http://informahealthcare.com/phd ISSN: 1083-7450 (print), 1097-9867 (electronic) Pharm Dev Technol, Early Online: 1–7 ! 2013 Informa Healthcare USA, Inc. DOI: 10.3109/10837450.2013.852571
RESEARCH ARTICLE
Solid-state stability and compatibility studies of clavulanate potassium
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Judyta Cielecka-Piontek1, Magdalena Paczkowska1, Przemysław Zalewski1, Kornelia Lewandowska2, and Bolesław Barszcz2 1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Poznan´, Poland and Department of Molecular Crystals, Institute of Molecular Physics, Polish Academy Sciences, Poznan´, Poland
2
Abstract
Keywords
The kinetic and thermodynamic parameters of degradation of clavulanate potassium in the solid state were studied by using a reversed phase high performance liquid chromatography (RP-HPLC) method. The degradation of clavulanate potassium was a first-order reaction depending on the substrate concentration at an increased relative air humidity (RH) and in dry air. The dependence ln k ¼ f(1/T) became the ln k ¼ (0.026 166.35)–(2702.82 1779.43)(1/T) in dry air and ln k ¼ (1.65 100.40) 103(5748.81 3659.67)(1/T) at 76.4% RH. The thermodynamic parameters Ea, DH6¼a, DS6¼a of the degradation of clavulanate potassium in the solid state were calculated. The dependence ln k ¼ f (RH%) assumed the form ln k ¼ (8.78 5.75) 10 2 (RH%) þ (2.64 108 40.41). The compatibility of clavulanate potassium with commonly used excipients was studied at an increased temperature and in dry air. The geometric structure of molecule, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals were also determined in order to predict the structural changes and reactive sites in clavulanate potassium during degradation and compatibility studies in the solid state. The ultraviolet (UV), Fourier transform infrared spectroscopy (FT-IR) and Raman spectra of degraded samples of the compound were analyzed.
Compatibility studies, pharmaceutical analysis, stability studies
Introduction The increasing resistance of bacteria is often a cause of the therapeutic failure of antibiotics, a problem that may be solved by applying chemotherapeutic agents with different mechanisms of antibacterial activity which are able to act synergistically. A combination of b-lactam antibiotics and b-lactamase inhibitors is known to produce a highly efficient bactericidal activity1–3, with clavulanic acid used as the most common inhibitor. Recent clinical studies demonstrated a particular efficiency of its connection with other antibiotics, such as analogs of carbapenem or oxazolidinone4–6. A similarity between the chemical structure of clavulanic acid and b-lactam antibiotics allows a molecule of the acid interact with the enzyme b-lactamase, while protecting the 4:5 fused b-lactam and heterocyclic rings, which are responsible for bactericidal activity. As a result, clavulanic acid, without showing any bactericidal activity, increases the effectiveness of antibiotics susceptible to degradation by b-lactamase7,8. Clavulanic acid is characterized by a significant enzymatic instability and, similarly to b-lactam analogs, is chemically unstable9–12. Several studies investigated the chemical stability of clavulanic acid in aqueous solutions10,13,14 and the products of its degradation (Figure 1) were analyzed15–17. Haginaka et al. reported that the degradation of clavulanic acid was a pseudofirst-order reaction under the conditions of acid–base hydrolysis13
History Received 11 April 2013 Revised 13 August 2013 Accepted 2 October 2013 Published online 8 November 2013
and proved a catalytic effect of degradation products on the rate of degradation of clavulanic acid18. The stability of clavulanic acid was found to be a function of pH (4.0–8.0) and temperature (293–318 K). Such salts as Na2SO44MgSO44CaCl24NaCl affected the stability of clavulanic acid and its degradation appeared to be a function of their ionic strengths9. Compatibility studies of clavulanic acid were reported for its binary systems with amoxicillin and ampicillin when molar enthalpies of b-lactam analogs in various solutions were established19. Nugrahani et al. suggested that the co-crystal system of clavulanic acid and amoxicillin in the solid state was a result of strong hydrogen bonding between the hydrates of b-lactam analogs and clavulanate potassium20. Such studies may be important to predict the behavior of the active pharmaceutical ingredient (API) during a process of technological process of development of new pharmaceutical formulations21,22. In light of the findings of previous studies reporting the problem of the significant susceptibility to degradation of clavulanate potassium, the aim of our studies was to evaluate its stability in the solid state and to examine its compatibility with excipients. To the best of our knowledge, such studies in the solid state for clavulanate potassium have not been reported.
Materials and methods Materials
Address for correspondence: Judyta Cielecka-Piontek, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan´, Poland. Tel: +48600288065. E-mail: [email protected]
Clavulanic acid potassium salt, provided by Chemos GmbH (Regenstauf, Germany), was a white or almost white powder23. All other chemicals and solvents were obtained from Merck KGaA (Darmstadt, Germany) and were of analytical grade. High
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H OH
O
N O
N
OH
COOK
O H2N
HO
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OH
N
HO
N
OH
HO
N
N
OH
N
COOK
Figure 1. Chemical structures of clavulanate potassium and its main degradation products in solutions.
quality pure water was prepared using an Exil SA 67120 purification system (Millipore, Billerica, MA). For the compatibility studies of clavulanate potassium, the following excipients were used: mannitol (Merck KGaA, Darmstadt, Germany), microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA), pregelatinized starch (Colorcon, Harleysville, PA), lactose anhydrous (DFE Pharma, Goch, Germany), lactose monohydrate (DFE Pharma, Goch, Germany), magnesium stearate (JRS Pharma, Rosenberg, Germany), sodium stearyl fumarate (Merck KGaA, Darmstadt, Germany), hydroxypropyl methylcellulose (HARKE Services GmbH, Muelheim an der Ruhr, Germany), colloidal silicon dioxide (Evonik Industries AG, Essen, Germany), talc (Merck KGaA, Darmstadt, Germany), croscarmellose sodium (FMC BioPolymer, Philadelphia, PA), Opadry White (Colorcon, Harleysville, PA) and Opadry Blue (Colorcon, Harleysville, PA). Analytical method Chromatographic separation and quantitative determination of clavulanate potassium were performed using a high-performance liquid chromatograph (HPLC) equipped with an LC-6A pump (Shimadzu, Kyoto, Japan), a ultraviolet visible (UV-VIS) (SPD6AV) detector (Shimadzu, Kyoto, Japan) and a Rheodyne with a 50 mL loop. As the stationary phase a LiChrospher RP-18 (Merck KGaA, Darmstadt, Germany), 5 mm particle size, 250 mm 4 mm was used. UV detection was performed at 220 nm. The mobile phase consisted of acetonitrile 12 mmol L1 solution of ammonium acetate (4:96 V/V). The flow rate was 1.0 mL min1. The first-derivative UV spectra of non-degraded samples of clavulanate potassium were recorded with a UV-VIS Lambda 20 (Perkin Elmer, Waltham, MA) spectrophotometer equipped with 1.0 cm-in-width quartz cells and controlled via the UV WinLab software. The infrared spectra of clavulanate potassium were recorded between 400 and 7000 cm1 in polycrystalline powder, at room temperature, with an FT-IR Bruker Equinox 55 spectrometer equipped with a Bruker Hyperion 1000 microscope. Raman scattering spectra were obtained with a Bruker IFS 66 FT-Raman spectrometer with laser excitation exc ¼ 1064 nm from the Nd:YAG laser. In each case, the power of the laser beam at the sample was less than 1 mW to avoid damages of the sample.
