Spectrophotometric estimation of linagliptin using ion-pair complexation and oxidative coupling reactions -A green approach

Abstract

Background: Two specific spectrophotometric methods (visible region) have been established using water as a solvent and validated for the analysis of linagliptin in API and pharmaceutical dosage forms. Materials and Methods: Method-A is established on the computation of the absorbance of green-colored chromogen complex at 660 nm, which is formed by the oxidative coupling reaction of linagliptin with 3-methyl-2-benzothiazoline hydrazine in the presence of ferric chloride. Method-B is established on the computation of the absorbance of an orange-colored ion-pair chromogen at 490 nm, which is formed by the charge transfer reaction of the primary amino group of linagliptin with picric acid. Results: Beer's law is obeyed in the drug concentration range of 2-12 µg/mL and 1-25 µg/mL for methods A and B, respectively, with a correlation coefficient of 0.999. The contemplated methods are validated statistically according to ICH Q2(R1) guidelines and results are found to be within the acceptable limits. The labeled amount of linagliptin in a marketed formulation (TRAJENTA ®) is determined without interference owed to excipients. Conclusion: The contemplated green approached spectrophotometric methods can prolifically be employed for analysis of linagliptin because of its easy access in most quality control laboratories.

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245 TJPS 2020, 44 (4): 245-250
http://www.tjps.pharm.chula.ac.th
Spectrophotometric estimation of
linagliptin using ion-pair complexation
and oxidative coupling reactions – A
green approach
Sunitha Gurrala, Panikumar Durga Anumolu, Sahitya Menkana,
Nikitha Gandla, Keerthi Toddi
Department of Pharmaceutical Analysis, Gokaraju Rangaraju College of Pharmacy,
Osmania University, Hyderabad, Telangana, India
ABSTRACT
Background: Two specific spectrophotometric methods (visible region) have been established
using water as a solvent and validated for the analysis of linagliptin in API and pharmaceutical
dosage forms. Materials and Methods: Method-A is established on the computation of the
absorbance of green-colored chromogen complex at 660 nm, which is formed by the oxidative
coupling reaction of linagliptin with 3-methyl-2-benzothiazoline hydrazine in the presence of
ferric chloride. Method-B is established on the computation of the absorbance of an orange-
colored ion-pair chromogen at 490 nm, which is formed by the charge transfer reaction of the
primary amino group of linagliptin with picric acid. Results: Beer’s law is obeyed in the drug
concentration range of 2–12 µg/mL and 1–25 µg/mL for methods A and B, respectively, with a
correlation coefficient of 0.999. The contemplated methods are validated statistically according
to ICH Q2(R1) guidelines and results are found to be within the acceptable limits. The labeled
amount of linagliptin in a marketed formulation (TRAJENTA®) is determined without interference
owed to excipients. Conclusion: The contemplated green approached spectrophotometric
methods can prolifically be employed for analysis of linagliptin because of its easy access in most
quality control laboratories.
Keywords: Linagliptin, spectrophotometric, 3-methyl-2-benzothiazoline hydrazine, picric acid
INTRODUCTION
Linagliptin is a competitive and reversible dipeptidyl
peptidase (DPP)-4 inhibitor that slows the breakdown
of insulinotropic hormone glucagon-like peptide
(GLP)-1 for better glycemic control in diabetes patients.
Chemically, it is known as 8-[(3R)-3-aminopiperidin-1-yl]-
7-(but-2-yn-1-yl)-3-methyl-1[(4-methylquinazolin -2-yl)
methyl]-3,7-dihydro-1H-purine-2,6-dione.
All-embracing literature survey revealed that few
analytical methods such as spectrophotometric,[1-7]
spectrofluorimetric,[8] and high-performance liquid
chromatography[9,10] were reported for the determination
of linagliptin. One colorimetric method using NQS
and vanillin as chromogenic reagents was reported.[11]
Colorimetry is found to be more sensitive and specific than
spectrophotometry (UV region). The advantage of using
chromogenic reagent in drug analysis is its selective
chemical reaction with an analyte to form a colored
derivative.[12-16] Visible spectrophotometric methods are
relatively inexpensive, simple, and faster (in terms of
sample preparation) than chromatographic techniques and
can prolifically be adopted for drug analysis due to easy
access in most quality control laboratories.
