AEGAEUM JOURNAL ISSN NO: 0776-3808

“Spectrophotometric Method Development and Validation for Anagliptin and HCl by Simultaneous Equation Method In Synthetic Mixture”

Ruchi H. Majithia*1 & Dr. Akruti Khodadiya2 *Assistant Professor, SAL Institute of Pharmacy, Ahmedabad, Gujarat- 380060 1Research Scholar, C. U. Shah College of Pharmacy and Research, Gujarat- 363030 1Mail ID: [email protected] 1Mobile No: +91-8155921801 2Associate Professor, C.U. Shah University, Wadhwan city, Gujarat- 363030 2Mail ID: [email protected]

*Corresponding Author

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ABSTRACT

A simple, accurate, precise, rapid and economical UV/VIS Simultaneous equation method was developed and validated for estimation of Anagliptin and Metformin HCl in synthetic mixture. Choice of a common solvent were essential so distilled water was selected as a solvent for the proposed method. Anagliptin and Metformin HCl showed maximum absorbance at 247 (λ1) nm and 233 (λ2) nm 2 respectively. Both drugs obey Beer Lambert’s law in the concentration range 2-12 µg/mL at λ1 (R = 2 0.9992) for Anagliptin, and in the range of 5-30 µg/mL for Metformin HCl found at λ2 (R = 0.9997). The % Recovery was 99.08 – 101.24 % of Anagliptin and 99.19 – 101.72 % of Metformin HCl by standard addition method. The LOD was found to be 0.103 µg/mL and 0.167 µg/mL for ANA and MET respectively. The LOQ was found to be 0.314 µg/mL and 0.506 µg/mL for ANA and MET respectively. The method was found to be precise as % RSD was less than 2.00 in Repeatability, Interday and Intraday precision for Anagliptin and Metformin HCl. The % assay of analyte drugs in synthetic mixture was found to be 99.72% of Anagliptin and 99.31 % of Metformin HCl which showed good applicability of the developed method.

KEYWORDS: Anagliptin (ANA), Metformin HCl (MET), UV – Spectrophotometry, Simultaneous estimation. INTRODUCTION Anagliptin, in form of Suiny® (100 mg tablets) is new drug formulation for therapy approved by the Japanese regulatory authority in 2014[01]. Anagliptin, chemically N- [2-[[2-[(2S)-2-Cyanopyrrolodin-1-yl]-2-oxoethyl] amino]-2-methylpropyl]-2-methylpyrazolo [1, 5-a] pyrimidine-6-carboxamide (Figure 1) is a Dipeptidyl Peptidase 4 (DPP 4) inhibitor which is used in treatment of type 2 NIDDM [02]. Dipeptidyl Peptidase 4 enzyme breaks down the incretins GLP-1 gastrointestinal hormones released in response to a meal. By preventing GLP-1 inactivation, they are able to increase the secretion of and suppress the release of glucagon by the alpha cells of the pancreas. This drives blood glucose levels towards normal level [03-04]. This drug is not official in any of the pharmacopeia. Anagliptin is a very effective pill with minimum risk profile for type 2 diabetus mellitus and longer duration of action for treatment of type 2 non-insulin dependent diabetus mellitus disease [05]. A literature survey revealed that few methods are reported for determination of ANA, either alone or in combination [06], by spectrophotometric [07-09], HPLC [10], LC/MS [11].

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Figure 1: Chemical Structure of Anagliptin

.HCl

Figure 2: Chemical structure of Metformin HCl

Metformin is chemically a 1-Caramimidamido-N, N-Dimethylmethanimidamide [12] (Figure 2) and has pharmacological action based on category [13-14]. It suppresses hepatic gluconeogenesis and glucose output from liver. This is the major action responsible for lowering blood glucose in diabetics. It is official in IP [15], BP [16], and USP [17]. Literature review revels that many spectrophotometric [18-19], HPLC [20-21], HPTLC [22] methods are reported for determination of Metformin hydrochloride (HCl), either alone or in combination [08]. The aim of the present work was to develop a Simultaneous equation method for estimation of ANA and MET in combination. It is pertinent to note that, some of the published methods enabled estimation of drugs in combination products containing two drugs via zero and first order derivative spectrophotometric method and HPLC however, so far, not any Simultaneous equation spectrophotometric method was reported for the same. Hence, to achieve this aim an accurate Simultaneous equation method has been developed and successfully applied to synthetic mixture. MATERIALS AND METHODS Apparatus

