Department of Medicinal Chemistry, Faculty of Pharmacy and Pharmaceutical Sciences Research Center,
University of Medical Sciences, Tehran (14155-6451), Iran
*Corresponding Author E-mail: souri@sina.tums.ac.ir
ABSTRACT:
Pantoprazole is a gastric proton pump inhibitor which is used for treatment of gastric and duodenal ulcers. In this report the stress degradation of pantoprazole was studied and an HPLC method was developed for the determination of pantoprazole in the presence of its degradation products. Pantoprazole was relatively stable in basic conditions but unstable under acidic condition, oxidative condition, heat, and light. Separation of pantoprazole and its degradation products was achieved on a Nova-Pak C18 column using a mixture of acetonitrile and KH2PO4 10 mM (pH 7.4) (25:75) as mobile phase. UV detection was performed at 290 nm. The method was linear over the range of 1-50 mg/mL pantoprazole (r2>0.999). The within-day and between-day precision values were also in the range of 0.7-3.1%. The proposed method was successfully used for the determination of pantoprazole in tablets and dissolution medium.
KEYWORDS: Pantoprazole, HPLC, Stability-Indicating, Stress Degradation
Inhibition of the gastric proton pump (H+ /K+ - ATPase) which is the final step in the pathway for acid secretion in the parietal cells will prevent production of acid. Pantoprazole, 5-(difluoromethoxy)-2-[[(3, 4-dimethoxy-2-pyridinyl) methyl]sulfinyl]-1H-benzimidazole in the form of sodium sesquihydrate (Figure 1), is a selective and irreversible inhibitor of H+/K+-ATPase1, 2. Pantoprazole is widely used in the treatment of gastric and duodenal ulcers or gastroesophageal reflux diseases and also hypersecretory diseases such as the Zollinger-Ellison Syndrome.1 Pantoprazole could be administered intravenously or as gastric-resistant tablets. Up to now, no pharmacopeial method has been published for determination of pantoprazole in pharmaceutical dosage forms. A literature search has revealed that a few HPLC methods were published for determination of pantoprazole enantiomers3, 4, pantoprazole and its sulphone metabolite5, or pantoprazole in human plasma6. A LC-MS/MS method was also reported for the determination of pantoprazole in human plasma7. Simultaneous determination of pantoprazole and domperidone was also reported by HPLC method8, 9.
A first-order derivative spectrophotometric method was also reported for the determination of omeprazole and pantoprazole and related impurities10.
The stability of pantoprazole under stressed conditions is not yet reported. According to the International Conference on Harmonization (ICH) guidelines11 and USP12 it was thought necessary to develop a stability-indicating HPLC method for determination of pantoprazole in the presence of its degradation products under acidic, alkaline, oxidative and photolytic conditions.
EXPERIMENTAL:
Chemicals:
Pantoprazole sodium sesquihydrate was from Hetero Drugs Limited (India, Lot No: PTH/WS/PS/07/18) and obtained from SAMISAZ Pharmaceutical Co. as a gift. Protium® tablets containing 40 mg of pantoprazole were from ALANTA Pharma AG (Germany, Batch No: 466631) and purchased from a local pharmacy. HPLC grade acetonitrile, potassium dihydrogen phosphate and other solvents and chemicals were analytical grade and purchased from Merck (Darmstadt, Germany). HPLC grade water was obtained by a Milli-Q system (Millipore, Milford, MA, USA).
Instrumentation:
The HPLC system (Waters, Milford, MA) was consisted of an isocratic pump (Model 515), an autosampler (Model 710 plus) and a variable UV-Vis detector (Model 480). The data processing system was a multi-channel Chrom and Spec software for chromatography, version 1.5 x. A water bath (Memmert, GmbH + Co. KG, Germany), a dry air oven (Melag, Germany), a sonicator (Tecna6, Tecno-Gas, Italy), an analytical balance (Mettler, Toledo, Switzerland) and a centrifuge (Hettich, Germany) were also used. The light sources were a 100 W tungsten lamp (visible light) and an E27 Mercury lamp 160 W (Narva, Germany) (UV light) with l max at around 360 nm.
