Arabian Journal of Chemistry (2019) 12, 1212–1218

King Saud University

Arabian Journal of Chemistry

www.ksu.edu.sa
www.sciencedirect.com

ORIGINAL ARTICLE

Applying green analytical chemistry (GAC)

for development of stability indicating HPLC
method for determining clonazepam and its related
substances in pharmaceutical formulations
and calculating uncertainty
Ahmed Badr Eldin a,*, Abdallah Shalaby b,1, Mahmoud Samy Abdallah a,2
,
Moataz Ahmed Shaldam a,3, Mohamed Adel Abdallah a,4

a
Sigma Pharmaceutical Industries, Quesna 32684, El Monofeya, Egypt
b
Analytical Chemistry Department, Faculty of Pharmacy, Zagazig University, Egypt

Received 26 February 2014; accepted 29 October 2014

Available online 18 November 2014

KEYWORDS Abstract Clonazepam contains one benzodiazepine ring in its chemical structure which makes it
Green analytical chemistry; vulnerable to degradation. In this study, green analytical chemistry approach was applied in
Clonazepam; attempts for the development of validated stability indicating RP-HPLC method for determining
Related substances; clonazepam and its related substances in pharmaceutical formulation. Validation has been per-
Uncertainty; formed according to ICH guidelines. Assay was capable of simultaneous monitoring of the intact
HPLC; drug in the presence of its related substances within the same run. HPLC assay involved an ODS
Stability indicating method column and a mobile phase composed of 2% sodium dodecyl sulfate, 0.05 M sodium acetate buffer
pH 3.5 and isopropanol in ratio (25:55:20) at a flow rate of 1.5 mL/min and detection was carried
out at 254 nm. HPLC method allowed good resolution between the peaks that corresponded to the

* Corresponding author. Tel.: +20 1223729152.

E-mail addresses: [email protected] (A.B. Eldin),
[email protected] (A. Shalaby), [email protected]
(M.S. Abdallah), [email protected] (M.A. Shaldam),
[email protected] (M.A. Abdallah).
1
Tel.: +20 1001748144.
2
Tel.: +20 1063340887.
3
Tel.: +20 1114954613.
4
Tel.: +20 1001577489.
Peer review under responsibility of King Saud University.

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http://dx.doi.org/10.1016/j.arabjc.2014.10.051
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This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).
Green analytical chemistry for development of HPLC method 1213

active pharmaceutical ingredients and its degradation products with good linearity, precision,
accuracy, specificity, LOD and LOQ. The expanded uncertainty (0.33%) of the method was also
estimated from method validation data. This analytical technique is not only ecofriendly but also
faster than the conventional liquid chromatographic system official in the USP-36.
ª 2014 The Authors. Production and hosting by Elsevier B.V. on behalf of King Saud University. This is
an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/3.0/).

