Utility of chromogenic property of 2, 2-dihydroxyindane-1, 3-dione for quantification of Memantine hydrochloride in pure, pharmaceutical preparation and application to uniformity testing

Article abstract of DOI:10.1016/j.saa.2019.117640

Accurate, simple, sensitive, and fast spectrophotometric assay was applied for the quantification of certain anti-Alzheimer drug namely; Memantine hydrochloride, in its bulk and pharmaceutical preparation. The described assay has been established on the reaction between the primary amino moiety of the cited drug and 2,2-dihydroxyindane-1,3-dione reagent in N,N′-dimethylformamide medium in boiling water bath. Which give Ruhemann's purple color that can be determined at λ_max = 595 nm. Beer's law has been obeyed within drug concentration range from 10-120 μg per milliliter. Detection limit and quantitation limit have been 1.6 & 4.9 μg per milliliter respectively. Developed procedure has been validated in agreement with International Conference of Horizon recommendations and applied successfully for detection of cited drug in bulk, pharmaceutical dosage form and content uniformity testing.

Full text of DOI:10.1016/j.saa.2019.117640

Journal Pre-proof
Utility of chromogenic property of 2, 2-dihydroxyindane-1, 3-dione for quantification
of Memantine hydrochloride in pure, pharmaceutical preparation and application to
uniformity testing
Noha N. Atia, Mustafa A. Marzouq, Ahmed I. Hassan, Walid E. Eltoukhi
PII:
S1386-1425(19)31030-3
https://doi.org/10.1016/j.saa.2019.117640
SAA 117640
DOI:
Reference:
To appear in: Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
Received Date: 20 July 2019
Revised Date: 6 October 2019
Accepted Date: 8 October 2019
Please cite this article as: N.N. Atia, M.A. Marzouq, A.I. Hassan, W.E. Eltoukhi, Utility of chromogenic
property of 2, 2-dihydroxyindane-1, 3-dione for quantification of Memantine hydrochloride in pure,
pharmaceutical preparation and application to uniformity testing, Spectrochimica Acta Part A: Molecular
and Biomolecular Spectroscopy (2019), doi: https://doi.org/10.1016/j.saa.2019.117640.
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Utility of chromogenic property of 2, 2-dihydroxyindane-1, 3-dione
for quantification of Memantine hydrochloride in pure,
pharmaceutical preparation and application to uniformity testing
IR spectra of ME (A) and its isolated reaction product with NHN (B)

Utility of chromogenic property of 2, 2-dihydroxyindane-1, 3-dione
for quantification of Memantine hydrochloride in pure,
pharmaceutical preparation and application to uniformity testing
a
b
b
b
Noha N. Atia , Mustafa A. Marzouq , Ahmed I. Hassan & Walid E. Eltoukhi
*
a
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Assiut
University, Assiut, Egypt
b
Department of Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Al-
Azhar University, Assiut branch, Assiut, Egypt
Abstract
Accurate, simple, sensitive, and fast spectrophotometric assay was applied for the
quantification of certain anti-Alzheimer drug namely; Memantine hydrochloride, in its
bulk and pharmaceutical preparation. The described assay has been established on the
reaction between the primary amino moiety of the cited drug and 2,2-
dihydroxyindane-1,3-dione reagent in N,N'-dimethylformamide medium in boiling
water bath. Which give Ruhemann's purple color that can be determined at λmax
=
5
95 nm. Beer’s law has been obeyed within drug concentration range from 10-120
microgram per milliliter. Detection limit and quantitation limit have been 1.6 & 4.9
microgram per milliliter respectively. Developed procedure has been validated in
agreement with International Conference of Horizon recommendations and applied
successfully for detection of cited drug in bulk, pharmaceutical dosage form and
content uniformity testing
Keywords:
Memantine
hydrochloride;
2,2-dihydroxyindane-1,3-dione;
Dimethylformamide; Ruhemann's purple; Spectrophotometry.

