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KP, U\i. y i *4 Porphyrinogens, Porphyrins, and the Porphyrias The porphyrias are consequences of impairment either in the formation of the porphyrinogens from porphobilinogen or in the transformation of the porphyrinogens to hem e.1 Such an impairment causes a pathologic accumulation of porphobilinogen or porphyrins (or both) in tissues and body fluids. ^ The manifestations of the porphyrias include jiiCtuiSnsitivity-of theSkin, persistent abdominal aching or pain, and neuropathy with widely variable expression that may affect the central, autonomic, and peripheral systems and include mental disturbance, motor dysfunction, and sensory loss. The cutaneous photosensitivity may range from very mild to very severe with mutilating lesions. The neuropathy ofporphyria is especially significant in that it can 1progress to life-threatening respiratory paralysis (Table ). The formation of the-porphyrinogens and heme from glycine and succinyl coenzyme A occurs in human cells of nearly all types and includes the following 15 major enzyme-catalyzed steps: Glycine + succinyl coenzyme A ' S-Aminolevulinate L' Porphobilinogen ,/ 3 .|UVr>ovp)wo>rpHhiyirr<ino>gvepnii II * .Uroporphyrinogen III t Heptacarboxyporphyrinogen I t Hexacarboxyporphyrinogen I t Pentacarboxyporphyrinogen I M Heptacarfooxyporphyrinogen III 6t !Hexacarboxyporphyrinogen III 7t Pentacarboxyporphyrinogen III iLoproporphyrinogen I 8 tCoproporphyrinogen III 190I1Protoporphyrinogen III (IX) Protoporphyrin IX ii | Heme Only the porphyrinogens of the series III are precursors of heme, and the isomeric porphyrinogens of the series I and the porphyrins of both series I and III are by-products with no known physiologic functions; the porphyrins are formed from the porphyrinogens by nonenzymic oxidation. As shown in Table 1, seven forms of porphyria are known to occur, and with one exception, each form is inheritable and is caused by or potentiated by an abnormally low activity of one of the several enzymes involved in the formation of the porphyrinogens and heme. Two forms of porphyria are expressed primarily in the erythropoietic system. Congenital erythropoietic porphyria is a rare disorder that is readily recognizable in neonatal life by the voiding of dark or wine-red urine; uroporphyrin I is primarily responsible for the color of the urine. Congenital erythropoietic porphyria is characterized also by moderate to extreme photosensitivity, hypertrichosis, hemolysis, splenomegaly, and increased uroporphyrin and coproporphyrin in the erythrocytes and blood plasma. Examination of the bone marrow in violet light (400 to 410 nm) shows red fluorescence of excessive porphyrins in the cytoplasm of red cell precursors. In addition, in most known cases uroporphyrin is deposited in the teeth and can be demonstrated by red fluorescence when exposed to violet light. Protoporphyria is relatively mild and is expressed in both the erythropoietic system and the liver. It is characterized by photosensitivity resulting in acute solar urticaria and chronic solar eczem a; the cutaneous lesions usually heal in hours or days without scarring. The chemical features include increased erythrocyte free protoporphyrin and increased fecal excretion of protoporphyrin. Urinary porphyrins and porphyrin precursors in most cases of protoporphyria have not been increased. A third and very rare form of erythropoietic porphyria has clinical characteristics like those of congenital erythropoietic porphyria and is expressed chem ically by increased protoporphyrin in