Stability indicating Fast LC method for the estimation of impurities of Pantoprazole in Tablet Dosage Forms

 

Paluru Rudra Mohan Reddy1*, J Sreeramulu1, Petla Y. Naidu2 and A. Rajasekhara Reddy2

1 Department of Chemistry, S.K. University, Anantapur 515003, India;

2Orchid Chemicals and Pharmaceuticals Ltd, SIPCOT Industrial Park, Irungattukottai, Sriperumbudur, Kancheepuram District, Tamilnadu 602105, India

*Corresponding Author E-mail: prudramohanreddy@rediffmail.com

 

ABSTRACT:

A fast, stability-indicating reversed phase liquid chromatographic method has been developed and subsequently validated for the estimation of impurities of Pantoprazole. The developed method utilizes sub 2-m C-18 column and mobile phase consisting of ammonium acetate buffer acetonitrile with gradient elution, at a flow rate of 0.8 mL per minute. The detection was carried out at 290 nm. The method was validated in terms of accuracy, precision, linearity, limit of detection, limit of quantification, ruggedness and robustness. This method has been successfully applied to pharmaceutical formulation and no interference from the tablet excipients was found. As the proposed method could effectively separate the drug from its degradants / impurities, ie, pantoprazole related compound A (PA), pantoprazole related compound B (PB), pantoprazole related compound C (PC) and pantoprazole related compound DandF (PDF), it can be employed as stability-indicating method for the determination of instability of the drug in bulk and pharmaceutical formulations.

 

KEYWORDS: Liquid chromatography; Pantoprazole; stability indicating; Drug product

 


 

INTRODUCTION:

Pantoprazole (5-(Difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridyl)methyl]sulfinyl]-benzimidazole), sodium salt, sesquihydrate1-2 a compound that inhibits gastric acid by blocking the H+/K+ adenosine triphosphate enzyme system of the gastric parietal cell. It is used for short-term treatment of erosion and ulceration of the esophagus. Pantoprazole sodium sesquihydrate molecular formula3-4 is C16H14F2N3NaO4S. 1.5H2O and its molecular weight is 432.37. Pantoprazole sodium is a white to off-white crystalline powder. It is freely soluble in water, in methanol, and in dehydrated alcohol and practically insoluble in hexane and in dichloromethane.

 

A method for the estimation of Impurities by High Performance Liquid Chromatography has been given in USP (United States Pharmacopoeia) and Different analytical procedures have been reported in the literature for the assay of Pantoprazole in dosage forms in biological fluids including Spectrophotometry5-10.

 

However to the best of our knowledge no Fast LC methods are reported for the estimation of impurities of Pantoprazole in dosage forms. So it is necessary to develop a Liquid chromatographic procedure which would serve as a simple, rapid and reliable method for the estimation of impurities of Pantoprazole. In the present study we report for the first time a reversed-phase Fast Liquid Chromatographic method for the determination of Pantoprazole sodium impurities ie, Pantoprazole Related compounds in Pharmaceutical Dosage forms were follows.

A:5-(Difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridyl)methyl]sulfonyl]-1H-benzimidazole (PRA),

 

B:5-(Difluoromethoxy)-2-[[(3,4-dimethoxy-2-pyridyl)methyl]thio]-1H-benzimidazole (PRB),

 

C: 5-(Difluoromethoxy)-1H-benzimidazole-2-thiol (PRC),

 

D: 5-(Difluoromethoxy)-2-[(RS)-[(3,4-dimethoxypyridin-2-yl) methyl] sulfinyl]-1-methyl-1H-benzimidazole (PRD)

 

F: 6-(Difluoromethoxy)-2-[(RS)-[(3,4-dimethoxypyridin-2-yl)methyl]sulfinyl]-1-methyl-1H-benzimidazole (PRF)

 

Since Pantoprazole Related compound D and Pantoprazole Related compound F are isomers these Impurities are quantified together. All the components can be estimated within a shorter span of 8 minutes run time. It costs the solvent consumption and precious time. Finally the method is thoroughly validated for the estimation of impurities.

