Development and Validation of RP-HPLC Method for the Estimation of Alogliptin in API And Tablet Formulation

 

Suyash Ingle*, Varsha Tegeli, Avinash Birajdar, Gajanand Nangare

D.S.T.S. Mandals College of Pharmacy, Solapur - 413004, Maharashtra, India.

*Corresponding Author E-mail: suyashingle18@gmail.com

 

ABSTRACT:

The analytical method was developed and validated for determination of Alogliptin in bulk and pharmaceutical dosage forms by High performance liquid chromatography. The separation was carried out on Zorbax SB-Aq (250 x4.6mm,5 ID) column. The mobile phase consists of 0.1% TFA Water : ACN in the ratio 62:38 at flow rate 1ml/min with diode array detector wavelength at 290nm.The column temperature was adjusted at 30 0.5C with injection volume 10l.The retention time of Alogliptin was 3.06min. The linearity of the calibration curve was linear over the concentration range 25-75g/ml (r2=1). The validation was carried out as per ICH guidelines. The development of method was easy, rapid, linear, precise, accurate and consistent.

 

KEYWORDS: Alogliptin, RP-HPLC, Validation, Method development, 290nm and diode array detector.

 

 


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 methods7,8, RPHPLCmethod9-15 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. 0.45m Millipore syringe filters(UltiporN66Nylon 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, 5 ID)

Mobile phase

0.1% TFA Water and Acetonitrile

Mobile phase ratio

62:38

Diluent

100% Water

Flow rate

1ml/min.

Injection Volume

10l

Detection

290nm in UV detector

Temperature

300 C

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. = 500g/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. = 50g 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 0.45 membrane filter twice.

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

 

3.0 VALIDATION OF ANALYTICAL METHOD9-16

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 :


 

Fig 2-Chromatogram of Alogliptin

 

Figure 3: Linearity overlay of Alogliptin


 

Table No.2 - Alogliptin concentrations

% Concentration

Alogliptin Conc.(g/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 30C0.5C. Sample and standard samples were injected in the volume of 10l 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 g/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(g/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

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

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

 

Std.Weight (mg)

Purity (%)

Potency (ug/m)

5

99.7

498.5

 


Table 9 : LOD and LOQ results

Parameter

g/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 25-75g/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 37.5g/ml,50g/ml, 62.5g/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 0.29g/ml, LOQ was found to be 0.89g/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.

 

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Received on 08.10.2020 Modified on 11.08.2021

Accepted on 31.12.2021 RJPT All right reserved

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

DOI: 10.52711/0974-360X.2022.00300