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Instrument used was of Shimandzu UV-1600 series with a pair of 1 cm matched quartz cells. Software used was UV Probe 4.2 series. A digital analytical balance (Wenstar DA14-222) and ultrasonic sonicator (Equitron) were used in the study. Validated pipette of 1, 2, 5 mL; volumetric flasks of 10,100 mL; beakers of 100, 250, 500 mL were made up of Borosil glass. Chemicals and Reagents Drug sample of ANA and MET were provided as a gift sample by Intas Pharmaceutical Pvt. Ltd., Ahmedabad, India. Biciphage tablets (Metformin Hydrochloride 500mg) were purchased from local pharmacy store. Solvents like Distilled water were from E. Merck, Mumbai. All the chemicals reagents were of analytical Grade. Preparation of standard stock solution Accurately weighed quantity of 10 mg of ANA and 10 mg of MET were transferred into 100 mL volumetric flask individually. Initially about 50 mL distilled water was added to the flask respectively and sonicated. The volume was made up to the mark with distilled water to prepare stock solutions correspond to 100 μg/mL of ANA and 100 μg/mL of MET. Methodology Selection of analytical Wavelength From standard solution of ANA (100 μg/mL) and MET (100 μg/mL), prepare 10 μg/mL for ANA and MET. The scanning for solution of ANA and MET were carried out in the range of

200 – 400 nm against distilled water as a blank. The maximum absorption (λmax) of ANA and MET was found at 247 nm and 233 nm respectively (Figure 3 and Figure 4). Absorption and absorptivity for a series of standard solutions were recorded at selected wavelengths. The two wavelengths selected should be such that at each wavelength the difference in the absorptivity between the two components should be as large as possible. Determination:

The standard solution of ANA was scanned in the range of 200 – 400 nm and the λmax was

found to be 247 nm as λ1 against distilled water (Figure 3). Similarly, the standard solution

of MET was scanned in the range of 200 – 400 nm and the λmax was found to be 233 nm as λ2 against distilled water (Figure 4). The absorbance of both the drugs were recorded at 247 nm and 233 nm and molar absorptivity (ε) for both the drugs which was calculated from the formula. ε = A/C

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Where, A= absorbance, C= concentration of analyte in gm/100 mL Simultaneous equation method:

If a sample contains two absorbing drug each of which absorbs at the λmax of the other, it may be possible to determine both drugs by the technique of simultaneous equation. Two wavelengths selected for the development of the simultaneous equation are 247 nm and 233 nm. The absorptivity values determined for ANA are 998.4 (ax1), 549.8 (ax2) and for MET are 249.1 (ay1), 770.8 (ay2) at 247 nm and 233 nm respectively. These values are means of six estimations. The absorptivity data are shown in (Table 1). The concentration of two drugs of mixture in 1:5 ratio at these wavelengths can be calculated using following equation: [23]

Cx = A1ay2 – A2ay1 …….. Equation no. – 1 ax1ay2 – ax2ay1 Cy = A1ax2 – A2ax1 ax2ay1 – ax1ay2 …….. Equation no. – 2

Where, A1 and A2 are absorbance of mixture at 247 nm and 233 nm; Cx and Cy are the unknown concentration of Anagliptin and Metformin HCl respectively in sample solution;

ax1= Absorptivity of ANA at λ1 (998.4)

ay1= Absorptivity of MET at λ1 (249.1)

ax2= Absorptivity of ANA at λ2 (549.8)

ay2= Absorptivity of MET at λ2 (770.8). Table 1: Absorptivity data for ANA and MET Co ANA Co MET S n. n. r. (µg ax1 ax2 at (µg ay1 ABS at ABS at ABS at ABS at ay2 at N / at 247 233 / at 247 247 nm 233 nm 247 nm 233 nm 233 nm o. m nm nm m nm L) L)

