The School of Pharmacy and Pharmaceutical

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Greatness Agwaze
Student name & number
21364229

Student Signature

PHU11103
Module

Balsam of Peru
Title of Assignment

17/04/2022
Date
Contents
Questions..............................................................................................................................................3
Calculations F/ Test solution Sudocrem/788.1mg...............................................................................5
References............................................................................................................................................8
 Detection and quantification of Balsam of Peru constituents in Proprietary
preparations using GCMS.

Questions
1. Why is isopropanol the preferred solvent to use for extraction of the analytes from the
proprietary preparations?
Isopropanol can dissolve a wide range of non-polar substances. It evaporates efficiently
under normal lab conditions. Ethanol can chemically react with constituents, for example, it
reacts with benzyl benzoate and benzyl cinnamate at the injection temperature (250 °C), to
form the ethyl ester derivatives e.g. ethyl benzoate. This is due to the polar OH groups and
the good leaving groups in benzyl benzoate and benzyl cinnamate. Isopropanol is more
sterically hindered and does not chemically react with constituents as easily as ethanol does.

2. How might you optimise the extraction efficiency of test analytes from a cream or
suppository-based formulation?
Multiple repeated extractions will be required on the test sample to optimise the extraction
efficiency of the test analytes. The sample should be sonicated, this way the analytes of
interest should dissolve in the solvent and the other constituents will not go into solution.

3. Benzophenone is used as an internal standard in this experiment. Why is it a suitable

choice in your opinion?
It is not a constituent of Balsam of Peru. It is stable and exhibits good resolution from the
analytes in question. Furthermore, it has similar physical and chemical properties to the
analytes of interest, for example, It is an aromatic compound like benzyl benzoate and
benzyl cinnamate and it also has poor solubility in water, like the analytes. It has a similar
retention time to the above-mentioned molecules.

4. The injection mode used in this experiment is split injection. What do you understand by
term split injection?
It is a type of injection where the sample is quickly evaporated in a hot chamber and the
vapour is split into a small portion that is added to the column, this prevents column
pollution. The remaining sample is vented from the system via the split opening. It is
commonly used with capillary columns, however, it is not suitable for dilute solutions.

5. The ideal chromatographic method should exhibit good resolution between individual
constituents present in a mixture. However, an equally important outcome is that each
signal produced for each analyte produces a tall, sharp signal. This is not the case in this
experiment for the analysis of benzoic and cinnamic acids. How might you resolve this
problem?
The reason for these smaller and less sharp peaks is due to the interaction (via hydrogen
bonding) of benzoic acid and cinnamic acid with the polar stationary phase. If the carboxylic
acids are derivatised to their corresponding esters this will decrease the interactions as
esters have dipole-dipole or pi-pi interactions with the stationary phase.
 6. What advantages does mass spectrometry hold over a flame ionisation detector in GC
analysis?
The mass spectrometer is a highly selective detector that detects molecular ions and
fragment ions, its minimal detectable quantity is 10^-12g and it can be used for the
detection of ionisable organic and inorganic compounds. However, a flame ionisation
detector is selective for organic compounds and has a minimum detectable quantity of 10^-
9g
7. What is meant by retention time of analyte in GC and HPLC analysis?
The retention time is the time that the solute spends in a column and is governed by the
solute’s affinity for the stationary and mobile phases. It is measured from the time of
injection to the time of detection.

8. In the context of this experiment, what do you understand by terms, limit of detection and
limit of quantitation?
The limit of detection is the amount of analyte molecule (benzyl benzoate, benzyl
cinnamate, benzoic acid and cinnamic acid) that produces a signal equal to or greater than
three times the baseline noise.
The limit of quantitation is the amount of analyte molecule that produces a signal equal to
ten times that of the base line noise.

