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Influence of pH, temperature and buffers on cefepime degradation kinetics and stability predictions in aqueous solutions
Influence of pH, Temperature and Buffers on Cefepime Degradation Kinetics and Stability Predictions in Aqueous Solutions JOSEPHINE O. FUBARA
AND
ROBERT E. NOTARI*
Contribution from The Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. Received April 17, 1998. Final revised manuscript received September 1, 1998. Accepted for publication September 1, 1998. Abstract 0 First-order rate constants (k) were determined for cefepime degradation at 45, 55, 65, and 75 °C, pH 0.5 to 8.6, using an HPLC assay. Each pH−rate profile exhibited an inflection between pH 1 and 2. The pH−rate expression was k(pH) ) kH1 f1(aH+) + kH2 f2(aH+) + kS + kOH(aOH−), where kH1 and kH2 are the catalytic constants (M-1 h-1) for hydrogen ion activity (aH+), kOH is the catalytic constant for hydroxyl ion activity (aOH−), and kS is the first-order rate constant (h-1) for spontaneous degradation. The protonated (f1 ) and unprotonated (f2 ) fractions were calculated from the dissociation constant, Ka ) (8.32 × 10-6 )e(5295)/RT where T was absolute temperature (T). Accelerated loss due to formate, acetate, phosphate, and borate buffer catalysis was quantitatively described with the catalytic constant, kGA (M-1 h-1) for the acidic component, [GA], and kGB (M-1 h-1) for the basic component, [GB], of each buffer. The temperature dependency for each rate constant was defined with experimentally determined values for A and E and the Arrhenius expression, kT ) Ae-E/RT, where kT represented kH1, kH2 , kS, kOH, kGA, or kGB . Degradation rate constants were calculated for all experimental pH, temperature, and buffer conditions by combining the contributions from pH and buffer effects to yield, k ) k(pH) + kGA[GA] + kGB[GB]. The calculated k values had <10% error for 103 of the 106 experimentally determined values. Maximum stability was observed in the pH-independent region, 4 to 6. Degradation rate constants were predicted and experimentally verified for cefepime solutions stored at 30 °C, pH 4.6 and 5.6. These solutions maintained 90% of their initial concentration (T90) for ∼2 days.
Cefepime (Scheme 1) is a fourth generation, semisynthetic cephalosporin. Its chemical, pharmacokinetic, and clinical characteristics have been reviewed.1-3 Like other fourth generation cephalosporins, cefepime demonstrates good activity against gram-negative organisms such as Pseudomonas aeruginosa, and Gram-positive organisms such as Staphylococcus aureus.1 It also exhibits increased stability against β-lactamase-overproducing bacteria. Cefepime hydrochloride for injection was recently introduced in the United States as a sterile dry mixture of cefepime dihydrochloride dihydrate and L-arginine. The L-arginine was included to adjust the pH of freshly constituted solutions to 4 to 6. Product instructions state that reconstituted solutions may be stored for 24 h at room temperature (20 to 25 °C) or for 7 days in the refrigerator (2 to 8 °C).4 Cefepime instability was studied in two infusion solutions (0.9% sodium chloride and 5% glucose) that were stored in polyethylene bags at 24 °C in the light and 4 °C in the dark.5 Solutions maintained 90% potency for ∼3 days at 24 °C and for more than 15 days at 4 °C. However, * Corresponding author. Phone (843) 792-8427. Fax (843) 792-0759. E-mail [email protected].
1572 / Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998
Scheme 1
a literature search did not disclose any studies that reported kinetic data for cefepime degradation. Therefore, the goal of this study was to provide investigators with the ability to predict cefepime degradation rate constants at any desired pH and temperature in the presence or absence of sufficient buffers to cover the range of the pH-rate profile. The specific aims of this investigation were (1) to study the kinetics of cefepime degradation in aqueous solution as a function of pH, temperature, and buffer concentration, (2) to develop equations that predict cefepime stability at any pH and temperature, in the presence or absence of buffer, (3) to predict and experimentally verify the 30 °C shelf life of buffered solutions at the pH of maximum stability, and (4) to examine the influence of L-arginine on cefepime degradation at the pH of a reconstituted parenteral solution, i.e., pH 4 to 6.
Experimental Section MaterialssCefepime dihydrochloride dihydrate was used as provided by Bristol-Myers Squibb (Princeton, NJ). All other chemicals were analytical or HPLC grade. Cefepime HPLC AssayssThe system consisted of a Waters M-510 solvent delivery module, a Waters M-481 variable wavelength detector, a Shimadzu C-R3A integrator, and a Rheodyne M-7125 manual injector. The injection volume was 20 µL. Chromatographic separations were achieved on a Nova-Pak C18 column (4 µm, 60 Å, 3.9-mm i.d. × 150 mm) at ambient temperature with a flow rate of 1.0 mL/min and UV detection at 257 nm. Mobile phases were filtered through a type HA, 0.45-µm membrane filter (Millipore Corporation) and deaerated under reduced pressure. The quantitative separation of cefepime from its degradation products was achieved with a mobile phase consisting of aqueous 0.05 M NaH2PO4 and 0.05 M Na2HPO4 (pH 6.8) with 4% (v/v) acetonitrile. The cefepime retention time was 6.3 min. Calibration plots of peak areas versus concentration were linear in the range of 1.87 to 15.0 × 10-5 M. The coefficient of variation (CV) for analysis was 1.4. Recovery was 99 ( 1%. Several methods were employed to ensure that assays were specific for cefepime in the presence of its degradation products.6 No residual peaks were found beneath the cefepime peak when reactions were allowed to proceed to completion. Following ∼80% degradation, the UV spectra at the leading edge, maximum, and trailing edge of the cefepime peak were superimposable with the reference standard (diode array detector, Model 996, Waters Inc.). No deviations from linearity were observed in the first-order plots.
