Validated spectral manipulations for determination of an anti-neoplastic drug and its related impurities including its hazardous degradation product

Article abstract of DOI:10.1039/d1ra03238k

Innovative and specific double dual wavelength, dual ratio subtraction spectrophotometric methods were carried out along with a successive ratio subtraction spectrophotometric method for determination of dacarbazine and its related impurities including toxic and hazardous ones. For determination of dacarbazine by the double dual wavelength method, the absorbance differences between 323 and 350 nm of the zero order absorption spectra of dacarbazine were used. The values of absorbance difference between 267.2 and 286.2 nm of the zero order spectra of 5-amino-imidazole-4 carboxamide were used for its determination by the dual ratio subtraction method. The zero order absorption spectrum of 2-azahypoxanthine at 235 nm was used for its determination after applying the successive ratio subtraction method. ICH guidelines were followed for validation of the developed methods, where linear relationships were obtained in the range of 4-20, 1-16, and 2-20 μg mL^?1for dacarbazine, 5-amino imidazole-4-carboxamide and 2-azahypoxanthine, respectively. Accurate, precise, and specific results were obtained upon applying the proposed methods according to ICH guidelines. Furthermore, the developed methods were successfully applied for determination of dacarbazine in its pharmaceutical formulation. Comparing the results of the developed methods with those of the official USP spectrophotometric method statistically showed no significant difference. The developed methods don't need any sophisticated techniques so they are considered cost effective methods. Moreover, the introduced methods have the advantages of being green where water was used as a solvent. The methods proved to be more economic, fast and simple than other reported HPLC methods.

Full text of DOI:10.1039/d1ra03238k

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Validated spectral manipulations for determination
of an anti-neoplastic drug and its related impurities
including its hazardous degradation product
Cite this: RSC Adv., 2021, 11, 21332
Ibrahim A. Naguib, a Eglal A. Abdelaleem,b Eman S. Hassan
b and Aml A. Emamb
*
Innovative and specific double dual wavelength, dual ratio subtraction spectrophotometric methods were
carried out along with a successive ratio subtraction spectrophotometric method for determination of
dacarbazine and its related impurities including toxic and hazardous ones. For determination of
dacarbazine by the double dual wavelength method, the absorbance differences between 323 and
350 nm of the zero order absorption spectra of dacarbazine were used. The values of absorbance
difference between 267.2 and 286.2 nm of the zero order spectra of 5-amino-imidazole-4 carboxamide
were used for its determination by the dual ratio subtraction method. The zero order absorption
spectrum of 2-azahypoxanthine at 235 nm was used for its determination after applying the successive
ratio subtraction method. ICH guidelines were followed for validation of the developed methods, where
linear relationships were obtained in the range of 4–20, 1–16, and 2–20 mg mLꢀ1 for dacarbazine, 5-
amino imidazole-4-carboxamide and 2-azahypoxanthine, respectively. Accurate, precise, and specific
results were obtained upon applying the proposed methods according to ICH guidelines. Furthermore,
the developed methods were successfully applied for determination of dacarbazine in its pharmaceutical
formulation. Comparing the results of the developed methods with those of the official USP
spectrophotometric method statistically showed no significant difference. The developed methods don't
need any sophisticated techniques so they are considered cost effective methods. Moreover, the
introduced methods have the advantages of being green where water was used as a solvent. The
methods proved to be more economic, fast and simple than other reported HPLC methods.
