1260 Mohamed & Lamie: Journal of AOAC International Vol. 99, No.

5, 2016

DRUG FORMULATIONS AND CLINICAL METHODS

Analytical Eco-Scale for Assessing the Greenness of a

Developed RP-HPLC Method Used for Simultaneous
Analysis of Combined Antihypertensive Medications
Heba M. Mohamed and Nesrine T. Lamie
Cairo University, Faculty of Pharmacy, Analytical Chemistry Department, Kasr Al-Aini St, Cairo 11562, Egypt

In the past few decades the analytical community Significant effort has been expended to quantify the greenness

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has been focused on eliminating or reducing of several organic solvents (4, 5). Currently, one of the main
the usage of hazardous chemicals and solvents, areas of focus being explored in greening chromatography is a
in different analytical methodologies, that have solvent-replacement approach, where the most common mobile
been ascertained to be extremely dangerous to phases containing acetonitrile and/or methanol are replaced by
human health and environment. In this context, less harmful and more ecofriendly supercritical or subcritical
environmentally friendly, green, or clean practices alternatives, such as water (6), ethanol or isopropanol (7), and
have been implemented in different research carbon dioxide (8, 9).
areas. This study presents a greener alternative The aim of this work is to highlight the benefits of
of conventional RP-HPLC methods for the applying green, environmentally friendly HPLC methods in
simultaneous determination and quantitative pharmaceutical analysis and the ease with which traditional
analysis of a pharmaceutical ternary mixture methods can be replaced without affecting method performance.
composed of telmisartan, hydrochlorothiazide, and In addition, the method’s greenness profile will be assessed
amlodipine besylate, using an ecofriendly mobile with the analytical Eco-Scale, a semiquantitative, green
phase and short run time with the least amount metric tool (10–12). A validated ecofriendly HPLC method
of waste production. This solvent-replacement was successively developed for the quantitative analysis and
approach was feasible without compromising simultaneous determination of a pharmaceutical ternary mixture
method performance criteria, such as separation of telmisartan (TL), hydrochlorothiazide (HZ), and amlodipine
efficiency, peak symmetry, and chromatographic besylate (AM), which is used for the management of hypertension
retention. The greenness profile of the proposed (see chemical structures in Figure 1). A review of the literature
method was assessed and compared with reported revealed that very few methods have used quantitative analysis
conventional methods using the analytical Eco-Scale on the studied ternary mixture by ultra-performance LC (UPLC;
as an assessment tool. The proposed method was 13) and RP-HPLC (14). Another method, using a different
found to be greener in terms of usage of hazardous chromatographic separation technique, the TLC-densitometric
chemicals and solvents, energy consumption, method, has also been reported (15). We used the analytical
and production of waste. The proposed method Eco-Scale to compare the greenness profile of the suggested
can be safely used for the routine analysis of the method with traditional HPLC and UPLC methods (13, 14).
studied pharmaceutical ternary mixture with a It was observed that, for the methods reviewed, threats to the
minimal detrimental impact on human health and the environment, when using or applying solvents and chemicals,
environment. and GAC principles were not deliberated.

T
he concept of green analytical chemistry (GAC) has Experimental
been a recent development (1). The concept foresees a
lessening or total removal of harmful chemicals used Materials and Solvents
in the analytical process, a reduction in energy consumption,
and a minimization of waste production, without affecting the (a) Pure TL standard.—The standard was generously
criteria for optimum performance of a method (2). LC is one of supplied by the National Organization of Drug Control and
the most harmful techniques, with risks to human health and Research (NODCAR, Cairo, Egypt), with a claimed purity of
environment alike as a result of its wide-spread application in 99.89%, according to the official method (16).
the analytical field and the usage of high amounts of dangerous (b) Pure HZ and AM standards.—The standards were
organic solvents (3). LC solvents are composed of mainly generously supplied by Al-Hekma Pharmaceuticals (Cairo,
volatile organic compounds, which diffuse directly into the Egypt), with a claimed purity of 100.45 and 99.93%,
environment, provoking both acute and chronic toxicity. respectively, according to official methods (16).
(c) Pharmaceutical formulations.—Telma-AMH, 40 tablets,
Batch No. FT0114029, Glenmark Pharmaceuticals Ltd
Received April 17, 2016. Accepted by JB June 23, 2016.
Corresponding author’s e-mail: [email protected] (Mumbai, India). It is claimed that each tablet contains 40 mg
DOI: 10.5740/jaoacint.16-0124 TL, 12.5 mg HZ, and 5 mg AM.
 Mohamed & Lamie: Journal of AOAC International Vol. 99, No. 5, 2016 1261