*
Corresponding author.
E-mail: walid20081@yahoo.com (Walid Eltoukhi)
1
. General introduction:
Alzheimer disease is the most frequently type of dementia and it can be contributed
by activation of N-methyl-D-aspartate (NMDA) receptor using amino acid glutamate
[
1]. ME used for treatment of dementia as it has low affinity, uncompetitive (NMDA)
receptor antagonist which binds to the (NMDA) receptor lead to operate the cation
channels. Recently ME was approved by Food and Drug Administration for the
treatment of moderate and severe Alzheimer disease [2]. ME is a 1‐amino‐3,5‐
dimethyladamantane derivative (Figure.1) [1]. Me is a saturated aliphatic tricyclic
structure with a primary amine moiety don’t has fluorescence or ultraviolet properties,
and not even electro active group [3]. Different analytical techniques were prescribed
for the detection of studied drug including spectrophotometry [4-6],
Spectrofluorimetry[2, 7], Potentiometry[8, 9] , LC-MS [10, 11], HPLC [12, 13]. Our
finding indicates that the reported methods include the use of expensive reagent (e.g.
1
, 2 - Naphthoquinone -4- sulphonate and 4‐chloro‐7‐nitro‐2, 1, 3‐benzoxadiazole),
use of toxic organic solvent for extraction (e.g. chloroform) [2, 4, 7]. Moreover,
chromatographic methods required sophisticated instrumentation, consuming large
volumes of organic solvents which elevate the cost and have bad impact on the
environment. 2,2-Dihydroxyindane-1,3-dione is a chromogenic reagent that
successfully applied for the assay of different pharmaceutical nitrogenous
compounds[14, 15], as it form a Ruhemann's purple color that can be detected
spectrophotometrically. The described assay aims to create accurate, sensitive,
cost effective and simple procedure for the detection of ME in bulk and

dosage form using spectrophotometric technique by utilizing readily available
chemical carbonyl reagent 2,2-dihydroxyindane-1,3-dione (NHN), which react
with primary aliphatic amine moiety of ME to give a purple color distinctively that
can be absorbed at λmax = 595nm. The described assay was validated according
to ICH recommendations [16], in addition, it has been used for content
uniformity testing of the studied tablet preparations according to USP
recommendations [17].
2
2
. Experimental
.1. Operating device
A double beam UV–visible spectrophotometer equipped to PC (Shimadzu, Japan)
with 1 cm quartz cuvettes, sensitive analytical balances (Glattbrugg, Switzerland),
ultrasonicator (Cole-Parmer, Chicago, USA), pH meter model 350 ( Jenwey, E.U)
was used for adjustments of pH ,and thermostatic water bath (Germany). All
®
calculation and statistic have been occurred by using GraphPad InStat version 3.05 .
2
.2. Pure powder and pharmaceutical pills
Memantine hydrochloride was supplied by Pharaonia Pharmaceuticals Industries Co.,
Borg El-Arab, Alexandria, Egypt.
Dosage form available in commercials product was determined:
Delmenda® tablet; containing 10 mg of ME (B.N # 82154) taken from Delta Pharma
Co., 10th of Ramadan, El-sharkeya, Egypt.
2
.3. Chemicals and reagents
All reagents that used have been of analytical grade (El Nasr chemical company, Abu
Zaabal, Cairo, Egypt). 2,2-Dihydroxyindane-1,3-dione (NHN), (Aldrich Chemical
Co. Ltd) 2% w/v solution was freshly prepared in DMF [14, 15].

2
.4. Preparation of standard and working solutions
An accurate amount equal to 100.0 mg of cited drug has transferred to 100-mL
calibrated flask. Drug powder has been dissolved in 25 mL dimethylformamide and
-1
complete to the final mark with the same solvent to get stock solution of 1.0 mg mL .
Another dilution was made with DMF to get working solution within the range of
calibration curve.
2
.5. General assay method
Accurate volumes of the selected drug, standard solution, equivalent to (100-1200 µg
-1
mL ) have been transferred to 10 mL; a screw capped test tube.1mL of 2% NHN was
added to series of 10- ml test tube. The solution has been mixed well and heated for
1
5 min in boiling thermostatic water bath. Cooling, the solutions quantitatively
transferred into 10-mL volumetric flask and completed to the final mark with
Dimethyl Formamide and the absorbance has been measured at 595 nm against blank
solution. To construct the calibration curve absorbance was plotted against the
,
-1
concentration of selected drug (µg mL ) and the regression equation has been derived
using GraphPad InStat version 3.05
2
2
.6. The prescribed assay application
.6.1. Application of pharmaceutics pills
Twenty pills of cited formulation containing ME was weighted accurately, finely
ground in a mortar. An accurately weighed quantity of the ground tablet equivalent to
1
00.0 mg of ME has been added to a 100- mL calibrated flask, an about 80 mL of
DMF have been added and the flask was sonicated for 20 minutes [18], complete with
DMF to the mark and then filter. The early amount of filtrate has been discarded.

