*Download the file to view the presentation with its effects. 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.

2.  Establish the importance of the experiment
◦ Handouts
◦ Surveymonkey.com
 62 participants

3. 63%
37%
Question 1: Do you purchases
pharmaceuticals in Mexico?
Yes No
14%
39%
18%
29%
Question 2: Why do you not purchase the
products in Mexico?
Do Not Trust Them
Unsafe to Travel There
Too Far Away
Other

4. Penacillin
31%
Amoxicillin
31%
Erythromycin
1%
Lisinopril
1%
Valium
3%
Vicodin
3%
Ambien
3%
Codeine
4%
Demerol
1%
Metformin
4%
Steriod
5%
Others
13%
Question 5: What product(s) do you purchase in Mexico?
*selected all that applied

5. Yes
62%
No
30%
They Are Better
8%
Question 11: Do you believe that they are
of the same quality?
Cheaper
69%
No Prescription
Is Needed
10%
I Live There
5% Other
16%
Question 9: What is the primary reason you
purchase Pharmaceuticals in Mexico?

11. C21H30O2
314.47 g/mol

12.  A method to chemically modify a compound
◦ Increase volatility
◦ Decrease volatility
 Focuses on O, S, N, and P functional groups
with an active Hydrogen available

13.  Silylation produces silyl derivatives to
increase volatility.
◦ Replaces active H with TMS.
◦ Solvent = Pyridine
 Acylation target carbohydrates and amino
acids
 Alkylation converts organic acids into esters
by transferring an alkyl group

14. C16H19N3O5S = 365.4 grams/mol

15.  MTBSTFA = C9H18F3NOSi
 (n-methyl-n-t-butyldimethylsilytrifluoroacetamide)
◦ 10,000 times more stable
◦ Suitable for GC/MS analysis
C6H15Si
115.27 g/mol
C9H18F3NOSi
241.33 g/mol
+

17.  0.0154 grams Amoxicillin
 100 µL 0.85 M Acetonitrile
 20 µL MTBSTFA
 Sat Overnight
 150 µL Hexane
 *Centrifuged 15 Minutes
 1 mL of Hexane (solvent)

19. GC Parameters:
 Inlet – 240 ºC
 Oven temp. start at 60 ºC,
 Ramp up to 300 ºC at 40 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 Scan – 50-500 amu

21. S1
 0.1002 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Pyridine (Solvent)
 20 µL of MTBSTFA
S2
 0.1012 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Methanol (Solvent)
 20 µL of MTBSTFA
S1 & S2
 *Centrifuged 20 min.
 Water bath 100 ºC 20 min.

22. GC Parameters:
 Inlet – 250 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 Scan – 50-500 amu

23. 60 ºC
 0.0983 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Pyridine (Solvent)
 20 µL of MTBSTFA
 *Centrifuged 20 minutes
 Water Bath 60 ºC 1 hour
40 ºC
 0.1012 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Methanol (Solvent)
 20 µL of MTBSTFA
 *Centrifuged 20 minutes
 Water Bath 40 ºC 1 hour

24. GC Parameters:
 Inlet – 250 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 Scan – 50-600 amu

28. Handedness
 Chiral Molecule is a
molecule that is the same
but arranged in a non-
superimposable way,
typically due to Carbon
 C8H12N2O3S
 MW = 216.0568
H

29. GC Parameters:
 Inlet – 250 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 Scan – 50-600 amu

30. GC Parameters:
 Inlet – 250 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 Scan – 50-600 amu

31. Standard 1
 0.1016 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Pyridine (Solvent)
 20 µL of MTBSTFA
Standard 2
 0.0751 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Pyridine (Solvent)
 20 µL of MTBSTFA
Standard 3
 0.0492 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Pyridine (Solvent)
 20 µL of MTBSTFA
Standard 4
 0.0246 g of Amoxicillin
 100 µL of Acetonitrile
 1 mL of Pyridine (Solvent)
 20 µL of MTBSTFA
Standards 1-4
 *Centrifuged 20 min.
 Water bath 100 ºC 20 min.
GC Parameters:
 Inlet – 300 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 SCAN - 50-600 amu

