- Email: [email protected]
Degradation kinetics of metronidazole in solution
Degradation Kinetics of Metronidazole in Solution DA-PENGWANG', AND MING-KUNGYEH Received December 26, 1990, from Tri-Service General Hospital, Department of Clinical Pharmacy, National Defense Medical Center, School of Pharmacy, Taipei, Taiwan, Republic of China. Abstract 0 The degradation kinetics of metronidazole in aqueous solutions of pH 3.1 to 9.9 under accelerated storage conditions were studied. The stability of metronidazole in solutions containing propylene glycol or polyethylene glycol 400 was also investigated. The reaction order for metronidazole in these aqueous and solvent systems followed pseudo-first-order degradation kinetics. The degradation rate of metronidazole was invariant under various total buffer concentrations at each specific pH within the investigated pH range. These results indicate that no general acidibase catalysis imposed by acetate, phosphate, and borate buffer species is responsible for the degradation of metronidazole. The catalytic rate constants for hydrogen ion, water, and hydroxyl ion for the degradation of metronidazole were 6.1 1 x M/s, 3.54 x 1O-' Us, and 4.10 x M/s, respectively. The pH-rate profile shows a pH-independent region of pH 3.9-6.6. Maximum stability of metronidazole was at pH 5.6 under zero total buffer species conditions. The ionic strength effect on metronidazole degradation in acetate and phosphate buffers followed the modified Debye-Huckel equation well. The Arrhenius plot showing the temperature dependence of metronidazole degradation indicates estimates of activation energy of 15.35 kcal/mol and a half-life of 963 h at room temperature in 0.1 M pH 3.1 acetate buffer solution (ionic strength = 0.5). Irradiationwith UV light (254 nm) of the metronidazole solutions (pH 3.1 acetate buffer) accelerated degradation in comparison with light-protectedsamples. Incorporation of propylene glycol into the metronidazole solution at pH 3.1 increased stability; however, an adverse effect on the stability of metronidazole was seen when polyethylene glycol 400 solvent system was used.
Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) is a synthetic antibacterial agent that i s used primarily in t h e treatment o f various anaerobic infections,l such as intraabdominal infections, s k i n and s k i n structure infections, gynecologic infections, bacterial septicemia, bone and j o i n t infections, central nervous system infections, lower respiratory tract infections, and endocarditis. Metronidazole was reported t o undergo hydrolysis in aqueous media due t o t h e presence of photolytically generated hydroxyl radicals.2 Light irradiation has more effect o n t h e degradation of metronidazole in solution than irradiation w i t h sonic energy.3 The objective o f t h i s investigation was t o study t h e degradation kinetics of metronidazole under various storage conditions such as pH, t o t a l buffer concentration, ionic strength ( p ) , temperature, light exposure, and cosolvent system.
Experimental Section Material-Metronidazole was obtained from Kingdom Pharmaceutical Company (Taipei, Taiwan, R.O.C.). High-performance liquid chromatography (HPLC) grade acetonitrile was used for all chromatographic determinations. The following analytical grade materials were of NF quality and used as received: propylene glycol, polyethylene glycol 400, ammonium carbonate, acetic acid, sodium acetate, potassium chloride, potassium phosphate monobasic, potassium phosphate dibasic, phosphoric acid, sodium hydroxide, boric acid, and sodium borate. Phenacetin was used as an internal standard and was obtained from Kingdom Pharmaceutical. Kinetic Studies-Eleven buffer solutions of varying buffer species with the pH ranges of 3.1to 9.9 (pH 3.1-4.9 acetatebuffer, pH 5.4-8.0 phosphate buffer, and pH 9.1-9.9 borate buffer) were prepared at the
0022-3549/93/0 100-0095$02.50/0 0 1993, American Pharmaceutical Association
c i p r2 I
0 ~ ~
Metronldazole
constant total buffer species concentration of 0.1 M and p of 0.5. Constant p was maintained by adjusting the amount of potassium chloride in solution. Sample solutions of metronidazole at 0.04 mg/mL were prepared by dissolving a specific amount of drug into a suitable amount of buffer and adjusted to volume with the same buffer solution. All sample solutions were sealed into 2-mL Type I flint glass ampules (Wheaton Scientific, Millville, NJ) and stored in a dark oven at 90 f 0.2"C for up to 85 h. At designated storage intervals, samples were removed from storage and immediately stored in a -20°C freezer until analysis. At the time of analysis, samples were removed from the freezer, thawed at room temperature, and mixed well before injecting onto an HPLC column. The concentration of metronidazole at each storage time interval was determined by a stabilityindicating reversed-phase HPLC method. The pH values were measured (model 6071, Jenco, San Diego, CA) for each sample at each storage condition to assure no appreciable pH change. Triplicate samples