Received 26th April 2021
Accepted 7th June 2021
DOI: 10.1039/d1ra03238k
rsc.li/rsc-advances
cause of the painful effect of DTIC peripheral intravenous
infusion.5 According to the literature survey, DTIC was deter-
mined by HPLC methods either alone6 or with other drugs.7,8
1. Introduction
Dacarbazine
(DTIC),
3,3-dimethyltriazeno-imidazole-4-
carboxamide (Fig. 1a),1 is an antineoplastic drug which is acti-
vated in the liver before acting as an alkylating agent. Dacar-
bazine is mainly used as a therapeutic agent for metastatic
malignant melanoma. It can also be used as a combination
therapy for so-tissue sarcoma, Hodgkin's disease, neuroblas-
toma, Kaposi's sarcoma, and different tumors.1 According to
USP,2 there are two related impurities for DTIC identied as 5-
amino-imidazole-4 carboxamide (AIC), Fig. 1b,2 and 2-azahy-
poxanthine (AHX), Fig. 1c,.2 The British pharmacopeia safety
data sheet revealed that AHX is a harmful substance which
causes skin irritations and serious eye irritations; moreover it
may cause cancer, genetic defects, and respiratory irritations.3
AHX is also considered the major degradation product of both
photolysis and hydrolysis of DTIC.4 AHX was found to be the
HPLC methods were also used for determination of the ternary
mixture but one of them required a very long time for analysis
exceeding 25 minutes,9 and the other one was gradient elution
using acetonitrile10 which is considered one of the most
hazardous solvents to human health and environmental safety
according to the environment, health and safety (EHS) and the
life cycle assessment (LCA) protocols.11 Chromatographic
methods like HPLC also have various limitations like
complexity, utilizing various expensive solvents, in addition to
tedious sample pretreatment steps. Therefore, development of
environmentally friendly, simple, economic, cost and time
saving spectrophotometric methods for determination of DTIC
and its related impurities, including the toxic and hazardous
one3 becomes of a great importance. Comparing the results of
the developed methods with those of the USP official spectro-
photometric method2 statistically showed no signicant differ-
ence. Validation of the developed methods was conrmed by
the results of the ICH guidelines parameters.12
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, Taif University, P. O.
Box 11099, Taif 21944, Saudi Arabia
bPharmaceutical Analytical Chemistry Department, Faculty of Pharmacy, Beni-Suef
University, Alshaheed Shehata Ahmad Hegazy St., Beni-Suef, 62514, Egypt. E-mail:
dr.emansherif@yahoo.com; Fax: +082/2317950; Tel: +2/01009158403
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1.1 Theoretical background
2. Experimental
1.1.1 Double dual wavelength spectrophotometric method
(DDW). Double dual wavelength (DDW) method depends on the
same concept of the previously established dual wavelength
spectrophotometric method which relies on the fact that the
difference in absorbance values between two wavelengths of the
absorption spectra is directly related to the concentration of
a denite component, while the other components showed no
interference.13 The cornerstone in the development of this
method is the appropriate choice of the two wavelengths; where
each of the two interfering components has the same absor-
bance values, while the determined component has a signi-
cant difference. The developed method is considered a new
successful application of the dual wavelength method on three
components not only two.
1.1.2 Dual ratio subtraction method (DRS). Dual ratio
subtraction (DRS) method depends on coupling of ratio
subtraction and dual wavelength spectrophotometric methods,
where the interference from the most extended component is
eliminated using ratio subtraction method, then dual wave-
length on the obtained spectra was applied by choosing two
wavelengths where the component to be measured shows
signicant absorbance difference while the absorbance of the
other interfering component is the same. First, ratio subtraction
method steps were followed;14 where division of the mixtures
spectra (X + Y) by a certain concentration of X (the most extended
component) was done, where new curves resulted (Y/X0 + [X/X0 ¼
constant1]). The constant values were then subtracted from the
resulted ratio spectra, and the new obtained curves were multi-
plied by X0 (the divisor). Dual wavelength on the developed spectra
was then applied to determine Y.
2.1 Instruments
UV-VIS spectrophotometer (SHIMADZU, Japan) model UV-1601
PC, and quartz cell with 1 cm path length, linked to IBM
adaptable computer were used. The soware used was UVPC
personal spectroscopy version 3.7.
2.2 Materials and reagents
2.2.1 Pure samples. Dacarbazine was bought from Sigma-
-Aldrich Chemie GmbH, Germany. The purity was 99.6% according
to the official USP spectrophotometric method.2 AIC was purchased
from Alfa Aesar, Kandel, Germany with a certied purity of 95%.
2.2.2 Pharmaceutical formulation. Dacarbazine medac®
powder for injection (Batch No. C180075B) was labeled to
contain 200 mg of DTIC per vial; it was manufactured by Medac
Gmbh, Wedel, Germany.
2.2.3 Chemicals. Methanol, HPLC grade (CHROMASOLVE,
Sigma-Aldrich, ChemieGmbH, Germany) and water for injection
(Egypt, Otsuka Pharmaceutical Co., 10th of Ramadan, Egypt), HCl
and NaNO2 (El-Nasr Pharmaceutical Chemicals Co., Cairo, Egypt).
