C H A P T E R
109
Assay of Kahweol and Cafestol in Coffee
Marta de Toledo Benassi1, Rafael Carlos Eloy Dias2
1Department of Food Science and Technology, Universidade Estadual de Londrina, Londrina, Paran, Brazil;
2Department of Chemistry, Instituto Federal Catarinense Cmpus Araquari, Araquari, Santa Catarina, Brazil
List of Abbreviations
max Wavelength of maximum absorption
cv. Coffee cultivar
CV Coefficient of variation
EI Electron impact ionization
GC Gas chromatography
HPLC High performance liquid chromatography
MS Mass spectrometry
MS/MS 2nd fragmentation of MS
MTBEMethyl tert-butyl ether
NMR Nuclear magnetic resonance spectroscopy
SPE Solid phase extraction
TMS Trimethylsilyl derivatives
109.1INTRODUCTION
Kahweol and cafestol are the main representatives of
the diterpenes class in coffee. They are produced only
by plants of the Coffea genus; cafestol is found in both
Coffea arabica and Coffea canephora; however, kahweol is
largely specific to arabica. They are classified as pentacy-
clic diterpenes alcohols based on the fusion of isoprene
(C5) to form a 20-carbon kauran skeleton. Kahweol dif-
fers from cafestol by a double bound between the C1 and
C2 carbon atoms, leading to a spectrum with maximum
absorption at different wavelengths: 230nm for cafestol
and 290nm for kahweol (Figure 109.1). Both compounds
exhibit low stability to heat, acids, and light, and kah-
weol is fairly instable in the free form. Because of the
small difference in their structures, it is difficult to sepa-
rate these compounds using a chromatographic process.
Other derivatives of these diterpenes, which occur
at lower levels, such as 16-O-methylcafestol and
16-O-methylkahweol are also mentioned. The first one
is described as exclusive to the C. canephora and Coffea
liberica species and is thermally stable. 16-O-methylca-
festol was proposed as a discriminator of arabica and
canephora species in commercial roasted coffees.15
Coffee in Health and Disease Prevention
http://dx.doi.org/10.1016/B978-0-12-409517-5.00109-1 2015 Elsevier Inc. All rights reserved.
Diterpenes are rarely present in the free form (less
than 4%); they are more frequently observed as fatty
acid monoesters esterified at the C17 hydroxyl group.
Up to 14 different fatty acids esters derived from cafestol
and 12 from kahweol are present in coffee beans; esteri-
fication with palmitic and linolenic acids are the most
common.68
During the roasting process, dehydrated byproducts
of kahweol and cafestol (dehydrocafestol and dehydro-
kahweol) are formed.711 Functional isomers have also
been detected, and the structures of degradation prod-
ucts as cafestal, kahweal, isokahweol, and dehydroiso-
kahweol have been elucidated.8
Interest in analyzing diterpenes has increased recently
due to their impact on health and the variability in their
contents when different methods of brew preparation are
used. Moreover, the possibility of using the diterpenes as
discriminators of the coffee species in commercial prod-
ucts (e.g., on the detection of accidental or fraudulent
addition of canephora to arabica coffee) and the interest
in elucidating the metabolic pathways involved in bio-
synthesis of diterpenes in plant and fruit tissues during
their development are also reasons to quantify kahweol
and cafestol in a wide range of coffee matrices.
In addition to the convenience of having a fast, robust,
and cost-effective method to evaluate diterpenes, the
method must be versatile, considering its possible appli-
cation to a variety of matrices. To conduct research in
different areas, it is important to establish effective meth-
odologies applicable to different coffee varieties and
species as well as different coffee tissues, green beans,
roasted or instant products, and coffee brews.
Several methods have been proposed for the iden-
tification and quantification of kahweol and cafestol.
Reports in the literature describe the use of spectromet-
ric techniques, such as spectrophotometry and Raman
spectroscopy, and gas chromatography (GC). However,
993
994 109. ASSAY OF KAHWEOL AND CAFESTOL IN COFFEE
FIGURE 109.1 Kahweol and cafestol spec-
tra (190400nm) (A) and structures (B), and
a typical chromatogram of roasted arabica
coffee (light roast degree) (C). Characteristics
(UV spectra and structures) and a chromato-
gram showing the separation of kahweol and
cafestol by HPLC. Unpublished. HPLC, high
performance liquid chromatography.
the current emphasis is on the use of high performance
liquid chromatography (HPLC). Mass spectrometry
(MS) and even nuclear magnetic resonance spectroscopy
(NMR) have been used in conjunction which other tech-
niques to elucidate the structures and confirm the iden-
tity of the compounds.
109.2 SAMPLE PREPARATION
AND EXTRACTION METHODS
Extraction with organic solvents is widely used to
determine the total lipids in food. However, it is a critical
step for analysis of lipid components. During the extrac-
tion, only a physical process is expected to occur, i.e., the
