A comparative study of two novel validated spectrophotometric techniques for resolution of four-component mixtures with severely overlapping spectra

Article abstract of DOI:10.1016/j.saa.2020.118213

Two new smart spectrophotometric methods are developed and validated for the determination of the quatertnary mixture of paracetamol (acetaminophen), diphenhydramine, p-aminophenol, and N-oxide degradate of diphenhydramine. Method A is the novel triple divisor ratio difference method, where the triple divisor ratio spectrum of the component of interest shows a significant amplitude difference at two selected wavelengths where the three interfering substances are used as triple divisor and give constant amplitude all over the spectrum. The triple divisors are normalized spectra of tertiary mixtures containing 40 μg/mL of each of the 3 interfering components. The selected wavelengths are 256–290 nm, 220-230 nm, 230–245 nm and 275–260 nm for the 4 components, respectively. Method B is double divisor - ratio difference-dual wavelength, where the double divisor ratio spectrum of the component of interest shows a significant amplitude difference at two selected wavelengths where two interfering substances are used as double divisor and give constant amplitude all over the spectrum, while the third one shows zero amplitude difference at these two selected wavelengths. The double divisors used are normalized spectra of diphenhydramine /N-oxide degradate of diphenhydramine binary mixture for both paracetamol and p-aminophenol and paracetamol/p-aminophenol binary mixture for both diphenhydramine hydrochloride and its N-oxide degradate. The double divisors binary mixtures contain 40 μg/mL of each component. The selected wavelengths are 243–233 nm, 223.8–270.2 nm, 237–250 nm and 265–234.6 nm for the 4 compounds, respectively. Both methods were successfully applied for the quantification of the four components in laboratory prepared quaternary mixtures and for the quantification of paracetamol and diphenhydramine hydrochloride in panadol night tablets. The results obtained by the developed methods were compared with those obtained by the United State Pharmacopeial method for the analysis of paracetamol and diphenhydramine, where no significant differences were found.

Full text of DOI:10.1016/j.saa.2020.118213

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
A comparative study of two novel validated spectrophotometric
techniques for resolution of four-component mixtures with severely
overlapping spectra
Nessreen S. Abdelhamid
Pharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmad Hegazy St., 62514 Beni-Suef, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 12 July 2019
Received in revised form 23 February 2020
Accepted 1 March 2020
Available online 4 March 2020
Two new smart spectrophotometric methods are developed and validated for the determination of the
quatertnary mixture of paracetamol (acetaminophen), diphenhydramine, p-aminophenol, and N-oxide
degradate of diphenhydramine. Method A is the novel triple divisor ratio difference method, where the triple di-
visor ratio spectrum of the component of interest shows a significant amplitude difference at two selected wave-
lengths where the three interfering substances are used as triple divisor and give constant amplitude all over the
spectrum. The triple divisors are normalized spectra of tertiary mixtures containing 40 μg/mL of each of the 3 in-
terfering components. The selected wavelengths are 256–290 nm, 220-230 nm, 230–245 nm and 275–260 nm
for the 4 components, respectively. Method B is double divisor - ratio difference-dual wavelength, where the
double divisor ratio spectrum of the component of interest shows a significant amplitude difference at two se-
lected wavelengths where two interfering substances are used as double divisor and give constant amplitude
all over the spectrum, while the third one shows zero amplitude difference at these two selected wavelengths.
The double divisors used are normalized spectra of diphenhydramine /N-oxide degradate of diphenhydramine
binary mixture for both paracetamol and p-aminophenol and paracetamol/p-aminophenol binary mixture for
both diphenhydramine hydrochloride and its N-oxide degradate. The double divisors binary mixtures contain
40 μg/mL of each component. The selected wavelengths are 243–233 nm, 223.8–270.2 nm, 237–250 nm and
265–234.6 nm for the 4 compounds, respectively. Both methods were successfully applied for the quantification
of the four components in laboratory prepared quaternary mixtures and for the quantification of paracetamol
and diphenhydramine hydrochloride in panadol night® tablets. The results obtained by the developed methods
were compared with those obtained by the United State Pharmacopeial method for the analysis of paracetamol
and diphenhydramine, where no significant differences were found.
