IJPRS/V7/I2/00028 - 02/05/2018 Development and Validation of Analytical Methods for Simultaneous Estimation of Sitagliptin Phosphate and Pioglitazone Hydrochloride Author's Affiliation

(Vaishnavi, K. G., Mallinath, S. K.)1,5, Department of Quality Assurance, D.S.T.S. Mandal’s College of Pharmacy, Solapur, Maharashtra, India.   (Varsha, S. T.)2, Department of Pharmaceutical Chemistry, D.S.T.S. Mandal’s College of Pharmacy, Solapur, Maharashtra, India.   (Nirmal, M. T.)3, Research Scholar, Parul University, Parul Institute of Pharmacy, P.O Limbda, Waghodia, Vadodara, Gujarat, India.   (Somnath, A. P.)4, Department of Pharmaceutical Analysis, D.S.T.S. Mandal’s College of Pharmacy, Solapur, Maharashtra, India.

Abstract

Simple, rapid, accurate and precise UV spectrophotometric and RP-HPLC methods had been developed for simultaneous estimation of Sitagliptin Phosphate (STG) and Pioglitazone Hydrochloride (PIO). The method applied was Simultaneous Equation Method (Vierodt’s Method), based on measurement of absorbance of Sitagliptin Phosphate and Pioglitazone Hydrochloride at λmax 267nm and 270nm respectively. Linearity was found in the concentration range of 50-250µg/ml for Sitagliptin and 5-25µg/ml for Pioglitazone with regression coefficient r2=09997 and r2=0.9995 respectively. RP-HPLC method was carried on Phenomenex C-18 column (150 mm × 4.6 mm, 5 m) by using a mobile phase acetonitrile: methanol: water (30:30:40) as a mobile phase at 1.0 ml/min flow rate at 270 nm. The linearity was found to be in the range of 10-50 µg/ml and 3-15µg/ml with regression coefficient of r2= 0.9998, and r2=0.9996 for Sitagliptin Phosphate and Pioglitazone HCl respectively. The peak obtained were sharp having clear baseline separation with a retention time 5.6 and 2.8 min for Sitagliptin Phosphate and Pioglitazone HCl. This method is accurate and precise and can be employed for routine analysis of Sitagliptin Phosphate and Pioglitazone hydrochloride in different pharmaceutical dosage forms.

Keywords

Sitagliptin Phosphate, Pioglitazone HCl, UV- spectrophotometry, RP-HPLC

Cite This Article

Vaishnavi, K. G., Varsha, S. T., Nirmal, M. T., Somnath, A. P., Mallinath, S. K. (2018). Development and Validation of Analytical Methods for Simultaneous Estimation of Sitagliptin Phosphate and Pioglitazone Hydrochloride. International Journal for Pharmaceutical Research Scholars (IJPRS), 7(2), 12-22.

INTRODUCTION

1Sitagliptin Phosphate and Pioglitazone Hydrochloride are antidiabetic drugs. Sitagliptin belongs to the class of

Figure 1: Structure of STG
Figure 1: Structure of STG

Dipeptidyl peptidase-4[DPP-4] inhibitors, a new class of anti-diabetic drugs which act by increasing glucose dependent insulin release. Therapeutically DPP-4 inhibitors are used to treat type 2 diabetes alone or combination with other drugs which increases the sensitivity of insulin at target site DPP-4 inhibitors act by inhibiting the inactivation of enteroendocrine incretins such as glucogon-like peptide-1(GLP-1) and glucose-dependent insulinotropic (GIP) polypeptide. 2Pioglitazone is a drug belongs to the class of thiazolidinedione, which is used to decreases insulin resistance. It is an Antidiabetic agent to manage NIDDM [non-antihyperglycemic insulin-dependent diabetes mellitus, sugar diabetes) called type-2 diabetes. There are various methods for

Figure 2: Structure of PIO
Figure 2: Structure of PIO

analysis of the drugs like UV, HPLC or HPTLC. But there is no method reported for simultaneous estimation of STG and PIO in bulk and dosage form. So, objective of the present study was to develop and validate simple, precise and accurate UV spectrophotometric and RP-HPLC method for simultaneous estimation of STG and PIO.