1 2 0.2210 0.1245 1105.0 622.9 5 0.1130 0.3499 226.08 699.8 2 4 0.4073 0.2262 1018.3 565.5 10 0.2465 0.7679 246.57 767.93 3 6 0.5976 0.3274 996.05 545.6 15 0.3661 1.1461 244.1 764.1 4 8 0.7668 0.4384 958.61 548.1 20 0.4890 1.5206 244.51 760.30

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5 10 0.9369 0.4939 936.95 493.9 25 0.6785 2.1203 271.41 848.14 6 12 1.1705 0.6269 975.45 522.4 30 0.7860 2.3541 262.00 784.72 Average 998.4 549.8 249.1 770.8

Figure 3: UV Spectrum of Anagliptin (Maximum wavelength 247 nm)

Figure 4: UV Spectrum of Metformin (Maximum wavelength 233 nm) Preparation of test solution for Assay Determination Anagliptin/Metformin Hydrochloride is used in the ratio of 100/500mg for treatment of diabetes. Due to non-availability of product the condition of mixture was simulated by using

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Biciphage tablets (Metformin Hydrochloride 500mg) and API of ANA. Twenty tablets of Biciphage 500 mg tablets were weighed and triturated in a mortar pestle and powder equivalent to 500 mg of MET was taken into a 100 mL volumetric flask. To this flask, 100 mg of ANA API was added, to make concentration of ANA/MET in ratio of 1:5. The volume was adjusted to mark with distilled water to prepare test stock solutions correspond to 1000 μg/mL of ANA and 5000 μg/mL of MET, respectively. The contents of the flask were sonicated for 15 min to dissolve the active ingredients completely. The solution was filtered through a Whatman filter paper no. 41. From this 0.1 mL aliquot was transferred into a 10 mL volumetric flask and the volume was made up with distilled water. This test solution containing working concentrations of 2 µg/mL ANA and 10 µg/mL MET respectively, in mixture was analyzed for assay determination. Preparation of Calibration Curve From working standard solution of ANA (100 µg/mL), aliquots of 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 mL were transferred into series of 10 mL volumetric flask with the help of validated 1 mL pipette accurately and diluted up to mark with Distilled Water with the use of validated 10 mL pipette. This yielded solutions of 2, 4, 6, 8, 10 and 12 µg/mL of ANA. From working standard solution of MET (100 µg/mL), aliquots of 0.5, 1.0, 1.5, 2.0, 2.5 and 3.0 mL were transferred into series of 10 mL volumetric flask with the help of validated 1 mL pipette accurately and diluted up to mark with Distilled Water with the use of validated 10 mL pipette. This yielded solutions of 5, 10, 15, 20, 25 and 30 µg/mL of MET. METHOD VALIDATION The proposed method was validated as per ICH guidelines Q2 (R1) [24]. Linearity and Range Linearity was studied by preparing standard solutions at 6 different concentrations. The linearity range for ANA and MET were found to be 2-12 µg/mL and 5-30 µg/mL

respectively. For each solution, the absorbance of ANA and MET were measured at λ1 and λ2. The calibration curves of absorbance versus concentration were plotted. The linearity of absorbance responses versus concentrations was demonstrated by linear regression analysis. Precision The precision of the proposed method was assessed as repeatability, intra-day precision and inter-day precision. Repeatability was performed by applying six replicates of sample

