Novel spectral manipulations for determinations of Tolnaftate along with related toxic compounds: Drug profiling and a comparative study

Article abstract of DOI:10.1016/j.saa.2019.117290

A comparative study using novel quadruple divisor and mean centering of ratio spectra spectrophotometric methods was developed for resolution of five- component mixture of Tolnaftate, β-naphthol (Tolnaftate alkaline degradation product and its toxic impurity), methyl(m-tolyl)carbamic acid (Tolnaftate alkaline degradation product), N-methyl-m-toluidine (Tolnaftate toxic impurity) and methyl paraben (as a preservative). For the novel quadruple divisor method, each component in the quinary mixture was determined by dividing the quinary mixture spectrum by a sum of standard spectrum of equal concentration of the other four components as a quadruple divisor. First derivative of each ratio spectra was then obtained which allowed selective determination of each component without interference from other components in the mixture. The second method was mean centering of ratio spectra that depended on utilizing the mean centered ratio spectra in four successive steps leading to enhancement of the signal to noise ratio. The absorption spectra of the five studied components were recorded in the wavelength range of 210–350 nm. The mean centered fourth ratio spectra amplitudes for each component were used for its determination. The developed methods were successfully applied for determination of laboratory prepared quinary mixtures to ensure method's specificity, then, were further applied on Tinea Cure cream where no interference from excipients. For the first time, Tolnaftate was determined along with its toxic impurity; β-naphthol, that could be absorbed by the skin, causing systemic toxic effects, unlike Tolnaftate that poorly absorbed, indicating the significance of this work. The proposed methods were statistically compared with each other and with the reference method. Furthermore, ICH guidelines were followed for their validation.

Full text of DOI:10.1016/j.saa.2019.117290

Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and Biomolecular
Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Novel spectral manipulations for determinations of Tolnaftate along with
related toxic compounds: Drug profiling and a comparative study
Raghda A. Emam ⁎, Maha M. Abdelrahman, Eglal A. Abdelaleem, Nouruddin W. Ali
Pharmaceutical Analytical Chemistry, Faculty of Pharmacy, Beni-Suef University, Alshaheed Shehata Ahmed 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:
A comparative study using novel quadruple divisor and mean centering of ratio spectra spectrophotometric
methods was developed for resolution of five- component mixture of Tolnaftate, β-naphthol (Tolnaftate alkaline
degradation product and its toxic impurity), methyl(m-tolyl)carbamic acid (Tolnaftate alkaline degradation
product), N-methyl-m-toluidine (Tolnaftate toxic impurity) and methyl paraben (as a preservative). For the
novel quadruple divisor method, each component in the quinary mixture was determined by dividing the
quinary mixture spectrum by a sum of standard spectrum of equal concentration of the other four components
as a quadruple divisor. First derivative of each ratio spectra was then obtained which allowed selective determi-
nation of each component without interference from other components in the mixture. The second method was
mean centering of ratio spectra that depended on utilizing the mean centered ratio spectra in four successive
steps leading to enhancement of the signal to noise ratio. The absorption spectra of the five studied components
were recorded in the wavelength range of 210–350 nm. The mean centered fourth ratio spectra amplitudes for
each component were used for its determination. The developed methods were successfully applied for determi-
nation of laboratory prepared quinary mixtures to ensure method's specificity, then, were further applied on
Tinea Cure® cream where no interference from excipients. For the first time, Tolnaftate was determined along
with its toxic impurity; β-naphthol, that could be absorbed by the skin, causing systemic toxic effects, unlike
Tolnaftate that poorly absorbed, indicating the significance of this work. The proposed methods were statistically
compared with each other and with the reference method. Furthermore, ICH guidelines were followed for their
validation.
Received 24 February 2019
Received in revised form 1 June 2019
Accepted 17 June 2019
Available online 19 June 2019
Keywords:
Tolnaftate
β-Naphthol
|N-Methyl-m-toluidine
Mean centering of ratio spectra
Quadruple divisor
Methyl paraben
©
2019 Elsevier B.V. All rights reserved.
1
. Introduction
referenced as TF impurity (A) and related substance [1]. Moreover, it
is formerly reported as TF hydrolytic degradation product [5]. It is pro-
duced from alkaline hydrolysis of TF along with methyl(m-tolyl)car-
bamic acid (MTC) [5]. N,3-dimethylaniline (N-methyl-m-toluidine
(NMT)), is referenced as TF impurity D [1]. It is TF related impurity aris-
ing during synthesis [6]. N-methyl-m-toluidine is toxic by different con-
tact routes; (inhalation, oral and dermal) and prolonged or repeated
exposure to it causes specific target organ toxicity [7]. Methyl paraben
Tolnaftate (TF) is chemically designated as O-naphthalen-2-yl-
methyl(3-methylphenyl)thiocarbamate [1]. It is an antifungal drug
that used topically in the treatment or prophylaxis of superficial derma-
tophyte infections and of pityriasis versicolor [2]. β-Naphthol (BN) is
chemically designated as naphth-2-ol [2]. It was formerly used as an an-
thelmintic in hookworm and tapeworm infections, but it has been su-
perseded by less toxic and more efficient drugs [2]. It has a potent
parasiticidal effect and has been used topically in the treatment of sca-
bies, ringworm, and other skin diseases [2]. BN is well absorbed by the
skin and chronic dermal application or prolonged contact with BN
may cause toxic effects such as skin sensitization, kidney impairment,
hemolytic anemia, hemoglobinuria and retina opacities [3,4]. BN is
(MP) is
a hydroxybenzoates preservative; alkyl esters of p-
hydroxybenzoic acid, with antibacterial and antifungal properties.
Methyl paraben is used as preservative in pharmaceutical preparations
in usual concentrations of up to 0.25% [2].
A review in the literature revealed few methods for quantitative de-
termination of TF including Pharmacopeial [1] and non-Pharmacopeial
spectrophotometric methods [5,8–10]. Different HPLC methods were
reported for determination of TF in bulk powder, different pharmaceu-
tical formulations or human skin samples [11–13], and in combination
with other drugs [14–16]. An isocratic supercritical fluid chromato-
graphic method was also reported for determination of TF and its re-
lated impurities (NMT and β-naphthol-1-chlorothiocarbamate) [8].
Abbreviations: BN, β-Naphthol; MTC, Methyl(m-tolyl)carbamic acid; MP, Methyl
paraben; NMT, N-Methyl-m-toluidine; TF, Tolnaftate.
⁎
Corresponding author.
E-mail addresses: raghdaemam88@gmail.com, Raghda011141@pharm.bsu.edu.eg
(R.A. Emam).
https://doi.org/10.1016/j.saa.2019.117290
386-1425/© 2019 Elsevier B.V. All rights reserved.
1

