Analytical Method Development and Validation of Lansoprazole By RP-HPLC
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1 Available online at SCIENTIFIC RESEARCH GATE International Journal of Multidisciplinary Papers, December 2017; 2 (1): International Journal of Multidisciplinary Papers Abstract Analytical Method Development and Validation of Lansoprazole By RP-HPLC Mounika 1, H.Padmalatha 1, Janmajoy Banerjee 1, Ranabir Chanda* 1 1Gyana Jyothi College of Pharmacy, Uppal Bus Dept. Hyderabad, India. In lansoprazole (LPZ) drug substance, 2,3-Dimethyl-4-nitropyridine N-oxide (DPN) and 2-Chloromethyl-3-methyl-4-(2,2,2- trifluoroethoxy)pyridine hydrochloride (CTP). These compounds are carried from raw materials. In literature, no method was reported for the determination of these two impurities. Hence, the present studies are aimed towards the determination of these two compounds in lansoprazole at the level of 16 ppm (lower end) by High Performance Liquid Chromatography. A simple Reverse phase liquid chromatographic method has been developed and subsequently validated for estimation of lansoprazole in tablet dosage form. The separation was carried out using a mobile phase consisting of disodium hydrogen phosphate buffer of ph 7.4. The column was used Inertsil ODS-3V, 150 x 4.6 mm, 5 µm (GL Sciences Inc, Japan, Part No ) with flow rate of 1.0 ml/min using UV detector at 210 nm. The described method was linear over a concentration range of µg/ml for the assay of Lansoprazole. The retention time of Lansoprazole was found to be 8.82 min. Results of analysis were validated statistically. The results of the study showed that the proposed RP-HPLC method is simple, rapid, precise and accurate, which is useful for the routine determination of Lansoprazole in tablet dosage form and in its pharmaceutical dosage forms. Keywords: Lansoprazole, RP-HPLC Citation to this Article: Mounika, Padmalatha H, Chanda R, Analytical Method Development and Validation of Lansoprazole By RP-HPLC. International Journal of Multidisciplinary Papers, December 2017; 2(1): Introduction Analytical chemistry is a branch of chemistry involved with the analysis of chemical substances. It encompasses the theory and practice of all means of acquiring information about the composition of matter. Pharmaceutical analysis is considered as interdisciplinary subject comprising of chemistry, pharmacy, physics, electronics, biology etc. It is used in identifying drug substances, called qualitative analysis, determining the concentration or amount of drug substances, called quantitative analysis and confirming the structures of drug substances. Analytical techniques like chromatography, spectroscopy, titrimetry etc. play a major role in these studies. The major areas of pharmaceutical analysis are assay of the drug from bulk drugs and formulations, detection and quantification of probable impurities and metabolites of drugs, accelerated stability studies, in-vitro dissolution studies etc [1] Impurity is defined as any substance coexisting with the original drug. Control of toxic impurities in drug substances has received more and more attention over the past years. These are to be determined based on the maximum daily dose. According to EMEA guidelines, a TTC value of 1.5 microgram/day intake of a toxic impurity is considered to be associated with an acceptable risk. The concentration limits in ppm of permitted toxic impurity in a drug substance is the ratio of TTC in microgram/day and daily dose in gram/day [2]. In lansoprazole (LPZ) drug substance, 2,3-Dimethyl-4-nitropyridine N-oxide (DPN) and 2-Chloromethyl-3-methyl- 4-(2,2,2-trifluoroethoxy)pyridine hydrochloride (CTP). These compounds are carried from raw materials. The maximum daily dose of lansoprazole is 90 mg /day. Hence, TTC is ppm [3]. *Corresponding author: MahmudaRatna ranabirchanda@gmail.com Page 16