 

MATERIAL & METHODS

Instruments

UV- VIS double beam spectrophotometer of Systronic 2201, Mumbai, with spectral bandwidth of 2nm and a pair of matched quartz cells of length 1cm were used for analytical work. Younglin Acme 9000 HPLC was used for identification and separation. All the weighing was carried out on the Electronic Balance AY220, Shimadzu, Japan. Sonication was carried out by Microclean-103.

Materials and Reagents

Pioglitazone Hydrochloride was purchased from Swapnroop Drugs and Pharmaceuticals, Aurangabad. Sitagliptin Phosphate was supplied as a Gift sample by Torrent Pharmaceutical Ltd, Ahmadabad, Gujarat. The reagents used were Hydrochloric acid and Distilled water for UV-spectrophotometric method and for RP-HPLC method, Acetonitrile LiChrosolv®, Methanol LiChrosolv® and water LiChrosolv® which were procured from Merck specialities Pvt. Ltd., Mumbai.

Methods

UV- Spectrophotometric Method

Preparation of 0.1N HCl

0.85ml of concentrated Hydrochloric acid was diluted with Distilled water to make the volume of 100ml to obtain 0.1N HCl.

Preparation of Standard Stock Solution

10 mg each of STG and PIO was weighed separately and transferred to two different 10 ml volumetric flasks. Both the drugs were dissolved in 5 ml of 0.1N HCl separately in volumetric flasks and volume made up to mark with 0.1N HCl to make the concentration of 1000µg/ml. From these solutions 1ml was pipetted out separately from both the volumetric flasks and transferred to two another 10ml volumetric flasks. Made up the volume up to the mark with the same solvent to obtain the final concentration of 100µg/ml for both the drugs.

Assay of Tablet

The fixed dose combination of these drugs is 100mg of STG with 30mg of PIO. Due to unavailability of the dosage form of this combination, standard STG drug 100mg is added to the tablets of PIO of 15mg (PIOZ 15) to simulate the condition of actual product. The required number of tablets were taken, weighed and their average weight was determined. The tablets were crushed to fine powder and from the triturate, tablet powder equivalent to 30mg of PIO was weighed and to this weighed powder, 100mg of standard STG drug was added to get 100mg:30mg combination. Now all this weighed powder was transferred to 100ml volumetric flask containing 50ml of 0.1N HCl, dissolved, made the volume up to 100ml to get the concentration of 300µg/ml and 1000µg/ml of PIO and STG respectively filtered through whatman filter paper. Now from this solution 1ml was diluted to 10 ml in other volumetric flask with same solvent to get the concentration of 30µg/ml and 100µg/ml. From above solution 5ml was diluted to 10 ml with the same solvent to get final concentration of 15µg/ml of PIO and 50µg/ml of STG.

Simultaneous Equation Method (Vierodt’s Method)

Figure 3: UV spectrum of STG
Figure 3: UV spectrum of STG

If sample contains two absorbing drugs (X and Y) each of which absorbs at the λmax of other. It may be possible to determine both the drugs by the technique of simultaneous equations if criteria below are met;

  1. The λmax of two components is reasonably dissimilar.
  2. The two components do not interact chemically.

iii. The two absorbing drugs (X and Y), each of which should absorb at the λmax of other.

Two equations are constructed based upon the fact that at λ1 and λ2 the absorbance of the mixture is the sum of the individual absorbance of X and Y.

Figure 4: UV spectrum of PIO
Figure 4: UV spectrum of PIO

Cx= A2ay1– A1ay2/ ax2ay1– ax1ay2 and

Cy= A1ax2– A2ax1/ ax2ay1– ax1ay2

Where, A1 and A2 are absorbances of diluted mixture at λ1 and λ2 respectively.

Cx and Cy are the concentrations of x and y respectively.

ax1 and ax2 are absorptivities of x at λ1 and λ2 respectively.

ay1 and ay2 are absorptivities of y at λ1 and λ2 respectively.

RP-HPLC Method

Standard Stock Solution of STG

10mg of standard STG was weighed and transfered to a 10ml volumetric flask then dissolved in the water and the volume was made up to the mark with water to obtain conc. of 1000µg/ml of STG and labelled as ‘Std Stock STG’.

Standard Stock Solution of PIO

10mg of standard PIO was weighed and transferred to a 10ml volumetric flask then dissolved in the methanol and the volume was made up to the mark with solvent to obtain conc. of 1000µg/ml of PIO and labelled as ‘Std Stock PIO’.