2
R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
Current ICH guidelines [17] recommend the determination of drug
minor components as impurities and degradation products along with
the active drug for developing and validating a stability indicating
assay method (SIAM). Hydrolysis is one of the most common routes of
degradation because water is used as a solvent for most drugs and ex-
cipients or due to moisture in the air [18]. Accordingly, TF was subjected
to alkaline hydrolysis as mentioned in the literature [5], and the ob-
tained degradation products (BN and MTC) were isolated, identified
and characterized by different spectroscopic techniques (IR, MS spec-
troscopy and 1H NMR). Moreover, NMT was synthesized by methyla-
tion of m-toluidine with dimethylsulphate [19]. The purity of the
methylated product was checked by TLC, IR, 1H NMR and mass
spectrometry.
To the best of our knowledge, there is no reported method for deter-
mination of TF in presence of its impurity along with degradation prod-
ucts. This determination has to be done due to the reported toxicity of
BN and NMT and the different pharmacological action of BN than the
parent drug. Also, unlike TF, BN is well absorbed by the skin then causes
systemic toxic effects that confirm the importance of this determination
due to BN toxicity. The UV spectra of related components are usually ex-
pected to be overlapped. Mean centering of ratio spectra spectrophoto-
metric methods for precise resolution of multi-component mixtures
were reported [20, 21]. The novel quadruple divisor method could be
considered as an extension of the recently reported triple divisor spec-
trophotometric method [20].
ꢀ
ꢁ
d
∝TF CTF
¼
þ Zero
dλ ∝BN C0BN þ ∝MTC C0MTC þ ∝NMT C0NMT þ ∝MP C0MP
The first derivative amplitudes measured in the last equation are
only dependent on concentration values, CTF, C , C , C
0
0
0
0
TF BN MTC NMT
and
, C
0
0
C
MP
(C is standard concentration), but are independent on the concen-
tration values CBN, CMTC, CNMT and CMP in the quinary mixture.
A calibration curve could be constructed by plotting the first deriva-
tive of the obtained ratio spectrum amplitude at any wavelength;
d
½
ðAm=∝BN C0BN þ ∝MTC C0MTC þ ∝NMT C0NMT þ ∝MP C0 MPꢀ
,
dλ
versus concentration of TF and a calculated regression equation was ob-
tained from which TF concentration in the quinary mixture could be de-
termined. The concentrations of the other components, BN, MTC, NMT
and MP, can be determined individually by similar procedures to that
of TF.
1.1.2. Mean centering of ratio spectra spectrophotometric method
Issa et al. [21] provided the MCR theoretical background used for re-
solving quinary mixtures as follow, if a mixture of five compounds, TF,
BN, MTC, NMT and MP obeyed Beer's law over the whole specified
wavelength range, then
Am ¼ ∝TFCTF þ ∝BNCBN þ ∝MTCCMTC þ ∝NMTCNMT þ ∝MPCMP
ð1Þ
The work in this manuscript was planned to determine and resolve
severely overlapped spectra of TF, toxic BN, MTC, toxic NMT and MP
by mean centering of ratio spectra and novel quadruple divisor spectro-
photometric methods.
The proposed spectrophotometric methods had the advantages of
being cost effective and could be used for resolution of complex drug
matrix without previous separation or extraction steps or need of com-
plicated apparatus like chromatographic methods.
m
where A is the absorbance vector of the mixture, ∝TF, ∝BN, ∝MTC, ∝NMT
and ∝MP are the absorptivity vectors, CTF, CBN, CMTC, CNMT and CMP are
the concentrations of TF, BN, MTC, NMT and MP in the mixture,
respectively.
For determination of TF, Eq. (1) is divided by ∝BN (the first divisor,
D1), corresponding to standard solution spectrum of BN, so the first
ratio spectrum is gained.
1
1
.1. Theoretical background
Am ∝TF CTF
∝MTC CMTC ∝NMT CNMT ∝MP CMP
¼
þ CBN þ
þ
þ
ð2Þ
∝BN
∝BN
∝BN
∝BN
∝BN
.1.1. Quadruple divisor spectrophotometric method
In the quadruple divisor spectrophotometric method, for determina-
If Eq. (2) is mean centered, then
tion of TF in a quinary mixture containing TF, BN, MTC, NMT and MP, the
spectra of the mixtures were divided by the sum standard spectra of
equal concentrations of BN, MTC, NMT and MP as (a quadruple divisor)
as following:
Am
D1
∝TF CTF
D1
∝MTC CMTC
∝NMT CNMT
∝MP CMP
þ mc
D1
mc
¼ mc
þ mc
þ mc
D1
D1
ð3Þ
As : Am ¼ ∝TFCTF þ ∝BNCBN þ ∝MTCCMTC þ ∝NMTCNMT þ ∝MPCMP
∝MTC
∝MTC
D1
If Eq. (3) is divided by mc
= mc
= the second divisor,
∝BN
D2, so the second ratio spectrum is obtained
and
Am
D1
D2
∝TF CTF
D1
D2
∝NMT CNMT
∝MP CMP
D1
D2
mc
mc
mc
mc
0
0
0
0
Aqd ¼ ∝BNC BN þ ∝MTCC MTC þ ∝NMTC NMT þ ∝MPC M
D1
D2
¼
þ CMTC þ
þ
ð4Þ
ð5Þ
Where, Am is the absorbance vector of the mixture, Aqd, is the ab-
sorbance vector of the quadruple divisor, ∝TF, ∝BN, ∝MTC, ∝NMT and
MP are the absorptivity vectors, CTF, CBN, CMTC, CNMT and CMP are the
If Eq. (4) is mean centered, then
∝
Am
D1
∝TF CTF
D1
D2
∝NMT CNMT
∝MP CMP
D1
D2
mc
mc
mc
mc
concentrations of TF, BN, MTC, NMT and MP in the mixture, respectively,
D1
D2
and C 0B N, CMTC, CNMT and CMP are the standard concentration of BN, MTC,
0
0
0
mc
¼ mc
þ mc
þ mc
D2
NMT and MP, respectively, in the quadruple divisor. The first derivative
of the resulted ratio spectra was then obtained as following:
∝
NMT
D1
∝NMT
D1
D2
mc
mc
d
Am
And if Eq. (5) is divided by mcð
Þ = mcð
Þ(the third
dλ ∝BN C0BN þ ∝MTC C0MTC þ ∝NMT C0NMT þ ∝MP C0 MP
∝MTC
mc
D1
d ∝TF CTF þ ∝BN CBN þ ∝MTC CMTC þ ∝NMT CNMT þ ∝MP CMP
¼
=
divisor, D3), this will results in the third ratio spectrum,
dλ ∝BN C0BN þ ∝MTC C0MTC þ ∝NMT C0NMT þ ∝MP C0 MP
Am
D1
∝TF CTF
D1
D2
∝MP CMP
D1
D2
mc
mc
mc
ꢀ
mc
=D3 ¼ mc
=D3 þ CNMT þ mc
=D3 ð6Þ
d
∝TF CTF
D2
¼
þ Constantꢀ
dλ ∝BN C0BN þ ∝MTC C0MTC þ ∝NMT C0NMT þ ∝MP C0MP