were used for each storage condition in this study. HPLC Analysis-The HPLC system was composed of a single piston pump (model 501, Waters Associates, Millipore Corporation, Milford, MA), a loop injector (model 7125, Rheodyne Incorporated, Cotati, CA), a fixed wavelength W absorption detector (model 441, Millipore) set at 254 nm, and a C,, column (NOVA-PAK, Millipore). The mobile phase was a 3070 mixture (viv) of acetonitrile and 0.01% ammonium carbonate at pH 8.0. The mobile phase was delivered at a constant rate of 0.9 mL/min. The absorbance of metronidazole and its internal standard, phenacetin (0.04 mg/mL), were recorded on an electronic integrator (Waters Modul745 Data Model, Millipore) at a speed of 1.0 cm/min. The concentration of metronidazole was determined by a method of peak area ratio when comparing the peak area ratio (drughnternal standard) of sample with standard solutions from the calibration curve. A representative chromatogram showing the stability-indicating nature of the developed HPLC method was demonstrated by forcibly degrading a 0.04-mg/mL metronidazole solution (pH 3.1, 0.1 M acetate buffer) at 90 f 0.2"C for 96 h (Figure 1).The peak of metronidazole decreased after the accelerated storage condition, but without apparent interference from the degradates. The retention times of metronidazole and phenacetin in the HPLC system were 4.5 and 8.8 min, respectively. Similar chromatograms were obtained with other solutions at neutral and basic media under stressed conditions. The homogeneity of the metronidazole peak was examined by collecting the elected metronidazole peak from several injections and performing a diode array spectral overlay analysis in the W range 191.0-401.0 nm (model SPD-MGA, Diode Array Spectrophotometer, Shimazu Corporation, Tokyo, Japan). No difference (curve fit > 0.99) in the U V spectra between the eluted and pure drug samples suggests the absence of any degradate or exogenous impurities eluting under the peak of interest. The linearity of the calibration curve of peak area ratio versus metronidazole concentration in the range 0.01-0.06 mg/mL showed an excellent correlation coefficient (r) of 0.998 (y = 2 3 . 9 5 ~+ 0.04). The intra- and interday precision (n = 5) of this HPLC method at the metronidazole concentration range 0.01-0.06 mg/mL were determined to have coefficients Journal of Pharmaceutical Sciences / 95 Vof. 82, No. 1, January 1993
A 1
4'700
h
i
~
2
d
-4
c
B
1
2
4.200
0
Figure 1-HPLC chromatogram of metronidazole (0.04mglmL) in pH 3.1,0.1 M acetate buffer solution (A) immediately after preparation and (B) after 4 days of storage at 90 2 0.2"C.Key: (1) metronidazole; (2) phenacetin; (3) degradation products.
of variation of 0.89 ? 0.46% and 1.16 2 0.48%, respectively. The sensitivity of the reported procedures was 50 ng/mL. The stability of metronidazole at each designated storage time interval in this study was expressed as a percentage of its initial concentration (100%at time zero). Buffer Effect Studies-At each pH buffer over the pH range 3.1-9.9, three buffer solutions (pH 3.1-4.9 acetate buffer, pH 5.4-8.0 phosphate buffer, and pH 9.1-9.9 borate buffer) of 0.05, 0.1, and 0.2 M total buffer concentration with constant p of 0.5 were prepared to study the catalytic effect of buffer species on the degradation of metronidazole (0.04 mg/mL) in aqueous solution. This study was done at 90 f 0.2 "C. Ionic Strength Effect Studies-Solutions of pH 3.1 acetate buffer and pH 7.4 phosphate buffer containing metronidazole at 0.04 mg/mL were prepared. While maintaining the pH, drug concentration, and total buffer concentration (0.1 M) constant, solutions with various p (0.1-0.7 for pH 3.1 acetate buffer and 0.3-0.9 for pH 7.4 phosphate buffer) were investigated for the salt effect on the stability of metronidazole at 90 t 0.2 "C. UV Photolysis Effect Studies-Solutions of 0.1 M pH 3.1 acetate buffer containing metronidazole at 0.04 mg/mL were prepared at constant p of 0.5. The solutions were sealed into 2-mL Type I flint glass ampules (Wheaton Scientific, Millville, NJ). Half of the solutions were wrapped with aluminum foil to protect the solutions from light and were used as the control group. Both wrapped and unwrapped solutions were placed under a UV light at a distance of 30 cm. The wavelength of the U V light was 254 nm and its intensity was controlled at 150 pW/cm. This stability study was conducted at 25 2 0.2 "C for up to 50 days. Thermal Effect Studies-Solutions of 0.1 M, pH 3.1 acetate buffer containing metronidazole at 0.04 mg/mL were prepared at constant p of 0.5. The stability of these solutions was investigated at 50 f 0.2, 60 f 0.2, 70 f 0.2, 80 2 0.2, and 90 f 0.2"C. Solvent Effect Studies-Various propylene glyco1:water and polyethylene glycol 400:water system with different ratios (10:90, 30:70, 50:50, 70:30)were prepared at a metronidazole concentration of 0.04 mg/mL. All solutions were buffered to a pH of 3.1 with acetate buffer species. The stability of these solutions was investigated at a constant temperature of 90 % 0.2 "C.
