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Directed Research CHEM 4502Fall 2010<br />Quantitation of Capsaicin Levels in Hot Peppers by Gas Chromatography/Mass Spect...
2<br />Background of Capsaicin<br /><ul><li> Derived from the genus Capsicum
 History of capsaicin
 Scoville Heat Chart</li></li></ul><li>Capsaicinoids<br />3<br /><ul><li> Alkyl vanillylamides</li></ul>Capsaicin (69%) MW...
4<br />It is a “hot” topic  <br /><ul><li> Food preparation
 Weight loss
 Alleviate pain & inflammation- inhibiting Substance P
 Protects heart health
 Cancer research
 Less-than-lethal weaponry</li></li></ul><li>Main Objectives<br />Development of a rapid, reproducible and simple method o...
6<br />Capsaicin Standard and GC/MS Analysis<br /><ul><li> Agilent 6890N Network GC System coupled with a 5975 inert XL Ma...
  Split injector with Zebron VF-5MS capillary column (15 m; i.d 0.25 mm; 0.25-μm film thickness)
  Initial temp was 125 °C at 5 °C/min with a Final Temp of 250 °C
  Run time: 27 minutes</li></li></ul><li>    Experimental Method<br />I. Derivatization<br />13.7 mg  of capsaicin standar...
TMAI
Ethylated derivatives- 9.4 mg</li></ul>7<br />Fig. 1. Capsaicin derivative, MW 333<br />Fig. 2. Dihydrocapsaicin derivativ...
TIC of derivatized product<br />8<br />
Spectrum of capsaicin derivative <br />9<br /><ul><li> Changed molecular mass from 305 to 333
 Ethylation of the common benzyl group caused the fragmentation of that group to increase from m/z 137 to 165</li></li></u...
 Method<br />II.	Analysis of capsaicin in hot pepper fruits<br />Prepare samples (Red Serrano and Caribbean Red)<br />Iden...
12<br />Red Serrano Pepper Results<br />
Caribbean Red Pepper Results<br />13<br />
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Quantitation of Capsaicin Levels in Hot Peppers by Gas Chromatography/Mass Spectrometry

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Quantitation of Capsaicin Levels in Hot Peppers by Gas Chromatography/Mass Spectrometry

  1. 1. Directed Research CHEM 4502Fall 2010<br />Quantitation of Capsaicin Levels in Hot Peppers by Gas Chromatography/Mass Spectrometry<br />Brandi VanAlphen<br />Dr. von Minden<br />December 14, 2010<br />
  2. 2. 2<br />Background of Capsaicin<br /><ul><li> Derived from the genus Capsicum
  3. 3. History of capsaicin
  4. 4. Scoville Heat Chart</li></li></ul><li>Capsaicinoids<br />3<br /><ul><li> Alkyl vanillylamides</li></ul>Capsaicin (69%) MW 305<br />Dihydrocapsaicin (22%) MW 307<br />
  5. 5. 4<br />It is a “hot” topic <br /><ul><li> Food preparation
  6. 6. Weight loss
  7. 7. Alleviate pain & inflammation- inhibiting Substance P
  8. 8. Protects heart health
  9. 9. Cancer research
  10. 10. Less-than-lethal weaponry</li></li></ul><li>Main Objectives<br />Development of a rapid, reproducible and simple method of quantitation using GC/MS, SIM and ChemStation.<br />Unique derivatization of capsaicin to be utilized as internal standards<br />5<br />
  11. 11. 6<br />Capsaicin Standard and GC/MS Analysis<br /><ul><li> Agilent 6890N Network GC System coupled with a 5975 inert XL Mass Selective Detector
  12. 12. Split injector with Zebron VF-5MS capillary column (15 m; i.d 0.25 mm; 0.25-μm film thickness)
  13. 13. Initial temp was 125 °C at 5 °C/min with a Final Temp of 250 °C
  14. 14. Run time: 27 minutes</li></li></ul><li> Experimental Method<br />I. Derivatization<br />13.7 mg of capsaicin standard<br />5 mL of N,N-DMA<br />1 mL of TMAH/25%<br />1 mL of Iodoethane<br />10 mL of 0.1 M Na2CO3<br />10mL of CCl4<br />Products<br /><ul><li>Water
  15. 15. TMAI
  16. 16. Ethylated derivatives- 9.4 mg</li></ul>7<br />Fig. 1. Capsaicin derivative, MW 333<br />Fig. 2. Dihydrocapsaicin derivative, MW 335<br />
  17. 17. TIC of derivatized product<br />8<br />