Theoretical calculations The optimization of the molecular geometry (MG), spatial electron distribution of frontier molecular orbitals (FMOs) analysis of clavulante potassium were obtained with density functional theory calculations using Becke’s three-parameter hybrid functional (B3LYP) implemented with the standard 6-31(d,p) as a basis set. The harmonic vibrational frequencies of Fourier transform infrared spectroscopy (FT-IR) and Raman spectra have been calculated using the same level of theory. All the calculations were made by using Gaussian 03 package and GaussView24. Kinetic and compatibility studies For the forced aging test, 5 mg samples of clavulanate potassium were weighed into 5 mL vials. To evaluate their stability at an increased air humidity, they were placed in heat chambers at 343, 353, 363 and 373 K in desiccators containing saturated solutions of inorganic salts: sodium bromide (50.9% RH), sodium nitrate (66.5% RH), sodium chloride (76.5% RH) and zinc sulfate (90.0% RH). To evaluate the stability of clavulanate potassium in dry air, the vials were immersed in a sand bath placed in heat chambers at 363, 373, 383 and 393 K. At specified time intervals, determined by the rate of degradation, the vials were removed, cooled to room temperature and their contents were dissolved in distilled water and analyzed by HPLC at a concentration 0.2 mg mL1. In order to study the compatibility of a 1:1 mixture of clavulanate potassium with commonly used excipients (mannitol, microcrystalline cellulose, pregelatinized starch, lactose anhydrous, lactose monohydrate, magnesium stearate, sodium stearyl fumarate, hydroxypropyl methylcellulose, colloidal silicon dioxide, talc, croscarmellose sodium, Opadry White, Opadry Blue), samples of clavulanate potassium were weighed into 5 mL vials. For the compatibility studies of potassium clavulanate, mixtures of it with selected excipient were prepared without water. Mixtures were stored under stressed conditions. The stability of the so-obtained mixtures was investigated at an increased RH (76.5%) at 303 and in dry air (0% RH) at 313 K. At specified time intervals, determined by the rate of degradation, the vials were removed, cooled to room temperature and their contents were dissolved in distilled water and analyzed by HPLC.
Solid-state stability and compatibility studies
DOI: 10.3109/10837450.2013.852571
3
100
333 K
CD (%) 10
343 K 353 K 363 K
1
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0
10
20 30 Time [min]
40
50
Figure 2. Semilogarithmic plots of cD(%) ¼ f(t) for the degradation of clavulanate potassium in the solid state in dry air, where cD is the concentration of clavulanate potassium, cD0 is the concentration of clavulanate potassium at 0 h, and cDt is the concentration of clavulanate potassium at t min.
Results and discussion To assess changes in the concentration of clavulanate potassium during degradation in the solid state, an HPLC-UV method was used and its selectivity was validated25. The lack of substituents in chromophores in clavulanate potassium made it difficult to apply HPLC coupled with a UV detector for the determination of degradation products formed during thermolysis. Therefore, the current stability study of clavulanate potassium was obtained by comparing changes in the UV and FT-IR, Raman spectra of non-degraded and degraded samples of clavulanate potassium. Stability studies of clavulanate potassium conducted at an increased RH (50.0–76.4% RH) and in dry air showed that its degradation was a first-order reaction depending on the substrate concentration described by the equation: ln Pcp ¼ ln Pcp0 kobs t During the degradation of clavulanate potassium, its concentration decreased in the time interval t0 ! t1 from (Pcp)max to (Pcp)0. The observed rate constants were equal to the slopes of the plots ln(Pcp) ¼ f(t) with a negative sign (kobs) (Figure 2). As expected, the exposure of the clavulanate potassium samples to an increased air humidity caused their faster degradation compared to when they were exposed to dry air. However, the kinetic mechanism of the degradation of clavulanate potassium under those conditions did not differ, which demonstrated that the degradation products formed in the solid phase did not have any catalytic effect. It should be noted that the acceleration of degradation of clavulanate potassium by products of hydrolysis was observed in its solutions17. A similar degradation mechanism was reported in the case of other b-lactam analogs that were influenced by the catalytic effect of solid-state degradation products26–28. A comparison of the observed solid-state degradation rate constants of clavulanate potassium with those of some b-lactam analogs obtained under the same experimental conditions demonstrated its lower stability relative to those carbapenems, which was especially notable in the range 313–333 K, in dry air. Regarding the technology of binary or multicomponent preparations, the findings of the current study suggest that clavulanate potassium will remain the key factor enhancing the stability of APIs. On the basis of the equilibrium geometry and the spatial distribution of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of clavulanate potassium established in this study, it was possible to predict the introduction of an oxygen atom at C-4 4:5 fused rings and the lack
Figure 3. HOMO and LUMO orbitals of clavulanate potassium. 1.00E-04
ki (s-1) 1.00E-05
76.4% RH 0% RH
1.00E-06 2.50E-03 2.70E-03 2.90E-03 3.10E-03 3.30E-03 3.50E-03 1/T (K-1)
Figure 4. Semilogarithmic relationship ki ¼ f(1/T) for the degradation of clavulanate potassium at 76.4% RH and at 0% RH in the solid state.
