KATO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 719 RESIDUES AND TRACE ELEMENTS Preprtion of Smples for Gs Chromtogrphy/Mss Spectrometry Anlysis of Phthlte nd Adipte Esters in Plsm nd Beverges by Stem Distilltion nd Extrction KAYOKO KATO,YOSHIHIRO YOSHIMURA, nd HIROYUKI NAKAZAWA 1 Hoshi University, Deprtment of Anlyticl Chemistry, Fculty of Phrmceuticl Sciences, 2-4-41, Ebr, Shingw-ku, Tokyo 142-8501, Jpn YUKO ITO nd HISAO OKA Aichi Prefecturl Institute of Public Helth, Deprtment of Chemistry, 7-6, Tsuji-mchi, Kit-ku, Ngoy Aichi 462-8576, Jpn Phthlte nd dipte esters re present in reltively lrge mounts in the environment, resulting in their lrge blnk vlues t nlysis nd mking precise nlysis difficult. We developed highly sensitive nlyticl method for phthlte nd dipte esters in plsm nd beverges by lowering the blnk vlues tht interfere with nlysis. The method uses closed distilltion clenup system in which stem distilltion nd extrction re performed simultneously. The recoveries from beverges nd plsm were both stisfctory, rnging from 90.2 to 118.3%, reltive stndrd devition (RSD) = 2.8 5.3%, nd 96.2 134.4%, RSD = 2.2 6.5%, respectively. The detection limits of dibutyl phthlte nd di-2-ethyl hexyl phthlte were 5 ng/ml, nd those of diethyl phthlte, butyl benzyl phthlte, nd di-2-ethyl hexyl dipte were 10 ng/ml in rbbit plsm nd beverges. Plstic products mde of mterils such s vinyl chloride resins re widely used not only s household goods but lso s medicl supplies, including blood tubing for rtificil dilysis, kits for blood trnsfusion (tubes nd bgs), nd ctheters. Inexpensive plsticizers, such s phthlte nd dipte esters, hve been dded to vinyl chloride resins to improve their qulity nd workbility; however, toxicologicl properties of severl phthltes tht hve eluted from plstic continers into food nd hve been tken into the body hve been described in the literture (1 3). Thus, highly sensitive nlyticl method is needed for such plsticizers in food nd biologicl smples. Phthlte nd dipte esters re present in reltively lrge mounts in the environment, e.g., in ir nd wter, resulting in their lrge blnk vlues t nlysis nd mking highly precise nlysis difficult. Conventionl smple preprtion methods for such plsticizers include complicted processes such s Received September 21, 2001. Accepted by JS December 22, 2001. 1 Author to whom correspondence should be ddressed; e-mil: nkzw@hoshi.c.jp. liquid liquid extrction nd solid-phse extrction, nd the high blnk vlues obtined due to contmintion t nlysis result in high detection limits (4 9). In stem distilltion, stem is injected into wter-insoluble orgnic compound to seprte voltile component tht is distilled with the stem (10). Even n orgnic compound hving high boiling point cn be distilled with stem t 100 C if it hs sufficient stem pressure. Stem distilltion hs been used for seprtion nd purifiction of compound in complicted mtrix, nd my be pplicble to nlysis of plsticizers in food nd in biologicl smples. However, becuse the processes of extrcting the distillte with n pproprite orgnic solvent nd concentrting the extrct involve risk of contmintion by plsticizers, the use of stem distilltion for nlysis of plsticizers in food nd in biologicl smples is difficult. Accordingly, it hs been suggested tht distilltor in which stem distilltion nd extrction re performed in closed system my be stisfctory for preprtion of such smples (11 14). Experimentl Regents () Solvents. Hexne, cetone, nd methnol were phthlte nlysis grde (Knto Kgku, Tokyo, Jpn). Toluene ws dioxin nlysis grde (Knto Kgku). (b) Chemicls. Anhydrous sodium sulfte ws pesticide nlysis grde (Wko Pure Chemicl Industries Ltd., Osk, Jpn). (c) Wter. Milli Q wter (Millipore, Bedford, MA). (d) Reference mterils. Diethyl phthlte (DEP), dibutyl phthlte (DBP), butyl benzyl phthlte (BBP), di-2- ethyl hexyl phthlte (DEHP), di-2-ethyl hexyl dipte (DEHA), DEP-d4, DBP-d4, BBP-d4, nd DEHP-d4 were environmentl nlysis grde (Knto Kgku). DEHA-d8 ws environmentl nlysis grde (Hyshi Pure Chemicl Industries, Osk, Jpn). (e) Stndrd solutions. All were prepred with hexne so tht ech of the stndrd solutions contined one of the phthlte nd dipte esters t 1000 ng/ml, nd were diluted when used.
