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Degradation Pathway of Pralidoxime Chloride in Concentrated Acidic Solution
Degradation Pathway of Pralidoxime Chloride in Concentrated Acidic Solution PETER
FYHR,'ARNEBRODIN,LENNART ERNEROT, AND J~RGEN LlNDQlJlST
Received December 30, 1983 from Astr8 Ldkemedel AB, S-757 85 SMertNje, Sweden.
Abrtract I3 The degradation products of pralidoxime chloride (1)(X- = Cl-) in concentrated aqueous solutions (550% w/v) were identified using one or more methods: HPLC, polarography, voltammetty, MS and/or NMR. The products found were the 2-cyano-, 2-carboxamidoand 2-carboxy-1-methyl-pyridinium chlorides, 1-methyl pyridinium chloride, cyanide ion, ammonia and carbon dioxide. 1-Methyl-2-pyridone was indirectly identifiedby the presence of cyanide ion. The degradation rate increased with increasing pH values between pH 1 and 3.2and with increasing concentrations between 1 and 50% w/v pralidoxime chloride. The results suggest that 1 (X- = Cl-) is dehydrated by a hydroxyl-ion catalyzed reaction to the nitrile 2 which is hydrolyzed to either the pyridone 6 and cyanide ion or to 2-carboxamido-1-methyl-pyridinium chloride 3. The amide is hydrolyzed to give the 2-carboxy derivative 4 which finally is decarboxylated to give 1-methylpyridinium chloride 5.
halidoxime salts (1, X- = C1-, I-, CH,S0,3, though considered too unstable for use under field conditions, have become increasingly popular as agents in the treatment of organophosphate poisoning. The treatment with an oxime antidote must be started immediately after exposure to the toxic agent and before acetylcholinesterase becomes permanently damaged.' To meet the requirements for rapid self administration, a n intramuscular solution administered by an automatic injector is the only alternative. A therapeutic dose of 1, X- = C1- is 0.5 - 1.0 g. Since the volume of existing injectors is limited to approximately 2 mL, the solution becomes very concentrated (25-+0%). Two routes for the degradation of 1 have been reported. At pH values below 4, a hydrogen-ion catalyzed hydrolysis to give the aldehyde 7 and hybxylamine has been suggested.28 The aldehyde 7 is then oxidized to the corresponding acid 4.8 Above pH 4, a hydroxyl-ion catalyzed dehydration to 2 occurs and this material is subsequently hydrolyzed either to 6 and cyanide ion or to 3 . 1 3 4 7 4 0 If reported at all, the concentration of 1 has been low. In a recently published paper," the degradation products have been identified in concentrated
NH.
3
Go 6
608 /Journal of Pharm8ceutical Sciences Vol. 75, No. 6, June 7986
Q),
4, R = OH (7. R = ti)
5
Accepted for publication March 31, 1986.
acidic solutions of 1, X- = C1-. This study deals mainly with analytical procedures and provides insufficient data for any conclusions on the degradation mechanism. We have reconsidered the degradation mechanism in a concentrated solution since formulating a parenteral solution for self administration would require knowledge of the stability in concentrated solutions.
Experimental Section Pralidoxime chloride solution was prepared by dissolving 1, X- = C1-, (Aldrich Chem. Co., Belgium) in water for injection to a concentration of 480 mg/mL. The pH was adjusted with either 2 M HC1 or 2 M NaOH to 3.5. Four solutions were used, unbuffered and buffered with m-alanine, citric acid and tartaric acid, 0.15 M. The solutions were filtered through 0.2-pm membrane filters (Millipore Corp., Mass., USA) and dispensed in 10-mL vials or 1.4-mL polypropylene ampules for autoinjectors (Astra AB,Sweden). The containers were stored at temperatures between 15 and 120 "C. All chemicals were of USP XX grade (E. Merck, Darmstad, BRD) unless otherwise specified. Routine 'H NMR were recorded on a Varian T-60 spectrometer. Chemical shifts are reported in parts per million relative to Me&. Notations are: (8) singlet; (d) doublet; (t)triplet; (q) quartet; (m) multiplet. Melting points were determined on a Mettler FP-2 melting point apparatus (Mettler GmbH, FRG). MS were performed on a LKB 9000 (LKB AB, Sweden), direct inlet, source temperature 135 "C, ionizing voltage 70 eV. IR spectroscopy was performed in KBr, pellets on a Jasco IRA-1. Pralidoxime Chloride (1, X- = C1-)-contained particulate contamination and was purified by filtering through a 0.2-pm membrane filter, and then recrystallizing three times from 75% 2propanol in 0.001 M HCl, mp 232 "C (lit.12 mp 235-238 "C). 2-Cyano-1-methylpyridiniurnIodide ( 2 b T h i s material was synthesized by the procedure of Kosower et al.,13 mp 169-173 "C (lit.ls mp 175-176°C). 'H NMR (D20): S 8.2-9.4 (m, 4, ArH), 4.6 (8, 3, CHJ. IR Conforms with reference substance (Ayerst Labs., Rouses Point, NY, USA). 