PHARMA SCIENCE MONITOR

PHARMA SCIENCE MONITOR AN INTERNATIONAL JOURNAL OF PHARMACEUTICAL SCIENCES DEVELOPMENT AND VALIDATION OF NEW ANALYTICAL METHOD FOR QUANTITATIVE ESTIMATION OF RACECADOTRIL AS AN ACTIVE PHAMACEUTICAL INGREDIENT BY RP-HPLC Patel YS 1, Sen AK 1, Shah B 2, Seth AK 1 1 Department of Pharmacy, Sumandeep Vidyapeeth, Piparia, Vadodara, Gujarat, India. 2 Lincoln Pharmaceutical Limited, Khatraj, Gandhinagar, Gujarat, India ABSTRACT A simple and sensitive spectroscopic method was developed for the estimation of Racecadotril in pharmaceutical dosage forms. Spectroscopic method is showing absorbance at 231 nm in methanol. This method obeys Beers law in the concentration range of 8 to100 μg ml -1 respectively. The proposed method is precise, accurate and reproducible and can be extended to the analysis of Racecadotril in bulk and tablet formulations. The quantitative determination of the drug was carried out using the second-derivative values measured at 223, 250 and 273 nm and Third-derivative spectrum values measured at 226and 240nm.Calibration graphs constructed at these wavelengths were linear in the concentration range of 8-100µg ml -1 for secondderivative and third-derivative spectrophotometry method. A HPLC assay utilized Phenomenex-Luna RP-18(2) (250X4.6mm, 5 µm) column, with mobile phase composition of Acetonitrile: 0.05M phosphate buffer (Potassium dihydrogen orthophosphate): triethylamine [80:19.95:0.05, (v/v/v)] of ph 3.953 ± 0.2 was used, and flow rate was 1.0 ml min -1 with UV detection at 231 nm. Atorvastatin calciumvastatin calcium (ATOR) was used as internal standard. The retention time of Racecadotril and ATORVASTATIN CALCIUM were 4.22 and 3.453 min respectively. The total HPLC run time was less than 6 min. Linearity was observed over concentration range of 8-80 µg ml -1 for Racecadotril. The proposed method was validated for various ICH parameters like linearity, limit of detection, accuracy, precision, ruggedness, robustness, and system suitability. Keywords: Racecadotril, Validation, RP-HPLC method. INTRODUCTION Racecadotril (Molecular Formula: C 6 H 12 F 2 N 2 O 2.HCl, Molecular Weight: 218.65 g/mol) belongs to Anti- Diarrheal category. [1] Racecadotril is acts by inhibition of enkephalinase, this produces an increases in the levels of enkephalins that act in the enterocyate, thus inhibits hypersecretion. Racecadotril is used in the treatment of acute water diarrhea in children and adults. [2] Racecadotril is chemically N-[2-[(Acetyl thio) methyl]-1-oxo-3-phenylpropyl] -glycine phenyl methyl ester (Fig.1). [3-6] The individual www.pharmasm.com IC Value 4.01 2007

determination of Racecadotril has been carried out in tablets by HPLC [7], in capsules by HPLC [8], in human plasma using solid-phase extraction by HPLC [9], in human plasma using a liquid chromatography/tandem mass spectroscopy [10], by NMR and mass spectroscopy [11] and Stability-indicating methods for the determination of Racecadotril in the presence of its degradation products. [12] The objective of the present work is to develop and validate new analytical methods for quantitative determination of Racecadotril in tablet dosage form. The method was validated as per ICH guidelines. [13] Figure 1 Structure of Racecadotril MATERIALS AND METHODS Experimental 1. Racecadotril, Active Pharmaceutical Ingredient (API) and working standard were supplied by Lincoln Pharmaceutical Limited (Gandhinagar, India). 2. Drug products of racecadotril and placebo (Batch No- 5+52/ F085) were manufactured and supplied by Lincoln Pharmaceutical Limited (Gandhinagar, India). Chemicals and reagents used 1. Milli-Q water 2. Acetonitrile (HPLC grade), Spectrochem 3. Methanol, Merck, INDIA 4. 0.05M Phosphate buffer 5. Triethylamine Apparatus and equipments used Hot air oven : Citizen precision. Analytical Balance : AX 205, METTLER TOLEDO. ph Meter : Libinidia, model PICO +. SonicAtorvastatin calcium : Oscar Ultra Sonics, OU- 72 (SPL). www.pharmasm.com IC Value 4.01 2008

