Application of Chromatography in Pharmaceutical and Biomedical Analysis

This document discusses the application of chromatography in pharmaceutical and biomedical analysis. It covers factors contributing to bacterial resistance to ciprofloxacin and measures to prevent it. It also explores methods for extracting ciprofloxacin from tablets and determining its amount. Additionally, it describes the setup of a Y-site assembly for investigating the chemical and physical incompatibility of amoxicillin, PN, and lipid.

APPLICATION OF CHROMATOGRAPHY IN
PHARMACEUTICAL AND BIOMEDICAL ANALYSIS
CSA743 – Assessment Task 2 (Practical Report 1)
PART A
1. Discuss THREE different factors that would have contributed to the emergence
of bacterial resistance to ciprofloxacin. Support your answer with suitable
examples.
There are various factors that might have contributed to the emergence of the resistance of
bacteria to ciprofloxacin. Among them include over prescription by the physicians of drugs and
antibiotics especially ciprofloxacin, patients opting for self-medication methods of treating
themselves and failure of hospitals to maintain proper and best health conditions, such as
cleanliness of the hospital, for treatment of ciprofloxacin (Snyder, 2014).
Many a time, physicians have prescribed a high amount of ciprofloxacin dosage to unsuspecting
patients. This behavior arises when the hospital and medical officers fail to do patient follow-up,
doctors failing to do proper diagnosis to the patient before administering treatment and lack of
proper information and skills by doctors handling the case concerning optimal therapies. In
addition, patient demand in the hospital overwhelms the medical staff hence chances of
misdiagnosis and over prescription becomes a possibility. This is also very evident in hospitals in
many developing countries such as Kenya where ciprofloxacin drugs are readily available in
chemists and various normal shops and kiosks and are sold without the proper prescription by the
physician or medical officer (Garrard, 2012).
Some behaviors which result to the resistance of the bacteria to ciprofloxacin drugs are caused
by patients themselves. This normally happens after the physician has given the patient a proper
ciprofloxacin dosage but the patient fails to follow instructions and proper ways of self-
administering the drug (Nipo, 2018). This is caused by forgetfulness of the patient, prematurely
stopping to take ciprofloxacin medication and inability of the patient to afford a full-course of
ciprofloxacin dose. In addition, in many developing countries patients are exposed to the risks of
accessing poorly manufactured, expired or counterfeit ciprofloxacin drugs (Pugo, 2018).
Hospitals also have their fair share in causing microbial resistance to ciprofloxacin. Various
practices in many hospitals such as treating large numbers of patients in a very long time in the
hospital environment may lead to bacteria becoming resistant to ciprofloxacin. This is because
the bacteria might have developed mechanisms to counter effects of the drug. In addition, failure
of hospitals to maintain high level of clean environment might to transmission of bacteria from
one patient to another if it is airborne. This problem is largely evident in poor nations where
there is insufficient hospital facilities and hospital staff.

2. What measures do you think the government/regulatory agencies should take to
prevent further increase in bacterial resistance to ciprofloxacin. Using suitable
examples, discuss how such measures can be implemented in various countries
where bacterial resistance to ciprofloxacin is high?
There are various measures a government or any regulatory agency can take to mitigate the rise
of bacterial resistance to ciprofloxacin drug. This measures of infection control programs to
counter infections in hospitals should be undertaken. The programs are important in hospitals in
ensuring safe patient care and proper risk management facilities for acute care. This measures
include proper surveillance, by hospitals, of patients, preparedness to investigation and control of
an outbreak and practicing proper patient care such as sterilizing medical equipment, isolating
patients and handwashing (Spence, 2018).
Firstly, one of the ways of effective infection is use of antimicrobial drugs such as ciprofloxacin
to prevent an infection. Although this is a trade-off between prevention of infections and start of
even more bacteria becoming resistant to drugs. This method has majorly been in European
countries where the success rate has been reported to be between 70% and 80%.
Secondly, hospitals should practice efficacy of infection control. This measure entails include
intensified washing of hands in hospitals using antiseptic, cohorting, closing hospital units to
admission of new patients to curb uncontrollable large numbers of patients needing ciprofloxacin
treatment and monitored intensive use of ciprofloxacin (Leo, 2018). This is from common
knowledge that these bacteria become more resistant to ciprofloxacin drugs when poor hospitals
are practiced such poor hygienic conditions and overwhelming numbers of patients.
In the case of the United States between 1970s and 1980s, it was reported that intensified
infection control measured mixed results in various hospitals. In some institutions, this method
had some success in preventing rise of microbial resistance to ciprofloxacin while other hospitals
reported no success in using the control measures in mitigating the resistance of the bacteria to
ciprofloxacin. Since then, for instance, the ciprofloxacin drug has been modified to increase its
effectiveness especially when used in acute situations (Harris, 2018).