Figure 1. Chemical structure of pantoprazole
Chromatographic Conditions:
The separation was achieved using a Nova-Pak C18, 4 mm column (150 mm ´ 3.9 mm, Waters). Mobile phase consisted of acetonitrile and 10 mM KH2PO4 (pH 7.4) (25:75) and used at a flow rate of 1mL/min. The mobile phase was prepared daily and degassed by filtration through a 0.45 mm filter and sonication for 10 min. UV detection was performed at 290 nm.
Standard Stock Solution:
An accurately weighed amount of pantoprazole sodium sesquihydrate was dissolved in methanol to reach a final concentration of pantoprazole equal to 500 mg/mL. The stock solution was stored at 4°C.
Table 1. The degradation conditions for neutral, acidic, alkaline, and oxidative stress tests
|
Degradation Condition |
Solvent |
Time |
Temperature |
|
Neutral |
Water Water Water Water |
5 days 1 h 2 h 4 h |
Room temperature Reflux Reflux Reflux |
|
Acidic |
0.1 M HCl 0.05 M HCl 0.01 M HCl 0.01 M HCl |
10 min 30 min 10 min 60 min |
Room temperature Room temperature Room temperature Room temperature |
|
Alkaline |
0.1M NaOH 1 M NaOH 1 M NaOH 1 M NaOH 1 M NaOH |
5 days 5 days 1 h 2 h 4 h |
Room temperature Room temperature Reflux Reflux Reflux |
|
Oxidation |
3% H2O2 3% H2O2 |
2 h 3 h |
Room temperature Room temperature |
Degradation Studies:
For neutral, acidic, alkaline and oxidative stress tests a solution of pantoprazole at a concentration of 500 mg/mL were used. The conditions of these stress studies are presented in Table 1. The product of acidic or alkaline degradation studies was neutralized by adding appropriate amount of sodium hydroxide or hydrochloric acid solution and injected to the HPLC system after dilution with mobile phase to yield a starting concentration of about 25 mg/mL of pantoprazole.
For heat stress studies pantoprazole powder was exposed to dry heat in an oven at 70°C and 95°C for 24 h.
Pantoprazole powder and pantoprazole solution (500 mg/mL in methanol in Pyrex flasks) were exposed to visible light and UV light to study the photostability of pantoprazole. For solid state about 100 mg of pantoprazole was spread on a watch glass in a layer less than 2 mm in thickness. The samples were exposed to light at 15 cm from the light source in a 40 ´ 30 ´30 cm chamber for 7 days. Samples were withdrawn at different time intervals and analyzed after appropriate dilution with mobile phase and compared with freshly prepared samples kept in normal conditions.
Linearity:
Linearity of the developed method was studied by injection of pantoprazole solutions in mobile phase at the concentrations of 1, 2.5, 5, 10, 20, 30, 40 and 50 mg/mL prepared by subsequent dilution of stock standard solution of pantoprazole in mobile phase. Calibration curves were constructed by plotting the measured peak areas over the concentration of standard samples and statistical analysis was performed.
System Suitability:
The HPLC system suitability parameters such as plate count, tailing factor and repeatability (RSD of retention times and peak areas for six repetitions) were evaluated by repeated injections of a pantoprazole solution (10 mg/mL in mobile phase).
Precision and Accuracy:
The within-day and between-day precision and accuracy were established by using three replicates of standard solutions of pantoprazole at three different concentrations (1, 10 and 50 mg/mL) on one day and three separate days.
Recovery:
Ten pantoprazole tablets were weighed and ground into fine powder using a glass mortar and pestle. An amount of tablet powder equivalent to 25% of one tablet weight (10 mg pantoprazole) was transferred to each of four 100 volumetric flasks. Approximately 50 mL of mobile phase and 20 mL methanol were added to each flask. After addition of standard solutions of pantoprazole to each flask to reach a concentration of 0, 100, 200 and 400 mg/mL, the solution was sonicated for 15 min. The volumetric flasks were made up to volume with mobile phase and injected to the HPLC system after filtration through a 0.45 mm polypropylene syringe filter (Teknokroma, Spain) and ten times dilution. The peak area of these solutions were compared with the same concentrations of standard solutions of pantoprazole and the relative recovery was calculated.