above-cited chromatographic conditions. The average peak area

ratios for each concentration of TL, HZ, and AM (using external
standards of TL, HZ, and AM 50 μg/mL) were plotted against
their corresponding concentrations and the regression equations
were computed.
(c) Synthetic mixtures.—Aliquots of TL, HZ, and AM
were transferred accurately from their corresponding standard
solutions (100 µg/mL) into a series of 10 mL volumetric flasks.
The prepared mixtures were analyzed as described in step (b),
the peak area ratios of the synthetic mixtures calculated, and
the concentrations from the corresponding regression equations
also calculated.
(d) Application to the pharmaceutical preparation.—Ten

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tablets of the drug formulation were accurately weighed and
finely powdered. An amount of powder equivalent to 20 mg
TL was accurately transferred into a beaker and 30 mL ethanol
Figure 1. Chemical structures of TL, HZ, and AM. added with continuous stirring for 10 min. The solution was
filtered into a 50 mL volumetric flask and diluted to volume with
(d) Chemicals and reagents.—Double-distilled water; ethanol. One milliliter of solution was accurately transferred
potassium monobasic phosphate (Sigma-Aldrich, Steinheim, into a 10 mL volumetric flask and diluted to volume with ethanol
Germany), 99.0% purity; potassium dibasic phosphate (Sigma- and analyzed as described in step (b). Drugs concentrations
Aldrich), 99.0% purity; and HPLC grade ethanol (Fisher were calculated from the corresponding regression equations.
Scientific, Loughborough, United Kingdom), 99.8% purity
were used.
Results and Discussion
Instruments
Method Validation
(a) Chromatographic analysis.—Analysis was performed
Validation of the developed method was carried out according
on a liquid chromatograph with an LC 10 AD pump connected
to International Conference on Harmonization guidelines (17).
to an SPD-10A detector (all Shimadzu, Kyoto, Japan).
Linearity ranges for each of the three studied drugs were
(b) Autosampler and degasser.—A SIL-20A autosampler
determined. Regression equations and parameters were calculated
and DGU-12A degasser were used (both Shimadzu).
and are presented in Table 1. LODs and LOQs were calculated
(c) The chromatographic instrument.—was connected to a
based on the SD of the response and the slope of the calibration
personal computer and P2055D laser-jet printer (both Hewlett
curve (S) using the following equations: LOD = 3.3 (σ/S) and
Packard, China).
LOQ = 10 (σ/S). Three different concentrations for each drug
(d) Intersil ODS-3 HPLC column.—4.6 mm × 250 mm,
were analyzed three times on the same day (for repeatability)
5 µm, purchased from GL Sciences, Inc. (Tokyo, Japan).

Standard Solutions Table 1. Linearity studies and characteristic regression

parameters for the proposed HPLC method
Standard solutions for TL, HZ, and AM (100 µg/mL) were Parameter TL HZ AM
prepared in ethanol. Linearity range, 2.0–50.0 5.0–50.0 5.0–50.0
µg/mL
Procedures Regression
Slope 0.02 0.02 0.019
(a) Chromatographic conditions.—Chromatographic
Intercept 0.0017 0.0014 0.014
separations were carried out on an Intersil ODS-3 HPLC column 2
(4.6 × 250 mm, 5 µm). An isocratic elution was used, with an r 0.9998 0.9998 0.9997

