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Journal of Analytical Toxicology, Vol. 28, April 2004

[ TechnicalNote Acute Poisoning with Phosphamidon: Determination of Dimethyl Phosphate (DMP) as a Stable Metabolite in a Case of Intoxication Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 F.A. Tarbah, B. Kardel, S. Pier, O. Temme, and T. Daldrup* Institute of Legal Medicine, Heinrich-Heine-University, P.O. Box 10 10 07, D-40001 Duesseldoff, Germany

Abstract rhea, urination, miosis, muscular twitching like bron- chiospasm, emesis, lacrimation, and salivation (mnemomic DUMBELS) (10). Many organophosphate pesticides (OP) such as phosphamidon are The relationship between OP concentrationsin body fluid and unstable in aqueous solutions and especially in blood in the presence of esterases. In a case of intoxication, the phosphamidon the severity of patients' reactions has not been thoroughly in- concentration in serum decreased from 10 mg/L to 4.4 mg/L after vestigated to date. This type of data is required to obtain a storage at -20~ for six months; nearly complete degradation was more accurate diagnoses,thus enabling the prediction of the ef- observed after three years. Dimethyl phosphate (DMP) is a ficacy of a clinical treatment (I i). metaboUte of phosphamidon, , , Many OPs are unstable in aqueous solution and especially in , , and trichlorfon. A gas blood becauseof the presence of esterases.Complete degrada- chromatographic-mass spectrometric method with deuterated tion of the OP during storage was frequently observed. OPs DMP-d6 as internal standard for the determination of DMP in are degraded more rapidly by esteraseactivities than by other biological material was validated. DMP was found in all of the chemical mechanisms (4,12-15). The organophosphorothioates patient's samples (3.9 and 4.9 mg/L in blood, 33.5 and 50.4 mg/L like ch]orpyrifos methy] are unstable in aqueous solution but in urine, and 8.1 mg/L in gastric fluid), even after storage at -20~ stable in blood for at least 72 h. A possible explanation is that for up to 3 years. No hints for a degradation of DMP when spiked in fresh blood and stored at 4~ for 1 week and stored in water proteins and lipids in biological specimens may effectively sta- over a time period of 10 months. Looking for the stable bilize methyl (16). metabolites like DMP in cases of suspected OP intoxication is A typical degradation product is para-nitrophenol, which can recommended. be found in blood and urine at high concentrations in the case of intoxication (17). Stability of OPs in serum sam- pies during storage was measured in 23 different OPs, which were mixed with serum containing 10 m~mL EDTA and stored Introduction up to 10 days at 4 and -20~ The recovery rates of the 23 OPs ranged between 50% () and 133% (dialifos) (18). The Organophosphate pesticides (OP) are responsible for nu- general population can be exposedto organophosphorus com- merous acute and even fatal poisonings (1-5), and therefore pounds because of the application of pesticides in household must always be considered when solving forensic and clinical and private garden. In general, the measurement of urinary cases (6-8). The mechanism of toxicity is the inhibition of alkyl phosphate metabolites is a more sensitive index of expo- esterase due to direct enzyme binding, resulting sure to OPs than cho]inesterase inhibition. The degradation in the accumulation of acetylcholine at the nerve synapses. In products of many OPs are the alkyl phosphates such as O,O- fatal cases, this leads to the disruption of nerve functions (cen- dimethyl phosphate (DMP), O,O-diethyl phosphate (DEP), O,O- trally and peripherally), tonic convulsion (limbs stretched and diethylthio phosphate (DETP), O,O-dimethyldithio phosphate rigid) or more likely clonic convulsion (rapid repetitive move- (DMDTP), O,O-diethyldithio phosphate (DEDTP), and O,O- ment), respiratory failure, and, when untreated, to asphyxiation dimethy]thio phosphate (DMTP). These phosphates are well (9). The muscarine and nicotine-like symptoms observed after known as markers of occupational OP exposure (19-25). Aprea poisoning with such anticholinesterase agents include diar- et al. (19) published a study in which 124 persons without oc- cupational exposure were examined in Italy. DMP was found to be of 10 ]J~JLurine. Hardt and Angerer (20) had investigated 54 "Author to whom correspondence should be addressed. E-mail: [email protected]. spot urine samples from non-occupational subjects in Ger-