1. Introduction by Sigma pharmaceutical industries (Egypt). All solvents and

reagents were of HPLC grade, and were purchased from
Due to scientific and public concern about the environment Sigma-Aldrich. HPLC grade water was obtained through a
pollution, environmentally friendly practices have been intro- Milli-Q system (Millipore, Milford, USA) and was used to pre-
duced in different areas of society and research. In green ana- pare all solutions.
lytical chemistry, sample preparation and LC analysis need Commercial samples of clonazepam tablets, manufactured
special attention because hazardous solvents are often used. by Sigma pharmaceutical industries – Egypt, were used in
European medicine agency (EMA) mentioned that solvents the applications.
like methanol and tetrahydrofuran are ranked by as hazardous
solvents (ICH Topic Q3C (R4), 2009) and because of their 2.2. Apparatus
inherent toxicity, safe detoxification of the waste solvents is
essential, which may lead to high to very high disposal costs. Shimadzu HPLC system (Shimadzu, Kyoto, Japan), set to
Possibilities toward green LC include reducing solvent use, recycle the mobile phase and was equipped with a System Con-
switching to more benign solvents and/or eliminating organic troller CBM-20A, Solvent Delivery Unit LC-20A, On-line
solvents (Armenta et al., 2008; Kerton and Marriott, 2013; Degassing Unit DGU-20A, and Photo-diode Array detector
Taylor, 2010; Clark and Tavener, 2006). Clonazepam SPD-M20A. The peak areas were integrated automatically
[5-(2-Chlorophenyl)-7-nitro-3H-1,4-benzodiazepin-2(1H)-one] by computer using a Shimadzu LC solution V1.24 SP1 soft-
is mainly used as anticonvulsant, muscle relaxant and anxio- ware program. A 20 lL volume of sample was introduced into
lytic agent. Clonazepam is slightly soluble in acetone, Auto-Sampler SIL-20A injector. Photo cabinet ‘‘Atlas Suntest
chloroform, acetic anhydride, hardly soluble in methanol, iso- CPS Plus’’ – Germany has been used for photodegradation.
propanol, ether, almost insoluble in water. In all of the afore-
mentioned LC techniques, including the USP-36 relevant 2.3. Analytical procedures
monograph, tetrahydrofuran and methanol have been used
as a part of mobile phase and extraction procedure. The pres- 2.3.1. Reference method
ent investigation describes a rapid, accurate and precise RP- According to the relevant monograph in the USP-36 (USP,
HPLC method for the determination of Clonazepam and its 2013) the elution was carried out on a BDS C8 Hypersil column
related compound A & B (Fig. 1) in pharmaceutical dosage (150 mm · 4.6 mm, 5 lm particle size) from Thermo scientific
forms within quality control laboratories without the need to (Massachusetts, USA). All analyses were performed at ambient
use of hazardous, toxic solvents since this drug is being temperature under isocratic conditions with a mobile phase of
marketed in domestic and international market. ammonium phosphate buffer solution pH 8.0: methanol: tetra-
hydrofuran at a flow rate of 1.5 mL/min in ration (60:52:13,
2. Experimental v/v), using UV detection at 254 nm. Diluent solution prepared
by mixing water, methanol, and tetrahydrofuran in ration
2.1. Materials and chemicals 60:52:13 was used for standard and sample preparation.

USP – reference standard materials of clonazepam (99.88%), 2.3.2. Proposed liquid chromatography method
related compound A and related compound B in addition to 2.3.2.1. Preparation of stock solutions and standards. Stock
excipient used for preparing the placebo were kindly provided standard solution of clonazepam, related compound A and

Figure 1 Structures of clonazepam and its related compounds (a) clonazepam (b) clonazepam related A and (c) clonazepam related B.
1214 A.B. Eldin et al.

related compound B was prepared in mobile phase at a con- prepared by addition of the suitable amount of standard and
centration of 0.2 mg/ml. Series of dilutions at concentration the related compounds (A&B) to the placebo. These samples
levels of 4–140 lg/mL were obtained from stock solution by were handled as descried under assay preparation to give the
the appropriate dilution in mobile phase. desired concentration.

