Journal of AOAC INTERNATIONAL, 104(4), 2021, 968–974

doi: 10.1093/jaoacint/qsab027
Advance Access Publication Date: 24 February 2021
Article

Downloaded from https://academic.oup.com/jaoac/article/104/4/968/6149119 by Chinese Academy of Medical Sciences user on 10 January 2022
DRUG FORMULATIONS

Comparative Study for the Assay of Plant Derived

Chemicals in Pharmaceutical Formulation Using
Methods Dependent on Factorized Spectra
Nesma M. Fahmy* and Adel M. Michael
Ahram Canadian University, Faculty of Pharmacy, Pharmaceutical Chemistry Department, Egypt

*Corresponding author’s e-mail: nesma_fahmy@acu.edu.eg

Abstract
Background: Modern built-in spectrophotometer software supporting mathematical processes provided a solution for
increasing selectivity for multicomponent mixtures.
Objective: Simultaneous spectrophotometric determination of the three naturally occurring antioxidants—rutin(RUT),
hesperidin(HES), and ascorbic acid(ASC)—in bulk forms and combined pharmaceutical formulation.
Method: This was achieved by factorized zero order method (FZM), factorized derivative method (FD1M), and factorized
derivative ratio method (FDRM), coupled with spectrum subtraction(SS).
Results: Mathematical filtration techniques allowed each component to be obtained separately in either its zero, first, or
R
derivative ratio form, allowing the resolution of spectra typical to the pure components present in Vitamin C ForteV tablets.
The proposed methods were applied over a concentration range of 2–50, 2–30, and 10–100 mg/mL for RUT, HES, and ASC,
respectively.
Conclusions: Recent methods for the analysis of binary mixtures, FZM and FD1M, were successfully applied for the analysis
of ternary mixtures and compared to the novel FDRM. All were revealed to be specific and sensitive with successful
application on pharmaceutical formulations. Validation parameters were evaluated in accordance with the International
Conference on Harmonization guidelines. Statistical results were satisfactory, revealing no significant difference regarding
accuracy and precision.
Highlights: Factorized methods enabled the resolution of spectra identical to those of pure drugs present in mixtures.
Overlapped spectra of ternary mixtures could be resolved by spectrum subtraction coupled FDRM (SS-FDRM) or by
successive application of FZM and FD1M.

Rutin (RUT), hesperidin (HES), and ascorbic acid (ASC) are natu-
ral plant derived antioxidants with the chemical structures that
are displayed in Figure 1. RUT is a flavonoid with antioxidant,
antiallergic, anti-inflammatory, anti-proliferative, and anti-
carcinogenic properties (1). It can be officially determined by ti-
tration (2). Other techniques for their quantitative analysis in-
clude spectrophotometry (3–5) and chromatography (5, 6). HES
is a flavanone glycoside found naturally in citrus fruits. It has
an antioxidant effect (7) and can help in reducing edema (8). It
can be analyzed by titration (2), spectrophotometry (9, 10),
fluorimetry (11), and chromatography (12). ASC, or vitamin C, is
an essential nutrient for tissue repair and enzymatic production
of neurotransmitters, with antioxidant properties (13). It can be
officially determined by titration (14). Moreover, ASC assay is
achieved by spectrophotometric methods (15–17) and chroma-
tography (18, 19). Determination of the binary mixture of RUT
and HES can be achieved by liquid chromatography (6). Both are
essential components in correcting bruising tendency and de-
creasing capillary permeability and fragility. They work best
when administered concomitantly with ASC (8) and they

Received: 27 September 2020; Revised: 6 February 2021; Accepted: 16 February 2021

