Novel Ion Pair HPLC Methods for the Assessment of Sitagliptin and Pioglitazone in Tablets

 

Linda Hammash, Yaser Bitar, Saleh Trefi

Pharmaceutical Quality and Pharmaceutical Chemistry Department, Faculty of Pharmacy,

University of Aleppo,  Syria.

 

ABSTRACT:

Objective: Developing simple, rapid and accurate ion-pair reversed phase high performance liquid chromatographic methods for the determination of pioglitazone hydrochloride, and sitagliptin phosphate in pure and tablet forms. These methods would be practical additional choices in quality control laboratories. Methods: pioglitazone hydrochloride and sitagliptin phosphateanalysis were performed depending on (Method A), the chromatographic conditions comprised of a classical C18-type stationary phase (250×4.6 mm, 5μm), The mobile phase consisting of 10^-2 sodium hexane sulfonic acid, in a mixture of 550ml of deionized water and 450ml of acetonitrile, and apparent pH of 2.5 was adjusted with phosphoric acid. (Method B) is an additional method for pioglitazone hydrochloride analysis, the mobile phase consisting of 10^-3 M cetrimide, in a mixture of 400 ml of deionized water and 600ml of acetonitrile, leading to measured pH=10. The flow rate was 1ml/min, the detection wavelengths were at 269nm for pioglitazone HCl (Method A,B), and 267nm for sitagliptin phosphate, the temperature was ambient temperature for both Methods A and B. Results: Two methods were validated in accordance to ICH guidelines. The developed methods given accepted linearity (R²>0.999) in the concentration range of (0.01875-0.21)mg/ml for pioglitazone hydrochloride and (0.1-0.6) mg/ml for sitagliptin phosphate, accuracy with mean recovery values between 98-102%, precision with relative standard deviations of the calculated concentrations were less than 2%. Conclusion: The results showed that the two developed methods were effective in separating and determining of pioglitazone HCl, sitagliptin phosphate in their pharmaceutical forms and routine analysis in quality control laboratories simply.

 

 

 

INTRODUCTION:

Thiazolidinediones (TZDs) represented a new class of oral agents with a novel mechanism of action that reduced insulin resistance and improved glycemic control. They actas agonists of nuclear receptors called peroxisome proliferator-activated receptors-gamma (PPAR-gamma) to enhance the actions of insulin^[1].

 

Pioglitazone is a TZD which is widely used for treating patients with type 2 diabetes^[2], itenhances tissue sensitivity to insulin by acting as a potent and selective agonist at peroxisome proliferator activated gamma receptor (PPAR-gamma) in adipose tissue, skeletal muscle, and liver^[3].

 

Sitagliptin is a dipeptidyl-peptidase inhibitor (DPP-4 inhibitor) that has recently been approved for the therapy of type 2 diabetes^[4], The inhibition of DPP-4 leads to an increase in the active levels of incretins such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP)^[5], this increases active increatin and insulin levels and decreases glucagonlevels and post-glucose-load glucose excursion^[6] (Figure.1).

 

 

Different HPLC methods have been applied to assay pioglitazone HCl in its pure and pharmaceutical forms ^[7-12]. Many of accurate UV-spectrophotometric methods have been also used in its assay^[13-16]. There were also HPTLC researches to determine pioglitazone HCl^[17-20]

 

Many HPLC methods have been applied to assay sitagliptinphosphate in its pure and pharmaceutical forms^[21-24] UV-spectrophotometric methods have been also used in its assay^[25-26]. Ion pair liquid chromatography is a new method is utilized to determine another drugs and pharmaceutical combination recently^[27-30].

 

 

pioglitazone hydrochloride

 

sitagliptin phosphate monohydrate

Figure. 1: chemical structure of pioglitazone hydrochloride and sitagliptin phosphate monohydrate.

