Spectrophotometric method for the determination, validation, spectroscopic and thermal analysis of diphenhydramine in pharmaceutical preparation

Article abstract of DOI:10.1016/j.saa.2010.05.031

A sensitive, simple and rapid spectrophotometric method was developed for the determination of diphenhydramine in pharmaceutical preparation. The method was based on the charge-transfer complex of the drug, as n-electron donor, with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), as π-acceptor. The formation of this complex was also confirmed by UV-vis, FTIR and ¹H NMR spectra techniques and thermal analysis. The proposed method was validated according to the ICH guidelines with respect to linearity, limit of detection, limit of quantification, accuracy, precision, recovery and robustness. The linearity range for concentrations of diphenhydramine was found to be 12.5-150 μg/mL with acceptable correlation coefficients. The detection and quantification limits were found to be 2.09 and 6.27 μg/mL, respectively. The proposed and references methods were applied to the determination of drug in syrup. This preparation were also analyzed with an reference method and statistical comparison by t- and F-tests revealed that there was no significant difference between the results of the two methods with respect to mean values and standard deviations at the 95% confidence level.

Full text of DOI:10.1016/j.saa.2010.05.031

Spectrochimica Acta Part A 77 (2010) 324–329
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectrophotometric method for the determination, validation, spectroscopic and
thermal analysis of diphenhydramine in pharmaceutical preparation
Sevgi Tatar Ulua,∗, Fikriye Tuncel Elmalib
a Department of Analytical Chemistry, Faculty of Pharmacy, Istanbul University, 34416 Istanbul, Turkey
b Department of Chemistry, Faculty of Arts and Sciences, Yildiz Technical University, 34210 Istanbul, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
A sensitive, simple and rapid spectrophotometric method was developed for the determination of diphen-
hydramine in pharmaceutical preparation. The method was based on the charge-transfer complex of the
drug, as n-electron donor, with 2,3-dichloro-5,6-dicyano-p-benzoquinone (DDQ), as ␲-acceptor. The for-
mation of this complex was also confirmed by UV–vis, FTIR and 1H NMR spectra techniques and thermal
analysis. The proposed method was validated according to the ICH guidelines with respect to linearity,
limit of detection, limit of quantification, accuracy, precision, recovery and robustness. The linearity range
for concentrations of diphenhydramine was found to be 12.5–150 ␮g/mL with acceptable correlation
coefficients. The detection and quantification limits were found to be 2.09 and 6.27 ␮g/mL, respectively.
The proposed and references methods were applied to the determination of drug in syrup. This prepara-
tion were also analyzed with an reference method and statistical comparison by t- and F-tests revealed
that there was no significant difference between the results of the two methods with respect to mean
