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these base stocks. Using the paraffinic blend no-lead cali-bration for the olefinic, aromatic, and the high- and low-vapor-pressure gasolines resulted in mean errors of 0.0001,0.0020, 0.0010, and 0.0009 g/USG, respectively. Applicationof the background correction resulted in correspondingmean errors of 0.0002, 0,0001, 0.0004, and 0.0007 g/USG.There fore variations in base stock composition have only asmall effect for no-lead gasolines. However, the small back-ground correction is significant at refineries where the ac-ceptable lead level is usually 0.01 g/USG, and at servicestations when the lead level is near the specification of 0.05g/USG maximum. Otherwise the background correctioncan be omitted for most no-lead gasoline monitoring appli-cations. Th e effect of base stock composition is insignifi-can t for low-lead and regula r/premium gasolines because ofthe greater dilutions involved, and no background correc-tion is therefore required.A simple diluti on is, of course, more rapid t han chemicalpretreatment methods such as ASTM D3237-73 and is tobe preferred for that reason. Including calibration andbackground correction, the analysis times are 30 min forone no-lead gasoline sample, and 2% hr for 20. The corre-sponding times for low-lead and regular/premium gasolinesare 20 min and 2 hr.

Flameless atomic absorption (9, 10) and more complexchemical pretreatment (11) methods have also been re-ported recently for the analysis of lead in gasoline. How-ever, flame atomic absorption methods adequately coverthe concentration range of current interest and are to bepreferred because of their b etter precision and speed.

In summary, the atomic absorption method describedhere is a simple dilution procedure that is not affected bydifferences in lead alkyls or gasoline base stock composi-tion. Neither chemical pretreatment nor unusually carefulattent ion to operating parameters is required. The methodis rapid and has very good precision and accuracy.

A C K N O WL E D G M E N TWe thank J. E. Coffey, P. L. Hettinga, D. Kulawic, and

W. M. Meston for experimental assistance, and T. Johnsonfor statistical discussions.L I T E R A T U R E C I T E D

(1) D. J. Trent, Atom. Absorp. News /.,4, 348 (1965).(2) N . Ouickert, A. Zdrojewski, and L. Dubois. Sci. Total Environ., 1, 309(1972).

Table 11. Comparison of Results with OtherMethods and Exchange Dat aLead Concentration, g / G a

This Work Colorimekic X -R a y Fxchan e Avebf stan% dev.0.008 0.0070.0201 0.02000.0270 0.02 640.0422 0.0426

0.31 * 0.05.3100.420 0.426 0.4310.426 0.423 0.4330.435 0.435 0.4330.483 0.46 1 0.4850.490 0.51 f 0.090.730 0.740.800 0.821.32 1.36 f 0.111.84 1.80 i 0.121.90 1.96 i 0.131.91 1.932.45 2.422.49 2.52 * 0.092 -93 2.93 f 0.134.59 4.56 i 0.34g /USG except fo r Canadian Cooperative Fuel Exchange datafo r which units are g / IG . Canad ian cooperative fuel exchange re -sults include standard deviation dat a. Other results are from theASTM fuel exchange. Result s were obtained in 1972 and 1973.

(3) Report EPS 1-AP-73-3, Air Pollution Control Directorate, EnvironmentalProtection Service, Ottawa, Canada, March 1973.(4) R. A. Mostyn and A. F. Cunningharn. J. lnst. Petrol., 53 , 101 (1967).(5 ) R. M. Dagnall and T. S . West, Talanta, 11, 1553 (1964).(6) M. Kashiki, S. Yarnazoe. and S . Oshirna. Anal. Chim. Acta, 53 , 95(7) Du Pont Petroleum Laboratory Test Method M112-71. E. i. Du Pont de(8) H. W. Wilson, Anal. Chem., 38 , 920 (1966).(9) M. P. Bratzel and C. L. Chakrabarti, Anal. Chim.Acta, 61, 25 (1972).

(1971).Nernours& Co., Inc.. Wilrnington, DE 19898.

(10) M. Kashiki, S.Yarnazoe, N. Ikeda, and S . Oshirna. Anal. Lett., 7 ( l ) ,53(11) K. Campbell and J. M. Palmer,J. lnst. Petrol., 58 , 193 (1972).RECEIVEDor review November 18, 1974. Accepted Febru-ary 27,1975.