I
40
60
100
80
Time (hrs)
Buffer Species Effect-The effect of general acidbase catalysis caused by acetate, phosphate, and borate buffers on the degradation kinetics of metronidazole was studied by varying the total buffer species concentration in solutions while maintaining the pH, p (0.5),and storage temperature (90 f 0.2 "C)constant. No significant difference was observed in the effects of acetate, phosphate, and borate species on the degradation rate constant of metronidazole under three different total buffer concentrations (0.05,0.1, and 0.2 M)of the same buffer species a t each designated pH solution in the range pH 3.1-9.9 (Table I). These results indicate that no effect of general acidbase catalysis of acetate, phosphate, and borate species on the metronidazole degradation in aqueous solution was observed and only specific acidbase catalysis occurred in this study. Degradation rate constants of metronidazole at zero buffer concentration under each designated pH were determined by extrapolating the linear regression line from the rate constants of three buffer concentrations to zero buffer concentration. pH-Rate Profile-The effect of pH on the degradation of metronidazole in aqueous solution under zero buffer concentration and constant p (0.5) at 90 f 0.2 "C is shown in plots of log Kobs(overall observed rate constant) versus pH (Figure 3). In the pH range 3.9-6.6 under these study conditions, metronidazole was more stable than in other pH regions. In this pH-independent region, the degradation of metronidazole was mainly due to the effect of water catalysis at pH 3.1-3.9. The degradation of metronidazole is described by the catalytic effect of specific acid and water; however, the specific base catalysis becomes more predominant as the pH goes up Table I-Degradation Rate Constants of Metronidaroie In Dlfferent Buffer Species Concentrations at Various pHs wlth Constant p (0.5) and Storage Temperature (90 2 0.2 "C)
k&
Results and Discussion
90 I Journal of Pharmaceutical Sciences Vof.82, No. 1, January 1993
\
Figure 2-Pseudo-first-order degradation kinetics of metronidazole in various buffer solutions (0.1 M) of different pHs at 90 ? 0.2 "C and p = 0.5. Key: (0)pH 3.1;(0) pH 4.4;(A) pH 9.1.
PH
Degradation Kinetics-Representative sets of stability profiles for metronidazole in different pH buffer solutions at 90 ? 0.2 "C are shown in Figure 2. The linear relationship between logarithmic percent remaining and storage time indicates a pseudo-first-order degradation kinetics for metronidazole in aqueous solution. The degradation rate constant was determined from the slope of the graph by a statistical regression analysis method. For all the pH buffers studied for metronidazole degradation, the regression lines were linear with r > 0.98.
20
3.1 3.3 3.9 4.4 4.9 5.4 5.9 6.6 8.0 9.1 9.9
-
x 10 (Us) at Buffer Concentration of:
0.05M
0.1 M
0.2M
0.98 0.72 0.38 0.16 0.19 0.29 0.41 0.45 0.90 4.36 10.04
1.04 0.81 0.27 0.18 0.21 0.23 0.50 0.56 0.98 4.46 10.32
1.01 0.74 0.28 0.15 0.20 0.27 0.38 0.55 0.90 5.64 11.32
-
-4.600r
1614-
/
I
A
12-
10W 1 0
d
8 -6 --
I
4 --
2
/
--
0 -r
-5.000 4 0.GOG
-2 0
2
4
6
&
10
12
14
Figure &The pH-rate profile of metronidazole in aqueous solution under zero buffer concentration and constant p (0.5)at 90 2 0.2 "C.The line shows the curve fit from eq 1.
from 6.6 to 9.9. Equation 1 is a general equation describing the degradation rate of metronidazole as a function of pH:
In eq 1, Kobsis the overall observed rate constant, KOis the water catalysis rate constant, KH+is the specific acid catalysis rate constant, KOH- is the specific base catalysis rate constant, and m and n are the orders of reaction with respect to [H+l and [OH-], respectively. Variables in this rate equation (eq 1)were determined by fitting the rate constants under zero total buffer concentration to the equation. The values of KH+ and KOH-were obtained from the intercepts of log Kobaversus pH/pOH plots in the low (pH c 3.9) and high (pH > 8.0) regions, respectively (KH+= 6.11 x M/s and KoH- = 4.10 x lop3M/s).The values of m and n were determined to be 0.57 and 0.61, respectively, from the slopes of the same plots. The nonintegral orders of m and n might imply the existence of some intermediate formations during the acidbase catalytic degradations sequences of metronidazole. The rate constant for the water catalysis (k,) was determined to be 3.54 x lo-' l/s from the intersections of the above two regression lines. The most stable pH based on this calculation was 5.6.The higher catalytic rate constant for hydroxyl ion than hydrogen ion on the degradation metronidazole indicates the faster degradation rate of metronidazole in alkaline regions than an acidic environment, which also explains the deeper slope in the high pH compared with low pH regions. The simulation pH-rate profile based on eq 1 in comparison with observed rate constants in the pH range 3.1-9.9 is also shown in Figure 3. Salt Effect-The modified Debye-Huckel equation4 is usually used in solutions with higher values of p (>0.01): log k = a + ~QZAZB [ 6 / ( 1 +6
1 1
0.200
0.43G
4
0.600
0.600
1.0'20
Figure 4-Solution salt effect on the degradation kinetics of metronidazole (0.04 mglrnl) in 0.1 M buffer solutions at 90 2 0.2 "C. Key: (0)pH 3.1 acetate buffer; (0)pH 7.4 phosphate buffer.