198 Reproduction(photocopying) of editorialcontent of this journal is prohibitedwithout publisher'spermission. Journal of Analytical Toxicology,Vol. 28, April 2004 many. Their analyses revealed a mean concentration of DMP in A quantitative, rapid, and sensitive gas chromatographic- urine of 30 IJg/L. mass spectrometric (GC-MS) method with deuterated DMP-d6 Stability studies carried out over a two-week period have as internal standard after derivatization with pentafluorobenzyl shown that DMP is stable in urine under storage at room tem- bromide PFB-Br is developed (Figure 2). perature, 4~ or -20~ (26). Further investigations carried out in our laboratory gave no hint of a degradation of DMP when stored in blood at 4~ for I week and stored in water for 10 months, whereas mevinphos was nearly completely de- Case History graded to DMP after storage for about 72 h in blood at 4~ (un- published data). A 19-year-old previously healthy female was admitted to Suspected criminal intoxication with OP was the reason a the emergency unit. She had attempted suicide by ingesting method to determine DMP as stable alkylphosphate in biolog- an unknown insecticide. The insecticide, blood (without addi- ical samples, especially in postmortem material, was devel- tion of any preservatives), urine, and gastric fluid were sent

oped. DMP is a stable metabolite and the degradation product to our laboratory. The medical investigation of the patient Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 of mevinphos, dicmtophos, monocmtophos, dichiorphos, phos- revealed aspiration pneumonia, acute pancreatitis, tachycardia, phamidon, and trichiorfon (Figure 1). and irritability. Skin and mucous membrane were normal.

Trade name Chemical name Chemical structure

1. Mevinphos O, O-dimethyI-O-(2-methoxycarbonyl- H3CO\ ~O 0 1-methylvinyl)-phosphate II Hsco/P~o_ C - CH-C- OCH5 I CHa

2. Dicrotophos O,O-dimethyI-O-(1-methyl-2-N,N- H3CO\ ~O

dimethylcarbamoylvinyl)-phosphate CH;I CH5

3. Monocrotophos O,O-dimethyI-O-(1-methyl-2- H=CO _ 0 \-// o N- methylcarbamoylvinyl)-phosphate H=CO/I"\O _ C = CH'C-II NH-CH~ I CH=

4. Dichlorvos O-(2,2-Dichlorovinyl)-O,O-dimethylphosphate H3CO\_// _ 0

H3CO \0 - CH = CCIz

5. Phosphamidon O,O-dimethyI-O-(2-chloro-2-N,N- H3CO . O \_6 CJ 0 /CzH= diethylcarbamoyl-l-methylvlnyl)-phosphate H3CO/I"\O _ I II C-C-C-N, \ CH3 C21"~

6. Trichlorfon O, O-dimethyl-(2,2,2-tdchloro-1 -hydroxyethyl)- H=CO . O

phosphonate H~CO/~\CH-CCI3 I OH Figure I. Examplesof organophosphatepesticides metabolizing to produce DMP as stable metabolite.