2.3.2.2. Sample preparation. Samples, of randomly selected clo- 2.3.2.7. Accuracy. Accuracy was calculated as the deviation of
nazepam tablets, were dissolved and diluted to the appropriate the mean from nominal concentration. To assess accuracy,
volumes to get a concentration of 0.04 mg/ml using mobile freshly prepared placebo of the clonazepam pharmaceutical
phase as solvent. All samples were filtered through nylon formulations was spiked with various amounts of clonazepam
sample filter (Whatman, 0.22 lm). and its related compounds (A&B) to obtain the concentration
levels of 30, 40 and 50 lg/ml. Each solution was injected in
2.3.2.3. Method development and chromatographic conditions. A triplicate.
variety of mobile phases were investigated in the development
of a stability-indicating HPLC method for the analysis of clo- 2.3.2.8. Limit of detection (LOD) and limit of quantitation
nazepam and its related compound in pharmaceutical prepara- (LOQ). The LOD is taken as the lowest concentration of an
tions. The suitability of mobile phase was decided on the basis analyte in a sample that can be detected, but not necessarily
of green analytical chemistry principles, selectivity, sensitivity quantified, under the stated conditions of the test while the
of the assay, stability studies, and separation of clonazepam LOQ is the lowest concentration of an analyte in a sample that
from the known related substances (A&B) beside the impuri- can be determined with acceptable precision and accuracy
ties formed during forced degradation studies. The elution under the stated conditions of test. They can be calculated
was carried out on a BDS C8 Hypersil column from the linear calibration curve where LOD is the 3 times
(250 mm · 4.6 mm, 5 lm particle size) from Thermo scientific of residual standard deviation of the regression line (at the
(Massachusetts, USA). All analyses were performed at ambi- lower concentration) divided by the slope, while LOD is ten
ent temperature under isocratic conditions with a mobile phase times the division product (Shrivastava and Gupta, 2011).
of isopropanol: 2% sodium dodecyl sulfate (SDS): 0.05 M
sodium acetate buffer (pH 3.5 ± 0.05) in ratio (20:25:55, v/v) 2.3.2.9. Robustness. In order to check the robustness, the effect
at a flow rate of 1.5 mL/min, using DAD detector at 254 nm. of small but deliberate variations in the chromatographic con-
ditions was evaluated. The conditions studied were flow rate
2.3.2.4. Stability indicating capabilities of the proposed method. (altered by ±0.01 mL/min), and pH of buffer solution (altered
The stability-indicating capability of the method was deter- by ±0.1). These chromatographic variations were evaluated
mined by its ability to separate the reference degradation sub- for retention time, peak area, number of theoretical plate
stances (A&B) from the drug of interest in addition to and asymmetry factor for each clonazepam and its related
subjecting reference solution of clonazepam (40 lg/mL) to compound.
forced degradation conditions by acidic, basic, oxidative, and
photolytic conditions to evaluate the interferences in the quan- 2.3.2.10. Selectivity and system suitability. Selectivity of this
titation of clonazepam. Standard solutions in 1 M hydrochlo- method was indicated by the absence of any excipient interfer-
ric acid and 1 M sodium hydroxide were used for the acidic ence at the retention times of the peaks of clonazepam and its
and basic hydrolysis, respectively. Both solutions were kept related compounds with an acceptable resolution in the system
at ambient temperature for 12 h. For oxidative degradation, suitability solution. The absence of interfering peak was eval-
solutions were prepared in hydrogen peroxide (3%) solution uated by injecting a blank sample consisting of diluent and pla-
and kept at ambient temperature for 4 h. Photodegradation cebo. The test is only valid if the resolution between
was induced by irradiating the neutral solution at 1.2 million clonazepam related A and clonazepam related B is more than
lux hours for 24 h. The distance between the light source and 2 as stated in the relevant monograph in the USP 36 (USP,
the sample was maintained at 25 cm. These solutions were 2013).
diluted with mobile phase to final concentration of 40 lg/ml
and were injected into chromatographic system. 2.3.2.11. Uncertainty of the method. Among the different
sources of uncertainty, the uncertainty associated with calibra-
2.3.2.5. Linearity. Linearity was evaluated by determining the tion appears to be the most important source in the overall
response of a series of dilutions of thirteen standard solutions uncertainty (ICH guideline Q2B, 2005).
from clonazepam and nine standard solutions of its related The quantification of clonazepam by was assess through the
compounds (A&B) at a concentration range of 4–140 lg/mL calibration curve equation (Y = aX + b), where Y is the
and 4–64 lg/mL, respectively. Each dilution was injected in response, a is the slope, X is the concentration of clonazepam
triplicate to plot the calibration curve. Slope, intercept, and and b is the intercept. Based on this information, the uncer-
regression coefficient (R2) of the calibration curves were calcu- tainty of HPLC’s result was estimated by the following
lated to ascertain linearity of the method. equation:
sffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2.3.2.6. Precision. For method repeatability, assay of authentic ðrY þ rb Þ2 ra 2
samples solutions was repeatedly performed six times on the UA% ¼ A%  t1a=n2  þ
ðY  bÞ2 a
same day (intra-day). For reproducibility, freshly prepared
solutions at aforementioned concentration level were analyzed where UA% is the expanded uncertainty, t1-a/n-2 is the t-stu-
at different days (inter-day), and results were statistically dent for confidence level of 1-a and n-2 degrees of freedom
evaluated in terms of % RSD. The authentic samples were (n correspond to the numbers of standard employed to obtain
Green analytical chemistry for development of HPLC method 1215

