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1 2 3 1 Stability-indicating LC method for the determination of cephalothin in lyophilized 4 5 2 powder for injection 6 7 3 8 9 10 4 11 12 13 5 Abstract 14 15 6 A stabilityindicating gradient reversed phase liquid chromatography (RPLC) method has 16 17 7 been developed for the quantitative determination of cephalothin (CET), an antimicrobial 18 8 compound, in the presence of its impurities and degradation products generated from forced 19 20 9 degradation studies. The developed method is also applicable for the related substances 21 Manuscript 22 10 determination in bulk drugs. The chromatographic separation was achieved on an Agilent 23 24 11 Eclipse XDBPhenyl, 250 mm x 4.6 mm, 5 µm column with mobile phase containing a 25 26 12 gradient mixture of solutions A (aqueous ammonium phosphate buffer, pH 4.5) and B 27 13 (acetonitrile) as mobile phase. The flow rate was 1.0 mL/min and the detection wavelength 28 29 14 was 238 nm. The drug substance was subjected to stress conditions of hydrolysis, 30

31 15 oxidation, photolysis, humidity and thermal degradation. Considerable degradation was Accepted 32 33 16 found to occur in base, acid and oxidative stress conditions. In the developed HPLC 34 17 method, the resolution between CET and its potential degradation products was found to be 35 36 18 greater than 2.4, further, the peak purity of CET in all conditions were more than 99% 37 38 19 proved the stabilityindicating power of method. The less active metabolite of cephalothin, 39 20 deacetylcephalothin (impurity B), was identified and is showed significant formation 40 Methods 41 42 21 especially in basic condition. This method is capable to detect the degradation products of 43 22 CET at a level of 0.05% with respect to test concentration of 500 µg/mL for a 10 µL 44 45 23 injection volume. The HPLC method developed was validated with respect to linearity, 46 47 24 accuracy, precision, specificity, limit of quantitation, limit of detection and robustness and 48 49 25 system suitability. To our knowledge, a rapid stabilityindicating LC method for CET has 50 26 not been published elsewhere and this method can be applicable to evaluate the quality of Analytical 51 52 27 product samples as well as in stability studies of cephalothin. 53 54 28 55 56 29 Keywords : Cephalothin, Degradation products, Liquid chromatography, Stability 57 58 30 indicating, Validation 59 60 Analytical Methods Page 2 of 22

1 2 3 1 Stability-indicating LC method for the determination of cephalothin in lyophilized 4 5 2 powder for injection 6 7 3 8 9 a,b b* 10 4 Karen de Souza Rugani ; Hérida Regina Nunes Salgado 11 12 a 13 5 Departamento de Fármacos Desenvolvimento e Validação, Bioagri Laboratórios – A 14 15 6 Mérieux NutriSciences Company, Rodovia Rio Claro – Piracicaba, SP 127, km 24, CEP 16 17 7 13412000 – Piracicaba, SP, Brazil 18 19 20 8 b Departamento de Fármacos e Medicamentos, Faculdade de Ciências Farmacêuticas, 21 Manuscript 22 23 9 UNESP – Univ Estadual Paulista, Rod. AraraquaraJaú, km 1, CEP 14801902 Araraquara, 24 25 10 SP, Brazil 26 27 11 28 12 29 13 * Corresponding author at: Universidade Estadual Paulista (UNESP), Faculdade de Ciências Farmacêuticas, 30 14 Departamento de Fármacos e Medicamentos, Campus Universitário, 14801902, Araraquara, SP, Brazil. Tel.:

31 15 +55 33016967; fax: +55 33016960. Accepted 32 33 16 E-mail address: [email protected] (H.R.N. Salgado). 34 35 17 36 37 38 18 Abstract 39

40 19 A stabilityindicating gradient reversed phase liquid chromatography (RPLC) method has Methods 41 42 20 been developed for the quantitative determination of cephalothin (CET), an antimicrobial 43 44 21 compound, in the presence of its impurities and degradation products generated from forced 45 46 22 degradation studies. The developed method is also applicable for the related substances 47 48 23 determination in bulk drugs. The chromatographic separation was achieved on an Agilent 49 24 Eclipse XDBPhenyl, 250 mm x 4.6 mm, 5 m column with mobile phase containing a 50 51 25 gradient mixture of solutions A (aqueous ammonium phosphate buffer, pH 4.5) and B Analytical 52 53 26 (acetonitrile) as mobile phase. The flow rate was 1.0 mL/min and the detection wavelength 54 55 27 was 238 nm. The drug substance was subjected to stress conditions of hydrolysis, 56 28 oxidation, photolysis, humidity and thermal degradation. Considerable degradation was 57 58 59 60 Page 3 of 22 Analytical Methods

1 2 3 29 found to occur in base, acid and oxidative stress conditions. In the developed high 4 5 30 performance liquid chromatography (HPLC) method, the resolution between CET and its 6 7 31 potential degradation products was found to be greater than 2.4, further, the peak purity of 8 9 32 CET in all conditions were more than 99% and proved the stabilityindicating power of 10 33 method. The less active metabolite of cephalothin, deacetylcephalothin (impurity B), was 11 12 34 identified and showed significant formation especially in basic condition. This method is 13 14 35 capable to detect the degradation products of CET at a level of 0.05% with respect to test 15 16 36 concentration of 500 g/mL for a 10 L injection volume. The HPLC method developed 17 18 37 was validated with respect to linearity, accuracy, precision, specificity, limit of quantitation, 19 38 limit of detection and robustness. To our knowledge, a rapid stabilityindicating LC method 20 21 39 for CET has not been published elsewhere and this method can be applicable to evaluate Manuscript 22 23 40 the quality of product samples as well as in stability studies of cephalothin. 24 25 41 26 42 Keywords : Cephalothin, Degradation products, Liquid chromatography, Stability 27 28 43 indicating, Validation 29 30 44

