Stability studies of cefoselis sulfate in the solid state

Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 222–226

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Stability studies of cefoselis sulfate in the solid state b a ´ ´ Przemysław Zalewski a,∗ , Robert Skibinski , Alicja Talaczynska , Magdalena Paczkowska a a a , Piotr Garbacki , Judyta Cielecka-Piontek a b

Department of Pharmaceutical Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Pozna´ n, Poland Department of Medicinal Chemistry, Medical University of Lublin, Jaczewskiego 4, 20-090 Lublin, Poland

a r t i c l e

i n f o

Article history: Received 30 March 2015 Received in revised form 8 May 2015 Accepted 28 May 2015 Available online 1 June 2015 Keywords: Cefoselis sulfate HPLC Q-TOF Stability in solid state

a b s t r a c t The process of degradation was studied by using an HPLC–DAD method. Two degradation products were identified with a hybrid ESI-Q-TOF mass spectrometer. The influence of temperature and relative air humidity (RH) on the stability of cefoselis sulfate was investigated. In the solid state at increased RH the degradation of cefoselis sulfate was an autocatalytic reaction of the first order with respect to substrate concentration while in dry air was first-order reaction depending on the substrate concentration. The kinetic and thermodynamic parameters of degradation were calculated. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Cephalosporins are semi-synthetic ␤-lactam antibiotics. They have a broad spectrum of antibacterial activity, desired pharmacokinetic parameters and produce low side effects [1–2]. The greatest limitation of a therapy using cephems is their significant chemical instability. The study of degradation of cephalosporins can help to understand the decomposition patterns of the drug molecule. Such information is used for determining storage and packaging conditions of the bulk substance and formulations. On the other hand the most of the side effects of ␤-lactams are caused by the generation of degradation products, so it is important to estimate the stability and mechanism of degradation in this group of medicinal compounds. Stability studies of cephalosporins antibiotics have been examined and the degradation under variable conditions has been performed [3–14]. Cefoselis sulfate (CSS) is a new, parenteral, fourth generation cephalosporin. It has a broad spectrum of antibacterial activity against Gram positive and Gram negative bacteria including Pseudomonas aeruginosa [15]. CSS is effective for various infections in obstetric and gynecologic field and also the prevention of surgical site infection [16]. The approved dosing regimen for intravenous CSS is from 2 g daily for 5 days. Cefoselis is a widely used ␤-lactam antibiotic, but occasionally induces seizures and convulsion in elder

∗ Corresponding author. Tel.: +48 618546649. E-mail address: [email protected] (P. Zalewski). http://dx.doi.org/10.1016/j.jpba.2015.05.033 0731-7085/© 2015 Elsevier B.V. All rights reserved.

and renal failure patients [17,18]. Chromatographic methods for the determination of CSS have proved the formation of many degradation products without their structural characterization [12,17]. CSS has been found markedly unstable in solutions under the influence of acids, bases, and increased temperature [12–14]. The aim of this work was to investigate the process of CSS degradation in the solid state and to identify degradation products. 2. Experimental 2.1. Standards and reagents CSS was obtained from Xingcheng Chempharm Co., Ltd., Taizhou, Zhejiang, China. It is white or light yellow crystalline 99.5% and conforms to the standards of Chinese Pharmacopoeia 2005. All other chemicals and solvents were obtained from Merck, Darmstadt, Germany and were of analytical grade. High-quality pure water was prepared by using a Millipore Exil SA 67,120 purification system (Millipore, Molsheim, France). 2.2. Kinetic analysis For the kinetic study, the Dionex Ultimate 3000 analytical system consisted of a quaternary pump, an autosampler, a column oven and a diode array detector (Sunnyvale (CA) USA). As the stationary phase a Kinetex with 5 ␮m core–shell particles, C18, 100A, 100 × 2.1 mm column was used (Phenomenex, Torrance (CA) USA). The mobile phase was composed of acetonitrile – 0.1% formic acid

P. Zalewski et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 222–226

H 2N

N

CH3 O O N H N

S

O

OH

COO 7

N

N N

3

S

H H

NH2

H2SO4 S

CSS – cefoselis sulfate (4th generation cephalosporin)

H 2N

N S

CH3 O O N H N O

CH3 O O N H N

N N

7

H H

N

3

H2N

CPS – cefpirome sulfate (4th generation cephalosporin)

N

N

3

N N HCl

S

H2N

H2SO4

S

7

CZH – cefozopran hydrochloride (4th generation cephalosporin)

COO N

COO

H H

O

223

N S

CH3 O O N H N O

COO 7

N

H H

3

S

N

CH2 2 HCl, 2H2O

CPH – cefepime dihydrochloride (4th generation cephalosporin)

O

H2N

N S

CH3 O O N H N O

COONa 7

N

H H

3

S

ONa

N S

N N CH3

Fig. 1. Chemical structures of CSS, CZH, CPS, CPH and CTD.

