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1
Department of Chemistry, Acharya Nagarjuna University, Nagarjuna Nagar-522510, India.
2
GITAM University – Bengaluru, Karnataka-562163, India.
3
Department of Science and Humanities, Vignan’s Foundation for Science,
Technology and Research, Vadlamudi-522213, India.
*Corresponding author E-mail: doctornadh@yahoo.co.in
http://dx.doi.org/10.13005/ojc/350145
ABSTRACT
A simple method is described to determine the amount of gemigliptin in bulk and tablet
formulation by visible spectrophotometry. Basis of the proposed method is the reaction of the primary
amine present on gemigliptin with ninhydrin in alkaline pH (alkaline borate buffer) medium to produce
a purple color (Ruhemann’s purple) which has maximum absorption at 558 nm. The method was
validated as per the current ICH guidelines. The obtained regression equation (y = 0.0148x+0.0011)
in the range of 5-30 μg mL-1 has a good correlation coefficient (> 0.999). As the method does not
require any separation, it is rapid and simple. The recovery levels of the drug were in the range of
99.73 – 99.96. This method is a green method as it no organic solvents were employed.
This is an Open Access article licensed under a Creative Commons license: Attribution 4.0 International (CC- BY).
Published by Oriental Scientific Publishing Company © 2018
Ratnakaram et al., Orient. J. Chem., Vol. 35(1), 363-369 (2019) 364
Mechanism involved in the formation of with ninhydrin reagent via oxidative deamination
Ruhemann’s purple depends upon the nature of of the primary amino group followed by the
substrate (amino acid, imino acid, primary amine, condensation of the reduced ninhydrin to form the
secondary amine, pyrrole and ammonium salt 37. coloured purple reaction product was reported with
In the case of amino acids, ninhydrin undergoes gabapentin38), lisinopril24, pregabalin15, amlodipine25,
tautomerization to form 1,2,3-indantrione which
famotidine24,39. The postulated reaction mechanism
reacts with amino acid to give Schiff’s base. An
involves the essential existence of ninhydrin and
aldehyde is yielded by decarboxylation of the formed
as well as its reduced form to form Ruhemann’s
ketamine along with formation of an intermediate
amine which condenses with another ninhydrin purple by amines40. Condensation with both forms
molecule to results in the product – Ruhemann’s of ninhydrin proceeds through either simultaneously
purple12. However, reaction of pharmaceutical drugs or sequentially (Figure 4).
O O H R1 O
OH H2N R1 N C H
+ CH N + R1OH
OH H2O OHR2
R2 CHR2
O O OH
Ninhydrin hydrate Gemigliptin
F
F F
O
N
N N F
R1 = R2 = N
F
F O F
F
O O O O
OH
N + N + R2CHO + H2O
OH
CHR2
OH O O O
Ruhemann Purule
Fig. 4. Mechanism for reaction between primary amine containing gemigliptin and ninhydrin
Validation of Method
Linearity and range
Linearity was observed in the concentration
range of 5 – 30 μg mL-1 (Table 1, Fig. 5). The
proposed analytical method is linear due to high
correlation coefficient (> 0.999) for the observed
linear regression equation y = 0.0148x+0.0011.
Different parameters (optical and regression) are
listed out in Table 2.
Table 1: Calibration values of
gemigliptin
Concentration
(µg mL-1) Absorbance*
Fig. 5. Calibration graph of gemigliptin
5 0.0751
Accuracy
10 0.1494
15 0.2242 Percent recovery values were determined in
20 0.2941 order to ascertain the accuracy of the proposed method.
25 0.3651 Various quantities of gemigliptin (5, 10 and 15 µg mL-1)
30 0.4482 were added to a nominal amount (10 µg mL-1) to
*Average of three determinations study recovery levels of 50%, 100% and 150%
Ratnakaram et al., Orient. J. Chem., Vol. 35(1), 363-369 (2019) 367
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