 

MATERIALS AND METHODS:

Chemicals and reagents:

Working standards of pioglitazoneHCl was gifted by BarakatPharmaceutical Industries, Aleppo-Syria, sitagliptin phosphate was gifted by Ibn Alhaytham Pharmaceutical Industries, Aleppo-Syria. The commercial drugs (brand name) Pioglet (pioglitazone HCl 15 mg Barakat Pharmaceutical Industries), Defast (pioglitazone HCl 30 mg UnipharmaPharmaceutical Industries), Actazone Asia (pioglitazone HCl 30 mg Asia PharmaceuticalIndustries), Sitacretin (sitagliptin phosphate 100 mg Ibn Alhaytham Pharmaceutical Industries), Gliptin (sitagliptin phosphate 100 mg Ultra Medica Pharmaceutical Industries), Glynuvia (sitagliptin phosphate 100 mg Diamond Pharmaceutical Industries) were purchased from Syria. All the otherused reagents were of HPLC grade: acetonitrile (Biosolve, France), methanol( Biosolve, France),phosphoric acid (Scharlau,EuropeanUnion), sodium hexane sulfonic acid (Abcr,Germany),cetrimide (Tnn, China),deionized water for HPLC and filters 0.45 μm.

 

Instrumentation:

The HPLC instrument used was an (Agilent 1260 infinity, Germany) equippedwith a UV detector, the pH meter (Crison, Spain), ultrasonic processor (Powersonic, model 405, Korea), analytical balance ±0.1mg (Sartorius, model 2215, Germany).

 

Pharmaceutical formulations:

Defast tablets, Unipharma. Damascus- Syria, (2018/2021), labeled 30mg of pioglitazone HCl.

 

Actazonetablets, Asia. Aleppo- Syria, (2018/2021), labeled 30 mg of pioglitazone HCl.

 

Pioglit tablets, Barakat. Aleppo- Syria, (2015/2020), labeled 15 mg of pioglitazone HCl.

 

Sitacretin tablets, Ibn Alhaytham. Aleppo-syria, (2018/2021), labeled 100 mg sitagliptin phosphate.

 

Gliptin tablets, Ultra Medica. Aleppo-syria, (2018/2021), labeled 100 mg sitagliptin phosphate.

 

Glynuvia tablets, Diamond. Aleppo-syria, (2015/2019), labeled 100 mg sitagliptin phosphate.

 

Reference solutions preparation:

·      The standard stock solution was prepared by dissolving 15 mg pioglitazone hydrochloride in methanol using 50mL volumetric flaskto give the final concentration of 0.3mg/ml.The working standard solutions of pioglitazone HClwereprepared by taking suitable aliquots of drug solution fromthe standard stock solution and the volume was made up to10ml with methanol to get concentrations between(0.01875-0.21) mg/ml.

·      The standard stock solution was prepared by dissolving 50 mg sitagliptinin deionized water using 50 mL volumetric flaskto give the final concentration of 1mg/ml. The working standard solutions of sitagliptin phosphate were prepared by taking suitable aliquots of drug solution from the standard stock solution and the volume was made up to 10 ml with deionized water to get concentrations between (0.1-0.6) mg/ml.

 

Method development and optimization of chromatographic conditions:

Selection of detection wavelength:

The utilized detection wavelengths wereλ _max269 nm (Method A,B) for pioglitazone hydrochloride, and λ _max267 nm for sitagliptin phosphate.

 

Column selection:

C18- reversed phase column, 250 x4.6 mm 5-µmwas utilized.

 

Mobile phase preparation:

Method (A):

The mobile phase consisting of 10^-2M sodium hexane sulfonicacid, in a mixture of 550ml of deionized water and 450ml of acetonitrile, and apparent pH of 2.5 was adjusted with phosphoric acid.

 

Method (B):

The mobile phase consisting of 10^-3M cetrimide, in a mixture of 400ml of deionized water and 600ml of acetonitrile, pH =10.