2-Carboxamido-1-methylpyridiniumChloride (3)-A 50% w/v eolution of 1, X- = C1- in water was heated in a closed glass vessel at 100 "C for 48 h. The solution was cooled in a ice bath. The material precipitated as orange needle-like crystals. This material was recrystallized three times from methanol: pinkish crystals, mp 250-256 "C; 'H NMR (D20): 6 8.0-9.0 (m, 4, ArH), 4.7 (8, 6, NH2 NHD, HDO, H20), and 4.5 ppm (s,3,CH,); MS: d z 137 (M+, 21,122 (4), 79 (loo), 78 (35),52 (481, 51 (29), 50 (58)zl.22;IR Conforms with a reference substance (Ayerat Labs., Rouses Point, NY, USA). A d . Calc. for C7H9NOCl*H20:C, 44.01; H, 5.81; N, 14.69. Found: C, 44.1; H, 5.9; N, 14.7. 2-Carboxyl-1-methylpyridiniumChloride (I)-This material was obtained from Aldrich Chem. Co., Belgium, and was used with further purification, mp 181-180 "C. 1-Methylpyridinium Iodide ( 5 b T h i s material was synthesized by the procedure of Kosower et al.", mp 117°C (1it.l' mp 116117 "C). 'H NMR S 9.0-7.9 (m, 5, ArH), 4.7 (s,6, HDO, H20), and 4.5 ppm (2,3, CHd. 1-Methylpyridone (GbThis material was obtained from Ayerst Laboratories, Rouses Point, NY, USA. 2-Formyl-1-methypyridiniumChloride ( 7 b T h i s material was obtained from Ayeret Laboratories, Rouses Point, NY, mp 178179 "C (lit.l6 mp 178-179 "C). OO22-3~9/86/0600-0608$0 1.OO/O 0 7986, American Pharmaceutical Association
Assay-1, X- = C1- and its degradation products were analyzed using HPLC. The system consisted of a reversed-phase column (Hibar-LiChrosorb RP-18 5 pm, 125 mm, i.d. 4 mm); a mobile phase of 2 mM sodium octylsulfonate (Eastman Kodak, NY), 50 mM Me,NCI (Fluka AG, BRD), 20 mM AcOH and, 10% MeOH in distilled water; a pump (LDC Constametric 111, LDC, FL) and a flow rate of 0.8 mL/min. Samples were injected using a Rheodyne 7120 injection valve with a 20-pL loop (Rheodyne, CA). Detection was performed with a variable wavelength UV detector (Perkin-Elmer LC-75). The chromatograms were recorded with a computing integrator (Hewlett-Packard 3390A) and a pen recorder (Philips PM8251, Holland). Peak heights were evaluated a t the wavelength of their absorbance maximum. Identification of Degradation Products-Reference solutions and degraded solutions of 1, X - = C1-, were analyzed by HPLC a t numerous wavelengths between 200300 nm. UV spectra were obtained by plotting peak height versus wavelength. The reference spectrum for peak 3 at maximum degradation was prepared from normalized 5 and 2 spectra by addition. The resulting spectrum was then normalized to the sample spectrum. Degraded solutions of 1, X- = C1-, and reference solutions were assayed separately and mixed in equal volumes at maximum absorbance. The change in peak absorbance8 and retention times were recorded. If the sample and reference solutions are identical, the mixed sample should have a peak height of (sample + referenceY2. Ammonia was detected and estimated with an ion-specific electrode. Cyanide ion was identified and estimated by cathodic stripping voltammetry (PAR Mod. 374 Polarographic Analyzer). Carbon dioxide was identified by MS (LKB 9000) using a saturated calcium hydroxide solution. The pH was measured with a combined glass-calomel electrode, calibrated against standard buffers. The measured values were regarded as the pH value even though the ionic strength of the solutions was > 0.1. Differential pulse polarography (PAR model 374 Polarographic Analyzer) at pH 4.5 was also used to identify the degradation products. Half-wave potentials found in decomposed solutions of 1, X- = C1-, were compared with potentials taken on reference substances and electrochemical literature data.16 Large rhombic crystals, up to 10 x 10 x 5 mm, precipitated slowly in highly degraded solutions during cold storage. They were extracted, washed and identified as 3. pH Profile-Solutions of 1 (X-= Cl-), 43% wlv, of pH 1.05, 1.34, and 2.02 buffered with HC1, of pH 1.57, 1.70, and 2.02, buffered with 0.1 M potassium hydrogen sulfate and of pH 2.40, 2.80, and 3.20 buffered with 0.1 M citric acid were treated a t 120 "C in an autoclave for 2 h. Five samples were taken during at least one half-life and assayed for 1, X- = C1- . The observed rate constants were calculated according to first-order kinetics. The pH-temperature dependence for each solution was established up to 80 "C, according to van't Hoff isochore by calibrating the electrode a t the actual temperatures against buffers with known pH temperature dependence. The pH was thereafter measured and the dependence was established by plotting measured pH versus 1/T, the plot was extrapolated to predict the pH at 120 "C.1' Observed Rate-Concentration Relationship-Solutions of 1, X= C1-, of different concentrations were heated in an autoclave at 120 "C for 1.5 h. Samples were taken a t 4 different times and assayed for 1, X- = C1-. The observed rate constants were calculated according to first-order kinetics. The solutions were not buffered and the pH was not adjusted. Since the ionic strength of the solutions varied from 0.06 to 2.90, adjustment to the same pH would not yield solutions of the same hydrogen-ion activity. The initial pH ranged from 5.0-3.5 for 0.1%-50% solutions respectively; the final pH was 2.6-2.8 for all solutions.