Keywords:
Triple divisor ratio difference
Double divisor ratio difference dual wavelength
Acetaminophen
Diphenhydramine
P-aminophenol
N-oxide degradate of diphenhydramine
© 2020 Elsevier B.V. All rights reserved.
1. Introduction
aminophenol (PAP) and the N oxide degradation product of DH
(NOD).
UV-spectrophotometry is one of the easiest and most convenient
methods of analysis. However the analysis suffers great difficulties
when the analyzed mixtures contain a number of components with se-
verely overlapping spectra.
Paracetamol (PC) or acetaminophen is N-(4-hydroxy phenyl) acet-
amide [1,2]. It is widely used as a minor analgesic and is used as an alter-
native to aspirin without the side effects of salicylates on gastric mucosa
[3].
This work aims to describe sensitive, precise and accurate spectro-
photometric methods for the simultaneous quantification of quaternary
mixtures with potentially overlapping components without interfer-
ence or preliminary separation.
Diphenhydramine hydrochloride (DH) is N,N-Dimethyl[2-
(benzhydryloxy)ethyl]amine [4]. It is an antihistamine used for
symptomatic relief of hypersensitivity reactions including allergic
rhinitis [5].
The suggested quaternary mixture consists of paracetamol (PC)
or acetaminophen, diphenhydramine hydrochloride (DH), para
Para aminophenol (PAP) is a potential impurity and a degradation
product of PC which is well known by its nephrotoxicity [6]. The N
oxide degradation product of DH (NOD) is diphenylmethoxy-N,N-
dimethyl ethanamine N-oxide. Fig. 1 shows the chemical structures of
the four components of the quaternary mixture.
Abbreviations: DD-RD-DW, double divisor - ratio difference-dual wavelength; DH, di-
phenhydramine; NOD, the N oxide degradation product of DH; PAP, para aminophenol;
PC, paracetamol; TD-RD, triple divisor ratio difference.
PC and DH are co-formulated in Panadol night® tablets for tempo-
rary relief of pain associated with sleeping disorders.
E-mail address: nesserinsalah@gmail.com.
https://doi.org/10.1016/j.saa.2020.118213
1386-1425/© 2020 Elsevier B.V. All rights reserved.

2
N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
Fig. 1. The structural formula of the four studied components.
PC and DH were simultaneously determined in their binary mixture
2. Experimental
by few analytical methods including HPLC methods [1,7–9], NIR spec-
troscopy [10], multivariate spectrophotometry [11] and capillary gas
chromatography [12].
2.1. Materials
Simultaneous quantification of PC and DH in presence of PAP was
described in the literature using first derivative spectrophotometric,
first derivative of ratio spectra, multivariate spectrophotometric and
HPTLC-densitometric methods, in bulk material and pharmaceutical
preparations [13].
2.1.1. Pure standards
Paracetamol and Diphenhydramine Hydrochloride were kindly sup-
plied by GlaxoSmithKline Company Cairo, Egypt. Their purities were
found to be 100.111.197% and 99.960.743%, respectively according to
the Pharmacopeial HPLC method [1].
A single published literature described the simultaneous quantifica-
tion of PC and DH in presence of PAP and NOD using double divisor sec-
ond derivative of ratio spectrum spectrophotometry, principle
component regression (PCR) and partial least squares (PLS) chemomet-
ric techniques [14].
Therefore, this work aims to describe new accurate selective and
time saving spectrophotometric methods for the simultaneous quantifi-
cation of the four components PC DH, PAP and NOD in their quaternary
mixture without interference or preliminary separation, with no need
for spectral derivatization steps.
P-aminophenol was purchased from Riedel-de Haen-AG-Germany;
its purity was certified to be 99%.
