FLAVOUR AND FRAGRANCE JOURNAL, VOL.

2, 1-12 (1987)

Determination of the Pungent Principles of Chillies and Ginger

by Reversed-phase High-performance Liquid Chromatography
with Use of a Single Standard Substance
A. B. Wad
Overseas Developmen! Administration, Tropical Development and Research Institute, 56-62 Gray’s Inn Road, London,
W C l X 8 L U , UK

Quantitative determination of the capsaicinoids and the gingerols, pungent constituents of chillies and ginger
respectively, is important for objective assessment of the pungency of these spices and their oleoresins.
High-performance liquid chromatography (HPLC) procedures applicable to these groups of compounds have
been reported from several laboratories. The principal difficulty with gingerol determinations hitherto has been
the lack of a stable and easily purified analytical standard, since the gingerols (and their dehydration products, the
shogaols) are labile, oily liquids which are difficult to purify. Since the capsaicinoids and the gingerols and their
analogues have closely related chemical structures, the possibility of selecting from among them one easily purified
crystalline compound to serve as an external standard for determination of both groups of compounds by HPLC
was investigated. It was found that caprylic acid vanillyl amide (CVA), one of the minor naturally occurring
capsaicinoids but an easily synthesized crystalline compound, was very suitable for this purpose, and that a
commercially available mixture of capsaicin and dihydrocapsaicin could be used as a slightly less convenient
alternative. Reversed-phase HPLC methods based on this principle are described, and shown to be suitable for the
analysis (without clean-up) of chillies and ginger or their oleoresins. An acceptable level of precision (relative
standard deviation less than 2% and 5% respectively) is demonstrated for the summed major pungent principles in
dried chillies and dried ginger, and it is shown that fair agreement between the HPLC and spectrophotometric
difference methods can be obtained for chillies of varying pungency levels.

KEY WORDS Chillies Ginger Oleoresins Pungency assessment HPLC analysis Capsaicinoids Gingerols
Shogaols

INTRODUCIION procedure and gives no information on the rela-

The degree of pungency (or ‘heat’) in dried chil-
lies (fruits of several species of Capsicum) or
dried ginger (from the rhizomes of Zingiber
officinale Roscoe), and in the extracts (oleore-
sins) prepared from these spices, is an important
determinant of the value and end uses of the
products. Traditionally, organoleptic methods
have been used for assessing their pungency, but
such methods are subjective and yield results with
a large variance. Chemical determination of the
concentrations of the substances responsible for
the pungency (capsaicinoids in the case of chillies,
gingerols and related compounds in the case of
ginger-see Table 1 and Figure 1) provides a
more objective ap roach. A useful spectro-
P
photometric method was developed in the early
1960s for the determination of total capsaicinoids
in chillies, but it involves a laborious clean-up
tive proportions of the individual capsaicinoids,
which differ somewhat in their No
similar method exists for the gingerols.
More recently HPLC methods, not requiring a
clean-up, have been r e p ~ r t e d ~
for
- ~the determi-
nation of individual and total capsaicinoids, and
these have found widespread application in the
quality assessment of chilli products.
A few HPLC methods for the gingerols have
been but in each case quantitation
has required the laborious isolation (from ginger
or ginger oleoresin) of some of the pungent com-
pounds for use as analytical standards. The ging-
erols and their dehydration products, the shogaols
(present in large amounts in ginger oleoresins),
are oily liquids at ambient temperature, and are
both difficult to purify and somewhat unstable.
In the early stages of the present work, [ 6 ] -
gingerol (V, n = 6) and [6]-shogaol (VI, n = 6)

Received I0 March 1987

0 Crown Copyright 1987 Accepted 2 7 April I 9 8 7
2 A. B. WOOD

Table 1. Structural relationships between three groups of pungent compounds

Molecular
Compound Structure weight
(refer to Molecular Molecular relative
Group Name Abbreviation Figure 1 ) formula weight to CVA

(a) The Caprylic acid

capsaicinoids vanillyl amide
(n-octanoic acid
vanillyl amide) CVA 1.000
n-Nonanoic acid
vanillyl amide NVA 1,050
n-Decanoic acid
vanillyl amide DVA 1.100
Capsaicin C 1.093
Di hydrocapsaicin DHC 1.100
Nordihydrocapsaicin NDHC 1.050
Homocapsaicin 1 HC 1
1.143
Homocapsaicin 2 HC 2
Homodihydrocapsaicin 1 HDHC 1 C19H31N03 321
= 1.151
Homodihydrocapsaicin 2 HDHC 2 lsomeric