(5:95 v/v). The flow rate of the mobile phase was 1.0 mL min−1 and the injection volume was 5 ␮L. The wavelength of the DAD detector was set at 260 nm. Separation was performed at temp. 30 ◦ C. The stability tests were performed according to the International Conference on Harmonization Guidelines [19]. 5 mg aliquots of CSS were weighed into glass vials. In order to test the influence of such environmental factors as temperature and humidity (RH), the samples were placed in desiccators containing saturated solutions of inorganic salts: sodium bromide (RH ∼ 50.9%), sodium nitrate (RH ∼ 66.5%), sodium chloride (RH ∼ 76.4%) and zinc sulfate (RH ∼ 90.0%) that were in incubators (Wamed, Warsaw, Poland) set to the desired temperatures (333, 343, 353, and 363 K). To evaluate the stability of CZH in dry air, the vials containing 5.0 mg of this substance were immersed in a sand bath placed in a heat chamber at 393 K. Each batch to be studied comprised 8–12 samples. At specific time intervals, determined by the rate of degradation, the vials were removed, cooled to room temperature and the contents dissolved in a mixture (1:1) of acetonitrile and water. The solutions obtained in that way were quantitatively transferred into volumetric flasks and diluted to a total volume of 25.0 mL with the same mixture of solvents. After filtration the 5 ␮L samples were injected onto the column.

2.3. LC–MS analysis The LC–MS analysis was performed with the use of an Agilent Accurate-Mass Q-TOF LC/MS G6520B system with a DESI ion source and an Infinity 1290 ultra-high-pressure liquid chromatography system consisting of a G4220A binary pump, a G1330B FC/ALS thermostat, a G4226A autosampler, a G4212A DAD detector, and a G1316C TCC module (Agilent Technologies, Santa Clara, USA). The chromatographic conditions were analogous to those described in the kinetic study. The MassHunter workstation software B.04.00 was used for the control of the system, data acquisition, and qualitative analysis.

The Q-TOF detector was tuned in the positive (4 GHz) and the main parameters were optimized as follows: gas temp. 300 ◦ C, drying gas 10 L min−1 , nebulizer pressure 276 kPa, capillary voltage 3500 V, fragmentor voltage 200 V, skimmer voltage 65 V, octopole 1 RF voltage 250 V. The data were acquired in the auto MS/MS mode with the mass range 50–1050 m/z and the acquisition rate 1.2 spectra/s (for MS and MS/MS data). The collision energy was calculated from the formula 2 V (slope) × (m/z)/100 + 10 V (offset) and maximum 2 precursors per cycle were selected with an active exclusion mode after 1 spectrum for 0.2 min. To ensure accuracy in mass measurements, reference mass correction was used. Masses 121.0508 and 922.0097 were used as lock masses.

3. Results and discussion Changes in the concentration of CSS under stress study conditions were evaluated using the HPLC method which was linear in the range 20–240 mg L−1 , accurate (RSD 99.90–100.15 %), precise (RSD 0.15–0.51%) and selective in the presence of CSS and its degradation products. The LOD and LOQ were 6.68 and 20.04 mg L−1 , respectively. In the chromatograms of CSS developed over a period of 0–2 min. the following compounds were eluted: CSS with a retention time of 1.42 min, degradation products (DP1) with retention time of 0.65 min, and degradation products (DP2) with retention time of 0.73 min. At an increased RH and temperature the dependence ln c = f(t) was not linear (c, the concentration of CSS at time t). First, an induction phase occurred with a very small substrate loss, which was followed by an acceleration phase with rapid degradation. Those findings indicated that the degradation of CSS at an increased RH and temperature was a first-order autocatalytic reaction relative to substrate concentration. The rate constants were calculated from the straight-line relationship ln ct /(c0 –ct ) = −kobs × t (c0 and ct , the concentrations of CSS at time t = 0 and t, respectively; kobs , the observed rate constant of the degradation reaction). The linearity of the dependence ln ct = f(t) for CSS degradation in hot dry air proved that the process followed the kinetics of a first-order reaction relative to substrate concentration.

