Capsaicin in Peppers

by Dan Iversen, Lucidity R&D Scientist

While doing inventory in the lab, I came across a vial of pure capsaicin. I wondered why we had capsaicin. Rather than throw it out, I decided to use it to know the level of capsaicin in the hot peppers that I eat. After a quick google search to see if capsaicin could be extracted, I decided to get started on this project.

I grabbed some jalapeno and pepperoncinis from home to bring into work. Not wanting the moisture content of the peppers to affect the extraction, I decided to dry the peppers in the toaster oven in the breakroom. I set the temperature to about 200 °F and set the timer to 60 minutes. That one hour did not dry the peppers completely, so I decided to keep the oven running for another 3 hours for a total of 4 hours of drying time. The air in the breakroom got quite spicy, and I got a few questions on what I was doing with peppers in the toaster oven.

For the extraction of the capsaicin I used 100 mL of ethanol per 1 g of dried pepper. Soxhlet extraction would be ideal, but I don’t have one, so I got a 250 mL erlenmeyer flask and added 100 mL of ethanol. I was going to add the pepper slices as is, but decided that smaller pieces would probably be better.

I broke the dried peppers with my fingers into fine pieces, and added them to the flask with the ethanol. I then added a stir bar and allowed the extraction to run for 4 hours. My goal at this point was to see if this drying and extraction method would work to extract the capsaicin, so I pulled up 1 uL of the solution and injected that into the miniGC and there was a peak at the same retention time as the standard.

Once the extraction time was finished I then filtered the extract into a 250 mL beaker and
allowed the ethanol to evaporate, if I had a rotovap I would have used that, but allowing the solution to evaporate in the fume hood overnight worked just fine. Once the alcohol had evaporated, I added 10 mL of ethanol to the beaker and swirled the solution to dissolve the capsaicin. I then added the solution to a sample vial.

My miniGC method was as follows:

miniGC Method:

Lucidity MiniGC Conditions
Carrier Gas: Hydrogen
Control: Pressure
Flow: 1 mL/min
Split: 10:1
Column: MXT-5 30 m x 0.25 mm, 0.25 μm
Injector: 250 °C
FID: 300 °C

Rate	Temp	Time
	150C	0 min
	250C	0 min
	280C	2 min

The capsaicin standard was made up to a 1000 ppm solution in ethanol and injected into the miniGC which gave the following chromatogram.

I then proceeded to inject the pepper solutions and they gave similar chromatograms. The
jalapeno pepper extract was quite easy to see, but the pepperoncini extract was difficult to see in the 1:10 dilution I performed. I then re-extracted the pepperoncini, but at the end instead of adding 10 mL of ethanol, I added 1 mL of ethanol. This time a small peak was observed at the capsaicin retention time.

Using the capsaicin standard and the chromatograms from the peppers I was able to determine that the capsaicin level in the pepperoncini to be 193 ppm and in the jalapeno to be 2,442 ppm.

So, I was able to see capsaicin in pepperoncini and jalapeno peppers, now it was time to ramp things up. I was out and about with my family and came across dried ghost peppers for sale at a very good price. Well, knowing that they are some of the hottest peppers in the world and that I could extract the capsaicin from them with relative ease, I had to buy them and test the amount of capsaicin in these ghost peppers.

Thankfully they were already dried because I don’t think my coworkers would have enjoyed being subjected to me drying them in the toaster oven in the break room. Being careful to not actually touch the peppers with any part of my hands I proceeded to follow the procedure outlined earlier.

Once the extraction was done, the solution evaporated, and reconstituted to 10 mL and then vialed, I injected the ghost pepper solution. I waited to see how large a peak would be observed. The ghost pepper peak was massive compared to the other peppers.

Even though the peak area is much, much larger than the standard peak, I decided to go ahead and calculate the amount of capsaicin anyway. The total capsaicin in the ghost pepper is 28,856 ppm. One of my colleagues brought in a package of ghost peppers they had and I ran the same analysis one of these. It turns out that their pepper has 32,766 ppm capsaicin. I think the reason for that is because the pepper I used had some moisture left and was pliable, whereas theirs was quite brittle, this would lead to a bit higher concentration of capsaicin. Although the capsaicin amount in peppers varies between peppers of the same kind.

This project was a proof of concept that could be done much better. Given more time I would have made a calibration curve for the capsaicin, which would have given more accurate results for the levels in the peppers. I would have also used SPE to clean my samples to remove some of the other peaks and clean up the chromatograms. And finally, I should have diluted the ghost pepper samples to make them closer in size to the standard peak to get a better result. Overall, this was an interesting and enjoyable experiment.