An Analysis of Amoxicillin Through GC/MS and Later FTIR
Comprehensive Studies Experiment
Kirk Teegardin
CTEC 2333
January 1 - April 29, 2016
Instructor: Hernandez
Table of Contents
Table of Contents……….………………………………………………………………….……2-3
Summary………………………………………………………………………………………...4-5
Introduction…………………………………………………………………………………..…5-9
Objective………………………………………………………………………………………......9
Equipment & Reagents………………………………………………………………………...9-10
Procedure………………………………………………………………………………….….10-18
Survey Data…………………………………………………………………………………..18-20
Data…………………………………………………………………………………………...21-25
Table 1: Standard 1 Data……………………………………….………………………...21
Table 2: Standard 2 Data…………………………………………………………………21
Table 3: Standard 3 Data…………………………………………………………………21
Graph 1: Amoxicillin % Versus Absorbance Height Ratio 1775:2150…….....................21
Graph 2: Amoxicillin % Versus Absorbance Height Ratio 1687:2150……………..…...22
Graph 3: Amoxicillin % Versus Absorbance Height Ratio 1686:2150……………….....22
Graph 4: Amoxicillin % Versus Absorbance Height Ratio 1685.5:2150….…………….22
Graph 5: Amoxicillin % Versus Absorbance Height Ratio 1519:2150……………..…...23
Table 4: Average Ratios for Sample 1…………………………………………………...23
Table 5: Sample 1 Determined Average Percentage Based of Multiple Calibration Curves
With Respective Height Ratios……….......……………………………………………...23
Table 6: Sample 1 Determined Concentration (mg) of Amoxicillin Determined from the
Weight Pill (g) & Percentage…………….......…………………………………………..23
Table 7: Average Ratios for Sample 2…………………………………………………...24
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Table 8: Sample 2 Determined Average Percentage Based of Multiple Calibration Curves
With Respective Height Ratios…...……………………………………………………...24
Table 9: Sample 2 Determined Concentration (mg) of Amoxicillin Determined from the
Weight Pill (g) & Percentage………...…………………………………………………..24
Table 10: Average Ratios for Sample 3……..…………………………………………...24
Table 11: Sample 3 Determined Average Percentage Based of Multiple Calibration
Curves With Respective Height Ratios………...………………………………………...24
Table 12: Sample 3 Determined Concentration (mg) of Amoxicillin Determined from the
Weight Pill (g) & Percentage…………...………………………………………………..25
Calculations…………………………………………………………………………………..25-27
Discussion…………………………………………………………………………………….27-34
References………………………………………………………………………………………..35
Appendix……………………………………………………………………………….…...36-109
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Summary:
The analysis various brands of Amoxicillin began with an investigation on how to
examine Amoxicillin in a Gas Chromatography/Mass Spectrum, GC/MS. A survey was
conducted locally to investigate the importance of examining any difference between
Amoxicillin samples from the Unites States and Mexico. There was not a set method on how to
achieve a comparison of the two; because of this, a bulk of this experiment involved attempting
different approaches to analyze Amoxicillin using GC/MS. Seven batches of Amoxicillin
samples in total were prepared for GC/MS analysis, with batch two showing the most promising
signs of Amoxicillin. The majority of Amoxicillin was viewable from this batch with seeing 6-
Aminopencillanic Acid, 35.2%. This compound was viewed several times and eventually a
sequence run was performed with four calibration standards. The sequence run did produce a
well-defined calibration line and it was eventually discovered that an obstruction had been built
up inside of the inlet of the GC. After the inlet was repaired, a few more batches were prepared
and tested but ultimately failed as well. Due to the continuous problems that were occurring, the
experiment then shifted to analyze Amoxicillin using Fourier Transform Infrared Spectroscopy,
FTIR. Various wavelength height ratios were analyzed and a total of 5 calibration lines were
created, all with a greater R2 than 0.940, Graph 1-5. The two calibration curves subsequently
used were created through the wavelength height ratios 1687:2150 and 1686:2150. The analysis
of sample 1, a purchased brand of Amoxicillin from CVS in the United States, was determined to
contain 465.14mg ± 46.48mg with the 1687:2150 ratio, which is within range of its labeled 500
milligrams, Table 6. Sample 2, a Mexico Amoxicillin sample from the company AMSA
Laboratorios operated in Mexico, was determined to have 292.90mg ± 4.46mg from the
1687:2150 ratio, well below the stated 500mg, Table 9. Sample 3, a second sample of
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Amoxicillin purchased from a Mexico pharmacy was from the company GlaxoSmithKline
(based out of the United Kingdom), had a large standard deviation for many of the created ratios
and so a different wavelength height ratio calibration curve was used to state the proper content
of the sample: 1519:2150 ratio with 529.07mg ± 7.95mg, Table 12. Sample 3 falls within range
of its stated 500mg, leaving Sample 2 the only sample to not contain the labeled amount of
Amoxicillin.
Introduction:
Gas Chromatography paired up with a Mass Spectrum detector can be one of the best
analytical tools to identify chemical compounds. GC/MS is commonly used for forensic drug
testing, engine exhaust analysis, petroleum product analysis, blood monitoring surgery, and
environmental contaminant identification. A GC instrument vaporizes the sample once injected
manually or through an autosampler, and separates the various compounds. Peaks are then
produced which are given a specific retention time to note for the elution from the point of first
being injected. The peak area is proportional to the concentration of that specific compound.
Separation of the compounds at various times is possible due to the GC column and the different
sizes of the injected compounds. The compounds are pushed through a set flow of carrier gas,
typically helium, to push the substance along the instruments column. The column itself is
usually packed with a material that assists in the partitioning of the compounds. Substances may
sometimes remain in the column during elution which can produce unexpected peaks in the
sequential GC run. Ideally in an experiment, the sample should fully elute from the GC column.
Using a Gas Chromatography system allows for many detectors to be used for various
experiments such as: an argon ionization detector, flame ionization detector, flame emission
detector, flame photometric detector, cross section detector, thermal conductivity detector,
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electron capture detector, and mass spectrum detector. When analyzing a compound, a response
factor is typically calculated by dividing the area of the spectral peak by the weight or volume of
the substance injected into the GC. The temperature of the inlet, where the sample first enters,
needs to be set to a high enough temperature to vaporize the liquid sample instantly. Too low of
temperature can result in having poor and broad peaks or the peaks may not even appear. A
temperature too high can result in the sample decomposing or changing its compound structure,
resulting in inconsistent data. The Mass Spectrum detector, used in this experiment, identifies
substances by electrically charging the molecules of the sample and accelerating them through a
magnetic field. While inside of this magnetic field, the sample’s molecules are fragmented and
the charge of each is then detected. These fragment pieces are unique and make it possible to
identify the compounds structure by piecing together the fragmented masses. A typical Mass
Spectrum instrument include: a sample inlet, an ionization source, a molecule accelerator, and a
detector. Like in regular Gas Chromatography, the GC/MS sample is injected into an inlet of
high temperature where the sample is vaporized into a gas. The sample then enters an ionization
chamber where a beam of electrons with a high voltage explodes the sample. The sample’s
molecules are shattered and then pass through the accelerator as individual particles. As these
charged sample particles pass through to the detector, intensified with electrons, the instrument
then records the fragment masses allowing for structural data to be produced. Each compound
has a mass spectrum which can be identified by comparing the data produced from the analysis
to online databases. The greatest mass detected for the compound is usually referred to as the
parent mass. Successful identification of the parent peak helps to identify the parent mass of the
compound, revealing the molecular mass of the compound. The parent mass reveals the mass of
the molecule while the subsequent peaks indicate the molecule’s structure. When analyzing high
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molecules mass compounds such as drugs and bodily fluids a parent peak is often not analyzed.
