1.0 INTRODUCTION:

Alogliptin-{2-[[6-[(3R)-3-aminopiperidine-1-yl]-3-methyl-2,4-dioxopyrimidin-1-yl]methyl]benzonitrile} an oral anti-diabetic drug in the DPP-4 inhibitor(gliptins) class that decreases blood glucose level in case of type-2 diabetes mellitus. It has been used in diabetes with dose of 25mg once a daily and dose may varies with any medical disorders, Where as it is soluble in methanol and water. Alogliptin does not undergo extensive metabolism. Two minor metabolites that were detected are N-demethylated alogliptin (<1% of parent compound) and N-acetylated Alogliptin (<6% of parent compound). The N-demethylated metabolite is active and an inhibitor of DPP-4. The N-acetylated metabolite is inactive.

Figure1: Structure of Alogliptin

Alogliptin inhibits dipeptidyl peptidase 4 (DPP-4), which normally degrades the incretins glucose-dependent insulin tropic polypeptide (GIP) and glucagon like peptide 1(GLP-1). The inhibition of DPP-4 increases the amount of active plasma incretins which helps with glycemic control. GIP and GLP-1 stimulate glucose dependent secretion of insulin in pancreatic beta cells. GLP-1 has the additional effects of suppressing glucose dependent glucagon secretion, inducing satiety, reducing food intake, and reducing gastric emptying. From the literature survey, it was found that development and validation of Alogliptin in bulk and pharmaceutical dosage form by analytical methods such as UV-vis. spectrophotometry methods^7,8, RPHPLCmethod^9-15 and HPTLC method¹⁶. Alogliptin is non-ionizable and retains long in reverse phase chromatography. The developed method was simple, precise, specific and accurate. The statistical analysis proved that method is reproducible and selective for the analysis of Alogliptin in bulk drug and tablet formulations.

2.0 MATERIALS AND METHOD:

2.1 Chemicals and reagents:

The drug Alogliptin was obtained as gift sample from Aadhar Life Sciences. RO Water and HPLC grade Acetonitrile and TFA(Tetra flouro-acetic acid) (Merck) Mumbai, India. 0.45µm Millipore syringe filters(Ultipor^®N₆₆^®Nylon Membrane) were also from Adhar Life Sciences, India.

2.2 Instruments:

Analytical balance (Aczet CY224C), HPLC (Agilent 1260 Infinity II autosampler), Vortex machine (Remi CM 101 plus), Sonicator (Labman).

2.3 Chromatographic equipment and conditions:

Table No. 1: optimized chromatographic conditions for the proposed method

Parameters	Optimized Conditions
Column	Zorbax SB-Aq (250 x4.6mm, 5µ ID)
Mobile phase	0.1% TFA Water and Acetonitrile
Mobile phase ratio	62:38
Diluent	100% Water
Flow rate	1ml/min.
Injection Volume	10µl
Detection	290nm in UV detector
Temperature	30⁰C
Retention time	3.06 min.
Run time	10 min.

2.4 Preparation of standard stock solution:

a. Initially Prepare a Standard Stock Solution (SSS-I) of Alogliptin by adding 5mg in 10ml volumetric flask and add 5ml diluent and Mix and sonicate for 5 minutes. Make up the volume to 10ml with diluent. (Conc. = 500µg/ml)

b. /Pipette out 1.0ml of SSS-I in 10ml volumetric flask. Add 5 ml diluent and vortex; make up the volume with diluent. (Conc. = 50µg ml)

2.5 Preparation of Mobile Phase:

a. HPLC grade Acetonitrile and purified RO water used for the preparation of mobile phase

b. Accurately measured the each component of the mobile phase (62% 0.1%TFA Water and 38%ACN).

c. Each component of mobile phase filtered through 0.45µ membrane filter twice.

d. Sonication is performed for 15mins.to degas the mobile phase.

3.0 VALIDATION OF ANALYTICAL METHOD^9-16

I. Specificity and Assay:

Blank, working standard and Drug product were injected and were observed for any diluent peak interfering with the main peak and assay was also calculated. This solution was assayed at 290nm.