31 Accepted 32 45 33 34 35 46 1. Introduction 36 37 38 47 Cephalothin (USAN, United States Adopted Names), also named cefalotin (INN, 39

40 48 International Nonproprietary Name), is a firstgeneration cephalosporin antimicrobial Methods 41 42 43 49 chemically described as (6R,7R)3[(acetyloxy)methyl]8oxo7[(2thienylacetyl)amino] 44 45 50 5thia1azabicyclo[4.2.0]oct2ene2carboxylic acid (Figure 1), it has an empirical 46 47 1 1,2 48 51 formula of C 16 H16N2O6S2, a molecular weight of 396.44 g mol and a pk a value of 2.22 49 3 50 52 . Cephalothin is given as the sodium salt by injection and 1.06 g of cephalothin sodium is 51 Analytical 52 53 equivalent to about 1 g of cephalothin 2. 53 54 55 54 56 57 58 59 60 Analytical Methods Page 4 of 22

1 2 3 55 Cephalosporins act by inhibiting synthesis of the bacterial cell wall and present a 4 5 6 56 larger spectrum of activity than penicillins due to be more stable to many bacterial β 7 8 57 lactamases 4,5 . Cephalothin was the first cephalosporin available for clinical use in the U.S. 9 10 58 and it is most active against Grampositive cocci, including oxacilinsusceptible 11 12 13 59 staphylococci and Streptococcus spp and has moderate activity against some Gram 14 15 60 negative bacilli 6. It is indicated for treatment of infections of skin and soft tissue 7. 16 17 8 18 61 Cephalothin remains the antibacterial of choice for treatment of uncomplicated celullits . 19 20 62 Cephalothin is given by intramuscular (IM) and intravenous (IV) route, however, 21 Manuscript 22 23 63 the IV is preferred because the IM is very painful. It is widely distributed in body tissues 24 25 64 and fluids except the brain and cerebrospinal where the concentrations achieved are low 26 27 65 and unpredictable. About 70% of the dose is bound to plasma proteins and the plasma half 28 29 30 66 life varies from about 30 to 50 minutes. Approximately 20 to 30% of CET is rapidly

31 Accepted 32 67 deacetylated in the liver (hepatic biotransformation) and about 60 to 70% of a dose is 33 34 2,9 35 68 excreted in the urine as cephalothin and the less active metabolite, deacetylcephalothin . 36 37 69 Cephalothin has been determined by spectrophotometry [BP, 2010] and by high 38 39 1113

40 70 performance liquid chromatographic (HPLC) with ultraviolet detection . Analysis of Methods 41 42 71 cephalothin and its deacylated metabolites in human urine 1415 , in human plasma by LC 43 44 16,17 18 45 72 , and its determination in rat blood were also reported in the literature. However, 46 47 73 there is no reported stabilityindicating LC method for the determination of cephalothin in 48 49 74 its lyophilized powder for injection dosage form. 50 51 Analytical 52 75 Several analytical methods are described in the literature for analysis of 53 54 19 55 76 cephalosporins in raw material and finished product. Among these are found titrimetry ; 56 2022 21,23,24 57 77 high performance liquid chromatography ; ultraviolet spectrophotometry and 58 59 60 Page 5 of 22 Analytical Methods

1 2 3 78 visible spectrophotometry 25 ; infrared spectrometry 26,27 , agar diffusion 2831 and 4 5 32 6 79 turbidimetry bioassay . 7 8 80 In quality control of pharmaceutical products, the identification and quantification 9 10 81 of the active substance and its impurities are very important due to reasons of safety and 11 12 13 82 efficacy. The impurities and possible degradation products which may be present in the 14 15 83 medicines may change their chemical, pharmacological, and toxicological properties 33,34 . 16 17 18 84 The forced degradation studies are performed to facilitate the development of 19 20 85 analytical methods to obtain a better understanding of active ingredient and stability of the 21 Manuscript 22 23 86 product, and to provide information on routes of degradation and formation of degradation 24 25 87 products 34,35 . In this context, the stress test (forced degradation) is defined as a stability test 26 27 88 performed with the finished product subjected to conditions that exceed those used in 28 29 36 33 30 89 accelerated stability studies . According to the ICH Q1AR2 , stress testing (forced

31 Accepted 32 90 degradation) performed with the addition of drug can help identify the likely degradation 33 34 35 91 products, contribute to the definition of the intrinsic stability of the molecule, and also 36 37 92 validate the ability of the analytical procedure used to be a stabilityindicating method. 38 39

40 93 Accordingly, the goal of the present work was to develop simple, sensitive and Methods 41 42 94 selective LC stabilityindicating method for the determination of CET in its lyophilized 43 44 45 95 powder for injection dosage form. Moreover, the present manuscript describes the 46 47 96 degradation behavior of cephalothin under acidic, basic, oxidative, thermal, humidity and 48 49 97 photolytic conditions with the identification of the main degradation product: 50 51 Analytical 52 98 deacetyltcephalothin. Stabilityindicating methods are traditionally performed using 53 54 99 gradient elution, in order to ensure that degradation products of several chemical 55 56 100 compositions are all separated. Optimization of LC conditions to separate the CET and its 57 58 59 60 Analytical Methods Page 6 of 22