 

Formulation solutions preparation:

·      Twenty tablets were crushed and powdered. An amount equivalent to labeled content (15 mg or 30 mg pioglitazone HCl) was taken and dissolved in 15 ml of methanol and sonicated for 10minutes. Then sample was transferred to 25 ml calibrated flask and diluted with methanol. Further dilutions were made to obtain an appropriate concentration (in the range of linearity) and then the general procedure was conducted.

 

·      Twenty tablets were crushed and powdered. An amount equivalent to labeled content (100 mgsitagliptin phosphate) was taken and dissolved in 15 ml of dionized water and sonicated for 10 minutes. Then sample was transferred to 25 ml calibrated flask and diluted with deionized water. Further dilutions were made to obtain an appropriate concentration (in the range of linearity) and then the general procedure was conducted.

Degraded starting solutions:

The starting standard solutions prepared as mentioned above were standing at room temperature and sunlight for 60 days. Then, they were analyzed for specificity tests demonstration.

 

Analytical method validation:

Method validation was performed in according toInternational Conference on Harmonization (ICH) recommended testcondition ^[31].

 

RESULTS AND DISCUSSION:

HPLC analysis:

The chromatographic conditions comprised of a C18 reversed phase column, 250x4.6 mm 5-µm, with a mobile phase consisting of 10^-2M of sodium hexane sulfonic acid in a mixture of 550ml of deionized water and 450ml of acetonitrile, and apparent pH of 2.5 was adjusted with phosphoric acid (Method A), 10^-3M cetrimide, in a mixture of 400ml of deionized water and 600ml of acetonitrile, leading to measured pH=10 (Method B). The flow rate was 1ml/min for both Methods A , B. The utilized detection wavelengths was 269 nm for pioglitazone HCl, and 267 nm for sitagliptin phosphate under ambient temperature. The reference solutions of pioglitazone HCl, sitagliptin phosphate were injected under the previous chromatographic conditions, the retention time for pioglitazone HCl was 4.4 min for Method A, and 6.7 min for Method B (Figure.2), and 3.7min for sitagliptin phosphate (Figure.3).

 

 

Method A

 

 

Method B

Figure.2:Chromatograms of pioglitazone HCl (Method A and B)

 

 

Method A

Figure. 3: Chromatograms of sitagliptin phosphate monohydrate (Method A).

 

Analytical method validation:

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 an analyte in the sample ^[29].

 

The linearity was evaluated by linear regression analysis, which was calculated by the least square regression method. Different concentrations of pioglitazone were prepared (0.01875, 0.0375, 0.75, 0.15, 0.21) mg/ml for Method A and B (Figure.3), and the concentrations of sitagliptin phosphate were prepared (0.1, 0.2, 0.3, 0.5, 0.6) mg/ml for Method A (Figure.4).

 

 

Methods A

 

Methods B

Figure.3: Linearity lines of pioglitazone HCl for both Methods A and B

 

 

Figure. 4: Linearity lines of sitagliptin phosphate (Method A)

 

 

 

Table 1: Accuracy of the proposed Method A

^amean±SD, n = 3.

 

 

Table 2: Accuracy of the proposed method B

^amean±SD, n = 3.

 

Accuracy:

Recovery studies were performed for three replicates of three concentrations within the range of linearity. Recovery results for pioglitazone HCl (99.2-100.8)% Method A, (98.13-101.33)% Method B, and for sitagliptin phosphate (98.8-101.67)% Method A were among the accepted range of accuracy (98-102%) which indicates that both of developed methods are accurate and applicable to the determination of pioglitazone HCl, and sitagliptin phosphate (Table1,2).

 

Precision:

Repeatability precision was established by injecting six replicate injections of standard solution (0.075mg/ml) pioglitazone HCl, and (0.3mg/ml) sitagliptin phosphate, the results are shown in table 3. Intermediate precision was determined by three replicate analyses ofthree concentrations levels during a period of two day. RSD% was less than 2% for both Methods A and B (Table 4,5).

Table 3: Repeatability precision for the proposed methods

^a mean, n = 6.