The present investigation aims to develop economic,
sensitive, and extraction-free visible spectrophotometric
methods for the estimation of the linagliptin subjugating
chemical derivatization technique. Two chromogenic reagents
(3-methyl-2-benzothiazoline hydrazine [MBTH] and picric
acid) are employed for chemical derivatization of linagliptin,
which were not reported earlier. The proposed methods
have high sensitivity and rely on the use of distilled water
as a solvent (eco-friendly) which dwindles the cost of the
experiment.
Corresponding Author:
Sunitha Gurrala,
Department of Pharmaceutical
Analysis, Gokaraju Rangaraju
College of Pharmacy, Osmania
University, Hyderabad,
Telangana, India. E-mail:
g.sunitha88@gmail.com
Received: Mar 08, 2020
Accepted: Jul 10, 2020
Published: Sep 30, 2020
Thai Journal of Pharmaceutical Sciences
Original Article

Gurrala, et al.: Colorimetric estimation of linagliptin
http://www.tjps.pharm.chula.ac.th246 TJPS 2020, 44 (4): 245-250
MATERIALS AND METHODS
Instrumentation and Chemicals
Double beam 1800 UV-Visible spectrophotometer (Shimadzu,
Japan), analytical balance (Shimadzu AUX 220, Japan), and
ultrasonic cleaner (Sonica) were used for the study. Linagliptin
standard gift sample was provided by Dr. Reddy’s Laboratories
Pvt. Ltd., Hyderabad, India. Methanol was purchased from
Qualigens, Mumbai. Ferric chloride, sodium hydroxide, and
chromogenic reagents (MBTH and picric) were purchased from
SD Fine-Chem Ltd., Mumbai. Double distilled water was used
throughout the study. Marketed dosage form (TRADJENTA®)
of linagliptin was procured from the local pharmacy.
Preparation of Solutions
The standard stock solution (1000 µg/mL) of linagliptin
was prepared by solubilizing accurately weighed 10 mg of
linagliptin in 10 mL of methanol. The solution was further
diluted with distilled water to get the required concentration
of linagliptin. Solutions of ferric chloride (3% w/v), MBTH
reagent (0.9% w/v), picric acid (0.4% w/v), and sodium
hydroxide (2M) were prepared in distilled water.
General Analytical Procedures
Method-A
Determination of linagliptin based on measurement of
oxidative-coupling derivative. Linagliptin standard solution
(100 µg/mL) of 1 mL was transferred into a volumetric
flask of 10 mL capacity. To this 2 mL of MBTH (0.9% w/v)
reagent, 2 mL of ferric chloride (3% w/v) was added, shaken
vigorously and kept aside for 10 min for color development.
The volume was contrived up to the mark with distilled water
and absorbance of green colored chromogen was measured at
660 nm against corresponding reagent blank.
Method-B
Determination of linagliptin based on the measurement of
ion-pair complex. Linagliptin standard solution (100 µg/
mL) of 1 mL was transferred into a volumetric flask of 10 mL
capacity. To this 2 mL of picric acid (0.4% w/v) reagent, 2 mL
of sodium hydroxide (2 M) was added, shaken vigorously and
kept aside for 20 min for color development. The volume was
contrived up to the mark with distilled water and absorbance
of orange-colored chromogen was measured at 490 nm against
corresponding reagent blank.
Evidence of Chemical Derivatization
The thin-layer chromatography (TLC) technique was used
with ethyl acetate:acetonitrile (8:2) as a mobile phase on pre-
coated TLC plates. Plates were spotted separately for method-A
and B with a freshly prepared solution of linagliptin, reagent
blank solution, and chromogen produced by that method.
Plates were developed in saturated chromatographic tanks
and spots were visualized in the UV chamber at 254 nm.
Validation of Methods
The proposed methods were validated for linearity, accuracy,
precision, LOD, and LOQ as per the ICH guidelines.[17]
Linearity studies
Method-A
Aliquots of standard drug solution of linagliptin (100 µg/mL)
ranging from 0.2 to 1 mL were taken into a series of 10 mL
volumetric flasks. To these flasks, 2 mL of MBTH (0.9% w/v)
reagent, 2 mL of ferric chloride (3% w/v) were added, shaken
vigorously and kept aside for 10 min for color development.