720 KATO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Figure 2. SIM chromtogrms of stndrd solution. GC conditions used for beverges. Figure 1. (f) Internl stndrd solutions. All were prepred with methnol so tht ech of the solutions contined one of the internl reference mterils t 1000 ng/ml, nd were diluted when used. (g) Stndrd solutions for clibrtion curves. All were prepred so tht ech of the stndrd solutions of phthlte nd dipte cid esters contined one of the internl reference mterils t 100 ng/ml. Instrumenttion () Gs chromtogrphy/mss spectrometry (GC/MS) system. HP5896 GC with Series HP5971 mss selective detector nd 7673 utosmpler (Hewlett-Pckrd, Plo Alto, CA). (b) Coolnt. Eyel Cool Ace CA-1110 (Tokyo Rikkiki Co. Ltd., Tokyo, Jpn). Apprtus Distilltion clenup system. () Essentil oil distilltion clenup system. See Figure 1. (b) Glsswre. All were wshed with cetone, followed by heting for t lest4hindryer t 200 C. Conditions for Gs Chromtogrphy/Mss Spectrometry Anlysis The column used in the experiment ws DB-5MS (J&W Scientific, Folsom, CA) 0.25 mm id 30 m, film thickness 0.25 µm. The crrier gs ws He (0.9 ml/min). For nlysis of plsm, initil column temperture ws 150 C, mintined for 3 min, nd then rised to 280 C t elevtion rte of 10 C/min. For nlysis of beverges, initil column temperture ws 50 C, rised to 280 C t elevtion rte of 10 C/min. Inlet temperture ws 280 C, nd detector temperture ws 280 C. A 1 µl mount of ech smple ws injected. Selected-ion monitoring (SIM) ws performed t m/z 149 for DEP, DBP, BBP, nd DEHP; t m/z 153 for DEP-d4, DBP-d4, BBP-d4, nd DEHP-d4; t m/z 129 for DEHA; nd t m/z 137 for DEHA-d8. For GC/MS conditions, we used different initil column tempertures between 50 nd 150 C for beverge nd plsm, respectively, becuse coexisting substnce from beverge sometimes ffected chromtogrphic resolutions when the smple ws nlyzed t initil column temperture of 150 C. Preprtion of Smples After ddition of wter (500 ml) into round-bottomed flsk (1000 ml), the flsk ws connected to the distilltor (Figure 1), nd smll mount of wter nd 4 ml toluene Tble 1. Accurcy dt Prmeter Linerity (ng/ml) 5 1000 5 1000 5 1000 5 1000 5 1000 Correltion (r) 0.998 0.997 0.995 0.998 0.997 Detection limit (ng/ml) 2 5 2 2 2 Retention time RSD, % 0.1 0.08 0.1 0.05 0.08 Pek re RSD, % 0.3 0.5 1.3 0.9 1.1 Retention time nd pek re RSDs re clculted t 50 ng/ml with n =6. GC conditions used for beverges nd plsm. Figure 3. Removl of plsticizers from wter.
