RP-HPLC Method Development and Validation for Pioglitazone in Bulk and Marketed Formulation

Received: 19-May-2021 Accepted Date: Jun 16, 2021 ; Published: 25-Jun-2021

Introduction

Pioglitazone an insulin sensitizer,S1] chemically a (RS)-5-(4-[2-(5-ethylpyridin-2-yl)ethoxy] benzyl)thiazolidine-2,4-dione an type 2 oral antdiabetic drug, sold under brand name Actoplus Met, Actos, Duetact, Glidipion, etc. Type 2 diabetes where the patients lack the capability of producing enough insuin in the body [1,2]. Pioglitazone activates a ligand-activated transcription factor PPAR-gamma, inducing cell differentiation and inhibiting cell growth and angiogenesis [3]. Pioglitazone inhibits macrophage and monocyte activation, adapts the transcription of insulin responsive genes, and stimulates adipocyte differentiation [4,5].

Pioglitazone enhances insulin sensitivity by making cells more responsive to it. In patients with type 2 diabetes, pioglitazone improves glycemic control mostly through enhancing peripheral insulin sensitivity, whereas metformin lowers hepatic glucose output. In hypoglycemic situations, pioglitazone masks symptoms such as increased heart rate, dizziness, and perspiration. It also has side effects such as edoema (when used with a sulfonylurea or insulin), heat failure, and respiratory infection. Due to the risk of urinary bladder cancer, pioglitazone was banned as an anti-diabetic medicine on July 18, 2013, however the prohibition was reversed on July 31, 2013 [6,7]. Literature survey reveals that, analytical and bio-analytical methods have been developed and validated for the estimation of Pioglitazone in bulk, pharmaceutical formulation and biofluids, which include techniques like HPLC, Spectrophotometry, and Polarography. [8-24] The current study has been undertaken to develop RP-HPLC method for the determination of Pioglitazone in bulk and pharmaceutical formulation.

Figure 1: Pioglitazone

Exprimental

Materials and Method

Pioglitazone was obtained as a gift sample from Dr. Reddy's Laboratories.Pioglitazone tablets were procured from the local market. HPLC grade Acetonitrile, AR grade Diammonium hydrogen orthophosphate, Potassium dihydrogen orthophosphate, orthophosphoric acid and triethylamine were used for the analysis, distilled water was utilized from Merkmilipore.

Instrument and Column

The Lachrom 2200 system equipped with auto sampler, and Lachrom elite control as the operating software. The chromatographic separation was carried out on C18 column, 300x3.9mm, 5μm (Bondapack column).

Preparation of Mobile Phase

Mixture of buffer (1.15 gm of diammonium hydrogen orthophosphate, 1.36 gm of potassium dihydrogen orthophosphate, 1ml triethylamine)and acetonitrile in the ratio of (50:50v/v)was filter, degased and used for analysis.

Preparation of Buffer

1.15gm of diammonium hydrogen orthophosphate, 1.36 gm of potassium dihydrogen orthophosphate and 1ml triethylamine was dissolved in 1000 ml water,pH was adjusted to 5.0 with orthophosphoric acid.

Preparation of Standard stock solution

30mg of Pioglitazone Hydrochloride was placed in 100ml volumetric flask and 10ml of dimethyl formamide was transferred to the volumetric flask and sonicated for 15 min. and the solution was made up to the mark using mobile phase. Then 2ml of the above solution was transferred to 25ml volumetric flask and the volume was made upto the mark using mobile phase.

Preparation of Sample Solution

Weighed and powder 20 tablets. Transferred equivalent weight 30mg of pioglitazone to a 100 ml volumetric flask. Add about 10 ml dimethyl formamide and sonicate for 5 min. Add 70 ml of mobile phase and sonicate for 10 min. Cool and dilute up to the mark with mobile phase . Filtered through whatman filter paper no.1. Transfer 2 ml of filtrate to 25 ml volumetric flask and dilute with mobile phase.

Chromatographic condition

The optimized chromatographic conditions and the optimized chromatogram for the newly developed method have been represented in Table 1 and Figure 2 respectively.

Figure 2:Optimized Chromatogram of Pioglitazone.

Method Validation

The method was validated as per ICH guidelines Q2(R1), in terms of System suitability, Linearity, Precision, Accuracy, and Robustness [25-30].

System Suitability Tests for Validation

System suitability tests is performed to ensure system performance before and duringthe analysis which demonstrates that the system is operating properly and ready todeliver results with acceptable accuracy and precision. Five replicate injections of a single standard solution were made on to a RP-HPLC system and the area of the pioglitazone peak was determined. USP Tailing for pioglitazone was recorded. The relative standard deviation of the peak area was calculated. The other parameters considered for system suitability were USP plate count for pioglitazone. The limit set and the values are reported given in Table 2.