Aliquot of this solution was transferred to a series of 10- mL calibrated flask to get
-1
sample solution (10-120 µg mL ) and analysis was completed as previous
3
3
Result and discussion
.1. Spectra
Ninhydrin react with primary amine moiety to yield Ruhemann purple color
14]. The descried assay depends on reaction of ME with Ninhydrin in
[
Dimethyl Formamide solution through oxidative deamination of aliphatic
primary amine moiety of ME. Dimethyl Formamide solution, Ninhydrin was
converted into o-carboxyphenylglyoxal which could reduce Ninhydrin into 2-
hydroxyindan-1,3-dione. The 2-hydroxyindan-1,3dione was combined with
primary amino moiety of ME to yield amino derivatives. Amino derivatives
that produced undergo condensation with other molecule of Ninhydrin to
yield a Ruhemann purple or diketohydrindylindene-diketohydrindamine,
which show absorption at 595nm (fig. 2). So we utilized the new absorption band
which is characteristic to ME to assay it in bulk, pharmaceutics formulation and
testing of uniformity
3
.2. Optimization of reaction condition
Several factors affecting the reaction condition (volume of NHN, condensation time,
temperature and diluting solution) have been investigated by changing each factor in
turn while keep the other factors still constant and notice its effect on the absorbance
maximum of the colored products which measured at 595 nm. Different amounts
(from 0.2 – 2.0 mL) of 2% w/v NHN in DMF medium have been utilized for general
assay; It has been found that maximum absorbance intensity has been result when the
reagent volume was from 0.8–1.2 ml. Higher or lower amount of volume result in

decrease in the absorbance intensity ( Fig.3), on the other hand after mixing the
reagent with ME, the mixture has been heated at different temperature degrees (°C).
The absorbances intensity depends on the degree of temperature of the reaction.
Maximum absorbance intensity has been yield at 100 °C. The heating time has been
also optimized. It was observed that absorbance increased by boiling till 10 minutes
and still constant for further 20 minutes; so the reaction must be heated at 100 °C for
1
0 minutes to give maximum absorbance intensity (Fig. 4 and 5). Lastly for diluting
solvents, Different diluting solvent has been examined including; DMF, ethyl alcohol,
methyl alcohol & water. Experiments indicate that DMF is proved to be the most
appropriate diluting solvent because it affords an excellent power of solvation for
NHN and gives the maximum absorbance value, and so, it has been chosen for further
investigation, (Figure. 6).
3
.3. Investigation of the molar ratio between NHN and ME
The stoichiometry between the investigated anti-Alzheimer drug and NHN has been
-2
detected by utilizing Job’s method of continuous variation [19] utilizing (10 M)
master equimolar solutions of both NHN and studied drug. The results showed a 2:1
ratio between NHN and ME (Fig. 7). This Ruhemann's purple colored product has
been established from via oxidative deamination of selected drug and then
condensation with two compounds of NHN, the proposed mechanism is showed in
(Fig. 8). In order to propose a possible reaction pathway, the product of the reaction
between NHN and ME has been isolated and determined by different techniques. The
product has Ruhemann's purple color with melting point 215 °C while Memantine
hydrochloride has melting point 258 °C [20]. FTIR spectroscopy has been utilized to
determine the functional groups present in the pure drug sample and the separated
product with NHN. The FTIR spectrum of Memantine was showed the expected

