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Analyzing the Heat

Levels of Spicy Foods

Using an Ultra C18 HPLC Column

As interest in spicy foods grows, so does the need

to test and classify products and raw materials for

their heat levels. In 1912, Wilbur Scoville developed

a method for quantitating the heat content of foods.

A Scoville Heat Unit (SHU) is defined as the number

of parts sugar water needed to neutralize the heat

of one part sample extract (e.g., if the heat of a

cayenne pepper is 30,000 SHU, that means 30,000

parts of sugar water are needed to dilute one part

of cayenne pepper extract to the last point that hot-

ness can be detected). However, this test is some-

what subjective because it relies on the tasters’

palates and sensitivities. In addition, tasters can

handle only a limited number of samples at one

time before "fatiguing of the palate" occurs.

This

makes it difficult to process a large number of sam-

ples in a reasonable amount of time, and can affect

reproducibility of the tests.

There are seven generally recognized capsaicinoids,

three of which are shown in Figure 1. The capsaici-

noid content, and thus the heat level, depends on

the type of pepper, maturity, growing conditions,

and processing methods. Starting in the 1970s, sev-

eral analytical methods for heat level measurement

were introduced. Of these, the HPLC procedures

have provided the greatest specificity while requir-

ing the least amount of sample preparation.

The

American Spice Trade Association (ASTA) and the

Association of Official Analytical Chemists (AOAC)

have both published methods for the determination

of capsaicinoids by HPLC.

AOAC Method 995.03

specifies the separation of

the three target capsaicinoids responsible for the

heat in chili peppers. This separation uses reversed

phase HPLC on a C18 column followed by quantita-

tion using either UV or fluorescence detection. The

AOAC method is performed isocratically with a

mobile phase consisting of 1% acetic acid in

water:acetonitrile (60:40). Standardization is per-

formed using synthetic capsaicin, N-vanillyl-

nonanamide, and the relative amounts of nordihy-

drocapsaicin, capsaicin, and dihydrocapsaicin are

calculated by applying the specified factors. Using

the appropriate calculations where 1ppm of total

capsaicinoids is approximately equal to 15 SHU, the

heat index then can be calculated.

Using a gradient elution program, an efficient sepa-

ration of the three target capsaicinoids is achieved

with the Restek Ultra C18 column (Figure 2). The

high percentage of organic at the end of the run

helps to elute any strongly adsorbing species pres-

ent in the samples and decreases analysis time. This

HPLC procedure provides an objective measure-

ment of the heat level of a wide range of samples.

By using a Restek Ultra C18 column and a gradient

elution program, the analysis can be performed

quickly and reproducibly with only minimal sample

preparation. The selectivity and lot-to-lot repro-

ducibility of the Ultra C18 column make it an excel-

lent choice for heat level measurements.

For more detail on HPLC detection of heat level,

including applications showing other products and

a table comparing hotness rankings by HPLC for

selected sauces, please request Applications Note

lit. cat.#59199.

nordihydrocapsaicin

dihydrocapsaicin

capsaicin

Figure 1

Structures of three capsaicinoids commonly

found in chili peppers

by Rebecca Wittrig, Ph.D., Food, Flavor, & Fragrance Innovations Group Leader

10 min.

Figure 2

The Restek Ultra C18 Column achieves fast, reproducible separation of capsaicinoids.

1. Bensinger, M. "How Hot is that 'Devil' Sauce?" Fiery Foods

Magazine (1997), Sept/Oct.

3. Chiang, G.H., Journal of Food Science (1986) 51 (2), 499-503.

2. AOAC Official Methods of Analysis (2000), method 995.03.

✔

Ultra C18 5µm HPLC Columns

For the complete HPLC column offering, refer to Restek’s

Annual Chromatography Products Catalog

(lit. cat.# 59960).

Length

1.0mm ID 2.1mm ID

3.2mm ID

4.6mm ID

30mm

9174531

9174532

9174533

9174535

50mm

9174551

9174552

9174553

9174555

100mm

9174511

9174512

9174513

9174515

150mm

9174561

9174562

9174563

9174565

200mm

9174521

9174522

9174523

9174525

250mm

9174571

9174572

9174573

9174575

for

more

info

Request Applications Note #59199

Sample:

habanero nugget extract;

Inj.:

10µL;

Conc.:

0.8g sample in 200mL;

Solvent:

ethanol;

Column:

Ultra C18 (cat.# 9174565)

Dimensions:

150x4.6mm;

Particle size:

5µm;

Pore size:

100Å

Mobile phase:

A: 1% acetic acid in water

B: acetonitrile

Time (minutes):

% B

Flow:

1.0mL/min.;

Temp.:

ambient;

Det.:

UV @ 280nm

✔

Fast, reproducible separation

of capsaicinoids

✔

Only minimal sample

preparation needed

1. nordihydrocapsaicin

2. capsaicin

3. dihydrocapsaicin

LC_0160