R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
Table 1
3
If Eq. (6) is mean centered, so,
The first, second, third and fourth divisors for TF, BN, MTC, NMT and MP in mean center of
0
1
0
1
ratio spectra method.
Am
∝TF CTF
mc
mc
B
D1
D2
C
B
D1
D2
C
First divisor Second divisor Third divisor (D3)
(D1) (D2)
Fourth divisor
(D4)
mc@mc
=D3A ¼ mc@mc
=D3A
0
B
1
C
∝
MP CMP
∝MTC
∝MP
mc
mc
mc
,
∝NMT
∝BN
using
0 μg mL−
mc
D1
D1
D2
BN,
10 μg mL
mcð
Þ,
þ mc@mc
=D3A
ð7Þ
D1
TF
−1
mcð
4
Þ, using
D3
1
D2
1
−1
using
μg mL NMT
−1
MTC
4 μg mL MP
∝
MTC
TF
∝MP
mc
MP
D1
mc
,
∝NMT
mc
∝
D1
mc
TF,
mcð
Þ,
D1
BN
6 μg mL−1
using
mcð
Þ, using
D3
and, if Eq. (7) is divided by mcð
=D3Þ, (the fourth divisor, D4), this
D2
−1
4 μg mL−
1
D2
will result in the fourth ratio spectrum:
using
4
μg mL NMT
−1
MTC
4 μg mL MP
MP
∝
mc
0
1
0
1
∝BN
∝NMT
D1
Am
D1
D2
∝TF CTF
D1
D2
mcð
using
Þ,
mc
,
using
−1
mc
mcð
mc
mc
TF,
10 μg mL
∝TF
D1
D3
B
C
B
C
MTC
NMT
MP
−1
Þ using
mc@mc
=D3A=D4 ¼ mc@mc
=D3A=D4 þ CMP
ð8Þ
ð9Þ
10 μg mL
D2
−1
−1
BN
10 μg mL NMT
10 μg mL
MP
∝
D1
D3
MP
∝MTC
mc
∝
∝TF
μg mL BN
BN
mc
mcð
If Eq. (8) is mean centered, so,
TF,
μg mL
mcð
using
Þ,
mc
,
using
−1
D1
Þ, using 4 μg
Þ, using 6 μg
−1
−1
8
D2
−1
4
mL MTC
μg mL−1 MP
X ¼ Z CTF
where
4
∝
NMT
D1
mc
∝MTC
D1
D2
∝
∝TF
6 μg mL BN
BN
mc
mcð
Þ
TF,
8 μg mL
mc
using
−1
D3
,
mcð
0
0
B
1
C
1
,using
6 μg mL
Am
D1
−1
−1
mL MTC
mc
B
C
NMT
X ¼ mc @mc@mc
=D3A=D4A and Z
D2
0
0
1
1
∝
D1
D2
TF CTF
mc
B
B
C
C
¼
mc @mc@mc
=D3A=D4A
-
-
Methanol, methylene chloride, toluene, potassium carbonate, so-
dium carbonate, m-toluidine, sodium hydroxide, hydrochloric acid
The mean centered fourth ratio spectrum signal obtained in Eq. (9) is
practically dependent only on CTF, but is independent on CBN, CMTC, CNMT
and CMP concentration values in the mixture. The concentrations of the
other components (BN, MTC, NMT and MP) can be individually deter-
mined by similar procedures to that of TF.
(
El-Nasr Pharmaceutical Chemicals Co., Abu Zabaal, Cairo, Egypt).
Dimethyl sulphate, Methanol HPLC grade: (CHROMASOLV®, Sigma-
Aldrich, Germany).
2
. Experimental
2.2.4. Preparation of degradation products
In the literature [5], TF was treated with 2 N NaOH solution in a boil-
ing water bath for about 20 min to produce a fluorescent solution due to
sodium β-naphtholate that was confirmed by UV and TLC in comparison
with pure sodium β-naphtholate. But, there was no confirmation or sep-
aration of the second assumed degradation product mentioned in their
work. Complete hydrolysis was done by heating TF solution in a water
bath at 90–100 °C with 5 N NaOH solution in a volume of 1 mL for 25
min [5]. In our work, 0.5 g of pure powdered TF was dissolved in meth-
anol and refluxed with 75 mL of 2 N NaOH solution at 80 °C. A fluores-
2
.1. Instruments
A double beam UV–visible spectrophotometer (SHIMADZU, Japan)
model UV-1601 PC with quartz cell of 1 cm path length, connected to
IBM compatible computer. The software used was UVPC personal spec-
troscopy software V. 3.7.
Data concerning MCR was performed using PLS-Toolbox 2.0 running
under MATLAB®, version (R2007b).
2
cent solution was produced after 30 min with smelling of H S gas
2
2
.2. Materials and reagents
odour and the degradation process was monitored by TLC using methy-
lene chloride: toluene (5.5:4.5, by volume) as a developing system. Two
new spots other than the drug spot were observed on TLC plate and the
degradation process was continued for 4 h till complete degradation of
TF. For separation of the two degradates from each other, the alkaline
degradation solution was neutralized by dropping 2 N HCl solution till
pH 6.8 at which degradates were precipitated, then isolated by filtration
.2.1. Pure standards
Tolnaftate (TF) and Methylparaben (MP) pure standards were
kindly supplied by Kahira Pharm. & Chem. Ind. Co. Cairo, Egypt as a
gift and were used as working standards. Their purity was found to be
9
cate and 99.97% for TF, according to the reference spectrophotometric
method.
9.49% and 99.22%, respectively, according to the manufacturer certifi-
2 3
and allowed to dry. 20 mL of 1 N Na CO solution was added to the
powdered mixture of the two degradates and stirred for 1 h. on the
magnetic stirrer at room temperature then the solution was filtered.
The precipitate was washed with distilled water and left to dry (a),
then the filtrate was dropped by 1 N HCl until precipitate was formed
which isolated and allowed to dry (b). both (a) and (b) degradation
products were subjected to IR, 1H NMR and MS spectroscopy to confirm
their chemical structures and the spectral data confirmed that,
degradate (a) was β-naphthol and degradate (b) was methyl(m-tolyl)
carbamic acid, the same as the reported scheme of degradation [5].
2
.2.2. Pharmaceutical formulation
Tinea Cure® Cream (B.N. 1670291) manufactured by Kahira Pharm.
Chem. Ind. Co. Cairo, Egypt, labeled to contain 1 g TF per 100 g cream.
&
2
.2.3. Chemicals and solvents
All chemicals used throughout this work were of analytical grade
and were used without further purification;