Identifying the parent mass and the structure are the most important aspect in GC/MS analysis
while also being the most difficult. Due to the sensitivity of the Mass Spectrum, a background
spectrum should typically be run in order to verify the no previous traces remain in the
instrument when switching between different samples. The GC/MS instrument combination
provides specific results but also produces uncertain qualitative results. Many scientists consider
Gas Chromatography Mass Spectrum to be an analytical tool for conclusive proof of identity.
Like with all other instruments used, GC/MS has several limitations in its operation. A GC
instrument may not fully separate the sample’s compounds resulting in the Mass Spectrum data
produced being inconsistent. This normally results in background noise in the Mass Spectrum
detector. Another limitation of GC/MS is the experience of the operator. The operator must
interpret the mass spectrum data that is not exact in practice. They must also use their data and
compare it to online databases using software to identify their specific compound. A third
limitation of GC/MS use is producing a false positive. When using GC/MS data, it is best to
cross reference with another instrument to confirm that it is in fact the compound it is believed to
be (Douglas). An example of confirming a false positive can be seen with drug testing. Certain
instances have occurred when someone tests positive for a drug, typically done through GC/MS
or LCMS, despite not being on the drug. A drug test should be crossed checked with another
instrument before being declaring someone positive for a specific drug. While unlikely, a person
taking Amoxicillin may sometimes test positive for cocaine (Haddard).
Sometimes compounds are not volatile or stable and therefore cannot be used for GC/MS
analysis. In this scenario, a possible remedy is Gas Chromatography Derivatization, a method of
chemically modifying a compound to create a new compound that makes it possible to run in a
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GC. Derivatization can increase the volatility of a compound by eliminating the presence of
polar OH, NH, and SH groups. The derivatization reaction usually focuses on O, S, N, and P
functional groups that have hydrogens available. Large compounds, such as Amoxicillin, tend to
have low volatility due to the size of the molecule and large dispersion forces holding the
molecule together. Derivatization can also be used to decrease a compounds volatility if the
compound has a very low molecular weight. This type of derivatization would be useful to help
separate the sample peaks from the solvent peaks. In GC derivatization, there are three types of
derivatization: silylation, alkylation, and acylation. Silylation produces silyl derivatives which
are more volatile, less stable, and more thermally stable. This derivatization process involves
replacing active hydrogen’s with trimethylsilane, TMS. Solvents need to be as pure as possible
to eliminate unnecessary peaks in the GC. Pyridine is the most common solvent when using
silylation derivatization because it tends to help the reaction take place. If the sample readily
dissolves in the reagent then it is usually a sign of the derivatization having completed. Many
reagents require heating, not in excess of 60 °C for about 15 minutes, for the derivatization to
occur. There are several silylating reagents available that can each be used for specific analysis
such as: HMDS (Hexamethyldislzane), TMCS (trimethylchlorosilane), TMSI
(Trimethylsilylimidazole), BSA (Bistrimethylsilylacetamide), BSTFA
(Bistrimethylsilyltrifluroacetamide), MSTFA (N-methyl-trimethylsilyltrifluoroacetamide), TMS-
DEA (Trimethylsilyldiethylamine), MTBSTFA (N-methyl-n-t-
butyldimethylsilyltrifluoroacetamide), and Halo-methylsilyl derivatization reagents. It is worth
noting that MTBSTFA, used in this experiment, dervatizes by replacing the active hydrogen with
a t-BDMS (tert-Butyldimethylsilyl ether) group. The t-BDMS derivatives are more resistant to
hydrolysis and can be up to 10,000 times more stable than other TMS derivatives. MTBSTFA is
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also suitable for GC/MS analysis. Acylation derivatization target highly polar, multifunctional
compounds, such as carbohydrates and amino acids. The third type of GC derivatization is
alkylation which is typically used to convert organic acids into esters (Regis Technologies, Inc.).
Through the use of Gas Chromatography derivatization, it is possible to analyze samples that
were previously unsuitable to be tested using GC/MS.
Objective:
The objective of this experiment was to analyze different brands of Amoxicillin and
determine if they contain the stated amount of Amoxicillin as labeled. This was to be
accomplished through the use of Gas Chromatography/Mass Spectrum, but resulted in having to
be completed through Infrared Spectroscopy due to the complexity of Amoxicillin.
Equipment & Reagents Used:
Equipment:
Agilent 7820A Gas Chromatography System Agilent 5977E Mass Spectrum Detector Column: HP-5MS MS 30mX25μm, 0.25μm Digital Balance Nicolet IR 100 FT-IR Instrument Mortar and Pestle Refrigerator Centrifuge Tissue Paper Wipes Test Tubes Test Tube Stoppers Parafilm Autosampler Autosampler Vials Micro Pipettes (20-1000 μL) 2 Volumetric Flasks (50 mL) 4 Syringe Filter System 2 Beakers (1000 mL) 2 Ring and Stands 2 Hot Plates
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3 Thermometers (Celsius) Heating Block
Reagents:
Amoxicillin Standard From Sigma-Aldrich (C16H19N3O5S) Potassium Bromide (KBr) Starch (C6H10O5) MTBSTFA Derivatization Reagent (C9H18F3NOSi) Acetonitrile (C2H3N) Hexane (C6H14) Deionized Water (H2O) Pyridine (C5H5N) Methanol (CH3OH) Potassium Hydroxide (KOH) Acetic Acid (CH3COOH) Acetone (C3H6O) 3 Amoxicillin Brand Samples
Procedure Conducted:
1. Initially, research was conducted to determine an appropriate way to analyze Amoxicillin
through using Gas Chromatography with a Mass Spectrum detector. During this research
period an FTIR run was conducted on an old Standard of Amoxicillin.
2. The old Amoxicillin standard was prepared in various amounts to be analyzed through FTIR:
Run 1 had 0.0995g of Amoxicillin and 0.9981 of Potassium Bromide, Run 2 had 0.0995
grams of Amoxicillin and 3.9955 grams of KBr, Run 3 had 0.0103 grams of Amoxicillin and
1.0019 grams of KBr, Run 4 had 0.0015 grams of Amoxicillin and 1.0019 grams of KBr.
Each of these runs produced better results but it was ultimately determined that the standard
of Amoxicillin has past the expiration date. It was also concluded that if FTIR is to be used,
a very small amount of Amoxicillin would be used to view Amoxicillin through FTIR.
