ORIGINAL ARTICLES

IVAX Drug Research Institute Ltd., Budapest, Hungary

HPLC separation of related impurities in etiprednol dicloacetate, a novel soft

M. Patthy, G. Seres, A´ . Csana´di, T. Szekeres, Z. Zubovics, N. Bodor

Received October 29, 2003, accepted November 12, 2003 Miklos Patthy, 1045 Budapest, Berlini u. 47-49, Hungary [email protected] Pharmazie 59: 382–386 (2004)

Etiprednol dicloacetate (ethyl 17a-dichloroacetoxy-11b-hydroxy-androsta-1,4-diene-3-one-17b-carboxy- late, code-named: BNP-166) has been prepared in a 3-step synthesis from as starting material. The primary aim of the present work was to develop HPLC methods for the separation of all the impurities found in experimental pilot plant batches of BNP-166 at concentrations 0.10 area %. Besides BNP-166, a total of 19 compounds, eight of them potential impurities, were involved in the HPLC studies in which several HPLC systems were examined and tested to optimize the separation. Of the parameters influencing chromatographic behaviour column type, the nature and composition of the mobile phase and column temperature were varied, while the pH of the eluent was kept constant at 4.5, a pH value at which stability of the BNP-166 ester bonds was found to be the highest. A comparison of the RRT values obtained allowed some conclusions to be drawn concerning the physi- co-chemical forces governing separation. The isocratic reversed-phase HPLC system (V02) chosen to be used for various GXP studies on BNP-166 affords baseline separation of nearly all the compounds concerned, and also the quantitation of the drug candidate (BNP-166). By means of this system, it was shown that the target compound prepared by the standardized synthesis method on a pilot plant scale, never contained more than 2–3 impurities with area % values higher than 0.10.

1. Introduction The aim of the present work was to develop an HPLC method capable of a baseline separation, in less than The soft corticosteroid etiprednol dicloacetate (ethyl 17a- 30 min, of all or almost all the actual and potential impu- dichloro-acetoxy-11b-hydroxy-androsta-1,4-diene-3-one- rities most likely to arise during the synthesis, purification 17b-carboxylate; code-named: BNP-166) was developed and/or storage of BNP-166. as a result of structural modification relying on the ‘‘in- active metabolite” of soft drug concept (Bodor 1984a; 1984b; 1988). It was developed for use in various clinical 2. Investigations, results and discussion fields, with indications such as asthma, allergic rhinitis, 2.1. Isolation and identification of the impurities Crohn’s disease and ulcerative colitis. Etiprednol dicloacetate (BNP-166) has been prepared in a After crystallization of BNP-166, synthetic contaminants 3-step synthesis from prednisolone as starting material (some of them derived from prednisolone and its contami- (see Scheme). nant as well as other commercial reagents) As several closely related 1,4-androstadiene-3-one deriva- were isolated from the mother liquor using conventional tives are also formed in the process of the BNP-166 syn- silica gel column chromatography for the contaminants thesis shown below, a systematic study had to be made of well separated by thin layer chromatography (TLC) on the relative merits of normal-phase and reversed-phase silica plates, and semi-preparative reversed-phase HPLC HPLC methods to achieve adequate separation. for the rest.

