau. Eo-, 15, 3S3 (1943). (20) White, W. C-, and Sweeney. T. R. U- S. Pub. Hoalih Service, Tu6. Htallh Rtpi., 60, 06 (1CM51(21) Woodard. G., O/ner. R. H.. nnd Montgomery, C. M., Science, 102. 177 (1045';. Tnlfi re*rcl> tefcs coaducifid as part of a program juppdt'ted by Irunsfer &f futids (toui xbe O&ct ol ibe QntrurmMtar Ceorre), U. 3. Army (C*S.< of the Surgeon Gooenl of Corps of Eoodpot*) :o ibe Bur^xu ol Entomology std P!sr.i Qoiondoe. PLTEXP009317 WATER PCB-SD0000054572