Linearity

The linearity of analytical procedure is its ability (within a given range) to obtain test results, which are directly proportional to the concentration of analyte in the sample [28]. 7 different solutions of pioglitazone ranging from 11.82 ppm to 35.46 ppm were prepared and analysed. Concentration was plotted on X-axis and area on Y-axis. Correlation coefficient and the equation of line were calculated. The data obtained and the graph of Linearity has been represented in Table 3 and Figure 3 respectively.

Range

Five replicates of each linearity level, 50% level (lower level) and 150% (upperlevel) was injected and %RSD for retention time and area were determined. The data is summarized in Table 4.

Precision

System Precision

Repeatability of pioglitazone standard in assay method.5 replicates of standard preparation were injected as per method parameters and the %RSD for the peak area and retention time was determined. The data has been summarized in Table 5.

Method Precision

The assay percentage for each test preparation and the mean assay of six test preparation and %RSD for the same was calculated and the data is summarized in Table 6.

Intermediate Precision

The intermediate precision was performed on different days, equipment and analyst. The data for interday precision has been given in Table 7, the data for intraday day has be en given in Tables 8 & 9 exhibits the results of one way ANOVA.

Equipment used: HPLC

Make: Shimadzu

Model: Class VP

Column: C18, 300*3.9 mm, 5 μm (Bondapack is suitable)

Reproducibility

Blank preparation: Mobile phase is used as blank preparation.

Placebo preparation

170.4 mg of placebo is weighed and transferred to a 100 ml volumetric flask. 10 ml of dimethyl formamide was added and sonicated for about 5 minutes. 70ml of mobile phase was added and sonicated for 10 min. Cooled and diluted upto the mark with mobile phase followed by transfer of 2.0 ml of filtrate to a 25 ml volumetric flask, diluted to volume with mobile phase.

Sample preparation

202.5 mg of sample (equivalent to 30 mg of pioglitazone) is weighed and transferred to a 100 ml volumetric flask. 10 ml of dimethyl formamide was added and sonicated for about 5 minutes. 70ml of mobile phase was added and sonicated for 10 min. Cooled and diluted upto the mark with mobile phase followed by transfer of 2.0 ml of filtrate to a 25 ml volumetric flask, diluted to volume with mobile phase. In continuation with the above experiment prepare and analyze six different independent samples as per method. The assay percentage for each test preparation and The mean assay of six test preparation and %RSD for the same was calculated. The data obtained is summarized in Table 10.

Accuracy

Known amount of the active ingredient at 3 levels each in triplicate, i.e. 3 x 80%, 3 x100% and 3 x 120% of the working concentration was spiked with placebo at 100 %level of 100 mg tablet samples were prepared in triplicate. Each sample was analyzedand calculated. The data for individual compound is summarized in Table 11.

Robustness

To demonstrate the robustness of the test method checked the method suitability by injecting test solution into RP-HPLC with slight variations in method parameters[31]. Standard solution and test solution were prepared as per method of analysis. Once blank and five replicate of standard injection and sample solution in duplicate were injected. The mean % assay for sample solution with slight variation in method parameter was calculated. Changes in chromatographic conditions were as follows:

1. Change in flow ± 10%

2. Change in organic phase ± 10%

3. Change in pH ± 0.2

The values for Robustness has been represented in Tables 12-17, and the chromatogram of blank solution has been represented in Figure 3.

Results and Discussions

To develop RP-HPLC method, several mobile phase and mobile phase compositions were tried. A satisfactorily separate and good peak symmetry was obtained with Bondapack 300*3.9mm 5μm C18 column using mobile phase Acetonitrile:Buffer (50:50%v/v) and flow rate 1.0 ml per min. The detection was carried out 267 nm and the retention time was found to be 8.08 min.

System Suitability for validation

Five replicate injections of a single standard solution were made on to a RP-HPLC system and the area of the pioglitazone peak was determined. USP Tailing for pioglitazone was recorded. The relative standard deviation of the peak area was calculated. The other parameters considered for system suitability were USP plate count for pioglitazone. The limit set and the values obtained are in below in Table 2.

Linearity and Range

Under optimised condition the different concentration vs area was plotted in the range from 11.82 ppm to 35.46 ppm. The graph was found to be linear for concentration range and has been given in Figure 3. The data has been given in Tables 3 and 4.

Figure 3: Calibration Curve (linearity of PiolitazoneHCl at 267nm)

For precision studies; system precision as one of the parameter the sample was injected in 5 replicates and area, standard deviation and % RSD was calculated, it was expected and found to be less than 2% [32]. The data for system precision has been summarized in Table5, method precision in Table 6, interday and intra day precision in Table 7 and 8, and reproducibility in Table 10.

Accuracy: For accuracy of the method, pioglitazone HCl was analyzed at three different levels in triplicates and the %RSD was calculated. The %RSD was found to be less than 2%.