199 Journal of Analytical Toxicology, Vol. 28, April 2004

Cholinesterase activity was reduced (100 U/L). The patient program: initial temperature 90~ for 6 min, 15~ to was conscious. The analysis of the unknown insecticide proved 250~ maintained for 1 rain; and carrier gas: nitrogen, 10 that it contained phosphamidon. The blood concentration mL/min constant flow. of phosphamidon determined by gas chromatography- GC--MS. The analyses were performed with a Hewlett-Packard nitrogen-phosphorus detection (GC-NPD) was 10 mg/L. The GC 5890 and MSD 5970 equipped with a Hewlett-Packard samples were stored at -20~ for 20 and 36 months before automatic liquid sampler HP 7673; column, HP-5 MS (30-m the DMP analyses. length, 0.25-ram i.d., 0.25-pro film thickness); carrier gas, helium (pressure 70 kPa); split/purge off time, 2 rain; injector temperature, 270~ transfer line temperature, 280~ and temperature program, initial temperature 60~ for Experimental 2 min, 40~ to 110~ 12~ to 170~ 40~ Materials to 300~ maintained for 3.5 rain (total run time: 17 min). All solvents used were of analytical grade with a purity greater The MS was in electron impact mode at 70 eV. using the selected ion monitoring mode, m/z 110, 116, 194, 306, and than 98%. Acetonitrile, acetone, ethanol, heptane, toluene, Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 water (HPLC grade), and potassium carbonate were obtained 312 for DMP and DMP-d6 were measured (quantitation ions from E. Merck (Darmstadt, Germany). Dimethyl phosphate are underlined). originated from Ultra Scientific (North Kingston, RI). Deuterated dimethyl phosphate (DMP-d6) was prepared by Extraction procedure for OP general screening Prof. Dr. G. Machbert (Institute of Legal Medicine, Erlangen, In an Eppendorf tube, an aliquot of the samples (0.7 mL Germany). PFB-Br was obtained from Sigma Chemical Co. (St. blood, serum, urine and/or gastric juice) was mixed with 1 mL Louis, MO), phosphamidon was from Dr. S. U. I. Ehrenstoffer toluene for 2 min. (vortex) and centrifuged at 14,000 rpm (Augsburg, Germany), and mevinphos was from Riedel-De (17,500 xg) for 5 min at 4~ to avoidthe problems of emulsion Hahn (Hannover, Germany). formation. A second aliquot was extracted in the same way, Biological materials (case of acute phosphamidon intoxica- but the internal standard (7 pL of 0.01% mevinphos in toluene) tion) include serum, urine, blood, gastric fluid, and the un- was added to the sample before extraction. The toluene layers known insecticide derived were from Remscheid Hospital, were transferred to glass vials. One microliter of each organic Germany phase was analyzed directly by GC-PND.

Standard preparation GC-MS method validation and reliability of the Phosphamidon stock solution was freshly prepared at a con- DMP determination procedure centration of i mg per 10 mL toluene (0.01%). Dimethyl phos- Acetonitrile (1.4 mL) and 20 pL 0.001% (200 ng/mL) deuter- phate stock solution was freshly prepared at a concentration of ated dimethyl phosphate DMP-d6 as internal standard were 1 mg per 10 mL water (0.01%), divided into 1-mL aliquots, and added to an aliquot of the samples (0.1 mL plasma spiked with stored at -20~ until usage. DMP at a concentration of 0-8 mg/L for the calibrations and at a concentration of 2 and 8 mg/L for precision tests). The sam- Internal standard ples were then mixed for 1 rain (vortex) and centrifuged at DMP-dG (0.01% in water) was used as the internal standard 14,000 rpm (17,500 xg) for 10 rain at 4~ A 1.2-mL portion of and prepared weekly. Mevinphos (0.01% in toluene) was used as the clear upper layer was transferred to a new dry glass vial and the internal standard and prepared weekly. evaporated to dryness using a gentle stream of nitrogen. The dry residue was suspended in 0.2 mL H~O and washed with 0.2 Instrumentation mL heptane. The heptane phase was discarded, and the aqueous GC-NPD. The analyses were performed with a Hewlett- phase was evaporated at room temperature under a stream of Packard GC-NPD, GC 5890 series II, injector: split/splitless, nitrogen. Ethanol (0.05 mL) wa~ added to improve the evapo- 270~ purge-off-time 1 min, column: Macherey-Nagel Per- ration. After adding 5 mg potassium carbonate, 0.2 mL ace- mabond OV-1-DF-1.80, 15-m length, 0.53-ram i.d.; temperature tonitrile and 0.01 mL PFB-Br, derivatization was performed in a sealed vial at 90~ for 30 rain. After cooling to ambient tem- F CH2--Br F CH,(*) -~- Br(-) perature, 0.05 mL of the derivatized extract were transferred in a high recovery glass vial, evaporated to dryness in a gentle stream of nitrogen, and reconstituted in 0.05 mL acetone. One microliter was analyzed by GC-MS. It should be noted that PFBBr must be handled in a hood be-

F F CH= -- 0 /OCH. cause it is a potent lachrymator.

F F "JI-~PX__H ' ~F Extraction procedure for DMP determination in human specimens F F F F In a glass vial, an aliquot of the samples (0.1 mL blood, Figure2. SuggestedSNI mechanismfor the reactionof PFB-Brwith DMP plasma, urine, and/or gastric fluid) was extracted in accordance (22). with the procedure described.