10.1021/js980170y CCC: $15.00 Published on Web 10/07/1998
© 1998, American Chemical Society and American Pharmaceutical Association
Table 1sExperimental Conditions and First-Order Rate Constants (102 k in h-1 ) for Degradation of Cefepime in Buffered Solutions 45 °C
buffer concn (M)a
55 °C
HCOOH
HCOONa
pH
102
0.764
0.076
2.53 ±0.06
1.40
0.382 0.191 0.382
0.038 0.019 0.382
0.191 0.095 0.038
0.191 0.095 0.381
0.019 0.009
0.191 0.095
1.20 1.03 1.09
3.54 ±0.04
pH
10 k
pH
102k
2.51 ±0.06
3.76
2.68 ±0.04
9.06
3.13 3.04 3.16
3.52 ±0.04
s
1.02 0.952 s
s
2.64 2.25 s
s s
s s
s s
s s
55 °C
buffer concn (M)a CH3COOH CH3COONa
k
65 °C 2
pH
102k
pH
0.038
s
s
0.189 0.095 0.379
0.019 0.009 0.379
s s 4.60 ±0.03
0.189 0.095 0.0379
0.189 0.095 0.379
0.0189 0.0095
0.189 0.095
s s s s 2.99 4.58 ±0.07 2.48 2.09 3.09 5.61 ±0.08 2.48 2.29
NaH2PO4 0.091
Na2HPO4 0.091
0.045 0.023 0.014
0.045 0.023 0.109
0.009 0.004
0.073 0.036
H3BO3 0.073
Na2B4O7 0.009
0.036 0.018 0.036
0.004 0.002 0.036
0.018 0.009
0.018 0.009
6.55 ±0.05 7.46 ±0.05
6.06 4.59 3.71 18.2 16.1 14.9
8.00 ±0.04
4.61 ±0.05
61.4
55.6 48.7 8.63 258 ±0.03 216 193
s
6.55 ±0.06 7.45 ±0.06
6.30 5.86 6.36 5.44 4.86
65 °C
0.379
5.63 ±0.03
3.65 ±0.03
8.04 7.26 7.74
75 °C
102k
pH
s
3.55 ±0.04
s s 8.16 6.24 5.94 8.46
4.60 ±0.04 5.65 ±0.04
6.90 6.36 15.78 12.8 10.6 51.4
15.2
reaction solution HCla
45 °C pH
0.48 0.78 1.19 1.58 formate 2.53 3.54 s acetate s s s phosphate s s borate s s
55 °C
102k(pH)
pH
102k
65 °C (pH)
pH
102k(pH)
6.90 0.48 15.3 0.49 32.8 5.28 0.79 12.5 0.79 26.3 4.00 1.19 9.27 1.19 18.9 2.66 1.59 6.36 1.59 13.8 0.936 2.50 2.73 2.68 6.75 0.884 3.52 1.99 3.65 5.14 s s s 4.61 4.40 s s s s s s 4.60 1.84 4.58 4.98 s 5.63 1.99 5.61 5.55 s 6.55 2.97 6.55 9.12 s 7.46 13.1 7.45 42.6 s 8.00 45.8 7.97 127 s 8.63 172 8.54 497
75 °C pH
102k(pH)
0.49 64.6 s s s s 1.59 32.8 s s s s s s 3.55 13.9 4.60 12.2 5.65 14.9 6.57 23.7 7.48 116 7.97 354 8.54 1237
a Mean values for duplicate kinetic studies in HCl. All reactions were adjusted to ionic strength of 0.5 with NaCl.
14.1 14.5 18.9 15.4 14.0 19.7 17.9 15.8
6.57 ±0.04 7.48 ±0.06
49.0 45.4 7.97 180 ±0.05 160 137 8.54 652 ±0.02 580 533
102k
Table 2sFirst-Order Rate Constants (102k(pH) in h-1 ) for Degradation of Cefepime in HCl and Those Corrected for Buffer Catalysis in the pH Range 2.5−8.6
7.97 ±0.02
40.1 32.6 27.5 123 124 118
Figure 1sSemilogarithmic plot of the percentage of cefepime remaining at 65 °C in (A) 0.18 M, (B) 0.09 M, and (C) 0.05 M phosphate buffer, pH 6.55.
464
Samples were removed as a function of time, transferred to glass tubes, and cooled with an ice and water mixture, and 0.5-mL aliquots were diluted with 1.0 mL of water or buffer to provide a pH of 4.6 to 6.5. Diluted samples were stored in an ice-water bath and usually assayed within 12 h after sampling. Those which were not assayed within 12 h were stored overnight in the refrigerator. No loss of cefepime was detected by HPLC when diluted samples were stored for 24 h in the refrigerator.
416 378 8.54 1,690 ±0.01 1,460 1,350
a Buffers were prepared at room temperature. All reactions were adjusted to ionic strength of 0.5 with NaCl.
Representative reactions in the acidic, basic, and neutral pH regions were analyzed in duplicate with the mobile phase described above and a second mobile phase that provided a cefepime retention time of 7.6 min. This mobile phase consisted of aqueous 0.125 M NaH2PO4 and 0.125 M Na2HPO4 (pH 6.7) with 5% (v/v) methanol. The concentrations and first-order plots obtained for both assays were similar. Cefepime Degradation KineticssThe temperatures, compositions of the reaction solutions, and the pH values are given in Tables 1 and 2. The pH values of the buffered reactions were measured before and after each reaction at the experimental temperatures that were stable to (0.1 °C. The ionic strength (µ) was adjusted to 0.5 with NaCl. The pH values for the reactions in HCl were calculated as a function of temperature with hydrogen ion activity coefficients determined from those reported at 0.5 µ by Harned and Owen.7 Reactions were initiated by placing 1.0 mL of a concentrated aqueous cefepime solution into 10 mL of HCl or buffer at the temperature of the study to provide 3.2 × 10-4 M cefepime. Reactions were quenched prior to analysis in the following manner.
Results and Discussion Determination of First-Order Rate Constantss Cefepime degradation rates were first order8,9 at constant pH, temperature, and ionic strength. Pseudo-first-order rate constants (k) were calculated by linear regression based on eq 1 (Tables 1 and 2):
ln [C] ) ln [C0] - kt
(1)
where the initial concentration of cefepime is [C0], the timedependent concentration is [C] and t is time. First-order plots were linear (r2 > 0.99) for g80% loss of [C0] (Figure 1). Each study was comprised of eight or more assays spaced to provide changes of ∼0.1[C0] per sampling interval. Duplicate studies provided k values with <5% differences. There was no significant difference between the rate constant for degradation of 10-4 M cefepime and that for a clinically employed concentration of 10 mg/mL (∼0.02 M) in 0.7 acetate buffer, pH 4.6, 45 to 75 °C. Journal of Pharmaceutical Sciences / 1573 Vol. 87, No. 12, December 1998
Figure 2sThe observed first-order rate constant for cefepime degradation (k) as a function of buffer concentration for acetate buffer at pH 5.61, 65 °C; formate buffer at pH 4.61, 65 °C; borate buffer at pH 8.63, 55 °C; and phosphate buffer at pH 6.57, 75 °C. Table 3sRate Constants (kcat in M-1 h-1) for Catalysis of Cefepime Degradation by Acidic (kGA) and Basic (kGB) Buffer Components components
kcat
45 °C
55 °C
65 °C
75 °C
HCOOH HCOOCH3COOH CH3COOHPO42B4O72-
kGA kGB kGA kGB kGB kGB
0.00534 0.00766 s s s s
0.0110 0.0201 0.00276 0.0283 0.269 24.6
0.0217 0.0498 0.00705 0.0745 0.691 45.7
s s 0.0488 0.127 1.60 123
Classical chemical kinetic analyses of the influence of pH, temperature, and buffer concentrations on the pseudofirst-order rate constants are discussed in the following sections.8-10 Buffer CatalysissThe rate constant for cefepime degradation increased when buffer concentration was increased at constant pH and temperature. All four buffers were catalytic (Figure 2). Pseudo-first-order rate constants in buffered solutions were described by eq 2:
k ) k(pH) + kGA[GA] + kGB[GB]
Figure 3sArrhenius plots for the natural log of the kGB values for (A) B4O72-, (B) HPO42-, and (C) CH3COO- as a function of the reciprocal of absolute temperature, K. Table 4sPreexponential Factors (A) and Activation Energies (E) for Catalytic Constantsof Buffer Componentsa components HCOOH HCOOCH3COOH CH3COOHPO42B4O72-
kcat
A (M-1 h-1)
E (cal/mol)
r
kGA kGB kGA kGB kGB kGB
1.039 × 4.186 × 1011 1.019 × 1019 6.772 × 109 8.322 × 1012 3.266 × 1013
14 978 20 129 32 498 17 044 20 251 18 247
0.999 0.999 0.977 0.989 0.999 0.989
108
a Linear regression using eq 4. Reported digits exceed experimental precision to minimize rounding errors in predictions, as suggested by Connors.8
(2)
where the rate constant in the absence of buffer catalysis is k(pH) and the catalytic constants for the acidic, [GA], and basic, [GB], buffer component concentrations are kGA and kGB in M-1 h-1, respectively. Factoring [GA] in eq 2 and substituting ratio for [GB]/ [GA], provides:
k ) k(pH) + {kGA + kGB(ratio)} [GA]
(3)
Three buffer concentrations were employed for each constant ratio. For each constant pH and ratio, a plot of k versus [GA] was linear with a slope equal to {kGA + kGB(ratio)} and an intercept equal to k(pH). The catalytic constants, kGA and kGB in Table 3, were calculated with simultaneous equations derived by substituting two or more ratios into slope ) {kGA + kGB(ratio)}. The basic species in phosphate and borate buffers were catalytic whereas the acidic components were not. Both the acidic and basic species of the formate and acetate buffers were catalytic. The temperature dependency for each buffer catalytic constant was determined by linear regression (Figure 3) based on the logarithmic form of the Arrhenius expression,
ln kT ) ln A - E/RT
(4)
where the kT value is kGA or kGB at absolute temperature, 1574 / Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998
Figure 4sThe pH−rate profiles for degradation of cefepime at 75 °C (b), 65 °C (0), 55 °C (O) and 45 °C (9). The solid curves were obtained by simultaneous nonlinear regression using eq 8 to fit the cefepime first-order degradation rate constants, k(pH), in Table 2. The dashed curve was predicted.
