A NOVEL RP – HPLC METHOD FOR THE QUANTIFICATION OF LINAGLIPTIN IN FORMULATIONS

Jamonline / 2(2); 2012 / 155–164 Lakshmi B & Reddy TV

All rights reserved© 2011 www.jamonline.in 155

Research Article

Journal of Atoms and Molecules An International Online JournalAn International Online JournalAn International Online JournalAn International Online Journal ISSN ISSN ISSN ISSN –––– 2277 2277 2277 2277 –––– 1247124712471247

A NOVEL RP – HPLC METHOD FOR THE QUANTIFICATION OF LINAGLIPTIN IN FORMULATIONS

Lakshmi B*1, TV Reddy2 1Kallam Harinadh Reddy College of Engineering, Guntur, Andhra Pradesh, India.

2Department 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

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 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. [1] 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, Glucagon-

like peptide-1 (GLP-1) and Glucose-

* Corresponding author

Lakshmi B,

Email: [email protected]



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]

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

Chromosil C18 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 equipment and

conditions

The development and validation of the assay

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.



Standard and sample solutions

A 10 mg amount of Linagliptin reference

substance was accurately weighed and

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 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.

Method validation

Method validation was performed following

ICH specifications for specificity, range of

linearity, accuracy, precision and robustness

Results and Discussions

System Suitability

Having optimized the efficiency of a

chromatographic separation the quality of the

chromatography was monitored by applying

the system suitability tests: capacity factor,

tailing factor and theoretical plates. The

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

Mobile phase Acetonitrile: Water:

Methanol (25:50:25 (v/v/v)

Pump mode Isocratic

PH 6.4 adjusted with TEA

Diluents Mobile phase

Column Zodiac C18 column (250 X

4.6 mm, 5µ)

Column Temp Ambient

Wavelength 238nm

Injection

Volume 20 µl

Flow rate 1ml/min

Run time 15 minutes

Retention

Time 7.0 minutes

Table.1 System suitability parameters



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



0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

0 20 40 60 80

Are

a

Concentration

Level Concentration of

Linagliptin in PPM Peak Area

Level 1 10 75698

Level 2 20 153278

Level 3 30 227831

Level 4 40 314961

Level 5 50 374682

Level 6 60 453678

Range 10ppm

to

60ppm

SLOPE

INTERCEPT

CORREALATION

COEFFICIENT

7573.8

- 4854.3

0.999

Table.2 Result of Linearity

Graph 1 Linearity

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.



Precision Results for Linagliptin:

Sample

Conc.

(in ppm)

Injection

No. Peak Areas

INTER DAY RSD

(Acceptance criteria

≤ 2.0%)

Linagliptin 40

1 315220

0.448

2 314458

3 312002

4 314141

5 315252

6 313056

Table.3 Result of Intraday Precision

Sample

Conc.

(in ppm)

Injection

No. Peak Areas

INTER DAY RSD

(Acceptance criteria

≤ 2.0%)

Linagliptin 40

1 315656

0.64

2 316296

3 314259

4 318001

5 311985

6 315623

Table.4 Result of Interday precision



Limit of Detection and Limit of

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.

Parameter Measured Value

Limit of

Quantification 0.03ppm

Limit of Detection 0.01ppm

Table.5 Result of LOD and LOQ

Robustness

Typical variations in liquid chromatography

conditions were used to evaluate the

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.

S.No Paramet

er

Condition Area % Of

change

1 Standard Standard

conditions 314961

2 Mobile

phase

Acetonitri

le: Water:

Methanol

(27:46:27

(v/v/v)

313995

99.69

3 Mobile

phase PH 6.1 314006 99.69

4 Wavelen

gth 240 nm 315201 100.07

Table.6 Rubustness results

Recovery

Recovery test was performed at 3 different

concentrations i.e. 20ppm, 40ppm, 60ppm.

Results are given in table.7

Recovery

Conc. of

sample

(ppm)

Recovery

(ppm)

% of

recovery

50% 20 19.94 99.7%

100% 40 39.68 99.2%

150% 60 60.04 100.06%

Table.7 Results of Recovery



Formulation Analysis

S.NO Tablet Dosage Sample

conc

Sample

estimated

% of Drug Estimated

in Tablet

1 TRADJENTA 5mg 40ppm 39.692 99.23

Table.8

Figure.3 chromatogram of Formulation



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

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.

http://www.genengnews.com/gen-news-

highlights/fda-approves-type-2-diabetes-

drug-from-boehringer-ingelheim-and-

lilly/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-news-

highlights/four-phase-iii-trials-confirm-

benefits-of-bi-s-oral-once-daily-type-2-

diabetes-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

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

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

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



s/fs312/en/. Accessed on: August 16,

2011.

9. International Diabetes Federation.

Diabetes Atlas. 3rd edn. Brussels:

International Diabetes Federation, 2006.

Documents

A NOVEL RP – HPLC METHOD FOR THE QUANTIFICATION OF LINAGLIPTIN IN FORMULATIONS