Research Article | Volume: 9, Issue: 12, December, 2019

Development of stability indicating method for the simultaneous estimation of alogliptin and pioglitazone in bulk and tablet dosage form by reversed-phase ultra-performance liquid chromatography method

Ramesh Dhani Harish Kumar Donthi Ramachandra Guptha Deveswaran Rajamanickam   

Open Access   

Published:  Dec 03, 2019

DOI: 10.7324/JAPS.2019.91208
Abstract

The objective of the work is to develop and validate a new reverse phased ultra-performance chromatography method and its stability studies for the simultaneous estimation of alogliptin and pioglitazone in bulk and tablet dosage form. The column of the method was BEH C18 (2.1× 50 mm, 1.7 μ) used as a stationary phase and the mobile phase was 45:55 v/v of phosphate buffer (pH 3) and methanol, respectively. The injection volume was 2 μl and flow rate was maintained at 0.3 ml/minute. The wavelength was 280 nm and the runtime was 3 minutes. The retention time of alogliptin was 0.4 minutes and pioglitazone was 0.529 minutes. The Linearity of the alogliptin was 6.25–37.5 μg/ml and pioglitazone was 15–90 μg/ml. The newly developed method could be used for the routine analysis of pure drug and its formulations in accordance with the ICH Q2 (R1) guidelines.


Keyword:     Alogliptin pioglitazone BEH C18 RP-UPLC phosphate buffer.


Citation:

Dhani R, Guptha HKDR, Rajamanickam D. Development of stability indicating method for the simultaneous estimation of alogliptin and pioglitazone in bulk and tablet dosage form by reversed-phase ultra-performance liquid chromatography method. J Appl Pharm Sci, 2019; 9(12):051–056.

Copyright: © The Author(s). This is an open-access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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INTRODUCTION

Alogliptin (Fig. 1) decreases the incretin glucose dependent insulinotropic polypeptide (GIP) and glucagon like peptide (GLP-2) (Cabrera et al., 2013) increases the plasma incretin concentration, which can controls the glucose levels in the blood (Marino and Cole, 2015). GIP and GLP-2 are stimulating the glucose dependent pancreatic beta cells. It inhibits the activity of GIP and GLP-2 (Cyrus and Vijay, 2010).

Pioglitazone (Fig. 2) is a thiazolidine class of anti-diabetic drug (Brunetti et al., 2010). It is an agonist of peroxisome proliferator activated receptor gamma and it activates the insulin responsive genes then increase the production insulin (Al-Majed et al., 2016; Lincoff et al., 2007).

On extensive survey of literature, very few reverse phased high-performance chromatography (RP-HPLC) methods have been reported for the estimation of alogliptin and pioglitazone combined dosage form (Vasanthi et al., 2017). The present developed reversed-phase ultra-performance liquid chromatography (RP-UPLC) method was accurate, precise, and robust for the simultaneous estimation of alogliptin and pioglitazone in Active Pharmaceutical Ingredient and tablet dosage form. The developed RP-UPLC method showed better resolution and low retention time, very good separation efficiency, and faster elution and tiny amount of sample consumed when compared to the (Vasanthi et al., 2017) reported RP-HPLC methods.


MATERIALS AND METHODS

Instruments used

The liquid chromatographic system was made up of Waters-Acquity, Japan, UPLC equipped with auto sampler and 2996 PDA detector with Empower 2 software. Chromatographic separation was performed on waters BEH C18 (2.1 × 50 mm, 1.7 μ) column.

Figure 1. Structure of alogliptin.

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Figure 2. Structure of pioglitazone.

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Chemicals used

Gift samples of alogliptin and pioglitazone are procured from Pharma Train, Hyderabad, India. HPLC grade water and methanol are purchased from Merck Laboratories, Mumbai, India. Potassium dihydrogen phosphate (K2H2PO4) was obtained from Finar Chemicals Pvt. Limited, Ahmedabad, Gujarat, India.