Combined Standard Stock Solution of STG and PIO

1ml of ‘Std Stock STG’ (1000µg/ml) and 0.3ml of ‘Std Stock PIO’ (300µg/ml) transferred to 10 ml volumetric flask and diluted to 10 ml with methanol to get ‘Std Stock MIX AP’ (100µg/ml STG and 30µg/ml PIO).

Selection of Analytical Wavelength

To investigate the appropriate wavelength for simultaneous determination of STG (10µg/ml) and PIO (10µg/ml) individual solutions in the mobile phase were scanned in the range of 200-400nm.

Selection of Mobile Phase and its Strength

The solutions of STG (10µg/ml) and PIO (10µg/ml) were prepared in water and methanol filtered through syringe filter, then injected into HPLC system. The chromatograms were analysed using different combination of Acetonitrile: Methanol: Water at a flow rate of 1ml/min for 10-30min at 270nm.

Mixed solution of STG (10µg/ml) and PIO (10µg/ml) was prepared in methanol and filtered through syringe filter, then injected into the HPLC system, after the column saturated with mobile phase and constant back pressure.

Selection of Column (Stationary Phase)

To get well resolved, symmetric peak with highest no. of theoretical plates the solution of the STG and PIO were analysed using C18 column as a stationary phase.

Chromatographic Conditions

  • Analytical Column: Phenomenex C18 column (150 mm ×6 mm, 5 mm)
  • Mobile Phase: Acetonitrile: Methanol: Water (30:30:40)
  • Flow Rate: 1ml/min
  • Injection Volume: 20 ml
  • Detection Wavelength: 270nm

Identification of Separated Peak of the Drugs

For identification of peak of the drugs; the

standard solutions of STG (10µg/ml) and PIO (10µg/ml) were injected separately into HPLC system and retention time were matched with retention time of mixture.

Method Validation

Method A

Figure 5: Calibration curve of STG of UV- Spectrophotometric method at λ1 (267nm) and λ2 (270nm)
Figure 5: Calibration curve of STG of UV- Spectrophotometric method at λ1 (267nm) and λ2 (270nm)
Figure 6: Calibration curve of PIO of UV- Spectrophotometric method at λ1 (267nm) and λ2 (270nm)
Figure 6: Calibration curve of PIO of UV- Spectrophotometric method at λ1 (267nm) and λ2 (270nm)

The UV spectrophotometric method was validated as per ICH guidelines Q2 R1 for method validation. The parameters evaluated were linearity, precision, accuracy, LOD and LOQ.

Linearity

This was studied by diluting standard stock solution (1000µg/ml) of STG to 50-250µg/ml and PIO (100µg/ml) to 5-25 µg/ml concentrations. Concentration curves of concentration against absorbance were plotted at their respective wavelengths for both the drugs and the obtained data was subjected to regression analysis. The standard curves for STG and PIO are shown in figure 5 and 6.

Accuracy

Recovery studies were carried out by standard addition method by adding known amount of SIT and PIO (working standard) to pre-analysed sample at three different concentration levels that is 80%, 100% and 120% of assay concentration and percent recoveries were calculated.

Table 1:  Standard absorptivity for STG

Concentration ((µg/ml) Absorbance Specific Absorptivity Values
λ1 (267 nm) λ2 (270 nm) λ1 (267 nm) λ2 (270 nm)
50 0.181 0.162 36.2 32.4
100 0.352 0.314 35.2 31.4
150 0.517 0.462 34.47 30.8
200 0.697 0.622 34.85 31.1
250 0.860 0.771 34.4 30.84
Mean 35.024 31.31

Table 2: Standard absorptivity for PIO

Concentration ((µg/ml) Absorbance Specific Absorptivity Values
λ1 (267 nm) λ2 (270 nm) λ1 (267 nm) λ2 (270 nm)
5 0.100 0.103 200 206
10 0.180 0.186 180 186
15 0.273 0.284 182 189.33
20 0.363 0.376 181.5 188
25 0.450 0.466 180 186.4
Mean 184.7 191.15

Precision

Precision was ascertained by determination of six replicates of same concentrations of sample and standard for method precision and system precision. Both intraday and inter-day precisions were carried out.

