Forced of metronidazole, oxytetracycline and furazolidone studies using High Performance Liquid Chromatography Studiul de degradare for at a metronidazolului, oxitetraciclinei i furazolidonei folosind Cromatografie de Lichid de Înalta Performan Violeta Tauber (Giugiu), Viorica Chiurciu Romvac Company S.A. Key words: metronidazol, oxytetracycline, furazolidone, forced studies, C 18 column. Cuvinte cheie: metronidazol, oxitetraciclina, furazolidona, studiu de degradare fortata, coloana C 18. Abstract The study describes the development of a HPLC -DAD method for the estimation of oxytetracycline and furazolidone in Enteroguard M - powder. The proposed method utilizes a Betasil C 18 column, (4.6 x 2 mm, 5 µm), at 3 C, optimum mobile phase consisted of 4 mm phosphate buffer (ph=7.5): methanol in gradient elution. The flow rate was optimized to 1mL/min and the detection was carried at 254 nm. The drug substance was exposed to temperature, light, oxidants, acid and basic hydrolysis, and the resultant samples were analyzed by the proposed method. Rezumat Studiul descrie dezvoltarea unei metode HPLC DAD pentru estimarea metronidazolului, oxitetraciclinei si furazolidonei din produsul Enteroguard M pulbere. In metoda propusa, separarea s-a facut pe o coloana Betasil C 18, (4.6 x 2 mm, 5 µm) la 3 C, folosind elutia in gradient a fazei mobile formata din tampon fosfat 4 mm (ph=7.5) si metanol. Achizitia s-a facut la lungimea de unda de 254 nm, folosind un debit de 1mL/min. Produsul a fost expus urm toarelor conditii de stres: temperatura, lumin, agen i oxidanti, hidroliza in mediu acid si mediu bazic, si probele rezultate au fost analizate prin metoda propusa. Introduction Oxytetracycline is a broad spectrum antibiotic cu used in the veterinary medicine to inhibit gram-positive and gram-negative bacteria synthesis. The European Committee has approved the use of oxytetracycline in a wide range of species: cats, dogs, sheep, goats and swine. Both oxytetracycline and oxytetracycline hydrochloride contain impurities that in the starting material should not exceed the European Pharmacopeia-required limits. Furazolidone is a nitrofuran antibacterial with a nitro group in its molecular structure. This group has a broad antibacterial and antiparasitic action and this is why nitrofurans are widely used in the treatment of gastrointestinal infections in cage birds, dogs and cats. Metronidazole is effective against protozoa and anaerobic bacteria, being used for prevention and treatment as well. The purpose of this study is to develop a new HPLC method for the assessment of furazolidone, oxytetracycline and related substances in veterinary products. 1. Materials and methods 1.1. Reference materials and reagents The oxytetracycline standard and the following related substances were purchased from the European Pharmacopeia: 4-epioxytetracycline, (4-EOTC); 73
tetracycline hydrochloride, (TC); -apo-oxytetracycline, ( -AOTC); -apo-oxytetracycline, ( -AOTC). The furazolidone standard was purchased from the USP. The metronidazole standard and impurity A were purchased from the European Pharmacopeia. The studied medicinal product, Enterogurad M powder was supplied by Romvac Company. Ultrapure water was used for the preparation of all solutions, obtained in-house with a Milli-Q system (Millipore, USA). The HPLC methanol was supplied by Merck. The HPLC dibasic potassium phosphate, hydrochloric acid and sodium hydroxide were supplied by Fluka. The oxygenated water was supplied by Chimreactiv SRL. The 85% orthophosphoric acid, used for ph adjustment, was purchased from Merck. Dimethylformamide used for sample preparation was purchased from Sigma. 1.2. Chromatographic system and The chromatographic separation of active substances (metronidazole, oxytetracycline and furazolidone) from their impurities (impurity A, 4-epioxytetracycline, tetracycline hydrochloride, alpha-apooxytetracycline, beta-apo-oxytetracycline) and from potential products has been carried out on a Betasil C 18 column (4.6 x 2 mm, 5 µm). The gradient elution of the mobile phase consisting of phosphate buffer 4 mm (ph=7.5) and methanol, is the appropriate condition for this chromatographic separation. Acquisition was at 254 nm wavelength, at 3 C with a 1mL / min flow rate. The elution gradient is described in table 1. Table 1 Gradient separation program Time (min) % solvent A METHANOL %solvent B HK 2PO 4. 25. 75. 9. 43. 57. 18. 43. 57. 22. 25. 75. 3. 25. 75. 