Results Figure 1 shows HPLC chromatograms at different times during the degradation of a 45% wlv solution of 1, X- = C1-. Four degradation products are clearly detectable. The appearance of the peaks, numbered according t o their retention times, is illustrated in Fig. 2. As can be seen, the material corresponding to peak 4 is formed first. It rapidly appears to attain a steady-state value. The next products to appear are
B
Figure 1-HPLC chromatograms of a 45% w/v solution of pralidoxime chloride (1) (K = Cl-) at an initial pH of 3.0, after (A) 10 min, (B) 30 min, (C) 120 min, . and (D)540 min at 80 "C. The numbers 1-4 with arrows refer to the four peaks.
noun
Flgure 2- Degradation product peak height at maximum absorbance from a 45% w/v solution of pralidoxime chloride 1 (K = CI- at an initial pH of 3.5 at 80 "C.A Peak 7, V Peak 2,O Peak 3 , O Peak 4, (see Fig. 1).
those corresponding to peaks 3 and 2. The material corresponding to peak 3 slightly before 2. The product represented by peak 1 is clearly the final material formed. The retention time ( t R ) of peaks 1, 2 and 5 corresponds to those of 4, 3 and 1,respectively ( t 1.8,4.4 ~ and 5.5 m id. Peak 3 corresponds to 2 (tR 4.2 min) and with 5 ( t 4.6 ~ min) at maximum degradation. Peak 4 ( t 5.0 ~ min) does not correspond to any of the reference substances. Compounds 6 ( t5.3 ~ min) and 7 ( t R 4.29 min) do not correspond to any of the peaks. By reference substance addition, the experimental value corresponds to the calculated value except when 5 is added a t minimal degradation. In this case, the addition also causes a change in t R from 4.40 to 4.60 min. The UV spectra of peaks 1, 2 and 3 a t minimal degradation conforms well with those of 4 , 3 and 2, respectively. The spectrum of peak 3 Journal of Pharmaceutical Sciences / 609 Vol. 75, No. 6, June 1986
at maximum degradation conforms to a mixture of 2 and 5. The spectrum of peak 4 does not correspond to any reference substance. Polarography of decomposed 1, X- = C1-, shows peaks at -0.48, -0.73, -0.90, -1.04 and -1.19 V. This conforms well with the half-wave potentials of 1at -0.48 and 1.19 V, 3 a t -0.90 V, 2 at -0.73 V and 4 at -1.04 V. Compound 7 has a half-wave potential of -0.31 V. This is not seen in the polarogram of decomposed solutions of 1, X- = C1-. The precipitate found upon cold storage was identified as 3. Assay for the final products showed an increase in the amount of ammonia and carbon dioxide as the degradation proceeded. Very low concentrations of cyanide ion were found. The followingdecomposition products have thus been identified: 2-cyano-, 2-carboxamido- and 2-carboxy- derivatives of 1, 1-methylpyridinium chloride, cyanide ion, ammonia, and carbon dioxide. Indirectly, by the presence of cyanide ion, 1methyl-2-pyridone 6 has been identified. Compounds 2, 3, 4 and 6 have recently been identified using a reversed-phase HPLC system.11 Using this system, 6 can be separated from the other degradation products and identified. The concentration-rate relationship (Fig. 3) shows an increased rate with increasing concentration. However, the pH decreased more rapidly in the more concentration solutions than in the dilute ones. Thus, since the degradation rate decreased with pH (Fig. 4), the slope of the concentration profile might be too small. The differences in measured pH might also be affected by the differences in ionic strength.
Discussion The HPLC data indicates that peak 3 is composed of 2 at an early stage of degradation. Later, the retention time of peak 3 approaches that of 5. The W spectrum of peak 3 at this stage seems to be a mixture of 2 and 5. Therefore, 5 apparently becomes a greater part of peak 3 as the degradation proceeds. Assuming similar molar abaorbances of the peaks, Fig. 2 indicates that 2 is the second product formed and that it seems to approach a steady-state value. As 5
'I
). 10
M
30
40
50
K wlv
Flgure 3-Observed rate constants at different concentrations, of pralidoxime chloride (X = Cl-) at an initial pH of 4.0 at 1 2 0 "C. 610 / Journal of Ph8fmaceutical Sciences Vol. 75, No. 6, June 7986
'
-3.0.
-3.5
1 2
1
3
PH Flgure +The pH profile of 43% w/v pralidoxime chloride 1 (X = Cl-) at 120 "C, (- -) slope 7.0.
-
successively becomes a greater part of peak 3, compound 2 reaches steady state. In the pH-profile (Fig. 4) the observed rate of 1, X- = C1-, degradation shows a positive dependence on pH, probably without a minimum or plateau, since studies in strong mineral acid show even lower degradation rates. Therefore, we suggest that the first step is a hydroxyl-ion catalyzed dehydration of the oxime moiety to a nitrile, which is the same mechanism as previously reported for a pH >4.4.6 The difficulties in defining pH in a 48% w/v solution of 1, X- = C1-, corresponding to an ionic strength of 2.8 have to be considered when attempting to explain the deviation from a slope of 1.The degradation reaction might be favored by the low water concentration (13 moles of water per mole of 1, X= C1-1. This is indicated by an observed increase in the rate of degradation with increasing concentration of 1, X- = C1(Fig. 3). Peak 2 is identified as compound 3. From Fig. 2, it can be seen that 3 is the third product formed, appearing slightly after 2. The second step is therefore suggested to be the hydration of 2 to 3.2.46 The reaction is probably hydrogen-ion catalyzed as it appears to be very fast. Compound 3 is the major decomposition product. It can be isolated in large amounts, approximately 50%of the initial amount of 1, X- = C1-, from decomposed solutions. Cyanide ion is detected in small amounts, less than 5 ppm after one half-life. This corresponds to 0.001% of the initial concentration of 1, X- = C1-. Thus, a very minor part of 2 is hydrolyzed to 6.7J8Compound 6 is not detected by HPLC since small amounts of this product will be completely masked by the peak attributed to 1, X- = C1-. The UV spectrum, retention time, and peak height of 4 are in close agreement with those of peak 1. This, and the formation of ammonia in equimolar amounts, strongly indicates that 3 is hydrolyzed to 4 by either hydrogen-ion or hydroxyl-ion catalysis. Since 4 is an acid with a positive charge close to the carboxyl function, it probably appears as a zwitterion even at low pH. Compound 4 readily loses carbon dioxide20 to form 5. During the degradation of solutions of 1, X- = C1-, at temperatures above 50 "C, carbon dioxide is also formed in large amounts. The presence of 5 in peak 3 has been discussed above. The identity of 5 has not been reported previously, and some authors" report that it is not present in measurable amounts.