32. GC Parameters:
 Inlet – 300 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 SCAN – 50-600 amu

33. y = 48.303x + 3E+06
R² = 0.468
0
2000000
4000000
6000000
8000000
10000000
0 20000 40000 60000 80000 100000
Area
Concentration (PPM)
Graph 1: Concentration (PPM) Versus Area
y = 48.303x + 3E+06
R² = 0.5253
0
2000000
4000000
6000000
8000000
10000000
0 20000 40000 60000 80000 100000
Area
Concentration (PPM)
Graph 2: Concentration Averages (PPM) Versus
Area Averages
Name Concentration (PPM) Area Average Areas
Standard 4 21964 3124602
Standard 4 21964 3059293
Standard 4 21964 3087453
Standard 3 43929 7201948
Standard 3 43929 6591084
Standard 3 43929 6425105
Standard 2 67054 8825625
Standard 2 67054 7083085
Standard 2 67054 6681811
Standard 1 90714 6788287
Standard 1 90714 5668981
Standard 1 90714 7278867
3090449.333
6739379
7530173.667
6578711.667
Table 1: Concentration Versus Areas

34. GC Parameters:
 Inlet – 250 ºC
 Oven temp. start at 40 ºC,
 Ramp up to 340 ºC at 20 ºC/min
 Flow rate of He = 1mL/min.
 Splitless
MS Parameters:
 Ion mode - 70 ev
 Solvent Delay 3 min.
 SIM – 114, 142.5, 160
amu

35.  Syringe filtered standards into new vials
 Ran sequence in SIM Mode - 160 m/z
 Results were inconclusive
 Build up suspected

36.  Stock Solution 1026 PPM Amoxicillin
 Made 1000 PPB solution of Amoxicillin
Standard 1
 2 mL of 1000 PPB Solution
 0.5 mL of 5 N KOH
Standard 2
 1 mL of 1000 PPB Solution
 1 mL of DI Water
 0.5 mL of 5 N KOH
Standard 3
 0.5 mL of 1000 PPB Solution
 1.5 mL of DI Water
 0.5 mL of 5 N KOH
Standards 1-3
 Water bath 50 ºC 20 min.
 Cooled for 60 Minutes
 + 1 mL of Acetic Acid
 + 5 mL of (7:1 ratio) Hexane: Ethyl Acetate solution
 Centrifuged for 15 minutes
 Top organic layer extracted to new test tubes
 Evaporated to dryness (several days)
 + 20 μL of MTBSTFA
 + 100 μL of Acetonitrile
 Heated in heating block at 110 °C for 15 minutes
 + 0.5 methanol solvent
 Each standard tested several times
 No signs of Amoxicillin or 6-
Aminopenicillanic Acid

37.  Batch 4 standard dilutions
 100 μL of each respective standard into new
vial
 + 900 μL of Methanol solvent
 No signs of Amoxicillin or 6-Aminopenllinc
Acid

38. Test Samples 1 & 2
 Water bath 60 ºC 15 min.
 Cooled for 60 minutes
 + 1 mL of Acetic Acid
 + 5 mL of (7:1 ratio) Hexane: Ethyl Acetate solution
 Centrifuged for 15 minutes
 Top organic layer extracted to new test tubes
 Evaporated to dryness (several days)
 + 20 μL of MTBSTFA
 + 100 μL of Acetonitrile
 Heated in heating block at 110 °C for 15 minutes
 + 0.5 methanol solvent
 New Stock Solution of 10,000 PPM Amoxicillin
Test Sample 1
 2 mL of 1000 PPM Stock Solution
 0.5 mL of 5 N KOH
Test Sample 2
 2 mL of 10,000 PPM Stock Solution
 0.5 mL of 5 N KOH
 Each test sample tested several times
 No signs of Amoxicillin or 6-
Aminopenllinc Acid

39.  Temperature affects the 8.10 peak of 6-
Aminopenicillanic Acid
 Amoxicillin as a whole cannot be seen in
GC/MS

41. Standard 1
 0.9916 grams of KBR
 0.0017 grams of Amoxicillin
 0.1026 grams of Starch
Standard 2
 0.9970 grams of KBR
 0.0034 grams of Amoxicillin
 0.1003 grams of Starch
Standard 3
 1.006 grams of KBR
 0.0073 grams of Amoxicillin
 0.1000 grams of Starch
Standard 1-3
 Each standard tested 3 times
 Starch Wavelength Height Ratios = 2150
 Amoxicillin's Wavelength Height Ratios = 1775,
1687, 1686, 1685.5, 1519

42. 2150
1775
1687
1686
1685.5
1519

43. 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%
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 1.2554 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%
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 1.7942 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%
Table 1: Standard 1 Data
Table 2: Standard 2 Data
Table 3: Standard 3 Data
Run
Amoxicillin At Absorbance Height
Run
Amoxicillin At Absorbance Height
Run
Amoxicillin At Absorbance Height
1.0940/0.8225 = 1.3301
(0.0017g Amox/ (0.0017g Amox + 0.1026 g Starch)) = 1.630%
Y Axis X Axis