C AOAC INTERNATIONAL 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com
V

968
 Fahmy & Michael: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021
Figure 1. The chemical structures of (a) RUT, (b) HES, and (c) ASC.
support ASC in the role it plays in the maintenance of healthy
collagen and cartilage for the normal function of the skin,
gums, and immune system. By combining the three antioxi-
R
dants, Vitamin C ForteV tablets can be used to treat the common
cold and to promote immunity defense, wound healing, and
joint and tissue support. The literature reveals the assay of the
three components in mixtures by chemometric (20) and chro-
matographic methods (21, 22).
The aim of the current study is to develop smart methods
for simultaneous determination of plant derived antioxidants
using factorized spectra to achieve spectral profiles identical to
those of the pure single drugs under investigation, thus en-
abling the measurement of each component at its kmax or Pmax
with maximum accuracy and precision.
Method
Theoretical Background
Factorized zero order method (FZM) coupled with spectrum
subtraction (SS) and factorized derivative method (FD1M) coupled
with SS
These methods can be used for mixtures of two analytes X and
Y showing no chemical interaction, with each obeying Beer’s
law. If their spectra show a wavelength where one component
has a contribution while the other is of no contribution, either
in their zero order or derivative form, then FZM or FD1M can
be applied, where extraction of the targeted spectra at their
corresponding order can be achieved as follows (23):
(a) Generation of factorized spectrum in FZM.—This factorized
spectrum is obtained by dividing the zero-order absorption
spectrum (D0) of any concentration of pure Y (throughout
all the scanned wavelengths) by its recorded absorbance
value at the chosen wavelength (kzero point) where X doesn’t
show any contribution (23–26).
(b) Generation of factorized spectrum in FD1M.—This factorized
spectrum is obtained by dividing the first derivative spec-
trum (D1) of any concentration of pure Y (throughout all
the scanned wavelengths) by its recorded peak amplitude
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value at the chosen wavelength (kzero point) where the first
derivative of X doesn’t show any contribution (23–26).
(c) Recovering the targeted spectra in the mixture and calculation of
their concentrations by SS-FZM or SS-FD1M.—The spectrum of
analyte (Y) in the mixture can be recovered by multiplying
this recorded response by the corresponding factorized
spectrum. While the spectrum of analyte (X) in the mixture
can be recovered by subtracting the recovered Y spectrum
from the mixture using the software, i.e., SS method.
Concentrations of each analyte are calculated using regres-
sion equations computed at the appropriate wavelength as the
pure form.
Similarly, the methods can be used for ternary mixtures of
X, Y, and Z, where the peak amplitude value at the chosen
wavelength (k zero point) in which only Y has a contribution,
while X and Z have zero crossing points intersecting the base-
line. Thus, the peak amplitude at k zero point is only correspond-
ing to the concentration of component Y. In this case the
spectrum of analyte Y in the mixture can be recovered by multi-
plying this recorded response by the corresponding factorized
spectrum and subtracting it from the ternary mixture, resulting
in a binary mixture of Z þ X.
Factorized derivative ratio method (FDRM) coupled with SS
This method can be used for ternary mixtures of three analytes
(X, Y, and Z), showing no chemical interaction, with each
one obeying Beer’s law, and with their zero order absorption
spectra showing severe overlap. One of the components is
chosen as a divisor (Z’) and its contribution is eliminated
through derivatization using a suitable order where Z/Z’ is
cancelled by derivatization. An appropriate wavelength is cho-
sen (k zero point) where X/Z’ spectrum has maxima or minima at
the zero point of the third component Y/Z’ (either zero crossing
or zero contribution point) (27).
(a) Generation of factorized spectrum in FDRM.—This factorized
derivative ratio spectrum of X is obtained by dividing
the derivative of the ratio spectra (DD1) of X by its
recorded peak amplitude value at this specified wavelength
(k zero point).
 970 | Fahmy & Michael: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021
(b) Recovering the targeted spectra in the mixture and calculation of
their concentrations by SS-FDRM.—First, the first derivative of
the ratio spectrum of the ternary mixture using component
Z’ as divisor is obtained. Second, the recovered DD1 spectra
of components X (X/Z’) in the mixture can be obtained by