 

Table 4: Intermediate precision for Method A

^a mean, n = 3, *mean, n = 6.

 

Table 5: Intermediate precision for Method B

^a mean, n = 3, *mean, n = 6.

 

Sensitivity:

LOD and LOQ were calculated to evaluate the sensitivity of the proposed methods. The calculated LOQ and LOD for pioglitazone HCl were 0.006 mg/ml and 0.002 mg/mL for Method A, and 0.012mg/ml and 0.0041mg/ml for Method B, respectively, The calculated LOQ and LOD for sitagliptin phosphate were0.039 mg/ml and 0.013mg/ml.

 

 

Specificity:

In order to demonstrate the specificity of the method, reference solutions were exposed to sunlight for 60 days at room temperature. The chromatograms of the reference solutions after degradation (60 days) indicated no additional peaks other than those ofpioglitazone HCl for Method A andMethod B (Figure4),sitagliptin phosphate (Figure 5),pioglitazone HCl and sitagliptin phosphate solutions are stable for 60 day in these conditions.

 

 

Method A

 

Method B

Figure. 4: Chromatogram of the standard reference solution degradation of pioglitazone HClMethod A, B.

 

 

Figure. 5: Chromatogram of the standard reference solution degradation of Sitagliptin phosphate.

 

Application of the methods on pharmaceutical formulations:

The two developed A, B methods were applied to determine the studied active pharmaceutical ingredients; (A,B) forpioglitazone HCl, (A) forsitagliptin phosphate.The results of quantitative analysis for Pioglitazone HCl, andsitagliptin phosphate were satisfactory(Table 6).It was observed that, all the tested formulations had concentrations within the specification, which recommended thatpioglitazone HCl tablets should contain not less than 95% and not more than 105% of the labeled amount of the active pharmaceutical ingredient (USP Pharmacopeia) ^[30], and not less than 90% and not more than 110% of the labeled amount of the active pharmaceutical ingredient forsitagliptin phosphate tablets ^[32].

 

Table 6: Results ofpioglitazone HCl and sitagliptine phosphate tablets assay by the developed HPLC methods

Pioglitazone hydrochloride						Sitagliptin phosphate
Formulation	Claim (mg/tab)	Recovery* ± SD		RSD%		Formulation	Claim (mg/tab)	Recovery* ± SD	RSD%
		Method A	Method B	Method A	Method B
Defast	30	101.67±0.64	102.17±0.34	0.63	0.33	Sitacretin	100	103.91±0.40	0. 38
Actazone Asia	30	97.35±0.45	96.13±0.36	0.46	0.37	Gliptin	100	102.34±0.36	0.35
Pioglit	15	102.62±0.40	103.24±0.46	0.39	0.45	Glynuvia	100	95.85±0.62	0.67

*mean,n=3

References indicate that the result of assaying Pioglitazone HCl tablets was 99.3% according to Lakshmi K.S et al.^[7], and 100.8% according to Prasad PS et al^[8]. For assaying sitagliptin phosphate tablets the result was 108.56% according to Qassas M.K et al.^[33], and 99.84% according to Raja T et al.^[23].

 

 

CONCLUSION:

Two Simple, accurate ion-pair HPLC methods have been developed and validated for the determination of pioglitazone HCl (Method A,B), and sitagliptin phosphate (Method A) drugs in their pharmaceutical forms. For pioglitazone HCl Method A is more sensitive depending on the values of LOQ and LOD. They can be applied to assaycommercial formulations (tablets). The methods are practical and valuable for routine application in quality control laboratories for the assay of pioglitazone HCl and sitagliptin phosphateas raw materials, active pharmaceutical ingredients and pharmaceutical formulations.

 

REFERENCES:

1.     Stanley S. Schwartz, Pioglitazone for the treatment of type 2 diabetes in patients inadequately controlled on insulin, Diabetes Metab Syndr Obes, 2010, 3: 243–252.