both test solutions of pH 3.1 (r = 0.9895) and pH 7.4 (r = 0.9616) follows the modified Debye-Huckel equation well, and the !@ZAZBvalue can be obtained from the slopes of these plots; they are 0.99 for the pH 3.1 acetate buffer and 0.34 for the pH 7.4 phosphate buffer. The decrease of the 2QZAZB value as the solution pH increases from 3.1 to 7.4 is due to the reduced fraction of ionized metronidazole and hydrogen ion concentration. The ideal degradation rate constant for metronidazole at zero p (infinite dilution) and 90 0.2 "C was estimated to be 0.600 L/s for pH 3.1 acetate buffer and 1.128 L/s for pH 7.4 phosphate buffer. Thermal Effect-The temperature dependence of metronidazole degradation at pH 3.1,O.l M acetate buffer solution ( p = 0.5) was studied at the temperature range 50-90 "C. An Arrhenius plot of log rate versus reciprocal of temperature (in K), as shown in Figure 5 , depicts the results of this investigation. The linearity (r = 0.9978) of the regression line in this plot shows a good indication of invariant activation energy for degradation of metronidazole in the temperature range 5090 "C. The activation energy for degradation was determined to be 15.35 kcdmol from the slope of this plot. If the activation energy for degradation remains constant in the temperature range 25-90 "C, the degradation rate constant of metronidazole in the acetate buffer at room temperature can be estimated to be 2.00 x L/s, which gives an estimation of half-life of 963 h. UV Light Effect-The stability of metronidazole under UV irradiation is depicted in Table 11. No determination of degradation order was made because there wasn't enough degradation in a period of 50 days for both light-exposed and
*
-4.700
Px
(2)
In eq 2, k is the overall reaction rate constant, a is the reaction rate constant at infinite dilution of a given solvent, 2Q is the constant for a given solvent and temperature, and Z,Z, is the charge on reactants A and B. To maintain a constant pH environment in the study of metronidazole degradation, higher concentration of buffering agents (0.1 M)were used. Values of p of 0.1-0.7 and 0.3-0.9 were employed for pH 3.1 and pH 7.4 solutions, respectively. A plot of log k versus (
3
-5.700
-6.200
I 4
2.700
2.800
2.900
3.000
3.100
3.200
1/T ( * 1 0 0 0 ) Figure S A r r h e n i u s plot of the degradation of metronidazole (0.04 mg/mL) in pH 3.1 acetate buffer (0.1 M) solution and at constant p (0.5). Journal of Pharmaceutical Sciences 1 97 Vol. 82, No. 1. January 1993
Table 11-Effect of UV Light on the Stability of Metronldarole In pH 3.1 Acetate Buffer Solution (0.1 M) at p of 0.5 and Temperature of 25 f 0.2 "C' Time (days)
% Remaining
Light-protected
Lig ht-exposed
Table Ill-Stability of Metronidazole in Propyiene G1ycol:Water or Polyethylene Glycol 400:Water Solvent Systems at pH 3.1 and 90 f 0.2
"C
qobs x 10 (Us) in:
Solvent Ratio
PGa:Water
PEG 400b:Water
~~
5 10
20 30 50
99.5 f 0.2 99.4 f 0.3 98.8 rl: 0.2 98.4 rl: 0.1 98.3 f 0.2
96.3 f 0.2 94.8 0.3 93.4 f 0.2 92.0 rl: 0.3 88.7 0.2
10:90 30:70 5050 70:30 ~
a a
Values are expressed as mean
2
standard deviation (n
~~
16.37 31.86 37.24 ~
Propylene glycol. Polyethylene glycol 400.
= 5).
light-protected samples. However, W irradiation did accelerate the degradation processes of metronidazole in the light-exposed samples in comparison with light-protected ones under otherwise identical experimental conditions. Solvent Effect-The effects of propylene glycol and polyethylene glycol 400 on the stability of metronidazole in pH 3.1 acetate buffer solutions at 90 5 0.2 "C are listed in Table 111. The degradation of metronidazole in these solvent systems followed pseudo-first-order kinetics. The degradation rate constant of metronidazole in the propylene glyco1:water system decreased as the content of propylene glycol increased. In contrast, the degradation rate constant of metronidazole in the polyethylene glycol 400:water system increased as the
98 J Journal of Pharmaceutical Sciences Vol. 82, No. 1, January 1993
~
15.72
10.82 8.43 7.03 6.81
polyethylene glycol 400 content increased. No explanation is proposed for the degradation mechanism of metronidazole in these solvent systems because of complicated factors, such as dielectric constant, surface tension, viscosity, activity coefficient of metronidazole, its transition products, etc.