(b) The gingerols Zingerone 0.695

and their [6l-Gingerol 1.054
analogues [8l-Gingerol 1.154
[ 101-Gingerol 1.254
[6]-Shogaol 0.989
[ 81-Shogaol 1.090
[ 101-Shogaol 1.190

(c) The paradols [4]-Paradol 0.896

[ 61-Paradol 0.996
[ 81-Paradol 1.097

were isolated from a commercial ginger oleoresin
by using some of the procedures described by
Connell and Sutherland,'" followed by preparative
HPLC. This was an extremely time-consuming
operation, and yielded only 30-70 mg quantities
of materials which were still not completely pure,
especially in the case of [6]-gingerol. Since the
intention was to use ultraviolet (UV) absorptio-
metric detection in the HPLC determination of
the gingerols, an investigation was undertaken to
ascertain whether a related (but crystalline and
more stable) compound, with similar UV absorp-
tion properties, could be used as standard.
Table 1 and Figure 1 show the structural relation-
ships of the gingerols and shogaols both to the
capsaicinoids and to the paradols. [61-paradol
and [6]-ginger01 are the major pungent principles
of 'grains of paradise' (seeds of Aframomurn
rnelegueta).
The three groups of compounds form a family
sharing a common chromophore, viz. the
4-hydroxy-3-methoxyphenyl group, and this
leads to each of them exhibiting a UV absorption
maximum near 280 nm, with little difference in
molar absorptivity ( E ) from compound to com-
pound, as shown by the values determined in the
course of this work for representatives of each
group and presented in Table 2. This leads in turn
to the expectation that, for a given UV detector
set at 280 nm, there should be little difference
between the HPLC responses of the compounds if
these are expressed in terms of peak area per
nanomole, so that it might be possible to select a
single compound to serve as standard for the
HPLC determination of any other members of
the family. The verification of this hypothesis, the
selection of a suitable standard, and the develop-
ment of HPLC methods for determination of the
pungent principles of chillies and ginger are
described in this paper. It is worth noting that the
shogaols (VI) differ from the other members of
the family in having an ab-unsaturated keto group
as an additional chromophore, and that this gives
rise to a much stronger UV absorption in the
 PUNGENT PRINCIPLES OF CHILLIES AND GINGER 3
0 gingerols or capsaicinoids by the single-standard
procedures without clean-up described here.
I H EXPERIMENTAL
HO
Solvents
1 (y.6) HPLC grade solvents were obtained from
Rathburn Chemicals Ltd, UK (acetonitrile,
HO I methanol, tetrahydrofuran), and Fisons Scientific
Apparatus, UK (water). Methanol for sample
m extraction and all other solvents were of ana-
lytical grade.
HO

/ Zingerone (n.0)
Reference Substances
m ('%)fl Poradols (n.4-8)

(Refer to Table 1 for explanation of abbrevia-

(CHJ n - 2
P Gingerols
PI Shogaols
HO
Fig. 1. Structures of some pungent compounds (see Table 1
for full identification)
lower wavelength region (see Table 2). This
results in considerable enhancement of the sho-
gaol responses relative to those of the other com-
pounds at 254 nm, a wavelength commonly used
in fixed wavelength detectors. Furthermore, in
both ginger and chilli extracts, interference from
coextractives is more severe at this wavelength
than at 280nm. Consequently 254nm is not a
suitable wavelength for the determination of
tions.)
(a) Capsaicinoids. CVA [N-(Chydroxy-3-
methoxybenzyl) octanamide] was synthesized by
procedures similar to those of Nelson" and
Crombie et al.,'* and was finally recrystallized
from diethyl ether-petroleum spirit. It had the
expected infrared spectrum, was pure by TLC,
and yielded essentially a single peak on HPLC
analysis under the conditions described here. Its
melting point was 42-44°C.
Small amounts of pure C and DHC were
donated by Dr J. Jurenitsch of the University of
Vienna, Austria. Commercially available C
(SO-SS%, Sigma London Chemical Co. Ltd, UK),
and NVA (Fluka AG, Switzerland, via Fluoro-
chem Ltd, UK) were recrystallized from hexane.
A natural mixture of capsaicinoids (Fluka AG)
was used without further purification.
(b) Gingerol and its Analogues. [6]-G and
[61-S were isolated from a commercial ginger

Table 2. Ultraviolet absorption characteristicsof some pungent compounds (as measured by the author)

Short wavelength band Long wavelength band

Compound
(refer to Amax Emax Amax Emax
Solvent Table 1) (nm) (nm)
Ethanol CVA 230 7440 281 3030
(96%v/v) Capsaicin 230 7500 280.5 3010
Dihydrocapsaicin 229 8000 280.5 3100
Zingerone 225 (inflection) 6 320 281.5 3090
[4l-Paradol 225 (inflection) 6 230 282 3060
[ 81-Paradol 225 (inflection) 6 270 282 3100