Table 4: Repeatability study data for STG and Pio (n=6)

Sr No. Concentration of STG (μg/ml) Absorbance of STG Concentration of PIO (μg/ml) Absorbance of PIO
1  

 

 

100

 0.352  

 

 

15

 0.273
2 0.345 0.271
3 0.355 0.267
4 0.349 0.270
5 0.353 0.269
6 0.349 0.267
SD 0.0036 0.0023
RSD 1.03 0.852

LOD

Detection limit was determined based on the standard deviation of peak areas of same concentrations that is standard solutions of STG (150µg/ml) and PIO (15µg/ml) prepared six times and LOD calculated by

LOD= 3.3x (SD/S)

Where, SD= Standard Deviation; S= Slope of Curve

LOQ

Detection limit was determined based on the standard deviation of peak areas of same concentrations that is standard solutions of STG (150µg/ml) and PIO (15µg/ml) prepared six times and LOQ calculated by

LOQ= 10x (SD/S)

Table 3. Accuracy for UV- spectrophotometric Method

Sr. No. Level of                   % Recovery Amount of ‘Sample Stock-A’ (ml) Amount of Standard Drug Added (µg/ml) Total Amount Found (µg/ml) Amount   Recovered (µg/ml) % Recovery
STG PIO STG PIO STG PIO STG PIO
1 0 0.5 0 0 4.88 1.52 0 0 0 0
2 80 0.5 4 1.2 8.8 2.7 3.92 1.18 98 98.33
3 100 0.5 5 1.5 10 3.1 5.12 1.58 102.4 105.33
4 120 0.5 6 1.8 10.92 3.34 6.04 1.82 100.66 101.11

Where, SD= Standard Deviation; S= Slope of Curve

Method B:

Specificity

Figure 7. Overlain Chromatograms of sample and standard solution of drugs
Figure 7. Overlain Chromatograms of sample and standard solution of drugs
Figure 8: Calibration curve of STG of RP-HPLC method
Figure 8: Calibration curve of STG of RP-HPLC method

The chromatogram of standard solution of mixture of STG and PIO was compared with formulation to observe the interference of excipient.

Linearity

1, 2, 3, 4 and 5ml of ‘Std Stock MIX AP’ were transferred in a series of 10ml volumetric flasks. The volume was made up to the mark with methanol to obtain the conc. of 10, 20, 30, 40 and 50µg/ml of STG and 3, 6, 9, 12 and 15µg/ml of PIO. The solutions were filtered through 0.45µ syringe filter and 20µl injected into the HPLC system and their chromatogram were recorded for 10mins under the chromatographic conditions as described above after getting a stable baseline. Peak areas were recorded for all the peaks. Calibration curves of STG and PIO were constructed by plotting the peak area of STG v/s conc. of PIO and peak area of STG v/s conc. of PIO, respectively. The correlation coefficient (r2) of least square linear regression for STG and PIO was calculated.

Table 5. Response of STG at various linearity

Conc. of STG (µg/ml) Peak Area (mV)
0 0
10 48
20 92
30 138
40 185
50 232

Table 6: Response of PIO at various linearity levels

Sr. No. Conc. of PIO (µg/ml) Peak Area (mV)
1. 3 54
2. 6 109
3. 9 160
4. 12 217
5. 15 266

Accuracy

Figure 9: Calibration curve of PIO of RP-HPLC method
Figure 9: Calibration curve of PIO of RP-HPLC method
Figure 10: Overlain Chromatograms of serial dilutions of STG and PIO in optimized chromatographic conditions
Figure 10: Overlain Chromatograms of serial dilutions of STG and PIO in optimized chromatographic conditions

20 Tablets (PIOZ 30) were weighed and finely powdered, an accurately weighed tablet powder (183.1mg) equivalent to 15 mg of PIO was dissolved and diluted to 100ml with methanol. 0.5 ml of above solution was transferred in four different 10ml volumetric flask labeled as 0%, 80%, 100% and 120%. Then 0, 0.8, 1, 1.2ml of ‘Std Stock MIX AP’ (100µg/ml STG and 30µg/ml PIO) were added and volume was made up to the mark with mobile phase. All the solutions were filtered through syringe filter and injected into the HPLC system and their chromatograms were recorded under the same chromatographic conditions after getting a stable baseline. Peak areas were recorded and percent recoveries were calculated.

