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Spectrophotometric Determination of Pantoprazole Sodium
in Pharmaceuticals Using N-Bromosuccinimide, Methyl
Orange and Indigo Carmine as Reagents
Basavaiah, K.*+, Anil kumar, U.R., Kalsang tharpa
Department of Chemistry, University of Mysore, Manasagangotri, Mysore 570 006, INDIA Vinay, K.B.
Process Analytical Laboratory, Advinus Therapeutics, Peenya, II Phase, Bangalore-58, INDIA ABSTRACT: Two sensitive spectrophotometric methods are presented for the assay of
pantoprazole sodium sesqui hydrate (PNT) in bulk drug and in formulations using N- bromosuccinimide (NBS) and two dyes, methyl orange and indigo carmine, as reagents. The methods involve the addition of a known excess of NBS to PNT in acid medium, followed by determination of unreacted oxidant by reacting with a fixed amount of either methyl orange and measuring the absorbance at 520 nm (method A) or indigo carmine and measuring the absorbance at 610 nm (method B). In both methods, the amount of NBS reacted corresponds to the amount of PNT and the measured absorbance is found to increase linearly with the concentration of PNT which is corroborated by the correlation coefficients of 0.9959 and 0.9985 for method A and method B, respectively. The systems obey Beer’s law for 0.1 - 2.0 µg mL-1 and 0.5 - 6.0 µg mL-1 for method A and method B, respectively. The limits of detection and quantification are also reported for both methods. Intra-day and inter-day precision, and accuracy of the methods have been evaluated. The methods were successfully applied to the assay of PNT in tablet preparations and the results were statistically compared with those of the reference method by applying Student’s t-test and F-test. No interference was observed from the common tablet excipients. The accuracy of the methods was further ascertained by performing recovery studies via standard-addition method. KEY WORDS: Pantoprazole sodium, Assay, Spectrophotometry, N-bromosuccinimide,
INTRODUCTION
Pantoprazole sodium sesqui hydrate (PNT) is sesqui hydrate [1]. Pantoprazole inhibits H+ K+ AT Pase chemically known as sodium 5-(difluoromethoxy)-2- pump function thereby healing the acid related [[(3,4-dimethoxy-2-p-methyl]sulfinyl]-1H-benzimidazole conditions. PNT is chemically more stable than omeprazole * To whom correspondence should be addressed.
+ E-mail: [email protected]
and lansoprazole in neutral to mildly acidic conditions, N-bromosuccinimide (NBS): An approximately 0.01 but under strongly acidic medium, active species is M NBS solution was prepared by dissolving about 1.8 g formed. PNT like omeprazole and lansoprazole also has a of chemical (SRL Research Chemicals, India) in water role in the eradication of Helicobacter Pylori [2]. with the aid of heat and diluted to one litre with water and The literature survey reveals that only few methods standardized [11]. The solution was kept in an amber are available for the determination of PNT in dosage coloured bottle and was diluted appropriately to get 80 forms and include HPLC [3-5], HPTLC [6], UV spectro- and 340 µg mL-1 NBS for use in method A and method B, respectively. The NBS solution was stored in a Visible spectrophotometry, because of its simplicity, cost-effectiveness, sensitivity, selectivity , fair accuracy Hydrochloric acid (5 M): Concentrated hydrochloric and precision, has remained competitive in an era acid (S.D. Fine Chem., Mumbai, India; sp. gr. 1.18) was chromatographic techniques for pharmaceutical analysis. diluted appropriately with water to get 5 M acid. However, only two visible spectrophotometric methods are found in the literature for the assay of PNT. In a was first prepared by dissolving accurately weighed 58.8 method reported by Salama et al. [9] PNT was quantified mg of dye ( S.D. Fine Chem., Mumbai, India, 85 % dye by stability-indicating procedure through chelation with content) in water and diluting to 100 mL in a calibrated iron(III) in aqueous-ethanol medium to form an orange flask and filtered using glass wool. It was further diluted chelate peaking at 455 nm. The method is applicable over to obtain a working concentration of 50 µg mL-1. Indigo carmine (200 µg mL-1 ): A 1000 µg mL-1 stock In a report by Monstafa et al. [10] two methods based on charge transfer complexation reaction using standard solution was first prepared by dissolving 2,3-dichloro-5,6-dicyano-1,4 benzo quinine (DDQ), a π accurately weighed 112 mg of dye (S.D. Fine Chem., acceptor and iodine as σ-acceptor with linearity ranges of Mumbai, India, 90 % dye content) in water and diluting to volume in a 100 mL calibrated flask. The solution was The same article describes one more procedure based on then diluted 5-fold to get the working concentration of ternary complex formation of PNT with eosin and copper (II) with a linear range of 4-26 µg mL-1. But all the Methanol-ammonia 4.0 % v/v: Methanol ( S.D. Fine methods involve the use of organic solvents and the last Chem., Mumbai) and ammonia ( S.D. Fine Chem., method involves liquid-liquid extraction step. Mumbai) were prepared as per the reference method [7]. The present investigation aims to develop simple, Standard solution of pantoprazole sodium: Pharma- sensitive and cost-effective methods for the determination ceutical grade PNT, certified to be 99.8 % pure was of PNT in pure form and in dosage forms using the procured from Cipla India Ltd, Mumbai, India, and was visible spectrophotometric technique. The methods utilize used as received. A stock standard solution containing NBS, methyl orange and indigo carmine as reagents. The 500 µg mL-1 PNT solution was prepared by dissolving proposed methods have the advantages of speed and accurately weighed 50 mg of pure drug in water and simplicity besides being accurate and precise, and can be diluting to 100 mL in a calibrated flask with water. The adopted by the pharmaceutical laboratories for industrial solution was diluted stepwise to get working concentrations of 5 and 20 µg mL-1 PNT for method A EXPERIMENTAL