KATO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 721 Tble 2. Recovery test of plsticizers from distilled wter Added, ng/ml Without I.S. b 100 83.9 (13.5) 45.6 (10.8) 67.4 (30.0) 46.2 (33.0) 110.5 (40.3) With I.S. 500 102.5 (1.9) 93.7 (2.8) 94.4 (4.3) 95.8 (1.8) 100.3 (2.8) 100 98.3 (0.5) 94.5 (1.1) 101.3 (2.9) 96.8 (1.3) 99.8 (2.2) b n = 4. GC conditions used for beverges. I.S. = internl stndrd. were plced in the distilltor, to which Dimroth condenser ws ttched. Before smple preprtion, the wter ws distilled for 8 h to remove plsticizer contminnts in the pprtus. During distilltion, toluene in the distilltor ws replced every 2 h. After 8 h, the toluene nd wter in the distilltor were discrded nd smple (either plsm or beverge, 4 ml) nd methnol solution (100 µl) contining one of the internl stndrds (1000 ng/ml) were dded into the flsk. A 1 ml mount of fresh toluene ws ccurtely mesured nd fed into the distilltor, to which Dimroth condenser ws ttched. After4hofdistilltion, the extrcted lyer of toluene ws tken into smll test tube, to which smll mount of nhydrous sodium sulfte ws dded for dehydrtion, nd smple ws obtined for GC/MS nlysis. Determintion The smples were prepred to hve specific concentrtions, the pek res were clculted by GC/MS, nd the clibrtion curves were obtined ccording to the internl stndrd method of using deuterted compounds. Results nd Discussion The present study describes n nlyticl method for DEP, DBP, BBP, DEHP, nd DEHA, which re frequently found in environmentl nd food smples. Determintion by GC/MS The retention time reltive stndrd devition (RSD) vlues, pek re RSD vlues, linerity, nd detection limit [signl-to-noise (S/N) rtio = 3] of ech smple re shown in Tble 1; SIM chromtogrms re shown in Figure 2. Becuse the RSD of the retention time of ech smple ws 0.1%, the following experiment ws conducted under the conditions for GC/MS nlysis described bove. Removl of Plsticizers from Wter In the present method, smple nd wter re heted nd circulted, nd the voltile component is collected in toluene. Thus, if plsticizer is present in the wter, it will be concentrted in toluene, resulting in high bckground vlue. Such plsticizers must be removed from the wter to be used before smple preprtion. Milli Q wter without further purifiction ws heted nd circulted for 4 h, nd toluene, which ws nlyzed by GC/MS, ws found to contin DBP t 110 455 ng/ml (295 ng/ml verge) nd DEHP t 120 280 ng/ml (200 ng/ml verge). However, s shown in Figure 3, these plsticizers in the Milli Q wter were completely removed fter 8 h of distilltion. Although DBP nd DEHP in the wter cn be removed s described bove, other plsticizers my still be present. Accordingly, 5 types of plsticizers (DEHA, DEP, DBP, BBP, nd DEHP, 1000 ng ech), were fed into the Milli Q wter fter 8 h ofdistilltion to study their removl. None of these plsticizers were detected fter 8 h of distilltion. Therefore, it ws confirmed tht 8 h of distilltion removes ll of these plsticizers. Tble 3. plsm Added, ng/ml Recovery test of plsticizers from rbbit 25 99.0 (2.2) 103.0 (6.5) 103.0 (4.2) 134.4 (6.3) 96.2 (5.2) 100 98.3 (2.2) 107.4 (5.3) 104.2 (4.2) 112.5 (4.8) 112.5 (4.8) 500 100.8 (0.9) 107.4 (1.8) 101.2 (1.0) 108.9 (3.2) 102.6 (1.3) Tble 4. Added, ng/ml Recovery test of plsticizers from beverge 25 103.2 (3.9) 90.2 (2.8) 102.4 (4.7) 118.3 (5.3) 101.3 (3.8) 100 97.3 (2.9) 93.5 (1.5) 100.3 (2.9) 105.8 (4.2) 100.8 (2.2) n = 4; concentrtion of internl stndrd, 100 ng/ml. n = 7; concentrtion of internl stndrd, 100 ng/ml.