Robustness

Under the selected experimental conditions, the standard and sample preparation run was carried out and % assay values were calculated. The data obtained for change in flow rate by +10% and -10% was summarised in Table 12 and Table 13 respectively. The data obtained for change organic phase by +10% and -10% has been summarised in table 14 and 15 respectively. And the data for change in pH +0.2 and -0.2 is summarized Table 16 and Table 17 respectively.

Conclusion

The validated RP-HPLC assay method for pioglitazone can be used for determination of its purity.The method has been shown to be specific, linear, precise and accurate across a suitable analytical range for pioglatazone. Solutions have been shown to be stable for at least 24 hours on ambient storagecondition.This method was found to be better in consideration of other reported methods for individual drugs, because of economical readily available mobile phase, UV detection and better resolution of peak.This method will be advantageous for rapid quantification of sample in routine and quality control analysis for bulk and pharmaceutical dosage form containing pioglitazone.

References

1. Nora Brettenthaler, Christian De Geyter, Peter R. Huber et al., J Clin Endocrinol Metab. 2004, 89(8): p. 3835–3840.
2. LoaiAljerf and Iyad Alhaffar. Biochemistry Research International. 2017, p. 1–12.
3. Jennifer L. Hatton, Lisa D. PPAR Research 2008.
4. https://pubchem.ncbi.nlm.nih.gov/compound/Pioglitazone-hydrochloride.2021.
5. https://go.drugbank.com/drugs/DB01132. 2021.
6. QuinnCE, HamiltonPK, Lockhart CJ et al., BJP. 2008, 153(4): p. 636–645.
7. SheetalVasundarMathai, Prabha M. Adhikari et al., J Diabetology. 2019, 10(2): p. 87-88.
8. Radhakrishna T, SreenivasRao D, Reddy GO. J Pharm Analy Biomed Analysis. 2002, 29: p. 598-607.
9. Sayed S, Thomas A, Kotapali L. J Pharm Research. 2009, 2(9): p. 1479-1480.
10. Lakshmi KS, Rajesh T, Sharma S. Inter J Pharm Tech Research. 2009, 1(3): p. 496-499.
11. Srinivasulu D, Sastry BS, Omprakash G. Inte J Chem Research. 2010, (1): p. 18-20.
12. Adukondalu D, Malathy PS, Venkateshwarrao J et al., Iner J Pharm Biological Sci. 2011, 1(4): p. 474-477.
13. Ravikanth CH, Anil Kumar A, UdayKiran V et al., Inter J Pharm Sci Drug Research. 2011, 3(1): p. 38-41.
14. Reddy UM , Reddy VP , Penumajji S et al., J Pharm Research. 2011, 4(4): p. 1209-1212.
15. Madhukar A,.Naresh K, Naveen Kumar CH et al., Der Pharmacia Lettre. 2011, 3(3): p. 128-132.
16. Ramakrishna Kommana, Rebecca Shiffali Devarapalli. Der Pharmacia Lettre. 2011, 5(1): p. 269-278.
17. Satheesh kumarN, Shanti kumarS, Srinivas R. J Pharmaceutical Analysis. 2014, 4(5): p. 295–302.
18. Kanakapura Basavaiah, Nagaraju Rajendraprasad. J Pharm Appl Chem. 2017, 3(2): p. 151-160.
19. Balaji N, Sayeeda Sultana. Int J App Pharm. 2017, 9(2): p. 34-41.
20. Sai Chandana R, Bhavya Sri K, Sumakanth M et al., IJSTR. 2019, 8(10): p. 3567-3578.
21. Pramila T, Alka Agarwal, Maya Sharma. WJPR. 2020, 9(8): p. 1966-1977.
22. Narsimha Rao, Doredla, et al. IntJ PharmTech Res.2012, 4(4): p. 1750-1757.
23. Bhavyasri K, Sai Chandana R, Sumakanth M.  PharmTech Res. 2019, 9(04): p. 110-117.
24. HasnaMandil, Amir Alhaj Sakur, Safa Alulu. Int J Pharm Pharm Sci. 2013, 5(4): 86-93.
25. ICH Harmonized Tripartite Guideline Validation of Analytical Procedures: Text and Methodology, Q2 (R1), 2005. p. 1-28.
26. Guideline for Submitting Samples and Analytical Data for Methods validation U.S Department of Health and Human Services, Food and Drug Administration, 1987: p. 7-28.
27. A Guide to Validation in HPLC, R.A. van Iterson Drenthe College, Holland: p. 1-1.
28. Breaux J, Jones K. and Boulas P. pharm tech, analytical chemistry & testing, 2003. p. 6-15.
29. Zayas J, Sanchez V, Talley M. Pham Tech. 2005,  p.154-162.
30. Loai Aljerf and Ammar Mashlah. Microchemical J. 2017, 132: p. 411–421.
31. Bharti Mangal, Sarwar Beg, OzairAlam et al. Arabian J Chem. 2020. 13(11): p. 7909-7920.
32. BushraTuwfeeq Alquadeib. Saudi Pharmaceutical J. 2019,  27(1): p. 66-70.