erythrocytes, increased urinary excretion of uroporphyrin III, heptacarboxyl porphyrin III, and an unidentified porphyrin, and increased fecal excretion of coproporphyrin, tricarboxyl porphyrin, and protoporphyrin.2 A fourth form of erythropoietic porphyria has been observed but has not been characterized fully in chemical terms. In one reported case3 and in one case of a neonate evaluated in the Mayo Medical Laboratories, extreme photosensitivity and porphyrinuria were observed, and erythrocytes Mayo Clin Proc 57:454-458,1982 454 CONFIDENTIAL SUBJECT TO PROTECTIVE ORDER. C232 Mayo Clin Proc, July 982f Vol 57 ___________________________. ' ._____________________ LABORATORY REPORT 455 Table 1.-- Acknowledged Forms of Porphyria Condition Mode of inheritance Enzyme deficient* Clinical expression Erythropoietic porphyrias Congenital erythropoietic porphyria Protoporphyria Hepatic pofphyrias Acute intermittent porphyria Hereditary coproporphyria Variegate porphyria Porphyria cutanea tarda Intoxication porphyria Autosomal recessive Autosomal recessive Autosomal dominant Autosomal dominant Autosomal dominant ? Acquired Uroporphyrinogen III cosynthase (step 4) Ferrochelatase (step 11) Uroporphyrinogen 1 synthase (step 3) Coproporphyrinogen oxidase (step 9) Protoporphyrinogen oxidase or ferrochelatase (step 10 or 11) Uroporphyrinogen decarboxylase (steps 5-8) Variable Photosensitivity, hemolytic anemia, hypertrichosis Photosensitivity Neuropathy, abdominal pain, psychosis Same as acute intermittent porphyria Same as acute intermittent porphyria + photosensitivity Photosensitivity Abdominal pain, neuropathy Numbered steps refer to the steps in the described reaction sequence for the formation of porphyrinogens. Table 2.-- Some Substances Reported to Induce Attacks of Acute Intermittent Porphyria4 porphyria is characterized by an increased urinary excretion of porphobilinogen during acute attacks and Barbiturates by low activity of uroporphyrinogen I synthase in Sulfonamides Phenytoin (Dilantin) Meprobamates (Miltown and Soma) Griseofulvin " ~~ - Methsuximide Dichloralphenazone erythrocytes. Coproporphyria may be expressed in part by increased coproporphyrin in erythrocytes and by increased urinary excretion of coproporphyrin; however, the pathognomonic finding is persistently increased fecal excretion of coproporphyrin III. Pyrazolones (antipyrenes) Methprylone Imipramine Eucalyptol Ergot substances Chlordiazepoxide (Librium) Similarly, variegate porphyria may cause increased urinary excretion of coproporphyrin, but the distinguishing characteristic is increased fecal excretion of coproporphyrin and protoporphyrin. In addition, variegate porphyria may cause increases in Estrogens Contraceptive steroids the concentrations of porphyrins in the blood plasma. Acute attacks of acute intermittent porphyria, coproporphyria, and variegate porphyria may be contained increased coproporphyrin and normal provoked by medications, and a variety of other concentrations of protoporphyrin. commonplace substances are suspect. Table 24 lists Acute intermittent porphyria, coproporphyria, some of the medications reported to induce attacks. variegate porphyria, and porphyria cutanea tarda have The sensitivity of affected persons to barbiturates is been classified as "hepatic porphyrias" because it is especially noteworthy. The use of pentothal as an assumed that errors in porphyrinogen synthesis are anesthetic during surgery or during dental procedures predominantly expressed in hepatocytes. Acute would be especially risky, and the use of barbiturates intermittent