Chromatographic conditions

Column: Waters BEH C18 (2.1 mm*50 mm, 1.7 μ)

Mobile phase ratio: Phosphate buffer (pH 3):Methanol (45:55)

Wavelength: 280 nm

Flow rate: 0.3 ml/minute

Injection volume: 2 μl

Run time: 3 minute

Preparation of mobile phase

450 ml (45%) of phosphate Buffer and 550 ml (55%) of Methanol was taken and ultra sonicated for 10–15 minutes for degassing, further filtered. pH was adjusted to 3 with orthophosphoric acid (Raval and Srinivasa, 2014).

Preparation of standard

12.5 mg of alogliptin and 30 mg of pioglitazone was taken in 100-ml volumetric flask then 70 ml of diluent was added and dissolved completely, the volume was made up to the mark (Stock solution). 1.5 ml of stock solution was pipette out into 10ml volumetric flask and the volume was made up to the mark with diluent (Neelima et al., 2014).

Validation of analytical method

The developed RP-UPLC method was validated for System suitability, Specificity, Linearity, Accuracy, Precision, Robustness, and stability studies. The validation in accordance with ICH guidelines Q2(R1).

System suitability

It was performed before each validation to check the retention time, theoretical plates, tailing factor, and resolution determined to five suitability injections (Manzoor et al., 2015).

Specificity

It was carried out to determine the analyte in the presence of other compounds, such as impurities, degradants, and matrix. In specificity study standard injection was compared to the running blank injection.

Linearity

From the standard stock solution, appropriate aliquots of alogliptin and pioglitazone were taken in different volumetric flask and make up the volume up to the mark with diluent to obtain different concentrations are 6.25, 12.5, 18.75, 25, 31.25, and 37.5 μg/ml for alogliptin and 15, 30, 45, 60, 75, and 90 μg/ml for pioglitazone, respectively (Haribabu et al., 2017). The solutions are injected into 2 μl fixed loop system and then chromatograms were recorded. The calibration curve was plotted by concentration Vs Peak area.

Precision

Both intraday and inter day was carried out for six injections in the day and between the days.

Accuracy

Recovery was obtained by adding known quantities of pure standard in three different concentrations 50%, 100%, and 150% to the pre-analyzed sample formulation. The amount of drug found, amount of drug recovered, and percentage recovery were calculated for the confirmation of the method accuracy (Komal and Amrita, 2015).

Robustness

It is an analytical procedure to measure the capacity to remain unaffected by small variations in the method parameter. Some typical variations impacts on method were flow rate, temperature, pH of mobile phase, and mobile phase composition.

As part of the Robustness, deliberate change in the Flow rate, Mobile Phase composition, Temperature Variation were made to evaluate the impact on the method. From the Standard solution, 18.75 ppm of alogliptin and 45 ppm of pioglitazone was prepared and analyzed using the varied flow rate and wave length change along with actual conditions.

Forced degradation studies

1.5 ml from the stock solution was taken in 5 different 10ml volumetric flasks. The required aliquots are prepared and the solutions are exposed to the stress conditions, such as acidic, alkaline, peroxide, thermal, and photolytic conditions (Mokhtar et al., 2016). Then, finally the amount of drug degraded in stress conditions was calculated. The data were shown in Table 10.


RESULTS AND DISCUSSION

System suitability

The retention time of alogliptin was 0.403 minutes and pioglitazone was 0.529 minutes. Three system suitability injections were injected into the chromatographic column. Then, the tailing factor, theoretical plates and resolution was calculated. The results are shown in Table 1 and the values were found to be within the limit.

Specificity

Comparing the results of standard solution along with running blank solution, there was no interference in blank.

Linearity

For the determining the linearity, plot the graph peak versus concentration and calculate the correlation coefficient. The correlation coefficient and regression equation of alogliptin (Fig. 4) was found to be Y = 797.9x – 228.7 (r2 = 0.9997) and pioglitazone (Fig. 5) was found to be Y = 2157.4x − 40.1 (r2 = 0.9998). The results are shown in Table 2 and Figure 3.