2.     Desouza C, Shivaswamy V. Pioglitazone in the Treatment of Type 2 Diabetes: Safety and Efficacy Review. Clin Med Insights Endocrinol Diabetes 2010, 3: 43-51.

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

4.     Gallowtiz B, Review of sitagliptin phosphate: a novel treatment for type 2 diabetes, Vascural and health risk management, 2007, 3(2):203-210.

5.     Dallumal R, Chua S, Vethakkan S, Sitagliptin: Is It Effective in Routine Clinical Practice, International Journal of Endocrinology, 2015, 2015:1-9.

6.     Chavhan V, GhanteM ,Sawant S, Development and validationof RP-HPLC method for simultaneous estimationof sitagliptin phosphate and simvastatin in bulk and tablet dosage form, 2014, 6(3):327-338.,

7.     Lakshmi KS, RajeshT, Sharma S, Determination of pioglitazone and glimepiride in pharmaceutical formulations and rat plasma by RP-LC, International Journal of PharmTech Research. 2009, 1(3): 496-499.

8.     Prasad PS, Imam SS, Aqil M, Rizwan M, Sultana Y, Ali A, Validated reversed phase hplc method for determination of pioglitazone hydrochloride in bulk drug and tablet formulations, Journal of Analytical Chemistry. 2015, 70(6):203–210.

9.     Dhole S, Khedekar P, Amnerkar N,RP- HPLC method for simultaneous estimation of pioglitazone hydrochloride metformin hydrochloride and glibenclamide in multicomponent tablet dosage form, International Journal of Pharma Research and Health Sciences. 2016, 4 (2):1059-66.

10.   Adukondalu D, P S Malathy P S, Venkateshwarrao J, Madhusudan Rao Y, Development and validation of HPLC method for detection of pioglitazone hydrochloride in dosage forms, International Journal Of Pharmacy and Biological Science. 2011, 4(1):474-478.

11.   Srinivasulu D, Sastry BS, Omprakash G, Development and validation of new RP-HPLC method for determination of pioglitazone hcl in pharmaceutical dosage forms, International Journal of Chemistry Research. 2010, 1(1):18-20.

12.   Deepa R, Laxmanbhai J., Madhabhai M.,Stability Indicating HPLC Method for Simultaneous Determination of Glimepride, Pioglitazone Hydrochloride and Metformin Hydrochloride in Pharmaceutical Dosage Form,Research Journal of Pharmacy and Technology, 2011, 4(4):606-611

13.   shanti kumar S, Krishnaveni Y, Ramesh G, Simultaneous estimation of sitagliptin and pioglitazone by uv-spectroscopic method and study of interference of various excipients on this combination of drugs, International Journal of Current Pharmaceutical Research. 2012, 4(2):113-116.

14.   Singhvi I, Mehta K,Kapadiya N, Analytical method development and validation for the simultaneous estimation of pioglitazone and glimepiride in tablet dosage form by multiwavelengthspectroscopy. 2011, 1(6):159-161.

15.   OnkarS,and Shweta S, Simultaneous determination of atorvastatin calcium and pioglitazone hydrochloride in its multicomponent dosage forms by UV spectrophotometry, International Journal of Pharmacy and Pharmaceutical Science Research. 2011, 1(2):75-79.

16.   Mali AD, Mali S, Tamboli A, Bathe R, Simultaneous uv spectrophotometric methods for estimation of metformin hcl and glimepiride in bulk and tablet dosage form, International Journal of Advances in Pharmaceutics. 2015, 4(6):117-124.

17.   Dhirender Singh SC, Kushnoor A, Development and validation of a HPTLC method for estimation of pioglitazone in bulk and tablet dosage form, International Journal of Biomedical and Advance Research. 2011, 2(9): 3919-3921.

18.   Miral Rank, Pankaj Kapupara, Ketan Shah, Development and Validation of Stability Indicating HPTLC Method for Pioglitazone Hydrochloride and Metformin Hydrochloride, Research Journal of Pharmacy and Technology, 2016, 9(10):1555-1561.