multiplying the recorded amplitude at the chosen wave-
length (k zero point) by the previously computed DD1 factor-
ized spectrum for X, whose concentration is calculated by
substitution in the regression equation representing the
linear relationship of the amplitudes of pure X using Z’ as a
divisor (Pmax–min) versus the corresponding concentrations.
Third, the DD1 spectrum of component Y (Y/Z’) is obtained
by its subtraction from the DD1 spectrum of the ternary
mixture then its concentration is calculated using the
corresponding regression equation. Similarly, the third
component (Z) can be obtained through the same proce-
dure by using either X or Y as a divisor.
Instrument and Software
A double-beam UV-Vis spectrophotometer, model V-760, Jasco
(Tokyo, Japan) connected to a compatible computer with
R
Microsoft Excel 2010 and Spectra ManagerV software, version 2
(JASCO Corp.) was used. The solutions were held in 1.00 cm
quartz cuvettes and were scanned in the range 200–400 nm at
room temperature.
Reagents
(a) Pure samples.—A pure standard of ASC was purchased from
BDH (England), while RUT and HES were purchased from
Sigma Aldrich (Spain). Their purity was checked using the
reported method (22) and was found to be 101.81 6 1.25,
99.55 6 1.25, and 100.82 6 0.84, respectively.
(b) Pharmaceutical formulation.—Tablets of Vitamin C Forte were
purchased from Australia. Each tablet claimed to contain
25 mg HES, 50 mg RUT, and 1000 mg calcium ascorbate
which is equivalent to 826 mg ASC.
(c) Solvents and reagents.—Deionized water, dimethyl formam-
ide (DMF), and 85% phosphoric acid of analytical grade.
(d) Standard solutions.—Standard stock solutions were prepared
with a concentration of 100 mg/mL of RUT and HES in a
mixture of DMF-0.1% phosphoric acid (50:50, v/v), and
a standard stock solution of ASC with a concentration of
1000 mg/mL was prepared in the same solvent mixture.
Procedures
RUT, HES, and ASC spectra
Zero-order absorption spectra of 10 mg/mL of RUT, 10 mg/mL of
HES, and 10 mg/mL of ASC were obtained by scanning over the
range of 200–400 nm against a mixture of DMF-0.1% phosphoric
acid (50:50, v/v) as a blank.
(a) Factorized spectrum of RUT.—The D0 spectrum of RUT was
divided by the recorded absorbance at 375 nm and stored
on the computer.
(b) Factorized spectra of HES.
(1) The D1 spectrum of HES was divided by the recorded
amplitude at 297 nm and stored on the computer.
(2) The DD1 spectra of HES using 10 mg/mL RUT as a
divisor was divided by the recorded amplitude at
271 nm and stored on the computer.
(3) The DD1 spectrum of HES using 70 mg/mL ASC as a
divisor was divided by the recorded amplitude at
272 nm and stored on the computer.
Linearity and calibration graphs
Portions equivalent to 2–50 mg/mL RUT, 2–30 mg/mL HES , and
10–100 mg/mL ASC were transferred from standard solutions
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(100 mg/mL) for RUT, HES, and (1000 mg/mL) ASC, into three series
of 10 mL volumetric flasks. These were diluted to volume with
DMF-0.1% phosphoric acid (50:50, v/v). The spectra of the pre-
pared standards were scanned against DMF-0.1% phosphoric
acid (50:50, v/v) as blank in the range of 200–400 nm and saved
on the computer.
(a) RUT.
(1) D0.—Zero order absorption spectra (D0) of RUT were
recorded and saved on the computer. A calibration
graph was made by relating the kmax of the scanned
spectra at 360 nm and the corresponding concentra-
tions to compute the regression equation.
(2) DD1.—The first derivative of the ratio spectra resulting
from dividing the D0 spectra of RUT by 70 mg/mL D0 of
ASC. A calibration graph was constructed by plotting
amplitude Pmax–min (P265–280) versus the corresponding
concentration of RUT to compute the regression
equations.
(b) HES.
(1) D1.—First derivative spectra (D1) of HES were recorded
and saved on the computer. A calibration graph was
designed by relating the Pmax–min of the scanned spec-
tra at P297–273 and the corresponding concentrations to
compute the regression equation.
(2) DD1.—The first derivative of the ratio spectra resulting
from dividing the D0 spectra of HES by 70 mg/mL D0 of
ASC. A calibration graph was constructed by plotting
amplitude Pmax–min (P294–275) versus the corresponding
concentration of HES to compute the regression
equations.
(c) ASC.
(1) D1.—First derivative spectra (D1) of ASC were recorded
and saved on the computer. A calibration graph was
constructed by relating the Pmax of the scanned spec-
tra at 262 nm and the corresponding concentrations to
compute the regression equation.
(2) DD1.—The first derivative of the ratio spectra resulting
from dividing the D0 spectra of ASC by 10 mg/mL D0 of
RUT. A calibration graph was constructed by plotting
