an analysis of amoxicillin through gcms and later ftir

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


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


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%




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%




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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21 | P a g e

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



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



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%



(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



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

23 | P a g e



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%



(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.

25 | P a g e

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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an analysis of amoxicillin through gcms and later ftir

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