porphyria, coproporphyria, and variegate to control psychiatric disturbances could cause porphyria are characterized by acute attacks of exacerbations of the symptoms. abdominal pain and by hypertension, neuropathy, and Porphyria cutanea tarda is characterized by mental disturbance that may last from several days to photosensitivity and increased urinary excretion of several months. Chronic photosensitivity and uroporphyrinogen I, uroporphyrin I, and the sensitivity of the skin to m echanical traurpa are usual heptacarboxyl porphyrinogen and porphyrin of series characteristics of variegate porphyria but not of acute III. Sensory neuropathy may occur in association with intermittent porphyria; in coproporphyria, sensitivity this disorder, but episodic abdominal pain, motor of the skin to light and to mechanical trauma may be dysfunction, and mental disturbance are not pronounced, mild, or nonexistent. In readily characteristics. Porphyria cutanea tarda appears to be measurable chemical terms, acute intermittent potentiated by a partial deficiency of CONFIDENTIAL C23201 c n o i c r T Tn PROTECTIVE ORDER. 456 LABORATORY REPORT Mayo Clin Proc, July 1982, Vpl 57 Fig. 1. Chromatogram of porphyrins standard. Injected sample con sisted of 5 pmol of each standard porphyrin: octacarboxyi (Uro), heptacarboxyl (Hepta), hexacarboxyl (Hexa), pentacarboxyl (Penta), tetracarboxyl (Copro), and dicarboxyi (Meso, Proto, and Zn Proto) porphyrins. Fig. 3. Porphyrins in urine from a male patient with porphyria cutanea tairia. Injected sample volume, 200 |U. Abbreviations are as in Figure 1. uroporphyrinogen decarboxylase, and its clinical expression is provoked by factors that include iron overload,5 chronic abuse of alcohol, and prolonged use of estrogens or contraceptive steroids. A porphyria cutanea tarda-like disorder has been caused in at least two cases by porphyrin-producing hepatic adenomas;6 in one of those cases, the porphyria disappeared after removal of the tumor. In addition, postmortem examinations of 36 persons with porphyria cutanea tarda revealed hepatocellular carcinomas in 17 cases (47% );7 the significance of that observation is uncertain. Early signs of porphyria cutanea tarda include enhanced facial pigmentation and hypertrichosis of the forehead, malar region, and forearms. - Intoxication porphyria is characterized by increased erythrocyte protoporphyrin (especially the zinc- complexed form), increased urinary excretion of S-aminolevulinate and porphobilinogen, and increased fecal excretion of protoporphyrin; the urinary excretion of porphyrinogens and porphyrins can be increased also. This condition can be caused by ingestion of or exposure to any of a variety of toxic substances; probably most of the recognized cases have been caused by ingestion of lead, hexachlorobenzene, or 2,3,7,8-tetrachlorodibenzo-p- dioxin. Heavy metals, halogenated aromatic hydrocarbons, and a variety of other chemicals can cause suppression of enzymes involved in porphyrinogen metabolism, with an accumulation of intermediates. Enzymes known to be suppressible by toxic substances include S-aminolevulinate dehydratase, uroporphyrinogen I synthase, and ferrochelatase. Probably other porphyrinogen-related enzymes, also, are susceptible. In the past, the separate quantitation of uroporphyrins, coproporphyrins, and protoporphyrins in patients' specimens involved tedious procedures for CON] SUBJECT TO PROTECTIVE ORDER. G2320* Mayo Clin Proc, July 1982, Vol 57 LABORATORY REPORT 457 Blood (fig porphyrins/dl) Erythrocytes Men (n 39) Women (n 50) Plasma Men and women Urine (lig porphyrins/24 h) Men (n * 33) Median Mean (SO) Range Women (n 24) Median Mean (SD) Range Men and women (n 57) Median Mean (SD) Range Porphyria cutanea tarda (n * Range 15) 24 h) Table 3.