Robustness:
The robustness of the method was evaluated by varying the amount of organic solvent, pH and ionic strength of buffer in the mobile phase.
Application of the Method:
Ten Protiumâ tablets were weighed and ground into fine powder using a glass mortar and pestle. An accurately weighed of tablet powder equivalent to 10 mg of pantoprazole was transferred to a 100 mL volumetric flask and 20 mL of methanol and 50 mL of mobile phase were added. After 15 min sonication the volumetric flask made up to volume with mobile phase and injected to the HPLC system after filtration and four times dilution to reach a concentration value about 25 mg/mL.
The dissolution profile of Pantoprazole tablets (SAMISAS, Batch NO: PAC004) was also compared with Protiumâ as standard tablet. A dissolution apparatus (Erweka, Heusenstamm, Germany) with six vessels in warm bath at 37±0.5°C was used. The paddle apparatus with rotation speed at 75 rpm and 1000 mL phosphate buffer (KH2PO4 50 mM, pH 6.8) as medium were used. Samples of 4 mL were drawn at 5, 10, 20, 30 and 45 min. The solutions were filtered using a 0.45 mm syringe filter and injected to the HPLC system. The changes due to the withdrawal of the samples were taken into account for calculating the concentrations. The percentage of released drug was calculated.
RESULTS AND DISCUSSION:
Chromatographic Conditions:
Satisfactory separation of pantoprazole and stress degradation products was achieved using a Nova-Pak C18 column and a mixture of acetonitrile and KH2PO4 10 mM (pH 7.4) (25:75) as mobile phase. Good peak shape without tailing and suitable baseline resolution was observed. Typical chromatograms are presented in Figure 2. Results of the system suitability tests are also summarized in Table 2 which was calculated for six replicate injections. The calculated parameters were within the acceptable range. The mean resolution (Rs) of the analyte peak with respect to the nearest degradation product peak was about 2.5.
Table 2. System suitability parameters
|
Parameters |
Found |
Acceptable limites |
|
Theoretical plates (n=6) |
11100 |
N>1500 |
|
Tailing factor (n=6) |
1.2 |
T<1.5 |
|
Repeatability (tR) (n=6) |
0.52 |
RSD<1% |
|
Repeatability (peak area) (n=6) |
0.37 |
RSD<1% |
tR: retention time (min); N: theoretical plates; T: tailing factor; RSD: relative standard deviation
Linearity:
Linear response for pantoprazole was observed in the concentration range of 1-50 mg/mL with r2>0.999. Statistical data are presented in Table 3. The limit of quantification with CV<3% was found to be 1 mg/mL for pantoprazole.
Table 3. Characteristic parameters for the calibration curves of pantoprazole (n=6)
|
Pantoprazole |
Results |
|
Linearity range |
1-50 mg/mL |
|
Regression equation |
y = 42.15x-12.21 |
|
Standard deviation of slope |
0.36 |
|
Relative standard deviation of slope (%) |
0.85 |
|
Standard deviation of intercept |
2.51 |
|
Correlation coefficient (r2) |
0.9997 |
Accuracy and Precision:
The accuracy and precision of the method were evaluated by repeated analysis of pantoprazole at three different concentrations (1, 10 and 50 mg/mL) on three separate days. The within-day and between-day accuracy and precision are presented in Table 4. The results showed that the assay method is reproducible in the studied concentration range. The intermediate precision determined by comparison of the assay results for pantoprazole formulation by two analysts and the CV values did not exceed 3%.