transfer of soluble constituents from an inert material to
a solvent with which the matrix is in contact. The lipids
must be recovered with no significant chemical changes,
particularly when the objective is to analyze specific
lipid-containing compounds.
The Soxhlet method, a classical approach, is recom-
mended by the Association of Analytical Communities
International. Lipids from solid material are extracted
by repeated percolation of an organic solvent in a heated
reflux in a Soxhlet device, followed by solvent removal.
The residue is composed of all of the components that can
be extracted under determined conditions and in a par-
ticular solvent, including free and esterified fatty acids,
waxes, pigments, vitamins, essential oils, phosphatides,
esterols, and diterpenes. The long period of heat expo-
sure (generally from 5 to 8h) may cause peroxidation and
hydrolysis reactions that could affect further analytical
results. Thus, cold extraction is an alternative approach.
One option is the Bligh Dyer method, a simplified version
of the well-known procedure proposed by Folch etal.,12
which consists of cold extraction with a mixture of chlo-
roformmethanol. Bligh Dyer extraction is faster than
IV. ANALYSIS AND METHODS
Soxhlet extraction, does not require special equipment or
lab devices, and does not involve heating, thereby avoid-
ing the degradation of thermosensitive compounds. Both
extraction procedures have been applied to extract coffee
lipids, aiming the analysis of diterpenes.1,5,11,1318
In addition to these more conventional techniques, alter-
native methods based on solvent volume reduction have
been proposed for lipid extraction, such as solid phase
extraction (SPE), matrix solid phase dispersion, supercriti-
cal fluid extraction, and microwave-assisted extraction.
Despite environmental advantages and relative safety,
only a few studies of coffee lipids have used them.17,19
Different solvents have been proposed for the extrac-
tion of coffee lipids, such as diethyl ether, methyl tert-
butyl ether (MTBE), petroleum ether with different
boiling point ranges, n-hexane, and a mixture of diethyl
ether and n-hexane. Pettit Jr.1 suggested the use of petro-
leum ether.7,20,21 Because diterpenes are relatively polar
compounds, diethyl ether and MTBE were more efficient
than the usual nonpolar solvents, such as petroleum
ether and n-hexane.7 Urgert etal.20 used diisopro-
pyl ether to extract kahweol and cafestol. Silva etal.24
compared the efficacy of diethyl ether and MTBE, and
observed better recovery and faster extraction of diter-
penes in coffee brews using diethyl ether. In their com-
prehensive review on the lipid fraction of coffee, Speer
and Klling-Speer8 suggested that MTBE should be used
instead of the more hazardous diethyl ether. Dias etal.,21
comparing extractions using hexane (dipole moment
0.08 D), diethyl ether (1.32 D), and MTBE (1.15 D), also
observed better results with the last and chose MTBE for
its operational effectiveness in extraction and low vola-
tility compared to diethyl ether.
There are two general ways for extracting kahweol
and cafestol. Some methods involve lipid extraction as
a first step, followed by separation of the diterpenes.
The advantage lies in the preliminary refining for later
 109.3 SEPARATION, IDENTIFICATION, AND QUANTIFICATION OF DITERPENES
assessment with less interference, but it is more time con-
suming. The second alternative does not include previ-
ous lipid extraction; direct saponification of the sample is
performed with the advantage of reduced experimental
error and less degradation of the compounds of interest.
Since diterpenes are components of the lipid unsa-
ponifiable fraction and they are present in the esterified
form, the saponification procedure must be well estab-
lished. Kahweol and cafestol esters are converted to their
corresponding alcohols, usually with potassium hydrox-
ide in methanol or ethanol.
In some studies, the saponification reaction was not
applied, since the aim was to determine the naturally occur-
ring free diterpenes or the kahweol and cafestol esters.7,15,22
Determination of free and esterified forms requires the use
of a preliminary separation step (as gel permeation chro-
matography) to remove the interfering compounds, and
even cleaning using SPE is suggested. A detailed descrip-
tion of these procedures is available in the literature.23
To evaluate the total kahweol and cafestol contents
of coffee matrices, some studies used direct saponifica-
tion4,11,20,2430 and others used lipid extraction and subse-
quent saponification.1,5,9,1317,31
Among the authors who applied preliminary lipid
extraction, Arajo and Sandi17 reported that the Soxhlet
method (hexane at boiling point for 16h) was more effi-
cient for extracting diterpenes from green and roasted
coffee than the supercritical fluid (CO2) extraction proce-
dure described (best condition defined as 70C/371bar).
Scharnhop and Winterhalter9 proposed a Soxhlet