The previously suggested degradation path at pH below 4 includes hydrolysis to 7 and hydroxylamine. However, we have not been able to detect 7 by HPLC or polarography. This hydrolysis attains equilibrium at higher concentrations of oxime, and the resulting aldehyde is oxidized to 4.8 The oxidation would shift the equilibrium toward 7,and thus, the degradation would proceed. The observed rate in our studies was, however, not affected by the presence or absence of oxygen. Thus, the previously described path is not applicable to concentration systems. Instead, the mechanism suggested here resembles the one described earlier for pH >4.= The first product to be formed during the degradation of 1, X- = C1-, is represented by peak 4. It does not correspond to any known reference substance. Peak 4 rapidly reaches its maximum height and thereafter retains a constant ratio to 1, X- = C1-. Thus, this product appears to be in equilibrium with 1, X- = C1-. The extent of the equilibrium seems to depend on temperature. In unbuffered systems, ita formation is accompanied by a marked increase in pH. The W spectrum shows a wide broad curve. This product could be the other isomer of 1.21 However, such a transformation would not cause a change in pH, and the W spectra of isomers are usually identical except for an eventual small lateral shift. Similar results have been reported by other investigators11 who found a product with similar properties eluting with 2hydroxymethyl-1-methylpyridiniumchloride 8. We have not considered this substance as a possible degradation product. A possible way for the formation of 8 is a disproportionation of 7 to 4 and 8. Even if this reaction were so fast that 7 could not be detected, hydroxylamine would be present in the solution. Thus, we believe that further evidence is necessary before 8 is definitely identified.
2. Ellin, R. I.; Easterday, D. E. J . Phurm. Phurmucol. 1961, 13, 370-373. 3. Ellin. R. I.; Carlese. J. S.; Kondritzer. A. A. J . Phurm. Sci.1962. 51, 141-146. 4. Barkman, R.; Edgren, B.; Sundwall, A. J . Pharm. Pharmcrcol. 1963,15,671-677. 5. Ellin, R. I.; Wills, J. H. J. Phurm. Sci. 1964, 53, 995-1007. 6. Cristensson, I. Forsvarsmedicin 1974,10, 109-113. 7. Ellin, R. I. J . Am. Chem.Soc. 1958,80,6588-6590. 8. Creasy, N. H.; Green, A. L. J.Pharm. Pharmrrcol. 1959,11,485490. 9. Fan, M. C.; Naim, J. G.; Walker, G. C. J . Pharm. Pharmacol. 1964,16,493-496: 10. Carter. C. W.:Naim. J. G.: Walker. G. C. Can. J . Pharm.Sci. 1968,3, 8-10; 11. h e , D. G.; Johnsson, R. N.; Kho, B. T. J . Phurm.Sci. 1983, 72, 7.51-7.56 .- - .- - . 12. T h e Merck Index”, 10th ed., Windholz, M., Ed.; Merck & Co., Inc.: Rahway, NJ, 1983; 1107. 13. Koeower, E. M.; Skon, A.; S c h w a , W. M.;Patton, J. W. J . Am. Chem. Soc. 1960,82,2188. 14. Kosower, E. M.; Skon, J. A. J . Am. Chem. Soc. 1960,82,21952203. 15. Ellin, R. I.; Kondritzer, A. A. A d . Chem.1959,31,200-201. 16. Meites, L.; Zuman,P. “Electrochemical Data”, vol. A-1; John Wiley & Sons: New York, 1974, p 292. 17. Martin, A. N.; Swarbrick, J.; Cammarata, A. “Physical Pharma, Lea & Febiger: Philadelphia, 1973; pp 125-126. 18. g k m a n , R. R. Rev. Znt. Santee Armees, 1963,36,91-98. 19. Ellin, R. I.; Wills, J. H. J . Phurm.Sci. 1964,53, 1143-1150. 20. Haake, P.; Mantecon, J. J . Am. Chem.Soc. 1964,86,5230-5234. 21. Ginsbur S ;Wilson, I. B. J . A m . Chem.Soc. 1957,79,481-485. 22. Larsen, .,* Ersgaard, . H. Or .Mass.Spec. 1978,13,417-424. 23. Holmen, H.; Ers aard, H.;& nck, J.; Larsen, E. Bwm. Mass. Spec. 1981, 8, 12%124.
References and Notes
The authors sincere1 thank Mrs. Miluse RenRel for skillful technical assistance, Helvi Scheinin for the preparation of reference substances, Dr. Garan Bondesson for advice in the synthesis of reference substances, Dr. Hans Thorin for the MS analysis, Dr. Inga Christensson for valuable discussions, and Ayerst Laboratories for providing their reference substances.
1. Sidell, F. R.; “Medical Protection Against Chemical Warfare
Agents”, papers from symposium of the Stockholm International Peace Research Institute; Herce Novi, Yugoslavia, 1974; Almquist & Wiksell International: f%xkholm, 1976; pp 22-35.
f
k
Acknowledgments
ds.