ethanol–0.02 M phosphate buffer, pH 7 (70 + 30, v/v) as the Accuracy, 100.26 ± 0.59 100.13 ± 0.69 100.48 ± 0.71
mean ± RSD, %
mobile phase and a flow rate of 0.7 mL/min. The mobile phase
was filtered using a 0.45 mm Teflon membrane filter (Millipore, Precision; RSD, %
Milford, MA) and degassed previously with ultrasonic vibrations Repeatability a
0.82 0.76 0.60
for 30 min. Detection was performed at 240 nm. All separations Intermediate 1.13 0.91 0.90
and determinations were performed at room temperature. precisionb
(b) Construction of the calibration graphs.—Aliquots of TL, LOD, µg/mL 0.55 1.25 1.41
HZ, and AM standard solutions (100 µg/mL) were transferred LOQ, µg/mL 1.66 3.78 4.27
into a series of 10 mL volumetric flasks and diluted to volume a
 Intraday (n = 9) RSD, % of concentrations (10.0, 30.0, and
with ethanol to yield solutions in the concentration ranges of 40.0 µg/mL) for TL, HZ, and AM, respectively.
2 to 50 µg/mL for TL, 5 to 50 µg/mL for HZ, and 5 to 50 µg/mL b
 Interday (n = 9) RSD, % of concentrations (10.0, 30.0, and
for AM. All prepared solutions were analyzed under the 40.0 µg/mL) for TL, HZ, and AM, respectively.
1262 Mohamed & Lamie: Journal of AOAC International Vol. 99, No. 5, 2016

and on 3 different days (for intermediate precision) to verify the Table 3. Parameters of system suitability for the proposed
precision of the method. RSD, % values are presented in Table HPLC method for the determination of TL, HZ, and AM
1, where good RSD, % of repeatability (0.82, 0.76, and 0.60) Reference
and intermediate precision (1.13, 0.91, and 0.90) are shown for Parameter HZ TL AM valuea
TL, HZ, and AM, respectively.
Retention time, min 3.24 5.01 7.17
To study the specificity of the proposed method, laboratory
Capacity factor, K′ 0.69 1.62 2.75 0–10
mixtures containing different concentrations of the three
drugs within the linearity range and the dosage form ratio Symmetry factor 1.10 1.00 1.14 ~1
were analyzed using the same chromatographic conditions. Resolution, RS 4.72    6.17 RS <2
The results are shown in Table 2 and exhibit good percentage Selectivity, α 2.33    1.69 α>1
recoveries (99.95 ± 1.26, 101.06 ± 0.88, and 100.26 ± 0.69) for
Theoretical plates, N 2014 4294 7126
TL, HZ and AM, respectively. a
 All parameters are according to the Center for Drug Evaluation and
The robustness of the suggested method was assessed by Research (18).