3. Procedures
3.1 Preparation of AHX
5-Amino-imidazole-4-carboxamide. HCL (AIC. HCL) (2 g, 0.012 mol)
in 6 M HCl (8 mL) was added drop wise to aꢁstirred solution of NaNO2
(6.45 g, 0.094 mol) in H2O (24 mL) at 0 C for 30 minutes. Aer
stirring of the solution for 1 hour, the mixture was ltered, and the
residue was washed with tetra hydro furan. 4-Diazo-4H-imidazole-5-
carboxamide (DICA) was formed as crude powder aer drying the
residue at room temperature. A suspension of the crude material
DICA (0.318 g, 0.0023 mol) in MeOH (5 mL) was stirred at 60 ꢁC for 10
hours. Aer cooling, activated carbon was added until the red solu-
tion was changed to black. Stirring of the resulted solution was done
for 12 hours at room temperature, followed by ltration, and evapo-
ration under reduced pressure to form 2-azahypoxanthine (AHX)
(0.282 g, 0.0021 mol) as a white powder,15 Fig. 2. 1H NMR, MS, and IR
analyses were done to conrm the formation of AHX, Fig. 3–5.
1.1.3 Successive ratio subtraction method (SRS). Succes-
sive ratio subtraction method was recently developed for sepa-
ration and quantitative determination of ternary mixtures.14 For
determination of the most un extended component [Z], the
following steps were followed. First ratio subtraction method
was applied as mentioned before in DRS, then the developed
spectra were divided by Y0, where the resulted curves represent
(X/Y0 + [Y/Y0 ¼ constant2]). Constant values were then subtracted
from the resulted ratio spectra, and then the new curves were
multiplied by Y0 (the divisor). There was a coincidence point
between the resulted spectra and the zero order spectra of AHX
at 235 nm which was used for its determination.
3.2 Preparation of standard solutions
Methanol was the solvent used for preparation of 1000 mg mLꢀ1
stock solutions of DTIC, AIC and AHX, and water for injection
Fig. 1 Chemical stuctures of (a) DTIC. (b) AIC, and (c) AHX.
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Fig. 2 Synthesis pathway of AHX from AIC. HCL.
Fig. 3 H1 NMR of AIC and AHX.
was used for preparation of 100 mg mLꢀ1 of their respective
3.4 Preparation of pharmaceutical formulation
working solutions.
A sample equivalent to 25 mg of DTIC was weighed, and 20 mL
methanol was added in 25 mL volumetric ask for dissolving
DTIC. The resulted solution was ultra-sonicated for 30 min.,
then lteration, cooling, and completing to volume with sterile
water were carried out. The working solution of DTIC (100 mg
3.3 Preparation of laboratory mixtures
Water for injection was used for preparation of mixtures con-
taining variable ratios of DTIC, AIC, and AHX using their
respective working solutions.
Fig. 4 Mass spectra of AIC and AHX.
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Fig. 5 IR spectra of AIC and AHX.
mLꢀ1) was obtained by diluting the resulted solution with absorbance differences between 323 and 350 nm of the zero
sterile water to get DTIC from its pharmaceutical dosage form. order absorption spectra of DTIC, Fig. 7, for DDW method,
against their respective concentrations was carried out, and
then its regression equation was computed. For DRS method,
plotting the absorbance differences between 267.2 and
286.2 nm of the zero order spectra of AIC, Fig. 8, against their
respective concentrations was carried out, and then its regres-
sion equation was computed. The calibration curve which
relates the zero order absorption spectra of AHX at 235 nm,
Fig. 9, and its corresponding concentrations for SRS method
was constructed, and then its regression equation was
computed.
3.5 Spectral characteristics
The absorption spectra of DTIC, AIC, and AHX were recorded
from 200 to 400 nm, where water was used as a blank, Fig. 6.
3.6 Construction of calibration curves
Variable portions equivalent to (40–200 mg) of DTIC (10–160 mg)
of AIC, and (20–200 mg) of AHX, were taken from their respective
working solutions (100 mg mLꢀ1), and completed to volume with
sterile water in a set of 10 mL volumetric asks. Plotting the
Fig. 6 Zero order absorption spectra of 12 mg mLꢀ1 of DTIC (—), AIC (---), and AHX (.) using sterile water as a blank.
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Fig. 7 Zero order absorption spectra of pure DTIC (4–20 mg mLꢀ1) using sterile water as a blank.
Fig. 8 Zero order absorption spectra of pure AIC (1–16 mg mLꢀ1) using sterile water as a blank.