1 2 3 101 degradation products included the identification of impurity B (deacetylcephalotin), on a 4 5 6 102 reversed phase Phenyl column, and method validation will be discussed in Results, section 7 8 103 3.1. 9 10 11 104 The system suitability method acceptance criteria set in each condition of CET 12 13 105 samples were: tailing factor ≤ 2.0, resolution between CET and any of the components > 14 15 16 106 2.0 and theoretical plates >2000. Hence, this method was successfully validated according 17 18 107 to the ICH guideline Q2 (R1) 37 . 19 20 108 21 Manuscript 22 23 109 2. Materials and Methods 24 25 26 110 2.1. Materials 27 28 29 111 Cephalothin sodium salt reference standard was assigned purity 99.2% and supplied 30

31 112 by Sigma–Aldrich (Germany). Cephalotin for impurity B identification reference substance Accepted 32 33 34 113 was supplied by European Pharmacopoeia (Strasbourg, France). All reagents used in the 35 36 114 experimental procedure were of analytical grade. Acetonitrile, hydrogen peroxide (H2O2), 37 38 115 phosphoric acid and concentrated hydrochloric acid (HCl) were obtained from Merck 39

40 Methods 41 116 (Darmstadt, Germany). Ammonium phosphate dibasic was supplied by Synth (Diadema, 42 43 117 Brazil) and sodium hydroxide pellets (NaOH) was supplied by Vetec (Rio de Janeiro, 44 45 46 118 Brazil). The cephalothin sodium lyophilized powder for injection, which was claimed to 47 48 119 contain 1 g of active drug (cephalothin), was kindly donated by União Química 49 50 120 Pharmaceutical Industry (Pouso Alegre, Brazil). Ultrapure water was obtained from a Milli Analytical 51 52 53 121 Q™ Plus apparatus (Millipore™, USA) and was used to prepare all solutions. Millex 54 55 122 PVDF membrane filters with a pore size of 0.45 m and diameter of 33.0 mm (Millipore™, 56 57 123 Tullagreen, Carrigtwohill, Ireland) was used. All other chemicals were of analytical grade. 58 59 60 Page 7 of 22 Analytical Methods

1 2 3 124 2.2. Chromatographic system 4 5 6 125 An Agilent model 1200 DAD/UV LC system, consisting of a quaternary pump 7 8 9 126 (G1311A), a degasser (G1322A), a column thermostat (G1316A), a diode array detector 10 11 127 (DAD) (G1315A), and an thermostatted autosampler (4 C) (G1329A), was used. The 12 13 128 chromatographic data were recorded using a computer system with ChemStation data 14 15 16 129 acquiring software (also from Agilent). The peak purity was determined using a diode array 17 18 130 detector (DAD). 19 20 131 Chromatographic separation was achieved on a Eclipse XDB Phenyl column (250 21 Manuscript 22 23 132 mm × 4.6 mm, 5 m particle size) – Agilent™ applying gradient elution based on a mobile 24 25 133 phase A (20 mM ammonium phosphate buffer, pH 4.5) and mobile phase B (acetonitrile). 26 27 134 The system was programmed at 35ºC for the column oven, 238 nm for detection 28 29 30 135 wavelength and 10 L for injection volume in a flow rate of 1.0 mL/min. The LC gradient

31 Accepted 32 136 program (Time (in min) / % mobile phase B) was set as 0.0/10; 2.0/10; 15.0/30; 15.1/10 33 34 35 137 and 30.0/10. 36 37 138 38 39 139 2.3. Preparation of sample for LC analysis

40 140 2.3.1. Standard preparation Methods 41 42 43 141 The cephalothin standard (10 mg) was accurately weighted and transferred into a 20 44 45 46 142 mL volumetric flask, diluted with purified water, and sonicated by 10 minutes to prepare 47 48 143 the standard solution at concentration of 500 g/mL. Solutions were filtered through a 0.45 49 50 144 m PVDF membrane filter (Millipore, Ireland) prior to injection. Analytical 51 52 53 145 54 55 146 2.3.2. Sample preparation 56 57 58 59 60 Analytical Methods Page 8 of 22

1 2 3 147 For the analysis of cephalothin, an amount of powder equivalent to 10 mg of CET 4 5 6 148 was transferred to a 20 mL volumetric flask with ultrapure water and sonicated for 10 min, 7 8 149 followed by the dilution to volume with ultrapure water obtained the concentration of 500 9 10 150 g/mL. These solutions were filtered through a 0.45 m PVDF filter before injections. 11 12 13 151 14 15 152 2.4. Forced degradation studies 16 17 18 153 Forced degradation conditions used are more severe than accelerated stability 19 20 154 testing such as temperature (50°C or 60ºC); relative humidity (≥75%); hydrolysis at high 21 Manuscript 22 37 23 155 and low pH; oxidation and photolysis . When performing forced degradation study, the 24 25 156 ideal range of degradation of the active ingredient is 520%, in order to cause degradation 26 27 157 but not so intense that generates secondary products 35,38 . The stress solutions were 28 29 30 158 prepared to obtain a final concentration of 500 g/mL of CET. Prior to injection, samples

31 Accepted 32 159 were withdrawn at appropriate time, neutralized (in case of acid and alkali hydrolysis), 33 34 35 160 diluted with purified water and passed through Millex 0.45 m membrane filter. The total 36 37 161 chromatographic run time was about two times higher than the retention time of the drug 38 39 162 peak in order to allow the elution of all possible degradation products formed. In all stress