Chromatographic condition The HPLC system (Shimadzu Corporation, Japan), model Shimadzu VP, consisted of a system controller (CLASS-VP), on-line degasser (LC 2010C, Shimadzu), low pressure gradient valve (LC 2010C, Shimadzu), solvent delivery module (LC 2010C, Shimadzu), auto injector (LC 2010C, Shimadzu), column oven (LC 2010C, Shimadzu), and CLASS-VP software version-spi, binary pump, auto injector (SIL-10AD VP, Shimadzu), column oven (CTO-10AS VP, Shimadzu) and PDA detector (PDA-SPD- M10A VP, Shimadzu Diode Array Detector) and Chem station (software). The chromatographic parameters The chromatographic conditions comprised a reverse-phase C 18 column (Phenomenex - Luna RP-18(2), 250 4.6 mm, 5 µm column with a mobile phase consisting of a mixture of solution ACN: Phosphate buffer: Triethylamine (80:19.95:0.05;v/v/v). The diluent was a methanol. The flow rate was kept 1.0 ml/min. and the detection was carried out at 231 nm wavelength & 30 C temperature. Buffer preparation 3.4 gm of potassium dihydrogen ortho-phosphate in 1000 ml volumetric flask, and dissolved in distilled water. The resulting solution was sonicated for ten minutes. ph of the resulting solution was adjusted to 3.953 ± 0.2 by using orthophosphoric acid (85%). Preparation of standard solution The stock solutions of 1 mg ml -1 Racecadotril in mixture of methanol were prepared. The working solution of 0.1 mg ml -1 prepared by transferring 5ml from respective stock solution to a 50 ml volumetric flask and completing to volume with the mixture of methanol. Calibration curve The calibration study was carried out for the drug at eight different concentration levels using Atorvastatin calcium as internal standard. Aliquots of standard Racecadotril working solutions were taken in different volumetric flasks and 20 µg ml -1 of Atorvastatin calcium was added to each flask as internal standard and diluted with mobile phase such that the final concentration of Racecadotril were in the range of 8-80 µg ml -1 (Fig 3). All stock and working solutions were sonicated for 5 min then filtered through www.pharmasm.com IC Value 4.01 2009

the nylon membrane filter (0.45 µ) prior to use. Triplicate 20 µl injections were made for each concentration and chromatographic under specified condition at ambient temperature (28 0 C). The peak area response ratio of the internal standard to pure analyte is determined beforehand and Calibration curve was constructed by plotting average peak area against concentration and regression equation was computed. Preparation of test solution An amount of the powder equivalent to 10 mg of Racecadotril (content of 1g Powder) was dissolved in 60 ml of Acetonitrile. The solution was sonicated for 10 min and filtered into a 100 ml volumetric flask through 0.45µ nylon membrane filter. The residue was washed 3 times with 10 ml of Acetonitrile, and then the volume was completed to 100 ml with the same solvent. Further dilutions were prepared within the linearity range. Validation of the method The developed method was validated in terms of linearity, accuracy, specificity, limit of detection, limit of quantification, precision and robustness. RESULTS AND DISCUSSION Literature review reveals individual methods for estimation of Racecadotril but newer method was reported for estimation of Racecadotril. A simple, precise, accurate, RP-HPLC method has been developed for the estimation of Racecadotril in bulk and in tablet formulation. The UV detection was carried out as 231 nm as Racecadotril showed very good absorbance at this wavelength. A Chromatogram of Racecadotril shown in fig- 2, Racecadotril with retention time of 4.22 min. Figure 2 www.pharmasm.com IC Value 4.01 2010