of an anomeric effect resulting from the presence of nitrogen and sulfur atoms the vinyl groups as the reasons for the greater susceptibility of clavulanate potassium to degradation in the solid state as compared to b-lactam analogs (Figure 3). The impact of temperature on the stability of clavulanate potassium was analyzed at 313–343 K, 76.5% RH and at 313– 343 K, in dry air (Figure 4). The rate of its degradation in the solid state as a function of temperature was expressed by the Arrhenius relationship: ln ki ¼ ln A Ea =RT where ki – reaction rate constants of clavulanate potassium (s1); A – frequency coefficient; Ea – activation energy (J mol1); R – universal gas constant (8.3144 J K1 mol1); T – temperature (K). A good correlation (r ¼ 0.9788 for degradation at 76.4% RH and r ¼ 0.9774 at 0% RH) was observed for the Arrhenius relationships, based on which the slopes (a) and the frequency coefficient (ln A) allowed the calculation of activation energy (Ea ¼ a R), enthalpy (DH6¼) and entropy (DS6¼) at 298 K (Table 1). A comparison of the activation energy values of degradation in the solid state with those obtained for the degradation of penam, cephem, carbapenem and thiopenem
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Table 1. Kinetic and thermodynamic parameters of the degradation of clavulanate potassium in the solid state at 0% and 76.4% RH. (k Dk) (s1)
Temperature (K)
Thermodynamic parameters
(74.47 0.43) 107 (109.26 0.89) 107 (119.70 0.40) 107 (152.84 0.80) 107
a ¼ 2702 1779 Sa ¼ 413 b ¼ 0.026 166.35, Sb ¼ 1.19 r ¼ 0.9774
Ea ¼ 22.47 14.79 (kJ mol1) DH6¼a ¼ 19.99 17.27 (kJ mol1) DS6¼a ¼ 274.52 202.43 (J K1 mol1)
(87.76 0.41) 107 (213.89 0.15) 107 (273.64 0.50) 107 (552.01 4.72) 107
a ¼ 5748 3659 Sa ¼ 850 b ¼ 1653 100 402, Sb ¼ 2.68 r ¼ 0.9788
Ea ¼ 47.80 30.42 (kJ mol1) DH6¼a ¼ 45.32 32.90 (kJ mol1) DS6¼a ¼ 183.31 149.14 (J K1 mol1)
Table 2. Effect of relative air humidity on the stability of clavulanate potassium at 313 K. Relative air humidity (%) 50.9 60.5 66.5 76.5
(k Dk) (s1)
Statistical evaluation of ln ki ¼ f (1/T)
(25.87 0.12) 107 (40.05 0,37) 107 (10.65 0.85) 107 (21.99 0.15) 107
a ¼ 0.088 0.058, Sa ¼ 0.013 b ¼ 2.64 108 40.41, Sb ¼ 0.86 r ¼ 0.9776
analogs indicated that clavulanate potassium was more prone to degradation than those b-lactam analogs. The entropy of the degradation of clavulante potassium in dry air was negative, which may indicate a bimolecular character of degradation. The values of the degradation rate constants of clavulanate potassium under the conditions of an increased temperature as well as increased RH were approximately twice as those recorded in dry air, suggesting that the attack of a water molecule on the carbonyl group of the b-lactam ring induced thermolysis. The influence of RH on the stability of clavulanate potassium was described thus: ln k ¼ ð8:78 5:75Þ102 ðRH%Þ þ 2:64 108 40:41 : The slope a expresses the effect of humidity on the stability of clavulanate potassium in the solid state and the value 10 b denotes its stability at 0% RH (Table 2). The following statistical parameters of the equation y ¼ ax þ b were calculated by using the least squares method: a Da, b Db, standard deviations Sa, Sb, Sy and the coefficient of linear correlation r. The values Da and Db were calculated for f ¼ n2 degrees of freedom and ¼ 0.05. A compatibility study of the impact of excipients on the stability of clavulanate potassium was conducted at an increased RH (T ¼ 303 K, 76.4% RH) and in dry air (T ¼ 313 K, 0% RH). Regardless of experimental conditions and excipients, the kinetic mechanism of degradation of clavulanate potassium did not differ. At an increased RH, a total degradation of clavulanate potassium was observed after a period of 12 d. The rate of its degradation in binary or multicomponent mixtures with excipients was greater than that in the absence of excipients and the difference was statistically significant. Brethauer et al. observed a catalytic effect in the degradation of clavulanate potassium in solutions at concentrations 0.25–2.0 mg mL1, which, in addition to the main degradants (1-amin-2-oxo-butan-4-ol, 2,5-bis(2-hydroxyethyl)pyrazine, 3-carboxyethyl-2,5-bis(2-hydroxyethyl)pyrazine, 3-ethyl-2,5-bis(2-hydroxyethyl)pyrazine), was tentatively attributed to carbon dioxide and acetaldehyde (intermediate and final degradation products)17. A similar process may be responsible for the rapid degradation of clavulanate potassium under the conditions of the current study. In order to determine changes in the structure of clavulanate potassium during its degradation at an increased RH and in dry
Transmitance
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0% RH 333 343 353 363 76.4% RH 303 313 323 333
Statistical evaluation of ln ki ¼ f (1/T)
400
600
800
1000
1200
1400
1600
1800
3000
3600
-1
Wavenumber (cm )
Figure 5. Experimental FT-IR spectra for non-degraded and degraded clavulanate potassium (1 – non-degraded sample; 2 – degraded samples at dry air; 3 – degraded samples at an increased relative air humidity).
air, the infrared and Raman scattering spectra of clavulanate potassium were recorded for non-degraded and degraded samples (Figure 5). The FT-IR and Raman scattering spectra clavulanate potassium were obtained and compared with the theoretical spectra based on the density functional theory. The differences between them were collected in Table 3. A comparison of the results for clavulanate potassium before and after degradation in dry air revealed slight alterations in band positions and considerable changes in the intensity of the vibrational bands (Figures 6 and 7). Namely, it was observed that the changes occurred in the bands corresponding to the stretching and bending vibrations of the C–N and C–N–C bonds in the b-lactam and the breathing b-lactam rings located in the experimental IR spectra at about 658, 944, 1229, 1308 and 1354 cm1. Similar changes in the Raman scattering spectra were also noted (Table 3). A complete disappearance of these bands did not occur as they contained some unbroken components associated with the stretching vibrations of the C–C bond between the b-penem ring and the COOH group, the stretching vibrations of the C–C bond in the b-penem ring and the vibrations of the C–H bond in the b-penem ring. No changes were evidenced in the region of the spectrum containing the bands corresponding to the stretching vibrations of the C–C and C–O bonds in the b-penem and the breathing b-penem rings (896, 1091, 1151 cm1), which suggested that this part of the molecule was not destroyed, analogous to the bands associated with the stretching vibrations of the C ¼ C and C ¼ O bonds located at 1603, 1702 and 1798 cm1.
896 944
1041 1091
1151 1229 1308 1354 1384 1603 1702 1798 2870 2911 2961 3015
885 936
1040 1084
1153 1230 1315 1351 1465 1777 1857 1916 2961 3042 3074 3101
1151 1230 1298 1357 1384 1606 1702 1799 2873 2912 2959 3015
1045 1095
897 944
661 768
Raman
1152 1229 1308 1354 1384 1603 1702 1798 2870 2911 2961 3015
1041 1091
896 944
658 766
IR
1151 1230 1298 – 1380 1606 1702 1799 2873 2911 2959 3015
1045 1095
897 944
661 768
Raman
RH ¼ 0%, T ¼ 313 K
Vibrational modes: s – stretching, b – bending, w – wagging, t – twisting.