  18. 18. Spectrum of capsaicin derivative <br />9<br /><ul><li> Changed molecular mass from 305 to 333
  19. 19. Ethylation of the common benzyl group caused the fragmentation of that group to increase from m/z 137 to 165</li></li></ul><li>Spectrum of dihydrocapsaicin derivative<br />10<br />
  20. 20. Method<br />II. Analysis of capsaicin in hot pepper fruits<br />Prepare samples (Red Serrano and Caribbean Red)<br />Identification and isolation of capsaicin<br />Internal standard was added to the extracted capsaicin<br />GC/MS Analysis using same parameters as the standard<br />11<br />
  21. 21. 12<br />Red Serrano Pepper Results<br />
  22. 22. Caribbean Red Pepper Results<br />13<br />
  23. 23. Higher level derivatization<br />14<br />I. Derivatization<br />102 mg of capsaicin standard<br />20 mL of N,N-DMA<br />5 mL of TMAH/25%<br />5 mL of Iodoethane<br />10 mL of 0.1 M Na2CO3<br />10mL of CCl4<br />II. Observations<br /><ul><li>Emulsion formed
  24. 24. White ppt formed after extraction of bottom layer and allowed to evaporate
  25. 25. Darkish yellow orange liquid
  26. 26. Added water- neon greenish crystals formed immediately
  27. 27. Added 10-mL of hexane and vacuum filtrated out white ppt.
  28. 28. Extraction of derivative with 10-mL of hexane and 10-mL of water
  29. 29. Analysis</li></li></ul><li>100-mg Derivatization Results<br />15<br />
  30. 30. Conclusions<br />An ethylated capsaicin derivative can be used as an internal standard by GS/MS techniques to quantify the amount of capsaicin and dihydrocapsaicin in hot peppers.<br />However, an optimal derivatization with 100 mg of capsaicin is needed to efficiently quantify capsaicin and dihydrocapsaicin in a variety of hot peppers.<br />Once an appropriate amount of internal standard is constructed and free of errors, a calibration curve can be constructed.<br />16<br />
  31. 31. Successes and Failures<br />Operation and method procedures<br />Capsaicin derivatization- Trial and Error<br />Suitable reaction guidelines<br />Advantages and disadvantages <br />Alkylating reagents and targeted functional group<br />Derivatization side reactions and contamination<br />NaOH on capsaicin and derivatized product<br />17<br />
  32. 32. Contamination<br />18<br />
  33. 33. Use of a different alkylating reagent such as DMF* for comparison<br />Experiment with different evaporation/drying methods<br />Use of derivatized internal standard on capsaicin products other than hot peppers <br />19<br />Future Studies<br />*Boger, Dale. Thermal Atropisomerism of TeicoplaninAglycon Derivatives: Preparation of the P,P,P and M,P,P Atropisomer of the TeicoplaninAglycon via Selective Equilibration of the DE Ring System, J. Am. Chem. Soc. 2000, 122, 10047-10055<br />
  34. 34. Acknowledgments<br />Dr. David von Minden <br />Dr. Steven Meier<br />Dean of College of Mathematics and Science and Dr. Cheryl Frech<br />Ryan Hays<br />Amanda Bridges, Will Watkins and Monkey Business from Lawton, OK<br />20<br />
  35. 35. 21<br />Literature Resources<br />1. Govindarajan V.S. and Sathyanarayana M.N. Capsicum-Production, Technology, Chemistry, and Quality. Part V: Impact on Physiology. Pharmacology, Nutrition, and Metabolism; Structure, Pungency, Pain, and Desensitization sequences. Food Sci. and Nutr. 1991, 29, 435–474.<br />2. Bucholz, C. F. ChemischeUntersuchungDerTrockenenReifenSpanischenPfeffers [Chemical Investigation of Dry, Ripe Spanish Peppers]. AlmanachoderTaschenbuchfürScheidekünstler und Apotheker (Weimar) [Almanac or Pocket-Book for Analysts (Chemists) and Apothecaries. 1816, 37, 1–30. <br />3. Thresh, J. C. Isolation of Capsaicin. The Pharmaceutical Journal and Transactions. 1876. 3rd series, 6, 941–947.<br />4. Späth, E. and Darling, S.Synthese des Capsaicins. Chem. Ber. 1930, 63B, 737–743.