The volume was contrived up to the mark with distilled water
to get a series of standard solutions containing 2, 4, 6, 8, 10,
and 12 µg/mL of linagliptin. The absorbance of the green-
colored chromogen was measured at 660 nm against the
corresponding reagent blank.
Method-B: Aliquots of standard drug solution of linagliptin
(100 µg/mL) ranging from 0.1 to 2.5 mL were taken into a
series of 10 mL volumetric flasks. To these flasks, 2 mL of picric
acid (0.4% w/v) and 2 mL of sodium hydroxide (2 M) were
added, shaken vigorously and kept aside for 20 min for color
development. The volume was contrived up to the mark with
distilled water to get a series of standard solutions containing
1, 5, 10, 15, 20, and 25 µg/mL of linagliptin. The absorbance
of the orange-colored chromogen was measured at 490 nm
against the corresponding reagent blank.
Precision
The intra-day and inter-day precision of the proposed
colorimetric methods were established with three different
concentrations of linagliptin within the linearity range. These
solutions were prepared in triplicate on the same day and 3
consecutive days over a period of 1 week. The percent relative
standard deviation (% RSD) values were calculated.
Accuracy
The accuracy of the methods was determined by calculating
recoveries of linagliptin by the standard addition method.
Standard solutions of linagliptin were added at 80, 100,
and 120% levels to the pre-quantified sample of linagliptin
and analyzed through proposed methods. Each sample was
prepared in triplicate at each level. The amount of linagliptin
was estimated by applying obtained values to the regression
equation.
Limit of detection (LOD) and limit of quantification (LOQ)
The sensitivity of the proposed methods was determined
concerning LOD and LOQ. These were separately determined
based on standard calibration curve using 3.3 σ/s and 10 σ/s,
formulae, respectively, where s is the slope of the calibration
curve and σ is the standard deviation of the y-intercept of the
regression equation.
Assay of Linagliptin Marketed Dosage
Forms
Twenty tablets of linagliptin (TRADJENTA®) were weighed
and powdered. The powder quantity equivalent to 10 mg of
linagliptin was dissolved in 10 mL methanol and filtered using
Whatman’s filter paper. Filtrate (0.1 mL) was transferred into
a 10 mL volumetric flask, 2 mL of MBTH and 2 mL of FeCl3
(method-A)/2 mL of picric acid and 2 mL of NaOH (method-B)
were added, shaken vigorously and kept aside for 10 min/20 min.
The volume was made up to the mark with water. The absorbance

Gurrala, et al.: Colorimetric estimation of linagliptin
247 TJPS 2020, 44 (4): 245-250
http://www.tjps.pharm.chula.ac.th
of the colored chromogen was measured at 660/490 nm against
the corresponding reagent blank. The amount of linagliptin was
computed from the Beer–Lambert’s plot.
RESULTS AND DISCUSSION
Basis of Chemical Derivatization
In method-A, linagliptin undergoes an oxidative coupling
reaction with MBTH giving green colored chromogen as a
product in the presence of oxidizing agent (ferric chloride).
Oxidation (loss of two electrons and one proton) of MBTH
by ferric chloride produces an electrophilic intermediate,
which coupled at the most nucleophilic site of linagliptin and
forms a green colored chromogen [Figure 1], which absorbs
visible light maximally at a wavelength of 660 nm in a
spectrophotometer.
Method-B involves recceing the charge transfer reaction
of linagliptin with picric acid. The hydroxy group of picric acid
(Lewis acid) transfers a proton to the amine group of linagliptin
(Lewis base) leads to the formation of an ion-pair complex.
This proton transfer reaction between linagliptin and picric
acid produces orange-colored complex [Figure 2] exhibiting
absorption maxima at 490 nm in a spectrophotometer.