N-oxide derivative of DH was prepared by oxidative degradation of
Diphenhydramine hydrochloride using 30% H2O2 for 6 h after liberating
the free base by sodium hydroxide as described by Nouruddin W. Ali
et al. [14] .The degradation was followed by application to TLC plates
and using ethyl acetate - acetone −20%methanolic sodium lauryl sul-
phate - acetic acid (5:5:1:0.25 by volumes) as a developing system.
The complete oxidative degradation and the purity of the product
were confirmed by the disappearance of diphenehydramine spot and
the presence of only one spot for its N-oxide degradation product. The
chemical identity was confirmed by mass and IR spectra (Fig. 2).
The double divisor - ratio difference – dual wavelength method DD-
RD-DW was used by Mohamed et al. for simultaneous determination of
drotaverine/caffeine/paracetamol and p-amino phenol quaternary mix-
ture [15].
The novel triple divisor- ratio difference method TD-RD described in
this work is an extension of triple divisor ratio spectrophotometric
method [16] coupling it with ratio difference dual wavelength method,
where there is no need for zero amplitude difference for one of the in-
terfering components, which is the main limitation in DD-RD-DW
method.
2.1.2. Pharmaceutical formulation
Panadol night® tablets (Batch No. 141118) labeled to contain
500 mg of Paracetamol and 25 mg of Diphenhydramine Hydrochloride,
manufactured by GlaxoSmithKline company.
These two methods were able to quantify PC, DH, PAP and NOD in
pure forms and quaternary mixtures and PC/DH binary mixtures in
pharmaceutical formulation without interference or prior separation.
2.1.3. Chemicals and reagents
Methanol of HPLC grade was obtained from ChromasolvW, Sigma-
Aldrich ChemieGmbh, Germany.
Fig. 2. The IR (a) and mass (b) spectra of N,N-Dimethyl [2-(benzhydryloxy)ethyl]amine N-oxide.

N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
3
2.2. Instruments
against the concentrations (μg/mL) to obtain linear calibration curves
and the regression equations were computed.
A UV lamp with short wavelength 254 nm (USA) and a double beam,
UV–visible spectrophotometer (SHIMADZU, Japan) model UV-1601 PC
with quartz cells of 1 cm path length, connected to IBM compatible com-
puter. The software was UVPC personal spectroscopy software version 3.7.
2.4.2. Analysis of laboratory prepared mixtures of PC, DH, PAP and NOD
Mixtures containing different concentrations of PC, DH, PAP and NOD
were analyzed by applying the above described procedure “Table 1”.
2.3. Prepared solutions
2.4.3. Application to pharmaceutical formulation; Panadol night ® tablets
Ten tablets were weighed, powdered and mixed well. An accurately
weighed portion of the powdered tablets equivalent to 100 mg of PC
and 5 mg of DH was transferred into 100-mL volumetric flask; dissolved
by sonication for 30 min with 75 mL of methanol, completed to volume
with the same solvent, and then filtered. Ten mL of the above solution is
diluted with methanol in 100mL volumetric flask to obtain a working
solution of the dosage form containing 100 μg/mL of PC and 5 μg/mL
of DH. From the prepared working solution, a volume of 4 mL is accu-
rately transferred into 10-mL volumetric flask and the volume is com-
pleted with methanol to obtain a solution of the tablets in which PC
concentration is 40μg/mL and DH concentration is 2μg/mL. The same
procedure for TD-RD and DD-RD-DW methods were applied, where
the concentrations of PC and DH in the tablets were calculated from
the corresponding regression equations.
2.3.1. Standard solutions
For stock standard solutions containing 1 mg/mL; 0.1 g of each of the
four components was accurately weighed into a separate 100 mL volu-
metric flask; dissolved in 50 mL of methanol and shaken to dissolve, and
then the volume was completed with methanol. For working standard
solutions containing 100 μg/mL, aliquots of 10 mL of each stock standard
solution were transferred into four separate 100 mL volumetric flasks,
and then the volume was completed with methanol.