45. 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 5.0084
3 - 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 4: Average Ratios for Sample 1
Amoxicillin At Absorbance Height
Run
Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5
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%
Run
Percentage Based Off of Calibration Curve With Respective Ratio
Table 5: Sample 1 Determined Average Percentage Based of Multiple Calibration Curves With Respective Height Ratios
Weight
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 - - - - - - - - - -
2 - 78.7612% 66.0248% 91.9826% 101.5664% - 498.0068 417.4748 581.6061 642.2041
3 - 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
- 46.4762 26.6496 34.0802 6.5008
- 465.14 ± 46.48 398.63 ± 26.65 557.51 ± 34.08 637.61 ± 6.50
(+) - 672.6441 517.6123 709.6647 666.6314
(-) - 257.6421 279.6491 405.3509 608.5833
Standard Deviation of Average Content of Amoxicillin (mg)
Concentration of Amoxicillin (mg) and Standard Deviation
95% Confidence Interval of Amoxicillin (mg) ±Range
Run
Percent Based Off of Calibration Curve With Respective Ratio
0.6323
Concentration (mg) Based Off of Wegiht of Pill and %
Table 6: Sample 1 Determined Concentration (mg) of Amoxicillin Determined from the Weight Pill (g) & Percentage
((7.4157 – 1.7325)/7.2158)) = 78.7612%0.6323 g X 78.7612% = 498.0068
3.9650/0.5347 = 7.4157

46. 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 4.1915
2 2.6050 2.4635 2.4862 2.4975 1.9185 0.4585 5.6816 5.3730 5.4225 5.4471 4.1843
3 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 7: Average Ratios for Sample 2
Run
Amoxicillin At Absorbance Height
Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5
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 8: Sample 2 Determined Average Percentage Based of Multiple Calibration Curves With Respective Height Ratios
Run
Percentage Based Off of Calibration Curve With Respective Ratio
Weight
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 50.1877% 51.6219% 45.8671% 64.3538% 76.6796% 289.6834 297.9614 264.7447 371.4502 442.5947
2 51.0439% 50.4512% 45.0473% 63.3241% 76.4599% 294.6255 291.2041 260.0129 365.5068 441.3266
3 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
2.7159 4.4633 4.5278 7.1704 0.9448
292.81 ± 2.72 292.90 ± 4.46 260.15 ± 4.53 364.71 ± 7.17 442.37 ± 0.94
(+) 297.3837 300.4235 267.7832 376.7993 443.9579
(-) 288.2265 285.3744 252.5166 352.6226 440.7722
Concentration of Amoxicillin (mg) and Standard Deviation
Concentration (mg) Based Off of Wegiht of Pill and %
Table 9: Sample 2 Determined Concentration (mg) of Amoxicillin Determined from the Weight Pill (g) & Percentage
Run
Percent Based Off of Calibration Curve With Respective Ratio
0.5772
95% Confidence Interval of Amoxicillin (mg) ± Range
Standard Deviation of Average Content of Amoxicillin (mg)

47. 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 - - - 4.5298
2 5.6195 3.4923 3.5105 3.5195 3.2560 0.7055 7.9653 4.9501 4.9759 4.9887 4.6152
3 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
Table 10: Average Ratios for Sample 3
Run
Amoxicillin At Absorbance Height
Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 5
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 11: Sample 3 Determined Average Percentage Based of Multiple Calibration Curves With Respective Height Ratios
Run
Percentage Based Off of Calibration Curve With Respective Ratio
Weight
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 52.2765% - - - 86.9853% 312.7182 - - - 520.3463
2 77.2359% 44.5911% 39.6474% 55.3989% 89.5862% 462.0250 266.7441 237.1710 331.3963 535.9046
3 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
97.2221 126.5877 121.3587 256.7109 7.9494
351.41 ± 97.22 356.26 ± 126.59 322.98 ± 121.36 512.92 ± 256.71 529.07 ± 7.95
(+) 515.3164 921.4278 864.8111 1659.0482 542.4719
(-) 187.5100 -208.9175 -218.8421 80.1389 515.6687
Concentration of Amoxicillin (mg) and Standard Deviation
95% Confidence Interval of Amoxicillin (mg) ± Range
0.5982
Standard Deviation of Average Content of Amoxicillin (mg)
Percent Based Off of Calibration Curve With Respective Ratio Concentration (mg) Based Off of Wegiht of Pill and %
Table 12: Sample 3 Determined Concentration (mg) of Amoxicillin Determined from the Weight Pill (g) & Percentage
Run

48. 465.14mg ± 46.48mg
292.81mg ± 2.72mg
Or
442.37mg ± 0.94mg
529.07mg ± 7.95mg

49.  Somewhat possible view Amoxicillin through
GC/MS
 Further testing of Amoxicillin Sample 2
through