doublet of primary NH2 at 2964 and 2933 cm-1. The wave number was observed at
505 may be assigned to the C - N stretching. Primary amines are reported to give
1
Ruhemann’s purple complex with NHN. The formation of the complex was evidenced
by comparing the spectra of complex with Memantine and NHN. NHN spectra
exhibited two broad bands at 3300 and 3250 cm-1 owing to two OH groups and 1061
cm-1 because of secondary alcohol C-O stretch. The carbonyl gave two peaks in the
region 1660-1760 cm-1. Aromatic resonance was recorded at 750 cm-1.
Many of the functionalities of NHN and Memantine were found to be absent; which
confirms the formation of complex. The twin peak of NH2 in Memantine disappeared
indicating that the primary amine has been changed to tertiary. The doublet of
carbonyl in NHN changed significantly into one single sharp peak at 1640 cm-1 and
-1
the broad band of O-H shifted to 3300 cm (Fig. 9).
3
.4. Validation of the prescribed method
The prescribed procedure was validated in agreement with International Conference
on Harmonization recommendations [21] in order to outline that the prescribed assay
agree with the demand of the cited analytical performance. All validation procedures
were examined through the defined calibration range of the prescribed procedure to
prove the validation of the prescribed assay.
3
.4.1. Linearity
Calibration graph for ME has been constructed by analyzing series of 6 concentrations
of ME, taking the mean of 3 determinations for each concentration to minimize the
error. Absorbance versus concentrations, have been plotted and treated statistically
by summation of a regression equation by method of least square [22]. Drug
-1
concentrations ranging from 10 – 120 µg mL were examined. Various analytical
factors as correlation coefficient (0.9999), intercept (0.1072), slope (0.004115) and

standard deviation of the intercept (0.002) were presented in (Table 1).
3
.4.2. Repeatability and Intermediate precision
The accuracy has been examined by 3 times assay for 6 different concentration of
ME. The values showed in (Table 2) prove the highest acceptance between the correct
results and the experimental results which indicate good accuracy for the prescribed
assay. Repeatability and Intermediate precision were detected by utilizing 3 different
concentrations for ME and3 determinations of each one. The (RSD) value was under
2
.0 % which indicate good repeatability & reliability for the prescribed method.
Results were showed in (Table 2).
3
.4.3. Limits of detection & limits of quantitation
LOD & LOQ have been summated in respect to International Conference on
Harmonization recommendation [21] according to detection limits = 3.3 * standard
deviation of the variables / slop. Where quantitation limits = 10 * standard deviation
of the variables / slop.
LOD & LOQ were 1.6 & 4.9 µg/mL orderly. Results were summarized in (Table 1).
3
.4.4. Robustness of the proposed assay
Volume of NHN and the condensation time have been checked. During those assay,
one parameter has been changed where other parameters still constant and the % of
recovery has been detected each time. Recoveries and standard deviations indicate a
small difference in each of selected parameter had no effect on the absorption
intensity of the product (Table 3).

3
3
.5. Application of the proposed assay
.5.1. Pharmaceuticals formulations
The prescribed assay has been used for the determination of pills formulations of
ME; the results that obtained were compared by those of reported method [23] by
applying of student's t-test & the variance ratio F-test [21] at 95% level. No
difference has been found between the values of the prescribed and the reported
method. This indicates high accuracy and precision in the assay of the investigated
compound (Table 4),
3
.5.2. Content uniformity testing
The method was applied to tablet uniformity testing that is a time-consuming assay
when applying normal techniques, according to the great accuracy of the prescribed
procedure and its ability to quickly detect the content of ME in only one pill obtained
with good precision. 10 different pills were detected by applying the same procedure
which used to analyze ME in tablet dosage form. The uniformity content was
examined by utilizing official United State Pharmacopeia guideline [17]. Acceptance
value (AV) was detected for selected pills and it was found to be very small than the
maximum value (L1). (Table 5).
4
. Summary
NHN has been selected as chromogenic reagent that form Ruhemann's purple colored
product with Memantine hydrochloride. The presented work has the advantage of
being rapid, sensitive ,as it has a small LOD as represented in table (1), simple, cost-
effective, as it not need an expensive or toxic reagent as reported methods, [2, 4, 7]
and accurate for analysis of anti-Alzheimer drug in its bulk and tablets. In addition, it
is a time-saving, no need for pre-treatment of sample and extraction free assy.
Moreover, the prescribed procedure is very suitable to be applied in content

uniformity testing and for routine assay and quality control inspection of the selected
anti-Alzheimer drug.
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Table 1: analytical parameters for the analysis of Memantine hydrochloride by
proposed assay
Parameter
Memantine (ME)
λmax (nm)
595
linear range ( µg/mL)
LOD (µg/mL )
10-120
1
.6
.9
LOQ (µg/mL)
4
Correlation coefficient (r)
0
.9999
.9998
2
Determination coefficient (r )
Slop(b)
0
0.004115
0.1072
Intercept(a)
SD of the intercept (Sa)
SD of slope (Sb)
0.002
0.00003
0.002781
SD of residual ( Sy.x)