3.6.1 Determination of DTIC in lab. prepared mixtures by
3.6.3 Determination of AHX in lab. prepared mixtures by
DDW method. The difference in values of zero order absorbance SRS method. Ratio subtraction was applied on the mixtures as
among 323 and 350 nm of the spectra of the laboratory prepared mentioned in DRS method and then the resulted spectra were
mixtures was used for determination of DTIC was determined divided by 6 mg mLꢀ1 of AIC. The constant of the plateau region
using where the absorbance difference values at these selected (260–272 nm) was subtracted, and then the resulted ratio
wavelengths for each of the related impurities equal zero.
spectra were multiplied by the same divisor (6 mg mLꢀ1 of AIC).
3.6.2 Determination of AIC in lab. prepared mixtures by The obtained spectra were used for calculating AHX concen-
DRS method. Division of the mixtures spectra by 16 mg mLꢀ1 of tration at 235 nm where there was a coincidence point with the
DTIC was carried out, followed by subtraction of the constant in zero order spectra of AHX aer application of the regression
the plateau region (305–330 nm), and then multiplication of the equation obtained from AHX zero order spectra at 235 nm.
resulted ratio spectra by the divisor (16 mg mLꢀ1 of DTIC), then
AIC concentrations were determined using the resulted spectra
aer applying the regression equation of the zero order absor-
4. Results
bance difference values of AIC between 267.2 and 286.2 nm
where the absorbance difference was zero for AHX at those
selected wavelengths.
2-Azahypoxanthine was prepared from AIC and the structure
was elucidated by 1H NMR, MS, and IR analyses, Fig. 3–5.
H1NMR revealed disappearance of hydrogens of NH2 groups of
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Fig. 9 Zero order absorption spectra of pure AHX (2–20 mg mLꢀ1) using sterile water as blank.
Fig. 10 Division spectra of variable lab. prepared mixtures if DTIC, AIC, and AHX using 16 mg mLꢀ1 of DTIC as a divisor and sterile water as a blank.
Fig. 11 The obtained spectra of variable lab. prepared mixtures of DTIC, AIC, and AHX using 16 mg mLꢀ1 of STIC as a divisor and sterile water as
a blank after subtracting the constant.
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Fig. 12 The obtained spectra of variable lab. prepared mixtures of DTIC, AIC, and AHX after subtraction of the constant and multiplication by the
divisor (16 mg mLꢀ1 of DTIC).
Fig. 13 Division spectra of lab. prepared mixtures obtained from RS method using 6 mg mLꢀ1 of AIC as a divisor.
AIC and appearance of triazinone single hydrogen of AHX, absorbance difference at those selected wavelengths equal zero
Fig. 3a and b. A base peak at m/z 137.1 appeared in MS chart for each of the two related impurities.
which correspond to AHX molecular weight, Fig. 4b. Structure
of AHX was further assured by disappearance of the forked peak
4.2 Determination of AIC using dual ratio subtraction
of NH2 groups as revealed by IR analyses, Fig. 5a and b.
method (DRS)
Determination of AIC in lab. prepared mixtures was done using
the newly proposed DRS method by applying ratio subtraction
4.1 Determination of DTIC using double dual wavelength
followed by dual wavelength for the resulted spectra. Division of
method (DDW)
the mixtures spectra by 16 mg mLꢀ1 of DTIC as a divisor was
Dacarbazine was determined by plotting the absorbance
differences between 323 and 350 nm of the zero order absorp-
tion spectra of DTIC against their corresponding concentra-
tions, Fig. 7, followed by computing its regression equation. It
was also determined in laboratory prepared mixtures by calcu-
lating the absorbance differences between 323 and 350 nm of
carried
out.
The
obtained
curves
represent
ꢀ
ꢁ
AIC þ AHX
þ constant ; Fig. 10. Subtraction of the constant
DTIC
in the plateau region (305–330 nm) was done, Fig. 11, and the
AIC þ AHX
resulted ratio spectrum
were multiplied by the
DTIC
the zero order absorption spectra of the mixtures where the divisor 16 mg mLꢀ1 of DTIC, Fig. 12. The absorbance difference
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Fig. 14 Division spectra of lab. prepared mixtures obtained from RS method using 6 mg mLꢀ1 of AIC as a advisor after subtraction of the constant.
Fig. 15 The spectra of lab. prepared mixtures using SRS method after multiplication by the divisor (6 mg mLꢀ1 of AIC).