extraction with MTBE at 80C for 5h. Applying Fou-
rier-transform Raman spectroscopy for discriminating
coffee species, Wermelinger etal.18 suggested a Soxhlet
extraction with MTBE at 55C for 5h, and Rubayiza and
Meurens16 reported efficient extraction by Soxhlet with
diethyl ether (6h). A similar procedure was used by
Pacetti etal.,5 before saponification and derivatization of
the unsaponifiable fraction for GC analysis.
To obtain significant amounts of cafestol and kahweol,
Oigman etal.31 applied an exhaustive Soxhlet extraction
(hexane, 90C, 16h) followed by a saponification process.
Standards of diterpenes have a restricted commercial
availability and are expensive, so the authors proposed
an optimized microwave-assisted methanolysis protocol
to achieve a fast and high yield reaction for obtaining a
mixture of cafestol and kahweol from green coffee beans.
Direct saponification has been described as an effi-
cient alternative for extracting unsaponifiable com-
pounds from different matrices because it avoids the
formation of artifacts. It is also considerably faster and
requires smaller amounts of solvent than methods that
apply pre-extraction of the lipids. Either cold or hot
direct saponification processes have been proposed.
Urgert etal.20 and De Roos etal.4 used an ethanolic
solution of sodium hydroxide to saponify (80C, 1h) the
IV. ANALYSIS AND METHODS
995
lipids from green coffee beans. The unsaponifiable mat-
ter was extracted with diisopropyl ether, and the extract
was cleaned with water.
Direct saponification with 2.5M KOH solution (in
ethanol) (80C, 1h), then extraction with MTBE fol-
lowed by cleaning with water, was recommended by
Dias etal.27 The extract was used for the analysis of kah-
weol in roasted coffee using a spectrometric method21
and for quantification of kahweol and cafestol in fruit
tissues and leaves, green beans, and roasted coffee by
HPLC.2528,30
Zhang etal.29 used the saponification conditions
described by Dias etal.27 but employed diethyl ether
as the solvent instead of MTBE. Silva etal.24 proposed
a similar procedure (with diethyl ether) but used a 2M
NaCl solution for cleaning instead of water.
Dias etal.11 compared four methods for extraction of
diterpenes: two using direct saponification, cold or hot
process, and Bligh Dyer and Soxhlet methods followed
by further saponification. The contents of diterpenes and
their dehydro derivatives (dehydrokahweol and dehy-
drocafestol) in roasted coffee were compared. Direct hot
saponification was more efficient for extraction, providing
better separation of the chromatographic peaks and a con-
tent of diterpenes that was 15% higher than that obtained
using the cold saponification process and 85% higher than
that obtained by the Soxhlet and Bligh Dyer methods.
The use of additional clean-up procedures after lipid
extraction or of preliminary fractionation steps before
analysis was described by some authors.
Del Castillo etal.22 proposed an off-line HPLC step to
fractionate the coffee compounds into different classes
followed by GC analysis of the selected fractions. Pettit
Jr.1 described the use of a bonded preparative HPLC C8
column with a gradient of acetonitrile:water prior to CG
analysis.
Scharnhop and Winterhalter9 proposed extraction by
Soxhlet method and hot saponification (KOH ethanolic
solution, 90C, 4h), followed by a separation step using
high-speed countercurrent chromatography with hexane
ethyl acetateethanolwater mixtures. The authors
reported efficient extraction with subsequent analysis by
HPLC.
For coffee brew analysis, some authors also described
the use of SPE.32
109.3 SEPARATION, IDENTIFICATION,
AND QUANTIFICATION OF DITERPENES
Several spectrometric and chromatographic tech-
niques have been described for identifying and quanti-
fying diterpenes.
A methodology for quantifying kahweol based on
colorimetric reactions was proposed by Wurziger.33 The
996 109. ASSAY OF KAHWEOL AND CAFESTOL IN COFFEE
aim was to differentiate arabica and canephora species
in green and roasted coffees. A petroleum ether extract
was mixed with potassium iodide or hydrochloric acid
(two reaction methods were tested) in glacial acetic acid.
The reaction occurred between KI or HCl and the double
bond of kahweol (see Figure 109.1). In an acidic medium,
the reaction products absorb at 620nm. The difference in
the absorbance measurements at 290nm (max for kah-
weol) and 620nm (max for the reaction products) is due to
the kahweol content, which is high in arabica coffee and
nearly absent in canephora coffee. The method is inter-
esting but quite empirical and only semi-quantitative,
and there is no mention of its later use.
Based on the principles of the Wurziger technique,
Dias etal.21 developed a spectrophotometric method for
quantifying kahweol in roasted coffee. The extraction con-
ditions suggested by Dias etal.27 were applied. The best
results were achieved by reacting the extract with KI, dilut-
ing it with 5% acetic acid, and determining the absorbance
