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IInntteerrnnaattiioonnaall JJoouurrnnaall ooff PPhhaarrmmaa SScciieenncceess Vol. 5, No. 6 (2015): 1307-1313 Research Article Open Access

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A Stability indicating RP-HPLC Method Development and Validation for Estimation of Related substances, Assay and degradants in Dexlansoprazole in Bulk and its Pharmaceuticals Dosage Form Sanjeeva Reddy Kallam, J. Srikanth and K. Vanitha Prakash Dr.Reddys Laboratories Ltd, IDA Bollaram, Hyderabad, Telangana, India.

* Corresponding author: Sanjeeva Reddy Kallam, e-mail: [email protected]

ABSTRACT A new simple, rapid, selective, precise and accurate isocratic reverse phase high performance liquid chromatography has been developed and validated for the estimation of Dexlansoprazole in bulk and pharmaceutical dosage form. The chromatographic separations were achieved using a High performance liquid chromatography (Inertsil ODS 3V 150 mm, 4.6 mm,5µm) employing water and Acetonitrile in the ratio of 500:500 %v/v as mobile phase with a 1.0 mL/min flow rate was chosen. Four impurities were eluted within 10 minutes of run time. The column temperature was maintained at 25oC and a detector wavelength of 285 nm was employed. Dexlansoprazole was exposed to thermal, photolytic, hydrolytic, basic and oxidative stress conditions. The stressed samples were analyzed by the proposed method. Complete degradation of the analyte was observed when it was subjected to oxidative, thermal, hydrolytic, basic and slight degradation was observed in photolytic conditions. Dexlansoprazole was found to be the major degradant. Peak homogeneity data of the Dexlansoprazole obtained by photodiode array (PDA) detection demonstrated the specificity of the method in the presence of degradants. The method was validated with respect to linearity, precision, accuracy, ruggedness, robustness, limit of detection and limit of quantification.

Keywords: Dexlansoprazole, RP HPLC, Degradation, Validation, Inertsil ODS 3V 150 mm, 4.6 mm, 5µm column.

1. INTRODUCTION Dexlansoprazole is the R-enantiomer of lansoprazole (a racemic mixture of the R- and S-enantiomers). It is chemically(R)-(+)2-([3-methyl-4-(2,2,2- trifluoroethoxy) pyridin-2-yl]methylsulfinyl)-1H- benzimidazole. Dexlansoprazole is a proton pump inhibitor that is marketed by Takeda Pharmaceuticals, Japan. Dexlansoprazole was approved by the U.S. Food and Drug Administration (FDA) on January 30, 2009 [3]. DLP DDR capsules approved for use of once-daily, oral treatment of heartburn associated with symptomatic non-erosive gastro esophageal reflux disease, the healing of erosive esophagitis and the maintenance of healed erosive esophagitis. Dexlansoprazole is marketed with a brand name ‘DEXILANT” (earlier known as KAPIDEX) [2]. Dexilant

is the first proton pump inhibitor with a Dual Delayed Release (DDR) formulation designed to provide two separate releases of medication upon oral administration. The capsules contain Dexlansoprazole in a mixture of two types of enteric-coated granules with different pH-dependent dissolution profiles. Dexilant is available in two dosage strengths: 30 mg and 60 mg, per capsule. The aim of this study is development of a simple, precise, rapid and accurate reverse phase HPLC method for the estimation of Dexlansoprazole in bulk and parenteral dosage form. The present work describes the development of a validated RP-HPLC method in bulk and pharmaceutical dosage form. The present RP-HPLC method was validated following the ICH guidelines.

Received: 28 September 2015 Accepted: 21 October 2015 Online: 02 November 2015

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Sanjeeva Reddy Kallam et al. / Int J Pharma Sci. 2015, 5(6): 1307-1313

2. MATERIALS AND METHODS 2.1 Chemicals and Reagents The reference samples of Dexlansoprazole were provided as gift samples from Everest organics Limited, Hyderabad. HPLC grade acetonitrile and all other chemicals were obtained from Merck chemical division, Mumbai. HPLC grade water obtained from Milli-Q water purification system was used throughout the study. Commercial tablets (Dexilant-30mg dosage) were purchased from the local pharmacy.