2.3.2. Laboratory prepared mixtures
Mixtures containing different ratios of four components were pre-
pared using their working solutions in methanol. The concentrations
of the four components in different mixtures are shown in Table 1.
N.B. The determination was done at room temperature and the spec-
trophotometric measurements were performed using scaling factor
equals 1 and Δλ equals 2.
2.4. Methodology
2.4.1. Linearity and construction of calibration curves
For TD-RD spectrophotometric method, the ratio spectra of different
concentrations of each drug were obtained by dividing the absorption
spectra by the normalized spectrum of a ternary mixture containing
equal concentrations (40 μg/mL) of the 3 interfering components. Two
wavelengths were selected in the ratio spectrum of each drug to
achieve a high amplitude difference. The selected wavelengths were
(256–290 nm), (220–230 nm), (230–245 nm) and (275–260 nm) for
PC, DH, PAP and NOD, respectively. The amplitude differences between
the 2 selected wavelengths in the ratio spectra of each drug were plot-
ted against the concentrations (μg/mL) to obtain linear calibration
curves and the regression equations were computed.
3. Results and discussion
3.1. Method development and optimization
The zero order absorption spectra of PC, DH, PAP and NOD show se-
vere overlap which hinders the use of direct spectrophotometry for the
simultaneous determination of the four compounds as shown in Fig. 3.
The aim of this work is to establish and validate spectrophotometric
methods which can easily resolve this overlap.
For DD-RD-DW spectrophotometric method, the ratio spectra of dif-
ferent concentrations of each drug were obtained by dividing the absorp-
tion spectra by the normalized spectrum of a binary mixture containing
equal concentrations (40 μ/mL) of two of the interfering components.
The used double divisors were DH/NOD mixture for PC and PAP, and
PC/PAP mixture for DH and NOD. Two wavelengths were selected in
the ratio spectra where the drug of interest gives considerable ampli-
tude difference, while the third interfering component gives zero am-
plitude difference. The selected wavelengths were (243–233 nm),
(223.8–270.2 nm), (237–250 nm) and (265–234.6 nm) for PC, DH,
PAP and NOD, respectively. The amplitude differences between the 2 se-
lected wavelengths in the ratio spectra of each drug were plotted
3.1.1. Double divisor – ratio difference – dual wavelength (DD-RD-DW)
spectrophotometric method
DD-RD-DW spectrophotometric method depends on dividing the
absorption spectrum of the quatertnary mixture by a double divisor
consisting of the normalized spectrum of a binary mixture of two of
the interfering components. Thus, these two interfering compounds
give constant amplitude all over the whole spectrum. In the ratio spec-
trum, two wavelengths are chosen at which the third interfering com-
ponent give equal amplitudes, such that the difference between the
amplitudes between these two wavelengths depends only on the con-
centration of one component, as these steps have been able to solve
the interference of the other three components.
Table 1
The simultaneous determination of paracetamol (PC), diphenhydramine (DH), p-aminophenol (PAP) and N-oxide degradate of diphenhydramine (NOD) in laboratory prepared mixtures
using DD-RD-DW and TD-RD.
Mixture Ratio % of degradation
Taken (μg/mL)
Recovery % obtained by DD-RD-DW
Recovery % obtained by TD-RD
PC:
DH
products
PC
25
DH PAP NOD PC
DH
PAP
NOD
PC
DH
PAP
NOD
1
2
3
4
5
6
7
1:2
1:2
1:1
2:1
2:1
2
2
5
10
20
50
0.5
2
1
4
99.323
–
100.822
–
–
98.286
99.929
100.286
98.857
99.214
99.714
101.269
99.651
0.982
98.983
–
100.786
–
–
98.153
99.876
100.154
98.754
99.456
99.342
101.134
99.553
0.968
100 200
98.947
–
98.851
–
20
40
40
40
40
20
20
20
2
1
4
8
20
36
1
2
4
1
99.935
100.641
100.542
101.115
101.051
100.657
0.690
101.833
99.796
100.389
101.852
99.944
100.763
0.903
99.875
100.562
100.653
101.097
100.843
100.336
0.779
101.687
99.951
100.451
101.768
99.874
100.753
0.826
100.263
101.250
99.763
97.763
99.597
1.321
100.103
101.390
99.540
97.901
99.557
1.313
20:1a 50
2:1
SD
90
20
18
Mean
a
The ratio present in Panadol night® tablets.

