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Taalas, John Spencer

Qualitative and Quantitative Chemistry, MTWTHF 9-10:45

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
Bibliographic citation:
Eldin A., Shalaby A., Abdallah M., Shaldam M., Abdallah M. (2014) 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. Arabian Journal of Chemistry


The environmental pollution has been a concern of the public and scientific
worlds. Using environmentally friendly practices have started in vast areas of the
society and research. Green analytical chemistry uses hazardous chemicals in
preparing the samples and in LC or liquid chromatography. This experiment or
investigation is leaned on producing a technique or method of RP-HPLC without
using hazardous and toxic chemicals.


The reagents and solvents are all HPLC grade and bought from Sigma-Aldrich.
The water is also HPLC grade. Commercial samples of clonazepam tablets are
manufactured in Sigma Pharmaceutical Industries. The apparatus that was used was
Shimadzu HPLC system.
The stock solutions of clonazepam, related compound A and related compound B
and the standard solutions were prepared. Next was the preparation of the sample
solution of randomly selected clonazepam tablets. Different mobile phases were tried

for developing a stability indicating HPLC method for the analysis of clonazepam and
its related substances. All analyses were performed at ambient temperature under
isocratic conditions. The mobile phase was isopropanol: 2% sodium dodecyl sulfate:
0.05 M sodium acetate buffer (pH 3.5 0.05) in ratio (20:25:55 v/v) at a flow rate of
1.5 mL/min, using DAD detector at 254 nm.
The use of the mobile phase isopropanol: 2% sodium dodecyl sulfate: 0.05 M
sodium acetate buffer (pH 3.5 0.05) in ratio (20:25:55 v/v) was found to be furnish
sharp, it has well defined peaks, it also has good symmetry and appropriate separation
between the peak of interest and the related compounds as a system suitability
requirement in the relevant monograph in the USP-36. Other organic modifiers and
surfactants did not produce results as good as the proposed method for the
optimization of the chromatographic conditions.
For the validation of the method, linearity, accuracy, precision, intermediate
precision, specificity, LOD, LOQ, robustness and uncertainty estimation was
determined one by one to make sure that the proposed method is valid. Overall the
results were good to make the proposed method acceptable for determining

clonazepam and its related substances in pharmaceutical formulations.

The proposed method to make a RP-HPLC procedure that does not use hazardous
and toxic chemicals in the sample preparation and LC analysis is important for the
field of Green analytical Chemistry (GAC) because using hazardous chemicals needs
to be detoxified safely and it may lead to a very high disposal costs. The authors
focused on making a method that can be environmentally friendly to lessen the
dangers of doing LC analysis with toxic reagents and chemicals.
The procedures of the said method were not discussed thoroughly. The RP-HPLC
analysis of clonazepam and its related substances was not shown in the journal. The

mobile phase was introduced but not the actual step by step analysis using HPLC. The
methodology was insufficient for the readers. The process of doing the HPLC
analysis was so short that the only thing youll know is the mobile phase it used to
separate the clonazepam and its related substances.
The ways to validate the method by using different statistical instruments was
mentioned and also the results. The explanation of these results was enough for me to
understand that this method is indeed valid, but the calculations to get the results were
not shown. This is very is important so that when someone will also do the same
experiment, they will be able to calculate the readings and get the result.
There were no repetitions of content or data in the journal. The tabulation of the
readings they got from the HPLC and the results of their calculations were shown.
They got good results and they were able to discuss it fairly to help the readers
understand the meaning of each result and its significance to the validation of the
development of a green HPLC method.
The article is timely because of the fact that being environmentally friendly in this
aspect is very practical. The use of hazardous chemicals leads to very high disposal
costs, so using non-hazardous chemicals can lessen this cost. Also it also helps with
the environmental pollution we experience today. The materials used are very
feasible and practical. Like I said earlier, they are more practical because it lessens the
disposal cost of the chemicals. The materials are also feasible as shown by the results
of the validation. They can successfully produce the same results of using hazardous

The article about proposing a RP-HPLC method is easy to understand in terms of
its results and how to make sure that this method can be used to make a routine

analysis of clonazepam and its related substances. This article is very timely and
applicable to its field of Green Analytical Chemistry (GAC) which is focused on
introducing green methodology in analyzing various kinds of chemicals and
substances. However, it lacks the step by step process of doing the RP-HPLC
analysis, which I think is one of the interests of the reader. Also the calculations for
the linearity, accuracy, precision, specificity, LOD, LOQ, robustness, and uncertainty
estimation are lacking.
Overall if you are just interested with the results of the experiment, this article is
good for you. But, if you are interested with how they were able to conceptualize the
experiment, the procedures to do the experiment, the calculations, the information in
this article is insufficient for you.