values and standard deviations at the 95% confidence level.
Received 4 February 2010
Received in revised form 21 May 2010
Accepted 24 May 2010
Keywords:
Diphenhydramine
Charge-transfer complex
2,3-Dichloro-5,6-dicyano-p-benzoquinone
Determination
Validation
Spectroscopic techniques
Thermal analysis
© 2010 Published by Elsevier B.V.
1. Introduction
The proposed method was based on the charge-transfer reac-
tion between n-electron donor and ␲-acceptor (DDQ) to give a red
Diphenhydramine hydrochloride (DPH) 2-(diphenylmethoxy)-
N,N-dimethylethanamine is a chemical mainly used as antihis-
taminic, antiemetic, sedative, and hypnotic [1] (Fig. 1).
colored DDQ•− radical anion. DDQ is a strong electron acceptor
and has been used for the determination of many electron donors
such as chloroquine and pyrimethamine [15], flucloxacillin and
dicloxacillin [16], lansoprazole and pantoprazole [17], meloxicam
[18], fluoxetine and sertraline [19], ciprofloxacin, enrofloxacin and
pefloxacin [20].
In the literature no method has been reported for the deter-
mination of DPH by using DDQ. In the present investigation, we
report that the development of accurate, reproducible, less time
consuming and adequately sensitive spectrophotometric method.
The solid complex has been synthesized and structure of the com-
plex has been confirmed by spectral techniques such as UV–vis,
FTIR, 1H NMR, and also by thermal analysis.
DPH works by blocking the effect of histamine at H1 receptor
sites. This results in effects such as the increase of vascular smooth
muscle contraction, thus reducing the redness, hyperthermia and
edema that occur during an inflammatory reaction. In addition,
by blocking the H1 receptor on peripheral nociceptors, diphenhy-
dramine decreases their sensitization and consequently reduces
itching that is associated with an allergic reaction. This is why
diphenhydramine is a popular choice for treatment of the symp-
toms of allergic rhinitis, hives, motion sickness, and insect bites
and stings [2].
Several published methods have been developed for the deter-
mination of DPH in pharmaceutical preparations and in biological
fluids including: spectrophotometry [3–6], flow injection analy-
sis [7], gas chromatography [8], atomic absorption spectrometry
[9], FT-Raman spectroscopy and high performance liquid chro-
matography [10] and capillary electrophoresis [11–13]. Liquid
chromatography/tandem mass spectrometry has been reported for
the determination of DPH in dog plasma [14].
2. Experimental
2.1. Materials
Pure powder of DPH was obtained from Sigma (St. Louis, MO,
USA). Fenotral syrup was obtained from commercial sources in
the local pharmacy. The DDQ was purchased from (Fluka, Switzer-
land). All solvents used were of analytical reagent grade (Merck,
Darmstadt, Germany).
∗
Corresponding author. Tel.: +90 212 4400000x13586; fax: +90 212 4400252.
E-mail address: sevgitatar@yahoo.com (S.T. Ulu).
1386-1425/$ – see front matter © 2010 Published by Elsevier B.V.
doi:10.1016/j.saa.2010.05.031