Molecular formula: C16H14F3N3O2S Molecular Weight : 369.36 IUPAC Name: (R) (+)-2-(((3-methyl-4-(2,2,2-trifluoroethoxy)pyridin-2-yl) methyl)sulfinyl)-1H-benzo[d]imidazole) Figure 1: Structure of Dexlansoprazole

Molecular formula: C7H6N2S Molecular Weight : 150.20 IUPAC Name: 1H-benzo[d]imidazole-2-thiol Figure 2: Mercapto-benzimidazole

Molecular formula: C14H12N4O3S Molecular Weight : 316.33 IUPAC Name: 2-(((3-methyl-4-nitropyridin-2-yl) methyl) sulfinyl)-1H-benzo[d]imidazole Figure 3: Structure of 2-Nitrosulphoxide

Molecular formula: C16H14F3N3O3S Molecular Weight : 385.36 IUPAC Name: [[(1H-benzimidazole-2-yl) sulfinyl] methyl]-3-methyl-4-(2,2,2- trifluoroethoxy)-pyridine 1-oxide Figure 4: Structure of Sulphone

Molecular formula: C16H14F3N3OS Molecular Weight : 353.36 IUPAC Name: 2-(((3-methyl-4-(2,2,2- trifluoroethoxy)pyridin-2- yl) methyl) thio)-1H-benzo[d]imidazole Figure 5: Structure of Sulphide

2.2 Instrument and chromatographic conditions The HPLC system used for the method development and validation consisted of gradient pumps from Agilent 1260 Technologies, Ultra violet detector from Agilent Technologies. USA, with auto sampler and auto injector. The HPLC system was equipped with data acquisition and processing software “EZ Chrome” Agilent Technologies. USA. The column used for separation of analytes is Inertsil ODS, 3V 150 mm, 4.6 mm,5µm column. Mobile phase consisting of, Water: Acetonitrile in the ratio of 500:500% v/v at a flow rate of 1mL/min. It was filtered through 0.45μm nylon filter and sonicated for 15 min in ultrasonic bath. Sample analyzed at 285 nm at an injection volume of 10 μL. 2.3 Preparation of Mobile phase Take Milli Q water and Acetonitrile in the ratio of 500: 500 % v/v. 2.4 Diluent: Mobilephase (Milli Q Water and Acetonitrile in the ratio of 500: 500 % v/v) and degas to sonicate for 15 minutes. 2.5 Preparation of Solutions: 2.5.1 Dexlansoprazole stock preparation (1000μg/mL): Accurately weighed and transferred 100 mg of Dexlansoprazole in to 100ml of clean dry volumetric flask, add 50mL of diluent (Mobilephase), then sonicated for 10min and make up the volume with diluent. 2.5.2 Impurity stock (four impurities) preparation (50μg/mL): Accurately weighed each impurity 5mg of 2-Mercapto-benzimidazole, 2- Nitrosulphoxide, sulphone & Sulphide transferred into 100mL of clean dry volumetric flask, add 50mL of diluent (Milli Q Water and Acetonitrile in the ratio of 500: 500 % v/v), then sonicated for 10 min and make up the final volume with diluent. 2.6 Sample Preparation: 2.6.1Dexlansoprazole sample preparation (100μg/mL): Accurately weighed and transferred 10 mg of Dexlansoprazole in to 100mL of clean dry volumetric flask, add 50mL of diluent (Milli Q Water and Acetonitrile in the ratio of 500: 500 % v/v), then sonicated for 10min and make up the volume with diluent.