References and Notes 1. Brogden, R. N.; Heel, R. C.; Speight, T. M.; Avery, G. S. Drugs 1978, 16, 387. 2. Barnes, A. R.; Sugden, J. K. Pharm. Acta Helv. 1986, 61, 218. 3. Kendall, A. T.; Stark, E.; Sugden, J. K. Int. J. Pharm. 1989, 57, 217. 4 . Carstensen, J. T. J. Pharm. Sci. 1970, 59, 1140. 5. Kaiho, F.; Nomura, H.; Makabe, E.; &to, Y. Chem. Pharm. Bull. 1987,35,2928.
Metronidazole (2-methyl-5-nitroimidazole-1-ethanol) is a synthetic antibacterial agent that i s used primarily in t h e treatment o f various anaerobic infections,l such as intraabdominal infections, s k i n and s k i n structure infections, gynecologic infections, bacterial septicemia, bone and j o i n t infections, central nervous system infections, lower respiratory tract infections, and endocarditis. Metronidazole was reported t o undergo hydrolysis in aqueous media due t o t h e presence of photolytically generated hydroxyl radicals.2 Light irradiation has more effect o n t h e degradation of metronidazole in solution than irradiation w i t h sonic energy.3 The objective o f t h i s investigation was t o study t h e degradation kinetics of metronidazole under various storage conditions such as pH, t o t a l buffer concentration, ionic strength ( p ) , temperature, light exposure, and cosolvent system.
Experimental Section Material-Metronidazole was obtained from Kingdom Pharmaceutical Company (Taipei, Taiwan, R.O.C.). High-performance liquid chromatography (HPLC) grade acetonitrile was used for all chromatographic determinations. The following analytical grade materials were of NF quality and used as received: propylene glycol, polyethylene glycol 400, ammonium carbonate, acetic acid, sodium acetate, potassium chloride, potassium phosphate monobasic, potassium phosphate dibasic, phosphoric acid, sodium hydroxide, boric acid, and sodium borate. Phenacetin was used as an internal standard and was obtained from Kingdom Pharmaceutical. Kinetic Studies-Eleven buffer solutions of varying buffer species with the pH ranges of 3.1to 9.9 (pH 3.1-4.9 acetatebuffer, pH 5.4-8.0 phosphate buffer, and pH 9.1-9.9 borate buffer) were prepared at the
0022-3549/93/0 100-0095$02.50/0 0 1993, American Pharmaceutical Association
c i p r2 I
0 ~ ~
Metronldazole
constant total buffer species concentration of 0.1 M and p of 0.5. Constant p was maintained by adjusting the amount of potassium chloride in solution. Sample solutions of metronidazole at 0.04 mg/mL were prepared by dissolving a specific amount of drug into a suitable amount of buffer and adjusted to volume with the same buffer solution. All sample solutions were sealed into 2-mL Type I flint glass ampules (Wheaton Scientific, Millville, NJ) and stored in a dark oven at 90 f 0.2"C for up to 85 h. At designated storage intervals, samples were removed from storage and immediately stored in a -20°C freezer until analysis. At the time of analysis, samples were removed from the freezer, thawed at room temperature, and mixed well before injecting onto an HPLC column. The concentration of metronidazole at each storage time interval was determined by a stabilityindicating reversed-phase HPLC method. The pH values were measured (model 6071, Jenco, San Diego, CA) for each sample at each storage condition to assure no appreciable pH change. Triplicate samples were used for each storage condition in this study. HPLC Analysis-The HPLC system was composed of a single piston pump (model 501, Waters Associates, Millipore Corporation, Milford, MA), a loop injector (model 7125, Rheodyne Incorporated, Cotati, CA), a fixed wavelength W absorption detector (model 441, Millipore) set at 254 nm, and a C,, column (NOVA-PAK, Millipore). The mobile phase was a 3070 mixture (viv) of acetonitrile and 0.01% ammonium carbonate at pH 8.0. The mobile phase was delivered at a constant rate of 0.9 mL/min. The absorbance of metronidazole and its internal standard, phenacetin (0.04 mg/mL), were recorded on an electronic integrator (Waters Modul745 Data Model, Millipore) at a speed of 1.0 cm/min. The concentration of metronidazole was determined by a method of peak area ratio when comparing the peak area ratio (drughnternal standard) of sample with