Acta Poloniae Pharmaceutica ñ Drug Research, Vol. 68 No. 6 pp. 823ñ829, 2011 ISSN 0001-6837 Polish Pharmaceutical Society

ANALYSIS

HPLC METHOD FOR IDENTIFICATION AND QUANTIFICATION OF BENZ- IMIDAZOLE DERIVATIVES IN ANTIPARASITIC DRUGS

ANNA KULIK1*, WANDA BIA£ECKA1, MARZENA PODOLSKA1, BARBARA KWIATKOWSKA-PUCHNIARZ1 and ALEKSANDER MAZUREK1,2

1Department of Basic and Applied Pharmacy, National Medicines Institute, 30/34 Che≥mska St., 00-725 Warszawa, Poland 2Department of Drug Chemistry, Medical University of Warsaw, 1 Banacha St., 02-097 Warszawa, Poland

Abstract: The subject of the study was to develop a versatile HPLC system for identification and determina- tion of four benzimidazole derivatives in the antiparasitic drugs. The tests covered: Zentel, Panacur, Vermox tablets and Systamex suspension. A satisfactory separation was obtained using the Nucleosil C8 column in the gradient system composed of mobile phase A: 85% orthophosphoric acid / water / acetonitrile in 0.05:75:25, v/v/v ratio, and mobile phase B: 85% orthophosphoric acid / water / acetonitrile in 0.05:50:50, v/v/v ratio. Both phases were adjusted to pH = 4.5 with 15% sodium hydroxide solution. A detection at 288 nm for oxfendazole and 254 nm for albendazole, fenbendazole and mebendazole was applied. The correlation coefficients in the range 0,9997 ñ 0,9999 proved that the calibration curves were linear. The method was validated in terms of selectivity, accuracy and precision.

Keywords: albendazole, fenbendazole, mebendazole, oxfendazole, HPLC method

Parasitosis is a human and animal disease in drugs (tablets and suspensions) intended both for caused by a parasitic infection. The most common humans and animals. parasitosis is helminthiasis, caused by the nema- The available literature contains only sparse todes (threadworms, ascarids, and whipworms) or studies related to the quantification of benzimidazole tapeworms. derivatives in drugs by HPLC. It concerns quantifi- Benzimidazole derivatives: albendazole, fen- cation of mebendazole with levamisol (1), mebenda- bendazole, mebendazole make one of the groups of zole with tiabendazole (2), mebendazole (3, 4), compounds used in treatment of parasitosis. Figure oxfendazole and oxyclosanide (5), albendazole (6) 1 shows structural and molecular formulas of the and albendazole with ivermectin (7). The rest of the benzimidazole derivatives. The substances widely publications concern quantification of various benz- affect adults and larvae of various intestinal and pul- imidazole derivatives in biological material: milk monary nematodes and tapeworms. They inhibit (8ñ11), meat (12ñ14) and plasma (15ñ21). microtubules synthesis in worms, disturbing glucose adsorption and digestion functions of a parasite. EXPERIMENTAL There are many drug products available on the market for treating parasitosis. The products are Materials available as tablets or suspensions with many auxil- Medical products: Zentel tablets by iary substances, such as methyl p-hydroxybenzoate, GlaxoSmithKline (albendazole 200 mg) ñ for propyl p-hydroxybenzoate, sorbic acid, benzoic acid humans, Panacur tablets by Intervet International or saccharin. B.V. (fenbendazole 250 mg) ñ for dogs and cats, The subject of the study was to develop a sin- Vermox tablets by Gedeon Richter Plc. (mebenda- gle, simple HPLC method for identification and zole 100 mg) ñ for humans, Systamex suspension by quantification of albendazole, fenbendazole, meben- Vetoquinol Biowet Sp. z o.o. (oxfendazole 2.265 dazole and oxfendazole g/100 g) ñ for cattle, sheep, goats, pigs and horses.

* Corresponding author: e-mail: [email protected]

823 824 ANNA KULIK et al.