Scheme

COCH OH CO H 2 CO2H 2 CO2Et OH OH OCOCHCl OCOCHCl HO HO HO 2 HO 2

NaIO EtI/K2CO3 4 CHCl2COCl

95% 93% 74% O O O O PREDNISOLONE ∆1-CORTIENIC ACID "M-COOH" BNP-166

382 Pharmazie 59 (2004) 5 ORIGINAL ARTICLES

Table: Chromatographic characteristics of BNP-166 and its potential contaminants

BNP-166 and its potential contaminants RRT values obtained in some HPLC systems

V01 V02 01/02/a 01/02/b 02/01

D1-Cortienic acid 0.17 0.17 0.14 0.22 0.12* M––COOH 0.19 0.22 n.a. 0.29 0.13* 11-Oxo- D1-cortienic acid ethyl ester 0.46* 0.40 0.33 0.45 0.31 17a-Dimethyloxamate, BNP analogue 0.46* 0.42* 0.36* 0.48* 0.33 D1-Cortienic acid ethyl ester (M––OH) 0.46* 0.42* 0.36* 0.50* 0.28 17a-Formate, BNP analogue 0.59 0.49 0.43 0.56* 0.44 11-epi BNP 0.67 0.58 0.50 0.56* 0.60 11-Deoxy M––OH 0.75 0.66 0.60 0.69 0.49 BNP-Methyl ester analogue 0.81 0.76 0.74 0.80 0.74 11-Deoxy-cortienic acid ethyl ester 1.00* 0.94* 0.93 1.01* 0.67 BNP-166 (ret. time: 6, 9, 21, 8,5 and 8 min, resp.) 1.00* 1.00 1.00 1.00* 1.00 Lumi BNP-166 1.00* 1.31 n.a. n.a. 1.22 11-Oxo BNP analogue 1.04* 0.86 0.83 0.84 1.10* 17a-Chloroiodoacetoxy BNP analogue 1.04* 0.94* n.a. 0.90 1.07 1,2-Dihydro BNP analogue 1.11 1.12 1.14 1.11 1.13* 11-Deoxy BNP analogue 1.74* 1.65 1.69 1.45 1.89* 17a-Trichloroacetoxy BNP analogue 1.74* 2.27 2.46 2.10 1.90* 1,2-Dihydro-11-deoxy BNP analogue 2.44 2.51 2.76 2.20 2.77

System 01/02/a Column: Eurospher-100, C18, 5 mm (125 4 mm I.D. with a 5 mm integrated pre-column); Eluent: methanol-aqueous buffer ¼ 60 : 40, v/v; Flow rate: 0.8 ml/min; Column temperature: 40 C. For the rest of the systems in the Table see the figure legends.

umns can often be separated by reversed-phase HPLC and vica versa. In preliminary separation experiments, there- fore, both normal-phase and reversed-phase HPLC sys- tems were tested in our laboratory for the separation of the compounds in the Table. In order to show the separation characteristics of the nor- mal-phase systems, a representative chromatogram ob- Structure elucidation of the compounds isolated was per- tained for a BNP-166 sample spiked with all the potential formed by NMR and MS. Structures were confirmed by impurities is presented in Fig. 1. synthesis and subsequent comparison of the NMR and Although the separation power of the silica system in MS spectra of the synthetically produced compounds with Fig. 1 appears to be good, the separation pattern suggests those of the isolated contaminants. some disadvantages: the separation takes almost one hour, The short, in-house names of the 17 isolated and identi- besides, the retention of BNP-166 is such that its peak is fied contaminants are shown in the Table. eluted into a major group of potential contaminants with similar retentions, meaning that a faster separation by gra- dient elution or by using an isocratic eluent with an in- 2.2. HPLC systems for resolving BNP-166 and related creased elution strength would not result in baseline se- impurities parations. Also, further difficulties can be anticipated with As was pointed out in earlier studies on the separation of such systems in developing a sensitive and reliable HPLC , normal-phase and reversed-phase HPLC systems method for the quantitation of the contaminants eluting can complement each other (Lin and Heftmann 1982): well after the BNP-166 peak, such as the intermediates compounds inseparable by adsorption HPLC on silica col- M-COOH and D1-cortienic acid. Therefore, subsequent ef-

10 RT(min) 0,70 1 3.81 1,2-dihydro-11-deoxy BNP analogue Silica system 2 4.14 Lumi BNP-166 0,65 3 4.37 11-deoxy BNP analogue (a peak marked with an asterisk 4 4.73 11-deoxy-cortienic acid ethyl ester means co-eluting peaks) 5 4.98 11-oxo BNP analogue 0,60 6 5.79 11-deoxy M-OH 7 6.42 17α-trichloroacetoxy BNP analogue 0,55 8 6.88 1,2-dihydro BNP analogue 9 7.46 11-oxo-D1-cortienic acid ethyl ester 0,50 10 7.68 BNP 166 11 8.29 17a-chloroiodoacetoxy BNP analogue + 17a-monochloroacetoxy BNP analogue 0,45 12 8.80 BNP-methyl ester analogue + 17a-monoiodoacetoxy BNP analogue + 17a-formate, BNP analogue 13 9.58 11-epi BNP 0,40 14 10.12 unknown U 15 12.50 D1-cortienic acid ethyl ester(M-OH) A 0,35 16 18.37 17a-dimethyloxamate, BNP analogue 0,30 17 19.21 unknown 18 24.32 M-COOH 0,25 3 9 19 44.13 D1-cortienic acid 12 15 0,20 2 4 16 0,15 1 6 13 11 18 Fig. 1: 0,10 5 78 Separation of BNP-166 and its potential con- 17 0,05 14 19 taminants in a normal-phase system. Column: 0,00 *** Nucleosil 100 Si, 5 mm, (250 4 mm I.D.); Eluent: n-hexane-dichloromethane-methanol-glac. 0 5 10 15 20 25 30 35 40 45 min acetic acid ¼ 80 : 16 : 4 : 0.1, v/v; Flow rate: 1 ml/min; Column temperature: 25 C