at 620nm. Good precision (coefficient of variation [CV]
below 5%) and recovery (116%), and low limits of detec-
tion (5.16mg 100/g) and quantification (17.2mg 100/g),
were observed. The kahweol contents determined were
similar to those obtained using an HPLC methodology.
Spectroscopic techniques were applied to distinguish
arabica and canephora coffees based on the kahweol
Raman bands.3,16 Characteristic bands of the arabica
spectra are associated with kahweol. The spectrum of
kahweol includes two Raman scattering bands at 1567
and 1478/cm, whereas the spectrum of cafestol exhibits
one scattering band at 1500/cm. These analyses usually
relied on statistical evaluation procedures, depended
on the calibration model, and no quantitative data were
presented. Wermelinger etal.18 described the use of the
intensity ratio between two Raman peaks, one character-
istic for kahweol and one characteristic for fatty acids,
to determine the content of canephora coffee in a blend.
Keidel etal.34 proposed a Fourier-transform Raman spec-
troscopic approach applicable to coffee beans and coffee
grounds that does not require the mechanical and chem-
ical pre-processing of samples. The aim was to create a
tool for classifying coffee beans and monitor its quality.
The relative contribution of kahweol was quantitatively
determined, resulting in an index value that is propor-
tional to the relative content of kahweol in the matrix.
GC1,4,5,14,20,22,35 and HPLC1,911,17,2332 are the most
frequently applied techniques for quantifying kahweol
and cafestol in roasted and green coffee beans and coffee
brews.
Chromatographic techniques have been commonly
used in conjunction with MS to elucidate the structures
and confirm the identity of the diterpenes and their
derivatives.1,4,5,811,14,20,23 Lercker etal.14 used electron
ionization (EI, 10mA, 70eV), but most of the research
applied the atmospheric pressure chemical ionization
IV. ANALYSIS AND METHODS
positive mode.4,5,911,23 Speer and Klling-Speer8 cited
the use of both ionization methods. In addition, some
articles also described the use of NMR as an auxiliary
technique to identify diterpenes and to help the elucida-
tion of the chemical structure of the compounds.1,9,31
Some authors used GC methods to analyze the fatty
acids esters of the diterpenes. Pettitt Jr.1 used a bonded
capillary column (15m) at 250C to analyze silyl deriva-
tives with detection by MS and confirmed the results
using NMR.
Regarding the esterified diterpenes, Lercker etal.14
analyzed trimethylsilyl derivatives (TMS) using a fused
silica capillary column (25m) with a stationary phase of
50% phenyl/50% methylpolysiloxane, a programmed
oven temperature (from 200 to 300C, 3C/min rate),
and a flame ionization detector (330C) coupled to a
EI-MS system.
Urgert etal.20 compared the diterpene composition
of coffee brews. After derivatization (with hexamethyl-
disilazane and trichloromethylsilane), a GC with flame
ionization detection (at 305C) coupled to a mass spec-
trometer was used to quantify kahweol and cafestol. A sil-
ica CP Sil5CB column (25m22mm) and a programmed
oven temperature (from 200C to 285C) were applied.
The authors reported CV below 6.3% and good recovery
(between 99% and 103%), for both compounds. The same
chromatographic conditions were used by De Roos etal.4
Pacetti etal.5 proposed an authentication method for
determination of the roasted coffee blend composition
based on the ratio of the absolute GC peak areas of the
TMS derivatives of kahweol and 16-O-methylcafestol.
A DB-5 MS fused silica capillary column (30m25mm)
with a programmed temperature (from 100C to 325C,
at a rate of 5C/min) and an MS detector were used.
Although highly used, the application of GC for diter-
pene analysis has been disputed. Speer and Klling-
Speer8 questioned the presence of dehydro diterpenes
in green beans, considering that they could be artifacts
developed during GC analysis, emphasizing the risk of
thermal degradation.
More recently, a large number of investigations
reported the use of HPLC.
To identify the cafestol and kahweol esters in arabica
coffee, Kurzrock and Speer23 used a Nucleosil 120-3 C18
column, an isocratic elution with acetonitrile:isopropanol
(70:30, v/v; flow rate of 0.6ml/min), UV detection
(220nm), and MS. Recovery of 94103% of the cafestol
esters was described.7
The chromatographic conditions for HPLC analysis of
esterified diterpenes are shown in Table 109.1.
As stationary phase, reverse phase C18 columns
have been used. Isocratic or gradient elution with
acetonitrile:water was the most usual mobile phase. UV
detection was carried out at 220/230nm for cafestol and
at 220/280/290nm for kahweol9,15,24,27,31,36,37 (Table 109.1).
 109.3 SEPARATION, IDENTIFICATION, AND QUANTIFICATION OF DITERPENES 997
TABLE 109.1 Sample Preparation and Chromatographic Conditions for HPLC Analysis of Kahweol and Cafestol in Coffee Matrices
Sample Stationary
Sample Preparation Phase Mobile Phase Detection Obs References

