Jamonline / 2(2); 2012 / 155–164 Research Article

Lakshmi B & Reddy TV

Journal of Atoms and Molecules
An International Online Journal
ISSN – 2277 – 1247

A NOVEL RP – HPLC METHOD FOR THE QUANTIFICATION OF LINAGLIPTIN IN FORMULATIONS Lakshmi B*1, TV Reddy2 Kallam Harinadh Reddy College of Engineering, Guntur, Andhra Pradesh, India. Department of Chemistry, P.B Siddhartha College of Arts and Science, Vijayawada, India. Received on: 06-03-2012 Revised on: 27-03-2012 Accepted on: 09–04–2012
2 1

Abstract: A simple, precise and accurate RP-HPLC method was developed and validated for rapid assay of Linagliptin in tablet dosage form. Isocratic elution at a flow rate of 1.0 ml/min was employed on a symmetry Chromosil C18 (250x4.6mm, 5µm in particle size) at ambient temperature. The mobile phase consisted of Acetonitrile: Water: Methanol (25:50:25 (v/v/v). The UV detection wavelength was 238 nm and 20µl sample was injected. The retention time for Linagliptin was 7 min. The percentage RSD for precision and accuracy of the method was found to be less than 2%. The method was validated as per the ICH guidelines. The method was successfully applied for routine analysis of Linagliptin in tablet dosage form and bulk drug. Key Words: Linagliptin, RP-HPLC, UV detection, recovery, precise, 238 nm. Introduction: Linagliptin is a DPP-4 inhibitor developed by Boehringer * Corresponding author Lakshmi B, Email: lakshmi_bumi@yahoo.com Ingelheim (German

Pharmaceutical Company) for treatment of type II diabetes. Linagliptin was approved by the US FDA on 2 May 2011 for treatment of type II diabetes.

It is being marketed by

Boehringer Ingelheim and Lilly. Linagliptin is an inhibitor of DPP-4

(dipeptidyl peptidase 4) an enzyme that degrades the incretion hormones, Glucagonlike All rights reserved© 2011 peptide-1 (GLP-1) and Glucose-

www.jamonline.in 155

Jamonline / 2(2); 2012 / 155–164 dependent Insulinotropic polypeptide (GIP). Both GLP-1 and GIP increase insulin biosynthesis and secretion from pancreatic beta cells in the presence of normal and elevated blood glucose levels. GLP-1 also reduces glucagon secretion from pancreatic alpha cells, resulting in a reduction in hepatic glucose output. Thus, Linagliptin stimulates the release of insulin in a glucose-dependent manner and decreases the levels of glucagon in the circulation. Linagliptin showed that the drug can effectively reduce blood sugar. [2] Chromosil C18

Lakshmi B & Reddy TV 250×4.6mm, Electronic

balance-Denver (SI234), a manual Rheodyne injector with a 20µl loop was used for the injection of sample. Peak LC software was used. UV 2301 Spectrophotometer was used to determine the wavelength of maximum absorbance. Determination of wavelength of maximum absorbance The standard solutions of Linagliptin were scanned in the range of 200-300 nm against mobile phase as a blank. Linagliptin showed maximum absorbance at 238nm. So the wavelength selected for the determination of Linagliptin was 238 nm. Chromatographic conditions The development and validation of the assay equipment and

Figure 1: Structure of Linagliptin Experimental Materials Working standard of Linagliptin was obtained from well reputed research laboratories. HPLC grade water, methanol, Acetonitrile was purchased from E. Merck (Mumbai, India). Apparatus A Series HPLC system PEAK LC7000, isocratic HPLC with PEAK 7000 delivery system. Rheodyne manual sample injector with switch (77251), Analytical column

was performed on A Series 200 HPLC system Peak LC7000 isocratic HPLC with Peak 7000 delivery system. Rheodyne manual sample injector with switch (77251), Analytical column Chromosil 100-5 C18. 250×4.6mm, manual injector Rheodyne valve) with 20µl fixed loop, PEAK LC software was used. The mobile phase consisted of Acetonitrile: Water: Methanol (25:50:25 (v/v/v). Injections were carried out using a 20µl loop at room temperature (20 + 2 °C) and the flow rate was 1 ml/min. Detection was performed at 238 nm with 15.0 min runtime.