(1974).

Spec rophotometr c andGas-Liqu d ChromatographicDeterminationof AmitriptylineHorace E. Hamilton, Jack E. Wallace, and Kenneth Blum2The Universityof Texas Health Science Center, 7703 Floyd Curl Drive, San Antonio, TX 78284

Amitriptyline, 10,11-dihydro-5H-5-(3-dimethylaminop-ropylidene)dibenzo(a,d]cycloheptene,nd its monomethy-lamino analog nortriptyline are psychotherapeutic agentswhose therapeutic value for the management of depres-

sions has been well established. The ultraviolet absorptionspect rum of amitri ptyline is nonspecific and difficult todistinguish in biologic extracts from the background ab-sorption contributed by normal biologic constituents. Anumber of spectrophotometric methods for the analysis ofamitriptyline in biologic specimens have been described( 1 - 5 ) ;however, these have generally lacked sufficient sensi-Department of Pathology.Department of Pharmacology.ANALYTICAL CHEMISTRY, VOL. 47 , NO. 7, JUNE 1975 1139


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Table I. Standard Curve of Amitriptyline Oxidation ProductCeric sulfate ulfuric acid, Alkal ine permanganate,

5 m l heptane 5 ml heptaneAmitr iptylin e Absorbance, Absorbance/concn , /m l Absorbance concn, ug/ ml Absorbance concn, ug/ ml

2 0.294 0.147 0.216 0 .1084 0.579 0.145 0.424 0.1066 0.874 0.146 0.623 0.1048 1.146 0.143 0.845 0.10610 1.429 0.143 1.043 0.1041 2 0 .7 1 1 0.143 1.241 0.103

a Absorbance at 250 nm . Values are means of triplica te determinatio ns.

Alkaline permanganate,50 m l hexane

Absorbance/Absorbance concn, e g / m l

0.144 0.0720.271 0.0680.411 0.0680.556 0.0700.691 0.0690.825 0.069

Table 11.Urine Amitriptyline Determination bySemimicroprocedureConcn, Amitriptyline foundi iglml Absorbance @g/ml8.0 0.842 * 0.010 7.45 * 0.09

0.414 i 0.018 3.66 i 0.16.02 .o 0.201 i 0.015 1.78 i 0.130.103 i 0.005 0.90 * 0.04o0.45 i 0.06.051 i 0.007.5aAbsorbance at 250 nm, adjusted fo r mean blank of 0.026.Mean of triplicate determinat ions f tandard deviation.

\ow ,220 240 260 280 300 3iC 340 360NANOMETERS

Figure 1. Ultraviolet absorption spectra of amitriptyline in water, 5/*g/ml(-), and of amitriptyline oxidation product corresponding toa sam ple of equivalent concentration in heptane (- - )tivity and specificity, or have required extensive purifica-tion procedures . Wallace an d Dah1 (6 ) described a spectro-photometric determination of amitriptyline based upon ox-idation of the drug by alkaline permanganate to anthraqui-none ( 7 ) , a product from the drug possessing a unique ul-traviolet absorption spectrum and a high extinction coeffi-cient. Although minor modifications of th at procedure havebeen published (8 ) , t is considered by several investigators(9-11) to be the method of choice for the analysis of ami-triptyline in urine. Th e current report describes a modifi-cation of the Wallace method that includes several innova-tive concepts not previously described. The present proce-dure provides enhanced sensitivity, increased stability ofoxidizing reagent, a decreased volume of solvent require-ment, and eliminates two steps, th us significantly reducingthe analysis time. The drug as its oxidation product can bequantitatively determined by either ultraviolet spectrome-tr y or gas-liquid chromatography. Both a semimicro andmicro adaptati on of the procedure are described.