calibration curve), A% is the result of sample (in percentage), involved. Considering the green chemistry fundamentals
rY is the standard deviation of the areas obtained in chro- (Clark et al., 2006), various solvent systems as mobile phases
matograms of sample, Y is the average of the areas obtained were tried for the development of a green and environmentally
in chromatograms of sample, rb is the error for the intercept benign HPLC method. Of several solvents and solvent mix-
and ra is the error for the slope. tures investigated, the mobile phase of isopropanol: 2%
SDS: 0.05 M sodium acetate buffer (pH 3.5 ± 0.05) in ratio
3. Results and discussions (20:25:55, v/v) was found to furnish sharp, well defined peaks
with very good symmetry (around 1) and appropriate separa-
3.1. Optimization of the chromatographic conditions tion (NLT 2) between the peak of interest and the related com-
pounds as a system suitability requirement in the relevant
monograph in the USP-36 (Fig. 2).
The HPLC procedure was optimized with a view to develop an
Other organic modifiers and surfactants were tried such as
ecofriendly stability-indicating method. No internal standard
Brij-L23, tween 20 and tween 80 did not resulted in chromato-
was used because no extraction or separation step was
graphic system as good as proposed method.

3.2. Method validation

3.2.1. Linearity
The calibration curves of clonazepam, clonazepam related A
and clonazepam related B were evaluated by the linear least
square analysis. The calibration curve plotted between concen-
tration of drug and peak area and the regression equations
were calculated and the correlation coefficients of the calibra-
tion curves were found to be R2 > 0.999 in all samples types.
Linearity was established in concentration range of 4–140 lg/
ml for clonazepam and 4–64 lg/ml for clonazepam related
compounds A & B as reported in Table 1. The linear regression
data for the calibration plot were indicative of a good linear
relationship between peak area and concentration over a wide
range. The correlation coefficient was indicative of high signif-
icance. The intercept of the ordinate showed the calibration
plot did not deviate from linearity.

3.2.2. Accuracy
Figure 2 Separation of system suitability solution using (a) the Accuracy was determined by the standard addition method.
proposed green method and (b) the USP-36 method (Clonazepam Three sets of placebo preparations were spiked with 32, 40
at 9.84, related compound A at 14.86 and related compound B at and 48 lg/ml of clonazepam and related substances reference
16.90) and (b) the USP-36 method (Clonazepam at 10.21, related standards and the mixtures were analyzed by the proposed
compound A at 21.41 and related compound B at 23.71). method. The experiment was performed in triplicate. Good

Table 1 Linearity of calibration curve for clonazepam and its related substances.
Clonazepam Clonazepam related compound A Clonazepam related compound B
Concentration (lg/ml) Peak area Concentration (lg/ml) Peak area Concentration (lg/ml) Peak area
4 354,436 4 43,266 4 116,673
8 714,285 8 87,160 8 240,323
16 1,438,491 16 176,453 16 493,393
24 2,153,360 24 273,040 24 739,427
32 2,880,450 32 367,352 32 978,949
40 3,594,755 40 451,953 40 1,232,962
48 4,323,766 48 545,139 48 1,475,217
56 5,042,151 56 638,419 56 1,715,512
64 5,775,803 64 734,168 64 1,926,860
80 7,304,113 R2 0.9998 R2 0.9997
100 9,080,737 Slope 11496.851 Slope 30429.064
120 10,822,764 Intercept 4458.937 Intercept 3781.023
140 12,556,717
R2 0.9999
Slope 90217
Intercept 231.86
1216 A.B. Eldin et al.