40 Methods 41 42 163 conditions the peak area of CET and its degradation products were obtained. The stress 43 44 164 samples were assayed against a qualified cephalothin reference standard and the peak purity 45 46 165 tests were carried out for the cephalothin and its degradation products peaks by using DAD 47 48 49 166 UV detector. The identification of the impurity B (deacetylcephalothin) was carried out. 50 51 167 Analytical 52 53 168 2.4.1. Acid and base hydrolytic degradation 54 55 56 57 58 59 60 Page 9 of 22 Analytical Methods

1 2 3 169 The cephalothin sample was treated separately with in 0.1 M HCl and 0.01 M 4 5 6 170 NaOH. The acid solution was exposed to a temperature of 60°C for 40 minutes and the 7 8 171 basic solution to a room temperature for 5 minutes. The forced degradation in acidic and 9 10 172 basic media was performed in the dark. The solutions were neutralized and then diluted 11 12 13 173 with purified water. 14 15 174 16 17 175 2.4.2. Oxidative degradation 18 19 20 176 To study the oxidative degradation on the drug sample, a solution of 3% H2O2 was 21 Manuscript 22 23 177 added and kept at room temperature for 20 minutes in the dark. The resultant solution was 24 25 178 diluted with purified water. 26 27 179 28 29 30 180 2.4.3. Humidity degradation

31 Accepted 32 33 181 The dry powder of the drug sample was placed at 30ºC / 75% RH (in a climate 34 35 182 chamber) for 10 days to study humidity degradation. After, the sample was dissolved and 36 37 183 diluted with purified water. 38 39 184

40 Methods 41 42 185 2.4.4. Thermal degradation 43 44 45 186 The dry powder of the drug sample was placed in oven at 60°C for 10 days to study 46 47 187 dry heat degradation. Powder was then dissolved and diluted with purified water. 48 49 50 188 51 Analytical 52 189 2.4.5. Photochemical degradation 53 54 55 190 The photochemical stability of the drug was studied by exposing the dry sample 56 57 191 powder to ultraviolet (UV) and cool white fluorescence (VIS) lamps for 10 days at 25ºC. 58 59 60 Analytical Methods Page 10 of 22

1 2 3 192 The illumination used was not less than 1.2 million lux hours (VIS) neither 200 watt h/m 2 4 5 6 193 (UV). The sample powder was then dissolved and diluted with purified water. 7 8 194 9 10 195 2.4.6. Standard preparation: Cephalothin for Impurity B identification 11 12 13 196 A standard solution containing 220 g/mL of cefalotin for Impurity B identification 14 15 197 (deacetylcephalothin) was prepared in purified water and analyzed in the forced 16 17 198 degradation test. This solution was filtered through a 0.45 m PVDF membrane filter 18 19 20 199 (Millipore, Ireland) prior to injection. 21 Manuscript 22 200 23 24 201 2.5 Analytical method validation 25 26 27 202 The developed chromatographic method was validated for the following 28 29 30 203 parameters: specificity, linearity, range, precision, accuracy, detection limit, quantitation

31 Accepted 32 204 limit and robustness 37 . 33 34 205 35 36 206 2.5.1. Specificity 37 38 39 207 The specificity was conducted during the forced degradation studies (section 2.4).

40 Methods 41 208 Placebo was also evaluated under the same conditions. Peak purity tests were used to show 42 43 44 209 that the CET chromatographic peak is only attributable to this component. 45 46 210 47 48 211 2.5.2. Precision 49 50 51 212 The precision of the method was evaluated by injection of six independent Analytical 52 53 213 preparations of cephalothin sample at a concentration of 500 g/mL quantified against a 54 55 56 57 58 59 60 Page 11 of 22 Analytical Methods

1 2 3 214 reference standard; the % RSD of assay was calculated. The intermediate precision of the 4 5 6 215 method was evaluated using two different analysts in different days. 7 8 216 9 10 217 2.5.3. Detection limit (LOD) and Quantitation limit (LOQ) 11 12 13 218 The LOD and LOQ were determined at a signaltonoise ratio of 3:1 and 10:1, 14 15 219 respectively, by injecting a series of dilute solutions of CET with known concentrations. 16 17 220 18 19 221 2.5.4. Linearity 20 21 Manuscript 22 222 Linearity standard solutions were diluted from cephalothin stock solution prepared 23 24 223 in purified water (1250 g/mL) at five concentration levels from 50 to 150% of assay 25 26 27 224 analyte concentration (250, 375, 500, 625 and 750 g/mL). Each concentration was 28 29 225 analyzed three times and the calibration curve was constructed using analyte standard peak 30

31 Accepted 32 226 area ratio versus concentration of analyte. Linearity was evaluated by linear leastsquares 33 34 227 regression analysis. 35 36 228 37 38 39 229 2.5.5. Accuracy

40 Methods 41 230 The accuracy of the method was evaluated in triplicate at three concentration levels 42 43 44 231 (375, 500 and 625 g/mL) by standard addition technique. The percentage of recoveries 45 46 232 was calculated at each level. 47 48 49 233 50 51 234 2.5.6. Robustness Analytical 52 53 54 235 To determine the robustness of the developed method, experimental conditions were 55 56 236 deliberately altered and the retention time and area of cephalothin were assessed. The flow 57 58 59 60 Analytical Methods Page 12 of 22