EXPERIMENTALReagents. A 5.5M sulfuric acid solution containing 25 mg ofceric sulfate per milliliter is prepared by adding 76 ml concentrat-ed sulfuric acid, very slowly and with constant stirring, to a largebeaker cont aining 6.25 g ceric sulfate and 174 ml water. Th e solu-tion is stable for two months at room temperature. The n-hexaneand n-hept ane utilized are of spectroanalytical quality.Apparatus. Reflux condensers were mounted on a Flexaframesupport. The semimicro determination utilizes a conventional Al-lihn condenser, the microdetermination utilizes a previously de-scribed external cold finger reflux condenser available fromKontes Glass Co., Vineland, N J (12). Heating mantles (Glas-Col,

270 watt, 500-ml capacity) were positioned upon magnetic stirrer sbeneath the condenser. Six reflux units were attached to a singleStaco variable transformer which applied voltage to each of theheating mantles through the use of a CRC Multi-lectric Outlet. Asimilar outlet box applied line voltage to each of t he six magneticstirrers. A convenient reflux system may be achieved by moun tingtwo sets of six reflux units each from a single support ma trix, oneset to the front and the other t o the rear. The use of BBs or shotpellets as a heat transfer media allows the heating mantles to beused with varying sizes of flasks, as required.Spectrophotometric measurements were performed on a Beck-man ACTA CIII ratio-recording spectrophotometer equipped witha 3-cell Multi-Positio n mount with microcuvette holder. Quartzmicrocuvettes, 10 mm X 2 mm X 25 mm, of 0.4-ml capacity, wereutilized for the microdeterminations. Quartz cuvet tes, 10 mm X 10mm X 45 mm, of 3.5-ml capacity, were utilized for the semimicroanalyses.Gas chromatographic determinations were carried out on a Shi-madzu GC-5A gas-liquid chroma tograp h equippe d with dualflame ionization dete ctor s, utilizing 3% OV-17, 100-120 mesh (2.0-meter X 4 mm-i.d. glass column) at a column tempera ture of 245C and ni trogen flow rate of 40 ml/min.Procedure. Semimicrodetermination. Five ml of urine, adjust-ed to pH 11-12, are extra cted into 25 ml n-hexane utilizing a two-minu te vigorous ma nual extract ion in a small separator y funnel or50-ml glass-stoppered tube. The hexane is transferred into a 50-mlglass-stoppered graduated cylinder, and the volume of recoveredsolvent measured. Te n ml of th e ceric sulfate-sulfuric acid solutionare added, and the cylinder is shaken vigorously for two t o threeminutes . Nine ml of th e acid extract are trans ferred to a 250-mlround bottom flask containing 5 ml of n-hep tane and a magneticstirring bar.Microde termina tion. Two ml of serum, plasma, or urine are ex-tracted into ten ml n-hexane and back-extracted into three ml ofceric sulfate-sulfuric acid solution as above. A measured quan tity ,i.e., 2 .8 ml, of th e acid extract and 1 ml n-heptane are pipetted intoa 50-ml round bot tom flask containing a magnetic stirr ing bar.Th e boiling flask is attached to t he reflux condenser and securedinto a heating mantle which is supported by the top surface of amagnetic stirre r. After the mixture has refluxed with vigorous stir -ring fo r 25 minutes (optimally achieved with 60 Vac applied to th eheating ma ntles for the macroreflux system and 40 Vac for the mi-croreflux system), the heating mantles are removed and the flasksallowed to cool. The flasks are removed and the heptane layer isremoved and scanned spectrophotometrically over the range 350-

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Table 111.Pla sma Amitriptyline Determination by MicroprocedureConcn, Absorbance, tangent Amitriptyline found,c Amitriptyline found,b*

Tota l absorbance techniqueb (total absorbance) (tange nt absorbance)uglml, added2.0 0.307 j: 0.024 0.133 j: 0.025 1.89 i 0.17 1.81 j: 0.231.5 0.202 j: 0.015 0.105 j: 0.010 1.15 * 0.10 1.41 j: 0.101 o 0.156 i 0.021 0.060 j: 0.004 0.83 i 0.15 0.79 * 0.040.5 0.106 j: 0.011 0.032 j: 0.004 0.48 j: 0.08 0.41 * 0.010.25 0.081 * 0.010 0.019 i 0.004 0.30 j: 0.07 0.23 i 0.03

a Mean of triplicate determinations f tandard deviation. Base line drawn between inflection point/minimums at approximate ly 235and 258 nm. Adjusted fo r mean blank plasma absorbanceof 0.038. Adjusted fo r mean blank plasma absorbanceof 0.004.