4
R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
2
.2.5. Synthesis of NMT impurity
As mentioned in the literature [19], N-alkylated aromatic amines
could be synthesized from the corresponding aromatic amines using
an alkylating agent like alkyl sulphate or alkyl halide. In a round bot-
tomed flask, a mixture of 0.01 M m-toluidine, 50 mL acetone (as a sol-
2 3
vent) and 0.02 M anhydrous powdered K CO (as a base and
dehydrating agent) was stirred for 1 h at 30–35 °C then 0.011 M of di-
methyl sulphate (DMS) (as alkylating agent) was added drop wise.
The progress of the reaction was followed up by TLC using methanol:
methylene chloride (4: 6, by volume) as a developing system and stir-
ring was continued for 4 h. till disappearance of m-toluidine spot on
TLC. The reaction mixture was filtered then the filtrate was dried at
room temperature to evaporate the solvent. The solid residue was
then collected and subjected to IR, 1H NMR and MS spectroscopy to con-
firm the formation of NMT.
2
•
.2.6. Solutions
Stock solutions of TF, BN, MTC, NMT and MP (1 mg mL−1): 100 mg of
each TF, BN, MTC, NMT and MP were accurately and separately
weighed into 100-mL measuring flasks and the volume was then
completed to the mark with methanol as a solvent.
Fig. 2. Degradation pathway of Tolnaftate.
•
Working solutions of TF, BN, MTC, NMT and MP (0.1 mg mL−1): They
were prepared by diluting 10 mL from their respective stock solutions,
into five separate 100-mL volumetric flasks and the volume was com-
pleted using methanol as a solvent.
3
3
5
.2. Linearity and construction of calibration curves
.2.1. Quadruple divisor spectrophotometric method (QD)
Accurately measured aliquots equivalent to 10–120, 5–150, 20–280,
–150 and 5–120 μg of TF, BN, MTC, NMT and MP, respectively, were
•
•
Laboratory prepared mixtures of TF, BN, MTC, NMT and MP: They
were prepared from their respective working standard solutions
−1
(
0.1 mg mL ) in different ratios using methanol as a solvent.
transferred from their corresponding working standard solution (0.1
mg mL ) into five separate sets of 10-mL volumetric flasks and the vol-
ume was completed to the mark with methanol. Zero order absorption
spectra of each set were recorded in the range of 215–350 nm.
For determination of TF, its absorption spectra were divided by sum
standard spectra of equal concentrations of BN, MTC, NMT and MP (6 μg
Pharmaceutical formulation standard solution: An accurately
weighed quantity of Tinea Cure® Cream equivalent to 100 mg TF
was transferred into a 100-mL volumetric flask containing 75 mL
methanol. The flask was then heated on a water bath until the
cream melted then the mixture was sonicated for 15 min and the vol-
ume was then completed to the mark with methanol to obtain 1000
−1
−1
mL of each) as a quadruple divisor. The first derivative (1D) of the re-
−1
μg mL TF pharmaceutical formulation stock solution from which a
sulted ratio spectra of TF was then obtained using Δλ = 8 and scaling
factor = 1000, the amplitude values measured at 256 nm were plotted
against the corresponding TF concentrations for constructing its calibra-
tion curve and the regression equation was calculated.
−
1
working solution of 100 μg mL
TF was quantitatively prepared
using methanol as a solvent.
Similarly, BN, MTC, NMT and MP were determined following the
abovementioned procedure for TF determination using a quadruple di-
visor of a mixture containing equal concentrations of the other four
3
. Procedure
−
1
3
.1. Spectral characteristics
components (8, 4, 8 and 6 μg mL of each, for determination of BN,
1
MTC, NMT and MP, respectively) and the D ratio spectra were then ob-
The absorption spectra of 10 μg mL−1 of each TF, BN, MTC, NMT and
tained using Δλ and scaling factor, 8 and 1000, for MTC and MP determi-
nations and 4 and 100 for BN and NMT determinations. The 1D ratio
spectra amplitudes were measured at 336.6, 288.4, 292 and 254 nm
for BN, MTC, NMT and MP, respectively. Calibration curves were con-
structed between the amplitude values at the aforementioned wave-
lengths against the corresponding concentrations of each component
and regression equations were then computed.
MP, were recorded over the range 200–400 nm using methanol as a
blank.
Fig. 1. Zero-order absorption spectra of 10 μg mL− each of Tolnaftate (TF), β-naphthol
BN), Methyl-tolylcarbamic acid (MTC), N-methyl-m-toluidine (NMT) and Methyl
paraben (MP) using methanol as a blank.
1
Fig. 3. Synthesis scheme of N-methyl-m-toluidine from m-toluidine.

Fig. 4. IR spectra of (a): Tolnaftate, (b): β-naphthol, (c): Methyl-tolylcarbamic acid, (d): m-toluidine and (e): N-methyl-m-toluidine.

Fig. 5. 1H NMR spectra of (a): Tolnaftate, (b): β-naphthol, (c): Methyl-tolylcarbamic acid, (d): m-toluidine and (e): N-methyl-m-toluidine.

Fig. 6. Mass spectra of (a): Tolnaftate, (b): β-naphthol, (c): Methyl-tolylcarbamic acid, (d): m-toluidine and (e): N-methyl-m-toluidine.

8
R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
3
.2.2. Mean centering of ratio spectra spectrophotometric method (MCR)
Different aliquots equivalent to 10–120, 5–150, 20–280, 5–150 and
–100 μg of TF, BN, MTC, NMT and MP, respectively, were accurately
from their respective working solutions (0.1 mg mL−1). The UV spectra
of prepared mixtures were recorded in the wavelength range of 215–
350 nm. The procedures explained under linearity for each method
were followed for calculation of the concentration of each component
in the mixture using their corresponding regression equation.
5
transferred from their corresponding working standard solutions (0.1
−1
mg mL ) into five separate series of 10-mL volumetric flasks, the vol-
umes were then completed with methanol. Zero order absorption spec-
tra of each set were recorded over the range of 215–350 nm. For TF
determination, the recorded spectra were divided by a standard spec-
4. Results and discussion
−
1
trum of 10 μg mL of BN, first divisor (D1), the obtained first ratio
Although chromatographic techniques are widely used methods of
analysis due to their high specificity and sensitivity [22], they are expen-
sive, time consuming and need tedious preliminary separation steps.
Spectrophotometric methods are simple and cost effective comparing
to chromatographic methods, especially in laboratories lacking expen-
sive chromatographic tools. Tolnaftate was subjected to alkaline hydro-
lysis producing BN and MTC. Both NMT and BN are TF toxic impurities
and BN has different pharmacological action than parent drug (TF),
Therefore, from the analytical point of view, it was important to develop
a highly specific stability indicating spectrophotometric methods for de-
termination of TF along with its toxic impurities (BN and NMT) and al-
kaline induced degradation product (MTC) without interference from
MP present as a preservative in the pharmaceutical formulation. The
work in this manuscript also aimed to provide an insight into alkaline
induced degradation pathway and degradation products of TF and to
elucidate the chemical structure of the degradation products and the
synthesized impurity. The developed methods are considered as a pro-
file for TF related components including toxic ones.
spectra were then mean centered. These vectors were then divided by
∝
∝
MTC
BN
mc
, second divisor (D2), and the second ratio spectra were then
∝
NMT
mc
∝BN
mean centered. By the same procedure, usingmc
as a third di-
D2
visor (D3), the third ratio spectra could be obtained then mean cen-
tered. Finally, the fourth ratio spectra were obtained by the same way,
MP
D1
D2
mc
using mcð
=D3Þ as a fourth divisor (D4). For BN, MTC, NMT and
MP determination, their respective recorded absorption spectra were
manipulated similarly as with TF determination using their correspond-
ing divisors concentrations mentioned in Table 1. The amplitudes of the
mean centered fourth ratio spectra at 285 and 279 nm for TF and NMT,
respectively, and at peak to peak 251–252, 310–311 and 312–313 nm
for BN, MTC and MP, respectively, were measured and plotted against
the corresponding concentrations of each component. Calibration
curves were obtained and regression equations were computed.
The UV-absorption spectra of TF and its related components along
with MP, Fig. 1, showed severe overlap in the zero order that hindered
their direct determination. Thus, from the analytical point of view, de-
velopment of new spectrophotometric methods for resolution of such
overlapped spectra without prior separation steps or using hyphenated
instruments such as HPLC, was a must.
3
.3. Application to pharmaceutical formulation
Proper dilution for the pharmaceutical formulation standard stock
−1
solution (1000 μg mL ) was performed to obtain concentration within
the linearity range of TF. The procedure illustrated under construction of
calibration curve for each method was then followed and concentration
of TF was determined using the computed regression equations.
4.1. Structure elucidation of TF degradation products and synthesized
impurity
Tolnaftate was subjected to alkaline hydrolysis under stress degra-
3
.4. Application to laboratory prepared mixtures
dation conditions giving two degradation products (BN and MTC) due
to cleavage of the thionoester bond in TF structure. Degradation path-
way of TF was illustrated in Fig. 2. NMT was synthesized by methylation
of m-toluidine using DMS as methylating agent in presence of
Different mixtures of TF, BN, MTC, NMT and MP in several ratios in
concentrations within their linearity ranges were laboratory prepared
Fig. 7. The coincident spectra of the first derivative of the ratio spectra of 4 μg mL pure TF (____) and a quinary mixture of TF, BN, MTC, NMT, and MP (4 μg mL−1 of each) (…..) using a
−1
mixture of 6 μg mL− each of BN, MTC, NMT and MP as a quadruple divisor and methanol as a blank.
1