standard solutions from the calibration curve. A representative chromatogram showing the stability-indicating nature of the developed HPLC method was demonstrated by forcibly degrading a 0.04-mg/mL metronidazole solution (pH 3.1, 0.1 M acetate buffer) at 90 f 0.2"C for 96 h (Figure 1).The peak of metronidazole decreased after the accelerated storage condition, but without apparent interference from the degradates. The retention times of metronidazole and phenacetin in the HPLC system were 4.5 and 8.8 min, respectively. Similar chromatograms were obtained with other solutions at neutral and basic media under stressed conditions. The homogeneity of the metronidazole peak was examined by collecting the elected metronidazole peak from several injections and performing a diode array spectral overlay analysis in the W range 191.0-401.0 nm (model SPD-MGA, Diode Array Spectrophotometer, Shimazu Corporation, Tokyo, Japan). No difference (curve fit > 0.99) in the U V spectra between the eluted and pure drug samples suggests the absence of any degradate or exogenous impurities eluting under the peak of interest. The linearity of the calibration curve of peak area ratio versus metronidazole concentration in the range 0.01-0.06 mg/mL showed an excellent correlation coefficient (r) of 0.998 (y = 2 3 . 9 5 ~+ 0.04). The intra- and interday precision (n = 5) of this HPLC method at the metronidazole concentration range 0.01-0.06 mg/mL were determined to have coefficients Journal of Pharmaceutical Sciences / 95 Vof. 82, No. 1, January 1993
A 1
4'700
h
i
~
2
d
-4
c
B
1
2
4.200
0
Figure 1-HPLC chromatogram of metronidazole (0.04mglmL) in pH 3.1,0.1 M acetate buffer solution (A) immediately after preparation and (B) after 4 days of storage at 90 2 0.2"C.Key: (1) metronidazole; (2) phenacetin; (3) degradation products.
of variation of 0.89 ? 0.46% and 1.16 2 0.48%, respectively. The sensitivity of the reported procedures was 50 ng/mL. The stability of metronidazole at each designated storage time interval in this study was expressed as a percentage of its initial concentration (100%at time zero). Buffer Effect Studies-At each pH buffer over the pH range 3.1-9.9, three buffer solutions (pH 3.1-4.9 acetate buffer, pH 5.4-8.0 phosphate buffer, and pH 9.1-9.9 borate buffer) of 0.05, 0.1, and 0.2 M total buffer concentration with constant p of 0.5 were prepared to study the catalytic effect of buffer species on the degradation of metronidazole (0.04 mg/mL) in aqueous solution. This study was done at 90 f 0.2 "C. Ionic Strength Effect Studies-Solutions of pH 3.1 acetate buffer and pH 7.4 phosphate buffer containing metronidazole at 0.04 mg/mL were prepared. While maintaining the pH, drug concentration, and total buffer concentration (0.1 M) constant, solutions with various p (0.1-0.7 for pH 3.1 acetate buffer and 0.3-0.9 for pH 7.4 phosphate buffer) were investigated for the salt effect on the stability of metronidazole at 90 t 0.2 "C. UV Photolysis Effect Studies-Solutions of 0.1 M pH 3.1 acetate buffer containing metronidazole at 0.04 mg/mL were prepared at constant p of 0.5. The solutions were sealed into 2-mL Type I flint glass ampules (Wheaton Scientific, Millville, NJ). Half of the solutions were wrapped with aluminum foil to protect the solutions from light and were used as the control group. Both wrapped and unwrapped solutions were placed under a UV light at a distance of 30 cm. The wavelength of the U V light was 254 nm and its intensity was controlled at 150 pW/cm. This stability study was conducted at 25 2 0.2 "C for up to 50 days. Thermal Effect Studies-Solutions of 0.1 M, pH 3.1 acetate buffer containing metronidazole at 0.04 mg/mL were prepared at constant p of 0.5. The stability of these solutions was investigated at 50 f 0.2, 60 f 0.2, 70 f 0.2, 80 2 0.2, and 90 f 0.2"C. Solvent Effect Studies-Various propylene glyco1:water and polyethylene glycol 400:water system with different ratios (10:90, 30:70, 50:50, 70:30)were prepared at a metronidazole concentration of 0.04 mg/mL. All solutions were buffered to a pH of 3.1 with acetate buffer species. The stability of these solutions was investigated at a constant temperature of 90 % 0.2 "C.