Ph. Eur. standards: albendazole, fenbendazole, mg/mL for albendazole and fenbendazole, 0.20 mebendazole and oxfendazole. mg/mL for mebendazole and 0.40 mg/mL for oxfendazole. A volume of 5 mL of each solution Reagents and apparatus was diluted to 25 mL with mobile phase B. Acetonitrile and methanol were of HPLC grade, all other chemicals were of analytical grade. Sample solutions A Shimadzu LC system with computer control and Ten tablets of Zentel, Panacur or Vermox were UV-VIS SPD-10AVVP detector, LC-10ATVP pulverized. A quantity of powdered tablet mass cor- pumps, DGU-14A degasser, SCL-10AVP controller responding to 50 mg albendazole for Zentel, 50 mg and SIL-10ADVP autosampler were used. fenbendazole for Panacur and 20 mg mebendazole for Vermox was weighed and transferred to 100 mL Standard solutions volumetric flasks. Systamex suspension was Standards of the active substances were dis- weighed in a quantity corresponding to 40 mg solved in 0.25% hydrochloric acid solution in oxfendazole and transferred to 100 mL volumetric methanol (solvent) to obtain concentration: 0.50 flask. The samples with portions of the solvent were

Figure 1. Structural and chemical formulae of the studied compounds HPLC method for identification and quantification of benzimidazole derivatives... 825

Figure 2. Chromatogram of a mixture of four analyzed compound at two different wavelengths. Retention times: oksfendazole 12,6; mebendazole 14,1; albendazole15,1; fenbendazole 16,8. The resolution between compounds: oksfendazole ñ mebendazole 9.8; mebenda- zole ñ albendazole 5.3; albendazole ñ fenbendazole 7.3

shaken in a mechanical shaker, diluted to 100 mL ing configuration was finally selected: Nucleosil C8 with the solvent, and filtered through a 0.45 µm fil- 5 mm, 250 × 4.6 mm column; mobile phase A: 85% ter. Five mL of each filtered sample solution was orthophosphoric acid, water and acetonitrile (0.05 : diluted to a 25 mL with mobile phase B. 75 : 25, v/v/v) adjusted to pH 4.5 (with 15% NaOH solution), phase B: a mixture of 85 % orthophos- RESULTS AND DISCUSSION phoric acid, water and acetonitrile (0.05 : 50 : 50, v/v/v) adjusted to pH 4.5 (with 15% NaOH solution) At first, an optimal system for identification ñ in the following gradient system: and separation of analyzed compounds and excipi- ents, which is especially important for suspensions, Time (min) Phase B (%) was searched. Several chromatographic systems 0ñ7 0 were tested (columns, mobile phases and gradient 8ñ9 0→100 runs). 10ñ20 100 An influence of a phase pH on separation of 21ñ22 100→0 mebendazole and albendazole was observed (the lower the pH, the worse the separation). The follow- 23ñ25 0 826 ANNA KULIK et al.

Figure 3. Chromatogram of a mixture of excipients in suspensions containing benzimidazole derivatives. Retention times of excipients: saccharin 2.7; benzoic acid 5.9; sorbic acid 6.5; methyl p-hydroxybenzoate 9.5; propyl p-hydroxybenzoate 14.4

Figure 4. Chromatograms of drug products. A ñ albendazole, F ñ fenbendazole, M ñ mebendazole, O ñ oksfendazole, Kñ sorbic acid, H ñ methyl p-hydroxybenzoate HPLC method for identification and quantification of benzimidazole derivatives... 827

Table 1. Calibration curve parameters, correlation coefficients, detection limits (LOD) and quantitation limits (LOQ) of the analyzed substances. Substance Calibration curve parameters LOD LOQ determined y = ax + b [µg/mL] [µg/mL] ∆ a ± a = 29441 ± 861; Sa = 335 ∆ Albendazole b ± b = 45334 ± 140885; Sb = 54806 9.1 27.7

Sy = 81551 r = 0.9997 ∆ a ± a = 48715± 601; Sa = 245 ∆ Fenbendazole b ± b = ñ52743 ± 91135; Sb = 37245 4.3 13.1

Sy = 63956 r = 0.9999 ∆ a ± a = 69005 ± 550; Sa = 225 ∆ Mebendazole b ± b = ñ9085 ± 84014; Sb = 34335 2.8 8.6

Sy = 59205 r = 0.9999 ∆ a ± a = 36806 ± 338; Sa = 138 ∆ Oksfendazole b ± b = ñ45166 ± 51873; Sb = 21199 3.3 10.0

Sy = 36754 r = 0.9999 a, b ñ regression coefficients; Sa, Sb ñ standard deviation of regression coefficients;