amplitude Pmax–min (P276–267) versus the corresponding
concentration of ASC to compute the regression
equation.
Applying the spectrophotometric methods for determining RUT, HES,
and ASC on laboratory prepared mixtures
The D0 RUT was obtained by multiplying the peak amplitude at
375 nm by the factorized spectrum of RUT. This was subtracted
from the mixture to get (HES þ ASC). After derivatization, the
peak amplitude at 297 nm was multiplied by the corresponding
factorized spectrum of HES to obtain its first derivative spec-
trum. SS of HES from the resolved binary mixture resulted in
the first derivative spectrum of ASC.
The peak amplitude at 272 nm on the derivative ratio spec-
trum of the ternary mixture using 70 mg/mL ASC as a divisor was
multiplied by the corresponding factorized spectrum to get the
DD1 of HES. By SS, the DD1 of RUT was obtained. The peak
 Fahmy & Michael: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021
amplitude at 271 nm on the derivative ratio spectrum of the ter-
nary mixture using 10 mg/mL RUT as a divisor was multiplied by
the corresponding factorized spectrum to get the DD1 of HES. By
SS the DD1 of ASC was obtained.
Application to pharmaceutical formulation
Ten Vitamin C Forte tablets were weighed and finely powdered
in a clean dry mortar. A portion of the powder equivalent to a
single tablet was quantitatively transferred to a 250 mL volu-
metric flask and dissolved in a mixture of 0.1% phosphoric acid
in water-DMF (50:50, v/v) to mark. The solution was ultra-
sonicated for 10 min and then filtered to obtain a stock solution
with claimed concentrations of 3304, 200, and 100 mg/mL for
ASC, RUT, and HES, respectively. Aliquots of 0.3 mL were
transferred from the prepared solution into 10 mL volumetric
flasks and these were diluted to volume with the same solvent
mixture so that the claimed concentrations of ASC, RUT, and
HES were 99.12, 6.00, and 3.00 mg/mL, respectively. The
assay was carried out as under analysis of laboratory
prepared mixtures.
Results and Discussion
Modern built-in spectrophotometer software that supports
mathematical processes such as subtraction, multiplication,
and division, in addition to derivatization of spectra, was a solu-
tion for increasing the selectivity for multicomponent mixtures.
The overlapping zero order absorption spectra of RUT, HES, and
ASC hindered the direct measurement of any one of them at its
kmax, so mathematical filtration of each component in the
mixture was obtained alone either in its zero, first, or derivative
ratio form, by the aid of factorized spectra, reproducing typical
spectra of the analytes under investigation from their synthetic
mixtures or their pharmaceutical formulation, confirming the
spectral profile of each drug.
Figure 2. Zero order absorption spectrum of 15 lg/mL RUT, 10 lg/mL HES, and 70 lg/mL ASC.
| 971
This smart study was conducted using two approaches; the
first one depended on applying SS-FZM and SS-FDM for resolv-
ing binary mixtures successively in order to resolve a ternary
mixture. The second approach depended on the severely
overlapped region of the spectrum where all the drugs under
investigation contributed and each one could be resolved by
SS-FDRM.
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Approach 1
The zero order absorption spectrum of the three drugs in combi-
nation showed that at 375 nm only RUT had a contribution as
shown in Figure 2. SS-FZM was applied, where multiplying the
absorbance at 375 nm by the corresponding factorized RUT
spectrum showed the D0 RUT in the mixture to be mathemati-
cally filtered from the ternary mixture alone allowing measure-
ment at its kmax (360 nm). Subtracting the obtained D0 RUT from
the ternary mixture (RUT þ HES þ ASC) resulted in obtaining
the resolved binary mixture of HES and ASC.
Although the resolved binary mixture by SS of (HES þ ASC)
showed an extended region for HES over ASC, trials for getting
the zero order absorption of HES by factorized spectrum failed
as at the apparently extended region of HES there was a very
small contribution of ASC. The first derivative of the resolved bi-
nary mixture showed a peak at 297 nm where only HES had a
contribution, as represented in Figure 3. SS-FDM was applied by
multiplying this peak amplitude at 297 nm by the corresponding
factorized spectrum to mathematically filter the D1 HES in mix-
ture alone where P297–273 was substituted in the corresponding
regression equation to get HES concentration, and then by SS
from the D1 resolved binary mixture. The D1 ASC was obtained
where P262 was substituted in the corresponding regression
equation to obtain ASC concentration.
 972 | Fahmy & Michael: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