S.T. Ulu, F.T. Elmali / Spectrochimica Acta Part A 77 (2010) 324–329
325
2.5. Determination of molar ratio
Applying the continuous variation method [21] for the reaction
of DDQ reagent with DP and refer to the stoichiometry of the drug
donor (D) to the reagent acceptor (A) of ratio 1:1 (D:A).
2.6. Method validation
Fig. 1. Molecular structure of diphenhydramine hydrochloride.
The method was validated according to International Con-
ference on Harmonization guidelines for validation of analytical
procedures [22,23].
2.2. Apparatus
2.6.1. Linearity
The linearity was evaluated by linear regression analysis, which
was calculated by the least square regression method. The cali-
bration curves were constructed by plotting concentration versus
absorbance, using linear regression analysis.
An UV-160A UV–visible spectrophotometer (Shimadzu, Japan)
was used for the absorbance measurements.
TGA–DSC (thermogravimetric analysis–differential scanning
calorimetry) curves were obtained with a TA SDT Q 600 thermal
analyzer apparatus using flowing nitrogen 100 mL min−1 temper-
,
ature range 25–750 and 25–1200 ◦C, at heating rate of 20 ◦C min−1
.
2.6.2. Limit of detection and limit of quantification
The FTIR spectra were recorded with ATR technique on a
PerkinElmer Spectrum One Bv 5.0 spectrophotometer. 1H NMR
spectra were recorded on a Varian UNITY INOVA 500 MHz spec-
trometer. TMS was used as internal standard in 1H NMR.
The limit of detection (LOD) and limit of quantification (LOQ)
were calculated as 3.3ꢁ/S and 10ꢁ/S, respectively, where S is the
slope of the calibration curve and ꢁ is the standard deviation of
intercept of regression equation.
2.6.3. Precision and accuracy
2.3. Solutions
DP samples (12.5, 75.0 and 150 ␮g/mL) in five replicates were
analyzed on the same day to determine the intra-day precision and
accuracy, and on each of five separate days to determine inter-
day precision and accuracy. Precision was expressed as the relative
standard deviation (RSD %). Accuracy was expressed as the mean
relative error (RME %).
10 mg mL−1 of DPH stock solution was prepared in water. DDQ
solution was prepared 2.0 mg mL−1 in acetonitrile. The DPH stock
and DDQ solutions were stored at 4 ◦C.
2.3.1. Preparation of diphenhydramine base solution
A 1 mL standard stock solution of DPH was transferred into the
12 mL each glass tube. Then, 1 mL of concentrated ammonia solu-
tion was added and shaken for 1 min. The alkaline aqueous layer
was extracted with three portions of chloroform each of 3 mL. The
chloroform phase was filtered through anhydrous sodium sulfate,
into a 10 mL calibrated flask and the volume. It was made up to
10 mL by adding chloroform (1.0 mg mL−1, calculated as diphenhy-
dramine base (DP)). From this solution, 5.0 mL was transferred into
10 mL calibrated flasks and evaporated to dryness in a water bath at
45 ◦C under a stream of nitrogen. Then, the residue completely dis-
solved in acetonitrile using an ultrasonic bath and then completed
to the mark with acetonitrile (0.5 mg mL−1, calculated as free base).
2.6.4. Recovery
The % recovery of the added pure drug was calculated as, %
recovery = [(Ct − Cs)/Ca] × 100, where Ct is the total drug concentra-
tion measured after standard addition, Cs the drug concentration
in the formulation sample and Ca is the drug concentration added
to formulation.
2.6.5. Robustness
The robustness of the spectrophotometric method was deter-
mined by analysis of samples under a variety of conditions such as
small changes in the reagent concentration and reaction time.
3. Results and discussion
2.4. Procedures
3.1. Absorption spectra and mechanism of the reaction
2.4.1. General procedure
The method is based on the formation of intensely red colored
DDQ•− radical anion by interaction of the drug with DDQ in acetoni-
trile at ambient temperature. As can be represented in the following
scheme, the radical anion results from dissociation of the original
charge-transfer complex (CT-complex) formed by interaction of the
drug (n-electron donor, D) with DDQ (␲-acceptor, A) (Scheme 1).
Interaction of DDQ with DP formed a red color product in ace-
tonitrile with absorption maxima at 460, 541 and 586.5 nm (Fig. 2).
Measurements were carried out at 460 nm, at which higher sensi-
tivity was achieved.
Transfer aliquots of 0.25–3.0 mL of diphenhydramine base solu-
tion (0.5 mg mL−1) into a series of 10 mL calibrated flasks. Bring
the volume to 3.0 mL with acetonitrile, added 1.0 mL DDQ reagent.
The reaction mixture was mixed and the volume was completed
to 10 mL with acetonitrile. The immediately formed dark red color
was measured at ꢀmax = 460 nm against a blank prepared in the
same manner except for an addition of drug and the calibration
graph was obtained.
2.4.2. Analysis of dosage forms
4 mL of fenotral syrup was transferred into stoppered glass
tubes. The solution was alkalized with 1.0 mL of concentrated
ammonia and then extracted with chloroform as indicated in Sec-
tion 2.3.1. After preparation of diphenhydramine base solution,
proceed as described in Section 2.4.1.
3.2. Optimization of reaction conditions
3.2.1. Effect of reagent concentration
The influence of the concentration of DDQ was examined by
addition of different volumes of 0.2% reagent in the range of