<br />5. Kosuge, S., Inagaki, Y., and Okumura, H. Studies on the Pungent Principles of Red Pepper. Part VIII. On the Chemical Constitutions of the Pungent Principles. Nippon Nogei Kagaku Kaishi. J. Agric. Chem. Soc. 1964, 35, 923–927.<br />6.New Mexico State University—College of Agriculture and Home Economics Home Page. "Chile Information—Frequently Asked Questions. http://web.archive.org/web/20070504035555/http://spectre.nmsu.edu/dept/academic.html?i=1274&s=sub.  (accessed Dec 1, 2010).<br />7. Razavi, R., Chan, Y., and Afifiyan, F.N. et al. TRPV1+ Sensory Neurons Control Beta Cell Stress and Islet Inflammation in Autoimmune Diabetes. Cell. 2006. 127, 6, 1123–1135.<br /> 8.Mori, A., Lehmann, S. and O’ Kelly J. et al. Capsaicin, A Component of Red Peppers, Inhibits the Growth of Androgen-Independent, p53 Mutant Prostate Cancer Cells. Cancer Research. (American Association for Cancer Research). 2006, 66, 6, 3, 222–3,229.<br />9. Which Treatment for Postherpetic Neuralgia? PLoS Medicine. 2005, 2, 7, e238. <br />10.Glinski, W., Glinska-Ferenz, M., and Pierozynska-Dubowska, M. Neurogenic Inflammation Induced by Capsaicin in Patients with Psoriasis. Actadermato-Venereologica (ActaDermVenereol). 1991, 71, 1, 51–54.<br />11. The Journal of the American Pharmacists Association. Note on Capsicums. 1912, 1, 453–454.<br />12. Cooper, T.H., Guzinski, J.A., and Fisher, C. Improved High-Performance Liquid Chromatography Method for the Determination of Major Capsaicinoids in Capsicum Oleoresins. J. Agric. Food Chem. 1991, 39, 2253–2256.<br />13. Li, H., Pordesimo, L.O., Igathinathane, C., and Vinyard. B. Physical Property Effects on Drying of Chile Peppers. International Journal of Food Properties. 2009. 12, 2, 316–330.<br />
  36. 36. 22<br />The End<br />Any Questions?<br />
  37. 37. 23<br />Literature Resources<br />Antonious, G., & Jarret, R. (2006). Screening Capsicum Accessions for Capsaicinoids Content. Journal of Environmental Science & Health, Part B -- Pesticides, Food Contaminants, & Agricultural Wastes, 41(5), 717-729. <br />Barbero, G., Liazid, A., Palma, M., & Barroso, C. (2008). Ultrasound-assisted extraction of capsaicinoids from peppers. Talanta, 75(5), 1332-1337<br />Li, H., Pordesimo, L., Igathinathane, C., & Vinyard, B. (2009). Physical Property Effects on Drying of Chile Peppers. International Journal of Food Properties, 12(2), 316-330.<br />Li, F., Lin, Y., Wang, X., Geng, Y., & Wang, D. (2009). Preparative isolation and purification of capsaicinoids from Capsicum frutescens using high-speed counter-current chromatography. Separation & Purification Technology, 64(3), 304-308. <br />Thompson, R., Phinney, K., Welch, M., & White V., E. (2005). Quantitative determination of capsaicinoids by liquid chromatography-electrospray mass spectrometry. Analytical & Bioanalytical Chemistry, 381(7), 1441-1451<br /> Thompson, R., Pennino, M., Brenner, M., & Mehta, M. (2006). Isolation of individual capsaicinoids from a mixture and their characterization by 13C NMR spectrometry. Talanta, 70(2), 315-322. <br />Von Minden, D. , & D’Amato, N. (1977) Simultaneous Determination of Cocaine and Benzoylecgonine in Urine by Gas-Liquid Chromatography. Analytical Chemistry, 49(13), 1974-1976.<br />
  38. 38. 24<br />What is Capsaicin?<br />Definition: Capsaicin is a colorless pungent crystalline compound derived from the genus Capsicum. Capsaicin is the source of the “hotness” found in peppers such as chili, cayenne and jalapeno. <br />Why is it a “hot” topic?<br /><ul><li>This chemical is a cancer fighter, a digestive aid and a powerful pain killer.
  39. 39. It is found in a high-dose dermal patch called Qutenza1 that is used to treat the pain of peripheral neuropathy (i.e. Shingles).