658 766
IR
628 740
Calculation (cm )
1
Non-degraded clavulanate potassium
1163 – 1315 – 1398 1626 – – – – – –
1034 –
896 –
659 –
IR
1151 – 1294 – 1380 1606 1702 1799 – 2908 – –
– 1095
900 944
661 –
Raman
RH ¼ 76.5%, T ¼ 303 K
Degraded clavulanate potassium
Experimental (cm1)
Band assignment O–H w in COOH group þ C–N–C b in b-lactam ring CH2 t at b-lactam ring þ breathing peneme ring þ C–C s between b-lactam ring and COOH group þ C–O–H b in COOH group C–C s þ C–O s in b-lactam ring C–C s between b-lactam ring and COOH group þ C–N s in b-lactam ring C–O s in 2-hydroxyethylidene group C–O s in b-lactam ring þ C–O s in 2-hydroxyethylidene group þ CH2 t at b-lactam ring C–O s in COOH group þ CH2 t at b-lactam ring C–N s in b-lactam ring þ C–C s in b-lactam ring þ CH2 t þ O–H b CH2 w/t at b-lactam ring þ C–N s in b-lactam ring C–H w at b-lactam ring þ C–N s in b-lactam ring CH2 w in 2-hydroxyethylidene group C¼C s C ¼ O s in COOH group C ¼ O s at b-lactam ring C–H s in acetaldehyde group C–H s in acetaldehyde group CH2 s at b-lactam ring C–H s at b-lactam ring
Table 3. Main characteristic experimental and calculated vibrational modes for non-degraded and degraded samples of clavulanate potassium.
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DOI: 10.3109/10837450.2013.852571
Solid-state stability and compatibility studies 5
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400
600
800
1000
1200
1400
1600
1800
3000
3600
-1
Wavenumber (cm )
Figure 6. Experimental Raman spectra for non-degraded and degraded clavulanate potassium (1 – non-degraded sample; 2 – degraded samples at dry air; 3 – degraded samples at an increased relative air humidity).
The analysis of the FT-IR and Raman spectra of the clavulanate potassium after degradation at an increased RH showed an almost complete disappearance of all characteristic bands, suggesting the destruction of not only the b-lactam ring but of the whole molecule under such conditions. Those findings were proved by analyzing the absorption spectra of degraded samples of clavulanate potassium. As a result of the use of derivative spectrophotometry it was possible to selectively determine clavulanate potassium relative to its degradation products. The spectrophotometric methods (first-derivative zero-absorption spectrum and FT-IR) used in this work showed that dimers were not formed during the degradation of clavulanate potassium in the solid state in contrast to the degradation of b-lactam analogs in the solid state when dimers appeared and produced characteristic shifts (Figure 7a and b)29–31. A compatibility study of clavulanate potassium in binary or multicomponent mixtures with excipients in dry air at T ¼ 313 K was performed over a period of 8 weeks. The greatest loss of clavulanate potassium was found as the result of first-reaction degradation, in the presence of hydroxypropyl methylcellulose, sodium stearyl fumarate and Opadry White (Figure 8), which was not necessarily caused by a catalytic effect and according to Waterman et al. could be a consequence of non-catalytic interactions between excipients and API molecules32. The observed rate constants of clavulante potassium in binary or
Figure 7. First-derivative spectra of clavulanate potassium during degradation in dry air at 323 K, t ¼ 0–20 h (A) and at increased relative humidity at 76.5% RH, 323 K, t ¼ 120 min (B). 100 95 90 % degradation
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Intensity
6
85 80 75 70 65
0
60
3 weeks
55
8 weeks
50
Figure 8. Diagram of compatibility studies of clavulanate potassium in mixture with excipients.
Solid-state stability and compatibility studies
DOI: 10.3109/10837450.2013.852571
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Table 4. Kinetic parameters of the degradation of clavulanate potassium in the solid state in mixtures with selected excipients. Excipient
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Mannitol Microcrystalline cellulose 101 Microcrystalline cellulose 102 Pregelatinized starch Pregelatinized starcap Lactose anhydrous Lactose monohydroate Talk Colloidal silicon dioxide (aerosol)
(k Dk) (s1) (3.31 1.02) (1.35 0.43) (4.38 0.59) (2.20 0.60) (2.23 0.70) (1.01 0.67) (3.12 0.69) (4.37 0.59) (3.19 0.69)
108 109 108 108 108 108 108 108 108
multicomponent mixture with selected excipients were collected in Table 4.
Conclusions Since clavulanate potassium is vulnerable to degradation in the solid state, special care should be taken during its storage in bulk and in pharmaceutical dosage form. A significant instability of the acid can also be key parameter during the technological processes of development of new pharmaceutical dosage forms. As expected, the solid-state stability and compatibility studies presented in this work proved air humidity to be the most important factor affecting the degradation of clavulanate potassium.
Declaration of interest This study was supported by a grant from the Foundation for Polish Science (no. VENTURES/2011-8/7).
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Excipient Magnesium stearate Sodium stearyn fumarate Croscarmellose sodium Crospovidone HMPC pharmacoat HPMC methocel Opadry White Opadry Blue
(k Dk) (s1) (2.02 0.44) (4.54 0.89) (2.87 0.87) (2.43 0.44) (4.03 1.32) (2.72 0.56) (4.04 0.86) (3.97 1.21)
108 108 108 108 108 108 108 108
14. Haginaka J, Yasuda H, Uno T, Nakagawa T. Degradation of clavulanic acid in aqueous alkaline solution – isolation and structural investigation of degradation products. Chem Pharm Bull 1985;33:218–224. 15. Lynch HC, Yang Y. Degradation products of clavulanic acid promote clavulanic acid production in cultures of Streptomyces clavuligerus. Enzyme Microbial Technol 2004;34:48–54. 16. Bersanettia PA, Almeidab RM, Barbozac M, et al. Kinetic studies on clavulanic acid degradation. Biochem Eng J 2005;23:31–36. 17. Brethauer S, Held M, Panke S. Clavulanic acid decomposition is catalyzed by the compound itself and by its decomposition products. J Pharm Sci 2008;97:3451–3455. 18. Martin J, Mendez R, Salto F. Studies on clavulanic acid 2. The catalytic effect of metal-ions on the hydrolysis of clavulanic acid. J Chem Soc Perkin Trans 1989;2:227–231. 19. Chadha R, Kashid N, Jain DV. Microcalorimetric evaluation of the in vitro compatibility of amoxicillin/clavulanic acid and ampicillin/ sulbactam with ciprofloxacin. J Pharm Biomed Anal 2004;36: 295–307. 20. Nugrahani I, Asyarie S, Soewandhi SN, Ibrahim S. The antibiotic potency of amoxicillin-clavulanate co-crystal. Int J Pharmacol 2007; 3:475–481. 21. Antovska P, Petrusevski G, Mareski P. Solid-state compatibility screening of excipients suitable for development of indapamide sustained release solid-dosage formulation. Pharm Dev Tech 2013;8: 481–489. 22. Parmar N, Amin S, Singla N, Kohli K. The solution, solid state stability and kinetic investigation in degradation studies of lercanidipine: study of excipients compatibility of lercanidipine. Pharm Dev Tech 2012;6:730–740. 23. The European Pharmacopoeia VIII. Strasbourg, France; 2010. 24. Frisch M, Trucks G, Schlegel H, et al. Gaussian 03, Revision B.05. Pittsburgh (PA): Gaussian, Inc.; 2003. 25. Cielecka-Piontek J, Paczkowska M, Zalewski P, et al. Theoretical and experimental analytical studies on potassium clavulanate. Curr Issues Pharmacy Med Sci 2012;25:317–321. 26. Sajonz P, Natishan TK, Wu Y, et al. Development and validation of a sensitive and robust wipe-test method for the detection and quantification of the antibiotic ertapenem and its primary degradates in a pharmaceutical manufacturing environment. J Liquid Chromatogr Relat Technol 2005;28:713–725. 27. Ito N, Suzuki M, Kusai A, Takayama K. Effect of initial concentration on stability of panipenem in aqueous solution. Chem Pharm Bull 2005;53:323–327. 28. Cielecka-Piontek J, Jelin´ska A. Catalytic effect of buffers on the degradation of doripenem in aqueous solutions. React Kinet Mech Cat 2011;102:37–39. 29. Hassan N, Abdel-Moety E, Elragehy N, Rezk M. Selective determination of ertapenem in the presence of its degradation product. Spectrochim Acta A 2009;72:915–917. 30. Cielecka-Piontek J, Lunzer A, Jelin´ska A. Stability-indicating derivative spectrophotometry method for the determination of biapenem in the presence of its degradation products. Cent Eur J Chem 2011;9:35–38. 31. Cielecka-Piontek J, Jelin´ska A. The UV-derivative spectrophotometry for the determination of doripenem in the presence of its degradation products. Spectrochim Acta A 2010;77:554–557. 32. Waterman KC, Gerst P, Dai Z. A generalized relation for solid-state drug stability as a function of excipient dilution: temperatureindependent behavior. J Pharm Sci 2012;101:4170–4177.