3. (a). Describe a method (other than the dissolution method; the one you used in
week 1) which can be used to extract ciprofloxacin from 500 mg ciprofloxacin
tablet.
(b). Discuss two advantages and two disadvantages of the method you describe
above to the method you used in week 1.
Other than dissolution we can use FTIR spectroscopy to extract and measure the amount of
ciprofloxacin in a 500mg ciprofloxacin tablet.
This method entails extracting the active ingredient by using methanol and then adding a
phosphate buffer of PH 6.0. in method we are able to undertake various processes such as
specificity, linearity, precision of the graph, detection limits, drug extraction and data
manipulation.
The method is based on the knowledge of interactions between IR radiation and molecules of the
CPX vibrating.
In this study the reagents are supposed to be of analytical reagent grade. Distilled water is
required and methanol. In addition, pharmaceutical ciprofloxacin tablet is required. The reactions
are supposed to be performed using glass standard volumetric flasks (Fogazzi, 2010).
After grinding the 500mg CPX into powder, dissolve it into a small amount of phosphate buffer
of PH 6 and the make the volume in the volumetric flask up to a certain volume with methanol
so that the concentration is 500mg/ml stock solution.
Thereafter, after round five minutes of stirring the solution centrifuge it with the aim of
separating excipients and then make determinations by testing the absorbance of the supernatant
solution. Finally, after dividing the supernatant solution into various volumetric flasks, using a
certain wavelength, the determination is done by validating selectivity, linearity, detection and
quantification limits and accuracy and precision using the graphs (P. Carmona, 2012).
After calculations and drawing graphs and tales it can be confirmed this method of spectroscopy
is suitable to be used in quantity analysis of the pharmaceutical drug ciprofloxacin. This is
because all validation measures can be ascertained to be correct thus indicating the parameters,
linearity, selectivity, precision, accuracy and detection and quantification limit are met.
The advantage of this method, FTIR spectroscopy, can be used in quantity analysis of other
substances in the pharmaceutical industry other than ciprofloxacin. Another advantage of this
infrared spectroscopy in quantifying the amount of ciprofloxacin in a tablet is that the method is
very fast in giving results and it is less expensive as compared to other methods of separating
ciprofloxacin from the CPX tablet (Grunenberg, 2011).
The disadvantage of using this method in separation of CPX from CPX tablet is that, one, it is
difficult to handle long and extensive procedures and maintenance of the sample cells to be used
and this method cannot be used for quantity analysis in atoms or monoatomic ions

4. A. Briefly discuss why did you use 10% of acidic acid to dissolve a tablet
containing 500 mg of ciprofloxacin.
B. Name two solvents (other than 10% acetic acid) that can be used to dissolve a
tablet containing 500 mg of ciprofloxacin.
C. Would you have used another type of solvent to dissolve a tablet containing
750 mg of ciprofloxacin? Yes, or no and why?
Ciprofloxacin is very insoluble in water. The CPX is also only slightly soluble in dehydrated
alcohol and dichloromethane. The CPX is very soluble in dilute acids. The reason why we
dissolve a tablet of ciprofloxacin using 10% of the acid is because the HCL salt contained in
ciprofloxacin tablet is readily dissolved in water containing an acidic PH of 5.0-6.7.
This salt is insoluble in neutral PH such as distilled water.
There are other solvents that can be used in dissolving the ciprofloxacin tablet or its powdered
form other than using acetic acid. They are dehydrated alcohol such as ethanol and
dichloromethane. Although these two solvents dissolve the CPX tablet slightly.
Despite the amount of ciprofloxacin drug increases to 750mg from 500mg, one does not need to
change the solvent used. Of all the three solvents discussed above, that is, acetic acid, ethanol or
dichloromethane, 10% acetic acid is the most suitable to be used in the increased quantity of
CPX tablet. This is because which more amounts it requires a solvent of better solubility to
dissolve al the 750 mg of ciprofloxacin.
In conclusion, the acetic acid has the best properties under which the ciprofloxacin will dissolve
readily and completely as compared to other solvents, ethanol and tetra chloromethane.
5. A. What was the amount of ciprofloxacin you found in a tablet that was used to
prepare 50 μg/ml of ciprofloxacin.
B. Provide a brief summary of the method you used to determine the amount of
ciprofloxacin present in a tablet containing 500 mg of ciprofloxacin.
When we did the experiment I found out that the amount of ciprofloxacin containing in 50
micrograms/ml is 52.52749/1000 = 0.05252749 mg.
We used the HPLC method to separate ciprofloxacin from ciprofloxacin tablet. This method
involves using distilled water. We weighed the ciprofloxacin in small amounts of 50 mg and
transferred it into the volumetric flasks containing methanol. Then we made the solutions into
100 ml stock solution. The solutions were later sonicated for around 30n minutes and the filtered
using a filtration membrane. Final concentrations were then diluted to 200 μg/ml. Finally, all the
solution in the volumetric flasks were diluted with mobile phase and we obtained final solutions
of concentrations 50 μg/ml.
We then validated this HPLC method by analyzing formulations, preliminary tests with the aim
of selecting optimum conditions such as PH, concentration of standardized solutions and other