T; the preexponential term is A; E is the energy of activation in cal/mol; and R is 1.987 cal/mol-deg.8-10 As suggested by Connors,8 the A and E values (Table 4) are reported with more digits than required to express the experimental precision to minimize rounding errors during predictions. Rate Constants as a Function of pH and TemperaturesThe observed rate constants at each pH were corrected for buffer catalysis using linear regression of k versus total buffer concentration (Figure 2) to determine the intercepts at each constant ratio. The first-order rate constants determined as a function of pH in HCl and from buffer intercepts at 45, 55, 65, and 75 °C (Table 2) were used to construct pH-rate profiles (Figure 4) for cefepime degradation.
Known pH-rate profiles vary in complexity from “V”or “U”-shaped to those with one or more inflections.8-10 The pH-rate profile for cefepime (Figure 4) is typical of many cephalosporin profiles that are “U”-shaped with an inflection in the acidic region.11,12 Such data are generally described by eq 5 or a kinetic equivalent.
k(pH) ) kH1 f1(aH+) + kH2 f2(aH+) + kS + kOH(aOH-)
(5)
In eq 5, kH1 and kH2 are the hydrogen ion activity (aH+) catalytic constants (M-1 h-1) for the protonated and unprotonated forms of cefepime, respectively; kOH is the catalytic constant (M-1 h-1) for hydroxyl ion activity (aOH) KW/aH+) and kS is the first-order rate constant (h-1) for spontaneous degradation. The values for KW as a function of temperature were taken from Harned and Owen.7 The fractions of the protonated (f1) and unprotonated (f2) forms of cefepime were calculated from the dissociation constant, Ka, and the hydrogen ion activity using eqs 6 and 7:
f1 ) (aH+)/[(aH+) + Ka]
(6)
f2 ) Ka/[(aH+) + Ka]
(7)
Table 5sPreexponential Factors (A),a Activation Energies (E in cal/mol), and Their 95% S-plane Confidence Limits (±SP) for Rate Constants in the pH−Rate Expression (eq 5) Determined with Equation 8b rate constant
A (±SP)
E (±SP)
kH1 kH2 ks kOH
(7.201 ± 1.134) × 10 (2.118 ± 0.104) × 1015 (1.071 ± 0.039) × 1015 (8.938 ± 0.366) × 1015 12
17 467 ± 105 19 231 ± 34 22 193 ± 24 13 814 ± 28
a In M-1 h-1 except for kS in h-1. b Determined by simultaneous nonlinear regression13 of the rate constants, k(pH), in Table 2. Reported digits exceed experimental precision to minimize rounding errors in predictions, as suggested by Connors.
At each temperature, k(pH) values were satisfactorily described using nonlinear regression13 with eqs 5-7 wherein kH1, kH2, kS, kOH, and Ka were adjustable parameters. All rate constants, except k(pH), were expressed as Ae-E/ RT, and these functions were substituted in eq 5 to give the following expression:
k(pH) ) (AH1e-EH1/RT) f1(aH+) + (AH2e-EH2/RT) f2(aH+) + (ASe-ES/RT) + (AOHe-EOH/RT) (aOH-) (8) The absolute temperatures corresponding to 45, 55, 65, and 75 °C were substituted for T. The resulting four simultaneous equations were used to effect simultaneous nonlinear regression on the 39 k(pH) values in Table 2.13 The dissociation constant, Ka, was described as a function of absolute temperature using the form of the van’t Hoff equation that applies to a limited temperature range where ∆Ha is nearly constant (eq 9).8 The constants were obtained by nonlinear regression with eqs 6-9.13 The Ka value equation was,
Ka ) (constant)e-∆Ha/RT ) 8.32 × 10-6e(5295)/RT
(9)
and the 95% S-plane confidence limits were ∆Ha ) 52585332 cal/mol and (constant) ) (7.85-8.80) × 10-6. Potentiometric titration of cefepime provided pKa values of 1.3 and 3.2. These are similar to the 1.5 and 3.1 values previously reported.5 Equation 9 predicted a kinetically determined pKa value of 1.2 at room temperature. This is consistent with the potentiometrically determined value of 1.3. Protonated thiazole has a pKa of 2.53 and protonated 2-aminothiazole has a pKa of 5.39.14 Therefore the cefepime pKa value of 1.3 is more likely that of the carboxylic acid than the protonated aminothiazolyl side chain. The values obtained for the remaining adjustable parameters in eq 8 (i.e. the A and E values) are shown in Table 5. Tables 1 and 2 contain 104 experimental k values that were calculated with eq 1 for ceftazidime degradation in the presence of HCl or buffers. Equations 6-9 and the parameter values in Table 5 were used to calculate k(pH) for all experimental pH and temperature values. The buffer A and E values in Table 4 were substituted in eq 4 to calculate kga and kgb values as a function of temperature. Pseudo-first-order rate constants (k) were calculated as a function of both pH and buffer catalysis with eq 2. An
Figure 5sLinear correlation (r2 ) 0.999) between the first-order rate constants (k) calculated with eqs 3 and 6−9 and the 106 experimental values determined at 45 to 75 °C in formate, acetate, phosphate, and borate buffers and in HCl. The log−log scale was used solely to facilitate visualization.