 

EXPERIMENTAL:

Instrumentation: Agilent SL 1200 series LC system equipped with DAD- detector -G1315C was used. The output signal was monitored and integrated using Chem32 software (Agilent technologies, USA).

 

Solutions:

Preparation of pH 4.5 buffer solution:

1.5g of Ammonium acetate was dissolved in 2000 mL of HPLC grade water in a suitable container and 2 mL of Triethylamine was added to this solution and mixed well. pH of the solution was adjusted to 4.5 0.05 with Orthophosphoric acid.

 

Preparation of Mobile phase-A:

A mixture of pH 4.5 buffer and Acetonitrile in the ratio 70:30 (v/v) was prepared and filtered through 0.2 m nylon membrane filter prior to use.

 

Preparation of Mobile phase-B:

A mixture of pH 4.5 buffer and Acetonitrile in the ratio 30:70 (v/v) was prepared and filtered through 0.2 m nylon membrane filter prior to use.

 

Diluent : A mixture of 0.02 N Sodium hydroxide solution and Acetonitrile in the ratio 50:50 (v/v).

 

Preparation of Standard solution (1.5 g/mL): About 57 mg of Pantoprazole sodium sesquihydrate (PSS) working standard was accurately weighed and transferred into a 100 mL volumetric flask, dissolved in about 70 mL of diluent and made up to volume with diluent and mixed well. 5 ml of this solution was further diluted to 50 mL with diluent. Further 3 mL of the resulting solution was pipetted out into a 100 mL volumetric flask and made up to volume with diluent and mixed well. The final solution was filtered through 0.45 m nylon membrane filter prior to use.

 

Preparation of Test solution: Twenty tablets were weighed and crushed to fine powder. Sample equivalent to about 50 mg of Pantoprazole was accurately weighed and transferred in to a 100 mL volumetric flask, about 70 mL of diluent was added, sonicated for 20 minutes with intermediate shaking and made up to volume with diluent and mixed well. The solution was filtered through 0.45 m nylon membrane filter prior to use.

 

Chromatographic conditions:

A Peerless HT C18 (50 x 4.6 mm, 1.8 m packing) column was used at column temperature 25C. The mobile phase was pumped through the column at a flow rate of 0.8 mL/min. The sample injection volume was 6 L. The Diode array detector was used to record the signal at a wavelength of 290 nm.

 

RESULTS AND DISCUSSION:

Method Development

Keeping the aim of best separation with shorter run time, it was decided to use sub-2 m particle size column. As the pKa of Pantoprazole is 3.8 the pH of the buffer selected as 4.5 using Ammonium acetate buffer for mobile phase preparation. Accordingly Zorbax Eclipse plus C18, 50 x 2.1 mm, 1.8 m column with a mobile phases consisting of pH 4.5 buffer, prepared by dissolving 1.5 g of ammonium acetate and 1 mL of Triethylamine in 2000 mL of water, pH was adjusted with Orthophosphoric acid, and Acetonitrile in different ratios were used for gradient elution at a flow rate of 0.2 mL min-1 and injected the Impurities spiked solution. Separation between Pantoprazole and PDF was not good. Experiment is continued with Zorbax Eclipse plus C18, 50 x 4.6 mm, 1.8 m column, the separation was not satisfactory, finally Peerless HT C18, 50 x 4.6 mm, 1.8 m column was used at a flow rate 0.8 mL min-1. Adequate resolutions and retention times were obtained. Triethylamine concentration is increased from 1 mL to 2 mL per 2000 mL of pH 4.5 Ammonium acetate buffer in mobile phase using simple gradient elution as below and obtained better peak shapes of impurities and Analyte than earlier.