Accuracy

The mean percentage recovery of alogliptin was 100.34% and pioglitazone was 100.30%. It was present within the limit, hence the method was accurate and it is shown in Tables 3 and 4.

Precision

It was carried out in intraday and intermediate day for the % Relative Standard Deviation (RSD) calculation. The results were found to be under accepted criteria, i.e., 2%. It is shown in Tables 5 and 6.

Table 1. System suitability parameters for alogliptin and pioglitazone.



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Table 2. Linearity results.



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LOD and LOQ

The Limit of Detection (LOD) of alogaliptin and pioglitazone found to be 2.91 and 2.96 μg/ml. The Limit of Quantification (LOQ) of alogliptin was 10.4 and 10.09 μg/ml for pioglitazone, respectively. The results are shown in Table 7.

Robustness

The variation of flow rate and wavelength was found to be (+10%), which was affected by the method. The standard solutions were injected under the selected robust conditions. The system suitability parameters, such as theoretical plates, tailing factor and resolution was observed and measured. Results shows >2000 theoretical plate count, < 2 of tailing factor and resolution was found to be > 2 (See Tables 8 and 9). Hence the method was robust and the results were shown in Figures 6 to 9.

Forced degradation studies

The working standards of alogliptin and pioglitazone were placed in different stress conditions, such as acid, base, thermal, peroxide, and photolytic conditions, and then observe the amount of drug degraded in above stress conditions.The results of degradation study of standard solutions were stable in all the selected stress conditions and there is no observe the much deviation. The results obtained were compared with existing methods and found to be stable in all stress conditions, but existing methods were stable in thermal and photolytic conditions only. The data are shown in Table 10.

Figure 3. Optimized chromatogram for alogliptin and pioglitazone.

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Figure 4. Linearity graph of alogliptin.

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Table 3. The accuracy results for alogliptin.



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Table 4. The accuracy results for pioglitazone.



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Table 5. Results for intraday precision.



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Table 6. Results for intermediate precision.



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Table 7. Results for LOD and LOQ.



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DISCUSSION

Present developed RP-UPLC method showed better results when compared to the reported RP-HPLC method by (Vasanthi et al., 2017). In the reported method, where C18 column (250 × 4.6 mm, 5 μm) was used, then slow elution of solutes at 2.234 minutes (alogliptin) and 3.294 minutes (pioglitazone) was observed, in newly developed RP-UPLC method the BEH C18 column (2.1 × 50 mm, 1.7 μ) was employed, the solutes are eluted rapidly and showed retention time at 0.403 minutes (alogliptin) and 0.529 minutes (pioglitazone), retention time difference between the two methods was noted of 2 minutes. In RP-UPLC method, mobile was prepared by ecofriendly chemicals such as 45 % of phosphate buffer (3.0 pH) and 55% of methanol, but in the reported RP-HPLC method, the mobile phase was buffer (pH 4.0) and acetonitrile with the ratio of 20:80%, it contains more organic phase then compared to the present method. When compared the stability indicating studies between both methods, in RP-HPLC method, when solution was placed in acid, base, thermal, oxidative, and photolytic stress conditions, it was stable in thermal and photolytic conditions. In acidic and basic conditions more amount of drug degraded. In the newly developed RP-UPLC method, there was not much amount of drug was degraded under selected stress conditions, such as acid, base, thermal, and photolytic. It was most stable in acidic conditions. In RP-HPLC method, high amount of sample was consumed because 20 μl of injection volume and flow rate was maintained at 1.0 ml/minute. But in the current RP-UPLC method, little amount of sample was consumed because 2 μl of injection volume and flow rate was maintained at 0.3 ml/minute. The advantages of RP-UPLC method was rapid analysis, faster elution when compared to the reported RP-HPLC methods. The data are shown in Table 11.