Table 4. Accuracy and precision of the method for determination of pantoprazole (n=9; three sets for 3 days)
|
Concentration added (mg/mL) |
Concentration founda (mg/mL) |
CV (%) |
Error (%) |
|
Within-day (n=3) 1.00 10.00 50.00 |
0.99 ± 0.02 10.17 ± 0.16 50.83 ± 0.37 |
2.02 1.57 0.73 |
-1.00 1.70 1.66 |
|
Between-day (n=6) 1.00 10.00 50.00 |
0.98 ± 0.03 10.19 ± 0.19 50.54± 0.64 |
3.06 1.86 1.27 |
-2.00 1.90 1.08 |
aData presented as mean ± SD
Robustness:
The influences of small changes in the mobile phase composition, buffer concentration and buffer pH were evaluated. Results are shown in Table 5. Peak area values were influenced less than ±2% in all different conditions used. The retention time of pantoprazole was decreased by increasing the pH value of the buffer and also by increasing the buffer strength. Also increasing the amount of organic solvent decreases the retention times. In spite of variations in retention time, no problem was observed for quantification.
Figure 2a
Figure 2b
Figure 2c
Figure 2d
Figure 2e
Figure 2f
Figure 2g
Figure 2. Typical chromatograms obtained from stability studies of pantoprazole. a pantoprazole standard solution (25 mg/mL); b solution in 0.01 M HCl after 30 min at room temperature; c solution in 1 M NaOH reflux for 4 h; d solution in water reflux for 1 h; e solution in 3% H2O2 after 2 h at room temperature; f pantoprazole powder after exposure to heat at 95° C for 24 h; g methanolic solution after exposure to UV light for 24 h.
Table 5. The influence of small changes in mobile phase composition (Method robustness)
|
Mobile phase composition |
Retention time |
Peak area |
|
Acetonitrile-KH2PO4 (pH=7.4) 10 mM (22:78) |
5.2 |
820.6 |
|
Acetonitrile-KH2PO4 (pH=7.4) 10 mM (28:72) |
3.5 |
826.7 |
|
Acetonitrile-KH2PO4 (pH=7.4) 9 mM (25:75) |
4.2 |
822.0 |
|
Acetonitrile-KH2PO4 (pH=7.4) 10 mM (25:75) |
4.5 |
825.7 |
|
Acetonitrile-KH2PO4 (pH=7.4) 11 mM (25:75) |
4.7 |
837.9 |
|
Acetonitrile-KH2PO4 (pH=7.1) 9 mM (25:75) |
4.3 |
827.3 |
|
Acetonitrile-KH2PO4 (pH=7.1) 10 mM (25:75) |
4.4 |
822.4 |
|
Acetonitrile-KH2PO4 (pH=7.1) 11 mM (25:75) |
4.5 |
838.9 |
|
Acetonitrile-KH2PO4 (pH=7.7) 9 mM (25:75) |
3.7 |
835.3 |
|
Acetonitrile-KH2PO4 (pH=7.7) 10 mM (25:75) |
4.2 |
825.1 |
|
Acetonitrile-KH2PO4 (pH=7.7) 11 mM (25:75) |
4.4 |
836.9 |
Relative Recovery:
The relative recovery of pantoprazole was evaluated by the addition of standard drug solution at three different concentrations to a tablet sample solution. The relative recovery ranged from 98.8 to 100.4% and no interferences were observed form tablet excipients.
Solution Stability:
Pantoprazole was found to be stable in methanol stored at 4°C for 7 days. The results showed that this sample compared with freshly prepared standard solution remained at 99.8% over a period 7 days. The pantoprazole solution in mobile phase was stable at least for 24 h at room temperature with a recovery of about 99%.
Degradation Studies:
The degradation behavior of pantoprazole under various conditions using proposed HPLC method was performed. Degradation samples were compared with freshly prepared standard solutions of pantoprazole and following results obtained.