-- Reference Value*7'9 Protoporphyrin Other porphyrins 17-52 16-65 <1 Uro porphyrin Heptacarboxylic Hexacarboxylic <1 <1 <1 Pentacarboxylic Coproporphyrin 16 20(11) 8-44 7 7(3) 0-12 11 11 (5) 4-22 4 5(2) 3-9 13 17(10) 4-44 6 6(3) 0-12 104-5,177 43-1,508 Uroporphyrins <1,000 3 3(1) 0-5 2 2(1) 0-5 2 2(1) 0-5 0-161 Coproporphyrins <200 2 2(1) 0-4 1 1 (1) 0-3 43 46 (25) 10-109 29 29 (14) 3-56 1 1 (1) 0-4 33 39 (23) 3-109 0-305 7-263 Protoporphyrins <1,500 differential extractions. Recently, the quantitative the instrument response (peak size on the chart) for a analysis has been improved greatly in terms of measured quantity of each porphyrin in the standard procedural convenience, accuracy, and specificity by mixture. Quantities of the porphyrins in the patients' the development of methods for simplified extractions specimens are determined from the sizes of the * of the total porphyrins and by applications of high- porphyrin peaks in the charts (Fig. 2 and 3) and the performance liquid chromatography to the analysis of instrument response data from the primary standard f buffered urine directly and of extracts of blood.8'10 mixture. The test data are converted to and reported Figures 1 to 3 illustrate some features of porphyrins as quantities of porphyrins excreted per 24 hours, analysis by high-performance liquid chromatography calculated by means of the volumes of the samples in our laboratories. Figure 1 presents a chromatogram analyzed (usually 100 to 200 m-D and the total of the standard mixture of purified porphyrins used for volumes of the timed collections. The various calibration of the chromatography system and porphyrins appear in the eluate at different times and illustrates the separational power and sensitivity of the are identified, therefore, by the respective retention ^ system. The system is calibrated in terms of the size of times (elution times); In cases in which the presence Suspected porphyria Congenital erythropoietic porphyria Protoporphyria Acute intermittent porphyria Hereditary coproporphyria Variegate porphyria Porphyria cutanea tarda intoxication porphyria Table 4.-- Recommended Tests Recommended tests Urinary porphyrins Urinary, fecal, erythrocytic porphyrins Urinary porphobilinogen, porphyrins; erythrocyte uroporphyrinogen I synthase Urinary porphobilinogen, porphyrins; fecal porphyrins; erythrocyte uroporphyrinogen I synthase Urinary porphobilinogen, porphyrins; fecal porphyrins; erythrocyte uroporphyrinogen I synthase Urinary porphyrins Erythrocytic porphyrins; urinary S-aminolevulinate, porphobilinogen, porphyrins; fecal porphyrins CONFIDENT5A11,SUBJECT TO PROTECTIVE ORDER. C23203 458 LABORATORY REPORT Mayo Clin Proc/July 1982, Vol 57 or quantity of porphyrin in a chromatographic fraction (peak) is doubtful, the fraction is trapped in a collector and evaluated by scanning spectrofluorometry. In the figures, the intervals (minutes) required for elution of the porphyrins are shown on the abscissas. Uroporphyrin is shown to elute from the chromatographic column at 1Q to 11 minutes after injection of the specimen sample, whereas elution of coproporphyrin requires 15 to 16 minutes and elution of protoporphyrin requires 18 to 19 minutes. Figure 3 presents an example of an analysis of a porphyric urine-- in this case a porphyrins profile typical of porphyria cutanea tarda-- showing large increases of uroporphyrin