Journal of Pharmaceutical Sciences / 811 Vol. 75,No. 6,June 1986
FYHR,'ARNEBRODIN,LENNART ERNEROT, AND J~RGEN LlNDQlJlST
Received December 30, 1983 from Astr8 Ldkemedel AB, S-757 85 SMertNje, Sweden.
Abrtract I3 The degradation products of pralidoxime chloride (1)(X- = Cl-) in concentrated aqueous solutions (550% w/v) were identified using one or more methods: HPLC, polarography, voltammetty, MS and/or NMR. The products found were the 2-cyano-, 2-carboxamidoand 2-carboxy-1-methyl-pyridinium chlorides, 1-methyl pyridinium chloride, cyanide ion, ammonia and carbon dioxide. 1-Methyl-2-pyridone was indirectly identifiedby the presence of cyanide ion. The degradation rate increased with increasing pH values between pH 1 and 3.2and with increasing concentrations between 1 and 50% w/v pralidoxime chloride. The results suggest that 1 (X- = Cl-) is dehydrated by a hydroxyl-ion catalyzed reaction to the nitrile 2 which is hydrolyzed to either the pyridone 6 and cyanide ion or to 2-carboxamido-1-methyl-pyridinium chloride 3. The amide is hydrolyzed to give the 2-carboxy derivative 4 which finally is decarboxylated to give 1-methylpyridinium chloride 5.
halidoxime salts (1, X- = C1-, I-, CH,S0,3, though considered too unstable for use under field conditions, have become increasingly popular as agents in the treatment of organophosphate poisoning. The treatment with an oxime antidote must be started immediately after exposure to the toxic agent and before acetylcholinesterase becomes permanently damaged.' To meet the requirements for rapid self administration, a n intramuscular solution administered by an automatic injector is the only alternative. A therapeutic dose of 1, X- = C1- is 0.5 - 1.0 g. Since the volume of existing injectors is limited to approximately 2 mL, the solution becomes very concentrated (25-+0%). Two routes for the degradation of 1 have been reported. At pH values below 4, a hydrogen-ion catalyzed hydrolysis to give the aldehyde 7 and hybxylamine has been suggested.28 The aldehyde 7 is then oxidized to the corresponding acid 4.8 Above pH 4, a hydroxyl-ion catalyzed dehydration to 2 occurs and this material is subsequently hydrolyzed either to 6 and cyanide ion or to 3 . 1 3 4 7 4 0 If reported at all, the concentration of 1 has been low. In a recently published paper," the degradation products have been identified in concentrated
NH.
3
Go 6
608 /Journal of Pharm8ceutical Sciences Vol. 75, No. 6, June 7986
Q),
4, R = OH (7. R = ti)
5
Accepted for publication March 31, 1986.
acidic solutions of 1, X- = C1-. This study deals mainly with analytical procedures and provides insufficient data for any conclusions on the degradation mechanism. We have reconsidered the degradation mechanism in a concentrated solution since formulating a parenteral solution for self administration would require knowledge of the stability in concentrated solutions.
Experimental Section Pralidoxime chloride solution was prepared by dissolving 1, X- = C1-, (Aldrich Chem. Co., Belgium) in water for injection to a concentration of 480 mg/mL. The pH was adjusted with either 2 M HC1 or 2 M NaOH to 3.5. Four solutions were used, unbuffered and buffered with m-alanine, citric acid and tartaric acid, 0.15 M. The solutions were filtered through 0.2-pm membrane filters (Millipore Corp., Mass., USA) and dispensed in 10-mL vials or 1.4-mL polypropylene ampules for autoinjectors (Astra AB,Sweden). The containers were stored at temperatures between 15 and 120 "C. All chemicals were of USP XX grade (E. Merck, Darmstad, BRD) unless otherwise specified. Routine 'H NMR were recorded on a Varian T-60 spectrometer. Chemical shifts are reported in parts per million relative to Me&. Notations are: (8) singlet; (d) doublet; (t)triplet; (q) quartet; (m) multiplet. Melting points were determined on a Mettler FP-2 melting point apparatus (Mettler GmbH, FRG). MS were performed on a LKB 9000 (LKB AB, Sweden), direct inlet, source temperature 135 "C, ionizing voltage 70 eV. IR spectroscopy was performed in KBr, pellets on a Jasco IRA-1. Pralidoxime Chloride (1, X- = C1-)-contained particulate contamination and was purified by filtering through a 0.2-pm membrane filter, and then recrystallizing three times from 75% 2propanol in 0.001 M HCl, mp 232 "C (lit.12 mp 235-238 "C). 2-Cyano-1-methylpyridiniurnIodide ( 2 b T h i s material was synthesized by the procedure of Kosower et al.,13 mp 169-173 "C (lit.ls mp 175-176°C). 'H NMR (D20): S 8.2-9.4 (m, 4, ArH), 4.6 (8, 3, CHJ. IR Conforms with reference substance (Ayerst Labs., Rouses Point, NY, USA). 2-Carboxamido-1-methylpyridiniumChloride (3)-A 50% w/v eolution of 1, X- = C1- in water was heated in a closed glass vessel at 100 "C for 48 h. The solution was cooled in a ice bath. The material precipitated as orange needle-like crystals. This material was recrystallized three times from methanol: pinkish crystals, mp 250-256 "C; 'H NMR (D20): 6 8.0-9.0 (m, 4, ArH), 4.7 (8, 6, NH2 NHD, HDO, H20), and 4.5 ppm (s,3,CH,); MS: d z 137 (M+, 21,122 (4), 79 (loo), 78 (35),52 (481, 51 (29), 50 (58)zl.22;IR Conforms with a reference substance (Ayerat Labs., Rouses Point, NY, USA). A d . Calc. for C7H9NOCl*H20:C, 44.01; H, 5.81; N, 14.69. Found: C, 44.1; H, 5.9; N, 14.7. 