METHOD VALIDATION: Validation was carried out with respect to various parameters, as required under ICH guidelineq2 (R1). [13] The developed method validated with respect to parameters such as linearity, precision, accuracy, specificity, ruggedness, robustness and solution stability. [13] System suitability and system precision System suitability and system precision was daily performed during entire validation of this method. The results of system suitability and system precision were presented. TABLE 1: SYSTEM SUITABILITY AND SYSTEM PRECISION Parameters Data obtained* Racecadotril Theoretical plates per meter 110465 Theoretical plates per column 5523 Symmetry factor/tailing factor 1.18 Resolution 2.861 Linearity Series of standard solutions were prepared containing Racecadotril (10-80 μg/ml) as per method given in assay. The solutions were prepared in triplicate and analyzed by using 10 μl into HPLC. The results related to linearity are presented in Table 2. TABLE 2: RESULTS OF LINEARITY Parameters Racecadotril Linearity Range 10-80 μg/ml Linearity equation y y = 0.0336x 0.0301 Correlation coefficient 0.9995 LOD 0.032 LOQ 0.099 www.pharmasm.com IC Value 4.01 2011

Figure 3 Linearity and calibration curve of Racecadotril Precision The method precision was done by preparing six different sample preparations by one analyst under the same conditions. The results are presented in Table 3. The results obtained were within 2% RSD. Ruggedness Ruggedness test was determined between two different analysts, instruments and columns. The value of percentage RSD was below 2.0%, showing ruggedness of developed analytical method. The results are presented in Table 3. TABLE 3: RESULTS OF METHOD PRECISION AND RUGGEDNESS Parameters Racecadotril %Assay % RSD Mean ± SD (n=6) Method Precision 98.3 ± 0.4 0.63 Ruggedness 99.9 ± 0.288 0.27 Accuracy The study was performed by increasing standard addition of known amounts of studied drugs to an unknown concentration (constant volume) of the commercial pharmaceutical formulations (Standard addition access the effect of a sample matrix changes the analytical sensitivity of the method). A constant volume of the unknown www.pharmasm.com IC Value 4.01 2012

solution is added to each of three 10 ml volumetric flasks. Then a series of increasing volumes of working standard solutions are added. Finally, each flask is made up to the mark with solvent and mixed well. The resulting mixtures were analyzed by the proposed HPLC method. The results obtained are compared with expected results. The excellent mean recoveries and standard deviation (Table 4) suggested good accuracy of the propose methods and no interference from formulations excipients. TABLE 4: RESULTS OF ACCURACY STUDY OF RACECADOTRIL Level of Formulation (µg ml -1 ) Conc. of standard addition in % Response factor* Racecadotril Conc. found (mcg) %Recovery %RSD 18 80 0.640368 7.9756 99.695 0.7549 20 100 0.717902 10.0503 100.503 1.0075 22 120 0.789854 11.9757 99.798 0.8822 TABLE 5: RESULTS OF PEAK PURITY IN SPECIFICITY STUDY Drug Racecadotril Standard solution 0.99932 Test solution 0.99814 Spiked sample solution 0.99795 Robustness Robustness of the method was carried out by deliberately made small change in the flow rate, ph, organic phase ratio and column oven temperature. Results are presented in Table 6. www.pharmasm.com IC Value 4.01 2013

Parameter TABLE 6: RESULTS OF ROBUSTNESS STUDY Retention time(rt) of Racecadotril Peak asymmetry Retention time(rt) of Atorvastatin calcium Flow rate 0.8 4.413 3.853 1.0 4.21 3.456 1.2 4.012 3.253 Acetonitrile % in mobile phase 15% 4.61 3.757 20% 4.213 3.454 25% 4.01 3.253 Change in ph 4.10 4.27 3.453 3.90 4.218 3.452 4.05 4.229 3.451 *Average of three experiments Solution stability Solution stability period for standard and sample preparation was determined by keeping the solution for 24 hours at room temperature. After 4, 8, 12, 16, 20, 24 hours the solutions were analyzed. Results related to solution stability are summarized in Table 7. No significant changes (<2%) were observed for the chromatographic responses for the solution analyzed, relative to freshly prepared standard. TABLE 7: RESULTS OF STANDARD AND SAMPLE SOLUTION STABILITY Time (hrs) Standard Solution (%RSD) Sample Solution (%RSD) RACECADOTRIL RACECADOTRIL Initial - - 4 hrs -0.5 0.6 8 hrs -0.2-0.4 12 hrs -0.2 0.1 16 hrs -0.8-0.3 20 hrs -0.6 0.2 24 hrs -0.1 0.5 www.pharmasm.com IC Value 4.01 2014