2 In literature, no method was reported for the determination of these impurities. Hence, the present studies are aimed towards the determination of these two compounds in lansoprazole at the level of 16 ppm (lower end) by High Performance Liquid Chromatography [4,5]. Lansoprazole is is chemically known as (RS)-2-([3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl]methylsulfinyl)- 1H-benzo[d]imidazole. CAS number is Molecular formula is C16H14F3N3O2S. Molecular weight is H N N O S N C H 3 O F F F Lansoprazole, an individual from the proton-pump-inhibitor class of gastric corrosive inhibitory operator, successfully raises intra gastric ph and is shown for the transient treatment of dynamic erosive reflux esophagitis, gastric ulcer, duodenal ulcer, and non erosive gastroesophageal reflux illness. Lansoprazole is likewise shown as a long haul upkeep treatment in patients with recuperated reflux esophagitis and mended duodenal ulcer and in the treatment of neurotic hypersecretory conditions, for example, Zollinger-Ellison disorder. As a proton-pump inhibitor, lansoprazole is additionally a vital segment of double and tripletherapy regimens for the destruction of Helicobacter pylori contamination. The most recent FDA-affirmed naming for lansoprazole incorporates the sign of mending and hazard diminishment in nonsteroidal calming drug-related gastric ulcers [6]. The ingestion of lansoprazole is fast, with mean Cmax happening around 1. 7 hours after oral dosing, and generally entire with supreme bioavailability more than 80%. There is no critical sustenance impact if the medication is given before dinners. Lansoprazole is 97% bound to plasma proteins. Lansoprazole is widely processed in the liver. Two metabolites have been distinguished in quantifiable amounts in plasma (the hydroxylated sulfinyl and sulfone subordinates of lansoprazole). Lansoprazole has a place with a class of antisecretory exacerbates, the substituted benzimidazoles, that suppress gastric acid discharge by particular hindrance of the (H, K )- ATPase + protein framework at the secretory surface of the gastric parietal cell. Since this chemical framework is viewed as the acid (proton) pump inside the parietal cell, lansoprazole has been portrayed as a gastric acid pump inhibitor, in that it obstructs the last stride of corrosive generation [7,8] For estimation of Lansoprazole, hence we wanted to develop a simple and accurate analytical method. This paper describes validated RP-HPLC for estimation of Lansoprazole using phosphate buffer of ph 7.4. The column was used Inertsil ODS-3V, 150 x 4.6 mm, 5 µm (GL Sciences Inc, Japan, Part No ) with flow rate of 1.0 ml/min using UV detector at 210 nm. 2. Materials and Methods 2.1 Materials (Chemicals and Solvents) Lansoprazole and the impurities were supplied by Hetero (R&D), Hyderabad. All the chemicals and solvents used were of analytical grade. Milli Q water was used throughout the experiment. 2.2 Instrument A water-alliance HPLC system (Alliance 2695) equipped with Photo Diode Array Detector (2996), UV-Visible detector (2487) and Empower2 software was used. 2.3 HPLC Method Development The method development was targeted to develop a RP-HPLC method that can separate LPZ from DPN, CTP and three impurities listed in USP (RCA, Imp-B and Imp-C). Page 17
3 2.4 Trial-I The method development was initiated with the USP method. 2.5 Chromatographic Conditions Column (GL Sciences Inc, Japan, Part No ) : Inertsil ODS-3V, 150 x 4.6 mm, 5 µm Flow rate Detector wavelength 1.0 ml/min 210 nm Injection volume 20 µl Column temperature 30 ºC Autosampler temperature 5 ºC Elution mode Gradient (Mobile phase A : Mobile phase B) Diluent Run time Methanol 50 min 2.6 Preparation of Buffer Dissolved 1.36 g of Potassium dihydrogen orthophosphate and 1.74 g Dipotassium hydrogen phosphate in 1000 ml of water. Adjusted ph to 7.4 with Triethyl amine and mixed. 2.7 Preparation of Mobile Phase A Mixed Buffer and Methanol in the ratio of 90:10 v/v. Filtered and degassed the mixture through 0.45 µm membrane filter paper. 2.8 Preparation of Mobile Phase B Mixed Acetonitrile and Methanol in the ratio of 90:10 v/v. Filtered and degassed the mixture through 0.45 µm membrane filter paper. 2.9 Preparation of Mobile Phase A and B Mobile phase A and B were prepared and their gradient programme was given in table 1. Table 1. Gradient Programme Time (Minutes) Mobile Phase A (% v/v) Mobile Phase (% v/v) Page 18
4 2.10 Preparation of DPN and CTP Individual Stock Solutions (100 ppm) Weighed accurately about 10.0 mg of DPN or CTP into separate 100 ml volumetric flasks, dissolved and diluted to volume with diluent and mixed. Diluted 1 ml of each solution separately to 50 ml with diluent Preparation of LPZ Test Solution (20 mg/ml) Weighed accurately about 200 mg of the LPZ test sample into a 10 ml volumetric flask, dissolved and diluted to the volume with diluent and mixed Observation In this condition, all the impurities except CTP were eluted, but DPN was found to degrade. Figure 1. Chromatogram Representing the Degradation of DPN 2.13 Trial-II In this trial, methanol is used as diluent because DPN