and heptacarboxyl porphyrin; those features are readily evident on comparison with the normal profile in Figure 2. The mesoporphyrin (a dicarboxyl ic porphyrin) and the mesoporphyrin derivative shown in the chromatograms were added to the specimens for use as internal standards; dicarboxyl ic porphyrins are usually not excreted in the urine. Reference values for porphyrins in blood, urine, and feces are given in Table 3 .8'9,n Table 4 is offeredas~a guide to the selection of tests to establish the diagnosis of porphyria and to identify the specific form for any individual case. REFERENCES 1. Meyer UA, Schmid R: The porphyrias, in The Metabolic Basis of Inherited Disease. Fourth edition. Edited by |B Stanbury, )B Wyngaarden, DS Fredrickson. New York, McGraw-Hill Book Company, 1978; pp 1166-1220 2. Hofstad F, Seip M, Eriksen L: Congenital erythropoietic porphyria with a hitherto undescribed porphyrin pattern Acta PaediatrScand 62:380-384, 1973 3. Heilmeyer L, Gotten R: Die kongenitale erythropoetische Coproporphyrie: eine dritte erythropoetische Porphyrieform. Dtsch Med Wochenschr 89:649-654, 1964 4. Tschudy DP, Valsamis M, Magnussen CR : Acute intermittent porphyria: clinical and selected research aspects. Ann Intern Med 83:851-864, 1975 5. Pimstone NR: The hepatic aspects of the porphyrias, in Modem Trends in Gastroenterology. Vol 5. Edited by AE Read. Boston, Butterworths, 1975, pp 373-417 6. Tio TH, Leijnse B, Jarrett A, Rimington C : Acquired porphyria from a liver tumour. Clin Sei 16:517-527, 1957 7. Kordai V: Frequency of occurrence of hepatocellular carcinoma in patients with porphyria cutanea tarda in long term followup. Neoplasma 19:135-139, 1972 8. Ford RE, Ellefson RD: Erythrocyte porphyrins. Clin Chem (in press) 9. Ford RE, Ou C-N, Ellefson RD: Liquid-chromatographic analysis for urinary porphyrins. Clin Chem 27:397-401, 1981 10. Piomdli S: Free erythrocyte porphyrins in the detection of undue absorption of Pb and of Fe deficiency. G in Chem k 23:264-269, 1977 IT . Mason HL: Unpublished data Ralph D. Ellefson, Ph.D. Section of Clinical Chemistry, Department of Laboratory Medicine SUBJECT TO PROTECTIVE ORDER. 023204 rr n f i d e n t i a l MONSANTO COMPANY \& _ j (T W 3 - y j ^ j_ - - j - j ~ j - j -i J r ' 8 U 2> 1 2 ^ x ^ re s o S 6r 8 ^^ %* V> f fi go VM vju ^ ) I r- ro ^ ^ N Q r -- >o co m(U "-J, Qf t 5rrf o^ (T (?/, 1 X- - ^ m -- -- X r -- " cv) M <\) m ^ mS ^f>c -4J N^ 0 0 T-* ^ r"\J -(NPJ & N KP J> & rT. n > o I aj o - j _a i u ,! II i ;' i ! ' ; ! 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D. Hileman, T. Ma'*.-- T IT LE: G C / M S ANALYSIS OF THE TETRACHLORODIBENZO-P-DIOXIN ISOMERS IN 2,4-DICHI.OROPHENOLS . 's * 4 Monsanto________ ____ . ''-....y M ONSANTO RESEARCH CORPORATION / DAYTON LABORATORY / DAYTON. OHIO 4 5 4 0 7 EASC - Davton (DEPT /SECTION! Analysis TYPE OF R E P O R T REPORT REPORT NO.: MDA-161 JOB/PROJECT NO.: 320.0100 DATE: 6 February 1981 PERIOD COVERED: T IT LE: GC/MS ANALYSIS OF THE TETRACHLORODIBENZO-P-DIOXIN ISOMERS IN 2,4-DICHLOROPHENOLS AUTHORS: F. D. Hileman CONFIDENTIAL SUBJECT TO PROTECTIVE ORDER ABSTRACT: Four 2,4-dichlorophenol samples were analyzed in order to identify and quantitate any tetrachlorodibenzo-p-dioxins present. Methodology, analytical results and quality assurance results are presented. Technical approval: f ,Af. ' Frederick D. Hileman Sr. Research Chemist , Approved by: Robert F . Ivory Environmental Analytical Sciences Center Service Project Manager Approval date: RESTRICTED DISTRIBUTION (see over) C O M P A NY CONFIDENTIAL This oocumem s rire properry of Monsanto