3. Further research was then conducted and GC Derivatization (see introduction section) was
discovered to be a possible way of analyzing Amoxicillin through GC/MS. The first batch 10 | P a g e
CH3COOH
KOH
C9H18F3NOSi
CH3OH
C6H14C2H3N
C5H5N
C3H6O
for GC/MS testing was prepared in the following manner: 0.0154 grams of Amoxicillin
standard (purchased from Sigma-Aldrich), mixed with 100 μL of 0.85 M Acetonitrile, 20 μL
of MTBSTFA, and then sat in a refrigerator overnight. The following day, 150 μL of
Hexane was added, centrifuged for 15 minutes, and finally an additional 1 mL of Hexane
solvent was added. *Whenever prepared samples were centrifuged, each of the small vials
would be wrapped in small tissue paper and placed inside of a test tube. The mixture then
had the organic top layer decanted into an autosampler vial. This was then tested in the
GC/MS with the following parameters: oven temperature start at 60 °C, hold for 3 minutes,
ramp up to 300 °C with a rate of 40 °C/minute, hold for an additional 6 minutes, splitless,
flow at 1 mL/min, carrier gas used was helium, inlet temp 240 °C. The Mass Spec
parameters used for this run were: ion mode – 70 ev, solvent delay for 3 minutes, scan run
from 50-500 atomic mass units (amu). Ultimately, this run conducted did not provide much
information other than MTBSTFA being present in the mixture.
4. Further research was then conducted before batch 2 was prepared. Batch 2 involved 2
different Amoxicillin solutions created, labeled as S1 and S2. S1 and S2 were prepared in
autosampler vials. S1 was prepared in the following steps: 0.1002 grams of Amoxicillin
standard, 100 μL of Acetonitrile, 1 mL of pyridine solvent, and 20 μL of MTBSTFA. S2 was
prepared with 0.1012 grams of Amoxicillin, 100 μL of Acetonitrile, 1 mL of Methanol
solvent, and 20 μL of MTBSTFA. S1 and S2 were then centrifuged for 20 minutes (see step
3 for centrifuge sample preperation) and then heated in a water bath at 100 °C for 20 minutes.
S1 went from a clear solution to a light yellow solution while S2 seemed to form a plastic
looking precipitate on the bottom of the vial. S2 had the solution poured into a new vial to
lose the plastic looking precipitate. The new parameters for the GC/MS were as follows:
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solvent delay for 3 minutes, scan mode with 50-600 amu, inlet temperature 250 °C, and GC
oven temperature starting at 40 °C up to 340 °C at a rate of 20 °C/ minute. These runs
initially did not seem meaningful but ended up being one of the most successful runs for
Amoxicillin in GC/MS, see discussion section.
5. S1 did not create that plastic looking precipitate on the bottom and pyridine was concluded to
be the primary solvent when conducting a GC/MS analyses with MTBSTFA; because of this
the following two samples were prepared very similarly to S1. Sample 1 from batch 3 was
prepared with 0.0983 grams of Amoxicillin standard, 100 μL of Acetonitrile, 1 mL of
Pyridine, and 20 μL MTBSTFA. Sample 2, from batch 3, was prepared with 0.0989 grams of
Amoxicillin standard, 100 μL of Acetonitrile, 1 mL of Pyridine, and 20 μL of MTBSTFA.
Both samples were then centrifuged for 20 minutes. Sample 1 was then placed inside of its
own water bath at 60 °C while Sample 2 was placed inside of a 40 °C water bath for 60
minutes. Each of these was then tested in the GC/MS with the previous parameters.
6. Batch 3 gave similar results to S1 from batch 2 with the increased temperature seeming to
produce better results; due to this, S1 was then retested and the results seemed to be different
from the original S1. The following day S1 was tested again and gave the desired and
expected results for S1 batch 2.
7. Batch 4 was then prepared, four different concentrated standards, since the previous batches
implied information the method to prepare the standards for analyzing Amoxicillin using
GC/MS. Standard 1 was prepared with the following: 0.1016 grams of Amoxicillin, 100 μL
of Acetonitrile, 1 mL of Pyridine, and 20 μL MTBSTFA. Standard 2 was prepared with
0.0751 grams of Amoxicillin, 100 μL of Acetonitrile, 1 mL of Pyridine, and 20 μL
MTBSTFA. Standard 3 was prepared with 0.0492 grams of Amoxicillin, 100 μL of
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Acetonitrile, 1 mL of Pyridine, and 20 μL MTBSTFA. Standard 4 was prepared with 0.0246
grams of Amoxicillin, 100 μL of Acetonitrile, 1 mL of Pyridine, and 20 μL MTBSTFA.
Each of the standards from batch 4 was then centrifuged for 20 minutes. Subsequently, they
were all then heated in a 100 °C water bath for 20 minutes, taken out of the water bath, and
then slightly agitated to stir up the solutions in each of the vials. Immediately then, the four
standards were placed inside of the water bath again for an additional 10 minutes. The
previous parameters from step 4 were used once again for the GC/MS.
8. The data from batch 4 began to be deviate from the expected results. A “clean run” was
performed of pure methanol through the instrument and heated up the oven to 300 °C. After
doing the “clean run,” the predicted results were produced from running S1 from batch 2.
The scan mode under the Mass Spectrum parameters was then switched to SIM Mode to
focus on the 113-115 and 159-161 mass to charge ratios, m/z. Standard 1, batch 4, was then
run in the GC/MS and produced similar results to S1.
9. Standard 4, batch 4, was then tested at specifically 160 m/z in SIM Mode. With the well-
produced 160 m/z peak a sequence run of batch 4 was then schedule to run overnight:
Standard 4 (3 runs), Standard 3 (3 runs), Standard 2 (3 runs), Standard 1 (3 runs), quality
check standard 1 (1 run) , quality check standard 3 (1 run), and finally sample runs of
standard 1-4. The data from the sequence run produced inconsistent results.
10. Each of the standards in batch 4 was then filtered through syringe filters to further clean the
standards. They were placed into new autosampler vials.
11. Another sequence was then set to run, similar to the sequence run from step 9 now that the
standards were further purified in the hope of producing consistent results. Results were
once again inconsistent, even more so then previous.
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12. Standard 4, batch 4 was then tested in SIM mode at 160 m/z. It was then tested again at a
split ratio of 25:1, then a 10:1 ratio, a 5:1 ratio, and lastly a 2:1 ratio. Data seemed to be
inconsistent and was theorized to be due to some kind of blockage within the GC inlet.
13. Several days later, it was confirmed that the inconsistencies were due to the gold seal having
a buildup of the solutions being run through it. The gold seal was then replaced and the
previous batches were too heavily concentrated and therefore can no longer be used in this
experiment.
14. A new method was determined to create the Amoxicillin samples and so batch 5 sample
preparations was undertaken. Batch 5 was then prepared by creating a 1026 PPM stock
solution by diluting 0.0513 grams of Amoxicillin in a volumetric flask mixing 50 mL of
deionized water. Batch 5 was then heated slightly to help homogenize the Amoxicillin in the
water. 50 μL of this 1026 PPM solution was then added to a new volumetric flask with 50
mL of deionized water making the solution 1000 PPB of Amoxicillin. Each of the following
standards for batch 5 was prepared in test tubes and not the autosampler vials. Standard 1,
batch 5, was prepared with 2 mL of the 1000 PPB Amoxicillin stock solution mixed with 0.5
mL of 5 N Potassium Hydroxide, KOH. Standard 2, batch 5, was prepared with 1 mL of
1000 PPM Amoxicillin stock solution, 1 mL of deionized water, and 0.5 mL of 5 N KOH.
Standard 3, batch 5, was prepared with 0.5 mL of 1000 PPB Amoxicillin stock solution, 1.5
mL of deionized water, and 0.5 mL of 5 N KOH. Each of these standards was then heated
for 20 minutes in a 50 °C water bath. 1 mL of glacial Acetic Acid was then added to each of
the standards in batch 5. 5 mL of a mixed Hexane:Ethyl Acetate solution (7:1 ratio) was then
added to each of the standards. The standards were then centrifuged for 10 minutes. The top
organic layer was then extracted into new test tubes. These test tubes were then heated in a
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low temperature water bath in an attempt to evaporate to dryness. This process took several
days. 20 μL of MTBSTFA and 100 μL of Acetonitrile were then added to the test tubes. This
solution was then poured into autosampler vials and sealed. These vials were then placed
inside of empty test tubes and heated in a heating block at 110 °C for 15 minutes. An
additional 0.5 mL of methanol was then added to these vials to bring the volume up to be run
in the GC/MS instrument. Standard 1, batch 5, was then tested in the GC/MS under Scan
Mode of 50-600 amu. The results were inconclusive and was reanalyzed with the splitless
parameter being switched to pulsed splitless. Results were again inconclusive.