excellent linear correlation (r2 ) 0.999) was found between the 106 experimentally determined k values (including the two 30 °C values discussed in the following section) and the k values calculated with eqs 2, 4, and 6-9. Although this correlation employed the k values, results are presented in Figure 5 on a log-log scale for visualization. These equations successfully calculated 103 out of the 106 experimental values with <10% error. There were three outliers with errors of 12, 15, and 20%. Shelf-Life PredictionssShelf life was defined as the time during which the concentration of cefepime equaled or exceeded 90% of its initial concentration (T90 ) 0.105/ k).8-10 The maximum stability for an unbuffered solution occurred at pH ∼4 to ∼6 where k(pH) was at a minimum (Figure 4). The T90 values that reflect cefepime degradation without buffer catalysis were calculated using k(pH) values obtained with eqs 6-9. Figure 6 shows these predicted T90 values at pH 4.6 as a function of temperature from 5 to 30 °C. Cefepime degradation was subject to acetate buffer catalysis in the pH region of maximum stability. Figure 6 also shows the T90 values predicted for cefepime in 0.758 M acetate buffer, pH 4.6, as a function of temperature based on degradation rate constants calculated with eqs 2, 4, and 6-9. The predicted degradation rate constants at 30 °C were 0.00241 h-1 at pH 4.6 and 0.00243 h-1 at pH 5.6. These rate constants were similar because they were determined within the pH-independent region, and both buffers contained the same concentration of the dominant catalytic species, i.e., 0.379 M CH3COO-. The experimentally determined degradation rate constants for cefepime stored at 30 °C in these acetate buffers were within 1 and 7% (respectively) of the predicted values. The experimental k values were 0.00239 h-1 (T90 ) 1.8 d) at Journal of Pharmaceutical Sciences / 1575 Vol. 87, No. 12, December 1998
constants at any pH and temperature in the presence or absence of buffers. Cefepime was most stable in the pH range 4 to 6. The equations also predicted the degradation rate at 30 °C, pH 4.6 and 5.6, in the presence of acetate buffer catalysis. This research provides a tool that allows an investigator to plan cefepime experiments in aqueous environments without having to first conduct degradation kinetic studies. An investigator wishing to expose cefepime to a buffered or nonbuffered solution at some desired pH and temperature can calculate the cefepime degradation constant for those conditions. This knowledge can be used to assess the feasibility of a research plan, to design experimental protocols and/or to calculate the loss of cefepime during a study.
References and Notes Figure 6sThe predicted shelf life (T90 ) 0.105/k) as a function of temperature (°C), in the pH region of maximum stability, for an aqueous solution of cefepime at pH 4.6 in the absence of buffer catalysis (curve A) and in the presence of 0.758 M acetate buffer (curve B). The experimental k values shown at 30 °C were determined for acetate buffer at pH 4.6 (0) and pH 5.6 (2). The insert shows the reduction in T90 values as L-arginine was added to acetate buffer at 30 °C, pH 4.6.
pH 4.6 and 0.00226 h-1 (T90 ) 1.9 d) at pH 5.6. Figure 6 further illustrates the agreement between these experimental T90 values at pH 4.6 and 5.6 and the predicted values at 30 °C. Cefepime is marketed as a mixture of the dry hydrochloride salt and sufficient L-arginine to provide reconstituted solutions of pH 4 to 6.4 The concentrations of L-arginine in the solutions for intravenous administration vary from 0.04 M (10 mg/mL cefepime) to 0.4 M (100 mg/ mL cefepime). The degradation of cefepime was studied at pH 4.6 in 0.2 M acetate buffer, 30 °C, wherein the L-arginine concentrations were varied from 0.1 to 0.4 M. The degradation rates were all first order, and the cefepime degradation rate constants were moderately increased by the addition of L-arginine. The insert in Figure 6 shows T90 as a function of L-arginine concentration at 30 °C, pH 4.6. The intercept of this plot is the T90 value (∼2.2 days) for cefepime degradation in the 0.2 M acetate buffer solution in the absence of L-arginine. The T90 value was reduced to ∼1.7 days in the most concentrated buffered L-arginine solution.
1. Okamoto, M. P.; Nakahiro, R. K.; Chin, A.; Bedikian, A.; Gill, M. A. Cefepime: A New Fourth-Generation Cephalosporin. Am. J. Hosp. Pharm. 1994, 51, 463-477. 2. Wynd, M.; Paladino, J. Cefepime: A Fourth-Generation Parenteral Cephalosporin. Ann. Pharmacother. 1996, 30, 1414-1423. 3. Okamoto, M. P.; Nakahiro, R. K.; Chin, A.; Bedikian, A. Cefepime Clinical Pharmacokinetics. Clin. Pharmacokin. 1993, 25, 88-102. 4. Physicians’ Desk Reference, 52nd ed., Medical Economics Company Inc.: Montvale, NJ, 1998; Maxipime, pp 800-804. 5. Rabouan-Guyon, M. S.; Guet, F. A. Courtois, P. Y.; Barthes, M. C. Stability Study of Cefepime in Different Infusion Solutions. Int. J. Pharm. 1997, 154, 185-190. 6. Fubara, J. O.; Ph.D. Dissertation, Medical University of South Carolina, Charleston, SC, 1998. 7. Harned, H. S.; Owen, B. B. The Physical Chemistry of Electrolytic Solutions, 3rd ed.; Reinhold: New York, 1958; pp 638, 748. 8. Connors, K. A. Chemical Kinetics; VCH: New York, 1990; pp 17-58, 245-292. 9. Carstensen, J. T. Drug Stability, 2nd ed.; Marcel Dekker: New York, 1995; pp 17-121. 10. Connors, K. A.; Amidon, G. L.; Stella V. J. Chemical Stability of Pharmaceuticals, 2nd ed.; John Wiley & Sons: New York, 1986; pp 8-31, 43-62, 302-321. 11. Wang, D.; Notari, R. E. Cefuroxime Degradation Kinetics and Stability Predictions in Aqueous Solutions. J. Pharm. Sci. 1994, 83, 577-581. 12. Yamaha, T.; Tsuji, A. Comparative Stability of Cephalosporins in Aqueous Solution: Kinetics and Mechanisms of Degradation. J. Pharm. Sci. 1976, 65, 1563-1573. 13. Scientist, MicroMath Scientific Software: Salt Lake City, UT. 14. Metzger, J. V. Thiazole & Its Derivatives, Part 1; John Wiley & Sons: New York, 1979; pp 91-94.
Acknowledgments Summary This research applied classical chemical kinetic analyses to the study of cefepime degradation in aqueous solutions. The completed study provides equations that successfully calculated the experimentally determined degradation rate
1576 / Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998
We thank Dr. Robert A. Lipper, Vice President, Biopharmaceutics R & D, Bristol-Myers Squibb Co., for supplying cefepime. Josephine Fubara gratefully acknowledges receiving the Earl Higgins Fellowship and an American Foundation for Pharmaceutical Education Predoctoral Fellowship.