224

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C14 H14 N5 O5 S2 C12 H14 N5 O2 S2 C7 H7 N2 OS C5 H10 N3 O 396.0428 324.05811 167.0270 128.08161

C5 H8 N3 O C3 H6 N3

NH2 3

H H

S

523.11859 3

CSS

1.42

523.11765

1.81

C19 H23 N8 O6 S2

S

H2N

N

O

CH3 O O N H N

NH H2 N N C5 H10 N3 O 4.64 128.08243 0.73 2

D2

201.04432 1

D1

0.65

128.08184

S

N

H3N C6 H9 N4 O2 S 1.22

OH

O

7

N

NH2

CH3 O N

201.04407

Mass error (ppm) Theoretical mass (m/z) Measured mass (m/z) Retention time (min) Name Comp. no.

Table 1 Q-TOF accurate mass elemental composition and MS/MS fragmentation of the analyzed substances.

Molecular formula [M + H+ ]

Chemical structures

COO

N N

OH

H2SO4

126.0124 84.0456

126.0116

MS/MS fragmentation (m/z)

C4 H4 N3 S

Fragmentation ions formulas

Table 2 The effect of relative air humidity on the stability of CSS at 363 K. Relative air humidity (%)

105 (k ± k) [s−1 ]

Statistical evaluation lnk = f(RH%)

50.9 66.5 76.4

(0.48 ± 0.04) (1.01 ± 0.14) (2.46 ± 0.22)

90.0

(4.94 ± 0.42)

a = 0.06 ± 0.02 Sa = 5.10 × 10−3 b = −15.42 ± 1.59 Sb = 0.37 r = 0.9932 Sy = 0.15

Previous studies demonstrated that the kinetic mechanism by which cephem derivatives degrade in the solid phase is determined by the chemical structure of compound and storage conditions [5–11]. For various cephalosporins, decomposition occurred according to the following reactions: the first-order reaction, the autocatalytic first-order reaction, the reversible first-order reaction, or the reversible autocatalytic first-order reaction relative to substrate concentration. The degradation mechanisms of ceftriaxone (CTD) [8] is analogous to that of CSS, whereas, cefpirome (CPS) [9], cefozopran (CZH) and cefepime (CPH) [11] degrade according to the first-order reaction model depending on substrate concentration both at an increased RH and in dry air. An analysis of those observations and of the chemical structure of the cephalosporins leads to the suggestion that the kinetics of their degradation is determined by the combined effect of the substituents at C-3 and C-7, which also has a major impact on drug stability (Fig. 1). Based on Q-TOF LC/MS analysis the main products of CSS degradation in the solid phase were derivatives of the substituents at C-3 and C-7 of the parent compound (Table 1). A similar pattern was observed for CPS [9]. As is known, the lactam ring is key for the antibacterial activity of the cephalosporins but it is also very unstable. Products of hydrolysis of the cephem ring were observed in solutions, thus indicating the preferred degradation path under such conditions [4]. However, the degradation of ␤lactam ring could be suggested as parallel reaction during the thermolysis. Despite close similarities between the chemical structures of CSS and CPS, different products of their degradation were identified. In the solid phase, a decarboxylated degradation product was registered only for CPS [9]. Based on the kinetic mechanism of degradation identified as an autocatalytic reaction, it may be assumed that products of CSS degradation (Table 1) have a catalytic effect on the degradation process in the solid phase. It has been reported that relative air humidity is a key factor determining the stability of the cephalosporins [5–11]. The degradation rate constant for CSS at RH ∼ 0% and 393 K equaled 1.01 × 10−7 and was 100 times less than at a 30 K lower temperature but at RH ∼ 66.5% (Table 2). That confirms the point of storing cephalosporins in airtight containers to ensure their optimal stability throughout shelf life. The influence of RH on the stability of CSS is presented in Table 2. The values of the reaction rate constants kobs were used to calculate the Arrhenius relationship in order to interpret the influence of the temperature on the reaction rate at 76.4% RH (Fig. 2). The energy of activation and the thermodynamic parameters, enthalpy and entropy for 298 K, were calculated from the parameters of the slope ln k = f(1/T) (Table 3). 3.1. The effect of chemical structure on the activation energy of the degradation reaction and the stability of selected cephalosporins In the solid state at RH ∼ 76.4% the highest activation energy (Ea ) was demonstrated by CPS and the lowest by CPH (Table 4). In structural terms, both have the same substituents at C-7 (Fig. 1).