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solution. For intermediate precision, Intraday and Interday precision was performed by determining corresponding responses of six replicates on same and different days for test solution containing ANA (2 µg/mL) and MET (10 µg/mL). The results were reported in terms of % RSD. Accuracy Recovery studies were carried out by standard addition method. A known amount of standard ANA (1, 2 and 3 µg/mL) and MET (5, 10 and 15 µg/mL) similar to 50%, 100% and 150% of the label claim were added to test solution of ANA (2 µg/mL) and MET (10 µg/mL). Same study was carried out three times, at each level of recovery. LOD and LOQ The LOD and LOQ of the developed method were calculated from the calibration curve using equation, LOD= 3.3* σ/S and LOQ= 10* σ/S. Where, σ = the standard deviation of y- intercepts of regression lines of six calibration curves, S = the average of the slopes of six calibration curves. RESULT AND DISCUSSION Linearity Aliquots of standard solution were applied in the concentration range 2-12 µg/mL and 5-30 µg/mL for ANA and MET respectively. The calibration curve obtained by the least square regression analysis between average absorbance and concentration showed linear relationship 2 with a correlation coefficient R nearer to 0.999 for ANA and MET at λ1 and λ2. The linear

regression equation obtained was y = 0.0925x + 0.0354 for ANA at λ1 (247 nm). The linear

regression equation obtained was y = 0.0788x - 0.0386 for MET at λ2 (233nm). (Table 2) (Figure 5-8)

Table 2: Linearity data of ANA at λ1 as 247 nm and MET at λ2 as 233 nm

ANA at λ1 MET at λ2 Sr Conc Conc No. Absorbance* ± SD %RSD Absorbance* ± SD %RSD (µg/mL) (µg/mL) 1 2 0.223 ± 0.002 1.003 5 0.349 ± 0.001 0.463 2 4 0.407 ± 0.003 0.838 10 0.767 ± 0.004 0.579 3 6 0.592 ± 0.005 0.957 15 1.140 ± 0.014 1.284 4 8 0.768 ± 0.005 0.773 20 1.527 ± 0.008 0.540

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5 10 0.946 ± 0.007 0.841 15 1.920 ± 0.013 0.710 6 12 1.159 ± 0.010 0.929 30 2.339 ± 0.027 1.182 *Average of six determinations, (SD = Standard Deviation, % RSD = Percentage Relative Standard Deviation)

Figure 5: Overlain spectra for linearity of ANA (2-12 µg/mL)

Figure 6: Overlain spectra for linearity of MET (5-30 µg/mL)

Calibration curveAVG of ANA at 247 nm 1.4

1.2 1 0.8 0.6 y = 0.0925x + 0.0354 0.4 R² = 0.9992 9

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Figure 7: Calibration curve of ANA at λ1 = 247 nm

Calibration curve of MET at 233 nm 2.5

2

1.5

1 y = 0.0788x - 0.0386 R² = 0.9997 0.5

0 0 5 10 15 20 25 30 35

Figure 8: Calibration curve of MET at λ2 = 233 nm Precision The % RSD of repeatability was found to be 1.083 and 1.098 of test solution containing ANA 2 µg/mL and MET 10 µg/mL. The % RSD of Intraday precision was found to be 1.213 and

1.294 at λ1 and λ2 respectively. The % RSD of Interday precision was found to be 1.291 and

1.310 at λ1 and λ2 respectively. Thus, confirming precision of the method. (Table 3) Table 3: Repeatability, Intraday and Interday Precision of ANA and MET Concentratio n of Test Absorbance* ± SD %RSD solution

(µg/mL)

At λ1 247 At λ2 233 ANA MET At λ1 247 nm At λ2 233 nm nm nm Repeatability 2 10 0.464 ± 0.005 0.867 ± 0.009 1.083 1.098 Intraday 2 10 0.468 ± 0.005 0.867 ± 0.011 1.213 1.294