parameters. This method is one of the simplest method of extracting ciprofloxacin from a tablet
containing the same because it is not time consuming in preparation of samples. And in addition
the method, high performance liquid chromatography, can be combined with other methods such
UV light to increase its efficiency and performance.
PART B
1. Describe how the Y-site assembly is set up to collect a sample containing
amoxicillin, parenteral nutrition (PN) and lipid that could be used to investigate
the chemical and physical incompatibility of amoxicillin, PN and lipid. In addition,
using a suitable clinical example discuss - why administration of total parenteral
nutrition (TPN – PN +lipid) and anidulafungin would be required to a patient
using a Y-site assembly.
There are various methods that can be used to set up Y-site assembly in order to collect samples
of amoxicillin, PN and lipid and they include dynamic light scattering, light obscuration,
turgidimetry, zeta potential and many others. Amoxicillin, parental nutrition and lipid can be
tested by using both a simulated and actual Y-site infusion methods.
In terms of the simulated Y-site compatibility, the tests were done using the history of the
previously known methods through visual inspection, evaluating the turgidity, determination of
the PH and many others (Kathrin Krohn, 2008) .
Evaluation was done in intervals of 60 minutes, 120 minutes and 240 minutes by mixing each
mixture and controlling the reactions. These mixtures are normally considered incompatible
physically if there is any evidence of a precipitate. In this case the absorbance value is known to
be over 0.01 A.
Actual Y-site infusion can be simulated to show infusion of antibiotics in the clinical setting. It
can be set using smart bumps and intravenous tubing (Skipper, 2012).
Doctors and medical officers administering parenteral nutrition should avoid co-infusion of
drugs. This is because these type of PN solutions have different composition and compatibilities
with other drugs. That is highly recommended that we use parenteral nutrition to administer
patients using a Y-site assembly. The drug should be administered through a different IV site and
if this site is not available the drug can be administered through a different line that a Y-
connection (Johnston, 2012).
In the cases of patients requiring continuous and uninterrupted PN administration can drugs
which do not lipid molecule be administered through same Y- connection with other drugs such
amino acids. In addition, using this formation of sharing same Y-connection, the patient should
be closely monitored for any side effect that might occur (Fan, 1997).

2. A. In week 2, you were provided with the samples containing amoxicillin, PN and
lipid. The concentrations of amoxicillin, PN and lipid were calculated based on
the doses of amoxicillin (infused over a period of 30 minutes) and TPN (infused
over a period of 24 hours) used in 0.5kg (Sample A) and 5kg (Sample B)
patients. The samples were then subjected to centrifugation. Report the results
obtained after centrifugation. If the results obtained from the two samples
(prepared based on 0.5 and 5kg body weights) were different from each other
then discuss the potential reasons for this difference.
The results obtained from experimenting using the two samples were different. This is because
different concentrations of amoxicillin were put in the two samples, sample A containing 0.5kg
while sample contained 5kg. in addition, amoxicillin is unstable at normal PH of 1.0-2.0 (Grant,
1992).
So since they used different quantities, the results would be different in regard to the two
samples.

2. B. In week 2, you prepared a lipid free sample (either sample C, D or E) containing
flucloxacillin or amoxicillin or cephazolin, and PN and water. The
concentration of each antibiotic was calculated based on the dose of
flucloxacillin, amoxicillin and cephazolin used in 0.5kg (Sample C), 0.5 kg
(Sample D) and 5kg (Sample E) patients, respectively.
Report and discuss the results obtained after high-performance liquid
chromatography and light microscopy. You are required to report and discuss
the results for the sample you prepared (either C or D or E). In addition, include
following in the discussion part of your answer:
a. Did the sample show the presence of particles through unaided vision
and/or through light microscopy? If so, whether the HPLC analysis should
have shown reduced concentration of antibiotic that was present in your
sample? Yes, or No and Why?
This method of hplc was used in our experiment, mixture containing amoxicillin, PN and water
were used to study using the high performance liquid chromatography. After setting the
experiment as it was discussed in part one of the report we waited for around 30 minutes to try
and observe the presence of particles.
After grinding the 500mg CPX into powder, dissolve it into a small amount of phosphate buffer
of PH 6 and the make the volume in the volumetric flask up to a certain volume with methanol
so that the concentration is 500mg/ml stock solution (Hsien-chang Meng, 1976).
Thereafter, after round five minutes of stirring the solution centrifuge it with the aim of
separating excipients and then make determinations by testing the absorbance of the supernatant
solution. Finally, after dividing the supernatant solution into various volumetric flasks, using a
certain wavelength, the determination is done by validating selectivity, linearity, detection and
quantification limits and accuracy and precision using the graphs (Silberman, 1989).
We observed presence of particles using the light microscope. This is because the particles were
minute and could not be seen with the naked eye. There were reduced concentrations of the
antibiotic (Robert Denio Baker, 1997).