II. Linearity and Range:

Linearity of an analytical procedure is its ability to obtain test results which is directly proportional to the concentration of analyte present in the sample whereas range of an analytical procedure is the differential interval between the upper and lower concentration of analyte present in the sample.5 samples of varying concentration ranging from 50 to 150% were prepared. The concentrations are given in Table No. 2 :

Table No.2 - Alogliptin concentrations

% Concentration	Alogliptin Conc.(µg/ml)
50	25
75	37.5
100	50
125	62.5
150	75

III. Instrument Precision and System Suitability:

Precision of an analytical parameter is the closeness of test results to that of the true value obtained from multiple sampling of a homogenous sample under the prescribed experimental conditions. Whereas here instrument precision was performed by preparing a single sample solution and 5 injections were made from same sample and checked for system suitability. The system suitability parameters are mentioned in table no.3

Table No.3

Parameters	Values
Theoretical plates	13434
Retention time	3.06 min.
Wavelength	290
Asymmetry(Tailing Factor)	1.09

IV. Accuracy:

The accuracy of an analytical parameter is the closeness of the test results to true value. Accuracy of the proposed method was done by standard addition method or recovery study by spiking out the standard stock solution.

a. Samples were made of 75%, 100% and 125% concentration as per table no.2 for Alogliptin.

b. Samples were injected in duplicate to calculate % RSD.

c. % recovery was also calculated.

V. LOD and LOQ:

The limit of detection (LOD) gives information about the lowest amount of analyte present in the sample which can be easily detected but not necessarily quantified.

Limit of quantitaion (LOQ) of an analytical parameter gives information about the lowest amount of analyte present in the sample which can be easily detected, and quantified with suitable precision and accuracy.

LOD and LOQ were determined using the following equations;

LOD= 3.3*σ/S

LOQ= 10*σ/S

Where, σ = Standard deviation, and S= slope of the regression coefficient.

4.0 RESULTS AND DISCUSSION:

4.1 Method Development:

The proposed HPLC method was developed and optimized for a series of trails in the terms of mobile phase selection, composition, wavelength, choice of stationary phase of column, flow rate and column temperature. Alogliptin showed the absorbance maxima 290nm. Hence this wavelength was chosen as a working wavelength for the proposed HPLC method.

4.2 Method Validation:

The optimum ratio of mobile phase for 0.1%TFA Water and Acetonitrile was finalized that 62:38 respectively, at the flow rate of 1ml/min. The separation was achieved using Zorbax XB-Aqs.column at the column temperature of 30°C±0.5°C. Sample and standard samples were injected in the volume of 10µl via auto sampler. The retention time of Alogliptin in this proposed method was found to be 3.06 minutes.

Table 4 :Specificity and assay results

Sample	Area	Assay
WS	1070248	-
Drug Product	1068217	99.81

Table 5 : Linearity results

% Concentration	Concentration µg/ml	Area
50	25	535388
75	37.5	802477
100	50	1070080
125	62.5	1332910
150	75	1600052

Fig.4 Calibration curve of Alogliptin

Table No.6- Regression analysis

Parameters	Values
Linearity range(µg/ml)	25-75
Regression Coefficient(r²)	1
Regression equation	y = 21278x +4277
Slope	21278
Intercept	4277

Table No.7: Precision results

Sample	Area	TP	ASY	RT
Reps1	1070080	13335	1.04	3.05
Reps2	1068587	13358	1.08	3.05
Reps3	1073240	13348	1.13	3.05
Reps4	1068833	13345	1.07	3.05
Reps5	1070500	13306	1.14	3.05
Average	1070248
STDEV	1857.75
%RSD	0.17

Table No.8-Accuracy results

Std Area	1070248
Sample ID	Conc. (ug/ml)	Area	Amount Recovered (ug/ml)	% Recovery	Average	STDEV	RSD
75% Rep 1	37.39	802477	37.38	99.97	99.96	0.0266	0.03
75% Rep 2	37.39	802175	37.36	99.94	99.96	0.0266	0.03
100% Rep 1	49.85	1070080	49.84	99.98	99.91	0.0986	0.10
100% Rep 2	49.85	1068587	49.77	99.84	99.91	0.0986	0.10
125% Rep 1	62.31	1332910	62.08	99.63	99.73	0.1332	0.13
125% Rep 2	62.31	1335431	62.20	99.82	99.73	0.1332	0.13

Std.Weight (mg)	Purity (%)	Potency (ug/m)
5	99.7	498.5

Table 9 : LOD and LOQ results

Parameter	µg/ml
LOD	0.29
LOQ	0.89

5.0 DISCUSSION:

1. Specificity and Assay:

The sample solution was assayed at 290nm and the results were found to be 99.81%w/w which fits within the range of 99.5-100.5%w/w. Hence the method was specific.

2. Linearity and Range:

Linearity of sample was performed for range of 25-75µg/ml, and the regression coefficient was found to be 1.0. Hence the method was linear within given range.

3. Instrument Precision:

As per ICH guidelines the limit for precision is NMT 2% RSD, the above developed method shows the precision of 0.17%RSD, which complies with the ICH guidelines. Hence the method was precise.

4. Accuracy:

Accuracy was performed for 37.5µg/ml,50µg/ml, 62.5µg/ml and the % recovery was found to be 99.96, 99.91, and 99.71% respectively. The developed method was accurate.