2.7 METHOD VALIDATION The validation of the method was carried out as per ICH Guidelines. The parameters assessed were specificity, linearity, precision, accuracy, stability, LOD and LOQ. 2.7.1 Specificity Specificity is the ability of the analytical method to measure the analyte response in the presence of interferences including degradation products and related substances. The specificity of the developed HPLC method for Dexlansoprazole was carried out in the presence of its impurities namely 2-Mercapto-benzimidazole, 2- Nitrosulphoxide, sulphone & Sulphide. Stress studies were performed for Dexlansoprazole bulk drug to provide an indication of the stability indicating property and specificity of the proposed method. Intentional degradation was attempted to stress conditions of UV light (254nm), heat (80°C), acid (0.5N HCl), base (0.5N NaOH), and Oxidation (3.0 % H2O2) to evaluate the ability of the proposed method to separate Dexlansoprazole from its degradation products. For all degradation studies, period was 24 hours. Assay and related substance studies were carried out for stress samples against qualified Dexlansoprazole reference standard. Assay was also calculated for Dexlansoprazole samples by spiking four impurities at the specification level (i.e., 0.1%). 2.7.2 Accuracy The accuracy of the assay method was evaluated in triplicate at three concentration levels 50%,100% and 150 % of test concentration (0.1 mg/mL).The percentage of recoveries were calculated. The accuracy study of impurities was carried out in triplicate at 50%, 100%, & 150% of specification level (0.1%) to the Dexlansoprazole analyte concentration (100 μg /mL).The percentages of recoveries for impurities were calculated from the slope and Y- Intercept of the calibration curve. 2.7.3 Precision The precision of the assay method was evaluated by carrying out six independent assays of Dexlansoprazole test samples against a qualified reference standard and calculate the %RSD of assay. The precision of the related substances method was checked by injecting six individual preparations of Dexlansoprazole (0.1mg/mL) spiked with 0.1 % of 2-Mercapto-benzimidazole, 2- Nitrosulphoxide, sulphone & Sulphide with respect to Dexlansoprazole analyte concentration. %RSD of area for each impurity of 2-Mercapto-benzimidazole, 2- Nitrosulphoxide, sulphone & Sulphide was calculated. The intermediate precision of the method was also evaluated using different

analyst and different instrument in the same laboratory. 2.7.4 Linearity The purpose of the test for linearity is to demonstrate that the entire analytical system (including detector and data acquisition) exhibits a linear response and is directly proportional over the relevant concentration range for the target concentration of the analyte. The linear regression data for the calibration plot is indicative of a good linear relationship between peak area and concentration over a wide range. The correlation coefficient was indicative of high significance. 2.7.5 Robustness Robustness of the method was investigated under a variety of conditions including changes of composition of buffer in the mobile phase, flow rate and temperature. This deliberate change in the method has no effect on the peak tailing, peak area and theoretical plates and finally the method was found to be robust. 2.7.6 Limit of Detection & Limit of Quantitation The LOD can be defined as the smallest level of analyte that gives a measurable response and LOQ was determined as the lowest amount of analyte that was reproducibly quantified. These two parameters were calculated using the formula based on the standard deviation of the response and the slope. LOD and LOQ were calculated by using equations, LOD=3.3×SD/S and LOQ=10×SD/S, where SD = standard deviation, S= slope of the calibration curve. 2.7.7 Solution stability and Mobile phase stability: The solution stability of Dexlansoprazole in the assay method was carried out by leaving both the test solutions of sample and reference standard in tightly capped volumetric flasks at room temperature for 24 hrs. The same sample solutions were assayed for 6 hrs. interval up to the study period. The mobile phase stability was also carried out by assaying the freshly prepared sample solution against freshly prepared reference standard solution for 6 hrs interval up to 48 hrs. Mobile phase prepared was kept constant during the study period. The % RSD for the assay of Dexlansoprazole was calculated during mobile phase and solution stability experiment. The solution stability of Dexlansoprazole and its impurities in the related substance method was carried out by leaving spiked sample solution in the tightly capped volumetric flasks at room temperature for 24 hrs. Content of imp - 2-Mercapto-benzimidazole, 2- Nitrosulphoxide, sulphone & Sulphide were checked in the test solutions.



Figure 6: Dexlansoprazole chromatogram.

Figure 7: Dexlansoprazole and impurities spiked.

Figure 8: Acid degradation chromatogram.