Table I V . Elimination Patte rn of Amitriptyline (and Nortriptyline) in UrineFollowing a Single Ora l Dose of 50 mg Amitriptyline HC1Subject 1 C 2 c 3 c 1 2 3

Amitriptyline level, u g/ml a Total amitriptyl ine excreted, u g b

A 0.38 2.91 0.71 439 2054 77B 0.28 0.90 0.58 224 368 268C 3.47 1.34 0.98 145 7 1038 859D (female) 0.99 1.54 1.57 536 635 557

a Mean of duplicate determinations. Mean concentration adjusted fo r ur ine volume. Three collective8-hrur ine spec imens per subject.

230 nm . Analysis at a single wavelength may be achieved by deter-mining the absorption at 250 nm. For a standard, an aqueous ami-triptyline solution was extracted and determined in a manneridentical to that described fo r the biologic specimen. The n-hep-tane f rom t h e reflux flasks can be directly injected into the columnof a gas-liquid chromatograph to provide a chromatographic anal-ysis if desired.

RESULTS AND DISCUSSIONThe amitriptyline oxidation product and anthraquinone

exhibited identical ultraviolet and infrared spectra as wellas gas chromatographic retention data. The spectral andgas chromatographic characterist ics of anth raquinone havebeen previously described (6, 7 ) . Consequently, oxidationof amitr iptyline with ceric sulfate yields anthraquinone,the product also obtained by the alkaline permanganateoxidation of the tricyclic compound (6). Derivatization toanthraquinone provides a markedly enhanced sensitivityand specificity for the determinat ion of amitriptyline by ul-traviolet spectrophotometry (Figure 1).A number of otheroxidants (13-18) which afford high yields of products fromvarious other drugs and have been utilized in other analyti-cal methods were examined, but all provided insignificantamounts of anthraquinone.

The ceric sulfate oxidation of amitriptyline resulted in amarked increase in sensitivity for the determination of thatdrug over that achieved with alkaline Permanganate. TableI presents data on the absorption of the oxidation productobtained by the Wallace method (6), a modification of theWallace procedure in which 5 ml n- hept ane was sub stit ut-ed for 50 ml n- hexane in the reflux step, and the macropro-cedure described in this paper. I t should be noted th at th epermanganate oxidation procedures for determining ami-triptyline (6, 8) require 50 ml hexane for the final solventas opposed to the proposed ceric sulfate oxidation methodthat requires only 5 ml heptane in the macromethod and 1ml heptane in the micromethod. In addition t o achieving alower absorbance per amount of concentration, previouslyreported methods required ten to f i f t y times as much drugto achieve an equivalent final concentration of anthraqui-none.To establish the efficiency and reproducibility of theproposed method, recovery studies were performed onurine containing 0.5 to 8 pg/ml ami triptyline and on plasma

containing 0.25 to 4 pglml. The urine assays were per-formed by utilization of the semimicro adaptation, and theplasma specimens were assayed by the microtechnique.These da ta are presented in Tables I1 and 111,respectively.The amitriptyline recovery over the concentration rangeexamined was 9 1 f 6% for urine and 88 f 8% for plasma(mean of triplicate determinations , f tandard deviation).Fo r absorbances


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2M 2M 270 290 310 330 350NANOMETERS

Flgure 2. Ultraviolet absorption spectra obtained from plasma ex-tracts, corresponding to plasma amitriptyline levels of ( a )0. (b) O ,(c )20, a50, (e)200, and (9 400 ng/ml

RATE OF ELIMINATION TOTAL ELIMINATION

1500-WI3 IOOOI0' 00.

I8 16 24 8 I6 24

HOURS HOURS

Flgure 3. Excretion patterns of amitriptyline (and metabolites) inadult humans following a single oral dose of 50 mg amitriptyline HCI.Urine was collected in three consecutive 8-hour pools during the ini-tial 24 hoursgency basis. The permanganate oxidation required a post-reflux acid wash to remove by-products of the reactionprior to spectrophotometr ic analysis, whereas the ceric sul-fate procedure requires no wash or further purification ofthe refluxed hept ane tha t contains the oxidation products.