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N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
Fig. 3. The absorption spectra of 30 μg/mL Paracetamol (line), 5 μg/mL Diphenhydramine hydrochloride (dashes), 5 μg/mL p-aminophenol (dashes & dots) and 5 μg/mL N oxide degradate
of diphenhydramine hydrochloride (dots).
3.1.1.1. Selection of the solvent. Several solvents were tried (e.g water,
ethanol, methanol),where the best solvent was methanol regarding sol-
ubility, stability and sensitivity.
two wavelengths of maximum amplitude difference are chosen. There
is no need to inspect the ratio spectrum for a zero amplitude difference
for one of the interfering components, making the method simpler and
less tedious.
3.1.1.2. Selection of the divisor. Several binary mixtures selections from
the components and different concentrations were tried as divisors.
The chosen double divisors which showed the best sensitivity and selec-
tivity were the normalized spectra of DH/NOD binary mixture for PC
and PAP and PC/PAP binary mixture for DH and NOD. The binary mix-
tures used as divisors contain 40 μg/mL of each component.
3.1.2.1. Selection of the solvent. Several solvents were tried (e.g. water,
ethanol, methanol), where the best solvent was methanol regarding
solubility, stability and sensitivity.
3.1.2.2. Selection of the divisor concentration. Tertiary mixtures with dif-
ferent concentrations were tried as divisors. The normalized spectra of
tertiary mixtures containing 40 μg/mL of each of the interfering compo-
nents gave the best results regarding selectivity and sensitivity.
3.1.1.3. Selection of the wavelengths. Different wavelength pairs in the
ratio spectra were tried. The chosen wavelength pairs which achieved
a zero amplitude difference for the third interfering component as
well as a high amplitude difference for the component of interest are
shown in “Figs. 4–7”.
3.1.2.3. Selection of the wavelengths. Different wavelength pairs were
tried. The chosen wavelengths for each of the four components are
shown in “Figs. 8–11”.
3.1.2. Triple divisor – ratio difference (TD-RD) spectrophotometric method
TD-RD spectrophotometric method depends on dividing the absorp-
tion spectrum of the quatertnary mixture by a triple divisor consisting of
the normalized spectrum of a mixture of the three interfering compo-
nents. All the interfering components give constant amplitude all over
the whole spectrum. In the ratio spectrum of the component of interest,
3.2. Method validation
The proposed methods were found to be linear in the ranges of
2–45 μg/mL, 2–50 μg/mL, 2–40 μg/mL and 1–50 μg/mL for PC, DH, PAP
Fig. 4. The ratio spectra of paracetamol (line) and p-aminophenol (dots) using the normalized spectrum of the binary mixture containing 40 μg/mL of each of diphenhydramine HCL and its
N-oxide degradate as a double divisor for the determination of paracetamol by DD-RD-DW spectrophotometric method.

N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
5
Fig. 5. The ratio spectra of diphenhydramine HCl (line) and its N-oxide degradate (dots) using the normalized spectrum of the binary mixture containing 40 μg/mL of each of paracetamol
and p-aminophenol as a double divisor for the determination of diphenhydramine HCl by DD-RD-DW spectrophotometric method.
Fig. 6. The ratio spectra of paracetamol (line) and p-aminophenol (dots) using the normalized spectrum of the binary mixture containing 40 μg/mL of each of diphenhydramine HCL and its
N-oxide degradate as a double divisor for the determination of p-aminophenol by DD-RD-DW spectrophotometric method.