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altering certain parameters, such as changing the ratio of
ethanol–buffer by ±2 units and adjusting the flow rate by and AM analysis by the suggested HPLC method with those
±0.1. For optimum study conditions, one factor was altered at obtained by applying the official method (16) and the reported
a time to evaluate its effect. The analysis was carried out in TLC-densitometric method (15), the calculated t- and F-values
triplicate (n = 3) at three different concentration levels for each were lower than the tabulated values, which indicates that for
component of the mixture. It was found that the minor variations accuracy and precision, no significant differences were found
in the above-mentioned parameters had no significant effect on (results are presented in Table 5).
the analysis of the ternary mixture using the suggested method.
The low RSD, % of peak area ratios (1.49, 1.36, 0.98) upon Method Development and Optimization
changing the mobile phase ratio and (1.27, 0.87, 1.66) upon
changing the flow rate, for TL, HZ, and AM, respectively, Several approaches were used to reduce the environmental
in addition to the closely retention times; indicated that the impact of the different analytical methodologies: For example,
proposed method has good robustness. using green sample pretreatment (19), environmentally friendly
solvents and reagents (20), and less hazardous solvents (3), as
System Suitability well as shortening chromatographic separation times (21) and
proper management of waste products (22). Use of all of these
System suitability testing is a critical part of an analytical approaches is spreading to different laboratories, which reflects
method, especially LC. Suitability tests are carried out to rising awareness of the significance of greening analytical
authenticate that the resolution and reproducibility of the methods.
chosen chromatographic system are suitable for the analysis to The 12 projected principles of green chemistry (10–12)
be undertaken. Parameters, such as retention time, resolution recognize that the use of hazardous reagents, solvents, and
(RS), symmetry, selectivity (α), and the number of theoretical auxiliary compounds is a colossal problem that needs to be
plates (N), were calculated and are shown in Table 3. addressed. An advisable solution is replacing harmful reagents
and solvents with greener substitutes.
Pharmaceutical Dosage Form Analysis
Table 4. Determination of TL, HZ, and AM in
pharmaceutical dosage form by the proposed method
The proposed HPLC method was efficaciously applied to and application of the standard addition technique
assay TL, HZ, and AM in Telma-AMH 40 tablets (Table 4).
Claimed Standard
Recovery value percentages were found to be high, which
Telma-AMH 40 Found, %a amount, µg/mL added, µg/mL Recovery, %
affirms the suitability of the proposed method for routine
TL 100.58 ± 0.57 10 10 101.46
QC testing and the determination of the three components in
their combined formulation. The validity of the method was 20 100.59

further confirmed by using a standard addition technique. 30 101.23

Upon statistically comparing the obtained results from TL, HZ, Mean 101.09
RSD % 0.44
HZ 99.28 ± 1.0 10 10 99.71
Table 2. Determination of TL, HZ, and AM in their synthetic 20 99.88
mixture using the proposed HPLC method
30 98.39
TL–HZ–AM Mean 99.32
concn, µg/mL TL HZ AM
RSD % 0.82
40 + 12.5 + 5 98.49 100.82 99.44
AM 101.07 ± 0.89 10 10 100.13
10 + 10 + 10 98.66 99.65 100.72
20 100.12
30 + 20 + 10 100.87 101.22 101.13
30 101.69
30 + 30 + 20 100.78 101.96 99.71
Mean 100.64
20 + 10 + 20 100.97 101.65 100.32
RSD % 0.89
Mean ± RSD, % 99.95 ± 1.26 101.06 ± 0.88 100.26 ± 0.69 a
Average of six different determinations.
 Mohamed & Lamie: Journal of AOAC International Vol. 99, No. 5, 2016 1263

Table 5. Statistical comparison between the results obtained by the proposed HPLC method and the official and reported
methods for the determination of Telmisartan, Hydrochlorothiazide and Amlodipine besylate in pure powder form

TL HZ AM

Official Reported Official Reported Official Reported

Parameter HPLC method methoda methodd HPLC method methodb methodd HPLC method methodc methodd
Mean 100.26 99.89 100.74 100.13 100.45 99.86 100.48 99.93 100.51
SD 0.59 1.03 0.82 0.69 0.52 1.09 0.72 0.51 1.14
% RSD 0.59 1.01 0.81 0.68 0.51 1.09 0.71 0.51 1.13
Variance 0.34 1.05 0.67 0.47 0.27 1.18 0.51 0.26 1.29
n 6 6 6 6 6 6 6 6 6
Student 0.76, 1.17 0.91, 0.51 1.53, 0.05
t-test (2.22)e (2.22)e (2.22)e

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F-test 3.08, 1.97 1.74, 2.51 1.96, 2.52
(5.05)e (5.05)e (5.05)e
a
Non aqueous titration (16).
b
Zero order spectrophotometric method at λmax 273 nm (16).
c
 HPLC method using C18 column and a mobile phase consisting of acetonitrile: methanol: buffer (15:35:50 by volume) at a flow rate of 1.0 mL /min
and detection at 237 nm (16).
d
 Reported TLC method using silica gel F254 plates and ethyl acetate: methanol: acetone (7.5: 2.5: 0.5 by volume) as the developing system (15).
e
 Figures in parentheses are the corresponding tabulated values at p = 0.05.