AHX
AIC
values between 323 and 350 nm of the resulted spectra repre-
senting AIC + AHX were used for calculating AIC concentration,
where the absorbance difference was zero for AHX at these
selected wavelengths, using the regression equation computed
aer construction of the calibration curve obtained by plotting
the absorbance difference values between 323 and 350 nm of
the zero order spectra of AIC against its corresponding
concentrations, Fig. 8.
the obtained ratio spectrum
was multiplied by the divisor
6 mg mLꢀ1 of AIC, Fig. 15. The new obtained spectra had
a coincidence point with the zero order spectra of AHX at
235 nm, Fig. 16. AHX concentrations were determined from the
regression equation computed aer calibration curve
construction which was obtained by plotting zero order absor-
bances of AHX at 235 nm against its corresponding concentra-
tions, Fig. 9.
4.3 Determination of AHX using successive ratio subtraction
method (SRS)
4.4 Methods validation
Ratio subtraction was applied as mentioned under DRS
method. The resulted spectra representing AIC + AHX were then
redivided by 6 mg mLꢀ1 of AIC. The new curves obtained
Application of methods validation parameters was carried out
in adherence with the ICH guidelines.13
4.4.1 Linearity and range. Linear relationships were illus-
trated by calibration curves resulted from application of double
dual wavelength for DTIC, dual ratio subtraction for AIC, and
ꢀ
ꢁ
AHX
AIC
represent
þ constant ; Fig. 13. The constant of the
plateau region (260–272 nm) was subtracted, Fig. 14, and then successive ratio subtraction for AHX where linearity was
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Fig. 16 The absorption spectrum of 6 mg mLꢀ1 of AHX (---) and the obtained absorption spectrum of lab. prepared mixture containing 6 mg mLꢀ1
of AHX (.) after multiplication by the divisor using SRS method.
Table 1 Regression and analytical parameters of the developed spectrophotometric methods for determination of DTIC, AIC and AHXa
Parameters
Range (mg mLꢀ1
Slope
DTIC
4–20
AIC
1–16
0.051
0.004
99.76 ꢂ 0.609
0.9999
1.196
1.453
0.128
AHX
2–20
0.010
0.019
100.44 ꢂ 0.738
0.9999
1.271
1.335
0.560
)
0.0351
ꢀ0.031
101.41 ꢂ 1.154
0.9996
1.363
Intercept
Accuracy (mean ꢂ SD)
Correlation coefficient (r)
Repeatability (RSD%)a
Intermediate precision (RSD%)b
LOD
1.776
—
LOQ
—
0.388
1.697
a
(RSD%)a and (RSD%)b; the intra- and inter-day relative standard deviation of concentrations (8, 12, 18 mg mLꢀ1) for DTIC, (2, 4, 10 mg mLꢀ1) for
AIC, and (6, 10, 16 mg mLꢀ1) for AHX.
Table 2 Determination of DTIC in its dosage form by the developed
4.4.3 Accuracy. Accuracy claries the closeness of agree-
method and standard addition technique results
ment between the obtained values and the real values. Calcu-
lation of pure DTIC, AIC, and AHX percentage recoveries was
done to assess accuracy, Table 1, which was more conrmed by
the standard addition technique, Table 2.
4.4.4 Precision. Precision claries the closeness of agree-
ment between sets of obtained values. Repeatability and
Dacarbazine medac® Taken
8
powder for injection
batch no: C180075B
labeled to contain
200 mg DTIC/vial
(mg mLꢀ1
)
Founda% ꢂ SD
100.85 ꢂ 0.595
Standard addition Pure added (mg Recovery%
technique
per band)
6
8
12
101.11
100.24
99.18
Mean ꢂ SD
100.18 ꢂ 0.967
Table 3 Assay results for the determination of DTIC, AIC, and AHX in
synthetic mixtures containing different ratios of the three components
a
Average of 6 determinations.
Lab. prepared
mixtures ratios of
obtained in the range of 4–20 mg mLꢀ1 for DTIC, 1–16 mg mLꢀ1 DTIC : AIC : AHX
for AIC, and 2–20 mg mLꢀ1 for AHX, Table 1.
DTIC
AIC
AHX
16 : 4 : 4
4.4.2 Range. Establishment of calibration ranges for DTIC,
18 : 1 : 4
99.29
98.51
98.54
99.02
99.41
99.63
101.35
99.88
100.93
AIC, and AHX was done through considerations of the practical
18 : 3 : 6
ranges necessary in adherence to Beer's Lambert law and the
concentration of DTIC present in pharmaceutical formulation
to get linear, specic and accurate results, Table 1.