200 Journal of Analytical Toxicology, Vol. 28, April 2004

Resultsand Discussion blood, urine, and gastric fluid. The mass spectra of DMP-d6-PFB and DMP-PFB are shown in The availability of deuterated dimethyl phosphate (DMP-d6) Figures 3A and 3B. The retention times were 8.86 rain for made it possible to develop a quantitative method to determine DMP-d6-PFB and 8.90 min for DMP-PFB. To improve the sen- DMP by GC--MS. Previous methods used large amounts of urine sitivity, we used the single ion mode with the following ions: samples or aqueous solutions and time consuming sample m/z 110, 194, and 306 for DMP-PFB and m/z 116 and 312 for preparations (19-25), whereas this method is fast and suitable DMP-d6-PFB. for very small amounts of any biological material, including Reliability of the analytical procedure Linec~ty. Detector response linearity studies were performed 116 A 100 by preparing five duplicate calibration samples covering the 90 80 range between 0.5 and 8.0 mg/L. A linear regression was fitted 70 after plotting the ratios of the DMP peak area and internal stan- 6O 181 312 dard peak area versus the concentration of the substance in mil- 50 Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 40 ligrams per liter (Figure 4). 84 98 Precision. In order to evaluate the repeatability (within-day 2O Ii 110 161 197 precision) of the method, duplicate analyses (n = 3) of plasma, ,.. 1. L . l,,J 4, l spiked with DMP, using five concentration levels (0.5, 1.0, 2.0, 0 011 811 100 I 140 180 180 200 220 240 260 '280 300 m/z 4.0, and 8.0 rag/L), were carried out. The reproducibility (be- tween-days precision) was evaluated with duplicate analyses t~o B 100| (n = 10) of plasma spiked with DMP using two concentration

80 Table II. Determination of Phosphamidon using GC-PDN 6O 50 8 1 3 Phosphamidon* Phosphamidon~

3O Sample mg/L mg/L

10 -. ,._ml, -, L .:.,.: ...... ,,.I .,__. , serum I0.0 4.4 9 , ,-.--, .... , .... ,_ ..,. 0 60 80 100 120 140 160 180 200 220 240 260 280 300 m/z * After 90 h storage. Figure 3. Scans of DMP-d6-PFB (A) and DMP-PFB (B). t After 6 months storage.

3.5 8OO0 DMP~6 Ion 110 0 2 ~ DMP 6OOO 0 ~

4000 0

2000 0 1 2 3 4 5 8 7 8 0 Amount ol DMP (mR/L)

Figure 4. Calibration curve of DMP in serum after extraction. ,,1,,,, h,,, h.., I,,,, I,, ,, I,,, ,I, *,,I, ,, ,J,,,,I.,., I ,.,, I,, i i1,,,,I 8.20 8.0 8.60 880 9.00 9.20 9.40 Time (rain) Table I. The Within-Day and Between-Day Precision for Figure 5. Determination DMP in blood in a case of phosphamindon in- the Analysis of DMP* in Spiked Plasma toxication.

DMP Found Within-day Belween.day Table III. Determination of DMP using GC-MS DMP Expected (mg/t) Precision(%CV) precision(%CV) (rag/L) n = 3 n = 3 n = 10 DMP Concentration* DMP Concentrationt Sample mg/t mg/L 0.5 0.44 6.0 n.d. 1.0 0.84 5.5 n.d. blood 3.9 4.9 2.0 1.85 6.2 8.9 urine 33.5 50.4 4.0 3.97 9.3 n.d. gastric fluid 8.1 n.d.* 8.0 8.10 1.9 6.8 * After 20 months storage. * Abbreviations: DMP, dimethyl phosphate; CV, coefficient of variation; and n.d., After 36 months storage. not determined. * Not determined.

