1. Determinationof Capsaicinoids in Jalapeno Peppers
using Reverse Phase High Pressure Liquid
Chromatography - Ultraviolet
Jake Unger, Dakoda Bazinet, Justin Allan, Kari Kramp and Greg Allen
Departmentof Bioscience,LoyalistCollege,Belleville,ON K8N 5B9, Canada; *
Jacobunger@loyalistc.on.ca
______________________________________________________________________________
ABSTRACT
The purpose of thisresearchwas to developasimple andinexpensivecapsaicinoidanalysismethod
usingreversedphase highpressure liquidchromatographywithultraviolet (RP-HPLC-UV)detectionof
twomajor capsaicinoids,capsaicin (C),anddihydrocapsaicin(DC) in freshjalapenopeppers.Soxhlet
extractionwasusedtoextractthe twocapsaicinoids withethanolasthe solvent;the extractwas
subjectedtosolid-phase extraction.The total capsaicinoidconcentration(C+ DC) in the extractis 0.2776
ppmwithits correspondingscoville heatunits(SHU) of 4470.
INTRODUCTION
Capsaicinoidsare agroupof chemical compounds thatproduce a sensationof burningincontactwith
tissue, foundinpeppersfruitsandproductsmade fromthe peppers. The twocapsaicinoids,C,andDC
are the mostpotentinterms of taste and concentrationof relative amount();the peppersplantsare
fromthe genus Capsicum(7).Peppersare animportantcommerciallyasa fruitandfor spices.The C in
peppersinlowconcentrationsisbeingusedasan analgesicinointments,nasal spraysetc.torelieve
pain(2) and hasbeenincreasingininterestforthese commercial applications.Manyextraction
techniquescanbe usedtoisolate the capsaicinoidcompoundssuchas,solventextractionetc.There is
alsoa varietyof methodstoquantitate capsaicinoidssuchas,gaschromatography,thinlayer
chromatography,etc. The purpose of thisresearchwas todevelopasimple,efficient,andinexpensive
capsaicinoidanalysismethodusingRP-HPLC-UV.
MATERIALS AND METHODS
Sampling
I obtainedthe freshjalapenopeppersfromthe local grocerystore the nightbefore the extractiontook
place.Colourof the pepperswasgreen.
Extraction
The pepperswere trimmedof theirstems,andwere blended until itwasauniformmixture.10.2401g of
blendedpepperswasweighedout andplacedina cellulose thimble,whichactsasa filter.The Soxhlet

2. equipmentwasrinsedwithethanoltoensure nocontaminates.The apparatuswas thenset-upwiththe
thimble containingthe peppersinside the apparatus.Then250 mL of ethanol wasaddedtothe round
bottomflask (RBF) of the Soxhletapparatusasthe extractionsolvent. The variance wasattachedtothe
heatingmantle andwassetto 95 to regulate the boilingtemperature of ethanol.The durationof the
extractionwas5 hours.The solventextractsvolumewasmeasured(195mL) witha graduatedcylinder
and thenwastransferredovertoa volumetricflask;leftoversolventextractwascollectedbyrinsingthe
RBF and the graduatedcylinderwiththe solventandtransferredtothe volumetricflask.The solvent
extractwas dilutedto250 mL with ethanol.
Cleanup
A C-18 (500mg) solid-phase extraction(SPE) cartridge wasconditionedwith10 mL of acetonitrile,
methanol,andwater(2 x 5 mL for eachcolumn).10 mL of the extractwasthenaddedto the
conditionedcolumn,Ithenadded4mL with1 mL of 1% aceticacid to finishthe elutionof the
capsaicinoids.The final extractwasstoredina HPLC vial.
Analysis
Analysiswasperformedona C-18 reverse-phase HPLCwithUV detectionat281 nm. The mobile phase
consistedof 77% methanol and23% water.Quantitationwasbyexternal standardsof Cand DC. The 3-
pointstandardcurve were linear(r= 0.999814109) and rangedfrom 0 mg/L to 100 mg/L total
capsaicinoid.See appendix Bforcalibrationcurve of standards.
RESULTS
The capsaicinoidextracthasa C concentrationof 0.16 mg/g and a DC concentrationof 0.13 mg/g. The
total capsaicinoidconcentrationinthe jalapenoextractis0.29 mg/g.The total concentration
representedinSHUis4470. See appendix A forcalculations.
Table 1: Shows the different units from concentration,Scovilleheatunits and Area for the standards,C and DC.
SHU Concentration
(mg/g)
Concentration (mg/L) Area
Standard NA NA 0.00 0
Standard NA NA 10.00 669781
Standard NA NA 100.00 8419162
Capsaicin 4470 0.15 0.913141088 35887
Dihydrocapsaicin 0.13 0.792720877 19929
DISCUSSION
There isnot a standardizedSHUfor jalapenopeppersdue tothe differentnumberof parameters suchas
moisture content,whichpartsof the pepperwere sampled,how ripe were the peppers,the amountof
pepper/solvent etc.Comparedto(3),a jalapenopepperhasa range between1000 – 4000 of SHU, I