Methanol and acetonitrile are the most broadly used solvents ethanol–0.02 M phosphate buffer mixture, pH 7 (70 + 30, v/v)
in most analytical methods, and it is worth mentioning that as the mobile phase, with a flow rate of 0.7 mL/min and UV
methanol and acetonitrile are rated by the U.S. Environmental detection at 240 nm.
Protection Agency as hazardous solvents (23), given their All experimental conditions affecting method performance
inherent toxicity and the fact that their disposal necessitates were investigated. According to a review of the literature,
specialized treatment steps, particularly for acetonitrile, where all the reported methods used a C18 column for the
detoxification through chemical treatment has to be carried out stationary phase to separate the studied drugs, (13, 14) and
because traditional disposal (i.e,. through combustion) produces all used acetonitrile as an organic solvent, therefore we
a highly toxic compound (hydrogen cyanide). began our environmentally friendly trials with a C18 column
Green organic solvents can be classified into two classes: and our system with ethanol–phosphate buffer. To improve
“traditional” and “nontraditional” or “newer.” Numerous chromatographic separation, we first used ethanol–0.02 M
comprehensive investigations of several solvents have been phosphate buffer, pH 7 (60 + 40, v/v) as the mobile phase,
done using Environmental, Health and Safety or Life-Cycle however poor resolution of TL, HZ, and AM resulted.
Assessment approaches or a combination of the two, where We then tested different ratios of the organic modifier
many of these studies (24–26) found that, besides water, ethanol (30–80%) and found that upon using >80% ethanol, TL
is one of several preferred solvents to use. Moreover, ethanol is and HZ together were poorly resolved and eluted, appearing
considered the solvent with the lowest environmental impact as overlapped peaks. On the other hand, decreasing the
according to criteria in the American Chemical Society Green ethanol ratio in the mobile phase (50%) did not affect the
Chemistry Institute solvents guide (27). resolution, but increasing the analysis time resulted in a
Ethanol can be a greener alternative to methanol and tailing of the peaks. In addition, we tested phosphate buffer
acetonitrile. The reported HPLC methods for the studied ternary at different pH values (pH 3–8), and found that optimum
mixture used harmful mobile phases, where acetonitrile was a separation with symmetric untailed peaks was obtained with
main component in both studies, which is environmentally not 0.02 M phosphate buffer, pH 7.
recommendable (28). The effect of the mobile phase flow rate (0.7, 1, and
On the other hand, high-temperature LC (HTLC) can ensure 1.5 mL/min) on the separation was also tested, where a flow rate
a greener and more rapid and efficient analysis. Yet HTLC is of 0.7 mL/min gave the optimum chromatographic separation
not regularly used because it has several drawbacks. Firstly, within a reasonable amount of analysis time and with minimal
a limited availability of stable high-temperature-resistant waste production. To improve the sensitivity of the developed
packing materials is a considerable problem. Secondly, the method, we tested several scanning wavelengths (220, 240, and
potential degradation of unstable compounds is highly likely: 270 nm) and found that 240 nm gave the best S/N.
“Amlodipline besylate is easily degradable” (29). And finally, a Although a conventional HPLC column was used, it did have
specific set up is a required for heating and cooling the mobile a high degree of separation efficiency and a short separation time
phase before and after its travel through the chromatographic (7 min) with a flow rate 0.7 mL/min, which led to a decrease
column (30). in the amount of waste produced. Other shorter columns were
A validated isocratic RP-HPLC method with UV detection tested, however overlapping peaks occurred with no effect on
was thus developed for the simultaneous quantitation of TL, the separation time. This substantiated the suggested method as
HZ, and AM. The method is based on the chromatographic a readily available, safer, and alternate method for laboratories
separation of the three components using a C18 column and an that do not have UPLC instruments or use monolithic or other
1264 Mohamed & Lamie: Journal of AOAC International Vol. 99, No. 5, 2016

