an analysis of amoxicillin through gc/ms and later ftir presentation

An Analysis of Amoxicillin Through GC/MS and Later FTIR

Establish the importance of the experiment◦ Handouts◦ Surveymonkey.com

62 participants

Survey Conducted

Primary Survey Questions

Question 1: Do you purchases pharmaceuticals in Mexico?

Yes No

Question 2: Why do you not purchase the products in Mexico?

Do Not Trust Them

Unsafe to Travel There

Too Far Away

Primary Survey Questions - Continued

Pe-nacillin

Amoxicillin31%

Erythromycin1%Lisinopril

Valium3%

Vicodin3%

Ambien3%

Codeine4%

Demerol1%

Metformin4%

Steriod5%

Others13%

Question 5: What product(s) do you purchase in Mexico?

*selected all that applied

Primary Survey Questions - Continued

Yes63%

They Are Better8%

Question 11: Do you believe that they are of the same quality?

Cheaper69%No Prescription Is

Needed10%

I Live There5%

Other15%

Question 9: What is the primary reason you purchase Pharmaceuticals in Mexico?

Amoxicillin Samples to TestCVS

Pharmacy

GlaxoSmithKline

AMSA Laboratorios

Purchased from Medicinesmexico.com

GC/MS - AutoSampler

SampleSolvent

GC/MS – Gas Chromatography

GC/MS – Mass Spectrum

Now What?

Tetrahydrocannabinol - THC

C21H30O2

314.47 g/mol

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

What is Chemical Derivatization?

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

Three Types of Derivatization

Amoxicillin Structure

C16H19N3O5S = 365.4 grams/mol

MTBSTFA = C9H18F3NOSi (n-methyl-n-t-butyldimethylsilytrifluoroacetamide)

◦ 10,000 times more stable◦ Suitable for GC/MS analysis

Derivatization Reagent - MTBSTFA

C6H15Si

115.27 g/mol

C9H18F3NOSi

241.33 g/mol

MTBSTFA Reaction with Amoxicillin

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)

Batch 1 Preparation – GC/MS

*Centrifuge Technique – GC/MS1. 2.

Batch 1 Run 1 Data – GC/MS

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

Batch 1 Run 1 Data – GC/MS

Batch 2 Preparation – GC/MSS1

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

Batch 2 Run 2, S1& Run 3, S2 Data – GC/MS

Batch 3 Preparation – GC/MS60 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

Batch 3 Run 4, 40ºC & Run 5, 60 ºC Data – GC/MS

40 ºC

60 ºC

Batch 3 Run 5, 60 ºC Data – GC/MS

Batch 2 & 3 Data – GC/MS40 ºC

60 ºC

S1, 100 ºC

Water Bath Temp. Better Peaks

Batch 2 S1 Data – GC/MS

Batch 2 & 3 Data – GC/MS( +) 6-Aminopenicillanic Acid 4-Thia-1azabicycloheptane-2-carboxylic acid, 6-amino-3,3-dimethyl-

7-oxo(-) 6-Aminopenicillanic Acid

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

Inconsistency Begins – GC/MS

What it should be

S1?, Run 6

Back to Working? – GC/MS

What it should be

S1, Run 19

Batch 4 – GC/MSStandard 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

Standards 1-4 *Centrifuged 20 min. Water bath 100 ºC 20 min.

MS Parameters: Ion mode - 70 ev Solvent Delay 3 min. SCAN - 50-600 amu

Batch 4 Sequence Run– GC/MS

MS Parameters: Ion mode - 70 ev Solvent Delay 3 min. SCAN – 50-600 amu

Standard 1Standard 2

Standard 3Standard 4

Batch 4 Sequence Run Calibration Curve – GC/MS

2000000

4000000

6000000

8000000

10000000

f(x) = 48.3028118594698 x + 3283814.61584145R² = 0.467955549500605

Graph 1: Concentration (PPM) Versus Area

Concentration (PPM)

2000000

4000000

6000000

8000000f(x) = 48.3028118594699 x + 3283814.61584144R² = 0.52529673457978

Graph 2: Concentration Averages (PPM) Versus Area Averages

Concentration (PPM)

Name Concentration (PPM) Area Average AreasStandard 4 21964 3124602Standard 4 21964 3059293Standard 4 21964 3087453Standard 3 43929 7201948Standard 3 43929 6591084Standard 3 43929 6425105Standard 2 67054 8825625Standard 2 67054 7083085Standard 2 67054 6681811Standard 1 90714 6788287Standard 1 90714 5668981Standard 1 90714 7278867

3090449.333

6739379

7530173.667

6578711.667

Table 1: Concentration Versus Areas

Batch 4, Standard 4 SIM Mode – GC/MS

S1, Run 30

MS Parameters: Ion mode - 70 ev Solvent Delay 3 min. SIM – 114, 142.5, 160

SIM Mode focuses on specific m/z

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

Batch 4 Sequence 2 – GC/MS

Batch 5 – GC/MS 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

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

Batch 6 – GC/MS

Batch 7 – GC/MS

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

Temperature affects the 8.10 peak of 6-Aminopenicillanic Acid

Amoxicillin as a whole cannot be seen in GC/MS

Conclusions from GC/MS Analysis

Amoxicillin - FTIRKBr + Amox

KBr + Starch

Standard Preparation - FTIRStandard 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

Amoxicillin Standard Run Example - FTIR

16871686

1685.5

Standard Data - FTIRStarch 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 Percentage1 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 Amox1775 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 Amox1775 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

RunAmoxicillin 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

Calibration Curves - FTIR

Sample 1 Ratio Interpretation - FTIR

Starch Abs. Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 51775 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 4: Average Ratios for Sample 1Amoxicillin At Absorbance Height

Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 51775:2150 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%

RunPercentage 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 642.20413 - 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

- 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 Amoxicil lin (mg) and Standard Deviation

95% Confidence Interval of Amoxicillin (mg) ± Range

RunPercent 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 Amoxicil lin 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

Starch Abs. Ratio 1 Ratio 2 Ratio 3 Ratio 4 Ratio 51775 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 7: Average Ratios for Sample 2

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%

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 442.59472 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 442.36512.7159 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 Amoxicill in (mg) and Standard Deviation

Concentration (mg) Based Off of Wegiht of Pill and %Table 9: Sample 2 Determined Concentration (mg) of Amoxicill in Determined from the Weight Pill (g) & Percentage

RunPercent Based Off of Calibration Curve With Respect ive Ratio

0.5772

Starch Abs. Ratio 1 Ratio 2 Ratio 3 Rat io 4 Rat io 51775 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

Table 10: Average Ratios for Sample 3

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%

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 - - - 520.34632 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 529.070397.2221 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 Amoxicill in (mg) and Standard Deviation

0.5982

Percent Based Off of Calibration Curve With Respect ive Ratio Concentration (mg) Based Off of Wegiht of Pill and %Table 12: Sample 3 Determined Concentration (mg) of Amoxicill in Determined from the Weight Pi ll (g) & Percentage

Conclusions from FTIR Analysis

GlaxoSmithKline

AMSA Laboratorios

CVS Pharmacy

465.14mg ± 46.48mg292.81mg ± 2.72mg

Or442.37mg ± 0.94mg

529.07mg ± 7.95mg

Somewhat possible view Amoxicillin through GC/MS

Further testing of Amoxicillin Sample 2 through

Final Remarks

AMSA Laboratorios

THE END

an analysis of amoxicillin through gc/ms and later ftir presentation

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