722 KATO ET AL.: JOURNAL OF AOAC INTERNATIONAL VOL. 85, NO. 3, 2002 Tble 5. Anlyticl results of plsticizers in commercil beverges in plstic continers Found, ng/g Beverge DBP DEHP A 71.0 25.8 B 51.0 22.3 C 35.0 18.8 D 7.8 3.3 E 9.0 4.7 F 5.8 6.8 G 105.3 30.0 H 82.3 36.3 Recovery from Wter Ech of the plsticizers with concentrtion of 500 or 100 ng/ml ws dded to 4 ml purified wter (Milli Q wter with further purifiction s described bove) to perform the recovery test. The recoveries fter 4hofdistilltion re shown in Tble 2. According to the bsolute clibrtion curve method, DEHP nd DEHA exhibited stisfctory recoveries, wheres DBP exhibited slightly low recovery of 67.4%, nd DEP nd BBP showed very low recoveries of 45.6 nd 46.2%, respectively. Also, the RSDs vried gretly, rnging from 10.8 to 40.3%. According to the distilltion method, the trget compound in the flsk boils t 100 C when heted with wter, vporizes with wter, nd then cools in the coolnt provided t the upper prt of the distilltor (Figure 1). The wter obtined nd the trget compound drip into the extrct solvent plced in the middle of the distilltor, nd the trget compound is then extrcted. The low recoveries of DEP nd BBP were ttributed to the low extrction efficiencies of these compounds in the extrct solvent with their reltively high wter-solubility. To improve recoveries, distilltion ws performed for more thn 4 h. However, recoveries were not significntly higher thn those obtined fter 4 h of distilltion, which suggests tht the concentrtion of plsticizer hs reched equilibrium between the extrct solvent nd the dripped solution fter 4 h of distilltion. However, ddition of stble isotopes (100 ng ech) serving s internl stndrd mterils into the flsk before distilltion improved the recoveries to 94.5 101.3% (RSD 2.9%) t concentrtion of 500 ng/ml, nd 93.7 102.5% (RSD 4.3%) t 100 ng/ml. Thus, we selected distilltion time of 4 h. Recovery from Plsm Whole blood ws collected from rbbit (Jpnese white, mle, 3000 3500 g) by venipuncture into glss tubes with heprin by using glss syringe; this ws followed by centrifugtion for 20 min t 3000 rpm. The plsm obtined Figure 4. SIM chromtogrms of plsticizers from beverge. ws subjected to recovery test. Tble 3 shows tht the verge recoveries of ech of the plsticizers in 7 mesurements rnged from 100.8 to 108.9% (RSD = 0.9 3.2%) t concentrtion of 500 ng/ml, nd from 98.3 to 112.5% (RSD = 2.2 5.3%) t 100 ng/ml. Even t lower concentrtion of 25 ng/ml, recoveries were s good s 96.2 134.4% (RSD = 2.2 6.5%). The detection limits of DBP nd DEHP were 5 ng/ml, nd those of DEP, BBP, nd DEHA were 10 ng/ml. Therefore, this distilltion method would be stisfctory for nlysis of plsticizers in biologicl smples. We nlyzed femle humn plsm. No DEHA, DEB, or BBP ws detected; however, DBP nd DEHP were detected, with DBP concentrtion of 30 ng/ml nd trce of DEHP. Recovery from Beverges Ech of the plsticizers with concentrtion of 100 or 25 ng/ml ws dded to 4 ml of beverge for the recovery test (Tble 4). The recoveries were stisfctory, rnging from 93.5 to 105.8% (RSD = 1.5 4.2%) t concentrtion of 100 ng/ml, nd from 90.2 to 118.3% (RSD = 2.8 5.3%) t 25 ng/ml. With this distilltion method, the detection limits of DBP nd DEHP in smples were 5 ng/ml, nd those of DEP, BBP, nd DEHA were 10 ng/ml. Becuse the results suggested tht this distilltion method is pplicble to food vilble on the mrket, we pplied the method to commercilly vilble smples. Eight commercil beverges in plstic continers were subjected to the nlysis. The results re shown in Tble 5, nd their typicl profile (beverge G) is shown in Figure 4. No DEHA, DEB, or BBP ws detected in ny of the smples; however, DBP nd DEHP were detected in ll of the smples, with the DBP concentrtion rnging from 5.80 to 105.3 ng/ml nd the DEHP concentrtion rnging from 18.8 to 36.3 ng/ml. Conclusions Opertionl blnk vlues were reduced by prepring Milli Q wter, followed by heting nd circulting the prepred wter in distilltor for t lest 8 h, with extrction nd removl of ny phthlte contminnts with toluene. Stndrd solutions of ech plsticizer were dded to beverges nd
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