Sy ñ standard error of the estimate; r ñ correlation coefficient

Table 2. Results and statistic evaluation of assay in Vermox, Zentel, Panacur tablets and Systamex suspension. Drug product Declared amount Found amount of active substance (active substance) of active substance n Mean S.D. %RSD Vermox (Mebendazole) 100.0 mg 6 97.62 ± 0.76mg 0.954 0.98 Zentel (Albendazole) 200.0 mg 6 202.74 ± 1.52mg 1.906 0.94 Panacur (Fenbendazole) 250.0 mg 6 254.63 ± 1.57mg 1.959 0.77 Systamex (Oxfendazole) 2.265 g/100 g 6 2.343 ± 0.019 g/100 g 0.023 1.00

Table 3. Results and statistical evaluation of recovery in Vermox, Zentel and Panacur tablets. Vermox (Mebendazole) Zentel (Albendazole) Panacur (Fenbendazole) Amount Amount Recovery Amount Amount Recovery Amount Amount Recovery added found [%] added found [%] added found [%] [mg] [mg] [mg] [mg] [mg] [mg] 16.20 16.16 99.75 40.30 40.47 100.42 40.00 39.26 98.15 16.50 16.37 99.21 39.00 39.40 101.02 39.20 39.15 99.87 20.50 20.41 99.56 49.90 49.32 98.84 50.00 49.36 98.72 20.00 19.75 98.75 47.80 47.37 99.10 49.70 48.89 98.37 24.50 24.56 100.24 59.10 58.38 98.78 60.90 59.68 98.00 24.80 24.39 98.35 58.80 58.01 98.66 60.30 59.28 98.31

Mean 99.31 ± 0.72 Mean 99.47 ± 1.05 Mean 98.57 ± 0.71 S.D. 0.68 S.D. 1.00 S.D. 0.68 %RSD 0.69 %RSD 1.00 %RSD 0.69 828 ANNA KULIK et al.

Table 4. Comparison of assay result of oxfendazole in Systamex Limit of detection and limit of quantitation suspension obtained by spectrophotometric method and proposed A limit of detection (LOD) and a limit of quan- HPLC method. titation (LOQ) were established based on the cali- Sample Spectrophotometric HPLC method bration curve parameters: LOD = 3.3◊Sy/a and No. method [% L.C.] LOQ = 10◊Sy/a, where Sy = standard error of esti- [% L.C.] mate, a = slope of the straight line. Table 1 shows 1. 105.6 102.5 the results. 2. 105.0 103.7 3. 107.1 104.9 Content determination 4. 104.1 102.5 Ten µL samples of prepared standard and sam- 5. 104.2 102.7 ple solutions were injected into the column. Figure 4 shows sample chromatograms. Table 2 shows the 6. 105.2 104.4 results and their statistical evaluation. Mean 105.2 103.4 S.D. 0.024 0.023 Accuracy %RSD 1.02 1.00 For Zental, Panacur and Vermox tablets estimated by sample analysis with known concentration of active substance Active substance standards (80%, 100%, and Mobile phase flow rate was 1.5 mL/min. 120% of declared content) were added to the mix- Detection at two wavelengths (UV-VIS detector) tures of excipients specific for each formulation. was applied: 288 nm for oxfendazole and 254 nm The samples were analyzed for content determina- for albendazole, fenbendazole, mebendazole and tion. Recovery of 98.0ñ100.2% (Table 3) confirmed identification of excipients in Systamex suspension. accuracy of the method. Figure 2 shows the chromatogram of the standardsí mixture. The retention times and resolution between For Systamex suspension estimated by comparison the neighboring peaks are also shown in the Figure with pharmacopoeial method caption. Oxfendazole, the active substance in The separation of several excipients present in Systamex, was also quantified by the spectrophoto- suspensions (for example in Zentel, Valbazen, metric method specified by British Pharmacopoeia. Systamex, Fenbesan) was checked. The results show The solutions of standard and sample at concentra- that the HPLC method can also be used for identifi- tion of 0.008 mg/mL were prepared in methanol. cation and determination of excipients in some sus- The samples were assayed spectrophotometrically at pensions. the wavelength 296 nm. The chromatogram of excipients used in sus- The results for HPLC method are very similar pensions including benzimidazole derivatives is to the values obtained by the reference (pharma- shown in Figure 3. copoeial) spectrophotometric method (Table 4). However, the HPLC results were closer to the Specificity declared content than those obtained from the spec- To verify the specificity of the method, the sol- trophotometric assay. This fact can be interpreted as vent, and the solutions of standards and excipients a slighty higher accuracy of the proposed HPLC were injected into the column. The chromatograms method. show that peaks of the solvent and excipients do not interfere with the peaks of active substances. CONCLUSION

