What is capsaicin? Capsaicin is a phytochemical found in chili peppers. Pure capsaicin is a crystalline white compound. Capsaicin was first extracted (albeit in impure form) in 1816 by Christian Friedrich Bucholz.He called it "capsicin," after the genus Capsicum from which it was extracted. Capsaicin was first isolated in pure, crystalline form in 1876 by John Clough Thresh,who gave it the name "capsaicin". Capsaicin was first synthesized in 1930 by E. Spath and S. F. Darling. In 1961, similar substances were isolated from chili peppers by the Japanese chemists S. Kosuge and Y. Inagaki, who named them capsaicinoids.The first method developed to assess the pungency of fruits was the Scoville organoleptic tests which estimates capsaicin content from the burning mouth sensation, using diluted samples and tasting panel of about 5 individuals. This test is subjective and does not determine the levels of individual capsaicinoids.
In 1961, similar substances were isolated from chili peppers by Japanese chemists who called them capsaicinoids. Capsaicinoids are a series of homologous branched and straight chain alkyl vanillylamides that are present in lesser concentrations than the parent compound capsaicin. The two major capsaicinoids I am interested in are capsaicin and dihydrocapsaicin. The only difference is the double bond. All capsaicinoids work together to produce the pungency but capsaicin and dihydrocapsaicin are twice as potent to the taste and nerves than the other ones.
Because we do not have difficulty digesting peppers, capsaicin is a hot topic. For centuries, we’ve used them for food preparation and because it is a thermogenic agent, you can burn calories up to 20 minutes after you eat them. Capsaicin was found to inhibit Substance P which aids in alleviating pain and inflammation. Capsaicin reduces cholesterol and triglycerides which can help protect your heart. Studies show that cultures who have hot peppers in meals have significantly lower rates of heart attacks and strokes than cultures who do not. The most familiar capsaicin product is found in less than lethal weaponry, or pepper spray.
My main objectives for my research are to develop a rapid, reproducible and simple method of quantitation using gas chromatography/mass spectrometry and SIM- selective ion monitoring. Because there is no flavor, color or odor- the precise amount of capsaicin present in chilis can only be measured by special lab procedures.Due to their similarity in structures, isolation of capsaicin and dihydrocapsaicin with traditional methods such as silica gel column chromatography, normal-phase thin-layer chromatography (TLC) becomes difficult The most recent methods for determination of capsaicinoids have used HPLC coupled to more selective techniques such as mass spectrometry. However, gradient elution methods were introduced to increase selectivity for separation of capsaicinoids for it is impossible to unequivocally identify individual compounds solely based on chromatographic behavior and UV data. Complex matrix and structural similarity. Identification of capsaicinoids thru GC/MS methods require tedious derivatization steps to increase volatility and LC-MS methods call for costly instruments. Past internal standards used for capsaicin were DMBMO (4,5 dimethoxybenzyl 4-methyloctamide) first offered by Copper et al. However it’s molecular weight is 307 which is the same as dihydrocapsaicin so separation is difficult.Octanoyl vanillyl amide is used as well. Because of the availability of standards and the variablity of capsaicinoids, a derivatized standard is unique for sensitivity, high selectivity, reproducibilty, and would provide absolute and relative quantities of the unknown compounds and give us structural information using ChemStation and SIM. Lower levels of detection can be achieved selected ion monitoring (SIM). Operation of a GC/MS in SIM mode allows for detection of specific analytes with increased sensitivity relative to full scan mode. In SIM mode the MS gathers data for masses of interest rather than looking for all masses over a wide range. When properly setup and calibrated, GC/MS-SIM can increase sensitivity by a factor of 10 to 100 times that of GC/MS-Full Scan. Because unwanted ions are being filtered, the selectivity is greatly enhanced providing an additional tool to eliminate difficult matrix interferences.Shown is a TIC, or total ion chromatogram of capsaicin. The largest peak is capsaicin and the second one is dihydrocapsaicin.
To analyze capsaicin, I used a standard purchased from Spectrum Laboratories which is 95% pure capsaicin. I used a Agilent 6890N Network GC System with a 5975 inert XL Mass Selective Detector. I used a split injector with a Zebron VF-5MS capillary column. The initial temperature was 125 degrees ramped 5 degrees per minute ending with a final temp of 250. The total run time was 27 minutes. This is a TIC of the capsaicin standard using SIM. SIM is selective ion monitoring. The red represents ion 137, which is the common benzyl fragment capsaicin and dihydrocapsaicin have.
To accomplish derivitization, I started with a small scale of 13.7 mg of capsaicin standard and dissolved it in 5 mL of dimethylacetamide. After it was dissolved, 1 mL of tetramethylammonium hydroxide (25 percent in methanol) and 1 mL of iodoethane was added. A precipitate formed indicating the end of the reaction. The derivative was extracted using 10 milliliters of sodium carbonate and 10 milliliters of carbon tetrachloride. Capsaicin was ethylated on the aryl phenol group for quantitation purposes. The phenol group was deprotonated by N,N-DMA and TMAH to form H2O and TMA+. By SN2 addition, the deprotonated phenol group attached to the ethyl group using iodoethane, removing the iodide ion to form TMAI.The crystallized product weighed 9.4 mg and was dissolved in 50 milliliters of ethyl acetate for analysis.