RESEARCH ARTICLE
Solid-state stability and compatibility studies of clavulanate potassium
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Judyta Cielecka-Piontek1, Magdalena Paczkowska1, Przemysław Zalewski1, Kornelia Lewandowska2, and Bolesław Barszcz2 1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Poznan´, Poland and Department of Molecular Crystals, Institute of Molecular Physics, Polish Academy Sciences, Poznan´, Poland
2
Abstract
Keywords
The kinetic and thermodynamic parameters of degradation of clavulanate potassium in the solid state were studied by using a reversed phase high performance liquid chromatography (RP-HPLC) method. The degradation of clavulanate potassium was a first-order reaction depending on the substrate concentration at an increased relative air humidity (RH) and in dry air. The dependence ln k ¼ f(1/T) became the ln k ¼ (0.026 166.35)–(2702.82 1779.43)(1/T) in dry air and ln k ¼ (1.65 100.40) 103(5748.81 3659.67)(1/T) at 76.4% RH. The thermodynamic parameters Ea, DH6¼a, DS6¼a of the degradation of clavulanate potassium in the solid state were calculated. The dependence ln k ¼ f (RH%) assumed the form ln k ¼ (8.78 5.75) 10 2 (RH%) þ (2.64 108 40.41). The compatibility of clavulanate potassium with commonly used excipients was studied at an increased temperature and in dry air. The geometric structure of molecule, highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) orbitals were also determined in order to predict the structural changes and reactive sites in clavulanate potassium during degradation and compatibility studies in the solid state. The ultraviolet (UV), Fourier transform infrared spectroscopy (FT-IR) and Raman spectra of degraded samples of the compound were analyzed.
Compatibility studies, pharmaceutical analysis, stability studies
Introduction The increasing resistance of bacteria is often a cause of the therapeutic failure of antibiotics, a problem that may be solved by applying chemotherapeutic agents with different mechanisms of antibacterial activity which are able to act synergistically. A combination of b-lactam antibiotics and b-lactamase inhibitors is known to produce a highly efficient bactericidal activity1–3, with clavulanic acid used as the most common inhibitor. Recent clinical studies demonstrated a particular efficiency of its connection with other antibiotics, such as analogs of carbapenem or oxazolidinone4–6. A similarity between the chemical structure of clavulanic acid and b-lactam antibiotics allows a molecule of the acid interact with the enzyme b-lactamase, while protecting the 4:5 fused b-lactam and heterocyclic rings, which are responsible for bactericidal activity. As a result, clavulanic acid, without showing any bactericidal activity, increases the effectiveness of antibiotics susceptible to degradation by b-lactamase7,8. Clavulanic acid is characterized by a significant enzymatic instability and, similarly to b-lactam analogs, is chemically unstable9–12. Several studies investigated the chemical stability of clavulanic acid in aqueous solutions10,13,14 and the products of its degradation (Figure 1) were analyzed15–17. Haginaka et al. reported that the degradation of clavulanic acid was a pseudofirst-order reaction under the conditions of acid–base hydrolysis13
History Received 11 April 2013 Revised 13 August 2013 Accepted 2 October 2013 Published online 8 November 2013
and proved a catalytic effect of degradation products on the rate of degradation of clavulanic acid18. The stability of clavulanic acid was found to be a function of pH (4.0–8.0) and temperature (293–318 K). Such salts as Na2SO44MgSO44CaCl24NaCl affected the stability of clavulanic acid and its degradation appeared to be a function of their ionic strengths9. Compatibility studies of clavulanic acid were reported for its binary systems with amoxicillin and ampicillin when molar enthalpies of b-lactam analogs in various solutions were established19. Nugrahani et al. suggested that the co-crystal system of clavulanic acid and amoxicillin in the solid state was a result of strong hydrogen bonding between the hydrates of b-lactam analogs and clavulanate potassium20. Such studies may be important to predict the behavior of the active pharmaceutical ingredient (API) during a process of technological process of development of new pharmaceutical formulations21,22. In light of the findings of previous studies reporting the problem of the significant susceptibility to degradation of clavulanate potassium, the aim of our studies was to evaluate its stability in the solid state and to examine its compatibility with excipients. To the best of our knowledge, such studies in the solid state for clavulanate potassium have not been reported.