Cyclohexane [6l-Shogaol 221 17400 281 3000

287 2600
4 A.
oleoresin (pre-washed with cold petroleum ether)
by extraction with hot etroleum ether followed
by cold precipitation,' and then preparative
HPLC on a column (250 x 16 mm) of Lichrosorb
RP-8 (10pm) with CH3CN-H20 (60:40) or
(70:30) as mobile phase. Their identities were
confirmed by mass spectrometry. The [6]-G
appeared to be only about 90% pure. Zingerone
was purchased from Phase Separations Ltd, UK.
(c) Paradols. Synthetic [4]-P and [8]-P were
donated by Mr A. M. Humphrey of Bush Boake
Allen Ltd, UK, and were recrystallized from hex-
ane-diethyl ether (1 :1) before use.
Reference Solutions
Solutions of reference compounds were pre-
pared in methanol or ethanol for the determina-
tion of retention times and response factors. Con-
centrations used were in the range 0.5 to
2.1 mmol l-', corresponding to 10-42 nanomoles
in a 2 pl injection. A solution of the purified
natural capsaicinoid mixture from Fluka A G (ca
1.O mg m1-I) was found to be very useful for peak
identification in sample chromatograms.
Standard CVA Solution
A stock solution was prepared by dissolving
CVA (0.12 g) in HPLC grade methanol (100 ml).
This solution was stored at ca. -1O"C, and was
diluted five-fold with methanol to yield a working
standard solution (0.24 mg ml-') when required.
Instrumentation
UV absorption data were measured in a Pye-
Unicam SP8-400 spectrophotometer with a
bandwidth of 1 nm. Two distinct HPLC instru-
mental assemblies were used, viz;
(1) Waters 6000 A pump, Waters autoinjector
(WISP), LDC UV 111 monitor (with 280 nm
filter), Waters Data Module (Model 730);
(2) Waters 6000 or Gilson 302 pump, Rheodyne
injector (with 20 pl sample loop), Cecil 212
variable wavelength detector (used on 2.0
absorbance range), and Perkin-Elmer LCI-
100 integrator.
All capsaicinoid determinations were carried out
on assembly (2); gingerol determinations were
carried out on either assembly.
B. WOOD
Sample Preparation
(a) Dried Chillies and Oleoresin Capsicum.
Chillies were ground to pass a sieve with 0.4 mm
apertures, and 5 g portions of ground material
were transferred to thimbles for Soxhlet extrac-
tion with methanol (120 ml), which was con-
tinued for 6 h. The extracts were concentrated to
about 50 ml, transferred through cottonwool fil-
ters into 100 ml volumetric flasks, and diluted to
volume with methanol rinsings.
Oleoresins (1.0 g) were dissolved in 100 ml of
CH30H-tetrahydrofuran (5050 or 80:20).
(b) Dried Ginger and Oleoresin Ginger.
Ginger was ground to pass a sieve with 1 mm
apertures, and 1.O g portions were transferred to
100 ml volumetric flasks. Methanol (ca. 99 ml)
was added up to the graduation, and the flasks
were stoppered and shaken vigorously. After 2 h,
they were again shaken and then left overnight
for solids to settle. Next day, a 20 ml aliquot of
supernatant solution was carefully withdrawn
from each flask and concentrated to 5.0 ml with
the aid of a vacuum rotary evaporator (water bath
temperature not exceeding 40°C).
Oleoresin (0.5 g) was simply dissolved in
methanol (100 ml).
HPLC Analysis
Mobile Phase. Acetonitrile was filtered through
a type FH Millipore filter (0.5 pm pores). Glacial
acetic acid was mixed with HPLC grade water in
the proportions 1:99 by volume, and the resulting
solution was filtered through a type H A filter
(0.45 pm pores). The filtered organic and aque-
ous components were mixed in the following
proportions: CH3CN-1% acetic acid (50:50)for
capsaicinoid determinations (mobile phase A);
CH3CN-1% acetic acid (65:35) for gingerol
determinations (mobile phase B).
After mixing, the mobile phase was degassed
under vacuum with magnetic stirring for 5 min.
Chromatography. Analyses were carried out on
ready-packed columns (250 x 4.6 mm) of 5 pm
Spherisorb ODS-2 (Phase Separations Ltd, UK),
without the use of guard columns. Separate col-
ums were reserved for capsaicinoid and gingerol
determinations. ODS-2 is a fully-capped CIS
bonded-phase packing.
Flow rates were 1.5 and 1.0 ml min-' for cap-
saicinoid and gingerol determinations respec-
tively. The injection volume was 2 0 p l and the
 PUNGENT PRINCIPLES OF CHILLIES AND GINGER
detection wavelength normally 280 nm (excep-
tions are indicated where appropriate).
Peak integration was on a ‘baseline-to-baseline’
basis, with the start of integration delayed for 3-4
minutes to exclude any negative peaks arising