Research Corporation and the racpient a responsible for its sefekeepnQ and dsposrtcn h contains CONFIDENTIAL INFORMATION which muff not be raproducad. revealed to unauthonieo persons or sent outside Monsanto Research Corporation and Monsanto Company without proper a ut hour aton _ C23172 REPT No.. /O COPY NO.: * n ssaa a COPY NUMBER 1-3 T. A. Taulli N1D 4 J. D. Wilson N3A 5 D. J. Dahm 1250 6 R, F. Ivory 1250 7 J. J. Brooks 1250 8 R. L. Haute 1250 9 T. Mazer 1250 10 F. D. Hileman 1250 11 Central Files 1250 12 Technical Reports Library R2C ABSTRACT ONLY R. K. Flitcraft/1250 R. M. Scott/1250 NOTE: Distribution of this report is restricted. Requests for additional copies should be made through the principal contact at the Monsanto location involved or the Manager, Environmental Analytical Sciences Center, Dayton Laboratory, Dayton, Ohio. ACKNOWLEDGEMENT Contributions of the- following Dayton Laboratory personnel to this project are gratefully acknowledged : Analyses: D. E. Kirk CONFIDENTIAL SUBJECT TO PROTECTIVE ORDER. C J 3 M P N Y o C . O N F 1 E IM ,T I A t When no longer needed, or upon request, return this report to Central Files, Dayton. 02317.5 INTRODUCTION Four 2,4-dichlorophenol samples were analyzed for the isomeric tetrachlorodibenzo-p-dioxins (TCDDs). The samples included two received from George Bautigam at NIC labeled KL01-7001:MB-833 and KK12-7069:MB-832 and two samples originally sent from the Krummrich Plant to us labeled KM01-7003 and KM07-7032. Sample 7003 had,been found in previous work (R. F. Ivory, October 10, 1980 memo to J.A. Gloeckner) to contain at least two TCDDs at levels of 4.5 and 9.5 ppb while sample 7032 was found in the same study to be free of TCDD at a lower detection limit of 0.9 ppb and was used in this study as a blank. All samples were spiked with 13C-labeled 2,3,7,8TCDD to assist in both the qualitative and quantitative analysis of the TCDDs. METHODOLOGY Samples were prepajred by dissolving in 20% KOH followed by extrac tion..with petroleum ether. The ether extract was then cleaned up using concentrated base and concentrated sulfuric acid washes. The extract was then put through a final clean up using a Woelm basic alumina column. A tetradecane keeper was added to the eluate from the column and the solvent was blown down to a level of 200 pb and submitted for GC/MS analysis. This clean up procedure was necessary to reduce the impurities associated with the TCDD to a level such that capillary column analysis was possible while retaining adequate sensitivity. The analysis was performed on a Hewlett Packard #5985B GC/MS system using a 50 meter Silar 10C capillary column for the TCDD separation as described by Buser et. a l . A n a l . C h e m . 52, 1980 pages 2257 to 2263. Standards of the twenty two dioxin isomers were obtained from Professor Christopher Rappe, University of Umea, Sweden. CONFIDENTIAL SUBJECT TO PROTECTIVE ORDER. 023174 Quality assurance was carried out by having one replicate (Sample 7001) and by spiking Sample 7069 at the 83 ppb level and at the 860 ppb level with unlabeled 2,3,7,8-TCDD. These spiking levels were established from the suggestion given by Jim Wilson that the TCDD levels in these samples could approach 100 ppb. As mentioned previously, Sample 7032 served as the method blank. RESULTS . The qualitative results of the analyses are graphically presented in the chromatographic traces of Figure 1. The top two traces are the standards of the 2:2 substituted TCDDs and the 3:1 and 4:0 substituted TCDDs. A typical selected ion chromatographic trace for Sample 7001 is given