Fig. 7. The ratio spectra of diphenhydramine HCl (line) and its N-oxide degradate (dots) using the normalized spectrum of the binary mixture containing 40 μg/mL of each of paracetamol
and p-aminophenol as a double divisor for the determination of the N-oxide degradate of diphenhydramine HCl by DD-RD-DW spectrophotometric method.

6
N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
Fig. 8. The ratio spectrum of paracetamol using the normalized spectrum of the ternary mixture containing 40 μg/mL of each of p-aminophenol, diphenhydramine HCl and its N-oxide
degradate as a triple divisor for the determination of paracetamol by TD-RD spectrophotometric method.
Fig. 9. The ratio spectrum of diphenhydramine HCl using the normalized spectrum of the ternary mixture containing 40 μg/mL of each of its N-oxide degradate, paracetamol and p-
aminophenol as a triple divisor for the determination of diphenhydramine HCl by TD-RD spectrophotometric method.
Fig. 10. The ratio spectrum of p-aminophenol using the normalized spectrum of the ternary mixture containing 40 μg/mL of each of paracetamol, diphenhydramine HCl and its N-oxide
degradate as a triple divisor for the determination of p-aminophenol by TD-RD spectrophotometric method.

N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
7
Fig. 11. The ratio spectrum of N-oxide degradate of diphenhydramine HCl using the normalized spectrum of the ternary mixture containing 40 μg/mL of each of paracetamol,
diphenhydramine HCl and p-aminophenol as a triple divisor for the determination of N-oxide degradate of diphenhydramine HCl by TD-RD spectrophotometric method.
and NOD, respectively. The regression equations were calculated and
found to be:
The accuracy was further confirmed by applying the standard addi-
tion technique to Panadol night® tablets through the addition of pure
PC and DH separately to the tablet solutions, where good recoveries
were obtained as shown in Table 3.
A = 0.091 C − 0.012 r = 0.999 for PC by DD-RD-DW
Selectivity of the proposed methods is ascertained through their ap-
plication for simultaneous determination of the four components in lab-
oratory prepared mixtures of different concentration ratios as well as
simultaneous determination of PC and DH in Panadol night® tablets,
as the good recoveries in Tables 1 and 3 show no interference from
other drugs, impurities, excipients or inactive ingredients.
A = 0.028 C − 0.010 r = 0.999 for DH by DD-RD-DW
A = 0.081 C + 0.007 r = 0.999 for PAP by DD-RD-DW
A = 0.043 C + 0.006 r = 0.999 for NOD by DD-RD-DW
A = 0.078 C − 0.037 r = 0.999 for PC by TD-RD
A = 0.027 C r = 0.999 for DH by TD-RD
A = 0.057 C + 0.004 r = 1 for PAP by TD-RD and
A = 0.035 C + 0.001 r = 0.9998 for NOD by TD-RD
The limits of detection and limits of quantification of the four studied
compounds by the two proposed methods were calculated and pre-
sented in Table 2.
The robustness of the two methods was studied by testing their abil-
ity to remain unchanged upon making some small intended changes in
the method conditions such as the concentrations of the divisors, sol-
vent suppliers (two different suppliers) and temperature (4 °C, 35 °C
& 45 °C). The results shown in Table 4 confirmed good robustness.
Comparing our methods with the double divisor second derivative
spectrophotometric method described by Nouruddin W. Ali et al. [14]
for determination of PC and DH in presence of PAP and NOD, the double
divisor second derivative method couldn't quantify PC or NOD. DD-RD-
DW and TD-RD are simpler than the double divisor second derivative
method as they need no derivatization steps after division. DD-RD-
Where A is the amplitude difference between the two chosen wave-
lengths, C is the concentrations in μg/mL and r is the correlation
coefficient.
Good linearities are evident from the closeness of the correlation co-
efficient values to 1 and low values of intercepts as shown in Table 2.