1.3. Preparation of standard solutions The standard metronidazole solution (C=.5 mg/ml) was prepared in methanol. The standard furazolidone solution (C=.5 mg/ml) was prepared in DMF. The standard stock oxytetracycline solution (C= 1mg/mL) was prepared in HCl.1 M. The working solutions of different concentrations (.8 mg/ml metronidazole,.5 mg/ml oxytetracycline,.17 mg/ml furazolidone) were prepared from the mentioned socks and diluted in HCl.1 M. The solutions were well mixed, filtered through PVDF.45 µm filter and injected in chromatographic system. The impurity stock was prepared by separately weighing 4-epioxytetracycline (C= 1 mg/ml), tetracycline hydrochloride (C= 1 mg/ml), alpha-apo-oxytetracycline (C=.8 mg/ml) and beta-apooxytetracycline (C=.8 mg/ml), followed by dissolution in HCl.1 M. Impurity A (C= 1mg/mL) of metronidazole was dissolved in methanol. The solutions were stirred in ultrasound bath for 1 minutes. The working solutions of different concentrations were prepared in HCl.1 M. The standard solution stocks were stored in refrigerator. 1.4. Preparation of sample solutions The amount of powder equal to 66.6 mg metronidazole, 4 mg oxytetracycline and 74
13.3 mg furazolidone was weighed in a ml volumetric flask. 15 ml HCl.1M, 5 ml methanol and 16 ml DMF were poured over this amount and the solution obtained was sonicated for 1 minutes. After cooling, the ml amount was brought to volume with HCl.1M. The working solutions were prepared from this solution in HCl.1 M, to obtain final concentrations of.8 mg / ml metronidazole,.5 mg / ml oxytetracycline and.17 mg / ml furazolidone. The solutions were well stirred, filtered by PVDF.45 µm filter and injected in chromatographic system. 1.5. Degradation study The study was conducted to assess the specificity of the analytical method in the presence of impurities and products. Sample stock solution In a ml volumetric flask were weighed.6666 g Enteroguard M - powder, equal to 66.6 mg metronidazole, 4 mg oxytetracycline and 13.3 mg furazolidone. 15 ml HCl.1M, 5 ml methanol and 16 ml DMF were poured over this amount and the solution obtained was sonicated for 1 minutes. After cooling, the ml amount was brought to volume with HCl.1M and the solution was filtered on quality filter paper type 1289. Degradation in acid medium In a 1 ml volumetric flask,.64 ml from the sample stock solution was treated with 6 ml HCl.1 N for 9 minutes at 25 C. After cooling the 1 ml amount was brought to volume with HCl.1M. Before being injected in the chromatographic system, the solution was filtered through PVDF.45 µm. Degradation in basic medium In a 1 ml volumetric flask,.64 ml from the sample stock solution was treated with6 ml NaOH.1 N for 9 minutes at 25 C. After cooling the 1 ml amount was brought to volume with HCl.1M. Before being injected in the chromatographic system, the solution was filtered through PVDF.45 µm. Oxidative In a 1 ml volumetric flask,.64 ml from the sample stock solution was treated with 6 ml H 2 O 2 3% for 9 minutes at 25 C. After cooling the 1 ml amount was brought to volume with HCl.1M. Before being injected in the chromatographic system, the solution was filtered through PVDF.45 µm. Thermal In a 1 ml volumetric flask,.64 ml from the sample stock solution was treated with 6 ml HCl.1 M for 6 at 6 C. After cooling the 1 ml amount was brought to volume with HCl.1M. Before being injected in the chromatographic system, the solution was filtered through PVDF.45 µm. Photolytic In a 1 ml volumetric flask,.64 ml was exposed at short wavelengths (253nm) for 12 h. At the end of exposure, the 1 ml amount was brought to volume with HCl.1M. Before being injected in the chromatographic system, the solution was filtered through PVDF.45 µm 2. Results and discussions The proposed method resulted appropriate for the determination of metronidazole, oxytetracycline and furazolidone in the presence of products. The mobile phase, consisting of methanol and dibasic potassium phosphate 4 mm at ph 7.5, is optimal, with an appropriate separation of: metronidazole (t R = 5.6 min), furazolidone (t R = 7.2 min), oxytetracycline (t R = 1.5 min) 75
in the presence of related substances at a flow rate of 1 ml/min. The 1 L volume, the 254 nm wavelength and the temperature of 35 C resulted appropriate for this method. The results of the study are illustrated in the chromatograms in figures 1-5 and the calculated parameters are shown in tables 3-5. Fig.1. Degradation of Enterogurd M powder in acid medium 76