JS980170Y
AND
ROBERT E. NOTARI*
Contribution from The Department of Pharmaceutical Sciences, College of Pharmacy, Medical University of South Carolina, 171 Ashley Avenue, Charleston, South Carolina 29425. Received April 17, 1998. Final revised manuscript received September 1, 1998. Accepted for publication September 1, 1998. Abstract 0 First-order rate constants (k) were determined for cefepime degradation at 45, 55, 65, and 75 °C, pH 0.5 to 8.6, using an HPLC assay. Each pH−rate profile exhibited an inflection between pH 1 and 2. The pH−rate expression was k(pH) ) kH1 f1(aH+) + kH2 f2(aH+) + kS + kOH(aOH−), where kH1 and kH2 are the catalytic constants (M-1 h-1) for hydrogen ion activity (aH+), kOH is the catalytic constant for hydroxyl ion activity (aOH−), and kS is the first-order rate constant (h-1) for spontaneous degradation. The protonated (f1 ) and unprotonated (f2 ) fractions were calculated from the dissociation constant, Ka ) (8.32 × 10-6 )e(5295)/RT where T was absolute temperature (T). Accelerated loss due to formate, acetate, phosphate, and borate buffer catalysis was quantitatively described with the catalytic constant, kGA (M-1 h-1) for the acidic component, [GA], and kGB (M-1 h-1) for the basic component, [GB], of each buffer. The temperature dependency for each rate constant was defined with experimentally determined values for A and E and the Arrhenius expression, kT ) Ae-E/RT, where kT represented kH1, kH2 , kS, kOH, kGA, or kGB . Degradation rate constants were calculated for all experimental pH, temperature, and buffer conditions by combining the contributions from pH and buffer effects to yield, k ) k(pH) + kGA[GA] + kGB[GB]. The calculated k values had <10% error for 103 of the 106 experimentally determined values. Maximum stability was observed in the pH-independent region, 4 to 6. Degradation rate constants were predicted and experimentally verified for cefepime solutions stored at 30 °C, pH 4.6 and 5.6. These solutions maintained 90% of their initial concentration (T90) for ∼2 days.
Cefepime (Scheme 1) is a fourth generation, semisynthetic cephalosporin. Its chemical, pharmacokinetic, and clinical characteristics have been reviewed.1-3 Like other fourth generation cephalosporins, cefepime demonstrates good activity against gram-negative organisms such as Pseudomonas aeruginosa, and Gram-positive organisms such as Staphylococcus aureus.1 It also exhibits increased stability against β-lactamase-overproducing bacteria. Cefepime hydrochloride for injection was recently introduced in the United States as a sterile dry mixture of cefepime dihydrochloride dihydrate and L-arginine. The L-arginine was included to adjust the pH of freshly constituted solutions to 4 to 6. Product instructions state that reconstituted solutions may be stored for 24 h at room temperature (20 to 25 °C) or for 7 days in the refrigerator (2 to 8 °C).4 Cefepime instability was studied in two infusion solutions (0.9% sodium chloride and 5% glucose) that were stored in polyethylene bags at 24 °C in the light and 4 °C in the dark.5 Solutions maintained 90% potency for ∼3 days at 24 °C and for more than 15 days at 4 °C. However, * Corresponding author. Phone (843) 792-8427. Fax (843) 792-0759. E-mail [email protected].
1572 / Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998
Scheme 1
a literature search did not disclose any studies that reported kinetic data for cefepime degradation. Therefore, the goal of this study was to provide investigators with the ability to predict cefepime degradation rate constants at any desired pH and temperature in the presence or absence of sufficient buffers to cover the range of the pH-rate profile. The specific aims of this investigation were (1) to study the kinetics of cefepime degradation in aqueous solution as a function of pH, temperature, and buffer concentration, (2) to develop equations that predict cefepime stability at any pH and temperature, in the presence or absence of buffer, (3) to predict and experimentally verify the 30 °C shelf life of buffered solutions at the pH of maximum stability, and (4) to examine the influence of L-arginine on cefepime degradation at the pH of a reconstituted parenteral solution, i.e., pH 4 to 6.
Experimental Section MaterialssCefepime dihydrochloride dihydrate was used as provided by Bristol-Myers Squibb (Princeton, NJ). All other chemicals were analytical or HPLC grade. Cefepime HPLC AssayssThe system consisted of a Waters M-510 solvent delivery module, a Waters M-481 variable wavelength detector, a Shimadzu C-R3A integrator, and a Rheodyne M-7125 manual injector. The injection volume was 20 µL. Chromatographic separations were achieved on a Nova-Pak C18 column (4 µm, 60 Å, 3.9-mm i.d. × 150 mm) at ambient temperature with a flow rate of 1.0 mL/min and UV detection at 257 nm. Mobile phases were filtered through a type HA, 0.45-µm membrane filter (Millipore Corporation) and deaerated under reduced pressure. The quantitative separation of cefepime from its degradation products was achieved with a mobile phase consisting of aqueous 0.05 M NaH2PO4 and 0.05 M Na2HPO4 (pH 6.8) with 4% (v/v) acetonitrile. The cefepime retention time was 6.3 min. Calibration plots of peak areas versus concentration were linear in the range of 1.87 to 15.0 × 10-5 M. The coefficient of variation (CV) for analysis was 1.4. Recovery was 99 ( 1%. Several methods were employed to ensure that assays were specific for cefepime in the presence of its degradation products.6 No residual peaks were found beneath the cefepime peak when reactions were allowed to proceed to completion. Following ∼80% degradation, the UV spectra at the leading edge, maximum, and trailing edge of the cefepime peak were superimposable with the reference standard (diode array detector, Model 996, Waters Inc.). No deviations from linearity were observed in the first-order plots.