Green coffee beans Extraction with Nucleosil 1203 Isocratic elution. UV 220nm Run time: 50min 15
MTBE (Soxhlet, (2504.6mm, Acetonitrile:water Rt: 22 and 23min for
5h); 5m) (50:40v/v). kahweol and cafestol
Clean up in FR: 0.6ml/min Method for free
a silica gel diterpenes
cartridge (Bio Recovery: 80.5% for
Beads S-X3) cafestol

Green coffee beans Extraction with Synergi Gradient elution. UV 280nm 9

(Coffea arabica, Coffea MTBE (Soxhlet, MaxRP 80 A Acetonitrile (A): Water (kahweol)
canephora) 80C, 5h); (2504.6mm, (B): 0min, 60%A; and 220nm
saponification 5m) 20min, 70%A; 40min, (cafestol)
(KOH, 90C, 4h) 100%A; 45min, 100%A.
FR: 0.8ml/min

Leaves, coffee tissues Direct Spherisorb ODS Isocratic elution. UV 290nm Run time: 20min 27 also
(perisperm, endosperm, saponification 1 (2504.6mm, Acetonitrile:water (kahweol) Rt: 16 and 17min for 10,25,28,30
pericarp), green and (2.5M KOH in 5m) (55:45v/v). and 220nm kahweol and cafestol
roasted coffee (different ethanol/80C, FR: 0.9ml/min (cafestol) Recovery: 99% and
degrees) 1h); extraction 94% for kahweol and
C. arabica, C. canephora, with MTBE, clean cafestol;
commercial roasted up with water Range: 501000mg
coffees 100/g; repeatability:
CV<6%;
DL: 2.3 and 3.0mg
100/g for kahweol
and cafestol

Plant parts (leaves, Extraction Nucleosil 100-5 Isocratic elution. UV 280nm 36

flower, endosperm, with MTBE (2504.6mm) Acetonitrile:water: (kahweol)
embryos), seedling, (Soxhlet, 4h); glacial acetic acid and 220nm
invitro cultures, green saponification (70:29:1v/v) (cafestol)
and roasted beans, coffee
brews
Several Coffea species
(C. arabica, Coffea
bengalensi, C. canephora,
Coffea dewevrei, Coffea
kapataka, Coffea salvatrix)

Roasted beans, coffee Extraction Nucleosil 120-3 Isocratic elution. UV 290nm 37

brews with MTBE (2504.6mm) Acetonitrile:water: (kahweol)
(Soxhlet, 6h); Glacial acetic acid and 230nm
saponification (70:29.5:0.5v/v). (cafestol)
FR: 0.6ml/min

Coffee brews Direct LichroCART Gradient elution. UV 220nm Run time: 30min 24
(commercial roasted saponification RP18 Acetonitrile (A): Rt: 6.9 and 7.2min
and instant coffees) (2M KOH/80C, (2504.6mm, water (B): for kahweol and
1h); extraction 5m) 010 min, 60% a; cafestol
with diethyl 15 min, 90% a; Validation just for
ether, clean up 1520 min, 90% a; cafestol:
with 2M NaCl 35 min, 60% a. Recovery: 96100%
FR: 1ml/min Range: 2.5250mg/l;
Repeatability:
CV<3%;
DL: 0.01mg/l

Commercial green Extraction Zorbax Isocratic elution UV 220nm Run time: 10min 31
coffee beans with hexane Eclipse XDB Methanol: Rt: 5.4 and 5.8min
(C. arabica) (Soxhlet, 16h); (1504.6mm, water (85:15, v/v). for kahweol and
saponification 5m) FR: 0.7ml/min cafestol

Describes the conditions to analyze kahweol and cafestol in coffee by HPLC. Unpublished. FR, flow rate; Rt, retention time; DL, detection limit, QL, quantification
limit; MTBE, methyl tert-butyl ether; HPLC, high performance liquid chromatography; CV, coefficient of variation.

IV. ANALYSIS AND METHODS

998 109. ASSAY OF KAHWEOL AND CAFESTOL IN COFFEE

Only a few studies reported validation data. Klling-
Speer etal.15 described recovery of 80.5% for cafestol.
Silva etal.24 reported good recovery (from 96% to 100%)
and repeatability (CV<3%) and a detection limit of
0.01mg/l for cafestol.
Dias etal.27 developed a reverse-phase HPLC method
for quantification of diterpenes in coffee fruit tissues,
leaves, and roasted coffee beans. Good recovery (99% for
kahweol and 94% for cafestol) and repeatability (CV<3%)
were obtained. Detection limits of 2.3 and 3.0mg 100/g
were observed for kahweol and cafestol, respectively.
Considering the confirmation of identity using MS,
Dias etal.11 proposed the MS parameters similar to the
described by Kurzrock and Speer,23 as follows: positive
mode; corona current of 4.0mA; source temperature of
450C; dry nitrogen gas; temperature, 350C; flow rate,
4l/min; nebulizer pressure, 60psi; MS/MS fragmentation
energy, 1.4V. The mass spectra were acquired over a mass-
to-charge ratio (m/z) scan range of 100700 (scan mode).
Scharnhop and Winterhalter9 proposed as MS parameters:
positive ion mode; capillary, 2500V; capillary exit offset,
70V; end plate offset, 500V; skimmer 1, 25V; skimmer 2,
10V; dry gas, N2, 11l/min; dry temperature, 355C; nebu-
lizer, 60psi; and scan range, 502200 m/z. Some works do
not report complete information about MS parameters.
Cafestol (C20H28O3, 316g/mol) was characterized by
the molecular ion peak [M+H]+ at m/z 317 and MS/MS
fragments at m/z 299 and m/z 281 due to the loss, respec-
tively, of one and two molecules of water and another
MS/MS fragment at m/z 147 [M+HC10H18O2]+.
A fragment at m/z 133 was also reported, referring
to [M+HC11H12O2]+ ion (Figure 109.2). Kahweol
(C20H26O3, 314g/mol), which contains two less hydro-
gen atoms than cafestol (Figure 109.1), exhibited analo-
gous fragments, with 2 u less than the fragments of
cafestol1,9,11,23 (Figure 109.2).
109.4 OCCURRENCE OF KAHWEOL
AND CAFESTOL IN DIFFERENT COFFEA
MATRICES
The contents of kahweol and cafestol were reported in
several coffee matrices (Tables 109.2109.4).
Only a few reports of the levels of diterpenes in cof-
fee plants are available.27,36,38 Low contents of these com-
pounds are described in coffee flowers of the arabica
and canephora species (up to 3.1mg 100/g). The cafes-
tol contents ranged from 45 to 125mg 100/g in arabica
leaves, whereas divergences were observed for contents
in canephora leaves (from not detected to 242mg 100/g).
Kahweol was not detected or present in small amounts
(up to 17.5mg 100/g) in the leaves of the two species
(Table 109.2).
Kahweol was almost absent in the tissues (pericarp,
perisperm, and endosperm) of canephora coffee. The
highest concentrations of kahweol were observed in
the endosperm of more developed fruits (up to 589mg
100/g) and the perisperm of immature arabica fruit
(up to 516mg 100/g). Cafestol was present in peri-
carp, perisperm, and endosperm (in ascending order
of concentration), with higher values reported for
arabica coffee. The highest level of cafestol (1022mg
100/g) was detected in the endosperm of C. arabica,