TABLE 8: SUMMARY OF VALIDATION PARAMETERS OF RP HPLC METHOD FOR ESTIMATION OF RACECADOTRIL Characteristics Acceptance criteria Results Specificity Peak purity Factor 995 999.32 Correlation coefficient 0.999 Linearity r 2 0.995 LOD (µg/ml) 0.032 LOQ (µg/ml) 0.099 Accuracy Level Recovery 98-102% (individual and mean) 50% % RSD 2% 99.69 ± 0.75 100% 100.5 ± 1.0 150% 99.79 ± 0.88 Method precision % RSD 2% 0.63 (Repeatability) Ruggedness % RSD 2% 0.27 Robustness % RSD of 3 replicates: 2% Complies Solution stability Difference in the response of standard and sample preparation is 2% at 24 hrs Standard : -0.1 mg Sample: 0.5 mg at 24 hrs Assay of tablet Batch number Assay of racecadotril 52+5/F085 98.85% CONCLUSION All these factors lead to the conclusion that the proposed Development and Validation for Assay Method for Racecadotril by RP-HPLC is accurate, precise, simple, selective, sensitive and rapid can be applied successfully for routine analysis in quality control department. www.pharmasm.com IC Value 4.01 2015

ACKNOWLEDGMENTS We are grateful to Lincoln Pharmaceutical Limited for the providing samples and all other facilities to complete the entire project. References 1. The Merck Index. 13th ed. White house Station (NJ): Merck & Co., Inc.; 2001. p. 651. 2. Thomos R: Physician s desk reference. U.S.A, PDR staff publication, 2003;53-54. 3. Gulhati CM: Monthly index of medical specialties (MIMS) India. New Delhi, Mims India publication, 2004;24:98. 4. Ministry of Health & Family Welfare, Government of India, Indian Pharmacopoeia, The Controller of Publications, Government of India, New Delhi 1996; Vol. 2: A-169. 5. Ministry of Health & Family Welfare, Government of India, Indian Pharmacopoeia, The Controller of Publications, Government of India, New Delhi 1996; Vol. 3: 1181. 6. United States Pharmacopoeia (USP-NF XXIV). Rockville MD 20852, United States Pharmacopoeia Convention Inc, 1985;2149-51. 7. Rao PS, Nappinnai M: UV and RP-HPLC Estimation of Racecadotril. Asian J Chem 2007;19(5):3697-702. 8. Dinesh, AS, Prashant SL: Determination of racecadotril by HPLC in capsules. Int.J pharm. chem 2007;69(6):819-21. 9. Xu F, Yang L, Xu G: A rapid and validated HPLC method to quantify Racecadotril metabolite, thiorphan, in human plasma using solid-phase extraction. Ana Tech Biomed Life Sci 2007;861(1):130-5. 10. Xu Y, Huang J, Liu F, Gao S, Guo Q: Quantitative analysis of Racecadotril metabolite in human plasma using a liquid chromatography/tandem mass spectrometry. Ana Tech Biomed Life Sci 2007;852(1-2):101-7. 11. Reddy K, Babu J, Sudhakar P, Sharma M, Reddy G, Vyas K: Structural studies of racecadotril and its process impurities by NMR and mass spectroscopy. Int J Chem Tech Res 2006;1:214-217. www.pharmasm.com IC Value 4.01 2016

12. Vetrichelvan T, Prabakaran S: New spectrophotometric methods for the determination of racecadotril in bulk drug and capsules. Indian J Pharm Sci 2007;69(2):307-9. 13. ICH, Q2B. Validation of Analytical Procedure: Methodology. International Conference on Harmonisation, IFPMA, Geneva, 2005. For Correspondence: PATEL YS Email: yagnik.2706@gmail.com www.pharmasm.com IC Value 4.01 2017