3. calculated4470 SHU inmy extractwhichisnot far off fromthat range.The data from(1) suggesta value
of 41000 SHU fora jalapenopepperwhichismuchgreaterthanthe calculated4470 SHU of total
capsaicinoids.The concentrationfrom(1) datashowsC witha 0.25 mg/g,DC with0.13 mg/g and a total
of 0.38 mg/g.The resultsare verysimilartothe concentrationsfrom(1),the C calculatedhasa lower
concentrationof 0.16 mg/g,the calculate concentrationof DCwas roundedto0.13 mg/gwhichisthe
same as the reference value. Soxhletextractionisaverysimple andcostefficientmethodbutthe
amountof time neededforextractionisadisadvantage whichcouldhave potentiallycome intoplayto
alterthe amountof capsaicinoidsthatcouldhave beenextractedif the Soxhletcouldrunlonger.ASEIsa
methodthatcan extractcapsaicinoidsusinghighpressure andtemperatureswhichalsoreducesthe
extractiontime to30 minutes anduseslesssolvent.The linearityof the standardsisrepresentedin
appendix Bwhichshowsthe reliabilityof the calibrationto findconcentrations. Capsaicinoidsare very
importantcommercial compoundsusedinavarietyof waysandwithbetterunderstandingof the pain
relievingpropertiesof the compound,the commercial usescanonlyincrease.
REFERENCES
(1) Huang,J, Mabury, S, Sagebiel,J,(2000). Hot Chili Peppers:Extraction,Cleanup,and
Measurementof Capsaicin. DesertResearchInstitute,Energy&EnvironmentalEngineering
Center,Reno,NV 89506J. Chem.Educ.,2000, 77 (12),p 1630DOI:
10.1021/ed077p1630.Publication Date (Web): December1,2000.
(2) McQuay HJ, Moore RA. Chapter33, Topical Capsaicin.An evidence-based resourceforpain relief.
Oxford.OxfordUniversityPress,1998.
(3) Kleiman,Dena(November8,1989). Rating Hot Peppers:Mouth vs.Computer.TheNew York
Times. Retrieved 2015-12-14.
(4) Kramp,K. 2015. Extraction Technologies –SoxhletExtraction.Pages:3-1to3-1. LoyalistCollege,
Belleville.
(5) Unger,J. (2015). Biochemistry Lab Book.Pages:20 – 25. LoyalistCollege,Belleville.
(6) Wagner,C, Cahill,T,andMarshall,P, (2011). Extraction,Purification,and Spectroscopic
Characterization of a Mixtureof Capsaicinoids. J.Chem.Educ.,2011, 88 (11), pp 1574–1579 DOI:
10.1021/ed1006025. PublicationDate (Web):August26, 2011.
(7) National PlantGermplasmSystem.(n.d.).RetrievedDecember14, 2015, from
https://npgsweb.ars-
grin.gov/gringlobal/taxonomylist.aspx?category=species&type=genus&value=agenus&id=2056

4. APPENDIX A: Sample Calculations (See (5) for all calculations):
Determiningthe dry weightof the peppersample assuming 85% moisture
Dry weightof peppersample =10.2401g freshsample x (85/100)
= 8.704085g of moisture
Dry weight= 10.2401g Freshsample – 8.704085g Moisture
=1.536015g
Linear RegressionofStandards
Slope:84948.25824 Intercept:-85121.8022 r = 0.999814109
Y = 84948.25824 x -85121.8022
FindingC in Extract by subbingin the area
Y = 84948.25824 x -85121.8022
35887 = 84948.25824 x -85121.8022
X = 1.424500098 mg/L
Scoville Heat UnitConversion
Thisis fortakingthe dilutionsintoaccount
C present= 1.424500098 mg/L x (5 mL/10 mL) x (250 mL/195 mL)
= 0.913141088 mg/L
Mass of C = 0.913141088 mg/L x 0.250 L
= 0.228285272 mg C
PPMC = 0.228285272 mg C / 1.536015g
= 0.1486217726 mg/g C
PPMof Total capsaicinoids=(0.198175 mg DC + 0.228285272 mg C)/1.536015
= 0.277640694 mg/g of Total capsaicinoids
SHU of Total capsaicinoids=(0.277640694 mg/g) x (16100SHU/mg/g)
= 4470.015188 SHU of total capsaicinoids

5. APPENDIX B: Calibration Curve of Standards
Figure 1: The graph shows the linearity of area as a function of concentration.
y = 84948x - 85122
R² = 0.9996
0
1000000
2000000
3000000
4000000
5000000
6000000
7000000
8000000
0 10 20 30 40 50 60 70 80 90 100
Area
Concentration (mg/L)
Area as a Function of Concentration (mg/L)