The precision of the results obtained by the proposed methods was
tested and the results are shown in Table 2.
The accuracy of the methods was verified by applying the methods
for determination of pure samples of the four components, where the
concentrations were obtained by substitution in the corresponding re-
gression equations and the results are shown in Table 2.
Table 2
Results of assay validation parameters of the proposed methods for the determination of paracetamol (PC), diphenhydramine (DH), p-aminophenol (PAP) and N-oxide degradate of di-
phenhydramine (NOD).
Parameters
Double divisor ratio difference dual wavelength DD-RD-DW
Triple divisor ratio difference TD-RD
PC
DH
PAP
NOD
PC
DH
PAP
NOD
Range (μg/mL)
Linearity
2–45
2–50
2–40
1–50
2–45
2–50
2–40
1–50
Slope
Intercept
Correlation coefficient
0.091
−0.012
0.999
0.028
−0.010
0.999
0.081
0.007
0.999
0.043
0.006
0.999
0.078
−0.037
0.999
0.027
0.000
0.999
0.057
0.004
1
0.035
0.001
0.999
Accuracy (mean
Precision (RSD%)
Repeatabilitya
SD)
99.636 1.168
100.488 1.327
100.094 0.740
100.660 1.141
100.465 0.675
100.612 0.913
100.084 0.921 99.792 0.728
0.429
0.457
0.596
0.625
1.875
0.595
0.630
0.621
1.863
0.512
0.591
0.267
0.800
0.353
0.456
0.521
1.564
0.391
0.468
0.580
1.741
0.516
0.612
0.634
1.903
0.498
0.551
0.301
0.903
Intermediate precisionb 0.542
LOD (μg/mL)c
LOQ (μg/mL)d
0.577
1.731
a and b: the intra-day and inter-day relative standard deviations of concentrations (5, 25 and 45 μg/mL).
c: LOD is calculated using the formula (LOD = 3.3 × SD/slope).
d: LOQ is calculated using the formula (LOQ = 10 × SD/slope).

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N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
Table 3
Determination of paracetamol (PC) and diphenhydramine hydrochloride (DH) in Panadol night® tablets (batch no. 141118) by the proposed double divisor ratio difference dual wave-
length (DD-RD-DW) and triple divisor ratio difference (TD-RD) methods and application of standard addition technique.
Panadol night® tablets (batch no.
141118)
Double divisor ratio difference dual wavelength (DD-RD-DW)
Triple divisor ratio difference (TD-RD)
Recovery%
SD
Standard addition techniquea
Recovery%
SD
Standard addition techniquea
(recovery%
SD)
0.592
0.392
(recovery%
SD)
0.575
0.416
PC (taken concentration 40 μg/mL)
DH (taken concentration 2 μg/mL)
98.325
103.743
0.206
0.479
100.479
100.507
98.337
103.557
0.221
0.458
100.457
100.497
a
Standard addition technique was performed by adding pure PC (2, 3 & 4 μg/mL) and DH (5, 20, 40 μg/mL) separately to tablet solution.
Table 4
Robustness study of the proposed double divisor ratio difference dual wavelength (DD-RD-DW) and triple divisor ratio difference (TD-RD) methods in terms of RSD%.
Robustness parameters
Double divisor ratio difference dual wavelength DD-RD-DW
Triple divisor ratio difference TD-RD
PC
DH
PAP
NOD
PC
DH
PAP
NOD
Two different methanol suppliersa
Different temperaturesb
0.578
0.891
0.682
0.725
0.609
0.654
0.593
0.652
0.525
0.825
0.614
0.689
0.673
0.696
0.632
0.678
(4 °C, 35 °C & 45 °C)
Divisor concentration (40
5 μg/mL)
0.312
0.341
0.590
0.456
0.303
0.314
0.610
0.415
a
ChromasolvW, Sigma-Aldrich ChemieGmbh, Germany & Al-Nasr pharmaceutical chemicals company, Abu Zaabal, Cairo, Egypt.