b. If the prepared sample did not show the presence of particles through
unaided vision and/or light microscopy then the HPLC analysis should have
shown reduced concentration of the active antibiotic present in the sample.
True or False and Why?
In the case whereby the presence of particles could not be observed through either naked eye or
using a light microscope, we could have done an HPLC analysis to determine if the
concentration of the antibiotic in the samples was reduced. This is because HPLC is the most
accurate method of determining concentrations of antibiotics by using various parameters (W.J.
Lough, 1995).
c. If the prepared sample did not show the presence of particles through
unaided vision and/or light microscopy then the concentration of active
antibiotic should have been the same as what was expected (in other words,
the same as what was labelled on the sample that was prepared for HPLC
analysis). True or False and Why?
Yes, the concentration of the antibiotics would have been the same as the first HPLC analysis
that was done. This is because this type of the analysis study has no errors thus meaning that the
results of the final HPLC were similar to the HPLC study done preparation.
2. C. In week 2, a lipid free sample containing amoxicillin, PN and water was
prepared. The concentrations of amoxicillin and PN were calculated based on
the doses of amoxicillin (infused over a period of 24 hours) and TPN (infused
over a period of 24 hours) used in 0.5 kg (Sample E) patients. Microscopic and
visual analyses of the sample did not show the signs of precipitation or
particles.
Answer the following questions:
1. Briefly describe the methodology you used to prepare the lipid free sample
(irrespective of the type of antibiotic you used).
In this study the reagents are supposed to be of analytical reagent grade. Distilled water is
required and methanol. In addition, pharmaceutical ciprofloxacin tablet is required. The reactions
are supposed to be performed using glass standard volumetric flask.
After grinding the 500mg CPX into powder, dissolve it into a small amount of phosphate buffer
of PH 6 and the make the volume in the volumetric flask up to a certain volume with methanol
so that the concentration is 500mg/ml stock solution.
Thereafter, after round five minutes of stirring the solution centrifuge it with the aim of
separating excipients and then make determinations by testing the absorbance of the supernatant
solution. Finally, after dividing the supernatant solution into various volumetric flasks, using a

certain wavelength, the determination is done by validating selectivity, linearity, detection and
quantification limits and accuracy and precision using the graph.
After calculations and drawing graphs and tales it can be confirmed this method of spectroscopy
is suitable to be used in quantity analysis of the pharmaceutical drug ciprofloxacin. This is
because all validation measures can be ascertained to be correct thus indicating the parameters,
linearity, selectivity, precision, accuracy and detection and quantification limit are met.
2. In clinical practice, would it be appropriate to infuse amoxicillin over a period
24 hours (also referred to as continuous infusion)?
This method is mostly appropriate for critically ill patients. This is because the continuous
infusion of time-dependent antibiotics has presumed benefits towards efficacy. This procedure is
suspected to have clinical benefits in patients where intermittent infusion is unable to achieve
desired results.
3. Which one of the two types of amoxicillin infusion (1- continuous and 2 -
intermittent infusion; e.g. three times a day) has a better clinical efficacy?
Support your answer using a suitable clinical example
Thorough research done overtime have shown that no clear differences can be seen when using
either of the method in treatment of patients. Factors usually considered when comparing this
two types of amoxicillin infusion include rate of mortality, cure and therapy, and it was shown
that there are no differences.
Statistically no evidence is found, though, since continuous infusion is a newer method than the
traditional intermittent infusion, the former should be adopted.
3. Why is it important to evaluate the pH of an admixture (antibiotic-PN-lipid)
collected at y-site? Support your answer with at least two suitable examples.
It is very important to evaluate the PH of the admixture because the stability of the admixture is
determined the PH. The PH depends on the type lipid in the admixture. This is because PH of
fats reduces with time and in addition, the addition of acidic compounds such as simple
carbohydrates will affect further the stability of the admixture since it reduces the PH level.
In addition, high concentrations of calcium and phosphate and low concentrations of amino acids
and glucose leads to reduced PH of the admixture. High PH of the admixture compounded with
increased temperature of the surrounding environment leads to a slower rate of infusion and
increased precipitation risk.

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