All rights reserved© 2011

www.jamonline.in 156

Jamonline / 2(2); 2012 / 155–164 Standard and sample solutions A 10 mg amount of Linagliptin reference substance was accurately weighed and

Lakshmi B & Reddy TV system suitability method acceptance criteria set in each validation run were: capacity factor >2.0, tailing factor ≤2.0 and theoretical plates >2000. In all cases, the relative standard deviation (R.S.D) for the analytic peak area for two consecutive injections was < 2.0%. A chromatogram obtained from reference substance solution is presented. System suitability parameters were shown in Table.1. Standard chromatogram was given in Figure.2

dissolved in 10 ml mobile phase in a 10 ml volumetric flask to obtain 1000ppm

concentrated solution. From standard solution by the serial dilution we prepared required concentrations of 100ppm. From this 4 ml was taken and made up to 10 ml using mobile phase. A composite of 20 tablets was prepared by grinding them to a fine, uniform size powder. 10 mg of Linagliptin was accurately weighted and quantitatively transferred into a 100 ml volumetric flask. Approximately 25 ml

Mobile phase

Acetonitrile: Water: Methanol (25:50:25 (v/v/v) Isocratic 6.4 adjusted with TEA Mobile phase Zodiac C18 column (250 X 4.6 mm, 5µ) Ambient 238nm

Pump mode PH Diluents

mobile phase were added and the solution was sonicated for 15 min. The flask was filled to volume with mobile phase, and mixed. After filtration, an amount of the solution was diluted with mobile phase to a concentration of 40µg/ml.


Column Temp Method validation Method validation was performed following ICH specifications for specificity, range of linearity, accuracy, precision and robustness Results and Discussions System Suitability Run time Having optimized the efficiency of a Retention Time chromatographic separation the quality of the chromatography was monitored by applying the system suitability tests: capacity factor, tailing factor and theoretical plates. The Wavelength Injection Volume Flow rate

20 µl

1ml/min 15 minutes

7.0 minutes

Table.1 System suitability parameters

All rights reserved© 2011

www.jamonline.in 157

Jamonline / 2(2); 2012 / 155–164

Lakshmi B & Reddy TV

Figure.2 Typical chromatogram of Linagliptin

Range of linearity Standard curves were constructed daily, for three consecutive days, using seven standard concentrations in a range of 10, 20, 30, 40, 50, 60µg/ml for Linagliptin. The linearity of peak area responses versus concentrations was demonstrated by linear least square regression analysis. The linear regression equation was y

= 7573- 4854x (r= 0.999). Linearity values can shown in Table: 2

All rights reserved© 2011

www.jamonline.in 158

Jamonline / 2(2); 2012 / 155–164

Lakshmi B & Reddy TV


Concentration of Linagliptin in PPM 10 20 30 40 50 60 SLOPE

Peak Area

Level 1 Level 2 Level 3 Level 4 Level 5 Level 6

75698 153278 227831 314961 374682 453678 7573.8 - 4854.3 0.999

Range 10ppm INTERCEPT to CORREALATION 60ppm COEFFICIENT Table.2 Result of Linearity

500000 450000 400000 350000 300000 Area 250000 200000 150000 100000 50000 0 0 20 40 Concentration 60 80

Precision To study precision, six replicate standard solutions of Linagliptin (40ppm) were

prepared and analyzed using the proposed method. The percent relative standard

deviation (% RSD) for peak responses was calculated and it was found to be which is well within the acceptance criteria of not more than 2.0%. Results of system precision studies are shown in Table.3 and Table.4.

Graph 1 Linearity

All rights reserved© 2011

www.jamonline.in 159

Jamonline / 2(2); 2012 / 155–164 Precision Results for Linagliptin:

Lakshmi B & Reddy TV

Conc. Sample (in ppm)

INTER DAY RSD Injection No. Peak Areas (Acceptance criteria ≤ 2.0%) 1 2 3 315220 314458 312002 0.448 4 5 6 314141 315252 313056



Table.3 Result of Intraday Precision

Conc. Sample (in ppm)

INTER DAY RSD Injection No. Peak Areas (Acceptance criteria ≤ 2.0%) 1 2 3 315656 316296 314259 0.64 4 5 6 318001 311985 315623



Table.4 Result of Interday precision

All rights reserved© 2011

www.jamonline.in 160

Jamonline / 2(2); 2012 / 155–164 Limit of Detection and Limit of
S.No Paramet er

Lakshmi B & Reddy TV

Quantification: To determine the Limit of Detection (LOD) sample was dissolved by using Mobile phase and injected until peak was disappeared. After 0.01 ppm dilution Peak was not clearly observed, based on which 0.01 ppm is considered as Limit of Detection and Limit of Quantification is 0.03 ppm.