Pharmazie 59 (2004) 5 383 ORIGINAL ARTICLES

1doycrincai ty ester ethyl acid 11-deoxy-cortienic System V01 0.6 (a peak marked with an asterisk means co-eluting peaks) uiBNP-166 Lumi 17 11-oxo- D 1 N 166 BNP a crincai ty ester(M-OH) ethyl acid -cortienic dmtyoaae N analogue BNP -dimethyloxamate,

0.5 17 17a-monochloro and 17a-monoiodoacetoxy BNP were synthetically D a

1 prepared as hypothetical contaminants. clrid ctx BNP acetoxy -chloroiodo crincai ty ester ethyl acid -cortienic 1ooBNP 11-oxo 0.4 17 17 a frae N analogue BNP -formate, a ,-iyr-1doiBNP 1,2-dihydro-11-deoxi 17 mnidaeoyBNP -monoiodoacetoxy M-COOH 17

0.3 M-OH 11-deoxy a a tihooctx BNP -trichloroacetoxy mnclraeoyBNP -monochloroacetoxy U 1doyBNP 11-deoxy A N ehlester methyl BNP 1p BNP 11epi D ,-iyr BNP 1,2-dihydro

0.2 1 crincacid -cortienic

Fig. 2: 0.1 Separation of BNP-166 and its potential contami- nants in a reversed-phase system. Column: Euro- 0 spher-100, C18, 5 mm (125 4 mm I.D. with a * ** * 5 mm integrated pre-column); Eluent: acetonitrile- 0123456789101112131415 aqueous buffer ¼ 60 : 40, v/v; Flow rate: 1 ml/min; --0.1 min Column temperature: ambient forts to optimize the separation concentrated on reversed- other systems tested, e.g. Systems 01/02/a, 01/02/b and 02/ phase HPLC systems. 01 can be regarded as mixtures of the basic versions. Two different types of reversed-phase column packing, a An inspection of these chromatograms reveals that System thoroughly end-capped packing material with a specific V01 (Fig. 2) resulted in a less than satisfactory separation surface area of 350 m2/g (Eurospher-100, C18, 5 mm) and of the main peak, with several co-elutions involving 10 a packing material with no special end-capping and a compounds, as indicated by the asterisks (*) at the base of specific surface area of 175 m2/g (Hypersil ODS, 3 mm) the peaks concerned. The run time, however, would be were tested. advantageous, less than 15 min. Because a change from a protic organic modifier to an Although the chromatogram obtained with System V02 aprotic eluent additive can offer a further separation alter- (Fig. 3) takes 24 minutes to run, the main peak is well native for steroids in reversed-phase systems (Francis and separated, and the number of co-eluting contaminants has Kinsella 1984), protic and aprotic mobile phase additives, been reduced to 4. such as methanol and acetonitrile, respectively, were used Systems 01/02/a and 01/02/b (and several other ‘‘mixed” in the eluent optimization studies. systems tested) offer no special advantages over System As there are no ionic groups in the molecules of BNP-166 V02. System 02/01, however, with 7 co-eluting contami- and most of the related potential contaminants, the pH of nants though, can still complement System V02 if resolu- the aqueous part of the eluent was kept constant at 4.5, a tion of M––OH and the BNP-166 analogue, 17a-dimethyl- safe compromise allowing satisfactory stability for the es- oxamate should be needed (the silica system in Fig. 1 can ter moieties in the BNP-166 molecule. also be considered here). Column temperature, as another factor influencing column Of the reversed-phase systems tested, System V02 was performance, was varied between ambient and 40 C. chosen as the best compromise for the various GXP stu- A selection of chromatograms from the reversed-phase dies in this Institute. All the more so as the main problem separation and optimization experiments is shown in here, the co-eluting diastereomer pair of the 17a-chloro- Figs. 2–5, with the respective relative retention time (RRT) iodoacetoxy BNP-166 analogue (one isomer co-elutes values in the Table. System V01 (with a packing of large with the potential contaminant 11-deoxy-cortienic acid surface area, almost fully end-capped, and an aprotic eluent ethyl ester, and the other, not shown, with the main peak) additive) and System V02 (with a packing of much smaller has been eliminated by replacing ethyl iodide with diethyl surface area, no special end-capping, and a protic eluent sulfate in the last step of the synthesis of the drug candi- additive) were the main alternative versions, whereas all the date (see Scheme).