FIGURE 109.2 Structures (A) of molecular ion peak (1), base peak (2) and typical fragments (3) of the first and second-order mass spectra (B) of
kahweol (peak 1, Figure 109.1(C)). Mass spectrograms and structures of characteristic ions of kahweol produced by mass spectra analysis. Unpublished.

IV. ANALYSIS AND METHODS

 109.4 OCCURRENCE OF KAHWEOL AND CAFESTOL IN DIFFERENT COFFEA MATRICES 999
and the great variation observed27,36 may be ascribed in the range of 110250mg of cafestol 100/g have been
to the diversity in the ripening stage of the products reported1,4,17,30,36,39,40 (Table 109.2).
(Table 109.2). It is probable that the wide range of concentrations
The kahweol contents for mature green beans (mainly reported in the literature is partly due to the different
endosperm) of C. arabica varied between 21 and 986mg methodologies used to extract the diterpenes. Prelimi-
100/g, whereas less variance was observed for the cafes- nary extraction followed by saponification leads to a
tol levels, from 168 to 604mg 100/g. For green beans of higher possibility of oxidation of diterpenes, mainly for
C. canephora, values below 14mg of kahweol 100/g and kahweol.

TABLE 109.2 Kahweol and Cafestol Contents (mg 100/g) in Different Plant Parts of Coffea species
Plant Parts Coffea Species and/or Cultivar Origin Kahweol Cafestol References

Flower Coffea arabica (invitro) India 1.1 3.1 36

Flower Coffea canephora (invitro) India 0.9 2.6 36

Leaves C. arabica <0.01 55 38

Leaves C. arabica cv. IAPAR 59 (mature) Brazil ND 45 27

Leaves C. arabica (young green) India 17.5 125 36

Leaves C. canephora NC 4.9 38

Leaves C. canephora cv. Apoat (mature) Brazil ND ND 27

Leaves C. canephora (young green) India 10.8 242 36

Perisperm C. arabica cv. IAPAR 59 (83 DAB) Brazil 516 132 27

Perisperm C. canephora cv. Apoat (120 DAB) Brazil ND 109 27

Periscarp C. arabica cv. IAPAR 59 (240 DAB) Brazil ND 49 27

Periscarp C. canephora cv. Apoat (214 DAB) Brazil ND 36 27

Endosperm C. arabica cv. IAPAR 59 (240 DAB) Brazil 589 304 27

Endosperm C. arabica (immature fruit, invitro) India 42 958 36

Endosperm C. arabica (mature fruit, invitro) India 142 1022 36

Endosperm C. canephora cv. Apoat (214 DAB) Brazil ND 94 27

Endosperm C. canephora (immature fruit, invitro) India 2 640 36

Endosperm C. canephora (mature fruit, invitro) India 1 216 36

Green bean C. arabica Brazil 300 600 39

Green bean C. arabica 431664 414534 40

Green bean C. arabica 110350 270670 7

Green bean C. arabica Brazil 476 384 17

Green bean C. arabica India 21 168 36

Green bean C. arabica (cv. Catuai, IPR100, Brazil 371986 221604 30

IPR102, IPR106)

Green bean C. canephora Togo, Burundi ND 200 39

Green bean C. canephora Philippines, 17 NC 1

Uganda

Green bean C. canephora 14 113 40

Green bean C. canephora cv. Robusta Ivory Coast 58 239250 4

Green bean C. canephora <10 150370 7

Green bean C. canephora India 3.4 110 36

Reports the contents of kahweol and cafestol in coffee plants. Unpublished. NC, not cited; ND, not detected; DAB, Days after bloom; cv.: Coffee cultivar.