1 2 3 237 rate of the mobile phase was changed to 0.9 and 1.1 mL/min, the column temperature was 4 5 6 238 studied at 30ºC and 40°C, the effect of the percent organic strength was studied by varying 7 8 239 initial proportion of acetonitrile by 8% and 12% and the pH of 20mM ammonium 9 10 240 phosphate buffer was changed to 4.4 and 4.6. Only one parameter was changed while the 11 12 13 241 others were kept constant. 14 15 242 16 17 243 18 244 3. Results 19 245 20 21

246 3.1. Optimization of chromatographic conditions Manuscript 22 23 24 247 The main objective of the chromatographic method is to separate cephalothin from 25 26 248 its degradation products. During the development of this method, different stationary 27 28 29 249 phases such as C18, C8, phenyl and cyano as well as different compositions of mobile 30

31 250 phase were tested (potassium phosphate buffer, triethylamine 0.1% (v/v), triethylamine Accepted 32 33 251 0.1% + acetic acid 0.1% (v/v), sodium perchlorate buffer, acetonitrile, methanol and 34 35 36 252 ethanol). The chromatographic separation was achieved on an Eclipse XDBPhenyl 37 38 253 (250 mm × 4.6 mm, 5m) column using mixture of aqueous 20 mM ammonium phosphate 39

40 254 buffer pH 4.5 and acetonitrile as a mobile phase in a gradient mode. The flow rate of the Methods 41 42 43 255 mobile phase was 1.0 mL/min, the column temperature was 35 ºC, at 238 nm, and the peak 44 45 256 shape of the cephalothin was found to be symmetrical. In optimized chromatographic 46 47 48 257 conditions CET and its degradation products were separated with resolution greater than 2. 49 50 258 The retention time of CET was found to be 14.7 min (Figure 2). 51 Analytical 52 53 259 54 55 56 260 3.2. System suitability 57 58 59 60 Page 13 of 22 Analytical Methods

1 2 3 261 System suitability tests are used to verify that the resolution and reproducibility 4 5 6 262 were adequate for the analysis performed. The parameters measured were tailing factor, 7 8 263 capacity factor, theoretical plates, retention time and repeatability as %R.S.D. of peak area 9 10 264 for six injections of a standard solution of a 500 g/mL of CET (100% concentration). The 11 12 13 265 tailing factor showed less than 2, the capacity factor was more than 2 and the theoretical 14 15 266 plates were more than 2000. The average of retention time was 14.8 minutes and the %RSD 16 17 18 267 of peak area was 0.71%. 19 20 268 21 Manuscript 22 269 3.3. Linearity 23 24 25 270 The linearity for detector response was observed in the concentration range of 250 26 27 271 750 g/mL for cephalothin. The calibration curve was constructed with concentration 28 29 30 272 against peak area. The regression equation for the calibration curve was found to be

31 Accepted 32 273 y=19.61312x + 32.45824 and the correlation coefficient (r 2) of 0.99999 was obtained. Good 33 34 35 274 linearity was found between the peak area and analyte concentration. 36 37 275 38 39 276 3.4. Precision

40 Methods 41 42 277 The repeatability of the method was assessed by six determinations of the 43 44 278 concentration of 500 g/mL representing 100%. The intermediate precision was carried out 45 46 47 279 by a second analyst in a different day. The % RSD of cephalothin during the repeatability 48 49 280 and the intermediate precision was 0.548% and 0.547%, respectively, confirming good 50 51 Analytical 52 281 precision of the method. 53 54 282 55 56 283 3.5. Limit of detection (LOD) and Limit of quantitation (LOQ) 57 58 59 60 Analytical Methods Page 14 of 22

1 2 3 284 The LOD and LOQ were determined experimentally for cephalothin and results 4 5 6 285 were found to be 0.0760 g/mL and 0.2532 g/mL, respectively. 7 8 286 9 10 11 287 3.6. Accuracy 12 13 288 The accuracy of the method was evaluated by recovery studies and was carried out 14 15 16 289 by standard addition method at three different levels (75%, 100% and 125%) described in 17 18 290 table 1. The percentage of recoveries for cephalothin and percent RSD were found to be, 19 20 21 291 respectively, 99.41% and 0.54%. Manuscript 22 23 292 24 25 26 293 3.7. Forced degradation studies (Specificity) 27 28 294 The specificity of the developed method was determined by injecting sample 29 30 295 solutions (500 g/mL) which were prepared by stress conditions. Degradation in 31 Accepted 32 33 296 cephalothin sample was observed when subjected to stress conditions like acid hydrolysis, 34 35 297 base hydrolysis and oxidation. Under oxidative conditions, photodegradation and humidity 36 37 38 298 conditions, in 10 days, cephalothin sample showed stable. The summary of forced 39

40 299 degradation results is given in Table 2 and 3. Typical chromatograms of stress testing are Methods 41 42 43 300 shown in Figure 3 and Figure 4. The placebo was also evaluated (Figure 5) and peak purity 44 45 301 test results confirmed that the cephalothin peak is homogenous and pure in all the analyzed 46 47 302 stress samples (Figure 6). The impurity B (deacetylcephalothin) was identified by 48 49 50 303 comparative analysis with a reference standard solution (Figure 7). 51 Analytical 52 304 Accounting for the fact that additional peaks of degradation products appeared in 53 54 305 the chromatograms in addition to the impurity B, the proposed method demonstrated 55 56 57 306 excellent selectivity and resolution between CET and the other compounds (minimum 58 59 60 Page 15 of 22 Analytical Methods