I
40
60
100
80
Time (hrs)
Buffer Species Effect-The effect of general acidbase catalysis caused by acetate, phosphate, and borate buffers on the degradation kinetics of metronidazole was studied by varying the total buffer species concentration in solutions while maintaining the pH, p (0.5),and storage temperature (90 f 0.2 "C)constant. No significant difference was observed in the effects of acetate, phosphate, and borate species on the degradation rate constant of metronidazole under three different total buffer concentrations (0.05,0.1, and 0.2 M)of the same buffer species a t each designated pH solution in the range pH 3.1-9.9 (Table I). These results indicate that no effect of general acidbase catalysis of acetate, phosphate, and borate species on the metronidazole degradation in aqueous solution was observed and only specific acidbase catalysis occurred in this study. Degradation rate constants of metronidazole at zero buffer concentration under each designated pH were determined by extrapolating the linear regression line from the rate constants of three buffer concentrations to zero buffer concentration. pH-Rate Profile-The effect of pH on the degradation of metronidazole in aqueous solution under zero buffer concentration and constant p (0.5) at 90 f 0.2 "C is shown in plots of log Kobs(overall observed rate constant) versus pH (Figure 3). In the pH range 3.9-6.6 under these study conditions, metronidazole was more stable than in other pH regions. In this pH-independent region, the degradation of metronidazole was mainly due to the effect of water catalysis at pH 3.1-3.9. The degradation of metronidazole is described by the catalytic effect of specific acid and water; however, the specific base catalysis becomes more predominant as the pH goes up Table I-Degradation Rate Constants of Metronidaroie In Dlfferent Buffer Species Concentrations at Various pHs wlth Constant p (0.5) and Storage Temperature (90 2 0.2 "C)
k&
Results and Discussion
90 I Journal of Pharmaceutical Sciences Vof.82, No. 1, January 1993
\
Figure 2-Pseudo-first-order degradation kinetics of metronidazole in various buffer solutions (0.1 M) of different pHs at 90 ? 0.2 "C and p = 0.5. Key: (0)pH 3.1;(0) pH 4.4;(A) pH 9.1.
PH
Degradation Kinetics-Representative sets of stability profiles for metronidazole in different pH buffer solutions at 90 ? 0.2 "C are shown in Figure 2. The linear relationship between logarithmic percent remaining and storage time indicates a pseudo-first-order degradation kinetics for metronidazole in aqueous solution. The degradation rate constant was determined from the slope of the graph by a statistical regression analysis method. For all the pH buffers studied for metronidazole degradation, the regression lines were linear with r > 0.98.
20
3.1 3.3 3.9 4.4 4.9 5.4 5.9 6.6 8.0 9.1 9.9
-
x 10 (Us) at Buffer Concentration of:
0.05M
0.1 M
0.2M
0.98 0.72 0.38 0.16 0.19 0.29 0.41 0.45 0.90 4.36 10.04
1.04 0.81 0.27 0.18 0.21 0.23 0.50 0.56 0.98 4.46 10.32
1.01 0.74 0.28 0.15 0.20 0.27 0.38 0.55 0.90 5.64 11.32
-
-4.600r
1614-
/
I
A
12-
10W 1 0
d
8 -6 --
I
4 --
2
/
--
0 -r
-5.000 4 0.GOG
-2 0
2
4
6
&
10
12
14
Figure &The pH-rate profile of metronidazole in aqueous solution under zero buffer concentration and constant p (0.5)at 90 2 0.2 "C.The line shows the curve fit from eq 1.
from 6.6 to 9.9. Equation 1 is a general equation describing the degradation rate of metronidazole as a function of pH:
In eq 1, Kobsis the overall observed rate constant, KOis the water catalysis rate constant, KH+is the specific acid catalysis rate constant, KOH- is the specific base catalysis rate constant, and m and n are the orders of reaction with respect to [H+l and [OH-], respectively. Variables in this rate equation (eq 1)were determined by fitting the rate constants under zero total buffer concentration to the equation. The values of KH+ and KOH-were obtained from the intercepts of log Kobaversus pH/pOH plots in the low (pH c 3.9) and high (pH > 8.0) regions, respectively (KH+= 6.11 x M/s and KoH- = 4.10 x lop3M/s).The values of m and n were determined to be 0.57 and 0.61, respectively, from the slopes of the same plots. The nonintegral orders of m and n might imply the existence of some intermediate formations during the acidbase catalytic degradations sequences of metronidazole. The rate constant for the water catalysis (k,) was determined to be 3.54 x lo-' l/s from the intersections of the above two regression lines. The most stable pH based on this calculation was 5.6.The higher catalytic rate constant for hydroxyl ion than hydrogen ion on the degradation metronidazole indicates the faster degradation rate of metronidazole in alkaline regions than an acidic environment, which also explains the deeper slope in the high pH compared with low pH regions. The simulation pH-rate profile based on eq 1 in comparison with observed rate constants in the pH range 3.1-9.9 is also shown in Figure 3. Salt Effect-The modified Debye-Huckel equation4 is usually used in solutions with higher values of p (>0.01): log k = a + ~QZAZB [ 6 / ( 1 +6
1 1
0.200
0.43G
4
0.600
0.600
1.0'20
Figure 4-Solution salt effect on the degradation kinetics of metronidazole (0.04 mglrnl) in 0.1 M buffer solutions at 90 2 0.2 "C. Key: (0)pH 3.1 acetate buffer; (0)pH 7.4 phosphate buffer.