Apparatus
A Systronics model 106 digital spectrophotometer Procedures
Spectrophotometry using methyl orange (method A). with 1-cm matched quartz cells was used for all absorbance measurements. Different aliquots (0.2 - 4.0 mL) of a standard 5 µg mL-1 PNT solution were transferred into a series of 10 mL Reagents and Standards
calibrated flasks by means of a micro burette and the total All chemicals used were of analytical purity grade and volume was adjusted to 4 mL by adding adequate quantity all solutions were prepared in distilled water. of water. To each flask were added 1 mL each of 5 M Spectrophotometric Determination of … HCl and 1 mL of NBS solution (80 µg mL-1), the last NBS after allowing the reaction between PNT and a being measured accurately. The flasks were stoppered, measured amount of NBS to be complete. The residual content mixed and let stand for 15 min with occasional NBS was determined by reacting it with a fixed amount shaking. Finally, 1 mL of 50 µg mL-1 methyl orange of either methyl orange or indigo carmine dye. The solution was added (accurately measured) and the volume methods make use of bleaching action of NBS on the was diluted to the mark with water and mixed well. The dyes, the decolouration being caused by the oxidative absorbance of each solution was measured at 520 nm PNT when added in increasing concentrations to Spectrophotometry using indigo carmine (method B). a fixed concentration of NBS, consumes the latter Varying aliquots (0.25-3.0 mL) of a standard 20 µg mL-1 proportionally and there occurs a concomitant fall in the PNT solution were transferred into a series of 10 mL concentration of NBS. When a fixed concentration of calibrated flasks by means of a micro burette and the total dye is added to decreasing concentrations of NBS, a volume was brought to 3 mL by adding water. To each concomitant increase in the concentration of dye results. flask were added 1 mL each of 5 M hydrochloric acid and Consequently, a proportional increase in the absorbance 340 µg mL-1 of NBS solution (by meansofamicro burette). The content was mixed well and the flasks were kept aside for 10 min with intermittent shaking. Finally, 1 mL Preliminary experiments were performed to fix the of 200 µg mL-1 indigo carmine solution was added to upper concentrations of the dyes that could be determined each flask, the volume was diluted to the mark with spectrophotometrically, and these were found to be 5 and water, mixed well and absorbance measured against a 20 µg mL-1 for methyl orange and indigo carmine reagent blank at 610 nm after 10 min. In either method, a respectively. A NBS concentration of 8.0 µg mL-1 was standard graph was prepared by plotting the absorbance found to bleach the red colour due to 5 µg mL-1 methyl versus the concentration of PNT . The concentration of orange whereas 34.0 µg mL-1 NBS was required to the unknown was read from the calibration graph or destroy the blue colour due to 20 µg mL-1 indigocarmine. computed from the regression equation derived using For both steps, i.e., the reaction between PNT and NBS, and the determination of the latter by reacting with the dye, HCl medium was found to be ideally suited. The Procedure for tablets
absorbance of the dye was not affected in 0.5 to 1.5 M A quantity of the finely ground tablet powder HCl concentrations. However, 1 mL of 5 M HCl was equivalent to 50 mg of PNT was accurately weighed into selected for oxidation of drug in both methods and the a 100 mL calibrated flask, 60 mL of water was added and same quantity of acid was maintained for bleaching step. shaken for 20 min; the volume was finally diluted to the The oxidation reaction was found to be complete in 15 mark with water, mixed well and filtered using a Whatman min for method A and 10 min for method B and contact No. 42 filter paper. First 10 mL portion of the filtrate was times upto 30 min had no effect on the absorbance of discarded and a suitable aliquot of the subsequent portion dyes. The absorbance of either dyes solution even in the (500 µg mL-1 PNT) was diluted appropriately to get 5 and presence of reaction product was found to be stable for 20 µg mL-1 concentrations for analysis by method A and method B, respectively. For comparison [8] the same tablet powder was extracted with 0.1 M NaOH, and after appropriate dilution to 10 µg mL-1 with 0.1 M Analytical data
NaOH, the absorbance was measured at 295 nm. A linear correlation was found between absorbance at λmax and concentration of PNT in the ranges given in RESULTS AND DISCUSSION
table 1. Regression analysis of the Beer’s law data using Method development
the method of least squares was made to evaluate the The proposed spectrophotometric methods are slope (b), intercept (a) and correlation coefficient (r) for indirect and are based on the determination of the residual each system and the values are presented in table 1. Table 1: Analytical and regression parameters of spectrophotometric methods.