Th e oxidation of amitriptyline to anthraquinone pro-vides t he ana lyst with a sensitive and specific gas chroma-tographic procedure as well as a spectrophotometric meth-od. Chromatographic examination of solutions of ami-triptyline and anthraquinone in heptane demonstratedthat the latter provides both a decreased retention timeand increased sensitivity (Figure 5 ) . Aliquots of the re-fluxed heptane are injected directly into the chromato-graph, with no further purification required, for the addi-tional quantitative determination of amitriptyline. Ex-tremely sensitive determinations may be achieved if the gaschromatograph is equipped with an electron capture detec-tor (20).

A number of structurally related pharmacologic com-pounds were examined for possible interference with thedetermination of amitriptyline. Desipramine, imipramine,chlopromazine, trifluoperazine, and phenanthroline exhib-ited no interference; the absorbances at 250 nm obtainedfrom extracts of urine containing 10 gglml of each of theabove drugs were indistinguishable from that obtained

2 4 6 8 1 0DAYS

Figure 4. Serum levels of amitriptyline (and metabolites) in a toxicoverdose case. ( 0 ) ndicates spectrophotometric determinations,(0 )ndicates gas chromatographic determinations

3 2 4 6 8 10 12TlME INMINUTES

Figure 5. Gas chromatogram of benzophenone ( A ) and anthraqui-none(4,eparated on 3 % OV-17 (2.0-meterX 4-mrn i.d. glass col-umn) at a column temperature of 200 'C. Quantities injected were50 and 300 ng of benzophenone and anthraquinone, respectively

from urine blanks. Nortriptyline, protriptyline, and cypro-heptadine are also converted to anthraquinone by the cericsulfate-sulfuric acid oxidation. However, upon extract ionand oxidation by the conditions described in this report,nortriptyline affords a yield of anthraquinone equivalent tothat of amitripty line, whereas protriptyline and cyprohep-tadine give yields of the product t ha t are approximatelyone half and one fifth of that obtained from amitriptyline.Since nortriptyline is a principal metabolite of amitripty-line, the proposed methods provide the analyst with a com-bined drug and metabolite level. Certain of the diphenyl -methanes are oxidized to benzophenone (6, 20 ) , which ab-sorbs maximally at 24 7 nm. However, the absorption char-acteristics of ant hraquinone are unique an d easily discern-ible from the abso rption associated with benzophenone. Inaddition, the sensitivity of the procedure for amitriptylinegreatly exceeds that achieved by oxidation of the diphenyl-methane type drugs (21). Anthraquinone is easily sepa-rated by gas-liquid chromatography from the more volatilebenzophenone derivatives obtained by oxidation of the di-phenylmethane compounds (Figure 6). Braithwaite et al.(22) have demonstrated that total plasma amitriptyline

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and nortriptyline levels exhibit greater correlation with theclinical amelioration of depression, as expressed by theHamilton rating scale ( 2 3 ) , than did plasma levels of ami-triptyline or nortriptyline alone. Braithwaite et al. furtherdemonstrated tha t significant correlation ( p < 0.02) existsbetween t otal plasma am itriptyline and nortriptyline levelsand weight gain, another index of clinical improvement indepression, whereas correlation between plasma amitripty-line levels and weight gain was nonsignificant. Th us, t he in-ability of the proposed methods to differentiate betweenparent and metabolite does not det er from the usefulnessof t he procedure to clinicians or toxicologists. Protr ipty lineand cyproheptadine, administered far less frequently thanamitriptyline, would not be expected to be administeredconcurrently with amitriptyline.

The procedures described in this report determine onlythe unconjugated drugs. Wallace (6 ) has demonstratedthat, if an acid hydrolysis step precedes the extraction ofurine specimens, the conjugated metabolites of a mitri pty-line may also be determined by conversion to anthraqui-none, thus resulting in an significant increase in the totalobserved urine drug level.