5. LOD and LOQ:

LOD was found to be 0.29µg/ml, LOQ was found to be 0.89µg/ml. Hence the developed method was validated for all the above parameters.

6.0 CONCLUSION:

The proposed method of validation for estimation of Alogliptin in Tablet formulation has proven to be very specific, linear, accurate, precise, sensitive, and economic. The method exhibits simplicity in terms of short analysis time, isocratic mode of elution of mobile phase, effective and clear resolution with low LOD and LOQ values. The proposed method was developed and validated as per the ICH guidelines.

7.0 ACKNOWLEDGEMENT:

We are thankful to the Principal College of Pharmacy, Solapur and Adhar life sciences pvt. limited, Solapur for providing required facilities for research work.

8.0 REFERENCES:

1. G. Srinivasa Rao, K. Mallesh, G. Vijay Kumar, Ch. Surekha and B, Venugopala Rao, A Validated Chiral HPLC Method for the Enantiomeric Purity of Alogliptin Benzoate, Der Pharma Chemica, 6(3), 234-239 (2014). https://www.derpharmachemica.com/abstract/a-validated-chiral-hplc-method-for-the-enantiomeric-purity-of-alogliptin-benzoate-1909.html

2. International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text on Validation of Analytical Procedures Q2A, 1994. https://www.ema.europa.eu/en/documents/scientific-guideline/ich-q-2-r1-validation-analytical-procedures-text-methodology-step-5_en.pdf

3. R. I. El-Bagary, E. F. Elkady and B. M. Ayoub, Liquid Chromatographic Determination of Alogliptin in Bulk and in its Pharmaceutical Preparation, Int. J. Biomed. Sci., 8(3), 1-5 (2012). https://eurekamag.com/research/054/140/054140476.php

4. International Conference on Harmonization (ICH), Validation of Analytical Procedures: Methodology Q2B, 1996. https://www.ikev.org/haber/stabilite/kitap/36%201.8%20%20Stability%20Workshop%20ICH%20Q2B%20C%20.pdf

5. P. J. Yadav, S. Jadhav amd K. Mohite, Development and Validation of RP-HPLC Method for Alogliptin Benzoate in Bulk Drug and Dosage Form, IJPPR. Human, 1(2), 1-9(2014). https://www.ijppr.humanjournals.com/wp,content/uploads/2014/08/Yadav_Priyanka_J.__Jadhav_Sayali_S.__Mohite_S._K..pdf

6. International Conference on Harmonization (ICH), Validation of Analytical Procedures: Text and Methodology Q2 (R1), 2005. https://database.ich.org/sites/default/files/Q2%28R1%29%20Guideline.pdf

7. Usami Yoshiko, Tsuyoshi Oki, Naka Masahiko, Sagisaka Masafumi, Kaneda Tsuguhiro. A simple HPLC method for simultaneous determination of Lopinavir, Ritonavir and Efavirenz. Journal-Chemical and pharmaceutical bulletin 2003; 51:715-718. https://pubmed.ncbi.nlm.nih.gov/12808252/

8. Mabrouk M. M., Hammad S. F., Mansour F. R., Amer M. M. 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(2):32–40.10.1155/2018/1902510

9. Y. Zhou, W. Zhou, L. Sun, Q. Zou, P. Wei and P. O. Yang, Characterization of Process-Related Impurities Including Forced Degradation Products of Alogliptin Benzoate and the Development of the Corresponding Reversed-Phase HighPerformance Liquid Chromatography Method, J. Sep. Sci., 37(11), 1248-1255 (2014). 10.1002/jssc.201301384

10. Hani Naseef, Ramzi Moqadi, and Moammal Qurt. Development and Validation of an HPLC Method for Determination of Antidiabetic Drug Alogliptin Benzoate in Bulk and Tablets 2018;4(3) | https://doi.org/10.1155/2018/1902510

11. Williams DA, Lemke TL, editors. Foye’s Principles of Medicinal Chemistry. 7th edition Philadelphia: Lippincott Williams and Wilkins, Baltimore: Wolters and Kluwer Business;2013:1283 https://creighton.pure.elsevier.com/en/publications/foyes-principles-of-medicinal-chemistry-seventh-edition

12. Suchita VG, Varsha M, Jadhav and Vilasrao JK High pressure liquid chromatographic method development and validation for estimation of Alogliptin in bulk and marketed formulation. International J.of pharmaceutical sciences and research 2016;7(5):2194-2199. RP-HPLC, Alogliptin benzoate, Glibenclamide, Validation.

13. Indian Pharmacopeia 2007. Vol. II: 376.

Received on 08.10.2020 Modified on 11.08.2021

Research J. Pharm.and Tech 2022; 15(4):1791-1794.

DOI: 10.52711/0974-360X.2022.00300