Materials and methods Materials
Address for correspondence: Judyta Cielecka-Piontek, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan´, Poland. Tel: +48600288065. E-mail: [email protected]
Clavulanic acid potassium salt, provided by Chemos GmbH (Regenstauf, Germany), was a white or almost white powder23. All other chemicals and solvents were obtained from Merck KGaA (Darmstadt, Germany) and were of analytical grade. High
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H OH
O
N O
N
OH
COOK
O H2N
HO
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OH
N
HO
N
OH
HO
N
N
OH
N
COOK
Figure 1. Chemical structures of clavulanate potassium and its main degradation products in solutions.
quality pure water was prepared using an Exil SA 67120 purification system (Millipore, Billerica, MA). For the compatibility studies of clavulanate potassium, the following excipients were used: mannitol (Merck KGaA, Darmstadt, Germany), microcrystalline cellulose (FMC BioPolymer, Philadelphia, PA), pregelatinized starch (Colorcon, Harleysville, PA), lactose anhydrous (DFE Pharma, Goch, Germany), lactose monohydrate (DFE Pharma, Goch, Germany), magnesium stearate (JRS Pharma, Rosenberg, Germany), sodium stearyl fumarate (Merck KGaA, Darmstadt, Germany), hydroxypropyl methylcellulose (HARKE Services GmbH, Muelheim an der Ruhr, Germany), colloidal silicon dioxide (Evonik Industries AG, Essen, Germany), talc (Merck KGaA, Darmstadt, Germany), croscarmellose sodium (FMC BioPolymer, Philadelphia, PA), Opadry White (Colorcon, Harleysville, PA) and Opadry Blue (Colorcon, Harleysville, PA). Analytical method Chromatographic separation and quantitative determination of clavulanate potassium were performed using a high-performance liquid chromatograph (HPLC) equipped with an LC-6A pump (Shimadzu, Kyoto, Japan), a ultraviolet visible (UV-VIS) (SPD6AV) detector (Shimadzu, Kyoto, Japan) and a Rheodyne with a 50 mL loop. As the stationary phase a LiChrospher RP-18 (Merck KGaA, Darmstadt, Germany), 5 mm particle size, 250 mm 4 mm was used. UV detection was performed at 220 nm. The mobile phase consisted of acetonitrile 12 mmol L1 solution of ammonium acetate (4:96 V/V). The flow rate was 1.0 mL min1. The first-derivative UV spectra of non-degraded samples of clavulanate potassium were recorded with a UV-VIS Lambda 20 (Perkin Elmer, Waltham, MA) spectrophotometer equipped with 1.0 cm-in-width quartz cells and controlled via the UV WinLab software. The infrared spectra of clavulanate potassium were recorded between 400 and 7000 cm1 in polycrystalline powder, at room temperature, with an FT-IR Bruker Equinox 55 spectrometer equipped with a Bruker Hyperion 1000 microscope. Raman scattering spectra were obtained with a Bruker IFS 66 FT-Raman spectrometer with laser excitation exc ¼ 1064 nm from the Nd:YAG laser. In each case, the power of the laser beam at the sample was less than 1 mW to avoid damages of the sample.
Theoretical calculations The optimization of the molecular geometry (MG), spatial electron distribution of frontier molecular orbitals (FMOs) analysis of clavulante potassium were obtained with density functional theory calculations using Becke’s three-parameter hybrid functional (B3LYP) implemented with the standard 6-31(d,p) as a basis set. The harmonic vibrational frequencies of Fourier transform infrared spectroscopy (FT-IR) and Raman spectra have been calculated using the same level of theory. All the calculations were made by using Gaussian 03 package and GaussView24. Kinetic and compatibility studies For the forced aging test, 5 mg samples of clavulanate potassium were weighed into 5 mL vials. To evaluate their stability at an increased air humidity, they were placed in heat chambers at 343, 353, 363 and 373 K in desiccators containing saturated solutions of inorganic salts: sodium bromide (50.9% RH), sodium nitrate (66.5% RH), sodium chloride (76.5% RH) and zinc sulfate (90.0% RH). To evaluate the stability of clavulanate potassium in dry air, the vials were immersed in a sand bath placed in heat chambers at 363, 373, 383 and 393 K. At specified time intervals, determined by the rate of degradation, the vials were removed, cooled to room temperature and their contents were dissolved in distilled water and analyzed by HPLC at a concentration 0.2 mg mL1. In order to study the compatibility of a 1:1 mixture of clavulanate potassium with commonly used excipients (mannitol, microcrystalline cellulose, pregelatinized starch, lactose anhydrous, lactose monohydrate, magnesium stearate, sodium stearyl fumarate, hydroxypropyl methylcellulose, colloidal silicon dioxide, talc, croscarmellose sodium, Opadry White, Opadry Blue), samples of clavulanate potassium were weighed into 5 mL vials. For the compatibility studies of potassium clavulanate, mixtures of it with selected excipient were prepared without water. Mixtures were stored under stressed conditions. The stability of the so-obtained mixtures was investigated at an increased RH (76.5%) at 303 and in dry air (0% RH) at 313 K. At specified time intervals, determined by the rate of degradation, the vials were removed, cooled to room temperature and their contents were dissolved in distilled water and analyzed by HPLC.
Solid-state stability and compatibility studies
DOI: 10.3109/10837450.2013.852571
3
100
333 K
CD (%) 10
343 K 353 K 363 K
1
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0
10
20 30 Time [min]
40
50
Figure 2. Semilogarithmic plots of cD(%) ¼ f(t) for the degradation of clavulanate potassium in the solid state in dry air, where cD is the concentration of clavulanate potassium, cD0 is the concentration of clavulanate potassium at 0 h, and cDt is the concentration of clavulanate potassium at t min.
Results and discussion To assess changes in the concentration of clavulanate potassium during degradation in the solid state, an HPLC-UV method was used and its selectivity was validated25. The lack of substituents in chromophores in clavulanate potassium made it difficult to apply HPLC coupled with a UV detector for the determination of degradation products formed during thermolysis. Therefore, the current stability study of clavulanate potassium was obtained by comparing changes in the UV and FT-IR, Raman spectra of non-degraded and degraded samples of clavulanate potassium. Stability studies of clavulanate potassium conducted at an increased RH (50.0–76.4% RH) and in dry air showed that its degradation was a first-order reaction depending on the substrate concentration described by the equation: ln Pcp ¼ ln Pcp0 kobs t During the degradation of clavulanate potassium, its concentration decreased in the time interval t0 ! t1 from (Pcp)max to (Pcp)0. The observed rate constants were equal to the slopes of the plots ln(Pcp) ¼ f(t) with a negative sign (kobs) (Figure 2). As expected, the exposure of the clavulanate potassium samples to an increased air humidity caused their faster degradation compared to when they were exposed to dry air. However, the kinetic mechanism of the degradation of clavulanate potassium under those conditions did not differ, which demonstrated that the degradation products formed in the solid phase did not have any catalytic effect. It should be noted that the acceleration of degradation of clavulanate potassium by products of hydrolysis was observed in its solutions17. A similar degradation mechanism was reported in the case of other b-lactam analogs that were influenced by the catalytic effect of solid-state degradation products26–28. A comparison of the observed solid-state degradation rate constants of clavulanate potassium with those of some b-lactam analogs obtained under the same experimental conditions demonstrated its lower stability relative to those carbapenems, which was especially notable in the range 313–333 K, in dry air. Regarding the technology of binary or multicomponent preparations, the findings of the current study suggest that clavulanate potassium will remain the key factor enhancing the stability of APIs. On the basis of the equilibrium geometry and the spatial distribution of the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of clavulanate potassium established in this study, it was possible to predict the introduction of an oxygen atom at C-4 4:5 fused rings and the lack
Figure 3. HOMO and LUMO orbitals of clavulanate potassium. 1.00E-04
ki (s-1) 1.00E-05
76.4% RH 0% RH
1.00E-06 2.50E-03 2.70E-03 2.90E-03 3.10E-03 3.30E-03 3.50E-03 1/T (K-1)
Figure 4. Semilogarithmic relationship ki ¼ f(1/T) for the degradation of clavulanate potassium at 76.4% RH and at 0% RH in the solid state.