Figure 11: Chromatogram of STG (10 µg/ml) in optimized chromatographic conditions
Figure 11: Chromatogram of STG (10 µg/ml) in optimized chromatographic conditions
Figure 12: Chromatogram of PIO (10 µg/ml) in optimized chromatographic conditions
Figure 12: Chromatogram of PIO (10 µg/ml) in optimized chromatographic conditions

 

 

 

 

 

 

 

Precision

The precision of an analytical method was studied by performing Repeatability and intermediate precision.

Figure 13: Chromatogram of combination of STG (10µg/ml) & PIO (10µg/ml) in optimized chromatographic conditions
Figure 13: Chromatogram of combination of STG (10µg/ml) & PIO (10µg/ml) in optimized chromatographic conditions

Repeatability

20µg/ml of STG and 6µg/ml of PIO solution was filtered through 0.45µ syringe filter and 20µl injected into the HPLC system and its chromatogram was recorded under the same chromatographic conditions after getting a stable baseline. Peak area was recorded. The procedure was repeated for six times.

Robustness        

Combined standard solution of STG (20µg/ml), PIO (6µg/ml) was prepared and analysed at different flow rates (0.9, 1.0, 1.1 ml/min) and different wavelengths (269, 270, 271nm) separately.

System Suitability

Chromatograms were studied for different parameters such as tailing factor, resolution and theoretical plates to see that whether they comply with the recommended limit or not.

Table 7. Recovery Studies

Sr. No. Level of                   % Recovery Amount of ‘Sample Stock-A’ (ml) Amount of Standard Drug Added (µg/ml) Total Amount Found (µg/ml) Amount   Recovered (µg/ml) % Recovery
STG PIO STG PIO STG PIO STG PIO
1 0 0.5 0 0 0 7.63 0 0 0 0
2 80 0.5 8 2.4 8.1 9.93 8.1 2.3 101.25 95.83
3 100 0.5 10 3 9.56 10.75 9.56 3.12 95.6 104
4 120 0.5 12 3.6 11.94 11.05 11.94 3.42 99.5 95

Table 8: Results of Repeatability Study for STG and PIO

Inj. Peak Area(mV) of STG Peak Area(mV) of PIO
1 95 110
2 93 109
3 93 106
4 96 108
5 95 109
6 92 109
SD 1.54 1.37
RSD 1.5 1.2

Table 9: Summary of UV- Spectrophotometric Method of STG and PIO

Sr. No. Parameters STG PIO
1. Linearity Range (μg/ml) 50-250 5-25
2. Regression Equation (y = mx+c) y=0.0034x+0.0059 y=0.0179x+0.004
3. Correlation Coefficient (r2) 0.9997 0.9995
4. LOD (μg/ml) 3.6 0.413
5. LOQ (μg/ml) 10.90 1.26
6. % Recovery 98-101 98-102
7. Repeatability(%RSD) 1.03 0.852

Table 10: Summary of RP-HPLC Method of STG and PIO

Sr. No. Parameters STG PIO
1. Linearity Range (μg/ml) 10-50 3-15
2. Regression Equation (y = mx+c) y=4.61x+0.7 y=17.73x+1.6
3. Correlation Coefficient (r2) 0.9998 0.9996
4. LOD (μg/ml) 1.1 0.25
5. LOQ (μg/ml) 3.34 0.77
6. % Recovery 95-104 95-102
7. Repeatability(%RSD) 1.5 1.2

RESULTS AND DISCUSSION

The method has been employed successfully for quantitative determination of STG and PIO by UV spectrophotometric method and Reverse Phase High Performance Liquid Chromatographic method for the simultaneous estimation of STG and PIO and to validate the developed methods according to ICH Q2 (R1) guidelines.

CONCLUSION

The UV- spectrophotometric and HPLC methods are simple, accurate, precise for estimation of STG and PIO in bulk and pharmaceutical formulation. The short chromatographic time makes this method suitable for processing of multiple samples in short time. The method shows no interference by the excipients. The statistical parameters and recovery data reveals the good accuracy and precision. This method can be useful and suitable for the estimation of the STG & PIO in bulk and pharmaceutical formulations.

ACKNOWLEDGEMENT

The authors are very thankful to the Principal of D.S.T.S. Mandal’s College of Pharmacy, Solapur, India and cooperative staff for providing the required facilities and guidance to carry out this research work.