*Y = a+bX, where Y is the absorbance and X concentration in µg mL-1. Sa=Standard deviation of intercept, Sb=Standard deviation of slope. Table 2: Evaluation of accuracy and precision.
RE: Relative error; RSD: Relative standard deviation * Mean value of seven determinations. The optical characteristics such as Beer’s law limits each day for five days with all solutions being prepared and Sandell sensitivity values for both methods are given afresh each day. The day-to-day relative standard in table 1. The limits of detection (LOD) and quantitation deviation values were less than 3.0 % and represent the (LOQ) calculated according to ICH guidelines [12] are best appraisal of repeatability of the proposed methods. also presented in table 1 and reveal the very high Application
In order to check the validity of the proposed methods, Method Validation
PNT was determined in some commercial tablets. Table 3 To evaluate the accuracy and intra-day precision of gives the results of the determination from which it is the methods, pure drug solution at three different clear that there is close agreement between the results concentration levels was analysed, each determination obtained by the proposed methods and the label claim. being repeated seven times. The relative error (%) and The results were also compared statistically by a relative standard deviation (%) were = 2 and indicate Student’s t- test for accuracy and variance ratio F- test for high accuracy and precision of the methods (table 2). For precision with those of the literature method [7] at 95 % a better picture of reproducibility on a day-to-day basis, a confidence level. The calculated t- and F-values (table 3) series of experiments were performed in which standard did not exceed the tabulated values (t=2.77, F=6.39) and drug solution at three different levels was determined indicate that there was no significant difference between Spectrophotometric Determination of … Table 3: Results of determination of PNT in tablets and statistical comparison with the reference method.
*Mean value of five determinations #Marketed by: a. Alkem Ltd.; b. Cipla Ltd.; c. Aristo Ltd. Tabulated t-value at 95 % confidence level is 2.77 Tabulated F-value at 95 % confidence level is 6.39. Table 4: Results of recovery experiments by standard addition method.
*Mean value of three determinations. the proposed methods and the literature method in respect CONCLUSIONS
Two useful micro methods for the determination of The accuracy and validity of the proposed methods PNT have been developed and validated. The methods were further ascertained by performing recovery studies. are simple and rapid taking not more than 20 min for Pre-analysed tablet powder was spiked with pure PNT at analysis. Besides, they are more sensitive than the three different levels and the total was found by the reported visible spectrophotometric methods [9,10] and proposed methods. Each determination was repeated also the chemometric methods reported by Wahbi et al. three times. The recovery of the pure drug added was [8]. Whereas the already reported methods [8] have a quantitative and revealed that co-formulated substances working range of 0.5 -3.5 µg mL-1 with detection limit of such as talc, starch, gelatin, gum acacia, calcium carbonate, 0.035 µg mL-1, the present methods (method A) has a calcium gluconate, calcium dihydrogen orthophosphate, detection limit of 0.02 µg mL-1 and has a wide linear sodium alginate and magnesium stearate did not interfere dynamic range of 0.1-2.0 µg mL-1. Precision wise the in the determination. However, ascorbic acid was found present methods (RSD, 0.54 -1.63 %) is comparable to to interfere strongly in the assay. Drugs such as that of the reported methods [8] (RSD, 0.5 %). The omeprazole and lansoprazole were also found to interfere. proposed methods rely on the use of simple and cheap The results of recovery study are compiled in table 4. chemicals and techniques but provide a sensitivity and Pantoprazole Sodium Salt and Corresponding Impurities, J. Pharm. Biomed., Anal., 32, 1019
[8] Wahbi Abdel-Aziz, M., Abdel-Razak, O., Gazy, A. A., Mahgoub, H., and Moneeb, M. S., Spectro- photometric Determination of Omeprazole, Lansoprazole and Pantoprazole in Pharmaceutical Formulations, J. Pharm. Biomed. Anal., 30, 1133
Fig. 1: Structure of pantoprazole sodium sesqui hydrate.
[9] Salama, F., El-Abasawy, N., Razeq, S. A. A., Ismail, comparable to that achieved by sophisticated and M. F. and Fouad, M. M., Spectrophotometric expensive technique like HPLC. Thus, they can be used Determination of Omeprazole and Pantoprazole as alternatives for rapid and routine determination of bulk Sodium via Chelates with Iron, Chromium, and sample and tablets as a part of industrial quality control. Cobalt, Bull. Fac. Pharm (Cairo University), 41, 185
Received : 1st January 2007 ; Accepted : 10th March 2009 [10] Moustafa, A. A. M., Spectrophotometric Methods for the Determination of Lansoprazole and Panto- REFERENCES
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