Table 2: Evaluation of accuracy and precision of the proposed assay
Parameter
ME (%found)*
100.0
Taken (µg/mL)
80.0
120.0
1
9
9
98.3
99.1
98.7
99.3
2
3
9
9
8.2
9.7
Repeatability
98.5
98.6
99.7
99.2
Mean
98.9
0.8
SD
0.4
0.5
%
RSD
0
9
9
9
.81
9.5
7.9
8.2
0.41
98.8
98.6
0.51
99.5
98.7
1
2
Intermediate
precision
3
99.2
98.8
97.9
98.7
Mean
98.5
0.9
SD
0.3
0.3
0.8
%
RSD
0
.91
0.81
*
Average of three determination. SD: standard deviation. RSD: relative standard
deviation

Table 3: Robustness of the proposed assay for the analysis of Memantine
hydrochloride
Parameter
% Recovery* ± SD
ME (100 µg/mL)
Optimum
condition
9
8.9 ± 0.9
1
0
1
- Volume of NHN (mL)
.8
.2
9
8.3 ± 0.5
8.8 ± 0.4
9
2- Heating time (minutes)
0
.4
.6
9
8.8 ± 0.9
0
98.6 ± 0.5
*
Average of three determinations

Table 4: Comparison between the proposed assay and the reported method for
determination of Memantine hydrochloride in its tablet dosage form
a
%
Recovery ± SD
Pharmaceutical
dosage form
b
b
Proposed method Reported method
t-value
F-value
®
Delmenda (10 mg
9
8.2 ± 0.2
98.3 ± 0.3
0.33
1.97
ME/tablet)
a Average of three determinations.
b Tabulated values at 95% confidence limit are t =2.306, F= 6.338

Table 5: Results of content uniformity testing of selected drug tablets using the
proposed assay
®
Delmenda 10 mg
Dosage form no.
%
labeled claim
1
2
3
4
5
6
7
8
9
98.2
97.8
98.3
99.1
96.8
98.6
99.2
98.5
98.6
98.8
10
Mean
98.4
0.7
SD
Acceptance value (AV)
Max. allowed AV(L1)
Acceptance value =2.4 × SD.
1.68
15.0

Figure 1: Chemical structure of Memantine hydrochloride

a
b
Wavelength (nm)
Figure 2: Absorption spectra of (a) (120 µg/mL) of ME after reaction with 2% w/v
NHN in DMF medium (b) blank

Volume of reagent (mL)
Figure 3: Effect of volumes of NHN (2 % w/v) on the absorption intensity of
1
00µg/mL Memantine hydrochloride

Time (min)
Figure 4: Effect of heating time on the absorption intensity of 100µg/mL of
Memantine hydrochloride

Temperature (°C)
Figure 5: Effect of different temperatures on the absorption intensity of 100µg/mL of
Memantine hydrochloride

Diluting solvent
Figure 6: Effect of different diluting solvents on the absorption intensity of
1
00µg/mL of Memantine hydrochloride
[
Drug]/ [Drug] + [NHN]

Figure 7: Plot of Job’s method for determination of molar ratio of studied drugs and
-2
NHN using 10 M equimolar solutions
.
Figure 8: suggested reaction mechanism between ME and NHN in DMF medium

Figure 9: IR spectra of ME (A) and its isolated reaction product with NHN (B)
\

Highlights
1
. Development of simple, sensitive, accurate and rapid spectrophotometric method
for detection of Memantine hydrochloride in pure powder and pharmaceutical dosage
form.
2
. The described method has been depend on the reaction of Memantine
hydrochloride with 2, 2-dihydroxyindane-1, 3-dione reagent in N, N'-
dimethylformamide medium in boiling water bath. Which give Ruhemann's purple
color that can be determined at λmax = 595 nm
3
4
. Validation of the described method according to the ICH and USP guidelines.
. Application of the proposed method on pharmaceutical dosage forms and content
uniformity testing.

Declaration of interests
☒
The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☒
The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests:
I have no known competing financial interests or personal relationships that could have appeared to
influence the work reported in this paper.