 

Time (minutes)

% Mobile phase-A

% Mobile phase-B

0-4

90-70

10-30

4-6

70-10

30-90

6-8

10

90

 

6 L injection volume was finalized for the proposed method based on the responses of impurities and Analyte peaks. Room temperature for column oven and 290 nm as detection wavelength were opted.

 

Column selection:

Based on the retention time, peak symmetry and separation of the compounds Peerless HT C18 (50 X 4.6 mm, 1.8 m) column was selected as suitable column for the estimation of impurities.

 

METHOD VALIDATION:11-14

The proposed method is extensively validated as per FDA and ICH guidelines.

 

Limit of Detection (LOD) and Limit of Quantification (LOQ):

The LOD and LOQ values were determined by injecting PA, PB, PC, PD and PF impurity solutions individually at the lowest concentrations with the test method conditions at which Signal-to-Ratio is about 3 for LOD and 10 for LOQ respectively. The established LOD and LOQ values for all the impurities are tabulated in Table-1.

 

PRECISION OF TEST METHOD: Precision was established for the developed method by spiking the impurities at target concentration level in the test solution containing the drug product.


Table 1. Method validation-Analytical data of proposed method

Parameter

PA

PB

PC

PDF

LOD (ppm)

0.016

0.022

0.005

0.015

LOQ (ppm)

0.049

0.070

0.016

0.048

Slope

0.9896

1.0078

0.9851

1.0112

Intercept

0.0032

0.0044

0.0112

-0.0077

Correlation coefficient

0.99

0.99

0.99

0.99

Precision (%RSD)

1.0

1.3

1.1

1.1

Intermediate precision (%RSD)

0.1

0.3

1.7

0.3

 

Accuracy (Recovery study)

Level-I (LOQ) 95.9

Level-II(100%) 98.6

Level-III(125%) 99.3

Level-I (LOQ) 96.3

Level-(100%) 102.2

Level-(125%) 100.3

Level-I (LOQ) 95.4

Level-II(100%) 99.5

Level-II(125%) 98.6

Level-I (LOQ) 93.2

Level-II(100%) 98.3

Level-III(125%) 102.0

 


 

(a)

 

 

(b)

 

 

(c)

 

 

(d)

 

(e)

Fig-1: Chemical structures; a: Pantoprazole, b: Pantoprazole Related compound A, c: Pantoprazole Related compound B, d: Pantoprazole Related compound C; e: Pantoprazole Related compound D and F.

Six spiked samples were prepared and injected to prove the precision of the method. The % R.S.D of all individual impurities was found to be satisfactory for all the six analytical measurements. This was also repeated on different day to determine inter-day precision. A different scientist established intermediate precision on a different chromatographic system. The small %RSD values presented in Table-1 prove the high precision of the method.

 

ACCURACY: The accuracy of the developed method was determined by spiking method. Different concentration levels ranging from LOQ to 125 % of target concentration of impurities were prepared by spiking in Test solution. At each level three solutions were prepared. The concentrations of PA in spiked solutions are 0.049, 1.078 and 1.3482-g mL -1 at LOQ (Level-I), 100% (Level-II) and 125% (Level-III) respectively of Target concentration (0.5%). The concentrations of PB in spiked solutions are 0.070, 1.000 and 1.250-g mL -1 at LOQ, 100% and 125% respectively of Target concentration (0.5%). The concentrations of PC in spiked solutions are 0.016, 1.080 and 1.350-g mL -1 at LOQ, 100% and 125% respectively of Target concentration (0.5%). The concentrations of PDF in spiked solutions are 0.048, 1.011 and 1.263-g mL -1 at LOQ, 100% and 125% respectively of target concentration (0.5%). The % recovery was found to be good and the values are presented in Table 1. Linearity of the method was checked by plotting calibration curves between the amount g mL -1 of component added in spiked solution versus the amount g mL -1 of component found (recovered). The slope, intercept and correlation coefficient were derived from least-square regression method. The values are presented in Table 1. It was found that the responses of impurities were linear from LOQ level to 125 % of target concentration. The correlation coefficient values of impurities indicate the best linearity of the method.