Fig.2. Degradation of Enterogurd M powder in basic medium. 5 4 4 pda-254nm PD H2O2 3% Pk # Name Retention Time Area ASTM LOD ASTM LOQ 5 4 4 mau 3 3 2 2 1 1-1 2.38 28465 2 excipienti 3.237 12227.. (imp A ).. 3 m etronidazol 5.673 3147183.3.11 4 furazolidona 7.27 236636.27.91 (4 epioxitetraciclina).. 5 oxitetraciclina 1.492 2941717.325.182 (alfa oxitetraciclina).. (tetraciclina).. (beta oxitetraciclina).. 3 3 2 2 1 1 - mau 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 Mi nutes Fig.3. Degradation of Enterogurd M powder in oxidant medium. 4 4 pda-254nm PD UV Pk # Name Retention Time Area ASTM LOD ASTM LOQ 4 4 mau 3 3 2 2 1 1-1 excipienti 3.235 9914.. (imp A ).. 2 m etronidazol 5.667 3128667.8.26 3 furazolidona 7.26 2172112.4.132 (4 epioxitetraciclina).. 4 oxitetraciclina 1.3 276348.293.978 (alfa oxitetraciclina).. (tetraciclina).. (beta oxitetraciclina).. 3 3 2 2 1 1 - m AU 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 Minutes Fig.4. Degradation of Enterogurd M powder under UV radiation 77
5 4 pda-254nm PD termic Pk # Name Retention Time Area ASTM LOD ASTM LOQ 5 4 4 4 m AU 3 3 2 2 1 1-1 ex c ip ien ti 3.2 4 8 7 2.. (im p A ).. 2 m etro n id az o l 5.6 8 2 3 2 1 2 9 4 8. 9. 3 1 3 fu ra z o lid o n a 7.2 8 2 2 2 6 4 1 6 4. 3 2. 1 6 (4 ep io x ite trac ic lin a ).. 4 o x itetra c ic lin a 1.5 2 3 2 8 7 8 9 6. 3 1 6. 1 5 2 5 1 2.6 3 8 9 5 3 5 (alfa o x itetrac iclin a ).. (te trac iclin a ).. (b e ta o x itetra c iclin a ).. 3 3 2 2 1 1 - m AU 1 2 3 4 5 6 7 8 9 1 11 12 13 14 15 16 17 18 19 2 21 22 23 24 25 26 27 28 29 3 Minutes Fig.5. Degradation of Enterogurd M powder under temperature. Results for Enteroguard M powder in various media Table 3 Stress Duration of Metronidazole Area % recovery Calculated parameters % Retention time Asymmetry Theoretical plates Enteroguard M powder 1 min 3228117 5.68 1.1263 18845. PD HCl.1 N 9 min 3157869 97.82 2.18 5.69 1.1227 18912. PD NaOH.1 N 9 min 3127616 96.89 3.11 5.68 1.1828 18763. PD H2O2 3% 9 min 3147183 97.49 2.51 5.67 1.11132 18667. PD thermal 6 min 3128667 96.92 3.8 5.67 1.1239 18871. PD UV 72 min 3212948 99.53.47 5.68 1.11635 1935. Results for Enteroguard M powder in various media Table 4 Stress Duration of Furazolidone Area % recovery Calculated parameters % Retention time Asymmetry Theoretical plates Enteroguard M powder 1 min 2481414 7.282 1.11455 27157 PD HCl.1 N 9 min 23596 94.91 5.9 7.37 1.127 27461 PD NaOH.1 N 9 min 123462 4.98 95.2 7.283 1.1625 26954 PD H2O2 3% 9 min 236636 92.96 7.4 7.27 1.9643 27164 PD thermal 6 min 2172112 87.54 12.46 7.26 1.11123 2748 PD UV 72 min 2264164 91.24 8.76 7.282 1.1136 27536 78
Results for Enteroguard M powder in various media Table 5 Stress Duration of Oxytetracycline Area % recovery Calculated parameters % Retention time Asymmetry Theoretical plates Enteroguard M powder 1 min 316282 1..9164 16222. PD HCl.1 N 9 min 322523 97.3 2.7 1.55.924 16476. PD NaOH.1 N 9 min 2915831 93.87 6.13 1.52.91773 16284. PD H2O2 3% 9 min 2941717 94.7 5.3 1.49.9556 15752. PD thermal 6 min 276348 88.95 11.5 1..95738 163. PD UV 72 min 287896 9.39 9.61 1.52.93563 1624. Conclusions The absence of additional peaks indicates that the product is stable in acid medium. Enteroguard M - powder is sensitive in alkaline medium because after exposure at 25 C in NaOH.1N for 9 minutes, furazolidone was approximately 95 % degraded and three additional peaks appear with retention at minutes: 3.8, 6.4, 9.4. After exposure of Enteroguard M powder to ultraviolet radiation, at 6 C (for 6 minutes) and in the presence of 3% oxygenated water as well, there were no peaks noticed which indicates that the product is stable in the presence of the following agents: oxidant, thermal and photolytic. The study indicates that furazolidone degrades 95% in basic medium while in photolytic medium it degrades12%, and oxytetracycline 11%. This study involved using a new HPLC method for the determination of impurities and active substances in Enteroguard M powder, in the presence of products. This method can be successfully applied to this product since there were no interferences of excipients or other products from the pharmaceutical product. References 1. European Pharmacopoeia 7. (Ed. 211), pp. 2651-2653. 2. Pietruszka K, Olejnik M, Sell B. (213). Forced study on dronedarone and application of validated stabilityindicating HPLC-UV method in stability testing of dronedarone tablets. 3. Pietruszka K, Olejnik M, Sell B. (27). Developement and validation of liquid chromatography method for the determination of nitrofurans in water. 4. Rajyalakshmi C, Benjamin T and Rambabu. C 5. Smyrniotakis CG, Archontaki HA. (27). C 18 columns for the simultaneous determination of oxytetracycline and related substances by reversed-phase high performance liquid chromatography and UV detection. 6. Wu Y, Fassihi R (25). Stability of metronidazole, tetracycline HCl and famotidine alone and in combination 7. www.ich.org 79