Table 2 Estimation of the accuracy as an item for validation of the proposed HPLC method for the determination of clonazepam and
its related substances.
Amount taken (lg) Amount found (lg) Percent recovery Mean percentage recovery RSD%
Clonazepam 32 31.87 99.59% 99.57% 0.11
40 39.78 99.45%
48 47.84 99.67%
Clonazepam related compound A 32 32.28 100.88% 99.77% 0.97
40 39.62 99.05%
48 47.71 99.40%
Clonazepam related compound B 32 32.16 100.50% 100.97% 0.42
40 40.53 101.33%
48 48.52 101.08%

Table 3 Repeatability and intermediate precision for clonazepam and its related substances.
Exp. No. Repeatability on day 1 Repeatability on day two
Clonazepam Related A Related B Clonazepam Related A Related B
1 99.90% 100.06% 100.92% 100.00% 100.16% 101.02%
2 99.93% 100.33% 101.16% 100.23% 100.63% 101.46%
3 99.96% 100.30% 100.99% 100.16% 100.50% 101.19%
4 99.97% 100.30% 101.76% 100.37% 100.70% 102.17%
5 99.79% 100.27% 101.91% 99.89% 100.37% 102.01%
6 99.93% 100.94% 101.65% 100.13% 101.14% 101.86%
Mean 99.91 100.37 101.40 100.13 100.58 101.62
SD 0.064 0.298 0.427 0.168 0.334 0.464
RSD (%) 0.064 0.297 0.421 0.168 0.332 0.456

recoveries (101.33–99.40%) were obtained at each concentra-

tion level with a very low % RSD (0.97–0.11%) which indi-
cated the accuracy of the proposed method (Table 2).

3.2.3. Precision
3.2.3.1. Analysis repeatability. It was evaluated by carrying out
the analysis of the six homogenous solutions of same test sam-
ple and content of related substances. The determinations were
carried out one after the other under conditions as similar as
possible. The relative standard deviation was calculated from
the results of the obtained observations (Table 3).
Figure 3 Clonazepam solutions under basic degradation condi-
3.2.3.2. Intermediate precision. The intermediate precision of
tions (Clonazepam at 9.28, basic degradation products at 10.52,
the method was checked by determining precision on a differ-
13.98, 19.34 and 20.73).
ent instrument, analysis being performed in different labora-
tory on a different day. The relative standard deviation was
calculated from the results of the obtained observations.
In all cases the RSD was lower than 2 (Table 3).

3.2.4. Specificity
The specificity of the method was evaluated through its ability
to discriminate between the peak of the parent drug and those
due to its related compounds A & B (Fig. 2).
In the same regard a forced degradation study was estab-
lished, clonazepam stock solution was exposed to various stress
conditions. On treating with 5 N NaOH extensive degradation
has been occurred, when treated with 1 N NaOH for one hour,
the height of peak was reduced and four new peaks of degrada- Figure 4 Clonazepam solution under acidic degradation condi-
tion products were observed (Fig. 3). Almost the same has been tions (Clonazepam at 9.26, acidic degradation products at 10.50,
happened on treating with 1 N HCl for one hour (Fig. 4). 13.55, 19.47 and 20.74).
Green analytical chemistry for development of HPLC method 1217