1 2 3 307 resolution of 2.4), being, therefore, applicable for routine cephalothin analysis, including 4 5 6 308 samples from stability studies. 7 8 309 9 10 310 3.8. Robustness 11 12 13 311 To determine the robustness of the developed method, experimental conditions were 14 15 312 deliberately altered, and in all the varied chromatographic conditions (flow rate, column 16 17 313 temperature, composition of organic solvent and pH value), no significant change was 18 19 20 314 observed and %RSD values showed less than 2, confirming the robustness of the method. 21 Manuscript 22 315 23 24 316 4. Discussion 25 26 27 317 The liquid chromatographic procedure was carried out to develop a stability 28 29 318 indicating method to characterize degradation products from the studied cephalosporin. 30

31 Accepted 32 319 Chromatographic conditions were elected after different mobile phases with different 33 34 320 organic solvent ratios were tested. Moreover, different brands of chromatographic columns 35 36 321 were also tested to optimize the separation, and the effects of organic solvent proportions 37 38 39 322 and compositions were examined.

40 Methods 41 323 The low LOD and LOQ values found are indicative of the high sensitivity of the 42 43 324 method. Besides, an analytical method can be considered specific, when it can demonstrate 44 45 46 325 drug separation and quantitation from the bulk physical mixture, finished product, its 47 48 326 degradation products and excipients. Our results obtained in the stress degradation are in 49 50 327 accordance with the current literature about the structural modifications of cephalothin. Analytical 51 52 53 328 54 55 329 5. Conclusions 56 57 58 59 60 Analytical Methods Page 16 of 22

1 2 3 330 HPLC methods have been widely used by pharmaceutical laboratories and they 4 5 6 331 have gained popularity in stability studies due to their sensitivity, specificity and high 7 8 332 resolution capacity. A validated stabilityindicating LC method was developed for 9 10 333 quantitative determination of cephalothin and also is applicable for the related substances 11 12 13 334 determination. The proposed LC method is found to be sensitive, precise, accurate, linear, 14 15 335 robust, specific and convenient for the separation of cephalothin in the presence of its 16 17 18 336 degradation products, including deacetylcephalothin, its less active metabolite. The method 19 20 337 was fully validated showing satisfactory data for all the method validation parameters 21 Manuscript 22 338 tested. Hence, the developed LC method can be used for the quality control and also for 23 24 25 339 analysis of stability samples. The HPLC method developed is very simple and results 26 27 340 confirm suitable precision, accuracy, and specificity. Therefore, the method could be useful 28 29 341 for both routine analytical and quality control assay of cephalothin in pharmaceutical form 30

31 Accepted 32 342 and it could be a very powerful tool to investigate chemical stability of this cephalosporin. 33 34 343 35 344 36 37 345 Acknowledgements 38 39

40 Methods 41 346 We thank the laboratory União Química Pharmaceutical Industry (Pouso Alegre, Brazil) for 42 43 347 the kind donation of medicines. This work was supported by Bioagri – a Mérieux 44 45 348 NutriSciences Company (Bioagri Laboratórios (Piracicaba, Brazil) and H.R.N. Salgado is 46 47 48 349 grateful to the PACDFCFUNESP (Araraquara, Brazil) and CNPq for financial supports. 49 50 Analytical 51 350 52 53 54 55 351 References 56 57 58 59 60 Page 17 of 22 Analytical Methods

1 2 3 352 1. M.J. O’Neil, The Merck index: an encyclopedia of chemicals, drugs and biologicals. 4 5 353 2006, 14 th ed, Whitehouse Station, pp.328. 6 7 8 354 2. S.C. Sweetman, Martindale : the complete drug reference, 2009, 36th ed. London, 9 10 355 Pharmaceutical Press. 11 12 356 3. Q.A Xu and L.A. Trissel, Stability-indicating HPLC methods for drug analysis. 2008, 3rd 13 14 357 Ed, Washington, Pharmaceutical Press, pp.168170. 15 16 17 358 4. J.G. Hardman and L.E. Limbird, Goodman & Gilman: The pharmacological basis of 18 th 19 359 therapeutics, 2003, 10 Ed, Rio de Janeiro, McGraw Hill, pp.903908. 20 21 Manuscript 22 360 5. B.G. Katzung, Basic and clinical pharmacology,. 2005, 9th Ed, Rio de Janeiro, 23 361 Guanabara Koogan, pp. 620621. 24 25 26 362 6. H.S. Sader and R.N. Jones, Historical overview of the cephalosporin spectrum: four 27 28 363 generations of structural evolution, Antimicrob. Newslett ., 1992, 8, pp.7584. 29 30

31 364 7. Brazil. Ministry of Health. 2007. 5th ed. Brasília, obtained from web site: Accepted 32 33 365 http://portal.saude.gov.br/portal/arquivos/pdf/rename2006.pdf (acessed 3 November 34 366 2013). 35 36 37 367 8. A.G. Vasconcellos, R.D. Leal, A. SilvanyNeto and C.M. NascimentoCarvalho, 38 39 368 Oxacilin or cefalotin treatment of hospitalized children with cellulitis, Jpn. J. Infect.