from the injection solvent (normally methanol).
Run times were typically 21 min and 17 min
respectively for chilli and ginger samples, and
about 10 min for the CVA standard solution.
Ideally, each sample solution was injected
twice, and the CVA standard solution at least
three times, during each day’s run. In the case of
dried ginger, both the primary extract (10 mg of
sample per ml) and the concentrated extract
(40 mg ml- ’) were analysed, to allow quantitation
of each pungent principle at an appropriate con-
centration level.
Column Maintenance. The columns were
washed with CH3CN (100-200 ml at 2 ml min-I)
after 2 or 3 days of use. If serious loss of resolu-
tion or peak symmetry was encountered, or
operating pressures became too high, a CH3CN
wash was carried out with the column inverted
(back-flushing).
Quantitation. The concentration of each ana-
lyte (capsaicinoid, gingerol, or shogaol) in each
sample solution (in mg ml-I) was calculated as
follows: analyte concentration = analyte peak
area/mean CVA peak area for standard solution
x CVA concentration in standard solution x
analyte mol. wt./CVA mol. wt. From these val-
ues, the concentrations (% m/m) in the original
sample were calculated, with correction to a
moisture-free basis in the case of the dried spices.
RESULTS AND DISCUSSION
Choice of Standard
Zingerone, a decomposition product of the
gingerols which occurs in ginger oleoresins and is
available commercially in crystalline form, at first
seemed a likely candidate. However, in mobile
phase B it was eluted very near the injection
solvent, making accurate measurement of its peak
area impossible. In contrast, CVA was found to
have ideal retention properties, being eluted well
after the injection solvent but before any of the
other capsaicinoids in mobile phase A, and hav-
ing almost the same retention time as [6]-G in
mobile phase B.
A specimen of commercial NVA was found to
5
contain several impurities even after recrystalliza-
tion, and was not considered further as a stan-
dard. A specimen of commercial capsaicin (nomi-
nally 80-85%) was found to contain DHC as the
only major impurity, and after a single recrystal-
lization from hexane was suitable for use as a
‘second-best’ standard, the proportions of C and
DHC being easily determined from the area ratio
of their HPLC peaks.
Basis of Quantitation
Solutions of CVA, C, Zingerone, [4]-P, and
[6]-S were prepared in HPLC mobile phase (A or
B as appropriate) and their molar absorptivities
( E ) were measured in a spectrophotometer set at
280 nm. The mean of these E values was 2960,
with a relative standard deviation of ?1.7%.
HPLC response factors (with peak areas cor-
rected where necessary to an abscissa scale of
1 min 1 ml), were determined for CVA, [4]-P,
[ 81-P, and [614 on instrumental assembly(1)
with detection at 280 nm and with mobile
phase B (except that for [8]-P an 80:20 mixture
of CH3CN and 1% acetic acid was used). The
overall mean value (in millions of integrator units
per nmol) for all four compounds was 3.72, with a
standard deviation of 20.06 (between com-
pounds). The mean value for CVA alone was
3.75, with a standard deviation of 20.06 (be-
tween injections, n = 9). Similar measurements
with CVA, C and DHC in mobile phase A on
instrumental assembly(2) yielded an overall mean
value (millions of integrator units per nmol) of
0.577, with a standard deviation of +0.007 (be-
tween compounds). The mean value for CVA
alone was 0.573, with a standard deviation of
+0.009 (between injections, n = 5).
These data provided strong support for the
assumption of equal responses per nmol at
280 nm for a given mobile phase and detection
system. Further support was sought by analysing a
solution of a purified natural capsaicinoid mixture
by both HPLC and GLC. The GLC method
involved conversion of the individual capsai-
cinoids to the methyl esters of their side-chain
acids, and was a modification of the procedure of
Jurenitsch and Leinm~eller.’~ The total capsai-
cinoids content of the mixture as determined by
HPLC, on the basis of equal responses per nano-
mole and with CVA as external standard, was
97.8% m/m (purity claimed by supplier: ca. 95%).
The percentages of individual capsaicinoids
6 A . B. WOOD

Table 3. Composition of a purified natural capsaicinoid mixture by GLC and HPLC methods
Method Individual capsaicinoids as percentage of total