IV. ANALYSIS AND METHODS

1000 109. ASSAY OF KAHWEOL AND CAFESTOL IN COFFEE

TABLE 109.3 Kahweol and Cafestol Contents (mg 100/g) in Roasted and Ground Coffees
Coffee Type Origin Kahweol Cafestol References

Coffea arabica, medium roast degree Mexico 736 573 20

C. arabica 371672 299584 40

C. arabica Brazil 398 329 17

C. arabica (two coffees with different proportion of defective Brazil 661866 360478 25
beans and geographic origin); three roasting degrees (light,
medium, dark)

C. arabica cv. IAPAR 59 (0, 10, 20, 30% of defective beans) Brazil 905 447 25

C. arabica; different roasting degrees (light, medium, dark, India 132453 187622 37
normal, city, full)

C. arabica (cv. Catuai, cv. Mundo Novo, cv. IAPAR 59, two Brazil 660930 280480 28
commercial coffee blends); dark roasting degree

C. arabica (cv. Red Catuai, cv IPR 100, cv. IPR 102, cv. IPR 106); Brazil 4391096 246668 30
medium roasting degree

Coffea canephora 413 76190 40

C. canephora cv. conilon (two coffees with different proportion of Brazil ND 163275 25
defective beans and geographic origin); three roasting degrees
(light, medium, dark)

C. canephora; three roasting degrees (light, medium, dark, India 146313 281363 37
normal, city, full)

C. canephora cv. conilon (three commercial coffee blends); dark Brazil ND 190240 28
roasting degree

Blend (C. arabica:C. canephora, 70:30 w/w); medium roasting Brazil 446 323 27
degree

Regular commercial products (20 brands) USA, Germany, the 50700 200700 20
Netherlands, Israel, (mean 469) (mean 486)
Norway, Belgium,
Finland, Egypt,
Indonesia, Malawi

Regular commercial products (38 brands) Brazil 100800 250550 26

Decaffeinated commercial products (5 brands) USA, Germany, the 250 a 600 450 to 650 20
Netherlands, (mean 411) (mean 485)

Reports the contents of kahweol and cafestol in roasted coffees. Unpublished. ND, not detected; cv., Coffee cultivar.

Even not taking into account differences in the effi-
ciency of the methods, there is still a wide variation in the
raw materials studied. The geographic origin, edapho-
climatic and agronomic conditions, and post-harvest
treatments as well as the use of different cultivars of each
species also explain the variability in the data reported.
Kurzrock and Speer7 and Lercker etal.40 reported signif-
icant variations in the contents of diterpenes of green coffee
beans of different origins. De Roos etal.4 observed that the
kahweol content was related to the geographic distribution
of the coffee species. Rubayiza and Meurens16 also reported
that arabica coffees from high-altitude/low-temperature
regions contained higher concentrations of kahweol.
Genetic diversity also greatly contributes to the diver-
sity in the profile of diterpenes for different species and
varieties of coffee, particularly in their kahweol content.
De Roos etal.4 noted that the diterpene profiles were
useful for taxonomic grouping of the Coffea species. The
authors reported that cafestol was present in all of the
wild coffee species studied. The kahweol levels were
low in canephora (58mg 100/g) and two varieties of
C. liberica (54152mg 100/g). High levels (5051065mg
100/g), comparable to arabica coffees (Table 109.2),
were observed in other species (Coffea stenophyla, Coffea
congensis, Coffea racemosa, Coffea salvatrix, Coffea pseudo-
zanguebariae, and Coffea sessiliflora).
Kitzberger etal.30 reported the kahweol and cafestol
contents of arabica cultivars grown in the same eda-
pho-climatic conditions. As the harvesting and post-
harvesting techniques were standardized, the influence
of genetic variability could be evaluated. Cultivars that
were developed to be more resistant to pests by the

IV. ANALYSIS AND METHODS

 109.4 OCCURRENCE OF KAHWEOL AND CAFESTOL IN DIFFERENT COFFEA MATRICES 1001
TABLE 109.4 Kahweol and Cafestol Contents in Coffee Brews (mg per cup)
Coffee Brews Coffea Species and/or Cultivar Origin Kahweol Cafestol References

Instanta Regular commercial products USA, Tanzania, the 0.050.6 0.010.7 20

(13 brands) Netherlands, South
Africa

Instanta Decaffeinated commercial products USA, the Netherlands 0.00.3 0.050.4 20

(6 brands)

Instantb Commercial products (2 blends) Switzerland 0.10.3 0.10.3 32

Instanta Regular and decaffeinated commercial Portugal 0.010.06 0.040.2 24

products (3 brands)

Turkish/Greekb Coffea arabica, medium roast; different Mexico 1.72.1 1.51.8 20

concentrations (50, 70, and 150g/l)

Turkish/Greek 11 brews (Restaurants, retail outlets) The Netherlands, 0.110.7 0.510 20

Greece, Egypt (mean 3.9) (mean 3.9)

Turkishc C. arabica Switzerland 5.3 5.4 32

Turkishc India 8.3 7.3 37

Turkishc C. arabica (5 roasting degrees) NC 1.42.5 29

French pressb 5 brews (Consumers) The Netherlands 2.68.0 2.35.5 20

(mean 4.4) (mean 1.2)