System V02

0.350 17 (a peak marked with an asterisk means co-eluting peaks) D a 1 dmtyoaae N analogue BNP -dimethyloxamate, crincai ty ester(M-OH) ethyl acid -cortienic N 166 BNP 1doycrincai ty ester ethyl acid 11-deoxy-cortienic

0.300 11-oxo- 17 17a-monochloro and 17a-monoiodoacetoxy BNP were synthetically 17 a a

mnidaeoyBNP -monoiodoacetoxy prepared as hypothetical contaminants. clrid ctx BNP acetoxy -chloroiodo D

0.250 1 17 crincai ty ester ethyl acid -cortienic 17 a frae N analogue BNP -formate, a 0.200 BNP -monochloroacetoxy ,-iyr-1doyBNP 1,2-dihydro-11-deoxy 17 D a 11-dezoxy-M-OH U 1 tihooctx BNP -trichloroacetoxy crincacid -cortienic

A 0.150 ,-iyr BNP 1,2-dihydro N-ehlester BNP-methyl M-COOH uiBNP-166 Lumi BNP 11-deoxy 1eiBNP 11-epi 0.100 BNP 11-oxo

Fig. 3: 0.050 Separation of BNP-166 and its potential contami- nants in a reversed-phase system. Column: Hy- 0.000 persil ODS, 3 mm (100 4 mm, I.D.); Eluent: * * methanol-aqueous buffer ¼ 60 : 40, v/v; Flow -- 0.050 0 1 2 3 4 5 6 7 8 910 11 12 13 14 15 16 17 18 19 20 21 22 23 rate: 0.8 ml/min; Column temperature: 40 C

384 Pharmazie 59 (2004) 5 ORIGINAL ARTICLES 17 D System 01/02/b a 1 crincai ty se (M-OH) ester ethyl acid -cortienic dmtyoaae N analogue BNP -dimethyloxamate, 0.6 ester ethyl acid 11-deoxy-cortienic (a peak marked with an asterisk means co-eluting peaks)

17a-monochloro and 17þ-monoiodoacetoxy BNP were synthetically

0.5 17 BNP-166

a prepared as hypothetical contaminants. mnclraeoyBNP -monochloroacetoxy N-ehlester BNP-methyl

0.4 BNP 11-oxo 1ooM-OH 11-oxo 17 17 a a frae N analogue BNP -formate, mnidaeoyBNP -monoiodoacetoxy 17 ,-iyr-1doyBNP 1,2-dihydro-11-deoxy D

0.3 BNP 17þ-trichloroacetoxy 1 a crincacid -cortienic clrid ctx BNP acetoxy -chloroiodo U 1doyM-OH 11-deoxy ,-iyr BNP 1,2-dihydro A M-COOH 1eiBNP 11-epi

0.2 BNP 11-deoxy

0.1 Fig. 4: Separation of BNP-166 and its potential contami- 0 nants in a reversed-phase system. Column: Euro- * * ** * spher-100, C18, 5 mm (125 4 mm I.D. with a 5 mm integrated pre-column); Eluent: methanol- 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 --0.1 min aqueous buffer ¼ 70 : 30, v/v; Flow rate: 0.8 ml/ min; Column temperature: ambient

