AOAC Method 995.03 - measuring Capsaicinoids in Capsicums

In the adjacent column is an outline of AOAC Method 995.03, this method requires the handling of extreme irritants and organic solvents.


The sample preparation takes over 5 hours and the testing requires a liquid chromatography equipment with a suitable detector.


For a quick and easy test for the hotness/chilliness of products please click the button below.

The following note an outline on how to perform the  AOAC Official Method 995.03 for determining Capsaicinoids in Capsicums and Their Extractives by Liquid Chromatographic Method


This methodology is applicable for the determination of  samples with 750-650000 Scoville Heat Units (SHU) of capsaicinoids in:

  • ground and crushed red pepper
  • chili pepper,
  • ground cayenne pepper,
  • ground jalapeno pepper,
  • red pepper oleoresins.

This method is not applicable to chili powders or products containing oregano or thyme.


Please note that this method uses chemical and solvents that should be used by a trained scientist, in an appropriate lab with the the necessary safety equipment and protection in place; including the handling of extreme irritants.


Please read safety notes for the safe handling of organic solvents and

special chemical hazards-acetone, acetonitrile, and ethanol, and see Material Safety Data Sheets, or equivalent, for each reagent. N-Vanillylnonanamide, which  is an extreme irritant and MUST not be inhaled.


You must dispose of waste solvents in an appropriate manner

compatible with applicable environmental rules and regulations.)



A. Principle

Your test sample is extracted in warm ethanol using a reflux condenser. The extract is filtered and injected into liquid chromatograph equipped with UV or fluorescence detector.


B. Apparatus

(a) Liquid chromatograph (LC), which is equipped with sample injector, and with a UV detector which is set at 280 run. wavelength or with a fluorometer with the excitation 280 nm and emission 325 nm.

The operating conditions: temperature,

ambient (20-25'C); flow rate, 1.5 mL/min., isocratic; relative retention times: 


(b) LC column.--A stainless steel column packed with  C18, dimensions 150 x 4.6 mm id, packed with 5 micrometer particle size. Use guard column.


(c) Reflux condenser.


(d) Syringe filter.- 0.45 micrometer.


(e) C18 solid-phase extraction cartridge.


C. Reagents

(a) Ethanol-95% or denatured, suitable for chromatography

(b) Acetone.-ACS grade.

(c) LC mobile phase@Acetonitrile-water. Use LC grade solvents, or equivalent. Please mix 400 mL acetonitrile with 600 mL H20 containing 1% acetic acid (v/v). De-gas with helium or by other suitable technique.


(d) (N-Vanillyl-n-nonanamide standard solutions. -N-Vanillyln-nonanamide standard)

Solution solution  A.-0. 15 mg/mL. Accurately weigh 75 mg N-vanillyl-n-nonanamide and transfer it into a 500 mL volumetric flask. Dilute to volume with ethanol, and mix. Please use standard solution A for analyzing all peppers except chili pepper.


Standard solution B.-0.015 mg/mL. Please transfer 10 mL of standard solution A into a I00 mL volumetric flask, and dilute to the volume with ethanol, and mix. Please use standard solution B when analyzing chili peppers.


D. Extraction

(a) Ground or crushed peppers.-Accurately weigh ca 25 g pepper into 500 ITIL boiling flask. Then place 200 mL pf ethanol into the same flask, then add several glass beads, and attach the flask to reflux condenser. Gently reflux the  sample for 5 hours and then allow to cool. Filter 1-4 mL sample through 0.45 gm syringe filter into small glass vial. Use the filtered sample for subsequent  LC analysis.


(b) Red pepper oleoresins.- Please accurately weigh 1-2 g of  oleoresin into a 50 mL volumetric flask. Increase weight of sample, if total capsaicinoid concentration is < 1 %.

Note: Do not allow any oleoresin to coat onto the sides of the  flask.

Please add 5 mL of acetone to the flask and swirl the contents of flask until test sample is completely dispersed (there should be no oleoresin on the  bottom of flask when tilting the flask neck to 45' angle). Please add five 3-5 mL portions ethanol, swirling flask during each addition.

Dilute contents of flask to volume with ethanol and mix well.