R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
9
Fig. 8. The coincident spectra of the first derivative of the ratio spectra of 5 μg mL BN (−.-.-.) and a quinary mixture of TF, BN, MTC, NMT, and MP (10, 5, 5, 5 and 5 μg mL−1, respectively)
−1
−1
(
__) using a mixture of 8 μg mL each of TF, MTC, NMT and MP as a quadruple divisor and methanol as a blank.
anhydrous K CO
2 3
as a base and dehydrating agent. The scheme of syn-
4.1.2. 1H NMR spectral changes
thesis was shown in Fig. 3. The degradation products and synthesized
impurity were isolated as mentioned before and then characterized by
IR, 1H NMR and MS analyses; Figs. 4-6.
Tolnaftate 1H NMR spectrum showed two singlet signals at δ 2.351
and 3.414 that were assigned to the N-substituted methyl group pro-
tons and meta-substituted methyl group protons on benzene ring.
These signals were disappeared in the 1H NMR spectrum of Deg. I
with the appearance of phenolic proton chemical shift at 9.807 ppm
that confirmed the formation of BN, as shown in Fig. 5(a & b). In the
1H NMR spectrum of Deg. II, the methyl groups protons were found to
resonate at δ 2.307 and 3.088 and a new very broad singlet signal was
appeared at δ 11.556 due to carboxylic group proton that confirmed
the formation of MTC as a degradation product as shown in Fig. 5(c).
The 1H NMR spectrum of m-toluidine, Fig. 5(d), showed singlet signal
at δ 2.262 due to meta substituted methyl group protons on the benzene
4
.1.1. IR spectral changes
Cleavage of the thionoester bond in TF structure by hydrolysis led to
the formation of the corresponding phenol (BN) (Deg. I) and carboxylic
acid (carbamic acid in MTC, Deg. II) accompanying by liberation of H
gas. The IR spectrum of TF, Fig. 4(a), showed aliphatic CH bands at
2
S
−1
−1
2
850–2930 cm and aromatic CH stretch at 3040 cm . The IR spec-
trum of Deg. I, Fig. 4(b), showed disappearance of the aliphatic CH
bands but not the aromatic one and appearance of new band at 3265
ring and a singlet signal at δ 3.254 due to two protons of the primary
−1
1
cm indicating the formation of new phenolic OH group of BN. Pres-
ence of the same CH bands of TF and appearance of OH broad band at
amine (NH
2
) (integration 2 protons). In the H NMR spectrum of the
synthesized impurity, Fig. 5 (e), another singlet signal at δ 1.785 was ap-
peared due to the new methyl group protons added by methylation and
conversion of the primary amine to a secondary one that was confirmed
by the appearance of a singlet signal at δ 4.768 due to only one proton of
NH (integration 1 proton) as shown in Fig. 5(e).
−
1
−1
3
352 cm and a characteristic amide C_O stretch at 1640 cm in
the IR spectra of Deg. II, Fig. 4(C), confirmed the presence of carbamic
acid group (\\N\\COOH) and formation of MTC. Meta-toluidine IR
spectrum, Fig. 4(d), showed a forked peak at 3327–3450 cm−1 charac-
2
teristic to NH group that disappeared in the IR spectrum of the synthe-
−1
sized product and a new sharp single peak at 3336 cm was appeared
instead, as shown in Fig. 4(e), indicating N-methylation of m-toluidine
and conversion of the primary amine to a secondary amine to form N-
methyl-m-toluidine.
4.1.3. Mass spectra
The electron impact showed mass ion peak at m/z 307 correspond-
ing to the intact drug, Fig. 6(a). The electron impact of Deg. I showed
Fig. 9. The coincident spectra of the first derivative of the ratio spectra of 10 μg mL MTC (− − -) and a quinary mixture of TF, BN, MTC, NMT, and MP (5, 5, 10, 5 and 5 μg mL−1
−
1
,
respectively) (__) using a mixture of 4 μg mL− each of TF, MTC, NMT and MP as a quadruple divisor and methanol as a blank.
1