of an anomeric effect resulting from the presence of nitrogen and sulfur atoms the vinyl groups as the reasons for the greater susceptibility of clavulanate potassium to degradation in the solid state as compared to b-lactam analogs (Figure 3). The impact of temperature on the stability of clavulanate potassium was analyzed at 313–343 K, 76.5% RH and at 313– 343 K, in dry air (Figure 4). The rate of its degradation in the solid state as a function of temperature was expressed by the Arrhenius relationship: ln ki ¼ ln A Ea =RT where ki – reaction rate constants of clavulanate potassium (s1); A – frequency coefficient; Ea – activation energy (J mol1); R – universal gas constant (8.3144 J K1 mol1); T – temperature (K). A good correlation (r ¼ 0.9788 for degradation at 76.4% RH and r ¼ 0.9774 at 0% RH) was observed for the Arrhenius relationships, based on which the slopes (a) and the frequency coefficient (ln A) allowed the calculation of activation energy (Ea ¼ a R), enthalpy (DH6¼) and entropy (DS6¼) at 298 K (Table 1). A comparison of the activation energy values of degradation in the solid state with those obtained for the degradation of penam, cephem, carbapenem and thiopenem
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Table 1. Kinetic and thermodynamic parameters of the degradation of clavulanate potassium in the solid state at 0% and 76.4% RH. (k Dk) (s1)
Temperature (K)
Thermodynamic parameters
(74.47 0.43) 107 (109.26 0.89) 107 (119.70 0.40) 107 (152.84 0.80) 107
a ¼ 2702 1779 Sa ¼ 413 b ¼ 0.026 166.35, Sb ¼ 1.19 r ¼ 0.9774
Ea ¼ 22.47 14.79 (kJ mol1) DH6¼a ¼ 19.99 17.27 (kJ mol1) DS6¼a ¼ 274.52 202.43 (J K1 mol1)
(87.76 0.41) 107 (213.89 0.15) 107 (273.64 0.50) 107 (552.01 4.72) 107
a ¼ 5748 3659 Sa ¼ 850 b ¼ 1653 100 402, Sb ¼ 2.68 r ¼ 0.9788
Ea ¼ 47.80 30.42 (kJ mol1) DH6¼a ¼ 45.32 32.90 (kJ mol1) DS6¼a ¼ 183.31 149.14 (J K1 mol1)
Table 2. Effect of relative air humidity on the stability of clavulanate potassium at 313 K. Relative air humidity (%) 50.9 60.5 66.5 76.5
(k Dk) (s1)
Statistical evaluation of ln ki ¼ f (1/T)
(25.87 0.12) 107 (40.05 0,37) 107 (10.65 0.85) 107 (21.99 0.15) 107
a ¼ 0.088 0.058, Sa ¼ 0.013 b ¼ 2.64 108 40.41, Sb ¼ 0.86 r ¼ 0.9776
analogs indicated that clavulanate potassium was more prone to degradation than those b-lactam analogs. The entropy of the degradation of clavulante potassium in dry air was negative, which may indicate a bimolecular character of degradation. The values of the degradation rate constants of clavulanate potassium under the conditions of an increased temperature as well as increased RH were approximately twice as those recorded in dry air, suggesting that the attack of a water molecule on the carbonyl group of the b-lactam ring induced thermolysis. The influence of RH on the stability of clavulanate potassium was described thus: ln k ¼ ð8:78 5:75Þ102 ðRH%Þ þ 2:64 108 40:41 : The slope a expresses the effect of humidity on the stability of clavulanate potassium in the solid state and the value 10 b denotes its stability at 0% RH (Table 2). The following statistical parameters of the equation y ¼ ax þ b were calculated by using the least squares method: a Da, b Db, standard deviations Sa, Sb, Sy and the coefficient of linear correlation r. The values Da and Db were calculated for f ¼ n2 degrees of freedom and ¼ 0.05. A compatibility study of the impact of excipients on the stability of clavulanate potassium was conducted at an increased RH (T ¼ 303 K, 76.4% RH) and in dry air (T ¼ 313 K, 0% RH). Regardless of experimental conditions and excipients, the kinetic mechanism of degradation of clavulanate potassium did not differ. At an increased RH, a total degradation of clavulanate potassium was observed after a period of 12 d. The rate of its degradation in binary or multicomponent mixtures with excipients was greater than that in the absence of excipients and the difference was statistically significant. Brethauer et al. observed a catalytic effect in the degradation of clavulanate potassium in solutions at concentrations 0.25–2.0 mg mL1, which, in addition to the main degradants (1-amin-2-oxo-butan-4-ol, 2,5-bis(2-hydroxyethyl)pyrazine, 3-carboxyethyl-2,5-bis(2-hydroxyethyl)pyrazine, 3-ethyl-2,5-bis(2-hydroxyethyl)pyrazine), was tentatively attributed to carbon dioxide and acetaldehyde (intermediate and final degradation products)17. A similar process may be responsible for the rapid degradation of clavulanate potassium under the conditions of the current study. In order to determine changes in the structure of clavulanate potassium during its degradation at an increased RH and in dry
Transmitance
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0% RH 333 343 353 363 76.4% RH 303 313 323 333
Statistical evaluation of ln ki ¼ f (1/T)
400
600
800
1000
1200
1400
1600
1800
3000
3600
-1
Wavenumber (cm )
Figure 5. Experimental FT-IR spectra for non-degraded and degraded clavulanate potassium (1 – non-degraded sample; 2 – degraded samples at dry air; 3 – degraded samples at an increased relative air humidity).
air, the infrared and Raman scattering spectra of clavulanate potassium were recorded for non-degraded and degraded samples (Figure 5). The FT-IR and Raman scattering spectra clavulanate potassium were obtained and compared with the theoretical spectra based on the density functional theory. The differences between them were collected in Table 3. A comparison of the results for clavulanate potassium before and after degradation in dry air revealed slight alterations in band positions and considerable changes in the intensity of the vibrational bands (Figures 6 and 7). Namely, it was observed that the changes occurred in the bands corresponding to the stretching and bending vibrations of the C–N and C–N–C bonds in the b-lactam and the breathing b-lactam rings located in the experimental IR spectra at about 658, 944, 1229, 1308 and 1354 cm1. Similar changes in the Raman scattering spectra were also noted (Table 3). A complete disappearance of these bands did not occur as they contained some unbroken components associated with the stretching vibrations of the C–C bond between the b-penem ring and the COOH group, the stretching vibrations of the C–C bond in the b-penem ring and the vibrations of the C–H bond in the b-penem ring. No changes were evidenced in the region of the spectrum containing the bands corresponding to the stretching vibrations of the C–C and C–O bonds in the b-penem and the breathing b-penem rings (896, 1091, 1151 cm1), which suggested that this part of the molecule was not destroyed, analogous to the bands associated with the stretching vibrations of the C ¼ C and C ¼ O bonds located at 1603, 1702 and 1798 cm1.