10.1021/js980170y CCC: $15.00 Published on Web 10/07/1998
© 1998, American Chemical Society and American Pharmaceutical Association
Table 1sExperimental Conditions and First-Order Rate Constants (102 k in h-1 ) for Degradation of Cefepime in Buffered Solutions 45 °C
buffer concn (M)a
55 °C
HCOOH
HCOONa
pH
102
0.764
0.076
2.53 ±0.06
1.40
0.382 0.191 0.382
0.038 0.019 0.382
0.191 0.095 0.038
0.191 0.095 0.381
0.019 0.009
0.191 0.095
1.20 1.03 1.09
3.54 ±0.04
pH
10 k
pH
102k
2.51 ±0.06
3.76
2.68 ±0.04
9.06
3.13 3.04 3.16
3.52 ±0.04
s
1.02 0.952 s
s
2.64 2.25 s
s s
s s
s s
s s
55 °C
buffer concn (M)a CH3COOH CH3COONa
k
65 °C 2
pH
102k
pH
0.038
s
s
0.189 0.095 0.379
0.019 0.009 0.379
s s 4.60 ±0.03
0.189 0.095 0.0379
0.189 0.095 0.379
0.0189 0.0095
0.189 0.095
s s s s 2.99 4.58 ±0.07 2.48 2.09 3.09 5.61 ±0.08 2.48 2.29
NaH2PO4 0.091
Na2HPO4 0.091
0.045 0.023 0.014
0.045 0.023 0.109
0.009 0.004
0.073 0.036
H3BO3 0.073
Na2B4O7 0.009
0.036 0.018 0.036
0.004 0.002 0.036
0.018 0.009
0.018 0.009
6.55 ±0.05 7.46 ±0.05
6.06 4.59 3.71 18.2 16.1 14.9
8.00 ±0.04
4.61 ±0.05
61.4
55.6 48.7 8.63 258 ±0.03 216 193
s
6.55 ±0.06 7.45 ±0.06
6.30 5.86 6.36 5.44 4.86
65 °C
0.379
5.63 ±0.03
3.65 ±0.03
8.04 7.26 7.74
75 °C
102k
pH
s
3.55 ±0.04
s s 8.16 6.24 5.94 8.46
4.60 ±0.04 5.65 ±0.04
6.90 6.36 15.78 12.8 10.6 51.4
15.2
reaction solution HCla
45 °C pH
0.48 0.78 1.19 1.58 formate 2.53 3.54 s acetate s s s phosphate s s borate s s
55 °C
102k(pH)
pH
102k
65 °C (pH)
pH
102k(pH)
6.90 0.48 15.3 0.49 32.8 5.28 0.79 12.5 0.79 26.3 4.00 1.19 9.27 1.19 18.9 2.66 1.59 6.36 1.59 13.8 0.936 2.50 2.73 2.68 6.75 0.884 3.52 1.99 3.65 5.14 s s s 4.61 4.40 s s s s s s 4.60 1.84 4.58 4.98 s 5.63 1.99 5.61 5.55 s 6.55 2.97 6.55 9.12 s 7.46 13.1 7.45 42.6 s 8.00 45.8 7.97 127 s 8.63 172 8.54 497
75 °C pH
102k(pH)
0.49 64.6 s s s s 1.59 32.8 s s s s s s 3.55 13.9 4.60 12.2 5.65 14.9 6.57 23.7 7.48 116 7.97 354 8.54 1237
a Mean values for duplicate kinetic studies in HCl. All reactions were adjusted to ionic strength of 0.5 with NaCl.
14.1 14.5 18.9 15.4 14.0 19.7 17.9 15.8
6.57 ±0.04 7.48 ±0.06
49.0 45.4 7.97 180 ±0.05 160 137 8.54 652 ±0.02 580 533
102k
Table 2sFirst-Order Rate Constants (102k(pH) in h-1 ) for Degradation of Cefepime in HCl and Those Corrected for Buffer Catalysis in the pH Range 2.5−8.6
7.97 ±0.02
40.1 32.6 27.5 123 124 118
Figure 1sSemilogarithmic plot of the percentage of cefepime remaining at 65 °C in (A) 0.18 M, (B) 0.09 M, and (C) 0.05 M phosphate buffer, pH 6.55.
464
Samples were removed as a function of time, transferred to glass tubes, and cooled with an ice and water mixture, and 0.5-mL aliquots were diluted with 1.0 mL of water or buffer to provide a pH of 4.6 to 6.5. Diluted samples were stored in an ice-water bath and usually assayed within 12 h after sampling. Those which were not assayed within 12 h were stored overnight in the refrigerator. No loss of cefepime was detected by HPLC when diluted samples were stored for 24 h in the refrigerator.
416 378 8.54 1,690 ±0.01 1,460 1,350
a Buffers were prepared at room temperature. All reactions were adjusted to ionic strength of 0.5 with NaCl.
Representative reactions in the acidic, basic, and neutral pH regions were analyzed in duplicate with the mobile phase described above and a second mobile phase that provided a cefepime retention time of 7.6 min. This mobile phase consisted of aqueous 0.125 M NaH2PO4 and 0.125 M Na2HPO4 (pH 6.7) with 5% (v/v) methanol. The concentrations and first-order plots obtained for both assays were similar. Cefepime Degradation KineticssThe temperatures, compositions of the reaction solutions, and the pH values are given in Tables 1 and 2. The pH values of the buffered reactions were measured before and after each reaction at the experimental temperatures that were stable to (0.1 °C. The ionic strength (µ) was adjusted to 0.5 with NaCl. The pH values for the reactions in HCl were calculated as a function of temperature with hydrogen ion activity coefficients determined from those reported at 0.5 µ by Harned and Owen.7 Reactions were initiated by placing 1.0 mL of a concentrated aqueous cefepime solution into 10 mL of HCl or buffer at the temperature of the study to provide 3.2 × 10-4 M cefepime. Reactions were quenched prior to analysis in the following manner.
Results and Discussion Determination of First-Order Rate Constantss Cefepime degradation rates were first order8,9 at constant pH, temperature, and ionic strength. Pseudo-first-order rate constants (k) were calculated by linear regression based on eq 1 (Tables 1 and 2):
ln [C] ) ln [C0] - kt
(1)
where the initial concentration of cefepime is [C0], the timedependent concentration is [C] and t is time. First-order plots were linear (r2 > 0.99) for g80% loss of [C0] (Figure 1). Each study was comprised of eight or more assays spaced to provide changes of ∼0.1[C0] per sampling interval. Duplicate studies provided k values with <5% differences. There was no significant difference between the rate constant for degradation of 10-4 M cefepime and that for a clinically employed concentration of 10 mg/mL (∼0.02 M) in 0.7 acetate buffer, pH 4.6, 45 to 75 °C. Journal of Pharmaceutical Sciences / 1573 Vol. 87, No. 12, December 1998
Figure 2sThe observed first-order rate constant for cefepime degradation (k) as a function of buffer concentration for acetate buffer at pH 5.61, 65 °C; formate buffer at pH 4.61, 65 °C; borate buffer at pH 8.63, 55 °C; and phosphate buffer at pH 6.57, 75 °C. Table 3sRate Constants (kcat in M-1 h-1) for Catalysis of Cefepime Degradation by Acidic (kGA) and Basic (kGB) Buffer Components components
kcat
45 °C
55 °C
65 °C
75 °C
HCOOH HCOOCH3COOH CH3COOHPO42B4O72-
kGA kGB kGA kGB kGB kGB
0.00534 0.00766 s s s s
0.0110 0.0201 0.00276 0.0283 0.269 24.6
0.0217 0.0498 0.00705 0.0745 0.691 45.7
s s 0.0488 0.127 1.60 123
Classical chemical kinetic analyses of the influence of pH, temperature, and buffer concentrations on the pseudofirst-order rate constants are discussed in the following sections.8-10 Buffer CatalysissThe rate constant for cefepime degradation increased when buffer concentration was increased at constant pH and temperature. All four buffers were catalytic (Figure 2). Pseudo-first-order rate constants in buffered solutions were described by eq 2:
k ) k(pH) + kGA[GA] + kGB[GB]
Figure 3sArrhenius plots for the natural log of the kGB values for (A) B4O72-, (B) HPO42-, and (C) CH3COO- as a function of the reciprocal of absolute temperature, K. Table 4sPreexponential Factors (A) and Activation Energies (E) for Catalytic Constantsof Buffer Componentsa components HCOOH HCOOCH3COOH CH3COOHPO42B4O72-
kcat
A (M-1 h-1)
E (cal/mol)
r
kGA kGB kGA kGB kGB kGB
1.039 × 4.186 × 1011 1.019 × 1019 6.772 × 109 8.322 × 1012 3.266 × 1013
14 978 20 129 32 498 17 044 20 251 18 247
0.999 0.999 0.977 0.989 0.999 0.989
108
a Linear regression using eq 4. Reported digits exceed experimental precision to minimize rounding errors in predictions, as suggested by Connors.8
(2)
where the rate constant in the absence of buffer catalysis is k(pH) and the catalytic constants for the acidic, [GA], and basic, [GB], buffer component concentrations are kGA and kGB in M-1 h-1, respectively. Factoring [GA] in eq 2 and substituting ratio for [GB]/ [GA], provides:
k ) k(pH) + {kGA + kGB(ratio)} [GA]
(3)
Three buffer concentrations were employed for each constant ratio. For each constant pH and ratio, a plot of k versus [GA] was linear with a slope equal to {kGA + kGB(ratio)} and an intercept equal to k(pH). The catalytic constants, kGA and kGB in Table 3, were calculated with simultaneous equations derived by substituting two or more ratios into slope ) {kGA + kGB(ratio)}. The basic species in phosphate and borate buffers were catalytic whereas the acidic components were not. Both the acidic and basic species of the formate and acetate buffers were catalytic. The temperature dependency for each buffer catalytic constant was determined by linear regression (Figure 3) based on the logarithmic form of the Arrhenius expression,
ln kT ) ln A - E/RT
(4)
where the kT value is kGA or kGB at absolute temperature, 1574 / Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998
Figure 4sThe pH−rate profiles for degradation of cefepime at 75 °C (b), 65 °C (0), 55 °C (O) and 45 °C (9). The solid curves were obtained by simultaneous nonlinear regression using eq 8 to fit the cefepime first-order degradation rate constants, k(pH), in Table 2. The dashed curve was predicted.