10
R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
Fig. 10. The coincident spectra of the first derivative of the ratio spectra of 5 μg mL NMT (….) and a quinary mixture of TF, BN, MTC, NMT, and MP (10, 5, 5, 5 and 5 μg mL−1, respectively)
−1
−1
(
__) using a mixture of 8 μg mL each of TF, MTC, NMT and MP as a quadruple divisor and methanol as a blank.
mass ion peak at 144 m/z corresponding to the molecular weight of the
expected degradate (BN), while that of Deg. II showed mass ion peak of
shown in Fig. 7-11. For determination of TF, the first derivative am-
plitude of the quinary mixture measured at wavelength maximum
of 256 nm were coincided with that obtained from pure TF of the
same concentration as in the quinary mixture, ensuring elimination
of the interference from the other four components. Similarly, BN,
MTC, NMT and MP were measured at 336.6, 288.4, 292 and 254 nm,
respectively; corresponding to a maximum or minimum wavelength
of the first derivative of the ratio spectra obtained using a mixture of
the other four components as a divisor, Fig. 7-11.
1
65 m/z corresponding to the molecular weight of the expected
1
degradate (MTC) and confirmed its identity and IR and H NMR inter-
pretations. Moreover, the synthesized impurity mass spectrum showed
parent peak at m/z 121 corresponding to NMT molecular weight and
confirming that, methylation had been done and affirming the IR and
1
H NMR interpretations. Mass spectra are shown in Fig. 6(a)(e).
4
4
.2. Methods development and optimization
.2.1. Quadruple divisor spectrophotometric method (QD)
For development of QD method, different concentrations of the
4.2.2. Mean centering of ratio spectra spectrophotometric method (MCR)
For MCR method optimization, selection of a suitable divisor concen-
tration was the key element in MCR method optimization for resolving
the highly overlapped spectra under study. Different divisors concentra-
tions were tested for each component determination in the quinary
mixture. The optimum divisors concentrations used, that gave optimum
specificity and results reproducibility, were listed in Table 1. As illus-
trated under linearity, for determination of TF, BN, MTC, NMT and MP
concentrations, mean centered fourth ratio spectra were obtained and
their amplitudes at 285 nm, Fig. 12(a), at peak to peak 251–252 nm,
Fig. 12(b), 310–311 nm, Fig. 12(c), 279 nm, Fig. 12(d) and at peak to
peak 312–313 nm, Fig. 12(e), respectively, were measured and plotted
against their corresponding concentrations.
quadruple divisor were tried out. Sum standard spectra of 6 μg
mL
quadruple divisor for specific determination of TF. For BN, MTC,
NMT and MP determinations, 8, 4, 8 and 6 μg mL
four components were used as quadruple divisors, respectively. An-
other key element in the QD method development was wavelength
selection. Different wavelengths were tried to select the optimum
one, at which, the first derivative signal maximum or trough ampli-
tude of the pure component, to be analyzed, synchronized with the
first derivative amplitude of its quinary mixture to get rid of the in-
terference of the other four components in the quinary mixture, as
−
1
each of BN, MTC, NMT and MP were chosen as the optimum
−
1
of the other
Fig. 11. The coincident spectra of the first derivative of the ratio spectra of 4 μg mL MP (-..-..-) and a quinary mixture of TF, BN, MTC, NMT, and MP (4 μg mL−1of each) (__) using a
−1
mixture of 6 μg mL− each of TF, MTC, NMT and MP as a quadruple divisor and methanol as a blank.
1

Fig. 12. The mean centered ratio spectra of (a): Tolnaftate, (b): β-naphthol, (c): Methyl-tolylcarbamic acid (d) N-methyl-m-toluidine and (e): Methyl paraben using methanol as a blank.

12
R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
Table 2
Regression parameters of the proposed QD and MCR spectrophotometric methods for determination of TF, BN, MTC, NMT and MP.
Parameters
QD method
TF
MCR method
TF
BN
MTC
NMT
MP
BN
MTC
NMT
MP
Linearity
Range
μg mL 1)
−
12–1
0.5–15
2–28
0.5–15
0.5–10
1–12
0.5–15
2–28
0.5–15
0.5–10
(
Slope
Intercept
Correlation Coefficient (r )
Accuracy (Mean)
Precision (%RSD)
1.9781
0.3396
0.9998
99.64
4.8289
0.7554
0.9999
99.58
1.0687
1.2752
0.9998
100.51
4.8249
0.7961
0.9999
99.56
1.9835
0.2933
0.9999
99.64
0.8665
−0.2357
0.9999
99.72
8.1095
0.5565
0.9999
100.03
5.1362
40.4734
0.9998
100.02
150.6880
55.8279
0.9999
99.86
2.8190
0.1550
0.9999
100.39
2
a
Repeatability
1.009
1.118
1.193
1.281
1.273
1.315
1.131
1.201
1.087
1.213
1.047
1.138
1.203
1.307
1.181
1.242
1.230
1.361
1.208
1.222
b
Intermediate Precision
LODc
μg mL−1)
0.333
1
0.167
0.5
0.667
2
0.067
0.5
0.167
0.5
0.333
1
0.167
0.5
0.667
2
0.167
0.5
0.167
0.5
(
LOQc
μg mL−1)
(
a
The intraday and:
b
c
The interday, average of three different concentrations repeated three times within day and three times in three successive days, respectively.
LOD and LOQ values were calculated using the following equations; LOD = 3.3 × SD / slope, LOQ = 10 × SD / slope [23].
4
4
1
.3. Methods application
Y5 ¼ 1:9835 C5 þ 0:2933 r5 ¼ 0:9999 for MP
where Y , Y , Y and Y are the first derivative amplitude values, C
.3.1. Quadruple divisor spectrophotometric method (QD)
2
3
4
5
2 3
, C ,
For determination of TF, different concentrations of TF in the range of
−1
−1
−1
C
4
and C are the concentrations in μg mL , r
5
2 3 4 5
, r , r and r are the cor-
–12 μg mL were divided by sum standard spectra of 6 μg mL each
relation coefficients of BN, MTC, NMT and MP, respectively.
of BN, MTC, NMT and MP, as a quadruple divisor, then the first deriva-
tive of the resulted ratio spectra were obtained and the amplitude
values at 256 nm, Fig. 7, were plotted against the corresponding concen-
trations, and the regression equation was calculated and found to be:
4.3.2. Mean centering of ratio spectra spectrophotometric method (MCR)
For application of the proposed MCR method, the absorption spectra
of different concentrations of TF, BN, MTC, NMT and MP in the ranges of
−1
1
–12, 0.5–15, 2–28, 0.5–15 and 0.5–10 μg mL , respectively, were re-
Y1 ¼ 1:9781C1 þ 0:3396; r1 ¼ 0:9998; for TF
corded in the wavelength range of 215–350 nm and the amplitude
values of the mean centered fourth ratio spectra, obtained after using
the corresponding divisors, Table 1, were plotted against the corre-
sponding concentration of each component to construct their corre-
sponding calibration curves. Regression equations were obtained and
found to be:
where Y
in μg mL and r
1
−
is the amplitude value at 256 nm, C
1
is the concentration of TF
1
1
is the correlation coefficient.
For determination of BN, MTC, NMT and MP, different concentra-
−
1
tions in the range 0.5–15, 2–28, 0.5–15 and 0.5–10 μg mL , respec-
tively, were divided by a sum standard spectrum of the other four
−1
components each of 8, 4, 8 and 6 μg mL , respectively, then the first
derivative of the resulted ratio spectra amplitudes at 336.6, 288.4, 292
and 254 nm, respectively, Fig. 8-11, were plotted against the corre-
sponding concentrations, and the regression equations were then ob-
tained and found to be:
Y6 ¼ 0:8665 C6−0:2357 r6 ¼ 0:9999 for TF
Y7 ¼ 8:1095 C7−0:5565 r7 ¼ 0:9999for BN
Y8 ¼ 5:1362 C8 þ 40:4734 r8 ¼ 0:9998for MTC
Y9 ¼ 150:6880 C9 þ 55:8279 r9 ¼ 0:9999for NMT
Y10 ¼ 2:8191 C10 þ 0:1550 r10 ¼ 0:9999for MP
Y2 ¼ 4:8289 C2 þ 0:7554 r2 ¼ 0:9999 for BN
Y3 ¼ 1:0687 C3 þ 1:2752 r3 ¼ 0:9998 for MTC
Y4 ¼ 4:8249C4 þ 0:7961 r4 ¼ 0:9999 for NMT
where Y
6
, Y
7
, Y
8
, Y
9
and Y10 are the amplitude values of the mean
Table 3
Determination of TF, BN, MTC, NMT and MP in laboratory prepared mixtures by the proposed methods.
Mix. TF
No.
BN
MTC
NMT
MP
⁎
⁎
⁎
⁎
⁎
Taken μg %Recovery
Taken μg %Recovery
Taken
μg mL
%Recovery
QD
Taken
%Recovery
QD
Taken
μg mL
%Recovery
QD
−
1
−1
−1
−1
mL
μg
mL
mL
QD
MCR
101.32
100.01
98.45
MCR
−1
MCR
MCR
98.63
101.16
100.24
101.84
100.09
99.296
100.21
± 1.175
QD
MCR
1
2
3
4
5
6
4
100.98
99.87
98.22
101.25
100.75
99.58
4
5
100.78
100.92
98.18
100.46
101.35
99.25
101.21
100.92
97.98
100.54
99.79
98.92
99.89
±
4
5
2
3
2
2
98.52
99.34
100.13
102.02
99.57
100.19
99.96 ±
1.178
99.53
98.93
101.01
100.92
98.18
99.35
4
5
0.5
3
2
2
99.96
100.64
98.17
101.82
99.23
101.18
100.17
± 1.334
4
5
0.5
3
2
100.28
101.78
99.86
98.87
98.42
100.39
99.93 ±
1.197
100.63
101.64
98.86
100.04
98.03
100.43
99.93 ±
1.197
10
12
12
10
12
0.5
99.39
3
2
2
101.48
100.65
100.22
± 1.170
2
Mean ± RSD %
100.11
Mean ±
RSD %
100.16
± 1.201
Mean
± RSD
%
99.65 ±
1.118
Mean ±
RSD %
Mean ±
RSD %
±
1.129
1
.251
⁎
Average of 3 determinations.