9. Step 1: Prepare calibration curves for all three analytes of interest (benzyl alcohol, benzyl
benzoate and benzyl cinnamate) using Excel. The following you tube video is helpful in this
regard: https://www.youtube.com/watch?v=d65jx4BhslA&t=164s
µg/L
Step 2: Determine the content of test analyte(s) in your assigned preparation. Please check
the GCMS video on this practical for an explanation of the process to follow.

Step 3: Determine the content of the test analytes in 20g of the proprietary preparation.

Calculations F/ Test solution Sudocrem/788.1mg

Table 1 Concentration of benzyl alcohol in µg/mL and the mean area ratio obtained by dividing the peak area of benzyl
alcohol divided by the peak area of benzophenone

Concentration Mean
µg/mL area
ratio
1 0.01
10 0.1
30 0.25
50 0.37
70 0.59
100 0.17
Figure 1 Calibration curve for Benzyl alcohol. The concentration 100 µg/mL has been excluded to improve linearity

Table 2 Concentration of benzyl benzoate in µg/mL and the mean area ratio obtained by dividing the peak area of benzyl
benzoate divided by the peak area of benzophenone.

Concentration Mean area

µg/mL ratio
1 0.03
10 0.2
30 0.51
50 0.76
70 1.2
100 0.35
Figure 2 Calibration curve for benzyl benzoate. The concentration 100 µg/mL has been excluded to improve linearity

Table 3 Concentration of benzyl cinnamate in µg/mL and the mean area ratio obtained by dividing the peak area of benzyl
benzoate divided by the peak area of benzophenone.

Concentration Mean
µg/L area
ratio
1 0.02
10 0.14
30 0.36
50 0.53
70 0.85
100 0.25

Concentration of benzyl cinnamate in mg/mL and the mean area ratio obtained by dividing the peak area of benzyl
benzoate divided by the peak area of benzophenone.
Figure 3 Calibration curve for benzyl cinnamate. The concentration 100 µg/mL has been excluded to improve linearity.

The calculation for benzyl alcohol in Sudocrem

Peak area ratio= Peak area of benzyl alcohol/ peak area of benzophenone (internal standard)
3217063/9466084=0.339852 this is y in the formula y=mx+c, where m is the slope, c is the y-
intercept and x is the unknown concentration
y = 0.008x + 0.0057.
0.339852=0.008x + 0.0057.
X= 42.41 µg/mL of benzyl alcohol in the Sudocrem preparation.

According to the HPRA Sudocrem contains 0.39g of benzyl alcohol per 100g (1).
(0.4g/100g) * 0.39g = 1.56mg. So, 0.4g of benzyl alcohol contains 1.56.mg of benzyl alcohol.
((1.56mg*1mL)/100mL)*1000= 15. 6µg/mL = Therefore, the theoretical concentration is
15.6µg/mL.
How much benzyl alcohol would 20g of Sudocrem contain?
Calculation= (20g/100g) * 0.39g= 78mg of benzyl alcohol in 20g of Sudocrem.

The calculation for benzyl benzoate in Sudocrem

Peak area ratio= Peak area of benzyl benzoate/ peak area of benzophenone (internal
standard.
13109229/9466084= 1.384863
y = 0.0163x + 0.0156
1.384863= 0.0163x + 0.0156
X= 84.00 µg/mL
According to the HPRA Sudocrem contains 1.02g of benzyl benzoate per 100g (1).
(0.4g/100g) * 1.02g = 4.08mg of benzyl benzoate in 0.4 g of Sudocrem.
((4.08mg*1mL)/100mL)*1000= 40.8µg/mL Therefore the theoretical concentration is 40.8
µg/mL
How much benzyl benzoate would 20g of Sudocrem contain?
(20g/100g)*1.02g= 204mg of benzyl benzoate in 20g of Sudocrem.
References
1. HPRA. [Online] [Cited: 04 15, 2022.]
https://www.hpra.ie/img/uploaded/swedocuments/Licence_PA0247-001-
001_15062021091154.pdf.