specific short columns. The proposed method is environmentally methods was considered according to four criteria: the use of
friendly and can be conducted using a traditional HPLC device persistent, bioaccumulative, and toxic chemicals; the use of
and conventional column, producing minimal waste. hazardous chemicals; corrosiveness; and the amount of waste
The suggested HPLC method showed good separation of generated (32).
TL, HZ, and AM using a green mobile phase composed of an The analytical Eco-Scale is one of several green metrics
ethanol–phosphate buffer, pH 7, in ratio (70:30, v/v) at a flow in use, and it has been found to be a good, semiquantitative
rate of 0.7 mL/min and detection of 240 nm. Retention times tool to evaluate the greenness of an analytical method. It
compares the various parameters and steps for the whole
were found to be 3.24, 5.01, and 7.17 min for HZ, TL, and AM,
analytical process.
respectively (Figure 2).
The analytical Eco-Scale is calculated by allotting penalty
points to any factor in the analytical procedure that disagrees
Assessment of Greenness: The Analytical Eco-Scale or does not match with perfect green analysis. A green analysis
is deemed ideal if it has an Eco-Scale value of 100, excellent if
Different approaches can be used for the assessment of

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>75, acceptable if >50, and inadequate if <50.
greenness (31). In one approach, the greenness of analytical Penalty points are assigned for each of the four main
parameters of the analytical procedure that departs from an
ideal green analysis: amount of reagents, hazardousness, energy
consumption, and waste production. For the reagents, penalty
points are assigned specific hazard categories—physical,
environmental, and health—that each reagent poses. Different
amounts of reagent will have differing penalty points. Penalty
points for hazards are based on the Globally Harmonized
System of Classification and Labeling of Chemicals (GHS).
Each chemical reagent is characterized by one or more of nine
pictograms, which are a graphic expression of their hazardous
properties. Two signal words are used in GHS: “danger”
(i.e., more severe hazard and equal to 2 penalty points) and
“warning” (i.e., less severe hazard and equal to 1 penalty point).
Other points are assigned based on the instruments used, energy
consumption, and amount and type of waste.
The comparison between the analytical Eco-Scale values of
the suggested method and reported methods is calculated as
shown in Table 6. According to Table 6, the proposed HPLC
method is greener than the reported methods, where the prior
scores higher on the Eco-Scale (13, 14), even though one of
the reported methods is UPLC. Notwithstanding, the proposed
method still scored higher, which proves that the proposed
method is an excellent green method of analysis with minimal
laboratory requirements, whereas the reported methods are less
acceptable.
Compared with a previously reported TLC-densitometric
method (15), which is considered a green alternative compared
with other TLC-densitometric methods, in this work we tried
to avoid all hazardous solvents by instead using solvents
that are greener than those used in the aforementioned study.
We avoided using methanol, a solvent that cannot be replaced
in the TLC method, which makes the proposed HPLC method
an even greener and more environmentally friendly alternate to
be used in QC laboratories.

Conclusions

The green chemistry movement has challenged researchers

and chemists in all fields to consider the environmental impact
of the entirety of their chemical procedures and to assess the
greenness of their processes. In this context, a green, validated
HPLC method was developed to replace existing traditional
methods for the analysis and quantitative determination
Figure 2. HPLC chromatogram of 40 μg/mL TL, 12.5 μg/mL HZ,
of pharmaceutical ternary mixtures used in hypertension
and 5 μg/mL AM in a synthetic mixture using a green mobile phase
management in both pure and dosage forms. The analytical
composed of ethanol–0.02 M phosphate buffer, pH7 (70 + 30 v/v), with
Eco-Scale—which takes into consideration the use and amount
a flow rate 0.7 mL/min.
 Mohamed & Lamie: Journal of AOAC International Vol. 99, No. 5, 2016 1265

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We express our sincere thanks to NODCAR and Al-Hekma
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