2-Carboxyl-1-methylpyridiniumChloride (I)-This material was obtained from Aldrich Chem. Co., Belgium, and was used with further purification, mp 181-180 "C. 1-Methylpyridinium Iodide ( 5 b T h i s material was synthesized by the procedure of Kosower et al.", mp 117°C (1it.l' mp 116117 "C). 'H NMR S 9.0-7.9 (m, 5, ArH), 4.7 (s,6, HDO, H20), and 4.5 ppm (2,3, CHd. 1-Methylpyridone (GbThis material was obtained from Ayerst Laboratories, Rouses Point, NY, USA. 2-Formyl-1-methypyridiniumChloride ( 7 b T h i s material was obtained from Ayeret Laboratories, Rouses Point, NY, mp 178179 "C (lit.l6 mp 178-179 "C). OO22-3~9/86/0600-0608$0 1.OO/O 0 7986, American Pharmaceutical Association
Assay-1, X- = C1- and its degradation products were analyzed using HPLC. The system consisted of a reversed-phase column (Hibar-LiChrosorb RP-18 5 pm, 125 mm, i.d. 4 mm); a mobile phase of 2 mM sodium octylsulfonate (Eastman Kodak, NY), 50 mM Me,NCI (Fluka AG, BRD), 20 mM AcOH and, 10% MeOH in distilled water; a pump (LDC Constametric 111, LDC, FL) and a flow rate of 0.8 mL/min. Samples were injected using a Rheodyne 7120 injection valve with a 20-pL loop (Rheodyne, CA). Detection was performed with a variable wavelength UV detector (Perkin-Elmer LC-75). The chromatograms were recorded with a computing integrator (Hewlett-Packard 3390A) and a pen recorder (Philips PM8251, Holland). Peak heights were evaluated a t the wavelength of their absorbance maximum. Identification of Degradation Products-Reference solutions and degraded solutions of 1, X - = C1-, were analyzed by HPLC a t numerous wavelengths between 200300 nm. UV spectra were obtained by plotting peak height versus wavelength. The reference spectrum for peak 3 at maximum degradation was prepared from normalized 5 and 2 spectra by addition. The resulting spectrum was then normalized to the sample spectrum. Degraded solutions of 1, X- = C1-, and reference solutions were assayed separately and mixed in equal volumes at maximum absorbance. The change in peak absorbance8 and retention times were recorded. If the sample and reference solutions are identical, the mixed sample should have a peak height of (sample + referenceY2. Ammonia was detected and estimated with an ion-specific electrode. Cyanide ion was identified and estimated by cathodic stripping voltammetry (PAR Mod. 374 Polarographic Analyzer). Carbon dioxide was identified by MS (LKB 9000) using a saturated calcium hydroxide solution. The pH was measured with a combined glass-calomel electrode, calibrated against standard buffers. The measured values were regarded as the pH value even though the ionic strength of the solutions was > 0.1. Differential pulse polarography (PAR model 374 Polarographic Analyzer) at pH 4.5 was also used to identify the degradation products. Half-wave potentials found in decomposed solutions of 1, X- = C1-, were compared with potentials taken on reference substances and electrochemical literature data.16 Large rhombic crystals, up to 10 x 10 x 5 mm, precipitated slowly in highly degraded solutions during cold storage. They were extracted, washed and identified as 3. pH Profile-Solutions of 1 (X-= Cl-), 43% wlv, of pH 1.05, 1.34, and 2.02 buffered with HC1, of pH 1.57, 1.70, and 2.02, buffered with 0.1 M potassium hydrogen sulfate and of pH 2.40, 2.80, and 3.20 buffered with 0.1 M citric acid were treated a t 120 "C in an autoclave for 2 h. Five samples were taken during at least one half-life and assayed for 1, X- = C1- . The observed rate constants were calculated according to first-order kinetics. The pH-temperature dependence for each solution was established up to 80 "C, according to van't Hoff isochore by calibrating the electrode a t the actual temperatures against buffers with known pH temperature dependence. The pH was thereafter measured and the dependence was established by plotting measured pH versus 1/T, the plot was extrapolated to predict the pH at 120 "C.1' Observed Rate-Concentration Relationship-Solutions of 1, X= C1-, of different concentrations were heated in an autoclave at 120 "C for 1.5 h. Samples were taken a t 4 different times and assayed for 1, X- = C1-. The observed rate constants were calculated according to first-order kinetics. The solutions were not buffered and the pH was not adjusted. Since the ionic strength of the solutions varied from 0.06 to 2.90, adjustment to the same pH would not yield solutions of the same hydrogen-ion activity. The initial pH ranged from 5.0-3.5 for 0.1%-50% solutions respectively; the final pH was 2.6-2.8 for all solutions.