T; the preexponential term is A; E is the energy of activation in cal/mol; and R is 1.987 cal/mol-deg.8-10 As suggested by Connors,8 the A and E values (Table 4) are reported with more digits than required to express the experimental precision to minimize rounding errors during predictions. Rate Constants as a Function of pH and TemperaturesThe observed rate constants at each pH were corrected for buffer catalysis using linear regression of k versus total buffer concentration (Figure 2) to determine the intercepts at each constant ratio. The first-order rate constants determined as a function of pH in HCl and from buffer intercepts at 45, 55, 65, and 75 °C (Table 2) were used to construct pH-rate profiles (Figure 4) for cefepime degradation.
Known pH-rate profiles vary in complexity from “V”or “U”-shaped to those with one or more inflections.8-10 The pH-rate profile for cefepime (Figure 4) is typical of many cephalosporin profiles that are “U”-shaped with an inflection in the acidic region.11,12 Such data are generally described by eq 5 or a kinetic equivalent.
k(pH) ) kH1 f1(aH+) + kH2 f2(aH+) + kS + kOH(aOH-)
(5)
In eq 5, kH1 and kH2 are the hydrogen ion activity (aH+) catalytic constants (M-1 h-1) for the protonated and unprotonated forms of cefepime, respectively; kOH is the catalytic constant (M-1 h-1) for hydroxyl ion activity (aOH) KW/aH+) and kS is the first-order rate constant (h-1) for spontaneous degradation. The values for KW as a function of temperature were taken from Harned and Owen.7 The fractions of the protonated (f1) and unprotonated (f2) forms of cefepime were calculated from the dissociation constant, Ka, and the hydrogen ion activity using eqs 6 and 7:
f1 ) (aH+)/[(aH+) + Ka]
(6)
f2 ) Ka/[(aH+) + Ka]
(7)
Table 5sPreexponential Factors (A),a Activation Energies (E in cal/mol), and Their 95% S-plane Confidence Limits (±SP) for Rate Constants in the pH−Rate Expression (eq 5) Determined with Equation 8b rate constant
A (±SP)
E (±SP)
kH1 kH2 ks kOH
(7.201 ± 1.134) × 10 (2.118 ± 0.104) × 1015 (1.071 ± 0.039) × 1015 (8.938 ± 0.366) × 1015 12
17 467 ± 105 19 231 ± 34 22 193 ± 24 13 814 ± 28
a In M-1 h-1 except for kS in h-1. b Determined by simultaneous nonlinear regression13 of the rate constants, k(pH), in Table 2. Reported digits exceed experimental precision to minimize rounding errors in predictions, as suggested by Connors.
At each temperature, k(pH) values were satisfactorily described using nonlinear regression13 with eqs 5-7 wherein kH1, kH2, kS, kOH, and Ka were adjustable parameters. All rate constants, except k(pH), were expressed as Ae-E/ RT, and these functions were substituted in eq 5 to give the following expression:
k(pH) ) (AH1e-EH1/RT) f1(aH+) + (AH2e-EH2/RT) f2(aH+) + (ASe-ES/RT) + (AOHe-EOH/RT) (aOH-) (8) The absolute temperatures corresponding to 45, 55, 65, and 75 °C were substituted for T. The resulting four simultaneous equations were used to effect simultaneous nonlinear regression on the 39 k(pH) values in Table 2.13 The dissociation constant, Ka, was described as a function of absolute temperature using the form of the van’t Hoff equation that applies to a limited temperature range where ∆Ha is nearly constant (eq 9).8 The constants were obtained by nonlinear regression with eqs 6-9.13 The Ka value equation was,
Ka ) (constant)e-∆Ha/RT ) 8.32 × 10-6e(5295)/RT
(9)
and the 95% S-plane confidence limits were ∆Ha ) 52585332 cal/mol and (constant) ) (7.85-8.80) × 10-6. Potentiometric titration of cefepime provided pKa values of 1.3 and 3.2. These are similar to the 1.5 and 3.1 values previously reported.5 Equation 9 predicted a kinetically determined pKa value of 1.2 at room temperature. This is consistent with the potentiometrically determined value of 1.3. Protonated thiazole has a pKa of 2.53 and protonated 2-aminothiazole has a pKa of 5.39.14 Therefore the cefepime pKa value of 1.3 is more likely that of the carboxylic acid than the protonated aminothiazolyl side chain. The values obtained for the remaining adjustable parameters in eq 8 (i.e. the A and E values) are shown in Table 5. Tables 1 and 2 contain 104 experimental k values that were calculated with eq 1 for ceftazidime degradation in the presence of HCl or buffers. Equations 6-9 and the parameter values in Table 5 were used to calculate k(pH) for all experimental pH and temperature values. The buffer A and E values in Table 4 were substituted in eq 4 to calculate kga and kgb values as a function of temperature. Pseudo-first-order rate constants (k) were calculated as a function of both pH and buffer catalysis with eq 2. An
Figure 5sLinear correlation (r2 ) 0.999) between the first-order rate constants (k) calculated with eqs 3 and 6−9 and the 106 experimental values determined at 45 to 75 °C in formate, acetate, phosphate, and borate buffers and in HCl. The log−log scale was used solely to facilitate visualization.