 

SPECIFICITY: The specificity defined as the ability of the method to quantify the Impurities and Analyte specifically in the sample matrix. The placebo samples were prepared in triplicates by following the procedure same as test solution. The possibility of excipient interference in the analysis was also studied. The drug product and Placebo was subjected to forced degradation under acidic (0.1N HCl), Basic (1N NaOH), water and oxidation (0.01% Peroxide) conditions to demonstrate the interference of Degradation products from Pantoprazole sodium. In acidic stress study 13% of analyte was degraded in the solution kept at Bench top for 5 min at room temperature. In base stress study 22% of analyte was degraded in the solution heated on water bath for 10 min at 70 C. In the case of peroxide stress study 7% of analyte was degraded in the solution kept at Bench top for 10 min at room temperature and in water the analyte was degraded 5% when heated on water bath at 70C for 30 minutes. The drug product is also subjected to visible light for about 288 hours and UV light for about 54 hours. 0.8 % degradation was found in the visible light stress and 0.4% degradation was found when subjected to UV light. The analyte was degraded 11% when the drug product is subjected to dry heat at 100C for 6 hours and 0.4% degradation was observed when exposed to humidity at 25C /90% RH for 288 hours. There are no interfering peaks at the retention times of impurities PA, PB, PC and PDF and Pantoprazole peaks from the chromatogram of placebo. From fig.2 good separation is also seen between impurities and Pantoprazole peaks from the spiked chromatogram. All the chromatograms of stressed samples were evaluated for peak purity of Pantoprazole using Chem32 software and found peak purity passes in all the degradation conditions, hence the proposed method is said to be stability indicating method consequently the method can be used for determining the stability of Pantoprazole.

 

Fig-2: Chromatogram of Impurities spiked solution

 

ROBUSTNESS: A study to establish the effect of variation in flow rate ( 0.2 mL min-1), column temperature ( 5C) and pH of the buffer in mobile phase( 0.2 units) was conducted. Impurity spiked solutions were prepared at target concentration as per the proposed method and injected into HPLC system. The selectivity and sensitivity were evaluated, and were not affected. From the above study the proposed method was found to be Robust.

 

RUGGEDNESS: The Ruggedness test for the method of analysis of impurities in Pantoprazole sodium Delayed-Release tablets was performed with analyst-to-analyst variability, system-to-system variability and column-to-column variability using six test solutions prepared by spiking of all known impurities at the target concentration level. The resolution between impurities and Pantoprazole is not significantly changed and the Tailing factor for Pantoprazole peak 1.4 0.1 is obtained from six test solutions in the study analyst-to-analyst variability, system-to-system variability and column-to-column variability. Conditions used in stability experiments should reflect situations likely to be encountered during actual sample handling and analysis. Stability data is required to show that the concentration and purity of analyte in the sample at the time of analysis corresponds to the concentration and purity of analyte at the time of sampling. Stability of Pantoprazole in test sample and standard solution was established for 2 days in refrigerator. A 500 g mL -1 test solutions were prepared in duplicate and injected immediately (0 hour). The aliquots are stored at refrigerator conditions and analyzed after 24 and 48 hours. The % of individual impurities and % total impurities and were calculated by using the developed method.

 

CONCLUSION

The developed fast LC method is linear, precise, specific and accurate and can be conveniently used for the determination of impurities of Pantoprazole in formulations and Active pharmaceutical ingredient (API). Hence, it can be concluded that the proposed method can be recommended for routine and quality control analysis owing to its simplicity rapidity and low cost.

 

ACKNOWLEDGEMENTS

The authors are highly thankful to the management of Orchid chemicals and Pharmaceuticals Ltd., Chennai for providing facilities to carry out this work.

 

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Received on 26.01.2010 Modified on 09.03.2010

Accepted on 17.04.2010 AJRC All right reserved

Asian J. Research Chem. 3(3): July- Sept. 2010; Page 600-603