Figure 9: Peroxide degradation chromatogram.

Figure 10: Thermal degradation chromatogram.



Figure 11: Photo degradation chromatogram.

Figure 12: Base degradation chromatogram.

2.8 Forced Degradation studies Stress studies were performed according to ICH guidelines under conditions of hydrolysis (acidic and alkaline), photolysis, oxidation, and thermal studies. 2.8.1 Oxidation: To 5 mL of stock solution of Dexlansoprazole, 5 mL of 3% hydrogen peroxide (H2O2) was added separately. The solutions were kept for 30 min at 60°C, for 24 hours at 25°C.For HPLC study, the resultant solution was diluted to obtain 100μg/mL solution then injected 10 μL into the system and the chromatograms were recorded to assess the stability of sample. 2.8.2 Acid Degradation Studies: To 5 ml of s tock s solution of Dexlansoprazole, 5ml of 0.5N Hydrochloric acid was added and refluxed for 30mins at 60 °C, after kept 24 hours at 25°C the resultant solution was diluted to obtain 100μg/mL solution then injected 10 μL into the system and the chromatograms were recorded to assess the stability of sample.

2.8.3 Alkali Degradation Studies: To 5 mL of stock solution of Dexlansoprazole, 5ml of 0.5N sodium hydroxide was added and refluxed for 30mins at 60°C, after kept 24 hours at 25oC. The resultant solution was diluted to obtain 100μg/mL solution then injected 10 μL into the system and the chromatograms were recorded to assess the stability of sample. 2.8.4 Dry Heat Degradation Studies: The standard drug solution was placed in oven at 80°C for 24 h to study dry heat degradation. For HPLC study, the resultant solution was diluted to 100μg/mL solution then injected 10µL into the system and the chromatograms were recorded to assess the stability of the sample. 2.8.5 Photo Stability studies: The photochemical stability of the drug was also studied by exposing the 100μg/mL solution to UV Light by keeping the beaker in UV Chamber for 24 hours in photo stability chamber. For HPLC study, the resultant solution was diluted to obtain 100μg/mL solution then injected 10 μL into the system and the chromatograms were recorded to assess the stability of sample.



Table 1: Optimized Chromatographic conditions.

Parameter Condition Mobile phase Water : Acetonitrile(500:500 % v/v) Column Inertsil ODS 3V 150 mm, 4.6 mm,5µm Wavelength 285nm Flow Rate 1.0mL/min Injection volume Run time

10 µL 10 min

Diluent Mobile phase (Milli Q water : Acetonitrile (500:500 % v/v))

Table 2: Forced degradation studies.

S.No Stress conditions Time (hours)

Assay of Active substance (%)

Total impurities (%)

Mass balance Assay impurities (%))

1 Normal 24 100 - 100 2 Acid hydrolysis 24 0.37 99.63 100

3 Base hydrolysis 24 0.0 100 100

4 Oxidation (3%H2O2) 24 0.93 99.07 100

5 Thermal at 80°C 24 0.22 99.78 100

6 UV light 24 94.34 5.66 100

Table 3: Limit of Detection and Limit of Quantification.

Table 4: Linearity data

Table 5: Accuracy data

Name Dexlansoprazole 2-Mercapto- benzimidazole

2-Nitrosulphoxide

Sulphone Sulphide

Accuracy %Recovery

100.4- 100.8 98.9-99.4 99.1-100.3 99.3-99.6 99.4-99.9

3. RESULTS AND DISCUSSION To establish and validate an efficient method for analysis of these drugs in bulk and pharmaceutical formulations, preliminary tests were performed. Different chromatographic conditions were employed for the analysis of the Dexlansoprazole and its impurities in both bulk and pharmaceutical dosage form. Finally the analysis was performed by using water and Acetonitrile in the ratio of 500:500 % v/v at a flow rate 1.0 mL/min. Samples were analyzed at 285nm at an injection volume of 10 μL and separation was carried by using Inertsil ODS 3V, (150 x 4.6 mm, 5µm) column. The proposed method was optimized to give very sharp peaks and meeting as per ICH guideline (Resolution greater than 2.0 between the peaks, theoretical plate count more than 2000 and asymmetry less than 2.0. The optimized conditions were given in table 1.