Derivatization/GLC

Direct HPLC
CVA

0.2
0.2
NDHC NVA

8.8
8.4
--
1.4
C

48.3
48.8
DHC
46.9 37.9
DVA
1.3
40.4
40.1
HC
1.2
HDHC

2.3
2.5

within the total as determined by the HPLC and
GLC methods are compared in Table 3, and the
agreement between the two methods is suffi-
ciently close to provide further validation of the
equal HPLC response assumption for the cap-
saicinoids.
Capsaicinoid Determinations
The chromatographic conditions used in this
work were similar to those described by Hoffman
et af.,' one difference being that the use of a guard
column was dispensed with in order to improve
resolution. The sample loading on the column was
lower than that used by Hoffman et a f . ,and it was
found that column performance could be
adequately maintained provided that the same
column was not used for ginger analysis, and that
the column was regularly washed with acetonitrile
(see Experimental section). Typical chromato-
grams of CVA, a chilli sample, a capsicum oleo-
resin, and a purified capsaicinoid mixture are
shown in Figure 2, and it can be seen that the
three major capsaicinoids (NDHC, C and DHC)
are well resolved from each other, with some of
the minor, capsaicinoids also being discernible.
Even I-IDHC can be determined in favourable
cases.
The extraction prodedure used for chillies was
that developed in the 1960s by the Phar-
maceutical Society/SAC Joint Committee for use
with their spectrophotometric difference
method,' since it was known that this procedure
extracted capsaicinoids exhaustively and because
it facilitated direct comparisons between the
HPLC and spectrophotometric methods. Chilli
extracts obtained in this way were normally ana-
lysed by HPLC without clean-up. Table 4 com-
pares the results obtained, for three chilli sam-
ples of differing pungencies, by three methods of
analysis: direct HPLC; HPLC after clean-up by
the ether-alkali extraction method;' and spectro-
photometric difference measurements' after the
same clean-up. There was fair agreement between
all three methods for these particular samples.
Sample 3 illustrates the difficulty which can be
experienced with the spectrophotometric method
in obtaining good agreement between determina-
tions at the two wavelengths. This may have
arisen from the relatively high pigment content of
this sample (ASTA colour value ca. 96).
The high recovery through the clean-up process
implied by the results in Table 4 does not always
seem to be attainable. The direct HPLC method
therefore has the advantage of avoiding the risk
of losses of capsaicinoids in addition to greatly
reducing the time required for analysis. Further-
more, information on the capsaicinpid composi-

Table 4. Comparison of HPLC and spectrophotometric analyses of chillies of

differing pungencies
~-
Total capsaicinoid content (%m/m) (on moisture-free basis)
By spectrophotometric
Sample no. By HPLC difference (after clean-up)
Without After
clean-up clean-up At 248 nm At 296 nm

1 1.51 1.44 1.45 1.47

2 0.89 0.90 0.91 0.91
3 0.28 0.28 0.28 0.24
 PUNGENT PRINCIPLES O F CHILLIES AND GINGER 7

3 4

3
I

t
0)

C
ln
ti 1 . 4

I/
0
Q
ln
(c) Attenuation 64 (d) Attenuation 32
z

1
0 5 10 15 20 min 0 5 10 15 20 min
Fig. 2. Chromatograms o f (a) a CHSOH extract of dried chillies (ca. 50 mg m1-l);(b) a purified natural capsaicinoid mixture
(1.0 mg ml-' in C2H OH); (c) a CVA standard solution (0.24 mg ml-' in CH30H); (d) a solution of a commercial oleoresin
capsicum [ 10 mg ml-' in CH3OH-THF (50:50)]. Instrumental assembly (2), mobile phase A at 1.5 ml min-', detection at
280 nm. Peak 1 = CVA, 2 = NDHC, 3 = C(+NVA), 4 = DHC, 5 and 6 = HC and DVA, 7 = HDHC 2, 8 = HDHC 1 (see
Table 1 for identification).

tion as well as the total content is obtained by the 51.1% was found for both total capsaicinoid con-
HPLC method. Although some of the minor c a p tent and total pungency, based on a single injec-
saicinoids are not fully resolved, their individual tion of each extract. In view of the errors inherent
pungencies are sufficiently similar, and their con- in determining the pungencies of the individual
tribution to the total sufficiently small, to enable a compounds, and of the fact that normally the
reasonably accurate calculation of the pungency mean values from two injections of each of two
of a sample to be made from the HPLC data on extracts would be taken for each sample analysed,
the basis of reported pun ency values for the pure this level of precision is very adequate.
5
individual capsaicinoids. This is illustrated for Capsicum oleoresins will not usually dissolve
one chilli sample in Table 5 , which also shows the completely in methanol, and it was found neces-
results of replicated extraction and analysis of the sary to add an ether to obtain satisfactory recov-
same sample. A relative standard deviation of ery of the capsaicinoids. This made meaningful
8 A. B. WOOD