P. Zalewski et al. / Journal of Pharmaceutical and Biomedical Analysis 114 (2015) 222–226

225

Fig. 2. Semilogarithmic relationship k = f(1/T) for degradation of CSS, CZH, CPS [9], CPH [11] and CTD [8] at RH ∼ 76.4%. Table 3 Kinetic and thermodynamic parameters of degradation of CSS in solid state at RH ∼ 76.4%. T [K]

106 (k ± k) [s−1 ]

Statistical evaluationlnk = f(1/T)

Thermodynamic parameters

333 343 353 363

(1.63 ± 0.20) (3.73 ± 0.48) (8.32 ± 1.62) (24.60 ± 2.15)

a = −10784.39 ± 3163.68 Sa = 735.23 b = 18.99 ± 9.11 Sb = 2.12 r = 0.9954Sy = 0.14

Ea = 89.67 ± 26.30 [kJ mol−1 ] H =/ a = 87.19 ± 28.78 [kJ mol−1 ] S =/ a = −87.04 ± 75.70 [J K−1 mol−1 ]

Ea , activation energy; H =/ , enthalpy; S =/ , entropy; Ea = −aR; H =/ = Ea −TR; S 1 = R(lnA–ln(kb T/h)) where: kB , Boltzmann’s constant (1.3807 × 10−23 J K−1 ); h, Planck’s constant (6.626 × 10−34 J s); R, universal gas constant (8.314 K−1 mol−1 ), T, temperature [K]; a, vectorial coefficient of Arrhenius relationship; A, frequency coefficient a Calculated for 298 K. Table 4 Kinetic and thermodynamic parameters of degradation of CSS, CZH, CTD [11], CPS [12] and CPH [16] in solid state at constant relative air humidity (RH ∼ 76.4%) and at T = 363 K. Cephem

Ea , [kJ mol−1 ]

CSS CZH CPH CTD CPS

90 95 52 79 167

a

± ± ± ± ±

26 30 12 9 [8] 16 [9]

Half-life (t0.5 ) [h]

Kinetic mechanism of degradation

34.5 0.4 0.4a 23.1 [8] 20.2 [9]

Autocatalytic reaction of the first order with respect to substrate concentration Airst-order reaction depending on the substrate concentration First-order reaction depending on the substrate concentration [11] Autocatalytic reaction of the first order with respect to substrate concentration [8] First-order reaction depending on the substrate concentration [9]

Calculated from extrapolated data.

The most similar Ea values were found for CSS and CZH despite their different substituents at C-3 and C-7. Thus, the combined impact of the substituents at C-3 and C-7 produces steric effects and determines the activation energy. In spite of a relatively low Ea at an increased RH, CSS proved the most stable of all the compared cephalosporins, most likely due to a different kinetic mechanism of degradation. The stability of CZH was very similar to that of CPH, although the two compounds have various substituents at C-3 and C-7. A marked difference was observed in the stability of CPH compared to CSS, CPS, and CTD despite the same substituents at C-7. There is a strong similarity between the substituents at C-3 in CZH and CPS. However, they differ significantly in their stability. Those findings further confirm that the combined impact of the substituents at C-3 and C-7 induces steric effects and determines the shelf life of the cephalosporins. 4. Conclusions The degradation of CSS in the solid phase in dry air follows the first-order reaction model, whereas, at an increased relative air humidity a first-order autocatalytic reaction relative to substrate concentration is observed. The degradation products are formed as a result of changes in the substituents at C-3 and C-7. The com-

bined effect of those substituents plays a key role in determining the kinetic mechanism and pathway of degradation as well as the stability of the investigated cephalosporins. Acknowledgements The paper was written with the use of equipment purchased within the project “The equipment of innovative laboratories doing research on new medicines used in the therapy of civilization and neoplastic diseases” within the Operational Program Development of Eastern Poland 2007–2013, Priority Axis I Modern Economy, Operations I.3 Innovation Promotion. References [1] Q.P. Shi, X.D. Jiang, F. Ding, M.L. Yu, S.Q. Zhang, Adverse drug reactions and pattern use of cephalosporins: a retrospective review of hospitalized patients during 5 years, Int. J. Pharm. 9 (2013) 66–73. [2] J.D. Campagna, M.C. Bond, E. Schabelman, B.D. Hayes, The use of cephalosporins in penicillin-allergic patients: a literature review, J. Emerg. Med. 42 (2012) 612–620. ´ [3] A. Jelinska, L. Dobrowolski, I. Oszczapowicz, The influence of pH, temperature and buffers on the degradation kinetics of cefetamet pivoxil hydrochloride in aqueous solutions, J. Pharm. Biomed. Anal. 35 (2004) 1273–1277.