Acidic Condition:
Pantoprazole totally degraded in 1 M HCl or 0.1 M HCl within about 10 min. In other experiments using 0.05 M HCl about 86% degradation was observed after 30 min at room temperature, and some degradation products were detected at retention times of 1.5, 3.3 and 8 min. Subsequent studies were performed in milder conditions using 0.01 M HCl at room temperature. Pantoprazole degraded in this condition about 35% and 92% after 10 min and 60 min respectively with same degradation products observed by using 0.05 M HCl. In all acidic degradations the solution turns to yellow (Figure 2b).
Alkaline Condition:
Pantoprazole was highly stable in the presence of 0.1 M NaOH for 5 days at room temperature. Upon 1 M NaOH, mild degradation (<5%) was observed after 5 days at room temperature. On the other hand about 18%, 39% and 69% degradation of pantoprazole was observed in the presence of 1 M NaOH after 1, 2 and 4 h reflux. Two other newly peaks were also observed in the chromatogram at retention times of 1.7 and 2.2 min (Figure 2c).
Neutral Condition:
No significant degradation (< 5%) was observed in neutral condition at room temperature after 5 days. On the other hand 26%, 47% and 78% degradation was observed upon refluxing pantoprazole in distilled water after 1, 2 or 4 h with appearance of two major peaks at retention times of 1.2 and 3.9 min (Figure 2d). The solution turns to a yellow brown color after about 1 h which becomes deeper in color with time.
Oxidative Degradation:
Pantoprazole was degraded about 53% after 2 h and 67% after 3 h exposure to 3% hydrogen peroxide at room temperature. Two major peaks were detected in the chromatogram at retention times of 1.4 and 2.1 min. The peak area of the former peak was not significantly changed with time but the peak area of the latter one was increased by time (Figure 2e).
Heat Stability:
The dry powder of pantoprazole was degraded about 10% upon exposure to dry heat at 70°C after 24 h and about 54% at 95°C after 24 h (Figure 2f).
Photostability:
The stability of pantoprazole in solid form was investigated in visible light. Negligible degradation (<1%) was observed upon exposure to visible light after 5 days. It seems that pantoprazole in solid form is relatively stable under light exposure. Upon exposure to UV light about 10% degradation was observed after 3 days.
Using pantoprazole solution more significant degradation was observed. Upon exposure of pantoprazole solution to UV light 36% degradation was observed after 24 h with a new major peak at retention time of 3.9 (Figure 2g). The degradation amount was about 64% after 60 h and the peak area of the degradation product was increased. It has to be mentioned that the temperature of the UV light chamber was reached to 50° C.
Pantoprazle solution was also relatively stable in visible light. About 6% degradation was observed after 5 days exposure to visible light.
Analysis of Pharmaceutical Product:
The proposed method was applied for determination of Protiumâ tablets. Satisfactory results were obtained (41.21 ± 1.17 mg per tablet) which was in good agreement with the label claims.
Figure 3. Dissolution profile of 40 mg Pantoprazole and Protiumâ tablets (n=6), using phosphate buffer 50 mM pH 6.8 as dissolution medium and paddle speed at 75 rpm.
Dissolution Test of Pantoprazole Tablets:
The dissolution profiles of Pantoprazole (SAMISAZ) and Protiumâ tablets using 1000 mL of phosphate buffer pH 6.8 as medium are shown in Figure 3. The stirring speed of 75 rpm leads to satisfactory results. The drug release profile obtained was satisfactory as more than 95% of the drug was released after 30 min.
CONCLUSION:
In this study a typical Stability-indicating HPLC method was developed under the recommendations of ICH guidelines. The proposed method is simple, accurate and reproducible for determination of pantoprazole in the presence of its degradation products in different conditions. The findings of this study showed that the drug is unstable in almost all conditions especially in acidic conditions. The proposed method was successfully used for determination of pantoprazole in pharmaceutical tablets and dissolution medium without any interference from the excipients.
ACKNOWLEDGEMENT:
The authors would like to thank the Iran National Science Foundation for the financial support of this project.
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Received on 10.06.2010 Modified on 02.08.2010
Accepted on 25.08.2010 © AJRC All right reserved
Asian J. Research Chem. 3(4): Oct. - Dec. 2010; Page 879-884