Table 5 . Precision of determination of pungency of a chilli sample by direct HPLC. (Extraction

replicated six-fold; each extract injected once. Pungencies of individual capsaicinoids are mean values
from references 2 and 3)
Nordihydrocapsaicin Total
and
Capsaicinoid Capsaicin Dihydrocapsaicin minor capsaicinoids capsaicinoids

Mean concentration in
moisture-free sample
(%m/m) 0.496 0.322 0.072 0.89

Standard deviation 20.007 ?0.005 20.007 20.01

Relative standard deviation of total capsaicinoid content 21.1%

Pungency of pure 16.6 x lo6 13.4 x lo6 8.9 x lo6 -

capsaicinoid (Scoville
units)

Contribution to 82 300 43 100 6 400 131 800

pungency of sample (21200) (2700) ( 5600) (21500)
(Scoville units)
(moisture-free basis)

Relative standard deviation of total pungency 21.1%

comparison between the HPLC and spectro-
photometric difference methods impossible in the
case of oleoresins, since the presence of the ether
caused losses of capsaicinoids and inadequate
removal of co-extractives in the clean-up proce-
dure used with the latter method. Two capsicum
oleoresin samples of very different ASTA colour
values were analysed by the HPLC method, using
both 80:20 and 50:50 mixtures of methanol and
tetrahydrofuran to dissolve them. No difference
between the two solvent mixtures was found in
respect of apparent capsaicinoids content but the
capsaicinoid peaks tended to be sharper with the
lower tetrahydrofuran content. The results of
these analyses, including six-fold replication for
one of the oleoresins with the 80:20 solvent mix-
ture, are presented in Table 6. A relative stan-
dard deviation of k0.896 was found for total
capsaicinoids content, based on mean results from
duplicate injections of each solution, again an
adequate level of precision.
Gingerol Determinations
The chromatographic conditions used were
adapted from those described by Smith, 7the prin-
cipal difference being the addition of acetic acid
rather than a buffer salt to the mobile phase to
suppress ionization of the gingerols. These condi-
tions gave better resolution of the gingerols than
several acetonitrile-water mixtures tried with var-
ious CB and CIS columns. As with the capsai-

Table 6. HPLC analysis of capsicum oleoresins dissolved in two solvent mixtures. (Relative standard
deviation (r.s.d.) values for the six-fold replicated analysis of oleoresin no. 2 are based on duplicate
injections of each solution)