in Figure 2 showing the ion traces characteristic for unlabeled and 13C-labeled TCDD. From Figures 1 and 2 it can be discerned that the peak eluting after the 1,3,6,8TCDD is most likely not a dioxin due to the incorrect 320/322 ratio. Note that this peak does appear in Sample 7032, a sample which was found to be free of TCDDs. Observe also that the peak between the 1,3,7,8- and 1,3,6,9-TCDD is in all likelihood not a dioxin, again from the 320/322 ratio and note that again this peak appears in the 7032 sample. The presence of the 2,3,7,8-TCDD in the 7001 sample (Figure 2) and in Samples 7069 and 7003 is confirmed by the, coelution with the 13C-labeled 2,3,7,8-TCDD. Further evidence for the presence of 2,3,7,8-TCDD is shown in Figure 3 where Sample 7069 has been spiked with 83 and 860 ppb of the 2,3,7,8-TCDD and the corresponding peak has grown appreciably. The cluster of TCDDs eluting after 2,3,7,8-TCDD consists of 3:1 substituted TCDDs. No attempt was made at this time to resolve this cluster, however, this can be done by using a different type of capillary column. CONFIDENTIAL. SUBJECT TO PROTECTIVE ORDER. C23175 KL01-7001 KK12- 7069 A _, KKOl-7003 KM07-7032 Figure 1. GC/MS analysis of TGDD isomers in 2,4-dichlorophenol. CONFIDfeNTI Ai, SUBJECT TO PROTECTIYE ORDER. Figure Figure 3. GC/MS analysis of unspiked and spiked 2,4-dichlorophenol Sample KK12-7032. CONFIDENTIAL. DonTFCTIVE ORDER. 023177 The TCDD peak eluting with either the 1,2,7,8-or 1,4,6,9-TCDD is either not a dioxin or contains a significant amount of an inter fering material. This is again evident from the 320/322 ratio (Figure 2) which is 1.94 rather than the actual value of 0.78. The quantitative results for these analyses are given in Table 1. These results were obtained using the 13C-labeled 2,3,7,8-TCDD as the internal standard to quantitate the 2,3,7,8-TCDD. All other TCDD isomers were then quantitated as if their response were iaential to 2,3,7,8-TCDD. The analysis of the replicate of 7001 was limited since a portion of the eluant from the Woelm column was spilled losing many of the TCDD isomers. The quantitation for the 2,3,7,8-TCDD is given since the presence of the internal standard would compensate for any losses in the 2,3,7,8-TCDD isomer. As a further indication of the reproducibility of the methods, the quantitative analyses of the other TCDD isomers in the 2,3,7,8-TCDD spiked samples are given in Table 1. Percent recoveries have not been calculated since an internal standard was used. The absolute recovery for the 13C-labeled 2,3,7,8-TCDD spiked into the samples at 93 ppb was 91% 10%. The lower detection limits that are given were based on an evaluation of the method blank. Note that the detection limits have been raised for the 3:1 mixed isomers and the 1469/1278 isomers due to interferences in- the method blank. 5 023178 TABLE 1. QUANTITATIVE ANALYSIS OF TETRACHLORODIBENZO-P-DIOXIN ISOMERS IN 2,4-DICHLOROPHENOL Sample Specific TCDD isomer concentration, nq/q (ppb) 3:1 1469 1368 1379 1378 1369 1268 2378 mix 1279 1278 KM01-7003 9a ND 0.8 4 ND 4 4 ND <8 KK12-7069 50 4 14 22 6 14 39 6 <15 KL01-7001 KL01-7001 15 1 5 5 2 _b - -- - 5 13 2 7 -- <9 - KM07-7032 ND ND ND ND ND ND ND ND ND De.tection limit 0.7 0.7 0 7 0.7 0.7 0.7 2 0.7 2 KK12-7069 (83 ppb 2,7,3,8-TCDD spike) 47 4 17 26 8 86 52 8 <17 KK12-7069 (860 ppb 2,3,7,8-TCDD spike) 26 4 14 20 r 9 840 tC t <18 a Retention time incorrect. ^Analysis of these isomers not carried out. cCould not be quantitated due to overlap from the high level 2,3,7,8-TCDD spike. CONFIDENTIAL SUBJECT TO PROTECTIVE ORDER. 023179