Evidence of Chemical Derivatization
Evidence of linagliptin chemical derivatization with proposed
reagents was ascertained by the TLC analysis of reaction
mixture. Three spots were observed with different Rf values on
both plates (method-A and method-B), indicate the presence
of three different compounds. Higher retardation factor values
were observed for derivatives [Table 1], denote the formation
of a new compound by the proposed reaction mechanism.
Optimization of Reaction Conditions
Optimization of reagent concentration, diluting solvent and
time for color development were established to accomplish
maximum absorbance and stability. The influence of variables
on absorption values of colored species was studied by varying
one parameter at a time and keeping others at constant.
Effect of reagent concentration
The effect of MBTH and picric acid concentration on their
reaction with linagliptin was studied by adding 2 mL of
various concentrations of MBTH (0.1, 0.3, 0.6, 0.9, 1.2, and
1.5 %w/v) in method-A/picric acid (0.1, 0.2, 0.3, 0.4, 0.5, and
0.6 %w/v) in method-B to a fixed concentration of linagliptin
(10 µg/mL). It was observed that absorbance values were
increased with increasing reagent concentration up to a
certain level, thereafter decreased absorbance may due to the
saturated level of reagent. In method-A, 0.9% MBTH and in
method-B, 0.4% picric acid was optimized, these levels were
adequate for reproducible and maximum color development.
Table 1: TLC analysis of linagliptin and reaction products
Sample Method-A Method-B
Rf value Rf value
Linagliptin standard 0.05±0.002 0.06±0.003
Reagent blank 0.42±0.01 0.53±0.02
Linagliptin-chemical derivative 0.68±0.03 0.77±0.02
TLC: Thin-layer chromatography
Figure 1: Oxidative coupling reaction between linagliptin and 3-methyl-2-benzothiazoline hydrazine

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Effect of solvents
Diluting solvent plays an important role in the stability of
the colored complex. The effect of different diluting solvents
such as acetone, acetonitrile, ethanol, methanol, and distilled
water have been studied [Figure 3] for measurement under
optimized time and reagent concentration. The best sensitivity,
maximum UV absorption, and product stability were attained
when water was used as a solvent for both the methods. Both
reagents were freely soluble in water. Hence, distilled water
was selected as a diluting solvent for proposed methods that
dwindle the cost of experiment and considered as a green
approach for spectrophotometric method development.
Effect of oxidizing agent/alkalinity
In method-A, ferric chloride was utilized as an oxidizing agent for
coupling reaction between linagliptin and MBTH reagent. It was
found that reagent and an oxidizing agent (3% of ferric chloride)
at a 1:1 ratio were adequate for the formation of an electrophilic
intermediate. In method-B, sodium hydroxide was employed as
an alkaline media for ion-pair complexation between linagliptin
and picric acid. The rate of reaction increases with an increase
in sodium hydroxide concentration (first-order rate kinetic
reaction). There was also a decrease in absorbance with an
increase in hydroxide concentration (more than 2 M) due to a
backward reaction. Hence, 2M sodium hydroxide was optimized
and 1:1 ratio of picric acid:sodium hydroxide was adequate for
the formation of orange-colored chromogen with linagliptin.
Optimization of reaction time
The optimum reaction time was determined by monitoring
the color development at different time intervals. Maximum
absorbance values were obtained at 10 min for method-A
and 20 min for method-B. The color developed for linagliptin
derivatives was stable up to 6 h with both reagents under
optimized conditions.
Stoichiometry of reaction
Stoichiometry of reaction in method-A and method-B was
studied by continuous variation method. Equimolar solutions
(2.11 × 10−5 M) of linagliptin and reagents (MBTH and picric
acid) were prepared in distilled water. The drug and reagent
(MBTH- method A/Picric acid-method B) were mixed in various
proportions to produce different mole ratio values (0, 0.2, 0.4,
0.5, 0.6, 0.8, and 1). The stoichiometric relationship exhibited
in Figure 4. A mole ratio of 0.5 gave the highest absorbance
value for both methods. This indicates that linagliptin has
one center (primary amino group) available for chromogenic
reaction with reagents at their optimum wavelengths.