Forced degradation studies were performed to establish the stability indicating property and specificity of the proposed method. Degradation studies were carried out at 24 hours under conditions of acid hydrolysis, base hydrolysis dry heat, oxidation, UV light and the drug substances were observed almost complete degradation in base hydrolysis, dry heat, oxidation and acid hydrolysis conditions and partially degradation observed in Photolysis. Oxidative degradation conditions were performed by the drug sample with 3% H2O2 at 25oC and the Dexlansoprazole degraded completely. Acid and base hydrolysis was performed by exposing the drug substances with 0.5N HCl and 0.5N NaOH at 25oC for 24 hours and it was completely degraded in both conditions And there was a small amount of degradation occurs under Photolysis. The results of forced degradation studies were given in table.2

Name 2-Mercapto- benzimidazole

2-Nitrosulphoxide Sulphone Sulphide

LOD 1.59 1.59 1.59 1.59 LOQ 4.81 4.81 4.81 4.80

Name Dexlansoprazole 2-Mercapto- benzimidazole

2-Nitrosulphoxide Sulphone Sulphide

Linearity (n=3) Intercept 526751.16 -1032.02 -1979.41 260.63 -240.54 Slope 73374211.10 182738.52 95436.02 39873.71 88597.01 r 0.99999 0.99987 0.99978 0.99991 0.99988



Precision was evaluated by a known concentration of Dexlansoprazole and its impurities was injected six times and corresponding peaks were recorded and % RSD was calculated and found within the limits. The low % RSD value was indicated that the method was precise and reproducible. Accuracy of the method was proved by performing recovery studies on the commercial bulk and formulation for Assay at 25%,50%, 100% 150%,200 and 250% level. For impurities 50%,100% and 150% level. Recoveries of Dexlansoprazole and its impurities ranges from 98.9% to 100.8% in proposed method and the results were shown in the (Table 5). Linearity was established by analyzing different concentrations for assay (25%,50%, 100% 150%,200% and 250% level and for impurities 50%,100% and 150% level) of Dexlansoprazole and its impurities respectively. The calibration curve was plotted with the area obtained versus concentration Dexlansoprazole and its impurities. In the present study six concentrations were chosen ranging between 25-250 μg/mL of Dexlansoprazole and its impurities. The linear regression data for the calibration plot is indicative of a good linear relationship between peak area and concentration over a wide range. The correlation coefficient was indicative of high significance and the results were shown in the (Table 4). Robustness of the method is the ability of the method to remain unaffected by small deliberate changes in parameters like flow rate, mobile phase composition and column temperature. To study the effect of flow rate of the mobile phase it was changed to 0.1 units from 1.0 mL to 0.9 mL and 1.1 mL. The effect of column temperature also checked by changing temperature to ± 5oC. This deliberate change in the above parameters has no significant effect on chromatographic behaviour of the samples. LOD and LOQ of Dexlansoprazole and its impurities were evaluated based on relative standard deviation of the response and slope of the calibration curve. The detection limits were found to be 0.05 μg/mL and 0.15 μg/mL of Dexlansoprazole and its impurities respectively. The quantitation limit were found to be 0.10μg/mL for Dexlansoprazole and its impurities .The results were given in the (Table 3).

4. CONCLUSION A new stability- indicating RP-HPLC method has been developed for estimation of Dexlansoprazole and its impurities in bulk and pharmaceutical dosage form. The developed method was validated and it was found to be simple, sensitive, precise, and robust and it can be used for the routine analysis of Dexlansoprazole in both bulk and pharmaceutical dosage forms. The forced degradation studies were carried out in accordance with ICH guidelines and the results revealed suitability of the method to study stability of Dexlansoprazole

under various degradation conditions like acid, base, oxidative, thermal, UV and photolytic degradations. Finally it was concluded that the method is simple, sensitive and has the ability to separate related substance the drug from degradation products found in the dosage form.

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