R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
13
Table 4
Determination of Tolnaftate in pharmaceutical formulation by the proposed methods with application of standard addition technique.
Pharmaceutical formulations
Taken (μg)
% Recovery ± RSD
Standard addition techniqueb
Pure added (μg mL−1)
Found
% Recovery
Tinea Cure® Creama B.N. 1,670,291 labeled to contain 1 g Tolnaftate/100 g
QD method
2
4
6
1.99
4.05
5.92
99.50
101.25
98.67
TF
4
93.97 ± 1.168
94.28 ± 1.394
Mean ± SD
99.81 ± 1.317
MCR method
2
4
6
2.03
3.95
5.92
101.50
98.75
98.67
TF
4
Mean ± SD
99.64 ± 1.611
a
Average of 6 determinations.
Average of 3 determinations.
b
centered fourth ratio spectra at 285 nm, peak to peak 251–252 nm,
10–311 nm, 279 nm and peak to peak 312–313 nm for TF, BN, MTC,
NMT and MP, respectively, C , C , C , C and C10 are the concentrations
and r10 are the correlation coefficients of them,
spectrophotometric methods. Good results, within the acceptable
criteria, were obtained confirming good repeatability as given in Table 2.
3
6
7
8
9
−1
in μg mL , r
, r , r , r
6 7 8 9
4.4.3.2. Intermediate precision. The above mentioned procedures under
repeatability were repeated inter-day on three different days for deter-
mination of the selected concentrations of TF, BN, MTC, NMT and MP.
Acceptable RSD % values were obtained as given in Table 2.
respectively.
4
.4. Methods validation
4
.4.4. Specificity
The recommendations of the international conference on harmoni-
zation (ICH) [23] had been followed.
Specificity of the proposed methods was evaluated by applying the
procedures mentioned under linearity of each method on different syn-
thetic laboratory prepared mixtures of the five studied components in
different ratios and calculating percent recovery and standard deviation.
Good assay results were obtained confirming specificity of the proposed
methods, Table 3.
4
.4.1. Range and linearity
Linearity of the proposed methods was evaluated and it was evident
in the ranges of 1–12, 0.5–15, 2–28, 0.5–15 and 0.5–10 μg mL− for TF,
BN, MTC and NMT, respectively, for QD method and 1–12, 0.5–15, 2–
2
for MCR method. Good linearity was evident from the high value of cor-
relation coefficient as listed in Table 2. The concentration ranges and the
other statistical parameters for the two proposed methods were listed
in Table 2.
1
8, 0.5–15 and 0.5–10 μg mL−1 for TF, BN, MTC and NMT, respectively,
4.4.5. Limit of detection (LOD) and limit of quantitation (LOQ)
Limit of detection and limit of quantitation were calculated for
degradates and impurity, according to ICH guidelines [23]. Low values
of LOD and LOQ of BN, MTC and NMT and were obtained, Table 2, indi-
cating high sensitivity of the proposed methods.
The proposed QD and MCR spectrophotometric methods were ap-
plied for determination of TF in its pharmaceutical formulation, Tinea
Cure® cream and the results obtained were acceptable with good recov-
ery and low SD values. The validity of the methods was further assessed
by applying the standard addition technique which confirmed the accu-
racy of the proposed methods, Table 4.
The results obtained by applying the proposed QD and MCR spectro-
photometric methods for determination of TF in Tinea Cure® cream
were statistically compared to those obtained by applying the reference
one [1] using Student's-t and F-tests where no significant difference was
found between them regarding both accuracy and precision, Table 5.
One way ANOVA test was performed for comparing between the devel-
oped QD and MCR spectrophotometric methods for determination of TF.
No significant difference between them was observed as shown in Table
6.
4
.4.2. Accuracy
To assess the proposed methods accuracy, they were applied for de-
termination of different blind TF, BN, MTC and NMT samples concentra-
tions from the corresponding calculated regression equations and the
results were recorded in Table 2. Accuracy was further checked by ap-
plying the standard addition technique on Tinea Cure® cream and
good recoveries were obtained indicating no interference from excipi-
ents, as given in Table 4.
4
4
.4.3. Precision
.4.3.1. Repeatability. Three different concentrations each of the studied
components were analyzed three times intra-day using the previously
mentioned procedure under linearity of the proposed
Table 5
Statistical comparison between the proposed and the reference methods results for determination of Tolnaftate in its pharmaceutical formulation.
Items
Proposed QD method
Proposed MCR method
Reference spectrophotometric [1]b method
Mean
SD
93.97
1.098
1.168
8
0.262
1.480
94.28
1.314
1.394
8
0.217
1.034
94.13
1.336
1.419
8
%
n
RSD
a
Student's t-test (2.144)
F-value (3.787)
a
a
Figures between parentheses represent the corresponding tabulated values of t and F at P = 0.05.
Spectrophotometric determination of Tolnaftate at zero order absorbance maximum at 257 nm using methanol as a solvent.
b