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S.T. Ulu, F.T. Elmali / Spectrochimica Acta Part A 77 (2010) 324–329
Scheme 1.
0.25–3.0 mL. The maximum absorbance was obtained when 1.0 mL
DDQ solution was utilized.
3.2.2. Effect of solvent
The formation of charge-transfer complexes where a reac-
tion occurs between ␲-acceptors and n-electron donors can be
influenced by the polarity of the solvent. Absorbance spectral char-
acteristics of DP with DDQ in different solvents were examined.
Double distilled water, methanol, ethanol, acetonitrile and chlo-
roform were tested for this investigation. Experimental results
indicated that acetonitrile gave the maximum absorbance and sta-
ble among the studied solvents.
3.2.3. Effect of time and temperature
The optimum reaction time was determined by monitoring the
color development at room temperature (25 5 ◦C). Complete color
intensity was attained 2 min.
Fig. 2. Absorption spectrum of CT-complex (different concentrations).

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327
Table 2
Results of standard addition method (n = 5).
Concentration of Concentration of Total concentration % analytical
drug in
formulations
(␮g/mL)
pure drug added of drug found
recovery
(
SD)a
(␮g/mL)
(␮g/mL)
25
25
25
25
75
125
49.23
97.03
150.06
96.92 1.2 × 10-2
96.04 1.0 × 10-2
100.04 1.5 × 10-2
a
Values are mean of five determinations
Table 3
Determination of DP in pharmaceutical preparations by the proposed and Ref. [3]
method.
Fig. 3. Mole ratio of DDQ to DP.
Drug
Proposed method Reference methodc
Fenotral syrups
(12.5 mg/5 mL)
% recovery SDa
98.48 3.1 × 10-1 98.12 3.9 × 10-1
3.2.4. Stoichiometry
tb
Fb
1.56
1.58
Job’s method of continuous variation was used for determining
the molar ratio of DP to each of the analytical reagent employed in
the charge-transfer reactions. The ratio was 1:1 in all cases (Fig. 3).
a
Values are mean of six determinations.
b
The tabulated values of t and F at 95% confidence limit are 2.23 and 5.05, respec-
tively.
3.3. Method validation
c
Ref. [3].
3.3.1. Linearity, detection and quantification limits
trations of acceptor reagent, and reaction time, on the analytical
performance of the method. In these experiments, one experimen-
tal parameter was changed while the other parameters were kept
unchanged, and the recovery percentage was calculated each time.
The small variations in any of the variables did not significantly
affect the results; recovery percentages were 97.72. This provided
an indication for the reliability of the proposed method during rou-
tine work.
Under the specified optimum reaction conditions, the calibra-
tion curves for DP with the reagent employed in the present work
were constructed. The regression equations for the results were
derived using the least-squares method. In all cases, Beer’s law plots
(n = 3) were linear with very small intercepts and good correlation
coefficients in the general concentration range of 12.5–150 ␮g/mL.
The LOD and LOQ were found to be 2.09 and 6.27, respectively
(Table 1).
3.4. Application
3.3.2. Precision and accuracy
The intra-day and inter-day relative standard deviation (RSD)
values obtained by the proposed method were found to be within
0.29–0.10%. Accuracy of the methods expressed as relative mean
error (RME) was below −4.6%.
Commercially available DP in syrup was subjected to analysis
both by the proposed method and by a reference method [3]. Sta-
tistical comparison of the results by the Student’s t-test and the
variance-ratio F-test at the 95% confidence level, revealed there was
no significant difference between the accuracy and precision of the
two methods (Table 3).
3.3.3. Recovery
Recovery studies were carried out by the standard addition
method. In this study, different concentrations of pure drug (50,
100 and 150 ␮g/mL) were added to a known preanalyzed formu-
lation sample and the total concentration was determined using
the proposed method (n = 5). The mean recovery was found to be
97.67% (Table 2).
3.5. Spectroscopic studies on the solid CT-complex
3.5.1. Infrared absorption spectra
Infrared spectra of electron donor DP, acceptor DDQ and its CT-
complex [(DP) (DDQ)] are shown in Fig. 4. In the spectra of the
CT-complex main bands of both the donor and the acceptor are
observed. From the data given in Table 4, decreases and increases
in the vibration frequencies of a particular band have been used
as evidence for a particular site of a CT interaction. For exam-
ple, ꢂ (C C), ꢂ (C–H) and ꢂ (C–O) stretching vibration bands of
the free base DP show some changes in both band intensities and
wave number values upon complexation. In the CT-complex, the
DDQ gains electrons from the donors and it changes to the ben-
3.3.4. Robustness
Robustness of the procedures was assessed by evaluating the
influence of small variation of experimental variables: concen-
Table 1
Optical characteristics, statistical data of the regression equations and validation
parameters for DP.
Parameters
Valuea
Molar absorptivity (L/mol cm)
1.2 × 103
Table 4
Sandell’s sensitivity (␮g cm−2/0.001 A)
2.11 × 10−2
FTIR spectral data of DP, DDQ and CT-complex.
Regression analysis
Slope
Intercept
Regression coefficient (r)
Linearity (␮g/mL)
LOD (␮g/mL)
Assignment (cm−1
)
DP
DDQ
2233
1551
1669
DP–DDQ
0.0045
0.0239
0.9997
12.5–150
2.09
ꢂ (C N)
ꢂ (C C)
ꢂ (C O)
–
2205, 2113
1573
–
2956, 2870
3028
1103
1492
–
2956
3032
1108
ꢂ (C–Halkyl
)
–
–
–
LOQ (␮g/mL)
6.27
ꢂ (C–Haryl
ꢂ (C–O)
)
a
Values are mean of three determinations.