Using the same parameters as the capsaicin standard, this is a TIC of my derivitized product. The capsaicin derivative was found at retention time 19.093 and the dihydrocapsaicin derivative was the peak at 19.432. Further analysis produces the mass spectra of the derivatives.
This is a mass spectrum of my capsaicin derivative. The molecular mass changed from 305 to 333. The benzyl group increased from 137 to 165 indicating ethylation of the aryl phenol group.
This occurred with dihydrocapsaicin as you can see on this mass spectrum. The original molecular mass of dihydrocapsaicin was 307 which increased to 335. The benzyl fragment with a mass to charge ratio of 137 originally increased to 165. This shows it was also ethylated.
Now to quantify capsaicin from hot peppers, the peppers are dried and minced in a food processor. The pepper sample is weighed out and the capsaicin was extracted with sodium carbonate and carbon tetrachloride. The carbon tet layer was removed and the 300 microliters of internal standard which was 9.4 mg per 50 mL of ethyl acetate was added. It was then analyzed using the same parameters as the capsaicin standard.
This is a total ion chromatogram of aRed Serrano pepper sample injected with the derivitized product. You can see the capsaicin and dihydrocapsaicin at RT of 22,23 and the derivitized products at the retention times of 24.
Now this is a chromatogram of a caribbean red pepper sample injected with the same amount of internal standard, the derivitized product. As you can see, there is a lot more capsaicin in this pepper than the serrano one so the derivatized products are too small for absolute quantification. The abundance of capsaicin in this pepper was around 3.2 million and serrano was around 750 thousand.
Now this is the TIC of the hexane layer confirming the derivatized product was present in that layer and not in the ppt.Underneath the TIC, is the mass spectrum of the unknown peak at RT 19.121 which has a MW of 333. This would be ethylated-capsaicin.
Alklyation derivatization of capsaicin for use as an internal standard using GC/MS is a unique method that can be used to quantify capsaicin. However, derivatization in higher levels with a low product loss, no side reactions or contaminations must be achieved.
Correct peak integration for quantification and higher sensitivity. Temperature-ramping. Capsaicin derivatization was complicated and time-consuming. To improve resolution, I had to play with the parameters to get better peak separation for capsaicin and dihydrocapsaicin. This did extend the run time but allowed for more accurate peak integration for quantitation. Split versus splitless. A very neat (and fundamental) aspect of this is that the amount of gas exiting the split vent can be varied while keeping the flow onto the column constant. This means that the AMOUNT of the split (called the split ratio) can be varied (in modern instruments via software control).95-100% derivatization• the reagent will not causeany rearrangements or structuralalterations during formationof the derivative• the reagent does not contributeto the loss of sampleduring the reaction and producesa derivative that willnot interact with the GCcolumn• the derivative is stable withrespect to timeThere are many advantages and disadvantages to an alkyl capsaicin derivative – The advantages are: 1. Wide range of alkylation reagents available (DMF-Dimethylformamide) 2. Reaction conditions can vary from strongly acidic to strongly basic. 3. Some reactions can be done in aqueous solutions 4. Alkylation derivatives are generally stable.Disadvantages-1. Limited to amines and acidic hydroxyls 2. Reaction conditions are frequently severe 3. Reagents are often toxicInternal standards- absolute and relative amounts and provides a control for extrinsic variation between different GC-runs.
Now this is a mass spectrum of stilbene which showed up on the previous TIC at the RT of 4.688.
These reagents work quickly, derivatizing upon dissolution.Suitable for flash alkylation, where derivatization takes placein the injection port· The different alkyl homolgues allow formation of a variety ofesters. polarity and volatility of the samples can be adjusted,thereby changing retention time· They will react with water to give the corresponding alcohol.Traces of water will not affect the rxn as long as you have an excess of acid.
Quantitation of Capsaicin Levels in Hot Peppers by Gas Chromatography/Mass Spectrometry
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<br />Background of Capsaicin<br /><ul><li> Derived from the genus Capsicum
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 />
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
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 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
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></ul><li>Spectrum of dihydrocapsaicin derivative<br />10<br />
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 />
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
White ppt formed after extraction of bottom layer and allowed to evaporate
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 />
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 />
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 />
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 />
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 />
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 />
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.
It is found in a high-dose dermal patch called Qutenza1 that is used to treat the pain of peripheral neuropathy (i.e. Shingles).
Capsaicin creams are primarily used to relieve pain and itching. Some conditions include back pain, fibromyalgia, psoriasis, and arthritis2.
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 />
25<br />Capsaicin Standard – 9/29/2010<br /><ul><li>10 mg of Capsaicin Standard/10 mL of ethyl acetate
Agilent 6890N Network GC System coupled with a 5975 inert XL Mass Selective Detector
Split injector; IT-50 °C at 10 °C/min with a FT of 265 °C
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 />
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%
Derivitized product at 3mg/50mL</li></ul>Step 5. Extraction<br />
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 />
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 />
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 />
36<br />Part 3. Quantitation of Capsaicin Levels<br /><ul><li>Internal Standard from alkylation reaction
Calibration curve of samples spiked with internal standard
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 />
38<br />Potential Problems<br /><ul><li> The TMAH might not be vigorous enough to replace the hydrogen on the carbamide group
Influence of reaction times and temperature on the recovery of capsaicin</li>