3.2.5. Detection (LOD), quantification (LOQ) limits and

system suitability
Limit of detection (LOD) and the limit of quantitation (LOQ)
were calculated as 3 S.D. and 10 S.D. of the blank, respec-
tively, according to the treatment by Miller and Miller
(Sadeghi et al., 2013). Before injecting solutions, the column
was equilibrated for at least 20 min with the mobile phase.
Their values prove the sensitivity of the proposed method
and that it can be used for detection and quantification of trace
amounts of clonazepam and its related compounds. The
obtained system suitability parameters were satisfactory
Figure 5 Clonazepam solution under oxidative degradation according to the relevant USP-36 monograph (Table 4).
conditions (Clonazepam at 9.27, oxidative degradation products
at 12.75 and 19.01). 3.2.6. Robustness
The robustness of an analytical procedure, as defined by the
ICH refers to its capability to remain unaffected by small
and deliberate variations in method parameters (Potka et al.,
2013). Robustness was determined by recording the effect of
slight change on the separation conditions of buffer and pH.
Table 5 shows that the theoretical plates, tailing factor and res-
olution of clonazepam and its related substances are not chan-
ged significantly which indicates the proper robustness of the
method.

3.2.7. Uncertainty estimation

Figure 6 Clonazepam solution under photo-irradiation condi- The expanded uncertainty amount (0.33% at confidence level of
tions (Clonazepam at 9.58, photo degradation product at 14.66). 95%), was estimated based on the error of the slope and the
Clonazepam was found to degrade more rapidly in oxidative intercept. Considering HPLC method, a six standard calibra-
conditions. Upon reflux with 30% hydrogen peroxide for 1 h, tion curve may decrease the uncertainty estimated, as it decrease
peak height was reduced, with the appearance of two new deg- the t-student multiplier. Increasing the number of injections per
radation peaks (Fig. 5). One degradation peak has been sepa- standard level may also contribute in the reduction of final
rated with dramatic change in the peak of the parent drug uncertainty (De Melo Abreu et al., 2005; Lourenço, 2012).
has been happened on exposing to photo-irradiation conditions Other sources of uncertainties, e.g. uncertainties of volumetric
in photo cabinet (Fig. 6).The above results show the method is glassware, were not considered due to its non significant contri-
stability indicating. bution into the total budget of the uncertainty.

Table 4 LOD, LOQ and system suitability parameters*.

Parameters Clonazepam Clonazepam related compound A Clonazepam related compound B
LOD (lg/ml) 0.024 0.386 0.357
LOQ (lg/ml) 0.0799 1.2875 1.1893
Theoretical plates (h) 18625.64 8854.82 9570.59
Tailing factor (T) 1.104 0.988 1.032
Resolution – 6.865 2.815
*
LOD, limit of detection; LOQ, limit of quantitation.

Table 5 Evaluation of robustness of the proposed method.

Drug name Variations Chromatographic parameters
Retention time Area Theoretical plates Asymmetry
Clonazepam Change in buffer pH 9.83 3594745 18620.52 1.103
Flow rate at 1.51 ml/min 9.75 3594760 18624.83 1.102
Clonazepam related compound A Change in buffer pH 14.66 451943 8840.77 0.998
Flow rate at 1.51 ml/min 14.56 451961 8857.94 1.009
Clonazepam related compound B Change in buffer pH 16.85 1232974 9511.48 1.026
Flow rate at 1.51 ml/min 16.75 1232950 9514.59 1.031
1218 A.B. Eldin et al.

3.3. Application Abdallah participated in the design and carried out the HPLC
analysis and wrote the paper. All authors read and approved
Samples of clonazepam 0.5 mg tablets (n = 6) were analyzed the final paper.
for the parent drug and its related substances by this method
and the results showed a percent recovery of 100.1% and a Acknowledgement
RSD of 0.60% for 6 replicates while the related substances
were within the accepted limits. The method was used for ana- The authors are grateful to Sigma pharmaceutical industries –
lyzing samples of the stability program of the drug product Egypt for providing the facilities to carry out this study.
also for analysis of raw materials samples which give results
between 99.6% and 100.2%. References

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