System V02 has been thoroughly tested for precision and phase systems the polarity difference between these pairs accuracy of BNP-166 determinations in various analytical of compounds is much smaller (see the acetonitrile-con- assignments. Detection was performed at 243 nm, the taining systems in Fig. 2 and Fig. 5), or can even be re- strong UV absorption maximum of the 4-en-3-one conju- versed (see the elution orders of BNP-166 and 11-oxo gated ketone structure [Emax: 15000, Kelce 1988; typi- BNP in the methanol-containing systems in Fig. 3 and cal RSD values: well below 1.0%]. A more complete Fig. 4). validation study of the method is under way. The 1,2-dihydro derivatives (coming mostly from the hy- drocortisone contaminant of the starting material predniso- lone) appeared to be less polar than the respective 1,4- 2.3. Some physico-chemical factors affecting the separa- diene derivatives (coming mostly from prednisolone; com- tion pare the retentions of 1,2-dihydro BNP and 1,2-dihydro- Our findings conform to some generalizations concern- 11-deoxy BNP with those of BNP-166 and 11-deoxy ing retention in normal- and reversed-phase HPLC sys- BNP, respectively, in Figs. 1–5). tems. The most powerful enhancer of molecule polarity, how- In this group of steroids, the least polar compound was ever, was the 17b-carboxylate moiety (if present and sol- 1,2-dihydro-11-deoxy BNP (eluting first in normal-phase vated; see the retentions of M––COOH and D1-cortienic systems and last in reversed-phase ones), showing the im- acid in Figs. 1–5). portance of the 11-hydroxy or 11-oxo group in increasing It is also worth noting that whereas the resolving power of the polarity (H-bonding capability) of such molecules. The most of the reversed-phase systems for D1-cortienic acid contribution of the 11a-hydroxy group to polarity is ap- ethyl ester and the 17a-dimethyloxamate BNP-166 analo- parently stronger than that of the 11b-hydroxy group (see gue approached zero, their resolution was the greatest of the retentions of 11-epi BNP and BNP-166). As expected, all in the normal-phase system. in normal-phase systems, 11b-hydroxy derivatives are re- The effect of elevated temperature was beneficial from the markably more polar than the respective 11-oxo deriva- point of view of system pressure in methanol-containing tives (thus BNP-166 and 11-oxo BNP as well as D1-cor- reversed-phase systems, but it had no real effect on elution tienic acid ethyl ester and 11-oxo-D1-cortienic acid ethyl orders or resolution (compare the RRT values obtained ester can be separated easily, see Fig. 1), but in reversed- with systems 01/02/a and 01/02/b in the Table).

1.0 System 02/01 (a peak marked with an asterisk means co-eluting peaks) 0.9 BNP-166 D

0.8 1 -cortienic acid ethyl ester (M-OH)

0.7 11-deoxy-cortienic acid ethyl ester 17 a 17

0.6 -dimethyloxamate, BNP analogue a -chloroiodo acetoxy BNP 17 a

0.5 11-oxo M-OH -formate, BNP analogue U 17 1,2-dihydro-11-deoxy BNP 1,2-dihydro BNP A a 11-oxo BNP -trichloroacetoxy BNP BNP methyl ester 0.4 D 1 -cortienic acid 11-deoxy M-OH

0.3 11-deoxy BNP M-COOH Lumi BNP-166 11-epi BNP 0.2

0.1 unkn. Fig. 5: 0.0 * Separation of BNP-166 and its potential contami- * * nants in a reversed-phase system. Column: Hy- --0.1 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 persil ODS, 3 mm (100 4 mm, I.D.); Eluent: min acetonitrile-aqueous buffer ¼ 48 : 52, v/v; Flow rate: 1 ml/min; Column temperature: ambient