Capsaicin Total capsaicinoids

ASTA Solvent No. of
colour mixture solutions mean r.s.d. Mean r.s.d.
Olcoresin no. value (CH,OH-THF) analysed %m/m %m/m
~~~ ~

1 ca. 790 50:50 4 1.57 - 3.63 -

80:20 2 1.57 - 3.64 -
-
2 ca. 60 50:50 2 3.18 - 5.85 -
80:20 6 3.77 ?0.5% 5.85 ?0.8%
 PUNGENT PRINCIPLES OF CHILLIES AND GINGER 9

cinoids, a guard column was not used, adequate
column performance being maintained by regular
washing with acetonitrile as described in the
Experimental section.
Chromatograms of a commercial ginger oleore-
sin and a dried ginger sample are shown ,in Fig-
ure 3. The former was run when the column was
almost new, the latter after it had been used for
I 5 10 15 20 min
Fig. 3. Chromatograms of: (a) a concentrated CH3OH laboratory, for dried ginger samples from several
extract of dried ginger (ca. 40 mg mr'); (b) a solution of a countries. For newly dried ginger, the ratio [6]-
commercial oleoresin ginger (5 mg mT' in CH30H). G : [6]-S gives some indication of how carefully
Instrumentd assembly (2), mobile phase B at 1.0 ml min-',
detection at 280 nm.Peak 1 includes zingerone, 2= [6]-G, 3=
_181-G.
- . 4= _1614.
- - 5= -1101-G: CVA has almost same retention dehydiates the gingerols to the corresponding
time as [6]-G in this sysiem-(see Table 1 for identification). shogaols. The values in Table 10 (10-50) suggest
about 13 months. Although some peak broaden-
ing and tailing is apparent in the dried ginger
chromatogram, [6]-G, [8]-G, [6]-S, and [ 101-G
(in retention time order) are still clearly sepa-
rated.
Chromatograms of a solution of ginger volatile
oil, at about the concentration likely to be present
in the ginger extracts, were obtained at three
detection wavelengths. These showed that the
most important interference from the volatile oil
would be that of the citrals (neral and geranial)
with the [8]-G peak, and that this would be
serious near 240 nm, and negligible at 290 nm.
Some interference appeared to be possible at
280 nm, so a ginger sample was analysed at the
same three wavelengths. The [6]-G and [ 101-G
reults were virtually unchanged with changing
wavelength, suggesting that these peaks were vir-
tually homogeneous. In addition, there was no
significant difference between the results for
[8]-G at 280 and 290 nm, indicating minimal
interference from the citrals at 280 nm. A solu-
tion of [6]-G, which had been calibrated against
CVA at 280 nm, was used as standard in these
determinations.
More serious, and unidentified, interferences
with [8]-G and [lo]-G were experienced with an
aged commercial sample of ginger oleoresin, so
that only [6]-G and [6]-S could be reliably
determined in this product.
The extraction method adopted for dried
ginger was compared with a more exhaustive
method, viz. three successive extraction/filtration
steps with combination of the filtrates. The results
in Table 7 show that there was no significant
difference in recoveries between the two
methods.
Tables 8 and 9 show the results of replicated
analyses of samples of dried ginger and ginger
oleoresin. The precision for [6]-G is adequate in
both cases, as is that for [6]-S in the oleoresin
sample. The precision for total gingerols in the
case of dried ginger is acceptable when allowance
is made for the fact that in routine analysis both
extraction and injection would be duplicated.
Finally, Table 10 shows the range of pungent
principle contents found, by HPLC analysis in this
the drying has been carried out, since overheating
10 A. B. WOOD

Table 7. Comparison of two extraction methods for HPLC analysis of a sample of dried
ginger (using instrumental assembly (1))

Apparent concentration in moisture-free sample (%m/m)

(range of results from four determinations by each method)

Method [ 61-Ginger01 [81-Ginger01 [ 101-Ginger01 [ 61-Shogaol

Single extraction 1.91-1 96 0.34-0.42 0.40-0.43 0.22-0.3 1

with cold methanol

Three successive 1.92-1 ‘99 0.3 1-0.38 0.36-0.42 0.20-0.29

extractions with
cold methanol

Table 8. Precision of determination of major pungent principles of a dried ginger sample by direct HPLC. (Extraction
replicated 10-fold; each extract injected once using instrumental assembly ( I ) )

Total Total G
Pungent principle [6]-Gingerol [81-Ginger01 [ 101-Ginger01 gingerols [ 61-Shogaol +[ 61-S

Mean concentration in 1.66 0.31 0.38 2.35 0.17 2.52

moisture-free sample
(%m/m)
Standard deviation 20.02 20.02 t0.09 20.11 20.03 z0.12

Relative standard deviation 2 1.2% t4.7% 24.8%

‘Table 9. Precision of determination of [6]-gingerol and these ranges show, total G varies considerably
[6]-shogaol in a sample (aged) of commercial ginger between varieties and origins, as well as being
oleoresin by HPLC. (Six solutions analysed; each injected affected by the drying and ageing processes.
twice using instrumental assembly (1). Unidentified
interferences prevented accurate determinations of l8J-G
and [ 101-G)
CONCLUSIONS
Pungent principle [61-Gingerol [ 61-Shogaol
The easily synthesized and purified minor capsai-
Mean concentration 5.94 5.96 cinoid CVA is well suited for use as an external
(Nm/m) standard in the determination of the pungent
Standard deviation ~0.04 20.07 principles of chillies and ginger by reversed-phase
~~~ ~
HPLC with UV detection at 280 nm. Methods
Relative standard 20.7% 21.2% based on this principle, and not reqiring sample
deviation clean-up, have been developed for chillies, ginger,
and their oleoresins, and shown to be capable of
that most of these samples had been carefully acceptable precision, with the possible exception
dried. The ratio tends to fall as the dried ginger of [ 81-G and [ 101-G determinations for oleoresin
ages. Steinegger and Stuckis found it varying be- ginger.
tween 3.2 and 9.9 in dried gingers examined by For chilli analysis, further validation of the
them, except for a 50-year-old sample in which it HPLC method was provided by comparison with
had fallen to 0.6. In contrast, fresh ginger samples the spectrophotometric difference method. The
which they examined (after freeze-drying) had method reported here is not suitable for detecting
ratios between 17 and 219. The total G concen- the adulteration of chilli products with synthetic
trations in Table 10 fall within the range NVA, since this compound is co-eluted with C.
(1.08-2.98%) found by Steinegger and Stucki for The only HPLC methods suitable for this purpose
fresh ginger samples, and are substantially above are those employing complexation with Ag’ ions
their values for dried ginger (0.65-1.17%). AS (added to the mobile p h a ~ e ) , ~which . ’ ~ also gives
 Table 10. Results of HPLC analysis of various dried ginger samples