Validation of Proposed Methods
The proposed methods were statistically validated as per the
ICH guidelines and results are depicted in Table 2. Linear
regression analysis was performed for the Beer’s Law data and
Figure 3: Effect of solvent on linagliptin reaction with 3-methyl-2-
benzothiazoline hydrazine and picric acid
Figure 2: Ion-pair complexation between linagliptin and picric acid

Gurrala, et al.: Colorimetric estimation of linagliptin
249 TJPS 2020, 44 (4): 245-250
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Table 3: Results of precision studies using proposed methods
Method Concentration
linagliptin (µg/mL)
Intra-day analysis Inter-day analysis
Amount found
(AM ±SD),
n
=3
% RSD Amount found
(AM ±SD),
n
=3
% RSD
Method-A 4 3.96±0.011 0.27 4.01±0.012 0.299
6 6.1±0.023 0.377 5.96±0.021 0.352
8 8.1±0.023 0.283 8.27±0.025 0.302
Method-B 5 5.06±0.004 0.079 5.2±0.002 0.038
10 9.92±0.021 0.211 10.2±0.01 0.09
15 14.9±0.019 0.127 14.8±0.02 0.135
calibration plots were drawn (correlation coefficient 0.999). A
linear increase in the absorbance was found with an increase in
linagliptin concentration at a range of 2–12 and 1–25 µg/ml of
linagliptin by methods A and B, respectively. Overlaid UV-Visible
spectra of linagliptin in the linearity range are shown in
Figures 5 and 6. The reproducibility of proposed methods was
evinced by precision studies [Table 3], where no significant
difference between intra- and inter-day precision values was
observed and % RSD values were less than 2. The results of
accuracy studies are demonstrated in Table 4. The % recoveries
of linagliptin denote the fair accuracy of proposed methods
with no interference of tablet excipients. A high value of molar
absorptivity and low values of Sandell’s sensitivity, LOD, and
LOQ signposts the good sensitivity of proposed methods.
Assay of Linagliptin Marketed Dosage
Forms
The proposed methods were applied for the assay of marketed
dosage forms containing linagliptin (label claim 5 mg). The %
assay of linagliptin found to be 99.8 and 100.3 by method-A
Table 2: Optimized characteristics of linagliptin
Parameters Value
MBTH Picric acid
Absorption
wavelength (nm)
660 490
Beers law range
(µg/mL)
2–12 1–25
Regression
equation (y)
Y=0.102x+0.011 Y=0.036x–0.002
Correlation
coefficient(r2)
0.999 0.999
Limit of Detection
(µg/ml)
0.35 0.091
Limit of
Quantification (µg/ml)
1.17 0.27
Molar Absorptivity
(L mole−1cm−1)
0.393×1050.189×105
Sandell’s sensitivity
(µg cm−2)
1.2×10−2 2.5×10−2
Stability of colored
species
6 h 7 h
MBTH: 3-methyl-2-benzothiazoline hydrazine
Figure 4: Job’s continuous variation plot for method-A and method-B
Figure 6: Overlaid UV-Visible spectra of linagliptin (1–15 µg/mL)
with picric acid
Figure 5: Overlaid UV-Visible spectra of linagliptin (2–12 µg/mL)
with 3-methyl-2-benzothiazoline hydrazine

Gurrala, et al.: Colorimetric estimation of linagliptin
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and method-B, respectively. There was no interference of
formulation excipients during the estimation of linagliptin in
tablets. The assay values were found to be within the limits
and % RSD was <2.
CONCLUSION
In this study, two spectrophotometric methods were developed
which evaded the use of organic solvents for analysis of
linagliptin in bulk and pharmaceutical dosage form. Chemical
derivatization mechanisms for linagliptin with MBTH and
picric acid were proposed. Proposed methods were passes
through validation parameters as per the ICH guidelines. The
assay values were in good agreement with the label claim and
suggested that no interference of formulation excipients during
the estimation of the drug. Contemplated methods are more
sensitive, less chemical perilous, and versatile over reported
methods. These have broad linearity range, high precision, and
accuracy. Hence, the proposed eco-friendly and economical
methods can be routinely employed in the quality control for
analysis of linagliptin in the pharmaceutical dosage forms.
ACKNOWLEDGMENTS
The authors express their gratitude the management of
Gokaraju Rangaraju College of Pharmacy for providing
research equipment and facilities.
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