Results Figure 1 shows HPLC chromatograms at different times during the degradation of a 45% wlv solution of 1, X- = C1-. Four degradation products are clearly detectable. The appearance of the peaks, numbered according t o their retention times, is illustrated in Fig. 2. As can be seen, the material corresponding to peak 4 is formed first. It rapidly appears to attain a steady-state value. The next products to appear are
B
Figure 1-HPLC chromatograms of a 45% w/v solution of pralidoxime chloride (1) (K = Cl-) at an initial pH of 3.0, after (A) 10 min, (B) 30 min, (C) 120 min, . and (D)540 min at 80 "C. The numbers 1-4 with arrows refer to the four peaks.
noun
Flgure 2- Degradation product peak height at maximum absorbance from a 45% w/v solution of pralidoxime chloride 1 (K = CI- at an initial pH of 3.5 at 80 "C.A Peak 7, V Peak 2,O Peak 3 , O Peak 4, (see Fig. 1).
those corresponding to peaks 3 and 2. The material corresponding to peak 3 slightly before 2. The product represented by peak 1 is clearly the final material formed. The retention time ( t R ) of peaks 1, 2 and 5 corresponds to those of 4, 3 and 1,respectively ( t 1.8,4.4 ~ and 5.5 m id. Peak 3 corresponds to 2 (tR 4.2 min) and with 5 ( t 4.6 ~ min) at maximum degradation. Peak 4 ( t 5.0 ~ min) does not correspond to any of the reference substances. Compounds 6 ( t5.3 ~ min) and 7 ( t R 4.29 min) do not correspond to any of the peaks. By reference substance addition, the experimental value corresponds to the calculated value except when 5 is added a t minimal degradation. In this case, the addition also causes a change in t R from 4.40 to 4.60 min. The UV spectra of peaks 1, 2 and 3 a t minimal degradation conforms well with those of 4 , 3 and 2, respectively. The spectrum of peak 3 Journal of Pharmaceutical Sciences / 609 Vol. 75, No. 6, June 1986
at maximum degradation conforms to a mixture of 2 and 5. The spectrum of peak 4 does not correspond to any reference substance. Polarography of decomposed 1, X- = C1-, shows peaks at -0.48, -0.73, -0.90, -1.04 and -1.19 V. This conforms well with the half-wave potentials of 1at -0.48 and 1.19 V, 3 a t -0.90 V, 2 at -0.73 V and 4 at -1.04 V. Compound 7 has a half-wave potential of -0.31 V. This is not seen in the polarogram of decomposed solutions of 1, X- = C1-. The precipitate found upon cold storage was identified as 3. Assay for the final products showed an increase in the amount of ammonia and carbon dioxide as the degradation proceeded. Very low concentrations of cyanide ion were found. The followingdecomposition products have thus been identified: 2-cyano-, 2-carboxamido- and 2-carboxy- derivatives of 1, 1-methylpyridinium chloride, cyanide ion, ammonia, and carbon dioxide. Indirectly, by the presence of cyanide ion, 1methyl-2-pyridone 6 has been identified. Compounds 2, 3, 4 and 6 have recently been identified using a reversed-phase HPLC system.11 Using this system, 6 can be separated from the other degradation products and identified. The concentration-rate relationship (Fig. 3) shows an increased rate with increasing concentration. However, the pH decreased more rapidly in the more concentration solutions than in the dilute ones. Thus, since the degradation rate decreased with pH (Fig. 4), the slope of the concentration profile might be too small. The differences in measured pH might also be affected by the differences in ionic strength.
Discussion The HPLC data indicates that peak 3 is composed of 2 at an early stage of degradation. Later, the retention time of peak 3 approaches that of 5. The W spectrum of peak 3 at this stage seems to be a mixture of 2 and 5. Therefore, 5 apparently becomes a greater part of peak 3 as the degradation proceeds. Assuming similar molar abaorbances of the peaks, Fig. 2 indicates that 2 is the second product formed and that it seems to approach a steady-state value. As 5
'I
). 10
M
30
40
50
K wlv
Flgure 3-Observed rate constants at different concentrations, of pralidoxime chloride (X = Cl-) at an initial pH of 4.0 at 1 2 0 "C. 610 / Journal of Ph8fmaceutical Sciences Vol. 75, No. 6, June 7986
'
-3.0.
-3.5
1 2
1
3
PH Flgure +The pH profile of 43% w/v pralidoxime chloride 1 (X = Cl-) at 120 "C, (- -) slope 7.0.
-
successively becomes a greater part of peak 3, compound 2 reaches steady state. In the pH-profile (Fig. 4) the observed rate of 1, X- = C1-, degradation shows a positive dependence on pH, probably without a minimum or plateau, since studies in strong mineral acid show even lower degradation rates. Therefore, we suggest that the first step is a hydroxyl-ion catalyzed dehydration of the oxime moiety to a nitrile, which is the same mechanism as previously reported for a pH >4.4.6 The difficulties in defining pH in a 48% w/v solution of 1, X- = C1-, corresponding to an ionic strength of 2.8 have to be considered when attempting to explain the deviation from a slope of 1.The degradation reaction might be favored by the low water concentration (13 moles of water per mole of 1, X= C1-1. This is indicated by an observed increase in the rate of degradation with increasing concentration of 1, X- = C1(Fig. 3). Peak 2 is identified as compound 3. From Fig. 2, it can be seen that 3 is the third product formed, appearing slightly after 2. The second step is therefore suggested to be the hydration of 2 to 3.2.46 The reaction is probably hydrogen-ion catalyzed as it appears to be very fast. Compound 3 is the major decomposition product. It can be isolated in large amounts, approximately 50%of the initial amount of 1, X- = C1-, from decomposed solutions. Cyanide ion is detected in small amounts, less than 5 ppm after one half-life. This corresponds to 0.001% of the initial concentration of 1, X- = C1-. Thus, a very minor part of 2 is hydrolyzed to 6.7J8Compound 6 is not detected by HPLC since small amounts of this product will be completely masked by the peak attributed to 1, X- = C1-. The UV spectrum, retention time, and peak height of 4 are in close agreement with those of peak 1. This, and the formation of ammonia in equimolar amounts, strongly indicates that 3 is hydrolyzed to 4 by either hydrogen-ion or hydroxyl-ion catalysis. Since 4 is an acid with a positive charge close to the carboxyl function, it probably appears as a zwitterion even at low pH. Compound 4 readily loses carbon dioxide20 to form 5. During the degradation of solutions of 1, X- = C1-, at temperatures above 50 "C, carbon dioxide is also formed in large amounts. The presence of 5 in peak 3 has been discussed above. The identity of 5 has not been reported previously, and some authors" report that it is not present in measurable amounts.