% Of change



Standard conditions Acetonitri



Mobile phase

le: Water: Methanol (27:46:27 (v/v/v) 313995 99.69

Parameter Limit of Quantification Limit of Detection

Measured Value 0.03ppm 0.01ppm 4 Wavelen gth 3 Mobile phase PH




240 nm



Table.5 Result of LOD and LOQ

Table.6 Rubustness results

Recovery Robustness Typical variations in liquid chromatography conditions were used to evaluate the Recovery test was performed at 3 different concentrations i.e. 20ppm, 40ppm, 60ppm. Results are given in table.7

robustness of the assay method. In this study, the chromatographic parameters monitored were retention time, area, capacity factor, tailing factor and theoretical plates. The robustness acceptance criteria set in the validation were the same established on system suitability test describe above. 50% 100% 150% 20 40 60 19.94 39.68 60.04 99.7% 99.2% 100.06% Recovery Conc. of sample (ppm) Recovery (ppm) % of recovery

Table.7 Results of Recovery

All rights reserved© 2011

www.jamonline.in 161

Jamonline / 2(2); 2012 / 155–164 Formulation Analysis

Lakshmi B & Reddy TV




Sample conc 40ppm Table.8

Sample estimated 39.692

% of Drug Estimated in Tablet 99.23




Figure.3 chromatogram of Formulation

All rights reserved© 2011

www.jamonline.in 162

Jamonline / 2(2); 2012 / 155–164 Conclusion The proposed method for the assay of Linagliptin in tablets or capsules is very simple and rapid. It should be emphasized it is isocratic and the mobile phase do not contain any buffer. The method was validated for specificity, linearity, precision, accuracy and robustness. Although the method could effectively separate the drug from its

Lakshmi B & Reddy TV controlled trial. Diabetes Obes Metab 2011;13:193-287. 4. Barnett AH, Harper R, Toorawa R,: Linagliptin monotherapy improves

glycaemic control in type 2 diabetes patients for whom metformin therapy is inappropriate, 46th Annual Meeting of the European Association for the Study of Diabetes. Stockholm, Sweden, 2010, pp Poster 823. 5. Owens DR, Swallow R, Woerle HJ, et al: Linagliptin improves glycemic control in type 2 diabetes patients inadequately

products, further studies should be performed in order to use it to evaluate the stability of pharmaceutical formulations. References 1. "FDA Approves Type 2 Diabetes Drug from Boehringer Ingelheim and Lilly". 3 May 2011.

controlled by metformin and sulfonylurea without weight gain and low risk of hypoglycemia, 70th American Diabetes Association Scientific Sessions. Orlando, Florida, U.S.A., 2010, ppPoster 548-P. 6. Lewin AJ, Arvay L, Liu D, : Safety and efficacy of linagliptin as add-on therapy to a sulphonylurea in inadequately controlled type 2 diabetes, 46th Annual Meeting of the European Association for the Study of Diabetes. Stockholm, Sweden, 2010, pp Poster 821-P. 7. Centers for Disease Control and

http://www.genengnews.com/gen-newshighlights/fda-approves-type-2-diabetesdrug-from-boehringer-ingelheim-andlilly/81245092/. 2. "Four Phase III Trials Confirm Benefits of BI’s Oral, Once-Daily Type 2 Diabetes Therapy". Genetic Engineering &

Biotechnology News. 28 June 2010. http://www.genengnews.com/gen-newshighlights/four-phase-iii-trials-confirmbenefits-of-bi-s-oral-once-daily-type-2diabetes-therapy/81243585 3. Del Prato S, Barnett AH, Huisman H, : Effect of linagliptin monotherapy on glycaemic control and markers of B-cell function in patients with inadequately controlled type 2 diabetes: a randomised All rights reserved© 2011

Prevention. National Diabetes Fact Sheet 2011. Available at

http://www.cdc.gov/diabetes/pubs/pdf/ndf s_2011.pdf. Accessed on: August 16, 2011. 8. World Health Organization. Fact Sheet No. 312: What is Diabetes? Available at: http://www.who.int/mediacentre/factsheet

www.jamonline.in 163

Jamonline / 2(2); 2012 / 155–164 s/fs312/en/. Accessed on: August 16, 2011. 9. International Diabetes Atlas. Diabetes 3rd edn. Federation. Brussels:

Lakshmi B & Reddy TV

International Diabetes Federation, 2006.

All rights reserved© 2011

www.jamonline.in 164