1.01.52.02.53.03.54.04.55.05.56.06.57.0 ppm

0.964

1.336

1.354

1.373

1.391

1.411

1.588

1.607

1.627

1.645

1.664

1.966

1.983

1.996

2.015

2.196

2.214

2.232

3.310

3.312

3.313

3.833

4.259

4.827

5.355

5.368

6.725

6.863

5.97

1.98

2.00

2.13

3.08

2.99

1.99

2.01

2.03

1.00

TheExtrac+onofCapsaicinandParallelQuan+ta+veAnalysisNicolleS.Jackson,AndrewR.Schroeder,

Dr.ChristopherNicholson,Dr.CarrieA.Simpson,Dr.KarenS.MolekDepartmentofChemistry,UniversityofWestFlorida,11000UniversityParkway,Pensacola,FL32514

Abstract

Acknowledgments

Conclusions

1HNMRQuan;ta;veAnalysis 1HNMRQualita;veCharacteriza;on

Introduc;on

LC-MSQuan;ta;veAnalysis

Several species of pepperwith varying intensity of heat on the Scoville Scalewereovendriedtoremoveexcesswater,andthecapsaicinwasextractedusinga vola+le organic solvent. ERETIC was used to analyze the 1H NMR spectraintensitytodeterminetheconcentra+onofcapsaicininthesamples.

Tradi+onal uses for Nuclear Magne+c Resonance (NMR), par+cularly inundergraduateseNngs, isspectralandconforma+onalanalysisofcompounds.The quan+ta+ve NMR (qNMR) was employed through the use of ERETIC, aprogram built into the Bruker NMR soRware, to determine the capsaicincontent of several peppers of the capsicum family. This soRware is used inconjunc+on with data collected from the Liquid Chromatography-MassSpectrometry (LC-MS) analysis of the samples to accurately determineconcentra+on.

CapsaicinExtrac;on

The1HNMRspectraof theextractedsampleswereobtained,andacalibra+oncurvewasgeneratedtodeterminetheconcentra+onofcapsaicininthesesamples.

The capsaicin standards were analyzed via the LC-MS using the photodiode array (PDA)detector.

y=0.9936x+0.1533R²=0.99915

0.0000

5.0000

10.0000

15.0000

20.0000

25.0000

30.0000

35.0000

40.0000

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0qNMRMeasuredCa

psaicinCo

ncen

tra;

on(m

M)

CalculatedCapsaicinConcentra;on(mM)

CapsaicinStandardqNMRCalibra;onCurve

qNMRMeasuredCapsaicinSampleConcentra;on(mM)Samples Run1 Run2 Run3 Average SD

Jalapeño1/15 7.2014 6.6108 6.6147 6.8090 0.3399Jalapeño2/20 19.5666 18.8677 18.8169 19.0837 0.4189Serrano1/24 8.7746 8.8701 8.7793 8.8080 0.0538Serrano2/21 28.8685 31.2117 28.8362 29.6388 1.3623Serrano2/24 6.834 6.8461 9.6195 7.7665 1.6047Habanero9/18 25.788 24.1948 24.7285 24.9038 0.8109

Habanero(1)1/16 27.7081 30.0199 30.5114 29.4131 1.4969Habanero(2)1/16 21.7927 24.0978 23.6707 23.1871 1.2263Habanero(3)1/16 15.1118 14.4291 14.5849 14.7086 0.3578Habanero2/20 20.1084 20.1244 20.2132 20.1487 0.0565Habanero2/21 21.0403 21.5236 21.6252 21.3964 0.3125Habanero2/24 23.5493 23.4652 23.502 23.5055 0.0422

Calibra;onCurveqNMRMeasuredCapsaicinConcentra;on(mM)Standard Run1 Run2 Run3 Run4 Run5 Run6 Average SD

1 33.9733 34.0307 34.0124 33.9797 33.9765 34.0184 33.9985 0.02492 15.0062 15.0423 15.0405 15.0171 15.027 15.0556 15.0315 0.01813 11.5520 11.5397 11.5733 11.4394 11.5091 11.5044 11.5197 0.04714 8.7888 8.7634 8.8360 8.7082 8.6951 8.7305 8.7537 0.05325 5.5581 5.5775 5.5237 5.5332 5.5357 5.5147 5.5405 0.0233

Extrac+onMethodsTried:•  wetextrac+on–excessiveamountofwaterinsamples•  freezedrying–instrumentalissues•  ovendrying–easeofprepara+on

Extrac+onSolventsTried:•  methanol–extractedbothchlorophyllandcapsaicin•  methylenechloride(DCM)–extractedonlycapsaicin

FinalExtrac+onMethodology:•  peppersamplescutthinandovendriedat120°Cforseveralhours•  ~100mLDCMwasusedtoextractthesamplesfor8hours

PercentCapsaicinbyMass

Sample CapsaicinbyMass(%)

1 Jalapeño1/15 0.0618712 Jalapeño2/20 0.132103 Serrano1/24 0.0623324 Serrano2/21 0.155065 Serrano2/24 0.0321226 Habanero9/18 0.0618537 Habanero(1)1/16 0.0393908 Habanero(2)1/16 0.0394259 Habanero(3)1/16 0.02840710 Habanero2/20 0.07097411 Habanero2/21 0.03925012 Habanero2/24 0.052205

Jalapeño Habanero Serrano

y=596.34x-8532.5R²=0.99561

0

50000

100000

150000

200000

250000

300000

350000

400000

0 100 200 300 400 500 600 700

PeakArea(a.u.)