excellent linear correlation (r2 ) 0.999) was found between the 106 experimentally determined k values (including the two 30 °C values discussed in the following section) and the k values calculated with eqs 2, 4, and 6-9. Although this correlation employed the k values, results are presented in Figure 5 on a log-log scale for visualization. These equations successfully calculated 103 out of the 106 experimental values with <10% error. There were three outliers with errors of 12, 15, and 20%. Shelf-Life PredictionssShelf life was defined as the time during which the concentration of cefepime equaled or exceeded 90% of its initial concentration (T90 ) 0.105/ k).8-10 The maximum stability for an unbuffered solution occurred at pH ∼4 to ∼6 where k(pH) was at a minimum (Figure 4). The T90 values that reflect cefepime degradation without buffer catalysis were calculated using k(pH) values obtained with eqs 6-9. Figure 6 shows these predicted T90 values at pH 4.6 as a function of temperature from 5 to 30 °C. Cefepime degradation was subject to acetate buffer catalysis in the pH region of maximum stability. Figure 6 also shows the T90 values predicted for cefepime in 0.758 M acetate buffer, pH 4.6, as a function of temperature based on degradation rate constants calculated with eqs 2, 4, and 6-9. The predicted degradation rate constants at 30 °C were 0.00241 h-1 at pH 4.6 and 0.00243 h-1 at pH 5.6. These rate constants were similar because they were determined within the pH-independent region, and both buffers contained the same concentration of the dominant catalytic species, i.e., 0.379 M CH3COO-. The experimentally determined degradation rate constants for cefepime stored at 30 °C in these acetate buffers were within 1 and 7% (respectively) of the predicted values. The experimental k values were 0.00239 h-1 (T90 ) 1.8 d) at Journal of Pharmaceutical Sciences / 1575 Vol. 87, No. 12, December 1998
constants at any pH and temperature in the presence or absence of buffers. Cefepime was most stable in the pH range 4 to 6. The equations also predicted the degradation rate at 30 °C, pH 4.6 and 5.6, in the presence of acetate buffer catalysis. This research provides a tool that allows an investigator to plan cefepime experiments in aqueous environments without having to first conduct degradation kinetic studies. An investigator wishing to expose cefepime to a buffered or nonbuffered solution at some desired pH and temperature can calculate the cefepime degradation constant for those conditions. This knowledge can be used to assess the feasibility of a research plan, to design experimental protocols and/or to calculate the loss of cefepime during a study.
References and Notes Figure 6sThe predicted shelf life (T90 ) 0.105/k) as a function of temperature (°C), in the pH region of maximum stability, for an aqueous solution of cefepime at pH 4.6 in the absence of buffer catalysis (curve A) and in the presence of 0.758 M acetate buffer (curve B). The experimental k values shown at 30 °C were determined for acetate buffer at pH 4.6 (0) and pH 5.6 (2). The insert shows the reduction in T90 values as L-arginine was added to acetate buffer at 30 °C, pH 4.6.
pH 4.6 and 0.00226 h-1 (T90 ) 1.9 d) at pH 5.6. Figure 6 further illustrates the agreement between these experimental T90 values at pH 4.6 and 5.6 and the predicted values at 30 °C. Cefepime is marketed as a mixture of the dry hydrochloride salt and sufficient L-arginine to provide reconstituted solutions of pH 4 to 6.4 The concentrations of L-arginine in the solutions for intravenous administration vary from 0.04 M (10 mg/mL cefepime) to 0.4 M (100 mg/ mL cefepime). The degradation of cefepime was studied at pH 4.6 in 0.2 M acetate buffer, 30 °C, wherein the L-arginine concentrations were varied from 0.1 to 0.4 M. The degradation rates were all first order, and the cefepime degradation rate constants were moderately increased by the addition of L-arginine. The insert in Figure 6 shows T90 as a function of L-arginine concentration at 30 °C, pH 4.6. The intercept of this plot is the T90 value (∼2.2 days) for cefepime degradation in the 0.2 M acetate buffer solution in the absence of L-arginine. The T90 value was reduced to ∼1.7 days in the most concentrated buffered L-arginine solution.
1. Okamoto, M. P.; Nakahiro, R. K.; Chin, A.; Bedikian, A.; Gill, M. A. Cefepime: A New Fourth-Generation Cephalosporin. Am. J. Hosp. Pharm. 1994, 51, 463-477. 2. Wynd, M.; Paladino, J. Cefepime: A Fourth-Generation Parenteral Cephalosporin. Ann. Pharmacother. 1996, 30, 1414-1423. 3. Okamoto, M. P.; Nakahiro, R. K.; Chin, A.; Bedikian, A. Cefepime Clinical Pharmacokinetics. Clin. Pharmacokin. 1993, 25, 88-102. 4. Physicians’ Desk Reference, 52nd ed., Medical Economics Company Inc.: Montvale, NJ, 1998; Maxipime, pp 800-804. 5. Rabouan-Guyon, M. S.; Guet, F. A. Courtois, P. Y.; Barthes, M. C. Stability Study of Cefepime in Different Infusion Solutions. Int. J. Pharm. 1997, 154, 185-190. 6. Fubara, J. O.; Ph.D. Dissertation, Medical University of South Carolina, Charleston, SC, 1998. 7. Harned, H. S.; Owen, B. B. The Physical Chemistry of Electrolytic Solutions, 3rd ed.; Reinhold: New York, 1958; pp 638, 748. 8. Connors, K. A. Chemical Kinetics; VCH: New York, 1990; pp 17-58, 245-292. 9. Carstensen, J. T. Drug Stability, 2nd ed.; Marcel Dekker: New York, 1995; pp 17-121. 10. Connors, K. A.; Amidon, G. L.; Stella V. J. Chemical Stability of Pharmaceuticals, 2nd ed.; John Wiley & Sons: New York, 1986; pp 8-31, 43-62, 302-321. 11. Wang, D.; Notari, R. E. Cefuroxime Degradation Kinetics and Stability Predictions in Aqueous Solutions. J. Pharm. Sci. 1994, 83, 577-581. 12. Yamaha, T.; Tsuji, A. Comparative Stability of Cephalosporins in Aqueous Solution: Kinetics and Mechanisms of Degradation. J. Pharm. Sci. 1976, 65, 1563-1573. 13. Scientist, MicroMath Scientific Software: Salt Lake City, UT. 14. Metzger, J. V. Thiazole & Its Derivatives, Part 1; John Wiley & Sons: New York, 1979; pp 91-94.
Acknowledgments Summary This research applied classical chemical kinetic analyses to the study of cefepime degradation in aqueous solutions. The completed study provides equations that successfully calculated the experimentally determined degradation rate
1576 / Journal of Pharmaceutical Sciences Vol. 87, No. 12, December 1998
We thank Dr. Robert A. Lipper, Vice President, Biopharmaceutics R & D, Bristol-Myers Squibb Co., for supplying cefepime. Josephine Fubara gratefully acknowledges receiving the Earl Higgins Fellowship and an American Foundation for Pharmaceutical Education Predoctoral Fellowship.
JS980170Y