REFERENCES

  1. Sitagliptin Phosphate: https://www.drugs.com/monograph/sitagliptin-phosphate.html Accessed on: 8th July 2017.
  2. Pioglitazone Hydrochloride: https://www.drugs.com/monograph/pioglitazone-hydrochloride.html Accessed on: 8th July 2017.
  3. Bailey, C. J., Green, B. D., & Flatt, P. R. (2010). Fixed-dose combination therapy for type 2 diabetes: sitagliptin plus pioglitazone. Expert opinion on investigational drugs, 19(8), 1017-1025. https://doi.org/10.1517/13543784.2010.505235, PMid:20629618
  4. Kommana, R., & Rebecca, S. D. (2013). Development and validation of HPLC and UV spectrophotometric methods for determination of pioglitazone hydrochloride in bulk and its formulations. Der Pharmacia Lettre, 5(1), 269-78.
  5. Mehta, R. S., Patel, D. M., Bhatt, K. K., & Shankar, M. B. (2005). UV and visible spectrophotometric analysis of pioglitazone hydrochloride in bulk and tablets. Indian journal of pharmaceutical sciences, 67(4), 487.
  6. Basniwal, P., Srivastava, P., & Jain, D. (2008). Spectrophotometric estimation of pioglitazone hydrochloride in tablet dosage form. Asian Journal of Pharmaceutics, 2(4), 225-227. https://doi.org/10.4103/0973-8398.45035
  7. Patil, S., Dwivedi, S., & Bagade, S. (2011). Development of spectrophotometric method for the estimation of pioglitazone HCl from two different marketed brands. American Journal of PharmaTech Research, 1, 264-275.
  8. Kulkarni, A., Mohd S., Zaheer, Z., and Dehghan, M. (2012). Spectroscopic Estimation of Pioglitazone Hydrochloride. Global Journal of Medical Research, 12(2), 57-61.
  9. Tarkase, K. N., Sarode, M. B., Gulve, S. A., & Gawade, A. (2013). Development and validation of UV spectrophotometric method for estimation of sitagliptin phosphate. Der Pharmacia Lettre, 5(3), 315-318.
  10. Jeyabalan, G., and Narendra, N. (2013). Development and validation of UV spectrophotometric method for Sitagliptin Phosphate in pure and tablet dosage form. Journal of Applied Pharmaceutical Science, 3 (01), 095-098.
  11. Yadav, P. J., Kadam, V. N., & Mohite, S. K. (2014). Development and validation of UV spectrophotometric method for alogliptin benzoate in bulk drug and tabletCurrent Pharma Research, 4(4), 1286-1290.
  12. Raval Kashyap, U. S., & Badodaria, K. (2014). First order derivative and dual wavelength spectrophotometry methods development and validation for simultaneous estimation of Alogliptin and Pioglitazone in bulk and dosage form. International Journal of Pharmacy and Pharmaceutical Sciences, 6(2), 730-738.
  13. Kumar, P., Manzoor, A., Shetty, S., and Aradhya, V. (2014). UV-visible Spectrophotometric Method for Simultaneous Estimation of Alogliptin Benzoate and Metformin Hydrochloride in Combine Tablet Dosage Form. International Journal of Universal Pharmacy and Biosciences, 3(6), 336-349.
  14. Chirag, P. A. (2014). Development and validation of UV spectrophotometric method for simultaneous estimation of metformin hydrochloride and alogliptin benzoate in bulk drugs and combined dosage forms. Der Pharma Chemica, 6(1), 303-311.
  15. Doredla, N., Yengisetty, B., Bojjagani, R., and Madasu, S. (2012). MethodDevelopment of Forced Degradation Studies of Pioglitazone Hydrochloride by Using UV Spectroscopy. International Journal of Pharmaceutical Technical Research, 4(4), 1750-1757.
  16. Naveed, S., Ashraf, Z., & Mukhtar, T. (2015). Assay of Pioglitazone HCL by using UV visible spectrophotometer. British Journal of Research, 2(1), 24-29.International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text and Methodology Q2 (R1), Geneva, 2005.

]]> https://www.ijprs.com/article/development-and-validation-of-analytical-methods-for-simultaneous-estimation-of-sitagliptin-phosphate-and-pioglitazone-hydrochloride/ Vaishnavi, K. G., Varsha, S. T., Nirmal, M. T., Somnath, A. P., Mallinath, S. K. http://www.ijprs.com/wp-content/uploads/2018/09/IJPRS-V7-I2-00028.pdf