CapsaicinConcentra;on(ppm)

CapsaicinStandardLC-MSCalibra;onCurve

PercentCapsaicinbyMass

Sample CapsaicinbyMass(%)

1 Jalapeño1/15 0.0405532 Jalapeño2/20 0.1335263 Serrano1/24 0.0528824 Serrano2/21 0.0700965 Serrano2/24 0.1115116 Habanero9/18 0.2364877 Habanero(1)1/16 0.3654118 Habanero(2)1/16 0.3356359 Habanero(3)1/16 0.23899310 Habanero2/20 0.20894511 Habanero2/21 0.19076612 Habanero2/24 0.133855

qNMRMeasuredPercentbyMassData

LC-MSMeasuredPercentbyMassData

1HNMRofChlorophyll*overlaidon1HNMRofCapsaicin

1.01.52.02.53.03.54.04.55.05.56.06.57.0 ppm

0.964

1.336

1.354

1.373

1.391

1.411

1.588

1.607

1.627

1.645

1.664

1.966

1.983

1.996

2.015

2.196

2.214

2.232

3.310

3.312

3.313

3.833

4.259

4.827

5.355

5.368

6.725

6.863

5.97

1.98

2.00

2.13

3.08

2.99

1.99

2.01

2.03

1.00

12

LC-MSMeasuredCapsaicinSamplePeakAreaandConcentra;on

Sample Run1 Run2 Run3 Average SD Concentra;on(ppm)

PeakArea(a.u.)

Jalapeño1/15 52668 29044 36621 39444 12062 80 39444Jalapeño2/20 273894 270684 271582 272053 1656 471 272053Serrano1/24 122354 122936 132868 126053 5909 226 126053Serrano2/21 133566 174196 133030 146931 23614 106 54834Serrano2/24 163688 177481 176538 172569 7706 261 146931Habanero9/18 301320 353761 360032 338371 32240 582 338371

Habanero(1)1/16 315096 266757 288098 289984 24225 501 289984Habanero(2)1/16 218306 216903 216465 217225 962 379 217225Habanero(3)1/16 174206 171975 166916 171032 3735 301 171032Habanero2/20 215573 203961 202260 207265 7245 362 207265Habanero2/21 322610 313673 311400 315894 5926 544 315894Habanero2/24 220286 203489 209125 210967 8549 368 210967

Calibra;onCurveLC-MSCapsaicinPeakArea(a.u.)Standard Run1 Run2 Run3 Average SD

1 38158 33398 36494 36017 24162 79011 80992 78415 79473 13493 127813 123294 124433 125180 23504 167417 158571 159803 161930 47915 246297 253207 248018 249174 35976 363777 345153 340962 349964 12145

Capsaicin

WewouldliketothanktheUniversityofWestFloridaDepartmentofChemistryforcon+nuingsupport.

References

*Org.Magn.Resonance,1984,22,779-783

y=596.34x-8532.5R²=0.99561

0

50000

100000

150000

200000

250000

300000

350000

400000

0 100 200 300 400 500 600 700

PeakArea(a.u.)

CapsaicinConcentra;on(ppm)

LC-MSCapsaicinCalibra;onCurveandSamples

Calibra+onCurve

Jalapeño1/15

Jalapeño2/20

Serrano1/24

Serrano2/21

Serrano2/24

Habanero9/18

Habanero(1)1/16

Habanero(2)1/16

Habanero(3)1/16

Habanero2/20

Habanero2/21

Habanero2/24

Linear(Calibra+onCurve)

y=0.9936x+0.1533R²=0.99915

0.0000

5.0000

10.0000

15.0000

20.0000

25.0000

30.0000

35.0000

40.0000

0.0 5.0 10.0 15.0 20.0 25.0 30.0 35.0 40.0

qNMRMeasuredCa

psaicinCo

ncen

tra;

on(m

M)

CalculatedCapsaicinConcentra;on(mM)

qNMRCapsaicinCalibra;onCurveandSamples Calibra+onCurve

Jalapeño1/15

Jalapeño2/20

Serrano1/24

Serrano2/21

Serrano2/24

Habanero9/18

Habanero(1)1/16

Habanero(2)1/16

Habanero(3)1/16

Habanero2/20

Habanero2/21

Habanero2/24

Linear(Calibra+onCurve)

0.000000

0.020000

0.040000

0.060000

0.080000

0.100000

0.120000

0.140000

0.160000

0.180000

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Percen

tCap

saicinbyMass

SampleNumber

qNMRPercentCapsaicinbyMassJalapeño1/15

Jalapeño2/20

Serrano1/24

Serrano2/21

Serrano2/24

Habanero9/18

Habanero(1)1/16

Habanero(2)1/16

Habanero(3)1/16

Habanero2/20

Habanero2/21

Habanero2/24

0.000000

0.050000

0.100000

0.150000

0.200000

0.250000

0.300000

0.350000

0.400000

0 1 2 3 4 5 6 7 8 9 10 11 12 13

Percen

tCap

saicinbyMass(%)

SampleNumber

LC-MSPercentCapsaicinbyMass

Jalapeño1/15

Jalapeño2/20

Serrano1/24

Serrano2/21

Serrano2/24

Habanero9/18

Habanero(1)1/16

Habanero(2)1/16

Habanero(3)1/16

Habanero2/20

Habanero2/21

Habanero2/24