Directed Research CHEM 4502

Fall 2010

Quantitation of Capsaicin Levels in Hot Peppers

by Gas Chromatography/Mass Spectrometry

Brandi VanAlphen

Dr. von Minden

December 14, 2010

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Background of Capsaicin

• Derived from the genus

Capsicum

• History of capsaicin

• Scoville Heat Chart

Capsaicinoids

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• Alkyl vanillylamides

Capsaicin (69%) MW 305

Dihydrocapsaicin (22%) MW 307

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It is a “hot” topic

• Food preparation

• Weight loss

• Alleviate pain & inflammation- inhibiting Substance P

• Protects heart health

• Cancer research

• Less-than-lethal weaponry

Main Objectives

• Development of a rapid, reproducible and simple

method of quantitation using GC/MS, SIM and

ChemStation.

• Unique derivatization of capsaicin to be utilized as

internal standards

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Capsaicin Standard and GC/MS Analysis• Agilent 6890N Network GC System coupled with a 5975 inert XL Mass

Selective Detector

• 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

I. Derivatization1. 13.7 mg of capsaicin standard

2. 5 mL of N,N-DMA

3. 1 mL of TMAH/25%

4. 1 mL of Iodoethane

5. 10 mL of 0.1 M Na2CO3

6. 10 mL of CCl4

Products Water

TMAI

Ethylated derivatives- 9.4 mg

Experimental Method

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Fig. 1. Capsaicin derivative, MW 333

Fig. 2. Dihydrocapsaicin derivative, MW 335

TIC of derivatized product

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Spectrum of capsaicin derivative

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• 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

Spectrum of dihydrocapsaicin derivative

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MethodII. Analysis of capsaicin in hot pepper fruits

1. Prepare samples (Red Serrano and Caribbean Red)

2. Identification and isolation of capsaicin

3. Internal standard was added to the extracted capsaicin

4. GC/MS Analysis using same parameters as the standard

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Red Serrano Pepper Results

Caribbean Red Pepper Results

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Higher level derivatization

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I. Derivatization1. 102 mg of capsaicin standard

2. 20 mL of N,N-DMA

3. 5 mL of TMAH/25%

4. 5 mL of Iodoethane

5. 10 mL of 0.1 M Na2CO3

6. 10 mL of CCl4

II. Observations•Emulsion formed

• White ppt formed after extraction of bottom

layer and allowed to evaporate

•Darkish yellow orange liquid

•Added water- neon greenish crystals formed

immediately

•Added 10-mL of hexane and vacuum filtrated

out white ppt.

•Extraction of derivative with 10-mL of hexane

and 10-mL of water

•Analysis

100-mg Derivatization Results

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Conclusions• 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.

• However, an optimal derivatization with

100 mg of capsaicin is needed to

efficiently quantify capsaicin and

dihydrocapsaicin in a variety of hot

peppers.

• Once an appropriate amount of internal

standard is constructed and free of

errors, a calibration curve can be

constructed.

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• Operation and method procedures

• Capsaicin derivatization- Trial and Error

– Suitable reaction guidelines

– Advantages and disadvantages

– Alkylating reagents and targeted functional

group

– Derivatization side reactions and

contamination

• NaOH on capsaicin and derivatized product

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Successes and Failures

Contamination

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• Use of a different alkylating reagent such as DMF* for comparison

• Experiment with different evaporation/drying methods

• Use of derivatized internal standard on capsaicin products other than hot peppers

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Future Studies

*Boger, Dale. Thermal Atropisomerism of Teicoplanin Aglycon Derivatives: Preparation of the P,P,P and M,P,P Atropisomer of the Teicoplanin Aglycon via Selective Equilibration of the DE Ring System, J. Am. Chem. Soc. 2000, 122, 10047-10055

• Dr. David von Minden

• Dr. Steven Meier

• Dean of College of Mathematics and Science and Dr. Cheryl Frech

• Ryan Hays

• Amanda Bridges, Will Watkins and Monkey Business from Lawton, OK

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Acknowledgments

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Literature Resources1. 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, 435–474.2. Bucholz, C. F. Chemische Untersuchung Der Trockenen Reifen Spanischen Pfeffers [Chemical Investigation of Dry, Ripe Spanish Peppers]. Almanach oder Taschenbuch für Scheidekünstler und Apotheker (Weimar) [Almanac or Pocket-Book for Analysts (Chemists) and Apothecaries. 1816, 37, 1–30. 3. Thresh, J. C. Isolation of Capsaicin. The Pharmaceutical Journal and Transactions. 1876. 3rd series, 6, 941–947.4. Späth, E. and Darling, S.Synthese des Capsaicins. Chem. Ber. 1930, 63B, 737–743.5. 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, 923–927.6.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).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, 1123–1135.8.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, 222–3,229.9. Which Treatment for Postherpetic Neuralgia? PLoS Medicine. 2005, 2, 7, e238. 10.Glinski, W., Glinska-Ferenz, M., and Pierozynska-Dubowska, M. Neurogenic Inflammation Induced by Capsaicin in Patients with Psoriasis. Acta dermato-Venereologica (Acta Derm Venereol). 1991, 71, 1, 51–54.11. The Journal of the American Pharmacists Association. Note on Capsicums. 1912, 1, 453–454.12. 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, 2253–2256.13. 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.

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The End

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