Approach 2 the derivative ratio spectrum of RUT was obtained and mea-
sured (P265–280).
This approach deals with the three spectra of the cited
On the other hand, the derivative ratio of the ternary mix-
antioxidants in their severely overlapped region (200–330 nm).
ture using RUT as a divisor, where RUT was eliminated from
SS-FDRM was applied twice using either a divisor (ASC 70 mg/mL
the mixture by derivatization, ASC and HES showed a peak at
or RUT 10 mg/mL).
271 nm where only HES had a contribution and ASC was
As shown in Figure 4A, the derivative ratio of the ternary
zero crossing, as presented in Figure 4B. Multiplying this peak
mixture using ASC as a divisor, where ASC was eliminated from

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by the corresponding factorized spectrum resulted in the
mixture by derivatization, RUT and HES showed a peak at
derivative ratio spectrum of HES, by SS from the DD1 mixture,
272 nm where only HES had a contribution and RUT was zero
the derivative ratio spectrum of ASC was obtained and
crossing. Multiplying this peak by the corresponding factorized
Pmax–min (P267–276) was substituted in the corresponding regres-
spectrum resulted in the derivative ratio spectrum of HES to be
sion equation to obtain its concentration.
measured from Pmax–min (P275–294), by SS from the DD1 mixture,

Figure 3. First derivative spectrum of 10 lg/mL HES and 70 lg/mL ASC.

Figure 4. (a) The first derivative of the ratio spectra of RUT and HES using ASC 70 lg/mL as a divisor. (b) The first derivative of the ratio spectra of ASC and HES using
RUT 10 lg/mL as a divisor.
 Fahmy & Michael: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021 | 973

Mathematical filtration successfully allowed obtaining RUT in
its D0 and DD1 forms while obtaining both HES and ASC in their
D1 or DD1 forms in both lab mixtures and Vitamin C Forte tablets.
Validation
Validation of the methods was carried out according to
range. Concentrations were obtained and good percentage
recoveries indicate accepted accuracy, as shown in Table 1.
(d) Precision.—Repeatability and intermediate precision for the
methods were assessed by analyzing three different con-
centrations of each drug repeated in the same day or on
three different days, respectively.
(e) Specificity.—The methods were shown to be specific by ana-

International Conference on Harmonization guidelines as fol-
lows (28):
(a) Linearity.—Linearity was calculated by constructing the dif-
ferent calibration graphs in three replicates for each data
point. The parameters of the regression equation for each
method and concentration ranges are listed in Table 1.
(b) LOD and LOQ.—LOD and LOQ for the proposed methods are
given in Table 1. LOD and LOQ were calculated using the slope
of calibration graph and SD of the residuals of the regression
line. The sensitivity of the proposed method was confirmed
by low LOD and LOQ values.
(c) Accuracy.—Accuracy of the results were assessed by apply-
ing the proposed methods to analyzing synthetic mixtures
of known quantities of RUT, HES, and ASC within linearity
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lyzing different laboratory prepared mixtures of RUT, HES,
and ASC in different ratios within the linearity range.
Satisfactory results of mean percentage recovery 6 SD are
displayed in Table 2.
(f) Application to the Australian formulation.—The developed
methods were applied to the assay of the cited drugs in
Vitamin C Forte, as presented in Table 2, without any inter-
ference of the pharmaceutical excipients.
(g) Statistical comparison.—Statistical results obtained by com-
paring the proposed methods and the reported method (20)
are shown in Table 3, revealing no significant difference.
Moreover, one-way analysis of variance (ANOVA) con-
firmed that all the methods had no significant difference
compared with the reported method for the determination
of RUT, HES, and ASC.

Table 1. Assay parameters and method validation obtained by applying the proposed spectrophotometric methods for the determination of
pure powdered forms of RUT, HES, and ASC

RUT HES ASC

0 1 1 1 1
Drug name D DD D DD D DD1
Method 360 nm P265–280 P297–273 P275–294 262 nm P276–267

Range, mg/mL 5–50 2–30 3–30 2–30 10–100

Slope 0.0214 0.0078 0.0021 0.0688 0.0006 0.001
Intercept –0.002 0.0004 0.0003 –0.0035 –0.0005 –0.0002
Correlation coefficient, r 1.0000 1.0000 0.9999 0.9999 0.9997 0.9999
Accuracy, R% 6 SD 100.18 6 0.20 99.57 6 0.28 100.94 6 1.02 100.25 6 1.02 99.69 6 1.95 100.17 6 0.85
Repeatability, RSDa 0.020 0.088 0.314 1.149 1.201 0.532
Inter-day precision, RSDa 0.471 1.555 0.875 1.158 1.639 1.423
LOD 0.02 0.05 0.40 0.57 2.11 1.13
LOQ 0.07 0.16 1.23 1.73 6.39 3.41

a
RSD of concentrations 10, 20, and 30 mg/mL for RUT, HESP, and ASC; each concentration was analyzed three times.