was degraded in USP diluent. CTP was not degraded in methanol. But, the peak shape is not good. Hence, injection volume was reduced to 20 µl to reduce overloading of the column. Slight improvement is achieved in peak shape, but a good peak shape is not observed. Hence, buffer is introduced to the mobile phase A to increase the peak shape. Since, most of the impurities and LPZ have the ph values about 7.0, ph value was slightly changed to 7.4 instead of 7.0 reported in USP method. Accordingly, buffer containing 0.01 M Potassium dihydrogen orthophosphate and 0.01 M Dipotassium hydrogen phosphate was prepared and its ph value was adjusted to 7.4 with Triethyl amine. Acetonitrile and Methanol in the ratio of 90:10 v/v was used as mobile phase B. USP gradient program is maintained. Since, 210 nm is showing high responses, when compared to 285 nm, 210 nm is selected Chromatographic Conditions Column (GL Sciences Inc, Japan, Part No ) : Inertsil ODS-2, 150 x 4.6 mm, 5 µm Flow rate Detector wavelength 0.8 ml/min 210 nm Injection volume 20 µl Column temperature 30 ºC Elution mode Gradient (Mobile phase A : Mobile phase B) Diluent Run time Methanol 60 min Page 19
5 2.15 Observation In this condition, peak shape of DPN is improved. But, the resolution between LPZ and RCA is very poor and CTP not eluted. Figure 2. Chromatogram Representing the Un-degraded of DPN Figure 3. Chromatogram Representing Retention Time of RCA Figure 4. Chromatogram representing retention time of LPZ Chromatogram representing CTP (Not eluted) Figure 5. Page 20
6 2.16 Trial-III In this trail, several gradients were tried to separated LPZ and RCA, but not achieved. Changing ph of mobile phase A is also unsuccessful. Hence, several C18 columns were tried and Inertsil ODS-3V column was resolved LPZ from RCA. Now, gradient program was slightly tuned to for the elution of CTP. When, mobile phase B ratio is increased in mobile phase, CTP was eluted. The finalized chromatographic conditions are given under the heading 4.iii HPLC Method Validation The proposed method was validated as per ICH guidelines. a) Specificity Specificty was conducted by spiking DPN and CTP along with RCA, Imp-B and Imp-C. DPN and CTP were eluted at retention times of 6.3 and 30.1 min., respectively. There is no interference due to blank at the retention times of DPN and CTP. DPN and CTP resolved from each other and from known impurities and LPZ. b) Limit of Detection (LOD): Limits of Detection for DPN and CTP have been established. LOD solution was prepared so as to obtain the S/N ratio is in between 3 to 5 for DPN and CTP the results are given below. Table 2. LOD Results Name of the Compound Conc. w.r.to. Test (ppm) S/N Ratio DPN CTP c) Limit of Quantitation (LOQ) Limits of Quantitation for DPN and CTP have been established. Based on the concentration obtained from LOD, the LOQ solution was prepared (3 times to LOD concentration) so as to obtain the signal to noise ratios are in between 10 to 15 for DPN and CTP, and the results are given below. Table 3. LOQ Results Name of the Compound Conc. w.r.to. Test (ppm) S/N Ratio DPN CTP d) Precision at Limit of Quantitation LOQ solution of DPN and CTP was injected six times and calculated % RSD. The results are summarized below. Table 4. Results for Precision at LOQ Level LOQ Solution Area of DPN Area of CTP Injection Injection Injection Injection Injection Injection AVERAGE Page 21
7 Average Area Malluri K. et al. International Journal of Multidisciplinary Papers, December 2017; 2 (1): Observation %RSD It was observed that the % RSD values for peak areas of DPN and CTP at LOQ level are less than 2.0. e) Linearity Linearity study was conducted for DPN and CTP and the range from LOQ level to 150% to specification as per the procedure mentioned in the protocol. Linearity graphs were obtained for DPN and CTP in the range of LOQ to 150% of specification. The results are given below. Table 5. Results of DPN Linearity Study Injection Level-1 (LOQ) Level-2 (8 ppm) Level-3 (12 ppm) Level-4 (16 ppm) Level-5 (20 ppm) Level-6 (24 ppm) Injection Injection Injection Injection Injection Injection Average % RSD Am ount of DPN (ppm ) Correlation Coefficient: Table 6. Results of CTP Linearity Study Figure 6. DPN Linearity Graph Injection Level-1 (LOQ) Level-2 (8 ppm) Level-3 (12 ppm) Level-4 (16 ppm) Level-5 (20 ppm) Level-6 (24 ppm) Injection Injection Page 22