14
R.A. Emam et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 223 (2019) 117290
Table 6
Results of ANOVA (one way) for comparison between the proposed methods and the reported ones [1] for determination of Tolnaftate (TF) in its pharmaceutical formulation.
Source of variation
TF
SS
df
MS
F
P-value
F crit
Between groups
Within groups
Total
0.369
33.014
33.383
2
21
23
0.185
1.572
0.117
0.900
3.4668
[
6] S.T. Patil, I.C. Bhoir, A.M. Bhagwat, M. Sundaresan, Assay of Tolnaftate and related
impurities by isocratic supercritical fluid chromatography, Fresen. J. Anal. Chem.
5
. Conclusion
3
67 (2000) 91–93.
This work provided an insight into alkaline degradation pathway
[7] National Center for Biotechnology Information, PubChem Compound Database,
CID=69675 https://pubchem.ncbi.nlm.nih.gov/compound/69675, Accessed date:
and degradates of TF then elucidation of their structures using IR,
H1NMR and MS spectroscopy. The proposed quaternary divisor and
mean centering of ratio spectra spectrophotometric methods were pre-
cise, accurate, reproducible, cost effective and specific and they can be
used for the routine analysis of Tolnaftate in pure form and in its phar-
maceutical formulation without interference from commonly encoun-
tered excipients. It is the first time to introduce such methods for
assaying Tolnaftate and its related substances (degradation products
and toxic impurities) with high sensitivity. Also, TF could be determined
in its pharmaceutical formulation without interference from MP, that
confirmed the high specificity of the proposed methods and recom-
mended their use in quality control analysis of Tolnaftate.
3
0 July 2018.
[
[
8] C.S.P. Sastry, B.S. Sastry, J.V. Rao, R. Krishna, Spectrophotometric methods for the de-
termination of Tolnaftate, Talanta 40 (1993) 571–576.
9] T. Tahssen, N.R. Ahmed, Indirect spectrophotometric determination of Tolnaftate in
pharmaceutical preparations, J. Educ. Sci. 22 (2009) 9–18.
[
10] N. Bhoyar, T.K. Giri, A. Alexander, D.K. Tripathi, K. Ajazuddin, Development of UV
spectrophotometric method for qualitative and quantitative estimation of
Tolnaftate in different formulations, Res. J. Pharm. Technol. 5 (2012) 207–209.
11] A.K. Dash, A liquid chromatographic method for the determination of Tolnaftate in
pharmaceutical formulations, J. Pharm. Biomed. Anal. 11 (1993) 847–853.
12] S.S. Zarapkar, K. Athalye, Reversed phase HPLC determination of Tolnaftate in cream
and topical solution, Ind. J. Pharm. Sci. 52 (1990) 16–17.
13] T. Kezutyte, O. Kornysova, A. Maruska, V. Briedis, Assay of Tolnaftate in human skin
samples after in vitro penetration studies using high performance liquid chromatog-
raphy, Acta Pol. Pharm. 67 (2010) 327–334.
[
[
[
[
14] S. Sheikh, S. Asghar, S.A. Patni, Liquid chromatographic technique for stability indi-
cating analytical method development and validation of salicylic acid and Tolnaftate
in pharmaceutical ointment by high performance, Int. J. Sci. Res. Pub. 2 (2012)
Declaration of Competing Interest
No conflict of interest to declare.
Acknowledgements
2
50–253.
[
[
15] C.N. Saha, S. Bhattacharya, A validated simultaneous RP-HPLC method for determi-
nation of betamethasone dipropionate and Tolnaftate in combined semisolid formu-
lation, Int. J. ChemTech Res. 1 (2009) 671–674.
16] M. Bapna, J.P. Balubhai, Development and validation of analytical method for simul-
taneous
Iodochlorhydroxyquin in pharmaceutical dosage form, Pharm. Chem. J. 2 (2015)
7–21.
estimation
of
betamethasone
dipropionate,
Tolnaftate,
1
The authors would like to thank Dr. Phoebe Farag Lamie (Assistant
professor of Organic Chemistry, Faculty of pharmacy, Beni-Suef Univer-
sity) who supported us with valuable information in synthesis and
structure elucidation of impurity.
[
17] ICH Q1A (R2), Stability testing of new drug substances and products, Proceeding of
the International Conference on Harmonization, 2003.
[18] K.C. Waterman, et al., Pharm. Dev. Technol. 7 (2002) 113–117.
[
19] U.R. Kalkote, A.R. Choudhary, A.A. Natu, R.J. Lahoti, N.R. Ayyangar, Synth. Commun.
(1991) 1889.
[
20] N.W. Ali, N.S. Abdelwahab, M. Abdelkawy, A.A. Emam, A comparative study of ICH
validated novel spectrophotometric techniques for resolving completely overlap-
ping spectra of quaternary mixtures, Spectrochim. Acta, Part A 154 (2016) 114–122.
References
[
[
1] BP, The British Pharmacopoeia, Her Majesty's, The Stationary Office, London, 2007.
2] Martindale, The complete drug reference, 35 ed. Pharmaceutical Press, London,
[21] M.M. Issa, R.M. Nejem, A.M. Abu shanab, N.T. Shaat, Resolution of five-component
mixture using mean centering ratio and inverse least squares chemometrics,
Chem. Cent. J. 7 (2013) 152–165.
th
2007.
[
[
[
3] F.N. Marzulli, H.I. Maibach, Dermatotoxicology, fourth ed. Hemisphere Publishing
Corp, New York, 1991 864.
[22] N.S. Abdelwahab, N.W. Ali, M. Abdelkawy, A.A. Emam, Validated RP-HPLC and TLC-
densitometric methods for analysis of ternary mixture of cetylpyridinium chloride,
chlorocresol and lidocaine in oral antiseptic formulation, J. Chromatogr. Sci. 54
(2015) 318–325.
4] J.B. Sullivan Jr., G.R. Krieger (Eds.), Hazardous Materials Toxicology-Clinical Princi-
ples of Environmental Health, Williams and Wilkins, Baltimore, MD 1992, p. 1098.
5] P.Y. Khashaba, S.R. El-Shabouri, K.M. Emara, A.M. Mohamed, Analysis of some anti-
fungal drugs by spectrophotometric and spectrofluorometric methods in different
pharmaceutical dosage forms, J. Pharm. Biomed. Anal. 22 (2000) 363–376.
[23] Validation of Analytical Procedures, Proceedings of the International Conference on
Harmonization, Geneva. , ICH, 2005 Q2 (R1).