CONCLUSIONTh e methods detailed in this report permit the rapid and

reliable quantitative determination of amitriptyline and itsprimary metabolite, nortriptyline, in biologic specimens.Sensi tivi ty is sufficient for reliable dete rmina tions in smallvolumes (1-2 ml) of urine following therapeut ic doses or inserum at high therapeutic or toxic levels. The method maybe amenable to the analysis of other antidepressants thathave the benzocycloheptadiene group as part of theirchemical structure. The combination of ceric sulfate andsulfuric acid provides an excellent medium for oxidation ofthe drugs to suitable derivatives tha n can be assayed by ei-ther ultraviolet spectrophotometry or gas-liquid chroma-tography. The combination of enhanced sensitivity, fewermanipulations, and less analysis time makes the methodpresented in this report a significant addition to the proce-dures available to the clinical and forensic laboratory.

A C KN OWLED GM EN TThe authors acknowledge the technical assistance of

David King, Ralph Grandberry, Linda Goggin, DianaBason, Michael Wallace, John Sulak, and Larry Earley, Jr .Appreciation is also expressed to Merck, Sharp andDohme, Inc., for providing the pharmacologic agents uti-lized in this study.

LI TER A TU R E C I TED(1)Huus, "Detection of Amitriptyline in Urine and Body Tissues", Merck,Sharp and Dohme Research Laboratories, 19 April, 1963.(2) . Sunshine and J. Baumler, Nature(London), 199, 1103 (1963).(3)G. Forbes, W. Pol lock Weir, H. Smith, and J. Bogan, J. forensic Sci.SOC . , , 183 (1965).(4)E. C.Munksgaard, Acta Pharmacol. Toxicob, 27, 129 (1969).

, . , . . .0 2 4 6TIME IN MINUTES

Flgure 6. Gas chromatogram of anthraquinone (A ) and amitriptyline(B),eparated on 3% OV-17 (2.0-meter X 4-mm i.d. glass column)at a column temperature of 245 OC and nitrogen f low rate of 40mllm in: Shimadzu GC-BA, FID, detector 300 OC. Quan tities injec tedwere 50 ng

(5) M. E. Amundson and J. A. Manthey, J. Pharm. Sci., 55, 277 (1966).(6) . E. Wallace and E. V. Dahl, J. forensic Sci., 12, 484 (1967).(7) . Bouche. J. Pharm. Sci , 61, 86 (1972).(8) . H. Cravey, "Amitriptyline", in "Manual of Analytical Toxicology", I,Sunshine, Ed., The Chemical Rubber Co., Cleveland, OH, 1971, p 14-16.(9) . G. C.Clarke, "Isolation and Identification of Drugs", The Pharmaceu-tical Press, London, 1969, 190.(10) . Sunshine, "Handbook of Analytical Toxicology", The Chemical RubberCo., Cleveland, OH, 1969, 7.(1 ) E. S. esell and G. T. Passananti. Clin. Chem .. 17,851 (1971).(12) . E. Wallace and H. E. Hamilton,J. Pharm. Sci,, 63, 1795 (1974).(13) . E. Wallace, J. D. Biggs, and E. V. Dahl, Anal. Chem .,38, 831 (1966).(14) . E. Wallace, Anal. Chem .,39, 531 (1967).(15) . E. Wallace, J. Pharm. Sci., 58, 1489 (1969).(16) . E. Wallace, J. forensic Scb, 14, 528 (1969).(17) . E. Wallace, H. E. Hamilton, J. T. Payte, and K . Blum, J. Pharm. Sci.,(18) . E. Wallace, H. E. Hamilton, J. A. Riloff , and K. Blum. Clin. Chem.,20,(19) . Allen and W. Rieman, Anal. Chem .,25, 1325 (1953).(20) . E. Wallace and H. E. Hamilton, manuscript in preparation.(21)H. E. Hamilton, J. E. Wallace, and K. Blum. J. Pharm. Sci , 73, 741(22) . A. Braithwaite, R. Goulding,J. Bailey, and A. Copper, Lancet, 1, 1297(23)M. Hamilton, J. Neurol. Ne urosurg. Psychiatry, 23, 56 (1960).RECEIVED or review December 31, 1973. Accepted Ja nu -ary 23, 1975. This research was supported by Grant R01DA00729 from the National Institute on Drug Abuse, NIH,Bethesda, MD, and in part by Grant 5 501 RR 05654-05from the National Inst itu te of Hea lth (General ResearchSupport), Bethesda, MD.

61 , 1397 (1972).159 (1974).

(1974).(1972).

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