8 Average Area Malluri K. et al. International Journal of Multidisciplinary Papers, December 2017; 2 (1): 7 15 Injection Injection Injection Injection Average % RSD Am ount of CTP (ppm ) Correlation Coefficient: Figure 7. CTP Linearity Graph 2.19 Observations The % RSD values for 6 replicate injections of Level-1 and Level-6 are less than 2.0 Correlation coefficient values of DPN and CTP are within the limit (Not less than 0.99) No systematic trend was observed (not more than 5 consecutive points on single side). f) Sample Analysis Three Lansoprazole Samples were analyzed (in duplicate injections). The results are given below. Table 7. Sample Analysis Results Sample No. Amount found (ppm) DPN CTP 1 Below the LOD Below the LOD 2 Below the LOD Below the LOD 3 Below the LOD Below the LOD Note: The limit of detection value for DPN and CTP are 1.0 ppm and 2.0 ppm. g) Accuracy at Limit of Quantitation: Page 23
9 Sample-1 was analyzed three times (from three individual preparations) for accuracy studies by spiking DPN and CTP at LOQ level to it and evaluated the % recoveries of DPN and CTP at LOQ solution in Lansoprazole. Results of accuracy at LOQ are given below. Table 8. Accuracy at LOQ Results S. No. Preparation % Recovery of DPN % Recovery of CTP 1 Preparation Preparation Preparation Figure A. Blank Chromatogram Figure 8. Analysis Chromatograms of Sample and Impurities (Representative Chromatograms) Figure B. Specificity Chromatogram C. LOD Chromatogram Figure Page 24
10 F igure D. LOQ Chromatogram Sample Chromatogram Figure E. Recovery Chromatogram Figure F. 3. Results and Discussions The developed RP-HPLC method for estimation of impurities in Lansoprazole using column Inertsil ODS-3V, 150 x 4.6 mm, 5 µm (GL Sciences Inc, Japan, Part No ) phosphate buffer 7.4 ph and methanol in the ratio of 90:10 v/v and (Mobile Phase A) and acetonitrile and Methanol in the ratio of 90:10 v/v (Mobile Phase B) as mobile phases. Detection of eluent was carried out using UV detector at 210 nm. The method was developed. The run time per sample is just 50 min. Pure Lansoprazole was compared with its impurities forms of 2,3-Dimethyl-4- nitropyridine N-oxide (DPN) and 2-Chloromethyl-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochloride (CTP) solutions and observations were recorded in figure 1-8. The method was validated as per ICH guidelines in terms of linearity, accuracy, specificity, intraday and interday precision, repeatability of measurement of peak area as well as repeatability of sample application. Since none of the methods is reported for estimation of 2,3 -Dimethyl-4- nitropyridine N-oxide (DPN) and 2-Chloromethyl-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochloride (CTP) in crude lansoprazole (LPZ)., this developed method can be used for routine analysis of crude lansoprazole (LPZ).. 4. Conclusions Observations from the present study indicate that the HPLC method meets the acceptance criteria for all the parameters selected for quantitation study. Hence, the method is suitable for the determination of 2,3-Dimethyl-4- nitropyridine N-oxide (DPN) and 2-Chloromethyl-3-methyl-4-(2,2,2-trifluoroethoxy)pyridine hydrochloride (CTP) in Lansoprazole. Page 25
11 Acknowledgement We cordially acknowledge authority of Gyana Jyothi College of Pharmacy, Hyderabad, India for providing laboratory facility for our research work. References 1. Fundamentals of Analytical Chemistry, 8th Edn., Skog, West, Holler, Crouch, BBN Printers, Haryana, India International Conference on Harmonisation Guideline on Stability Testing of New Drug Substances and Products; Q1A(R2) (2003). 3. The Merck Index (Merck & Co., Inc. Whitehouse Station, NJ, USA), 2006, 14, International Conference on Harmonisation Guideline on Impurities in New Drug Products; Q3B(R2), International Conference on Harmonisation Guideline on Specifications: Test Procedures and Acceptance Criteria for New Drug Substances and New Drug Products: Chemical Substances; Q6A, S. Muthu Kumar, D. Selva Kumar, T. Rajkumar3, E. Udhaya Kumar, A. Suba Geetha, Dinesh Diwedi, Development and validation of RP-HPLC method for the Estimation of Lansoprazole in tablet dosage form, J. Chem. Pharm. Res., 2010, 2(6): S.S. Raman, K.A. Harikrishna, A.V.S. Prasad, K. Ratnakar Reddy, K. Ramakrishna, Development and validation of a stability-indicating RP-LC method for famciclovir, J. Pharma. Biomed. Analy., 2009, 50 (8), Sarojini Sarangapani, Manavalan Rajappan, Lansoprazole Release from a Floating Dosage Form based on the Natural Polymer of Delonixregia, Int. J. Pharm. Tech. Res., 4 (3): Page 26
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