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Fig. 5. 1H NMR spectra of (A) DP and (B) CT-complex in CDCl3.
protons (–CH3) at nitrogen atom gave one sharp singlet NMR sig-
nal at 2.2 ppm while methylene protons (–CH2–) gave a triplet at
2.5 and 3.5 ppm. Moreover, methine proton (–CH–) gave a singlet
signal at 5.3 ppm. In the CT-complex, while signals of methyl and
methylene protons shifted to 2.9, 3.3 and 3.7 ppm, respectively, sig-
nal of methine proton did not change. All the observed proton peaks
in the spectra of the donor also exist in the CT-complex spectra with
small shift to higher ppm values. These changes in the ı (ppm) val-
ues of the donor (DP) and CT-complex support the charge migration
from the donor (base) toward the acceptor (acid).
Fig. 4. FTIR spectra of (A) DP, (B) DDQ and (C) CT-complex.
zenoid form having the unshared electrons on the oxygen atoms.
In the FTIR spectrum, the lack of the ꢂ (C O) absorption provides
further evidence the presence of [DDQ]•− radical anion for this
complex. The ꢂ (C N) and ꢂ (C C) stretching vibration bands for
the free DDQ occur at 2233 and 1551 cm−1, respectively, and at
2205–2113 and 1573 cm−1 upon complexation. The split of the ꢂ
(C N) into two bands in the case of CT-complex could be related to
such as symmetry and dipole moment changes in the complexed
DDQ compared with the free molecule. All these changes could be
related to both electronic and symmetry changes upon complexa-
tion.
3.5.3. Thermal analysis measurements
Thermogravimetric (TGA) and differential scanning calorimetric
(DSC) analysis were carried out in order to confirm the decomposi-
tions and structures of the formed solid CT-complex. TGA and DSC
curves of the DP, DDQ and CT-complex are shown in Fig. 6. The
DP donor shows one degradation step at 190–330 ◦C corresponds
to the loss of DP. The found weight loss of this step is 92%. The
DDQ acceptor has one degradation step at 170–290 ◦C correspond-
ing to the loss of DDQ. The found weight loss of this step is 95%. The
thermal decomposition of the complex in inert atmosphere pro-
ceeds approximately with three main degradation steps. The first
stage and second stage of degradation occur within a temperature
range of 25–160 and 160–380 ◦C. The found weight loss associ-
ated with these stages of decomposition is 26.2% and 52.0% (78.2%)
may be attributed to loss of the DDQ and diphenyl group of donor
molecules, which is in good agreement with the calculated value
of 79.0%. The third decomposition temperature at 380–1200 ◦C is
related to the decomposition of the C6H11NO group of the donor.
The weight loss of this step is 20.1% in agreement with the calcu-
lated value of 20.9%.
3.5.2. 1H NMR spectra
1H NMR spectra of the donor (DP) and the CT-complex are
measured in CDCl3 and are shown in Fig. 5A and B (Table 5). The
spectrum of CT-complex was compared with the donor. Methyl
Table 5
1H NMR for DP, DDQ and CT-complex.
1H NMR (ı, ppm)
DP
DP–DDQ
–CH3
–CH2
–O–CH2
–CH
–CHaryl
2.2 (s)
2.9 (s)
2.5 (t)
3.3 (t)
3.5 (t)
3.7 (t)
5.3 (s)
5.3 (s)
7.15–7.30 (m)
7.16–7.26 (m)

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329
4. Conclusion
The proposed method was simple, rapid, accurate, precise and
inexpensive and can be used for routine analysis of DP in pharma-
ceutical preparation. The proposed method for DP determination
has many advantages over other analytical methods due to its sen-
sitivity, simplicity, rapidity and low cost [8–14]. It does not require
any extraction, heating, buffer or any other agent. Unlike the gas
chromatographic and HPLC procedures, the instrument is simple
and is not of high cost. The proposed method is suitable for routine
quality control.
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Fig. 6. TGA and DSC diagrams of (A) DP, (B) DDQ, and (C) CT-complex.