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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]
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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.
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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
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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
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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.
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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
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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
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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
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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
Jamonline / 2(2); 2012 / 155–164 Lakshmi B & Reddy TV
All rights reserved© 2011 www.jamonline.in 164
s/fs312/en/. Accessed on: August 16,
2011.
9. International Diabetes Federation.
Diabetes Atlas. 3rd edn. Brussels:
International Diabetes Federation, 2006.
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