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[4] T. Sugioka, T. Asano, Y. Chikaraishi, E. Suzuki, A. Sano, T. Kuriki, M. Shirotsuka, K. Saito, Stability and degradation pattern of cefpirome (HR 810) in aqueous solution, Chem. Pharm. Bull. 38 (1990) 1998–2002. ´ ´ [5] P. Zalewski, J. Cielecka-Piontek, P. Garbacki, A. Jelinska, M. Karazniewicz-Łada, Stability-indicating HPLC method for the determination of cefcapene pivoxil, Chromatographia 76 (2013) 387–391. ´ ˛ M. Bałdyka, K. Juszkiewicz, I. [6] B. Medenecka, A. Jelinska, M. Zajac, Oszczapowicz, Stability of the crystalline form of cefaclor monohydrate and its pharmaceutical preparations, Acta Pol. Pharm. 66 (2009) 563–569. ´ ˛ M. Knajsiak, The stability of cefprozil in [7] A. Jelinska, B. Medenecka, M. Zajac, oral suspension cefzil, Acta Pol. Pharm. 65 (2008) 261–265. ˛ A. Jelinska, ´ [8] M. Zajac, P. Zalewski, Stability of ceftriaxone disodium in biotrakson and tartriakson, Acta Pol. Pharm. 62 (2005) 89–94. ´ [9] P. Zalewski, R. Skibinski, J. Cielecka-Piontek, Stability studies of cefpirome sulfate in the solid state: identification of degradation products, J. Pharm. Biomed. Anal. 92 (2014) 22–25. ˛ A. Jelinska, ´ [10] M. Zajac, L. Dobrowolski, I. Oszczapowicz, Evaluation of stability of cefuroxime axetil in solid state, J. Pharm. Biomed. Anal. 32 (2003) 1181–1187. [11] W. Musiał, M. Zajac, Stability of cefepime dihydrochloride monohydrate in solid state, Acta Pol. Pharm. 59 (2002) 243–246. ´ [12] P. Zalewski, J. Cielecka-Piontek, A. Jelinska, Development and validation of the stability-indicating LC–UV method for the determination of cefoselis sulphate, Cent. Eur. J. Chem. 10 (2012) 121–126.

´ [13] P. Zalewski, J. Cielecka-Piontek, A. Jelinska, Stability of cefoselis sulfate in intravenous solutions, Asian J. Chem. 25 (2013) 7596–7598. ´ [14] P. Zalewski, J. Cielecka-Piontek, A. Jelinska, Stability of cefoselis sulfate in aqueous solutions, React. Kinet. Mech. Cat. 108 (2013) 285–292. [15] T. Kuriyama, T. Karasawa, K. Nakagawa, S. Nakamura, E. Yamamoto, Antimicrobial susceptibility of major pathogens of orofacial odontogenic infections to 11 beta-lactam antibiotics, Oral Microbiol. Immunol. 17 (2002) 285–289. [16] T. Chimura, N. Hirayama, K. Morisaki, T. Murayama, F. Sato, K. Akatsuka, M. Numazaki, Y. Igarashi, Clinical effects of cefoselis (CFSL) on infections in obstetric and gynecologic field and prevention of postoperative infections, Jpn. J. Antibiot. 53 (2000) 637–641. [17] K. Ohtaki, K. Matsubara, S. Fujimaru, K. Shimizu, T. Awaya, M. Suno, K. Chiba, N. Hayase, H. Shiono, Cefoselis, a beta-lactam antibiotic, easily penetrates the blood–brain barrier and causes seizure independently by glutamate release, J. Neural. Transm. 111 (2004) 1523–1535. [18] M. Sugimoto, I. Uchida, T. Mashimo, S. Yamazaki, K. Hatano, F. Ikeda, Y. Mochizuki, T. Terai, N. Matsuoka, Evidence for the involvement of GABA(A) receptor blockade in convulsions induced by cephalosporins, Neuropharmacology 45 (2003) 304–314. [19] ICH, Stability Testing of New Drug Substances and Products (Q1AR). International Conference on Harmonization, IFPMA, Geneva (2000).