Table 8. Robustness results for change in flow rate.



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Table 9. Robustness results for change in wavelength.



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Table 10. Degradation results for alogliptin and pioglitazone.



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Table 11. Stastical comparison of previous method and present method.



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Figure 5. Linearity graph of pioglitazone.

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Figure 6. Less flow chromatogram of alogliptin and pioglitazone.

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Figure 7. More flow chromatogram of alogliptin and pioglitazone.

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Figure 8. Less wave chromatogram of alogliptin and pioglitazone.

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Figure 9. More wave chromatogram of alogliptin and pioglitazone.

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CONCLUSION

In the present study, a new RP-UPLS method was developed for the simultaneous estimation of alogliptin and pioglitazone. The new method was validated according to the ICH guidelines. The method was consumed less solvents with high resolution and short run time was observed. When compared to reported RP-HPLC methods, the current RP-UPLC method was found to precise, accurate, and robust, and it can be used for the routine analysis of pharmaceutical formulations.


ACKNOWLEDGMENT

The authors thank M.S. Ramaiah University of Applied Sciences, MSR Nagar, Bangalore for their support.


FINANCIAL SUPPORT AND SPONSORSHIP

None.


CONFLICT OF INTEREST

All the authors declared that they have no conflict of interest.


REFERENCES

Al-Majed A, Bakheit AH, Abdel Aziz HA, Alharbi H, Al-Jenoobi FI, Pioglitazone. Profiles Drug Subst Excip Relat Methodol, 2016; 41:379–438. CrossRef

Brunetti L, Kalabalık J. Management of type-2 diabetes mellitus in adults. Focus on individualizing non-insulin therapies. P & T, 2012; 37:687–96.

Cabrera A, Jarvis CI, Charron D. Alogliptin: a new dipeptidyl peptidase-4 inhibitor for type 2 diabetes mellitus. Ann Pharmacother, 2013; 47:1532–9. CrossRef

Cyrus VD, Vijay S, Pioglitazone in the treatment of type 2 diabetes: safety and efficacy review. Clin Med Insights Endocrinol Diabetes, 2010; 3:43–51. CrossRef

Haribabu B, Rama Krishna Veni P, BalaMurali Krishna K, Lakshmi Prameela K. Determination of alogliptin benzoate and pioglitazone simultaneously in tablet dosage forms by RP-HPLC. Marmara Pharm J, 2017; 21:345–54. CrossRef

ICH Q2(R1). International Conference on Harmonization, validation of analytical procedure, text and methodology. IFMA, Geneva, Switzerland, 2005.

Komal S, Amrita P, Development and validation of HPTLC method for simultaneous estimation of alogliptin benzoate and pioglitazone hydrochloride in bulk drugs and combined dosage forms, Int J Pharma Res Rev, 2015; 4:35–42.

Lincoff AM, Wolski K, Nicholls SJ, Nissen SE. Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus: a meta-analysis of randomized trials. JAMA, 2007; 298:1180–8. CrossRef

Marino AB, Cole SW. Alogliptin: safety, efficacy and clinical implications, J Pharm Pract, 2015; 28:99–106. CrossRef

Manzoor A, Anusha M, Satishkumar SA, Kuppast IJ, Siddalingaswamy MS, Ravi MC, RP-HPLC method development and validation for simultaneous estimation of alogliptin and pioglitazone in combined tablet dosage form, World J Pharm Pharm Sci. 2015; 4:863–74.

Mokhtar MM, Sherin FH, Fotouh RM, Mona MA. Development and validation of a reversed phase HPLC method for simultaneous determination of antidiabetic drugs alogliptin benzoate and pioglitazone HCl. Der Pharmacia Sinica, 2016; 7:32–40.

Neelima B, Kumar PR, Bindu VH, Prasad YR. A validated stability indicating RP-HPLC method for simultaneous determination of alogliptine and pioglitazone in bulk and pharmaceutical formulations. Int J Pharm, 2014; 4:458–64.