Linearity The results show that the developed HPLC Relation between peak areas and concentra- method is versatile and can be applied for four ana- tions of each compound was investigated for the fol- lyzed active substances: albendazole, fenbendazole, lowing concentration ranges: fenbendazole, meben- mebendazole and oxfendazole. It allows identifica- dazole or oxfendazole 0.03 to 0,3 mg/mL and alben- tion and quantification of the compounds in various dazole 0.05 to 0.3 mg/mL. Table 1 shows the linear formulations (tablets and suspensions). The advan- regression parameters with the correlation coeffi- tage of the method is that it can be used for identifi- cients. The latter prove the linearity of the calibra- cation and determination of several excipients in tion graphs. suspensions (saccharin, benzenecarboxylic acid, HPLC method for identification and quantification of benzimidazole derivatives... 829 sorbic acid, methyl p-hydroxybenzoate, and propyl 7. Anil W., Shubash G., Roshan J. et.al.: Indian J. p-hydroxybenzoate) which is not possible by a spec- Chem. Technol. 15, 617 (2008). trophotometric method. 8. De Ruyck H., Van Renterghem R., De Ridder The method is specific because no interference H. et al.: Food Control 11, 165 (2000). of the peaks was observed. Values of the correlation 9. Balizs G., Hewitt A.: Anal. Chim. Acta 492, coefficients, being near unity, prove that the method 105 (2003). is linear. Accuracy of the method was checked for 10. Aguilera-Luiz M.M., Vidal J.L.M., Romero- the albendazole, fenbendazole and mebendazole Gonz·lez R. et al.: J. Chromatogr. A 1205, 10 tablets by means of recovery analysis and for the (2008). Systamex suspension by comparison with a spec- 11. Ortelli D., Cognard E., Jan P. et al.: J. trophotometric (pharmacopoeial) method. In all Chromatogr. B 877, 2363 (2009). samples of the suspension, assay of the active sub- 12. Gentili A., Peret D., Marchese S.: Trends Anal. stance determined by HPLC generated values closer Chem. 24, 704 (2005). to the declared content than those obtained by spec- 13. Dowling G., Cantwell H., O¢Keeffe M. et al.: trophotometry. The results showed good accuracy of Anal. Chim. Acta 529, 285 (2005). the elaborated HPLC method. 14. Kaufmann A, Butcher P., Maden K. et al.: J. Chromatogr. A 1194, 66 (2008). REFERENCES 15. Mirfazaelian A., Dadashzadeh S., Rouini M.R.: J. Pharm. Biomed. Anal. 30, 1249 (2002). 1. Zarapkar S.S., Halkar U.P., Rane S.H.: Indian 16. Kitzman D., Cheng Kuei-Ju, Fleckenstein L.: J. Drugs 36, 517 (1999). Pharm. Biomed. Anal. 30, 801 (2002). 2. Orsine E.M.A., Kedor-Hackmann E.R.M., 17. Chen X., Zhao L., Xu H., Zhong D.: J. Pharm. Santoro M.J.R.M.: Drug Dev. Ind. Pharm. 26, Biomed. Anal. 35, 829 (2004). 879 (2000). 18. Batzias G.C., Theodosiadou E., Delis G.A.: J. 3. Xiang Y.Z., Chen M.S., Xia J., Gao H., Sun Pharm. Biomed. Anal. 35, 1191 (2004). W.J.: Yaowu Fenxi Zazhi 19, 331 (1999). 19. Chiap P., Evrard B., Bimazubute M.A.: J. 4. Al-Kurdi Z., Al- Jallad T., Badwan A., Jaber Chromatogr. A 870, 121 (2000). A.M.Y.: Talanta 50, 1089 (1999). 20. Bonato P.S., Oliveira A.R.M de, Santana F.J.M. 5. Khan A.R., Akhtar M.J., Mahmood R. et al.: J. et.al.: J. Pharm. Biomed. Anal. 44, 558 (2007). Pharm. Biomed. Anal. 22, 111 (2000). 21. Belaz K.R.A., Cass Q.B., Oliveira R.V.: Talanta 6. Fregonezi-Nery M.M., Baracat M.M., Kedor- 76, 146 (2008). Hackmann E.R.M.: 34, 1255 (2001). Received: 7. 09. 2011