15. Batch 6 was then prepared using the original standards of batch 4 and diluting them to run in
the GC/MS. Each of these prepared standards were labeled DStandard to signify the
respective dilution of the standard. 100 μL of the batch 4 standards were each placed in a
new vial mixed with 900 μL of Methanol solvent to create DStandards of 1 mL solution.
DStandards, batch 6, were then centrifuged in their vials for 20 minutes. DStandard 4, with
the lowest concentration of Amoxicillin, was then tested in the GC/MS under Scan Mode 50-
600 amu to see if the previous information from S1, batch 2, could be detected. Data was
inconclusive. DStandard 1, the highest concentrated diluted standard, was then tested using
the same parameters and the information was again unfamiliar.
16. Batch 7 was then prepared. Batch 7 was prepared with two different samples. A new 10,000
PPM Amoxicillin stock solution was prepared by diluting 0.5004 grams of Amoxicillin in a
50 mL volumetric flask with deionized water. Sample 1 was prepared with the previously
created 1000 PPM Amoxicillin stock solution created for use in batch 5. Sample 1 had 2 mL
of the 1000 Amoxicillin PPM stock solution mixed with 0.5 mL of 5 N KOH. Sample 2 had
2 mL of the freshly prepared 10,000 Amoxicillin PPM stock solution mixed with 0.5 mL of 5
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N KOH. Each of these samples were prepared in test tubes. Both of these samples, batch 7
were then heated in a water bath of 60 °C for 15 minutes. They were then allowed to sit and
cool off for 60 minutes. 1 mL of glacial Acetic Acid was then added to each of the samples.
5 mL of a 7:1 ratio mixed solution of Hexane: Ethyl Acetate extraction solvent was then
added to each of the samples. The samples were then centrifuged for 15 minutes. The top
organic layer was then extracted an placed inside new test tubes. These test tubes were then
placed inside of individual water baths at a low temperature and heated to evaporate to
dryness over the course of a few days. Once they had evaporated to dryness, 100 μL of
Acetonitrile and 20 μL of MTBSTFA were added to each of the samples. These samples
were then poured into vials. The vials were sealed and placed inside of test tubes to be
heated on a 110 °C hot plate for 15 minutes. 0.5 mL of Methanol solvent was then added to
each of the samples to bring them up to volume for GC/MS analysis. Each of these samples
were then tested and no sign of Amoxicillin was detected. MTBSTFA seemed to be the
primary structure appearing in the Mass Spec. Due to time limitations, GC/MS was no
longer used to analyze Amoxicillin and so the backup plan of FTIR was then used for the
remainder of the experiment.
17. A mixture of KBr and Amoxicillin was first prepared by homogenizing 0.9949 grams of
KBr with 0.0016 grams of pure Amoxicillin standard, from Sigma Aldrich, in a mortar and
pestle. This was then tested in the FTIR. A mixture of Starch and KBr was then prepared by
homogenizing 1.0032 grams of KBr with 0.0987 grams of pure starch in a mortar and pestle.
This sample then tested in the FTIR. Specific wavelengths were then selected for the
Amoxicillin mixture that did not appear with the Starch mixture. Standard 1 was then
prepared by homogenizing 0.9916 grams of KBr, 0.0017 grams of Amoxicillin, and 0.1026
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grams of Starch in a mortar and pestle. Standard 2 was prepared similarly by homogenizing
0.9970 grams of KBr, 0.0034 grams of Amoxicillin, and 0.1003 grams of Starch. Standard 3
was prepared by homogenizing 1.0006 grams of KBr, 0.0073 grams of Amoxicillin, and
0.1000 grams of Starch.
18. Each of these FTIR standards were tested in the FTIR instrument using the disc plate
apparatuses and the compressor that applied 10,000 psi to form the disc of the standard.
Each standard was ran three times to determine an average for each of the selected
wavelengths. Height ratios were created through the wavelengths: 1775, 1687, 1686, 1685.5,
and 1519 all compared to 2150. A total of 5 calibration curve were then created of the ratios
created for each of the wavelengths.
19. Sample 1 was Amoxicillin Sample purchased in the United States from CVS Pharmacy. It
was cut and 0.1007 grams of the contents were then added with 0.9993 grams of Potassium
Bromide, KBr. The sample was then compressed in similar fashion, as done with the FTIR
standards. Sample 1 was then run 3 times in the IR with focusing on creating height ratios of
wavelengths: 1775, 1687, 1686, 1685.5, and 1519 all compared to 2150.
20. Sample 2 was the first Amoxicillin sample from Mexico. The brand for this Amoxicillin
sample was AMSA Laboratorios. The capsule was cut at the top and 0.1004 grams of the
contents were then added with 1.0023 grams of KBr. The sample was then compressed in
similar fashion, as done with the FTIR standards. Sample 2 was then ran 3 times in the IR
with focusing on creating height ratios of wavelengths: 1775, 1687, 1686, 1685.5, and 1519
all compared to 2150.
21. Sample 3 was the second brand of Amoxicillin purchased from a Mexico pharmacy
company. The brand for this Amoxicillin sample was GlaxoSmithKline, a pharmaceutical
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company based out of United Kingdom. The pill was cut at the top and 0.1022 grams of the
contents were then added with 1.0037 grams of KBr. The sample was then compressed in
similar fashion, as done with the FTIR standards. Sample 3 was then run 3 times in the IR
with focusing on creating height ratios of wavelengths: 1775, 1687, 1686, 1685.5, and 1519
all compared to 2150.
22. The weight of the powder was needed to compare all of the results. Each of the Amoxicillin
brand samples were weighed in the same fashion. Each sample was weighed initially. Then
they were cut and dumped of all of the drug content. The capsules were then rinsed
thoroughly with Acetone to remove the remnants of the drug from the inside of the pill
capsule. The empty capsules were then allowed time to have the Acetone evaporate before
being weighed again. The difference was then subtracted to determine the weight of the drug
inside of each of the samples.
Survey Data:
These are the questions and answers asked to people that took part in the survey for this
experiment:
Q1: Do You Purchases Pharmaceuticals in Mexico:Yes 39 62.90%No 23 37.10%Total 62 100.00%
If no, in Q1, the respondents were to answer only these, and the demographic section, but some
people that responded yes still responded to these questions.
Q2: Does Not Purchase in Mexico Because:Do Not Trust Them 4 14.29%Unsafe to Travel There 11 39.29%
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Too Far Away 5 17.86%Other 8 28.57%Total 28 100.00%
The respondents that answered yes, in Q1, were to answer these but some participants that answered
no, in Q1, also answer these questions from the survey.