19.   Sharma MC, Sharma S, Kohli D, HPTLC method development and validation for the estimation of atorvastatin calcium and pioglitazone HCl in pharmaceutical combined tablet dosage form, Annals of Biological Research.2010, 1(1):124-129.

20.   Anand P, Senthilkumar KL, Senthilkumar B, Saravanakumar M,Thirumurthy R, HPTLC method for determination of telmesartonHCl with pioglitazone HCl in pharmaceutical dosage form, International journal pharma research and development.2010, 2(9):185-190.

21.   Karimulla S K, Vasanth P M, Ramesh, Ramesh M, Method development and validation of sitagliptin and metformin using reverse phase HPLC method in bulk and tablet dosage form, Der Pharmacia Lettre, 2013, 5 (5):168-174.

22.   Nancy Veronica B, Krishnamoorthy B, Muthukumaran M, Development and validation of a new simple RP-HPLC method for estimation of Metformin HCl and Sitagliptin phosphate simultaneously in bulk and dosage forms, International Journal Advanced Pharmceutical Genuine Research, 2014, 2(1):1-14.

23.    Raja T , Lakshmana Rao A, Validated rp-hplc method for simultaneous estimation of metformin hydrochloride and sitagliptin phosphate in bulk drug and pharmaceutical formulation, International Journal of Pharmaceutical, Chemical and Biological Sciences, 2012, 2(4): 696-702.

24.   PratapPulla R, Sastry BS, Rajendra Prasad Y, Appala N, Simultaneous Estimation of Metformin HCL and Sitagliptin Phosphate in Tablet Dosage Forms by RP-HPLC, Research Journal of Pharmacy and Technology,2011;4(4):646-649

25.   Patil S, Ramesh B, Hareesh AR, Patil K, Validated UV Spectrophotometric Method for Estimation of Sitagliptin Phosphate in Tablet Dosage Form, Research Journal of Pharmacy and Technology,2010; 3 (3):798-800.

26.   PallawGhodeswar, Avnish Jain, Sanjay Jain,Development of First Order Derivative UV –Spectrophotometric Method for the Estimation of Sitagliptin Phosphate, Research Journal of Pharmacy and Technology,2011;4(4): 631-633.

27.   Shasho H, Alhajsakur A, Trefi S, separation and assay of four antihistamine drugs diphenhydramine, chlorpheniramine, cyproheptadine and fexofenadine in pharmaceuticalforms by a single HPLC method, International Journal of Pharmacy and Pharmaceutical Sciences. 2018; 4(10):53-60.

28.   Trefi S, Bitar Y, Separation and analysis of amlodipine/benazepril combination in capsules by a novel ion pair liquid chromatography, International Journal of Pharmacy and Pharmaceutical Sciences. 2019;1(11):107-112.

29.   Trefi S, Bitar Y,Gilard V, Separation and Quantification of Sacubitril-Valsartan combination in Tablets by a New ion-HPLC, Research Journal of Pharmacy and Technology. 2019;12(3):1117-1022.

30.   Trefi S, Assay of four psychotropic drugs chlorpromazine, clomipramine, amitriptyline and nortriptylinein tablets by asingle HPLC method,Int J Pharm PharmSci, 2016;8(8):182-188.

31.   ICH Q2(R1). Validation of analytical procedures: text and metholodogy. International Conference on Harmonisation of Technical Requirements for registration of Pharmaceuticals for Human Use. 2005.

32.   United States Pharmacopeia- National Formulary andits supplements, USP38- NF 33, 2015.

33.   Qassas MK, Mardini MA, Ghazal H, A Validated HPLC Stability Indicating Method for the Determination of Sitagliptin in Bulk Drug Substance and Tablets,2015;32(1):194-198.

 

 

 

 

 

Accepted on 20.05.2019          © RJPT All right reserved

Research J. Pharm. and Tech 2019; 12(8):3713-3719.