  40. 40. Capsaicin creams are primarily used to relieve pain and itching. Some conditions include back pain, fibromyalgia, psoriasis, and arthritis2.
  41. 41. The American Association for Cancer Research reports studies suggesting capsaicin is able to kill prostate cancer cells by causing them to undergo apoptosis3. </li></ul>(2010). QUTENZA. Monthly Prescribing Reference, 26(6), A.16. Retrieved from Academic Search Complete database<br />Welland, D. (2008). Spice Up Your Cuisine To Help Protect Against Heart Disease, Cancer, Diabetes. (Cover story). Environmental Nutrition, 31(10), 1-4. Retrieved from Academic Search Complete database.<br />(2006). HOW HOT PEPPERS LEAD TO SUICIDES. Maclean's, 119(13), 41. Retrieved from Academic Search Complete database.<br />
  42. 42. 25<br />Capsaicin Standard – 9/29/2010<br /><ul><li>10 mg of Capsaicin Standard/10 mL of ethyl acetate
  43. 43. Agilent 6890N Network GC System coupled with a 5975 inert XL Mass Selective Detector
  44. 44. Split injector; IT-50 °C at 10 °C/min with a FT of 265 °C
  45. 45. Run time: 20 minutes</li></li></ul><li>26<br />Ion 137<br />Ion 305 (Capsaicin)<br />SIM (Selective Ion Monitoring) for m/z 305, 165, 137<br />
  46. 46. 27<br />Derivitization-First Run <br />12 mg of Capsaicin <br />35 mL of N,N-DMA ; 5 mL of TMAH/25%<br />2 mL of Ethyl-Iodide; <br />50 mL of 0.1 M Na2CO3<br />20mL CCl4; 50 mL of Ethyl acetate<br />Step 3. Precipitation<br />Results<br /><ul><li> Low recovery -25%
  47. 47. Derivitized product at 3mg/50mL</li></ul>Step 5. Extraction<br />
  48. 48. 28<br />First Derivitization<br />200 μL of Capsaicin Standard; evaporate<br />200 μL of N,N-DMA and 50 μL of TMAH (25%)<br />10 μL of Methyl-Iodide; (45 °C for 30 minutes)<br />500 μL of 0.1 M Na2CO3<br />200 μL CCl4, vortex.<br />Figure 1. TIC of Derivitized Product (CH3-R); 9/29/2010<br />Abundance<br />Time<br />
  49. 49. 29<br />Capsaicin Analysis<br />1. Peak at RT 16.793 yields Capsaicin<br />
  50. 50. 30<br />Capsaicin Analysis Cont’d<br />2. Peak at RT 16.974 yields Dihydrocapsaicin <br />
  51. 51. 31<br />Methyl-Capsaicin Analysis<br />1. Peak at RT 16.968 <br />
  52. 52. 32<br />Methylated-Capsaicin Analysis Cont’d<br />2. Peak at RT 17.143 <br />
  53. 53. 33<br />Overlap of TIC<br />
  54. 54. 34<br />Part 1. The Isolation and Identification of Capsaicin in Hot Peppers<br />A Variety of Hot Peppers <br />preparation of samples by a<br /> drying processes<br /> Extraction<br />food processor; cyclohexane<br /> pipettes with glass wool<br /> GC/MS Analysis <br />Agilent Technologies 6890N GC System with a 5975 inert XL Mass Selective Detector<br />
  55. 55. 35<br />Part 2. Alkylation of the Hydrogens to Produce an Internal Standard<br /> 200 μL Capsaicin <br />(95% Pure Pharmaceutical Grade)<br /> 1. NNDMA<br /> 2. TMAH<br /> 3. Methyl Iodide<br /> 4. Na2CO3/CCl4<br /> Alkylated Capsaicin<br />
  56. 56. 36<br />Part 3. Quantitation of Capsaicin Levels<br /><ul><li>Internal Standard from alkylation reaction
  57. 57. Calibration curve of samples spiked with internal standard
  58. 58. Concentration amounts from peak area ratios</li></li></ul><li>37<br />Desired Result from an Internal Standard<br />151<br />CH3<br />CH3<br />333<br />
  59. 59. 38<br />Potential Problems<br /><ul><li> The TMAH might not be vigorous enough to replace the hydrogen on the carbamide group
  60. 60. Influence of reaction times and temperature on the recovery of capsaicin</li>

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