Q4: Who Purchases The Products in Mexico:Yourself 11 27.50%Mother 12 30.00%Father 7 17.50%Grand Parent 2 5.00%Sibling 4 10.00%Other 4 10.00%Total 40 100.00%
Q6: Do You Have a Prescription for the Products in Mexico?:Yes 16 40.00%No 21 52.50%No Answer 3 7.50%Total 40 100.00%
The following questions were to be filled out by all participants but not everyone completed it since they
asked for demographic information.
Q12: Age:18-25 6 11.32%26-35 5 9.43%36-44 10 18.87%45-50 12 22.64%51-59 10 18.87%60+ 10 18.87%Total 53 100.00%
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Q3: Are You Aware of Cheaper Prices in Mexico:Yes 35 97.22%No 1 2.78%Total 36 100.00%
Q5: What Product Is Purchased in Mexico:*selected all that appliedPenicillin 23 30.67%Amoxicillin 23 30.67%Erythromycin 1 1.33%Lisinopril 1 1.33%Valium 2 2.67%Vicodin 2 2.67%Ambien 2 2.67%Codeine 3 4.00%Demerol 1 1.33%Metformin 3 4.00%Steroid 4 5.33%Others 10 13.33%Total 75 100.00%
Q9: What Is The Primary Reason For Purchasing Them In Mexico?
Cheaper 27 69.23%No Prescription Needed 4 10.26%I Live There 2 5.13%Other 6 15.38%Total 39 100.00%
Q11: Do You Believe That They Are of The Same Quality?:Yes 25 62.50%No 12 30.00%They Are Better 3 7.50%Total 40 100.00%
Q15: Ethnicity:White 12 22.64%Hispanic 37 69.81%African American 1 1.89%Native American 2 3.77%East Asian 1 1.89%Total 53 100.00%
Q14: Marital Status:Single 11 20.75%Married 32 60.38%Divorced 8 15.09%Widowed 1 1.89%Domestic Partnership 1 1.89%Total 53 100.00%
Income:$0-$10,000 5 9.43%$10,000-$20,000 11 20.75%$20,000-$30,000 7 13.21%$30,000-$40,000 3 5.66%$40,000-$80,000 12 22.64%$80,000+ 7 13.21%No Answer 8 15.09%Total 53 100.00%
Data:
Table 1: Standard 1 Data
RunAmoxicillin At Absorbance Height Starch Abs. Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5 Amox
1775 1687 1686 1685.5 1519 Height 2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 Percentage
1 1.0940 1.5688 1.5560 1.5855 1.4680 0.8225 1.3301 1.9074 1.8918 1.9277 1.7848 1.630%2 1.1990 1.5670 1.5550 1.5827 1.4540 0.8651 1.3860 1.8114 1.7975 1.8295 1.6807 1.630%3 1.1150 1.5706 1.5570 1.5881 1.4740 0.8749 1.2744 1.7952 1.7796 1.8152 1.6848 1.630%
Averages 1.1360 1.5688 1.5560 1.5854 1.4653 0.8542 1.3302 1.8380 1.8230 1.8574 1.7168 1.630%
Table 2: Standard 2 Data
RunAmoxicillin At Absorbance Height Starch Abs. Ratio 1 Ratio 2 Ratio 2 Ratio 4 Ratio 5 Amox
1775 1687 1686 1685.5 1519 Height 2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 Percentage
1 1.2554 1.5946 1.6020 1.6060 1.4403 0.7923 1.5845 2.0126 2.0220 2.0270 1.8179 3.2787%2 1.2709 1.5905 1.5599 1.6060 1.4439 0.8025 1.5837 1.9819 1.9438 2.0012 1.7993 3.2787%3 1.2735 1.5934 1.6031 1.6084 1.4416 0.8105 1.5713 1.9659 1.9779 1.9845 1.7787 3.2787%
Averages 1.2666 1.5928 1.5883 1.6068 1.4419 0.8018 1.5798 1.9868 1.9812 2.0042 1.7986 3.2787%
Table 3: Standard 3 Data
RunAmoxicillin At Absorbance Height Starch Abs. Ratio 1 Ratio 2 Ratio 2 Ratio 4 Ratio 5 Amox
1775 1687 1686 1685.5 1519 Height 2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 Percentage
1 1.7942 2.2060 2.2900 2.2400 1.8740 1.0290 1.7436 2.1438 2.2255 2.1769 1.8212 6.8034%2 1.7975 2.2030 2.2180 2.2260 1.8710 1.0395 1.7292 2.1193 2.1337 2.1414 1.7999 6.8034%3 1.7856 2.2005 2.2165 2.0140 1.8935 0.9207 1.9394 2.3900 2.4074 2.1875 2.0566 6.8034%
Averages 1.7924 2.2032 2.2415 2.1600 1.8795 0.9964 1.8041 2.2177 2.2555 2.1678 1.8926 6.8034%
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Table 4: Average Ratios for Sample 1
RunAmoxicillin At Absorbance Height Starch Abs. Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5
1775 1687 1686 1685.5 1519 Height 2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 - - - - - 0.5355 - - - - -
2 - 3.9652 3.8270 3.7990 2.6780 0.5347 - 7.4157 7.1573 7.1049 5.00843 - 3.5608 3.5601 3.5599 2.6500 0.5342 - 6.6657 6.6644 6.6640 4.9607
Averages - 3.7630 3.6936 3.6795 2.6640 0.5348 - 7.0407 6.9108 6.8845 4.9846
Table 5: Sample 1 Determined Average Percentage Based of Multiple Calibration Curves With Respective Height Ratios
RunRatio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5 Percentage Based Off of Calibration Curve With Respective Ratio
1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 - - - - - - - - - -
2 - 7.4157 7.1573 7.1049 5.0084 - 78.7612% 66.0248% 91.9826% 101.5664%3 - 6.6657 6.6644 6.6640 4.9607 - 68.3662% 60.0643% 84.3602% 100.1124%
Averages - 7.0407 6.9108 6.8845 4.9846 - 73.5637% 63.0445% 88.1714% 100.8394%
Table 6: Sample 1 Determined Concentration (mg) of Amoxicillin Determined from the Weight Pill (g) & Percentage
RunWeight Percent Based Off of Calibration Curve With Respective Ratio Concentration (mg) Based Off of Weight of Pill and %of Pill
(g) 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 0.6323 - - - - - - - - - -
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2 - 78.7612% 66.0248% 91.9826% 101.5664% - 498.0068 417.4748 581.6061 642.20413 - 68.3662% 60.0643% 84.3602% 100.1124% - 432.2795 379.7866 533.4094 633.0106
Averages - 73.5637% 63.0445% 88.1714% 100.8394% - 465.1431 398.6307 557.5078 637.6074
Standard Deviation of Average Content of Amoxicillin (mg) - 46.4762 26.6496 34.0802 6.5008
Concentration of Amoxicillin (mg) and Standard Deviation - 465.14 ± 46.48
398.63 ± 26.65
557.51 ± 34.08
637.61 ± 6.50
95% Confidence Interval of Amoxicillin (mg) ± Range(+) - 672.6441 517.6123 709.6647 666.6314
(-) - 257.6421 279.6491 405.3509 608.5833
Table 7: Average Ratios for Sample 2
RunAmoxicillin At Absorbance Height Starch Abs. Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5
1775 1687 1686 1685.5 1519 Height 2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 2.5618 2.4935 2.5085 2.5160 1.9151 0.4569 5.6069 5.4574 5.4903 5.5067 4.19152 2.6050 2.4635 2.4862 2.4975 1.9185 0.4585 5.6816 5.3730 5.4225 5.4471 4.18433 2.5980 2.4507 2.4546 2.4560 1.9208 0.4579 5.6737 5.3520 5.3606 5.3636 4.1948
Averages 2.5883 2.4692 2.4831 2.4898 1.9181 0.4578 5.6541 5.3941 5.4244 5.4391 4.1902
Table 8: Sample 2 Determined Average Percentage Based of Multiple Calibration Curves With Respective Height Ratios