896 944
1041 1091
1151 1229 1308 1354 1384 1603 1702 1798 2870 2911 2961 3015
885 936
1040 1084
1153 1230 1315 1351 1465 1777 1857 1916 2961 3042 3074 3101
1151 1230 1298 1357 1384 1606 1702 1799 2873 2912 2959 3015
1045 1095
897 944
661 768
Raman
1152 1229 1308 1354 1384 1603 1702 1798 2870 2911 2961 3015
1041 1091
896 944
658 766
IR
1151 1230 1298 – 1380 1606 1702 1799 2873 2911 2959 3015
1045 1095
897 944
661 768
Raman
RH ¼ 0%, T ¼ 313 K
Vibrational modes: s – stretching, b – bending, w – wagging, t – twisting.
658 766
IR
628 740
Calculation (cm )
1
Non-degraded clavulanate potassium
1163 – 1315 – 1398 1626 – – – – – –
1034 –
896 –
659 –
IR
1151 – 1294 – 1380 1606 1702 1799 – 2908 – –
– 1095
900 944
661 –
Raman
RH ¼ 76.5%, T ¼ 303 K
Degraded clavulanate potassium
Experimental (cm1)
Band assignment O–H w in COOH group þ C–N–C b in b-lactam ring CH2 t at b-lactam ring þ breathing peneme ring þ C–C s between b-lactam ring and COOH group þ C–O–H b in COOH group C–C s þ C–O s in b-lactam ring C–C s between b-lactam ring and COOH group þ C–N s in b-lactam ring C–O s in 2-hydroxyethylidene group C–O s in b-lactam ring þ C–O s in 2-hydroxyethylidene group þ CH2 t at b-lactam ring C–O s in COOH group þ CH2 t at b-lactam ring C–N s in b-lactam ring þ C–C s in b-lactam ring þ CH2 t þ O–H b CH2 w/t at b-lactam ring þ C–N s in b-lactam ring C–H w at b-lactam ring þ C–N s in b-lactam ring CH2 w in 2-hydroxyethylidene group C¼C s C ¼ O s in COOH group C ¼ O s at b-lactam ring C–H s in acetaldehyde group C–H s in acetaldehyde group CH2 s at b-lactam ring C–H s at b-lactam ring
Table 3. Main characteristic experimental and calculated vibrational modes for non-degraded and degraded samples of clavulanate potassium.
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DOI: 10.3109/10837450.2013.852571
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400
600
800
1000
1200
1400
1600
1800
3000
3600
-1
Wavenumber (cm )
Figure 6. Experimental Raman spectra for non-degraded and degraded clavulanate potassium (1 – non-degraded sample; 2 – degraded samples at dry air; 3 – degraded samples at an increased relative air humidity).
The analysis of the FT-IR and Raman spectra of the clavulanate potassium after degradation at an increased RH showed an almost complete disappearance of all characteristic bands, suggesting the destruction of not only the b-lactam ring but of the whole molecule under such conditions. Those findings were proved by analyzing the absorption spectra of degraded samples of clavulanate potassium. As a result of the use of derivative spectrophotometry it was possible to selectively determine clavulanate potassium relative to its degradation products. The spectrophotometric methods (first-derivative zero-absorption spectrum and FT-IR) used in this work showed that dimers were not formed during the degradation of clavulanate potassium in the solid state in contrast to the degradation of b-lactam analogs in the solid state when dimers appeared and produced characteristic shifts (Figure 7a and b)29–31. A compatibility study of clavulanate potassium in binary or multicomponent mixtures with excipients in dry air at T ¼ 313 K was performed over a period of 8 weeks. The greatest loss of clavulanate potassium was found as the result of first-reaction degradation, in the presence of hydroxypropyl methylcellulose, sodium stearyl fumarate and Opadry White (Figure 8), which was not necessarily caused by a catalytic effect and according to Waterman et al. could be a consequence of non-catalytic interactions between excipients and API molecules32. The observed rate constants of clavulante potassium in binary or
Figure 7. First-derivative spectra of clavulanate potassium during degradation in dry air at 323 K, t ¼ 0–20 h (A) and at increased relative humidity at 76.5% RH, 323 K, t ¼ 120 min (B). 100 95 90 % degradation
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Intensity
6
85 80 75 70 65
0
60
3 weeks
55
8 weeks
50
Figure 8. Diagram of compatibility studies of clavulanate potassium in mixture with excipients.
Solid-state stability and compatibility studies
DOI: 10.3109/10837450.2013.852571
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Table 4. Kinetic parameters of the degradation of clavulanate potassium in the solid state in mixtures with selected excipients. Excipient
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Mannitol Microcrystalline cellulose 101 Microcrystalline cellulose 102 Pregelatinized starch Pregelatinized starcap Lactose anhydrous Lactose monohydroate Talk Colloidal silicon dioxide (aerosol)
(k Dk) (s1) (3.31 1.02) (1.35 0.43) (4.38 0.59) (2.20 0.60) (2.23 0.70) (1.01 0.67) (3.12 0.69) (4.37 0.59) (3.19 0.69)
108 109 108 108 108 108 108 108 108
multicomponent mixture with selected excipients were collected in Table 4.
Conclusions Since clavulanate potassium is vulnerable to degradation in the solid state, special care should be taken during its storage in bulk and in pharmaceutical dosage form. A significant instability of the acid can also be key parameter during the technological processes of development of new pharmaceutical dosage forms. As expected, the solid-state stability and compatibility studies presented in this work proved air humidity to be the most important factor affecting the degradation of clavulanate potassium.
Declaration of interest This study was supported by a grant from the Foundation for Polish Science (no. VENTURES/2011-8/7).
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Excipient Magnesium stearate Sodium stearyn fumarate Croscarmellose sodium Crospovidone HMPC pharmacoat HPMC methocel Opadry White Opadry Blue
(k Dk) (s1) (2.02 0.44) (4.54 0.89) (2.87 0.87) (2.43 0.44) (4.03 1.32) (2.72 0.56) (4.04 0.86) (3.97 1.21)
108 108 108 108 108 108 108 108
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