15 : 2 : 3
17 : 3 : 3
Mean ꢂ SD
99.52
98.10
98.79 ꢂ 0.592
101.08
98.39
99.51 ꢂ 0.998
97.75
101.33
100.25 ꢂ 1.519
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Table 4 Parameters of statistical analysis of the results of the devel-
oped DDW spectrophotometric method compared with the official
spectrophotometric method for determination of DTIC in Dacarbazine
medac® powder for injection
column and a mobile phase consisting of 2-propanol: phos-
phate buffer (pH 7, 0.02 M) 4 : 96 v/v which required very long
time for analysis exceeding 25 minutes.9 The second reported
HPLC method used a mobile phase consisting of 100% 0.5 M
sodium acetate (pH 7.0) and 25% acetonitrile in 0.05 M sodium
acetate (pH 5.5) by gradient elution;10 acetonitrile is considered
one of the most hazardous solvents to human and environ-
ment.11 Moreover, the developed methods don't need any
sophisticated techniques and don't require any sample
pretreatments.
Official spectro-
DDW spectro-
photometric method
photometric
Parameters
methodb [USP]
Mean
SD
n
100.85
0.595
6
0.4198
3.320
100.67
1.084
6
—
—
Student's t-testa (2.228)
F-testa (5.050)
6. Conclusion
a
Figures in parenthesis are the corresponding tabulated values at p ¼
b
0.05. Direct spectrophotometric determination at 323 nm (The
The main aim of the proposed research is the development and
validation of novel, simple, cost effective, time saving, and eco-
friendly spectrophotometric methods for determination of the
antineoplastic drug, DTIC in bulk powder and in dosage form
without the need of sophisticated techniques or tedious sample
pre-treatment steps. The main advantage of the developed
methods arises from using the zero order absorption spectra
without needing any derivatization steps. The proposed
methods have the capability to determine DTIC along with its
related impurities including the very hazardous and toxic
degradation product, AHX3 with high accuracy and precision.
The developed methods have the merits of being more simple,
economic, time saving and eco-friendly compared to the previ-
ously published HPLC methods using the most ever green
solvent, water, as a blank. Additionally, successful application
was done for determination DTIC in Dacarbazine medac®
powder for injection, where no interference from any pharma-
ceutical formulation excipients.
United States Pharmacopeia, 2011).
intermediate precision were calculated to assess precision,
values of RSD% were illustrated in Table 1.
4.4.5 Specicity. The proposed methods were successfully
applied on variable laboratory prepared mixtures containing
variable ratios of DTIC, AIC, and AHX. Specicity was conrmed
and the obtained results were illustrated in Table 3.
4.4.6 Limits of detection and quantitation (LOD and LOQ).
Limits of detection and quantitation indicate the lowest
amount that can be detected and quantied. LOD and LOQ were
calculated for the developed methods using the following
equations; LOD ¼ 3.3 ꢃ SD/slope, and LOQ ¼ 10 ꢃ SD/slope,
Table 1.
Upon comparing the results achieved aer application of the
proposed spectrophotometric method for determination of
DTIC in Dacarbazine® powder for injection and the results of
the USP official spectrophotometric method2 statistically. The
resulted values of t-test and F-test were less than theoretical
values, which conrms accuracy and precision at 95% con-
dence level, Table 4.
Conflicts of interest
There are no conicts of interests to declare.
Acknowledgements
5. Discussion
The authors would like to extend their sincere appreciation to
Taif University Researchers Supporting Project number (TURSP-
2020/56), Taif University, Taif, Saudi Arabia.
The challenge to develop green, simple, and economic spec-
trophotometric methods to resolve components with over-
lapping spectra is the main goal for any pharmaceutical
analyst.16 The literature survey showed that no spectrophoto-
metric methods were reported for determination of the studied
mixture; hence, the main focus of the presented research was on
developing novel and green spectrophotometric methods for
determination of the anti neoplastic drug, dacarbazine, and its
related impurities, including its hazardous and toxic one, AHX.3
Regarding the greenness prole,17 water successfully passes the
four acceptance criteria (no hazards, no corrosion, no PBT
(persistence, bio accumulation, and toxicity)), and there isn't
any harmful wastes produced from using it, so it is a completely
green and is considered the rst choice of green solvents.18 The
developed methods have the merits of being more time saving
and green compared to the previously published HPLC
methods.9,10 The rst HPLC method used an avidin protein
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21342 | RSC Adv., 2021, 11, 21332–21342
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