Pharmazie 59 (2004) 5 385 ORIGINAL ARTICLES

3. Experimental The retention time of BNP-166 in the respective reversed-phase system, the eluent flow rate, column temperature, as well as all the system para- meters of a representative normal-phase chromatogram are specified in the 3.1. Materials respective figure or figure legend. All the major eluent components (distilled water included) were of HPLC BNP-166 sample concentrations (from 200 mg/ml solutions in the eluent grade. Acetonitrile and distilled water were purchased from Riedel-deHae¨n or, preferably, acetonitrile – aqueous buffer mixtures of 1 : 1, v/v to avoid (Seelze, Germany), methanol, ethyl-acetate and dichloro-methane were the even the slightest ester decomposition) were determined by comparing the product of Merck (Darmstadt, Germany), n-hexane was from Carlo Erba sample peak area with calibration standard peak areas. (Milano, Italy) and glacial acetic acid was obtained from Reanal (Buda- pest, Hungary). All other chemicals, such as sodium hydroxide, were com- This research paper was presented during the 4th Conference on Retrome- mercially available products of special reagent grade. The Hypersil ODS tabolism Based Drug Design and Targeting, May 11–14, 2003, Palm and Nucleosil 100 Si chromatographic columns were purchased from Coast, Florida, USA. GROM (Herrenberg-Kayh, Germany), while the Eurospher-C18 column was from Knauer (Berlin, Germany). BNP-166 was synthetized in-house, and all the impurities concerned were References isolated and/or synthetized in our laboratories. Lumi BNP-166 (ethyl 17a- dichloroacetoxy-11b-hydroxy-1a,5b-cycloandrost-3-ene-2-one-17b-carbox- Bodor N (1984a) Novel approaches to the design of safer drugs: Soft ylate), also prepared here (Csana´di et al. 2004), was made by analogy with drugs and site-specific chemical delivery systems. In: Testa B (ed.) Ad- the known photo-rearrangement of prednisolone into lumiprednisolone vances in Drug Research, Academic Press, London vol. 13 p. 255–331. (Williams et al. 1980). Bodor N (1984b) Soft drugs: principles and methods for the design of safe drugs. Med Res Rev 3: 449–469. Bodor N (1988) A novel approach to safer drugs. In: Christophers E et al. 3.2 Analytical method (eds.) Topical corticosteroid therapy, Raven Press Ltd. New York, The HPLC equipment used in the separation studies and for the BNP-166 p. 13–25. assay and purity tests consisted of a Waters 1525 binary solvent pump, a Csana´di A´ , Horva´th Gy, Szekeres T, Hasko´ T, Ila L, Ivanics J, Patthy M, Waters 2487 dual wavelength absorbance detector (operated at 243 nm), a Sala´t J, Seres G, Pallagi I, To´th G, Szederke´nyi F, Ko´nya A, Tegdes A, Rheodyne 7725i manual injector with a 20 ml sample loop, a Waters col- Zubovics Z and Bodor N (2004) Etiprednol dicloacetate, a new soft umn thermostat (an attachment to the pump) and a Waters Breeze software drug candidate. Development of chemistry. Pharmazie, for system control and data handling/processing. (submitted for publication). Two reversed-phase HPLC columns were used (as shown in the legends of Francis FE, Kinsella Jr. RA (1984) Reversed-phase high-performance Figs. 2–5): either a Hypersil ODS, 3 mm column (100 4 mm, I.D.) or a liquid chromatography of C21 metabolites of . J Chromatogr Eurospher-100, C18, 5 mm column (125 4 mm I.D. with a 5 mm inte- 336: 361–367. grated pre-column). Kelce WR, Ganjam VK (1988) Radiometric quantitation of D4-3-ketoster- Reversed-phase HPLC elution solvents consisted of an aqueous buffer to oid-5a-oxidoreductase utilizing high-performance liquid chromatogra- which either methanol or acetonitrile was admixed at varying volume ra- phy. Steroids 52: 217–235. tios. Lin JT, Heftmann E (1982) Comparison of adsorption and reversed-phase Preparation of the aqueous buffer (1 liter): to 990 ml distilled water 1.0 ml partition high-performance liquid chromatography for the separation of of glacial acetic acid was added and the pH of the solution was adjusted to androgens. J. Chromatogr. 237: 215–224. 4.5 0.05 using 2 N NaOH and a pH meter equipped with a glass elec- Williams JR, Moore RH, Li R, Weeks CM (1980) Photochemistry of 11a- trode. The buffer was then diluted to volume with distilled water. The and 11b-hydroxy steroidal 1,4-dien-3-ones and 11a- and 11b-hydroxy eluent (aqueous buffer þ organic modifier) was filtered and degassed be- steroidal bicyclo[3.1.0]hex-3-en-2-ones in neutral and acidic media. J fore or during use. Org Chem 45: 2324–2331.

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