201 Journal of Analytical Toxicology, Vol. 28, April 2004 levels (2 and 8 rag/L). The within day and between days preci- References sion data are presented in Table I. The coefficients of variation ranged between 1.9 and 9.3%. 1. F.W. Sorensen and M. Gregersen. Rapid lethal intoxication caused Recovery. The average recovery of DMP after extraction from by the herbicide glyphospate-trimesium (Touchdown). Hum. Exp. plasma samples (n = 5) was 60%. Toxicol. 18:735-737 (1999). Limit of detection (LOD). The LOD, defined as the lowest 2. H. Jamil. Acute poisoning--a review of 1900 cases. J. Pak. Med. Assoc. 40:131-133 (1990). concentration of the analyte that can be clearly detected above 3. A. Kamenczak, K. Jasinska-Kolowa, D. Targosz, 8. Szkolnicka, the baseline signal, is estimated as being three times the signal- and K. Sancewicz-Pach. Acute pesticides poisoning in the Krakow to-noise ratio. The LOD was determined (n = 5) by injection of Department of Clinical Toxicology in 1986-1995. Przeglad spiked plasma with DMP in decreasing concentrations. The lekarski, 1997, pp 54671-54676. LOD was below 0.06 mg/L. 4. R.D. O'Brien. Toxic Phosphorous Esters, Chemistry, , and Biological Effects. Academic Press, New York, NY, 1960, pp 115-133 and 175-194. Toxicological finding after acute phosphamidon intoxication 5. P. Casey and J.A. Vale. Deaths from pesticides posoning in England The results of the toxicological analyses proved that phos- and Wales (1945-1989). Hum. Exp. ToxicoL 13:95-101 (1994). Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021 phamidon was the main component of the unknown insecticide 6. E. Meyer, D. Barrey, W. Lambert, C. Van Peteghem, M. Piette, solution. The result was confirmed by GC-MS. and A. De Leenheer. Analysis of in postmortem samples by HPLC with diode-array detection and GC-MS using solid- Patient serum and urine samples were first stored at 4~ for phase extraction. J. Anal Toxicol. 22:248-252 (1998). 90 h (samples arrived out of the work hours) before analyses for 7. G. Estela and P. Marques. Acute intoxication by azinphos-ethyl. OP. Phosphamidon was only found in serum at a concentration J. Anal. ToxicoL 14:243-246 (1990). of 10 rag/L, but not in the urine sample. The reanalyses of the 8. P. Kintz, C. Jamey, S. Doray, and B. Ludes. Acute fatal poisoning blood sample, stored at -20~ after 6 months revealed a re- with dichlorophen. Int. J. Legal Meal. 110:95-96 (1997). 9. T.S.Thompson, R.G. Treble, A. Magliocco, and J.R. Rottger.Case study: duction of more than 50% of the phosphamidon concentration fatal poisoning by . Forensic Sci. Int. 95:89-98 (1998). (Table II). Looking for DMP as a stable metabolite of phos- 10. M.J. Ellenhorn and D.G. Barceloux. Medical Toxicology--Diag- phamidon was the aim of this study. No data were published nosis and Treatment of Human Poisoning, 1st ed. Elsevier, New about the concentration of DMP in blood or serum. A GC-MS York, NY, 1988, pp 625-641. method with DMP-d6 as internal standard was developed for this 11. Y. Cho, N. Matsuoka, and A. Kamiya. Determination of organo- phosphorus pesticides in biological samples of acute poisoning by purpose. DMP could be detected in blood, urine, and gastric HPLC with diode-array-detector. Chem. Pharm. Bull. 45:737-740 fluid, stored at -20~ even after 3 years (Figure 5). To our (1997). knowledge, we presented the first study in the determination of 12. B.T. Bowman and W.W. Sans. Stability of parathion and DDT in di- DMP in blood in an acute case of OPs intoxication. The subse- lute iron solution. J. Environ. Sci. Health. Bull. 15:233-246 (1980). quently analyses of the blood and urine samples were repeated 13. A. Bouaid, A. Martin-Esteban, P. Fernandez, and C. Camara. Degra- dation of atrazine and several organophosphorus pesticides in 16 months later, after storage under the same conditions. Re- oranges. Ann. Chim. 91: 93-102 (2001). sults showed no degradation of DMP (Table III). Instead, we ob- 14. A.K. Singh, R.J. Zeleznikar, and L.R. Drewes. Analysis of served an increase of the DMP concentrations. This increase and in blood utilizing a sensitive gas chromatography-mass could be due to the degradation of phosphamidon as shown in spectrometry method. J. Chromatogr. 324:163-172 (1985). the degradation experiment with mevinphos mentioned be- 15. V. Drevenkar, B. Stengl, B. Tkalcevic, and Z. Vasilic. Occupa- tional exposure control by simultaneous determination of N- fore. The reference values for unexposed populations ranged be- methylcarbamates and organophosphorus pesticide residues in tween 0.01 and 0.03 mg/L in urine (19,20). In the present study, human urine. Int. J. Environ. Anal. Chem. 14:215-230 (1983). the concentrations of DMP in urine were 33.5 to 50.4 mg/L. 16. E Moriya, Y. Hashimoto, and T. Likuo. Pitfalls when determining These concentrations and the DMP concentrations in blood tissue distributions of OP chemical: sodium fluoride accelerates (3.9 to 4.9 mg/L) indicate that our clinic case was an OPs in- chemical degradation. J. Anal. Toxicol. 23:210-214 (1999). 17. P. Michalke and T. Daldrup. Freier und gebundener Anteil von p- toxication after extensive overdose of phosphamidon. Nitrophenol in Blut und Urin nach E 605-1ntoxikation. Xllth Congress of the International Academy of Forensic and Social Medicine. H. Egermann, Vienna, Austria, 1982, pp 427-430. 18. F. Tarbah, H. Mahler, O. Temme, and T. Daldrup. Analytical method for the rapid screening of organophosphate pesticides in Conclusions human biological samples and foodstuffs. Forensic Sci. Int. 121: 126-133 (2001). In the summary, the described method provides a reliable 19. C. Aprea, G. Sciarra, and L. Lunghini. Analytical method for the de- quantitative analysis of DMP in singular or more than one bio- termination of urinary alkylphosphates in subjects occupationally exposed to organophosphorus pesticides and in the general pop- logical specimens. The GC-MS method is specific and sensitive ulation. J. Anal. Toxicol. 20:559-563 (1996). to detect even very low concentrations of DMP. This case of 20. J. Hardt and J. Angerer. Determination of dialkylphosphates in phosphamidon intoxication proofs that DMP is easily detectable, human urine using gas chromatography-mass spectrometry. even after three years of storage. J. Anal. ToxicoL 24:678-684 (2000). In case of OP intoxications, especially in case of a suspected 21. A.N. Oglobline, G.E. O'Donnell, R. Geyer, G.M. Holder, and B. Tattam. Routine gas chromatographic determination of di- criminal act, it is recommended to look for the stable metabo- alkylphosphate metabolites in the urine of workers occupationally lites, like DMP, to avoid any false-negative results of stored bi- exposed to organophosphorus. J. Anal. Toxicol. 25:153-157 ological samples (2001).