both test solutions of pH 3.1 (r = 0.9895) and pH 7.4 (r = 0.9616) follows the modified Debye-Huckel equation well, and the !@ZAZBvalue can be obtained from the slopes of these plots; they are 0.99 for the pH 3.1 acetate buffer and 0.34 for the pH 7.4 phosphate buffer. The decrease of the 2QZAZB value as the solution pH increases from 3.1 to 7.4 is due to the reduced fraction of ionized metronidazole and hydrogen ion concentration. The ideal degradation rate constant for metronidazole at zero p (infinite dilution) and 90 0.2 "C was estimated to be 0.600 L/s for pH 3.1 acetate buffer and 1.128 L/s for pH 7.4 phosphate buffer. Thermal Effect-The temperature dependence of metronidazole degradation at pH 3.1,O.l M acetate buffer solution ( p = 0.5) was studied at the temperature range 50-90 "C. An Arrhenius plot of log rate versus reciprocal of temperature (in K), as shown in Figure 5 , depicts the results of this investigation. The linearity (r = 0.9978) of the regression line in this plot shows a good indication of invariant activation energy for degradation of metronidazole in the temperature range 5090 "C. The activation energy for degradation was determined to be 15.35 kcdmol from the slope of this plot. If the activation energy for degradation remains constant in the temperature range 25-90 "C, the degradation rate constant of metronidazole in the acetate buffer at room temperature can be estimated to be 2.00 x L/s, which gives an estimation of half-life of 963 h. UV Light Effect-The stability of metronidazole under UV irradiation is depicted in Table 11. No determination of degradation order was made because there wasn't enough degradation in a period of 50 days for both light-exposed and
*
-4.700
Px
(2)
In eq 2, k is the overall reaction rate constant, a is the reaction rate constant at infinite dilution of a given solvent, 2Q is the constant for a given solvent and temperature, and Z,Z, is the charge on reactants A and B. To maintain a constant pH environment in the study of metronidazole degradation, higher concentration of buffering agents (0.1 M)were used. Values of p of 0.1-0.7 and 0.3-0.9 were employed for pH 3.1 and pH 7.4 solutions, respectively. A plot of log k versus (
3
-5.700
-6.200
I 4
2.700
2.800
2.900
3.000
3.100
3.200
1/T ( * 1 0 0 0 ) Figure S A r r h e n i u s plot of the degradation of metronidazole (0.04 mg/mL) in pH 3.1 acetate buffer (0.1 M) solution and at constant p (0.5). Journal of Pharmaceutical Sciences 1 97 Vol. 82, No. 1. January 1993
Table 11-Effect of UV Light on the Stability of Metronldarole In pH 3.1 Acetate Buffer Solution (0.1 M) at p of 0.5 and Temperature of 25 f 0.2 "C' Time (days)
% Remaining
Light-protected
Lig ht-exposed
Table Ill-Stability of Metronidazole in Propyiene G1ycol:Water or Polyethylene Glycol 400:Water Solvent Systems at pH 3.1 and 90 f 0.2
"C
qobs x 10 (Us) in:
Solvent Ratio
PGa:Water
PEG 400b:Water
~~
5 10
20 30 50
99.5 f 0.2 99.4 f 0.3 98.8 rl: 0.2 98.4 rl: 0.1 98.3 f 0.2
96.3 f 0.2 94.8 0.3 93.4 f 0.2 92.0 rl: 0.3 88.7 0.2
10:90 30:70 5050 70:30 ~
a a
Values are expressed as mean
2
standard deviation (n
~~
16.37 31.86 37.24 ~
Propylene glycol. Polyethylene glycol 400.
= 5).
light-protected samples. However, W irradiation did accelerate the degradation processes of metronidazole in the light-exposed samples in comparison with light-protected ones under otherwise identical experimental conditions. Solvent Effect-The effects of propylene glycol and polyethylene glycol 400 on the stability of metronidazole in pH 3.1 acetate buffer solutions at 90 5 0.2 "C are listed in Table 111. The degradation of metronidazole in these solvent systems followed pseudo-first-order kinetics. The degradation rate constant of metronidazole in the propylene glyco1:water system decreased as the content of propylene glycol increased. In contrast, the degradation rate constant of metronidazole in the polyethylene glycol 400:water system increased as the
98 J Journal of Pharmaceutical Sciences Vol. 82, No. 1, January 1993
~
15.72
10.82 8.43 7.03 6.81
polyethylene glycol 400 content increased. No explanation is proposed for the degradation mechanism of metronidazole in these solvent systems because of complicated factors, such as dielectric constant, surface tension, viscosity, activity coefficient of metronidazole, its transition products, etc.
References and Notes 1. Brogden, R. N.; Heel, R. C.; Speight, T. M.; Avery, G. S. Drugs 1978, 16, 387. 2. Barnes, A. R.; Sugden, J. K. Pharm. Acta Helv. 1986, 61, 218. 3. Kendall, A. T.; Stark, E.; Sugden, J. K. Int. J. Pharm. 1989, 57, 217. 4 . Carstensen, J. T. J. Pharm. Sci. 1970, 59, 1140. 5. Kaiho, F.; Nomura, H.; Makabe, E.; &to, Y. Chem. Pharm. Bull. 1987,35,2928.