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Precision Interday 2 10 0.469 ± 0.006 0.868 ± 0.011 1.291 1.310 Precision *Average of six determinations Accuracy Accuracy of the method was confirmed by recovery study from marketed formulation at three level of standard addition method. Percentage recovery for ANA was in range of 99.08 – 101.24 %, while for MET, it was found to be in range of 99.19 – 101.72 %. (Table 4) Table 4: Accuracy data of ANA and MET Amount Amount Total of Recovered of Test Absorbance* Amount % % Drug Standard amount Solution ± SD Found Recovery RSD added (µg/mL) (µg/mL) (µg/mL) (µg/mL) 2 0 0.446 ± 0.002 2.001 --- 100.09 1.317 2 1 0.528 ± 0.003 3.008 1.008 100.29 1.462 ANA 2 2 0.614 ± 0.004 4.049 2.049 101.24 1.259 2 3 0.688 ± 0.005 4.954 2.954 99.08 1.290 10 0 0.874 ± 0.003 9.919 --- 99.193 1.104 10 5 1.206 ± 0.012 15.151 5.151 101.01 1.267 MET 10 10 1.541 ± 0.018 20.344 10.344 101.72 1.320 10 15 1.835 ± 0.024 25.147 15.147 100.35 1.566 *Average of three determinations LOD & LOQ The LOD was calculated by standard formula as given in ICH guidelines was found to be 0.103 µg/mL and 0.167 µg/mL for ANA and MET respectively. The LOQ was calculated by standard formulae as given in ICH guidelines was found to be 0.314 µg/mL and 0.506 µg/mL for ANA and MET respectively. Analysis of ANA and MET in test solution

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The developed methods was applied to sample solution of synthetic mixture. The % Assay of ANA and MET was 100.601% and 100.206 % respectively of the labelled amount. (Figure 9, Table 5) Table 5: Analysis of Tablet formulation Amount of Amount of % Label claimed* ± Drug drug drug % RSD SD Actual Estimated ANA 2 1.994 99.720 ± 0.972 0.974 MET 10 9.931 99.311 ± 1.455 1.465 *Average of six determinations

Figure 9: Spectra of Test solution of ANA (2 µg/mL) and MET (10 µg/mL) Table 6: Summary of Validation Parameters Sr. ANA (247 nm) MET (233 nm) Parameter No. λ1 λ2 1 Specificity Specific Specific 2 Linearity Range 2-12 µg/mL 5-30 µg/mL 3 Regression Line equation y = 0.0925x + 0.0354 y = 0.0788x - 0.0386 4 Correlation Coefficient R² = 0.9992 R² = 0.9997 Precision % RSD 5 Repeatability 1.083 1.098 Intraday Precision 1.213 1.294

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Interday Precision 1.291 1.310 6 Accuracy (% Recovery) 99.08 – 101.24 99.19 – 101.72 7 LOD (µg/mL) 0.103 µg/Ml 0.167 µg/mL 8 LOQ (µg/mL) 0.314 µg/mL 0.506 µg/mL