40 Methods 41 369 Dis., 2012, 65 , pp.712. 42 43 370 9. A. Korolkovas, Guanabara Therapeutic Dictionary, 2010, Guanabara Koogan, 17 th ed. 44 45 371 Rio de Janeiro, Brazil. 46 47 48 372 10. Brazilian Pharmacopoeia, 2010, 5th ed. 2010, ANVISA, Brasília, Brazil. 49 50 51 373 11. V.D. Gupta and K.R. Stewart, Quantitation of carbenicillin disodium, cefazolin sodium, Analytical 52 53 374 cephalothin sodium, nafcillin sodium, and ticarcillin disodium by highpressure liquid 54 375 chromatography. J. Pharm. Sci . 1980, 69 , pp.12641267. 55 56 57 58 59 60 Analytical Methods Page 18 of 22

1 2 3 376 12. European Pharmacopoeia, 2011, 7th ed, Strasbourg, Council of Europe, 2, pp.1603 4 5 377 1604. 6 7 th 8 378 13. US Pharmacopoeia 36 , 2013, US Pharmacopeial Convention, Rockville, MD, 9 379 pp.2906−2907. 10 11 380 14. M.J. Cooper, M.W. Anders and B.L. Mirkin,. Ionpair extraction and highspeed liquid 12 13 381 chromatography of chephalothin and deacetylcephalothin in human serum and urine. 14 382 Drug Metab. Dispos ., 1973, 1, pp.659662. 15 16 17 383 15. R.P. Buhs, T.E. Maxim, N. Allen, T.A. Jacob and F.J. Wolf, Analysis of cefoxitin, 18 19 384 cephalothin and their deacylated metabolites in human urine by highperformance 20 21 385 liquid chromatography, J. Chromatogr. , 1974, 99 , pp.609618. Manuscript 22 23 386 16. I. NilssonEhle, T.T. Yoshikawa, M.C. Schotz and L.B. Guze, Quantitation of 24 25 387 antibiotics by highpressure liquid chromatography: cephalothin, Antimicrob. Agents 26 27 388 Chemother ., 1978, 13 , pp.221227. 28 29 30 389 17. B.C. McWhinney, S.C. Wallis, T. Hillister, J.A. Roberts, J. Lipman and J.P.J. Ungerer,

31 Accepted 32 390 Analysis of 12 betalactam antibiotics in human plasma by HPLC with ultraviolet 33 391 detection, J. Chromatogr. B, 2010, 878 , pp.20392043. 34 35 36 392 18. Y.L. Chang, Y.F. Chen, C.F. Chen and T.H. Tsai, Determination of unbound 37 38 393 cephalothin in rat blood by online microdialysis and microbore liquid 39 394 chromatography, J. Chromatogr. B, 2000, 742 , pp.125130. 40 Methods 41 42 43 395 19. A.H. Moreno and H.R.N. Salgado, Comparison of high performance liquid 44 396 chromatography and three titrimetric methods for the determination of ceftazidime in 45 46 397 pharmaceutical formulations, Adv. Anal. Chem., 2012, 2, pp.613,. DOI: 47 48 398 10.5923/j.aac.20120203.01 49 50 51 399 20. A.H. Moreno and H.R.N. Salgado, Development and validation of HPLC method for Analytical 52 400 determination of ceftazidime, J. AOAC Int., 2007 , 90 , pp.739743. 53 54 55 401 21. D.C.M. Vieira and H.R.N. Salgado, Comparison of HPLC and UV spectrophotometric 56 57 402 methods for the determination of cefuroxime sodium in pharmaceutical products, J. 58 59 60 Page 19 of 22 Analytical Methods

1 2 3 403 Chromatogr. Sci., 2011, 49 , pp.508511. 4 5 6 404 22. A.H. Moreno and H.R.N. Salgado, Stability study and degradation kinetics of 7 8 405 ceftazidime in pharmaceutical preparations, Adv. Analytical Chem., 2012, 2, pp.15. 9 10 406 DOI: 10.5923/j.aac.20120201.01 11 12 407 23. A.H. Moreno and H.R.N. Salgado, Rapid and selective UV spectrophotometric method 13 14 408 for the analysis of ceftazidime, J. AOAC Int ., 2009, 92 , pp.820823. 15 16 17 409 24. T.M. Pedroso and H.R.N. Salgado, Validation of cefazolin sodium in pharmaceutical 18 19 410 dosage forms by UVspectrophotometric method, Phys. Chem., 2013, 3, pp.1120. 20 21 411 doi:10.5923/j.pc.20130301.03 Manuscript 22 23 412 25. A.H. Moreno and H.R.N. Salgado, Spectrophotometric determination of ceftazidime in 24 25 413 pharmaceutical preparations using neocuproin as a complexing agent, 2008, Anal. 26 27 414 Let., 41 , pp.21432152. DOI: 10.1080/00032710802240818 28 29 30 415 26. A.H. Moreno and H.R.N. Salgado, Development and validation of quantitative analysis

31 Accepted 32 416 of ceftazidime in powder for injection by infrared spectroscopy, Phys. Chem., 2012, 2, 33 417 pp.611. DOI: 10.5923/j.pc.20120201.02 34 35 36 418 27. D.C.M. Vieira, P.C. Ricarte and H.R.N. Salgado, Development and validation of the 37 38 419 quantitative analysis of cefuroxime sodium in powder for injection by infrared 39 420 spectroscopy, Adv. Anal. Chem ., 2012, 2, pp.8087.