RunRatio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5 Percentage Based Off of Calibration Curve With Respective Ratio
1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 5.6069 5.4574 5.4903 5.5067 4.1915 50.1877% 51.6219% 45.8671% 64.3538% 76.6796%2 5.6816 5.3730 5.4225 5.4471 4.1843 51.0439% 50.4512% 45.0473% 63.3241% 76.4599%3 5.6737 5.3520 5.3606 5.3636 4.1948 50.9540% 50.1613% 44.2987% 61.8808% 76.7800%
Averages 5.6541 5.3941 5.4244 5.4391 4.1902 50.7285% 50.7448% 45.0710% 63.1862% 76.6398%
Table 9: Sample 2 Determined Concentration (mg) of Amoxicillin Determined from the Weight Pill (g) & Percentage
RunWeight Percent Based Off of Calibration Curve With Respective Ratio Concentration (mg) Based Off of Weight of Pill and %of Pill
(g) 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1
0.5772
50.1877% 51.6219% 45.8671% 64.3538% 76.6796% 289.6834 297.9614 264.7447 371.4502 442.59472 51.0439% 50.4512% 45.0473% 63.3241% 76.4599% 294.6255 291.2041 260.0129 365.5068 441.32663 50.9540% 50.1613% 44.2987% 61.8808% 76.7800% 294.1063 289.5313 255.6922 357.1758 443.1740
Averages 50.7285% 50.7448% 45.0710% 63.1862% 76.6398% 292.8051 292.8989 260.1499 364.7109 442.3651
Standard Deviation of Average Content of Amoxicillin (mg) 2.7159 4.4633 4.5278 7.1704 0.9448
Concentration of Amoxicillin (mg) and Standard Deviation292.81 ±
2.72292.90 ±
4.46260.15 ±
4.53364.71 ±
7.17442.37 ±
0.94
95% Confidence Interval of Amoxicillin (mg) ± Range(+) 297.3837 300.4235 267.7832 376.7993 443.9579
(-) 288.2265 285.3744 252.5166 352.6226 440.7722
Table 10: Average Ratios for Sample 3
RunAmoxicillin At Absorbance Height Starch Abs. Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5
1775 1687 1686 1685.5 1519 Height 2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 4.0998 - - - 3.2080 0.7082 5.7890 - - - 4.52982 5.6195 3.4923 3.5105 3.5195 3.2560 0.7055 7.9653 4.9501 4.9759 4.9887 4.61523 3.7330 5.0030 5.1712 5.9810 3.2286 0.7037 5.3048 7.1096 7.3486 8.4994 4.5880
Averages 4.4841 4.2477 4.3409 4.7503 3.2309 0.7058 6.3530 6.0298 6.1622 6.7440 4.5777
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Table 11: Sample 3 Determined Average Percentage Based of Multiple Calibration Curves With Respective Height Ratios
RunRatio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5 Percentage Based Off of Calibration Curve With Respective Ratio
1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1 5.7890 - - - 4.5298 52.2765% - - - 86.9853%2 7.9653 4.9501 4.9759 4.9887 4.6152 77.2359% 44.5911% 39.6474% 55.3989% 89.5862%3 5.3048 7.1096 7.3486 8.4994 4.5880 46.7229% 74.5179% 68.3380% 116.0883% 88.7596%
Averages 6.3530 6.0298 6.1622 6.7440 4.5777 58.7451% 59.5545% 53.9927% 85.7436% 88.4437%
Table 12: Sample 3 Determined Concentration (mg) of Amoxicillin Determined from the Weight Pill (g) & Percentage
RunWeight Percent Based Off of Calibration Curve With Respective Ratio Concentration (mg) Based Off of Weight of Pill and %of Pill
(g) 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150 1775:2150 1687:2150 1686:2150 1685.5:2150 1519:2150
1
0.5982
52.2765% - - - 86.9853% 312.7182 - - - 520.34632 77.2359% 44.5911% 39.6474% 55.3989% 89.5862% 462.0250 266.7441 237.1710 331.3963 535.90463 46.7229% 74.5179% 68.3380% 116.0883% 88.7596% 279.4965 445.7662 408.7980 694.4403 530.9601
Averages 58.7451% 59.5545% 53.9927% 85.7436% 88.4437% 351.4132 356.2551 322.9845 512.9183 529.0703
Standard Deviation of Average Content of Amoxicillin (mg) 97.2221 126.5877 121.3587 256.7109 7.9494
Concentration of Amoxicillin (mg) and Standard Deviation351.41 ±
97.22356.26 ± 126.59
322.98 ± 121.36
512.92 ± 256.71
529.07 ± 7.95
95% Confidence Interval of Amoxicillin (mg) ± Range(+) 515.3164 921.4278 864.8111 1659.0482 542.4719(-) 187.5100 -208.9175 -218.8421 80.1389 515.6687
Calculations:
Serial Dilution
V1 = M2V2 / M1
V1 = the volume of the solution that must be taken out from M1 to create the new solution M2 = the desired concentration for the solution you are creating V2 = the volume of the new sample desired M1 = the original concentration of the sample taking volume out of
This formula is used to create a new solution with a lower amount of concentration. This
formula was used when preparing the Amoxicillin samples in batch 5 to create the 1000 PPB of
Amoxicillin.
Amoxicillin (%) Based off of Calibration Curves
A% = (y-b)/m
A% = Amoxicillin percentage y = Absorbance height ratio of (Wl:2150)
24 | P a g e
b = Normal b value from the Calibration Curve m = Slope for Particular Calibration Curve
This formula is used to determine the percentage of Amoxicillin (x-value) for the
particular Wavelength, Wl, ratio to Wavelength 2150. All of the Calibration Curves are created
by setting a particular wavelength height and then comparing it to the wavelength height of 2150.
It is important to select the appropriate Calibration Curve, created from the standards, that
coincides with the samples wavelength ratios to calculate the correct Amoxicillin percentage.
Pill Weight
Pi - Pe
Pi = Is the weight of the entire sample pill Pe = Is the weight of capsule itself after cleaned
This formula is used to calculate the weight of the powder inside of the sample. This is
necessary in order to determine the concentration of Amoxicillin in milligrams for each of the
samples. It is important to make sure that before weighing the pill capsules that they are
thoroughly cleaned and time is given to allow for the cleaning liquid to evaporate from the pill
capsule.
Concentration of Amoxicillin (mg) in Samples
A% X W
A% = Percentage of Amoxicillin calculated in a previous formula W = Determined weight of the powder inside of the sample
This formula is uses the determined % of Amoxicillin and converts the value into
milligrams in order to be compared to the label which states that each of the samples should
contain 500 mg. The previously calculated A% is multiplied by the weight of the sample pill. It
is important to match up the respective data with correct sample pill weight.