Concentration in moisture-free Individual gingerols

sample (% m/m) as percentage of total G
Country of Sample Ratio
origin Type no. Total G [6]S Total G + [6]S [6]-G/[6]-S [6]-G [8]-G [10]-G

Jamaica Sliced, 1.87 0.04 1.91 30 65.2 12.8 21.9

artificially dried I: 1.56 0.03 1.59 34 64.7 14.1 21.2
Sliced, sun-dried 1.80 0.04 1.84 30 65.6 13.9 20.5
1: 1.94 0.06 2.00 23 69.6 12.9 17.5

Dominican Sliced, artificially

Republic dried 5 1.55 0.02 1.57 50 63.9 13.5 22.6
(experimental scale)

Ivory Coast Sliced, artificially 6 1.51 0.04 1.55 27 71.5 10.6 17.9
dried

Malawi Split, atificially 7 2.74 0.13 2.87 15 69.0 11.3 19.7

dried 8 2.54 0.14 2.68 13 73.2 13.0 13.8
9 2.35 0.17 2.52 10 70.6 13.2 16.2 >
z
India Dried whole 10 1.50 0.06 1.56 21 82.0 10.7 7.3 W
(Cochin) rhizomes 9
~
z
Peru Sliced, artificially 11 1.47 0.06 1.53 15 61.9 15.6 22.4
dried 12 1.48 0.04 1.52 25 68.2 14.9 16.9
8P
12 A. B. WOOD
more complete separation of other minor capsai-
cinoids. First indications are that the basis of
quantitation described here is at least approxi-
mately valid with this type of HPLC when detec-
tion is at 280 nm. However, the need for pre-
cautions to avoid silver deposition in the
apparatus makes Ag’ complexation HPLC less
convenient in routine use than the proposed
method which, although it does not resolve all of
the minor capsaicinoids, permits at least the esti-
mation of NDHC, C, and D H C in addition to the
determination of total capsaicinoids. These four
parameters appear to be of some value in the
chemotaxonomic classification of capsicums,Is
and enable a predicted Scoville index to be cal-
culated. Comparison of pungencies so calculated
with those determined sensorially would be inter-
esting, but has not yet been carried out in this
laboratory.
One of the methods used hitherto for assessing
the pungency of ginger has been to determine its
non-volatile ether extract (NVEE). In this inves-
tigation, only a tenuous correlation was found
between NVEE and pungent principles content.
When the NVEE from one sample was re-
dissolved in methanol and analysed by HPLC, it
was found that only about 60% of the pungent
principles in the ginger had been recovered, and
that these accounted for only about 20% of the
NVEE. It would be interesting to compare results
obtained by the HPLC method with those from
organoleptic testing. Denniff et a1.,I6 have shown
that synthetic [6]-, [8]-, and [ 101-gingerols all
have pungencies in the region of 100 000 Scoville
units (S.U.), with [6]-shogaol having a pungency
of about 130 000 S.U. The dried ginger samples
whose gingerol contents are shown in Table 10
would thus be expected to have pungencies in
the range 1 500-3 000 S.U. (moisture free basis).
In the case of ginger oleoresins, which have an
appreciable content of zingerone, the proposed
method might not give an adequate estimate of
pungency, since this compound is obscured by
co-extractives. Zingerone has been shown to
have a pungency of about 50 000 S.U.” Chen ef
d.,”carried out a detailed study of a liquid
carbon dioxide extract of ginger and found, in
addition to [ 61-, [8]-, and [ 101-G, small amounts
of [12]-G, methyl [6]-G, methyl [8]-G, and
methyl [ 101-G. These additional compounds,
however, accounted for only about 2.4% of the
total gingerols, and it is probable that they do not
contribute greatly to the overall pungency.
Acknowledgements-I should like to thank: Dr J. Jurenitsch
of the University of Vienna, Austria, for gifts of pure capsaicin
and dihydrocapsaicin; Mr A. M. Humphrey of Bush Boake
Allen Ltd, UK, for gifts of [4]- and [8]- paradols; and my
colleagues as follows: Dr D. R. Hall for synthesizing the CVA
used in this investigation, Ms M. L. Barrow and Mr D. J.
James for carrying out much of the analytical work, and Ms
J. M. Robinson for mass spectrometry of the isolated [6]-
gingerol and [6]-shogaol.
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