The previously suggested degradation path at pH below 4 includes hydrolysis to 7 and hydroxylamine. However, we have not been able to detect 7 by HPLC or polarography. This hydrolysis attains equilibrium at higher concentrations of oxime, and the resulting aldehyde is oxidized to 4.8 The oxidation would shift the equilibrium toward 7,and thus, the degradation would proceed. The observed rate in our studies was, however, not affected by the presence or absence of oxygen. Thus, the previously described path is not applicable to concentration systems. Instead, the mechanism suggested here resembles the one described earlier for pH >4.= The first product to be formed during the degradation of 1, X- = C1-, is represented by peak 4. It does not correspond to any known reference substance. Peak 4 rapidly reaches its maximum height and thereafter retains a constant ratio to 1, X- = C1-. Thus, this product appears to be in equilibrium with 1, X- = C1-. The extent of the equilibrium seems to depend on temperature. In unbuffered systems, ita formation is accompanied by a marked increase in pH. The W spectrum shows a wide broad curve. This product could be the other isomer of 1.21 However, such a transformation would not cause a change in pH, and the W spectra of isomers are usually identical except for an eventual small lateral shift. Similar results have been reported by other investigators11 who found a product with similar properties eluting with 2hydroxymethyl-1-methylpyridiniumchloride 8. We have not considered this substance as a possible degradation product. A possible way for the formation of 8 is a disproportionation of 7 to 4 and 8. Even if this reaction were so fast that 7 could not be detected, hydroxylamine would be present in the solution. Thus, we believe that further evidence is necessary before 8 is definitely identified.
2. Ellin, R. I.; Easterday, D. E. J . Phurm. Phurmucol. 1961, 13, 370-373. 3. Ellin. R. I.; Carlese. J. S.; Kondritzer. A. A. J . Phurm. Sci.1962. 51, 141-146. 4. Barkman, R.; Edgren, B.; Sundwall, A. J . Pharm. Pharmcrcol. 1963,15,671-677. 5. Ellin, R. I.; Wills, J. H. J. Phurm. Sci. 1964, 53, 995-1007. 6. Cristensson, I. Forsvarsmedicin 1974,10, 109-113. 7. Ellin, R. I. J . Am. Chem.Soc. 1958,80,6588-6590. 8. Creasy, N. H.; Green, A. L. J.Pharm. Pharmrrcol. 1959,11,485490. 9. Fan, M. C.; Naim, J. G.; Walker, G. C. J . Pharm. Pharmacol. 1964,16,493-496: 10. Carter. C. W.:Naim. J. G.: Walker. G. C. Can. J . Pharm.Sci. 1968,3, 8-10; 11. h e , D. G.; Johnsson, R. N.; Kho, B. T. J . Phurm.Sci. 1983, 72, 7.51-7.56 .- - .- - . 12. T h e Merck Index”, 10th ed., Windholz, M., Ed.; Merck & Co., Inc.: Rahway, NJ, 1983; 1107. 13. Koeower, E. M.; Skon, A.; S c h w a , W. M.;Patton, J. W. J . Am. Chem. Soc. 1960,82,2188. 14. Kosower, E. M.; Skon, J. A. J . Am. Chem. Soc. 1960,82,21952203. 15. Ellin, R. I.; Kondritzer, A. A. A d . Chem.1959,31,200-201. 16. Meites, L.; Zuman,P. “Electrochemical Data”, vol. A-1; John Wiley & Sons: New York, 1974, p 292. 17. Martin, A. N.; Swarbrick, J.; Cammarata, A. “Physical Pharma, Lea & Febiger: Philadelphia, 1973; pp 125-126. 18. g k m a n , R. R. Rev. Znt. Santee Armees, 1963,36,91-98. 19. Ellin, R. I.; Wills, J. H. J . Phurm.Sci. 1964,53, 1143-1150. 20. Haake, P.; Mantecon, J. J . Am. Chem.Soc. 1964,86,5230-5234. 21. Ginsbur S ;Wilson, I. B. J . A m . Chem.Soc. 1957,79,481-485. 22. Larsen, .,* Ersgaard, . H. Or .Mass.Spec. 1978,13,417-424. 23. Holmen, H.; Ers aard, H.;& nck, J.; Larsen, E. Bwm. Mass. Spec. 1981, 8, 12%124.
References and Notes
The authors sincere1 thank Mrs. Miluse RenRel for skillful technical assistance, Helvi Scheinin for the preparation of reference substances, Dr. Garan Bondesson for advice in the synthesis of reference substances, Dr. Hans Thorin for the MS analysis, Dr. Inga Christensson for valuable discussions, and Ayerst Laboratories for providing their reference substances.
1. Sidell, F. R.; “Medical Protection Against Chemical Warfare
Agents”, papers from symposium of the Stockholm International Peace Research Institute; Herce Novi, Yugoslavia, 1974; Almquist & Wiksell International: f%xkholm, 1976; pp 22-35.
f
k
Acknowledgments
ds.
Journal of Pharmaceutical Sciences / 811 Vol. 75,No. 6,June 1986