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95% Confidence Interval of Amoxicillin (mg) ± Range
X ± (tϭ/ )
X = The average concentration of Amoxicillin (mg) in a particular sample t = The t-test value according Student’s t-Test Table ϭ = Standard Deviation for particular concentration of Amoxicillin (mg) Wl:2150 ratio G = The square root of n (number of runs for particular sample)
This formula is used to determine the range that the sample ± standard deviation should
fall between. In one case, the high value of the range is determined by adding the average
concentration of Amoxicillin (mg) in a particular sample to the initially calculated t-test value
multiplied by the standard deviation for the concentration of Amoxicillin (mg) for the adjacent
W1:2150 ratio and divided by the square root of runs used for that particular sample. The low
value is nearly the same except the initially calculated t-test value. This is then multiplied by the
standard deviation, for the concentration of Amoxicillin (mg), for the adjacent W1:2150 ratio.
This value is then divided by the square root of the runs used for that particular sample is instead
subtracted from the average concentration of Amoxicillin (mg). This formula is calculated for
each of the samples to see if our determined data falls within these set ranges.
Discussion:
This analysis of Amoxicillin using the Gas Chromatography System with the Mass
Spectrum detector involved producing seven different batches of samples in an attempt to
identify Amoxicillin with each of the batches preparation as stated in the procedure section. The
numerous batches being prepared were due to there not being a readily available method on the
analysis Amoxicillin using GC/MS. There were plenty of methods on the analysis of
Amoxicillin using High Performance Liquid Chromatography Mass Spectrum, LC/MS, but that
26 | P a g e
instrument was not available for the conducting of this experiment. This led to the problem and
question of “can Amoxicillin be analyzed using GC/MS?”
Before conducting an experiment, it is important to establish the reason and purpose of
conducting an experiment. Initially, I conducted a survey which was distributed through
Facebook, SurveyMonkey.com, and through handouts which give reason as to the importance of
the experiment conducted. A total of 62 people participated in the survey with 39 people, 62.9%,
stating that they purchase pharmaceuticals in Mexico, Q1. The reasons the other 23 people don’t
purchase pharmaceuticals in Mexico is mostly due to the fear of violence, 39.29%, possibly
arising if they do visit Mexico, Q2. In the initial stages of creating this experiment, it was
determined that the most purchased product from Mexico would be experimented on to see if it
is compares of what the label states. Question 5, Q5, asked participants what product do they
purchase in Mexico; with a tie at 30.67% between Penicillin and Amoxicillin as being the top
purchased products from Mexico. For this reason, it was decided that Amoxicillin would be
analyzed for this experiment. The primary reason people would go to Mexico to purchase these
products, with 69.23% being that they are cheaper, Q9. Many of the purchasers of the products
also believed the products to be of the same quality at 62.50% and some believe that the products
are even better, 7.50%, Q11.
The Amoxicillin Standard and MTBSTFA derivatization reagent was purchased from
Sigma Aldrich. Sample 1 of Amoxicillin 500mg was purchased from CVS Pharmacy in the
United States of Amoxicillin. Sample 2 and 3 were different 500mg brands of Amoxicillin
purchased from a Mexico pharmacy company, medicinesmexico.com. Sample 2 was
Amoxicillin of the AMSA Laboratorios brand. Sample 3 was of the GlaxoSmithKline brand, a
United Kingdom pharmaceutical company. The first seven batches created for GC/MS analysis
27 | P a g e
were all prepared with the Amoxicillin Standard, Sigma-Aldrich, in trying to create an
appropriate method for detecting Amoxicillin. The first batch involved using the GC/MS
instrument for the first time. During the first runs, I was learning the many aspects of the
programs that are involved with a single run such as: the autosampler injector, Mass Hunter, and
the online database software. The first batch was prepared with Hexane as the solvent due to
research suggesting that Hexane would not interact with Amoxicillin. After fully preparing this
first batch (see procedure), the sample was tested in the GC/MS in Scan Mode at 50-500 amu
where only tert-Butyldimethysilanol was primarily being detected, despite only using 20 µL of
MTBSTFA. Batch two was then prepared differently with Pyridine as the solvent for S1 after
researching that Pyridine is typically the used solvent in silyation derivatization as it assist in the
reaction of MTBSTFA. S2, also part of batch 2, was prepared with methanol as the solvent in
hope of determining the best solvent to use for the remainder of the experiment. Batch two
showed significantly different data from batch one. S1 and S2, tested in Scan Mode at 50-600
amu, showed many similar peaks to each other. S1, the Pyridine prepared sample, had stronger
peaks produced. When selecting the peaks, with the use of the online databases, I was able to
view the selected peak and identify what the compound, based off of a certain probability from
the online databases. When I first looked at this data, I did not realize that the peak produced at
8.10 minutes, 6-Aminopencillanic Acid at a 35.2% probability, was of importance. This same
peak’s second highest probability at 25.5% was 4-Thia-1-azabicyclolheptane-2-carboxylic acid,
6-ami-3, 3-dimethyl-7-oxo. These two structures are the same, with the exact same molecular
weight of 216.056863. The only difference between the two is the placement a carbon that alters
the position of the nitrogen, hydrogen, and sulfur attached to the carbon. This change in
positioning is typically called handedness, with one being left handedness and the other right
28 | P a g e
handedness. What makes these two particular structures important in this experiment was they
make up a large portion of the Amoxicillin structure; only missing a benzene ring with an
additional OH bond attached to this benzene ring. As mentioned, this data didn’t seem useful
and so a third batch was then prepared. Batch three had two samples prepared identically except
in the heating stage of preparation of each of the two samples. Drastically lower temperatures
were used in heating the samples in the hope of making sure that the compound structure would
not change, since it was believed that the high temperature used in batch 2 may have skewed the
S1 and S2, batch 2, samples. The two prepared samples for batch 3, tested in Scan Mode 50-600
amu, showed nearly identical results, but with the lower temperature, 40 °C sample, showing
very small peaks. The 60 °C sample showed slightly higher peaks than the 40 ºC sample. While
analyzing batch 3, the discovery of the 4-Thia-1-azabicyclolheptane-2-carboxylic acid, 6-ami-3,
3-dimethyl-7-oxo structure with a probability of 42% at 8.10 minutes was made. The
temperature changes from batch 3 showed a direct correlation of increased temperature yielding
a more pronounced signal of the 8.10 peak; with S1, batch 2, having the best peak produced at
this time. S1, batch 2, was then tested in SIM mode with the selection of specific mass to charge
ratios, m/z, selected: 30, 44, 98, 114, and 160. After seeing inadequate data produced, S1 was
then tested in Scan Mode once again with a range of 50-600 amu. The expected outcome was to
see identical results to the previous run of S1 with the same parameters; this was not outcome. A
few days then passed, and S1 was again in the same manner as previous. This time, S1 showed
the expected results and again the signs of 4-Thia-1-azabicyclolheptane-2-carboxylic acid, 6-
ami-3, 3-dimethyl-7-oxo with a probability of 35.4%. S1 was then tested in SIM mode with a
focus on the 216 m/z. Again, S1 was tested with the selection of 44 m/z. S1 was then tested
with 44, 75, and 160 selected as the m/z. The next run include m/z’s of 74.9, 75, and 75.1. S1
29 | P a g e
was then tested with 159.9, 160, and 160.1 m/z’s. After several more runs of mass to charge
ratios, it was decided to up the inlet temperature and perform a “clean run” of the GC with pure
methanol. After reconfirming of S1, batch 2, showing 6-Aminopencillanic Acid; four standards,
batch 4, were then prepared identically to S1 with varying concentrations to create a calibration
curve. The highest created standard was then tested to verify the produced results were similar
to S1, with the 6-Aminopencillanic Acid. Due to them matching up, a sequence run was set to
run overnight testing each of the standards three times with two quality controls runs and created
data readily available to be exported into a quantitative program. After using this program, and
Microsoft Excel, it was concluded that the linearity for the calibration curve were not good, with
an R2 values of 0.468 and 0.5253, Appendix Table 1 and Graph 1-2. The standards were then
resequenced after being filtered through syringe filters. Again the data produced was inadequate.