Table 2. Determination of RUT, HES, and ASC in laboratory prepared mixtures and pharmaceutical dosage form by the proposed methods

RUT HES ASC

Mixture number
Ratio FZM FDRM FDM FDRM SS-FDM SS-FDRM
(RUT: HESP: ASC) 360 nm P265–280 P297–273 P275–294 262 nm P276–267

Mixture 1 99.90 102.34 101.30 100.13 102.90 98.91

6 0.58 6 0.48 6 0.11 6 1.06 6 0.43 6 1.40
Mixture 2 100.38 98.83 102.35 98.89 100.12 102.14
6 0.44 6 0.34 6 1.57 6 0.13 6 0.12 6 1.87
Mixture 3 100.76 99.74 102.97 101.66 98.50 101.78
6 1.67 6 1.28 6 0.60 6 0.37 6 0.88 6 0.69
Mixture 4 99.33 98.43 102.49 101.04 98.24 98.09
6 0.30 6 0.65 6 1.57 6 0.32 6 1.98 6 0.22
Mixture 5 99.98 98.01 100.69 100.68 101.39 99.78
6 0.01 6 0.06 6 0.41 6 1.03 6 1.92 6 1.07
Vitamin C Forte tablets, batch number: (B)065619 99.18 100.85 102.68 103.28 104.16 102.80
6 0.62 6 0.88 6 1.22 6 1.59 6 0.42 6 0.92
 974 | Fahmy & Michael: Journal of AOAC INTERNATIONAL Vol. 104, No. 4, 2021

Table 3. Statistical comparison between results obtained by the proposed methods and the reported method (20) for the determination of RUT,
HES, and ASC in pure powder form

RUT HES ASC

0 1 1 1
D DD D DD D1 DD1
Parameters Reported methoda 360 nm P265–280 Reported methoda P297–273 P275–294 Reported methoda 262 nm P276–267

Mean 99.30 99.73 99.68 100.09 100.67 99.57 101.10 100.85 99.69

Downloaded from https://academic.oup.com/jaoac/article/104/4/968/6149119 by Chinese Academy of Medical Sciences user on 10 January 2022
SD 1.24 0.82 0.82 1.37 1.33 1.59 1.47 1.88 1.40
n 15 6 6 15 6 6 15 6 6
Variance 1.54 0.67 0.67 1.88 1.78 2.53 2.16 3.53 1.96
F —— 2.30 2.30 —— 1.06 1.35 —— 1.63 1.10
(2.96)b
Student’s t —— 0.78 0.69 —— 0.88 0.75 —— 0.34 2.03
(2.09)b
ANOVA 0.47 0.47 1.79
F
(3.40)b

a
Partial least square (PLS) method.
b
Figures between parentheses represent the corresponding tabulated values of t and F at a ¼ 0.05

Conclusions
The novel spectrophotometric methods were found to be rapid
and economic. They also have the advantage of high selectivity
for determining mixtures with overlapping spectra. They do not
need sophisticated software or expensive solvents. SS could be
used for mixtures showing partial or complete overlap, and its
advantage is cancelling the whole spectrum of the interfering
substance, accordingly the choice of wavelength selected for
calibration is not critical. It is simple and does not require
any special software, so was successfully coupled with
FZM, FDM, and FDRM. Mathematical filtration steps on the spec-
trophotometric spectra could resolve the partial overlap and
then resolution of the ternary, severely overlapped mixture was
overcome by the novel SS-FDRM. Robustness of the methods is
very good as the proposed methods were measured at their
maxima with accepted accuracy and precision. The proposed
methods accordingly could be successfully applied for routine
analysis of the studied drugs, either in their pure bulk powders
or pharmaceutical formulations, in quality control laboratories.
Acknowledgments
The authors would like to show their deep everlasting thanks to
Hayam Lotfy for her continuous support.
Conflict of interest
The authors declare that there is no conflict of interest in the
presented work.
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