The samples have been kept at the mentioned temperatures for 3 h before analysis.
b
3.3. The comparison of the method validation results of the two proposed
methods
Table 5
Statistical comparison of the results obtained by the proposed methods and the
Pharmacopoeal HPLC method when applied to pure Paracetamol (PC) and
Diphenehydramine hydrochloride (DH).
The comparison between the method validation results of the two
proposed methods showed that; TD-RD is more accurate than DD-RD-
DW for the determination of PC, DH and NOD, while DD-RD-DW is
more accurate for the determination of PAP.
TD-RD was found to be more precise for the determination of the
four components.
Items
Double divisor
ratio difference
dual wavelength TD-RD
DD-RD-DW
Triple divisor
ratio difference
Pharmacopoeal
HPLC method
[1]
PC
DH
PC
DH
PC
DH
The results of the robustness studies of the two methods showed
that TD-RD is more robust than DD-RD-DW for the determination of
PC and DH, while DD-RD-DW is more robust for the determination of
PAP and NOD.
The future research plan is to develop and validate a successful HPLC
method for separation and quantification of the four components and to
study the factors or parameters affecting the stability of the drugs and
the presence of any other related compounds.
Mean
SD
N
Variance
Student's t-test
(2.262)a
F-value (3.18)b
99.967 100.207 100.225 100.375 99.997 99.897
1.076 1.131 0.668 0.888 0.833 0.756
10 10 10 10 10 10
0.694 0.572
1.158
0.945
1.279
0.482
0.446
0.508
0.789
0.211
0.457
0.246
0.521
0.639
a
Figures in parentheses represent the corresponding tabulated values of t at P = 0.05.
Figures in parentheses represent the corresponding tabulated values of F at P = 0.05.
b
4. Conclusion
DW and TD-Rd are somewhat less accurate and have nearly the same
linearity ranges as the double divisor second derivative method.
Statistical comparison of the results obtained by the proposed
methods to those obtained by the pharmacopeial HPLC method [1],
when applied for determination of PC and DH in pure samples and phar-
maceutical formulation are shown in Tables 5 and 6, respectively. No
significant differences were found with respect to accuracy and preci-
sion, as revealed by the calculated t and F values.
The present work provides new sensitive, accurate and selective
spectrophotometric techniques for the simultaneous determination of
PC, DH, PAP and NOD in pure forms, laboratory prepared quaternary
mixture and in pharmaceutical formulation. The novel method TD-RD
is simpler and less tedious. Both methods need no spectral derivatiza-
tion, which recommends the use of the proposed methods for routine
quality control analysis.
CRediT authorship contribution statement
Table 6
Statistical comparison of the results obtained by the proposed methods and the
Pharmacopoeal HPLC method when applied to Panadol night® tablets (batch no. 141118).
Nessreen S. Abdelhamid: Conceptualization, Methodology, Soft-
ware, Validation, Formal analysis, Investigation, Resources, Writing -
original draft, Writing - review & editing.
Items
Double divisor
ratio difference
Triple divisor
ratio difference
Pharmacopoeal
HPLC method [1]
dual wavelength TD-RD
DD-RD-DW
Declaration of competing interest
PC
DH
PC
DH
PC
DH
The authors declare that they have no known competing financial
interests [13–16] or personal relationships that could have appeared
to influence the work reported in this paper. [3]
Mean
SD
N
98.325 103.743 98.337 103.557 98.360 103.665
0.206
6
0.479
6
0.221
6
0.458
6
0.218
6
0.472
6
Variance
0.042
0.780
0.901
0.229
0.782
0.974
0.049
0.859
0.981
0.210
0.696
0.948
0.048
0.223
Student's t-test (2.23)a
F-value (4.28)b
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a
Figures in parentheses represent the corresponding tabulated values of t at P = 0.05.
Figures in parentheses represent the corresponding tabulated values of F at P = 0.05.
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b

N.S. Abdelhamid / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 233 (2020) 118213
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