This led me to believe that a buildup of some type could have resulted in producing the
inconsistent data that was occurring. Several days later, it was confirmed that a buildup had
occurred on the gold inlet and turning it black. This broken part was replaced shortly after. Due
to the destruction of the gold inlet, batch 5 was prepared with a newly researched method of
hydrolysis and extraction. Batch 5 showed zero signs of Amoxicillin, including no signs of 6-
Aminopencillanic Acid. Batch 6 was then prepared with the previous standards from batch 4,
but was highly diluted in solvent. These standards from batch 6 were tested using the GC/MS
instrument and showed no signs of Amoxicillin. The final sample batch was then prepared,
batch 7; again using a hydrolysis and extraction method but with a higher concentration of
Amoxicillin. These results proved to be useless. As time began to be an issue, the brand
samples of Amoxicillin had yet to be tested and a determined method for analyzing Amoxicillin
with GC/MS had seemed to be unachievable. Several things were learned and experienced with
30 | P a g e
the GC/MS instrument: the temperature affected the data acquired for locating 6-
Aminopencillanic Acid, and Amoxicillin as a full compound cannot be fully identified using
GC/MS. Further studies with analyzing Amoxicillin with GC/MS would include extensive
investigation in the method to produce consistent results from batch 2 without affecting the
instrument. Although the objectives of this experiment was not accomplished using GC/MS,
Fourier Transform Infrared Spectroscopy was used to analyze the differences in the Amoxicillin
brands and to see if they are properly labeled with the appropriate amount of Amoxicillin.
Using the FTIR instrument, KBr and Amoxicillin were mixed together and compared to
an Amoxicillin and Starch mixture. It was determined that starch displayed a peak at wavelength
2150 and Amoxicillin’s various peaks: 1775, 1687, 1686, 1685.5, and 1519 are to be compared
to the 2150 peak. The instrument was set to display the produced information in absorbance
mode as opposed to percent transmittance. Three standards with varying amounts of Amoxicillin
Standard were created and tested using the FTIR instrument. Each standard was tested three
times and the heights were recorded for each of the 2150, 1775, 1687, 1686, 1685.5, and 1519
wavelength peaks. The following height ratios were then created for each standard and run:
1775: 2150, 1687: 2150, 1687:2150, 1686:2150, 1685.5:2150, and 1519:2150, Table 1-3. The
average of each respective ratio was then used to create a calibration curve for each of the ratios,
Graph 1-5. The top two calibration curve’s created were Graph 2 with an R2 value of 0.9932 and
Graph 3 with an R2 value of 0.9972. Each of the samples were then prepared in similar fashion
as the standards. Sample 1 was the United States brand of Amoxicillin purchased from CVS
Pharmacy, Sample 2 was a Mexico Brand of Amoxicillin from AMSA Laboratorios, and Sample
3 was also a Mexico purchased sample of Amoxicillin but from GlaxoSmithKline. All three
samples are labeled to contain 500mg of Amoxicillin and should produce near identical results
31 | P a g e
using FTIR. The same ratios were performed for each of the samples. The weight of the powder
was also determined for each of the samples in order to acquire the milligrams of Amoxicillin
contained in each of the sample capsules. For Sample 1, the determined results of Amoxicillin
for each respective ratio are as follows: 1775:2150 = N/A, 1687:2150 = 465.14mg ± 46.48mg,
1686: 2150 = 398.63mg ± 26.65mg, 1685:2150 = 557.51mg ± 34.08mg, and 1519:2150 =
637.61mg ± 6.50mg, Table 6. It is important to recall that the calibration curve created from
Graph 2 and 3 provide the most accurate data; therefore, it is safe to claim that the Amoxicillin
Brand from America contains 465.14mg ± 46.48mg or 398.63mg ± 26.65mg of Amoxicillin.
The values for Sample 1 are within range of the stated label of 500mg. Sample 2’s determined
amount of Amoxicillin for each respective ratio are the following: 1775:2150 = 292.81mg ±
2.72mg, 1687:2150 = 292.90mg ± 4.46mg, 1686: 2150 = 260.15mg ± 4.53mg, 1685:2150 =
364.71mg ± 7.17mg, and 1519:2150 = 442.37mg ± 0.94mg, Table 9. The data determined for
Sample 2 displays that for all of the wavelengths the values are well under the labeled 500mg of
Amoxicillin except for results produced from the 1519:2150 ratio. Due to the low standard
deviation for this particular ratio, also using the worst R2 to determine this value, it is determined
that Sample 2 is still below the stated 500mg that AMSA Laboratories states. Sample 3, the
GlaxoSmithKline brand, determined content of Amoxicillin for each respective wavelength are
as stated: 1775:2150 = 351.41mg ± 97.22mg, 1687:2150 = 356.26mg ± 126.59mg, 1686: 2150 =
322.98mg ± 121.36mg, 1685:2150 = 512.92mg ± 256.71mg, and 1519:2150 = 529.07mg ±
7.95mg, Table 12. According to the data produced through experimentation, the values state this
brand of Amoxicillin also contains less then 500mg but it is worth noting that there is a wide
range of standard deviation for this particular sample ratio’s of: 1775: 2150, 1687:2150,
1686:2150, and 1685.5:2150. For sample 3, it is best to use the 1519:2150 ratio calibration
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curve, Graph 5, to determine the amount of Amoxicillin in Sample 3, 529.07mg ±7.95mg. The
large range of standard deviation could indicate the sample may in fact contain the stated amount
of 500mg of Amoxicillin. For each of the samples, a 95% confidence interval test was also
performed to see if the data determined falls within the range of the confidence interval test,
Tables 6, 9, and 12. For Sample 3, the range from this confidence interval test has a very large
range for several of the height ratios created; providing further evidence to suggesting the use of
the 1519:2150 ratio to state the true concentration, milligrams, of Amoxicillin in this Mexico
Brand sample, 529.07 ± 7.95 mg of Amoxicillin, Table 12.
Many people believe that pharmaceuticals from Mexico are of the same quality as of
those in the United States. While this may be true for some it is not for others. As seen with
Sample 2, using the FTIR instrument, the values of Amoxicillin are nearly 200mg below the
stated label, when looking at most of the wavelength height ratios. The United States sample and
Mexico purchased sample, GlaxoSmithKline pharmaceuticals, seem to have the stated values of
500mg of Amoxicillin. Despite the problems with GC/MS, the use of the FTIR instrument
provided the essential information to prove the importance of where one purchases their
pharmaceuticals. It is also worth noting that with all experiments there is always a chance of
mistakes occurring. The data produced displays the Amoxicillin sample from AMAS
Laboratorios is below 500mg, but when conducting this experiment there were many areas where
error may have occurred resulting in the declared conclusions.
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