CONCLUSION The proposed spectrophotometric method is precise, specific, linear and accurate for the estimation of ANA and MET in synthetic mixture. The developed method is validated as per ICH guidelines Q2 R1. The method was successfully used for simultaneous estimation of both drugs in presence of each other. AKNOWLEDGEMENT The authors are graceful to INTAS Pharmaceuticals, Ahmedabad for providing a gift sample of Anagliptin and Metformin. CONFLICT OF INTEREST: Nil REFERENCES 1. “Anagliptin Tablet” (Accessed on March, 2020) https://medical.kowa.co.jp/asset/item/24/35/7-pse_163.pdf 2. “Anagliptin Compound ” (Accessed on April, 2020) https://pubchem.ncbi.nlm.nih.gov/compound/44513473 3. H. Mohan, “Textbook of Pathophysiology”, 6th Edn, Medical publishers Pvt Limited, (2005), pp. 818 - 828. 4. K. D. Tripathi, “Essential of Medical Pharmacology”, 6th Edn, Jaypee Brother Mesical Publishers Ltd, (2008), pp. 254 - 274. 5. Y. Takeuchi, M. Namiki, Y. Kitahara, “Safety, Tolerability, Pharmacokinetics and Pharmacodynamics of E3024, a Novel and Selective Dipeptidyl Peptidase-IV Inhibitor, in Healthy Japanese Male Subjects: Rash Development in Men and Its Possible Mechanism,” Pharmacology & Pharmacy. vol. 4, (2013), pp. 663-678. 6. P. Bhatti, H. Panchal, “Stability Indicating Chromatographic Method Development and Validation for the Simultaneous estimation of Metformin HCl & Anagliptin in its synthetic mixture by HPLC”, World J. of Pharmaceutical Res., vol. 6, (2017), pp. 956-973. 7. R. Majithia, J. Shah, “Development and validation of analytical method for estimation of Anagliptin tablet dosage form by UV spectrophotometric method”, International J. of Pharmacy and Technologies, vol. 6, (2015), pp. 7765–7771. 8. S. Shah, D. Maheshwari, “First order Derivative Spectrophotometric method for Simultaneous Estimation of Anagliptin and Metformin HCl in Bulk and Synthetic Mixture”, J. of Glob. Tr. in Pharmaceutical Sci., vol. 6, (2015), pp. 2925-2929. 9. A. Patil, A. Shirkhedkar, “Development and Validation of Five Simple UV-Spectrophotometry Methods for Estimation of Anagliptin in Bulk and in-house Tablets”, Pharm. Methods., vol. 7, (2016), pp. 127-131. 10. C. Pandya, S. Rajput, “Stress Degradation Studies of Anagliptin, Development of Validated Stability Indicating Method, Degradation Kinetics Study, Identification and Isolation of Degradation Products”, Chromatographia, An International J. of sep. sci. (2018), pp. 1-21. 11. S. Baira, D. Sigalapalli, N. Bathini, “LC/QTOF/MS/MS characterization, molecular docking and in silico toxicity prediction studies on degradation products of Anagliptin”, J. of Pharmaceutical and Biomedical Anal., vol. 159, (2018), pp. 92-99. 12. “Metformin Hydrochloride Compound ” (Accessed on March, 2020) https://www.drugbank.ca/drugs/DB00331 13. W. Roger, Cate W, “Clinical Pharmacy & Therapeutics”, 4th Edn, Churchill Livingstone Elsevier, (2008), pp. 685 - 710. 14. H. Rang, M. Dale, Rang & Dale’s pharmacology, 7th Edn, Elsevier Publication, (2012), pp. 402 - 408

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15. IP: The Indian Pharmacopoeia Commission, 2014, Ghaziabad, India. 16. BP: The British Pharmacopoeia, Medicine & Healthcare Products Regulatory Agency, 2010, London. 17. USP: The United States Pharmacopoeia Convention, 2010, Rockville, MD, USA. 18. C. Parle, A. Parle, “Development and validation of UV spectrophotometric method for simultaneous estimation of metformin hydrochloride and benzoate in bulk drugs and combined dosage forms”, Der Pharma Chemica., vol. 6, (2014), pp. 303-311. 19. J. Chopade, S. Deshpande, S. Shah, “Simultaneous estimation of Metformin HCl and by Q- Analysis Method”, International J. for Pharmaceutical Res. Scho., vol. 2, (2013), pp. 66- 73. 20. P. Umapathi, J. Ayyappan, “Quantitative Determination of Metformin Hydrochloride in Tablet Formulation Containing Croscarmellose Sodium as Disintegrant by HPLC and UV Spectrophotometry”, Tropical J. of Pharmaceutical Res., vol. 11, (2012), pp. 107-116. 21. M. Kar, P. Chaoudhary, “HPLC Method for Estimation of Metformin HCl in Formulated Microspheres and Tablet Dosage Form”, Indian J. of Pharmaceutical Sci., vol. 71, (2009), pp. 318-320. 22. C. Reddy, G. Mubeen, M. Pal, “HPTLC method for estimation of metformin hydrochloride, Biomed. & Pharmacol. J., vol. 1, (2008), pp. 445-448. 23. A. G. Davidson, A. H. Beckett, J. B. Stenlake, “Practical Pharmaceutical Chemistry”, 4th Edn, CBS Publisher, New Delhi, (2002), pp. 270-296. 24. ICH Harmonized Tripartite Guideline, Validation of Analytical Procedures: “Text and Methodology Q2 (R1)”, International Conference on Harmonization, Geneva, Switzerland, (2005), pp. 1 – 12.

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