Raval K, Srinivasa U. Development and validation of HPLC method for the simultaneous estimation of pioglitazone and alogliptin in bulk and dosage form. Int J Curr Res, 2014; 6:10201–7.

Vasanthi R, Noori K, Shyam Sundar P, Alagar Raja M, Dutt R. Development of rapid stability indicating method for simultaneous estimation of alogliptin and pioglitazone in bulk and combined dosage form by RP-HPLC method, Indo Am J Pharm, 2017; 3(5):234–44.

Reference

Al-Majed A, Bakheit AH, Abdel Aziz HA, Alharbi H, Al-Jenoobi FI, Pioglitazone. Profiles Drug Subst Excip Relat Methodol, 2016; 41:379-438.https://doi.org/10.1016/bs.podrm.2015.11.002

Brunetti L, Kalabalık J. Management of type-2 diabetes mellitus in adults. Focus on individualizing non-insulin therapies. P & T, 2012; 37:687-96.

Cabrera A, Jarvis CI, Charron D. Alogliptin: a new dipeptidyl peptidase-4 inhibitor for type 2 diabetes mellitus. Ann Pharmacother, 2013; 47:1532-9.https://doi.org/10.1177/1060028013504076

Cyrus VD, Vijay S, Pioglitazone in the treatment of type 2 diabetes: safety and efficacy review. Clin Med Insights Endocrinol Diabetes, 2010; 3:43-51.https://doi.org/10.4137/CMED.S5372

Haribabu B, Rama Krishna Veni P, BalaMurali Krishna K, Lakshmi Prameela K. Determination of alogliptin benzoate and pioglitazone simultaneously in tablet dosage forms by RP-HPLC. Marmara Pharm J, 2017; 21:345-54.https://doi.org/10.12991/marupj.300864

ICH Q2(R1). International Conference on Harmonization, validation of analytical procedure, text and methodology. IFMA, Geneva, Switzerland, 2005.

Komal S, Amrita P, Development and validation of HPTLC method for simultaneous estimation of alogliptin benzoate and pioglitazone hydrochloride in bulk drugs and combined dosage forms, Int J Pharma Res Rev, 2015; 4:35-42.

Lincoff AM, Wolski K, Nicholls SJ, Nissen SE. Pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus: a meta-analysis of randomized trials. JAMA, 2007; 298:1180-8.https://doi.org/10.1001/jama.298.10.1180

Marino AB, Cole SW. Alogliptin: safety, efficacy and clinical implications, J Pharm Pract, 2015; 28:99-106.https://doi.org/10.1177/0897190014522063

Manzoor A, Anusha M, Satishkumar SA, Kuppast IJ, Siddalingaswamy MS, Ravi MC, RP-HPLC method development and validation for simultaneous estimation of alogliptin and pioglitazone in combined tablet dosage form, World J Pharm Pharm Sci. 2015; 4:863-74.

Mokhtar MM, Sherin FH, Fotouh RM, Mona MA. Development and validation of a reversed phase HPLC method for simultaneous determination of antidiabetic drugs alogliptin benzoate and pioglitazone HCl. Der Pharmacia Sinica, 2016; 7:32-40.

Neelima B, Kumar PR, Bindu VH, Prasad YR. A validated stability indicating RP-HPLC method for simultaneous determination of alogliptine and pioglitazone in bulk and pharmaceutical formulations. Int J Pharm, 2014; 4:458-64.

Raval K, Srinivasa U. Development and validation of HPLC method for the simultaneous estimation of pioglitazone and alogliptin in bulk and dosage form. Int J Curr Res, 2014; 6:10201-7.

Vasanthi R, Noori K, Shyam Sundar P, Alagar Raja M, Dutt R. Development of rapid stability indicating method for simultaneous estimation of alogliptin and pioglitazone in bulk and combined dosage form by RP-HPLC method, Indo Am J Pharm, 2017; 3(5):234-44.

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