40 Methods 41 42 421 28. A.H. Moreno and H.R.N. Salgado, Microbiological assay for ceftazidime injection, J. 43 44 422 AOAC Int ., 2007, 90 , pp.13791382. 45 46 47 423 29. Tozo, G.C.G. and Salgado, H.R.N. 2007. Microbiological assay for cefoxitin sodium in 48 49 424 dosage form. J. AOAC Int . 90, 452455 . 50 51 Analytical 52 425 30. D.C.M. Vieira, P.C. Ricarte and H.R.N. Salgado, Validation of microbiological assay 53 426 for determination of cefuroxime in injectable preparations. Latin Am. J. Pharm ., 2012, 54 55 427 31 , pp.746750. 56 57 58 59 60 Analytical Methods Page 20 of 22

1 2 3 428 31. P.V. Aléssio and H.R.N. Salgado, Development and validation of a successful 4 5 429 microbiological agar assay for determination of ceftriaxone sodium in powder for 6 7 430 injectable solution, Pharmaceutics , 2012, 4, pp.334342, DOI: 8 9 431 10.3390/pharmaceutics4030334. 10 11 432 32. T.M. Pedroso and H.R.N. Salgado, Development and validation of a microbiological 12 13 433 assay by turbidimetry to determine the potency of cefazolin sodium in lyiphilized 14 15 434 powder, Anal. Methods , 2014. in press . 16 17 18 435 33. ICH, Stability testing of new drug substances and products, Q1A (R2), 2003, 19 20 436 International Conference on Harmonization of Technical Requirements for 21 437 Registration of Pharmaceuticals for human use, IFPMA, Geneva. Manuscript 22 23 24 438 34. L.C. Vaucher, C.S. Paim, A.D. Lange and E.E.S. Schapoval, LC method for 25 26 439 telithromycin in tablets: A stabilityindicating assay, Int. J. Pharmaceutics , 2009, 33 , 27 28 440 pp.8287. 29 30 441 35. K.M. Alsante, A. Ando, R. Brown, J. Ensing, T.D. Hatajik, W. Kong, and Y. Tsuda,

31 Accepted 32 442 The role of degradant profiling in active pharmaceutical ingredients and drug products, 33 34 443 Adv. Drug Deliv. Rev ., 2007, 59 , pp.29–37. 35 36 37 444 36. S. Klick, P.G. Muijsellar, J. Waterval, T. Eichinger, C. Korn, T.K. Gernding, A.J. 38 39 445 Debets, C.S. Griend, C. Beld, G.W. Somsen and Jong, G.J ., Toward a generic approach

40 446 for stress testing of drug substances and drug products. Pharmaceutical Technol ., Methods 41 42 447 2005, pp.4866. 43 44 448 37. ICH Q2 (R1), 2005. Validation of analytical procedures: text and methodology. In: 45 46 449 Proceedings of the International Conference on Harmonization. 47 48 450 38. D.W. Reynolds, K.L. Facchine, J.F. Mullaney, K.M. Alsante, T.D. Hatajik and M.G. 49 50 451 Motto, Available guidance and best practices for conducting forced degradation 51 Analytical 52 452 studies, Pharm.Tech., 2002, 26 , pp.4855. 53 54 453 55 56 57 58 59 60 Page 21 of 22 Analytical Methods

1 2 3 4 1 Table 1 5 6 7 2 Results of recovery for cephalothin. 8 9 Amount of Standard 10 Total Found Recovery Mean Recovery 11 Cephalothin added 12 (µg/mL) (%)* (%) 13 (µg/mL) 14 R1 15 123.15 123.07 99.93 16 R2 246.30 243.50 98.86 17 99.41 18 R3 369.46 367.44 99.45 19 20 21 3 * Mean value of three replicates Manuscript 22 4 23 24 5 25 6 26 7 27 28 8 29 9 30 10

31 Accepted 32 11 Table 2 33 34 35 12 Summary of forced degradation results of cephalothin. 36 37 38 Stress condition Time % Degradation Purity factor 39

40 Methods 41 Acid hydrolysis (0.1 M HCl, 60 °C) 40 minutes 16.10 99.92 42 43 Base hydrolysis (0.01 M NaOH, 60 °C) 5 minutes 14.92 99.91 44 45 46 Oxidation (3% H 2O2, room 20 minutes 99.98 47 20.24 48 temperature) 49 50 Photodegradation (UV/VIS) 10 days 0.10 99.98 51 Analytical 52 Thermal (60 °C) 10 days -0.81 99.98 53 54 Humidity (30 ºC / 75% RH) 10 days -1.32 99.91 55 56 57 13 58 59 60 Analytical Methods Page 22 of 22

1 2 3 14 Table 3 4 5 6 15 Summary of degradation products. 7 8 Acid hydrolysis Basic hydrolysis Oxidation 9 RRT 10 (%) area (%) area (%) area 11 12 13 0.26 ND ND 0.13 14 0.29 ND 0.11 ND 15 16 0.37 ND 0.06 ND 17 18 0.39 ND ND 0.13 19 20 0.44 0.73 2.86 1.54 21 0.49 ND ND 0.25 Manuscript 22 23 0.58 0.23 ND ND 24 25 0.60 ND ND 1.80 26 0.72 (impurity B) 3.05 10.49 0.12 27 28 0.76 ND ND 9.50 29 30 0.77 ND ND 1.26

31 0.82 0.04 ND ND Accepted 32 33 0.87 1.54 ND ND 34 35 0.89 0.13 0.16 0.14 36 0.92 1.80 ND ND 37 38 1.04 ND 0.05 ND 39

40 1.18 ND 0.05 ND Methods 41 42 1.28 ND 0.04 ND 43 16 RRT: Retention relative time; ND: not detected 44 45 46 17 47 18 48 49 50 51 Analytical 52 53 54 55 56 57 58 59 60