202 Journal of Analytical Toxicology,Vol. 28, April 2004

22. W.C. Lin, C.H. Kuei, H.C. Wu, C.C. Yang, and H.Y. Chang. Method 25. F. Tarbah, B. Kardel, S. Pier, O. Temme, and T. Daldrup. Quantifi- for the determination of dialkyl phosphates in urine by strong cation of dimethyl phosphate (DMP) as a metabolite in case of anion exchange disk extraction and in-vial derivatization. J. Anal acute OP insecticide intoxication. Proceedings SOFT-TIAFT Toxicol. 26:176-180 (2002). meeting, Albuquerque, NM, 1998, pp 498-508. 23. R. Bravo, W.J. Driskell, R.D. Whitehead, Jr., L.L. Needham, and 26. J.L. Lewalter. Determination of dimethylphosphate in urine by D.B. Barr. Quantitation of dialkyl phosphate metabolites of GLC. Bayer company Leverkusen, Germany, personal communi- organophosphate pesticides in human urine using GC-MS-MS cation (1991 ). with isotopic internal standard. J. Anal. Toxicol. 26:245-252 (2002). 24. S.J. Reid and R.R. Watts. A method for the determination of dialkyl Manuscript received January 28, 2003; phosphate residues in urine. J. Anal. Toxicol. 5:126-132 (1981 ). revision received June 13, 2003. Downloaded from https://academic.oup.com/jat/article/28/3/198/701920 by guest on 01 October 2021

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