French pressb C. arabica, medium roast; different Mexico 3.212.6 3.09.8 20

concentrations (35, 50, 70 and 150g/l)

French pressd Commercial C. arabica Spain 266 279 22

French pressb India 17.4 19.7 37

French presse Commercial product (1 brand) Portugal 0.01 0.6 24

Frenchf C. arabica (5 roasting degrees) NC 4.18.5 29

Scandinavian 14 brews (Regular consumers) Norway, Finland 1.114.6 0.812.1 20

boiledb (mean 3.9) (mean 3.0)

Scandinavian C. arabica, medium roast; different Mexico 514 3.810.6 20

boiledb concentrations (35, 50, 70, and 150g/l)

Boiledb C. arabica Switzerland 7.2 7.2 32

Scandinaviand Commercial C. arabica Spain 666 615 22

Boiledf C. arabica (5 roasting degrees) NC 4.17.7 29

Espressoc 10 brews (bars and restaurants) Italy 0.23.9 0.22.9 20

(mean 1.8) (mean 3.9)

Espresso 21 brews (bars and restaurants) The Netherlands, 0.03.9 0.03.1 20

Spain, Switzerland (mean 1.4) (mean 1.5)

Espresso C. arabica, medium roast; different Mexico 1.83.6 1.62.4 20

concentrations (100, 200, and 400g/l)

Espressoc C. arabica and commercial capsules Switzerland 0.061.0 0.091.0 32

(7 brands)

Espressoc Commercial product (1 brand) India 5.1 6.0 37

Espressoc Commercial product (4 brand) Portugal 0.060.4 0.20.8 24

Espresso Brew from a vending machine (bar) Portugal 0.2 0.45 24

Mochac C. arabica Switzerland 2.3 2.3 32

Mochac India 4.6 6.9 37

Mochac C. arabica (5 roasting degrees) NC 1.12.0 29

Continued

IV. ANALYSIS AND METHODS

1002 109. ASSAY OF KAHWEOL AND CAFESTOL IN COFFEE
TABLE 109.4 Kahweol and Cafestol Contents in Coffee Brews (mg per cup)contd
Coffee Brews Coffea Species and/or Cultivar Origin
Indian filterb India
Filter paperd Commercial C. arabica Spain
(two filter types)
Filter paperb C. arabica Switzerland
Filter paperb India
Filter Commercial product (1 brand) Portugal
Electric dripd Commercial C. arabica Spain
(three filter types)
Electric dripb India
Reports the contents of kahweol and cafestol in coffee brews. Unpublished.
aBrew concentration: brew with 2g of soluble granules.
bCup of 150ml.
cCup of 60ml
dCup of 50ml, 7.5g 100m/l.
e7.5g 100m/l.
fCup of 160ml.
introgression of genetic material from canephora coffee
displayed higher contents of kahweol than of cafestol.
The largest amount of information available con-
cerns roasted and ground coffee and the concentrations
of diterpenes vary greatly. Besides, there are controver-
sies regarding the presence or absence of kahweol in
canephora (Table 109.3).
In general, higher cafestol contents were observed for
arabica (187668mg 100/g) compared to canephora cof-
fee (76363mg 100/g) considering products subjected to
different roasting processes. A wider range is described
for the kahweol contents of arabica roasted coffees (132
1096mg 100/g)17,20,25,28,30,37,40 (Table 109.3).
Regarding the content of kahweol in canephora
roasted coffee, some authors described that it is
absent,25,28,39 whereas others reported only traces or low
concentrations (lower than 13mg 100/g),7,40 and unex-
pectedly high levels have also been cited37 (Table 109.3).
In addition to the sources of variation already men-
tioned (differences in analytical methods and raw mate-
rials), another factor that could account for the variations
in the content of diterpenes is the roasting process.
There is no consensus regarding the stability of these
compounds. Some authors emphasized that dehy-
droderivatives of diterpenes were formed7,8 or reported
that diterpenes were lost during roasting.29,37 In gen-
eral, the literature points out that despite the formation
of degradation products, no significant changes occurs
in the content of diterpenes in coffee produced by dif-
ferent roasting processes.5,25,30 Urgert etal.20 assessed
the behavior of kahweol and cafestol in arabica coffee
and concluded that a high degree of roasting (26.5%
weight loss) did not reduce the concentrations of these
compounds.
IV. ANALYSIS AND METHODS
Kahweol Cafestol References
0.86 1.2 37
TR to 25 TR to 11 22
0.02 0.02 32
0.37 1.3 37
0.010.4 0.040.8 24
16153 TR to 173 22
0.62 1.8 37
It is interesting to observe that lipid concentration is
also influenced by roasting degree, even if dry base data
are used. Total lipid content increases proportionally to
the roasting intensity mainly due to the destruction of
carbohydrates. Dias etal.10 reported that roasting pro-
cess similarly affected the levels of diterpenes in arabica
and canephora coffees and that the effect depended on
the intensity of the process. Cafestol and kahweol were
degraded to dehydrocafestol and dehydrokahweol,
respectively, after 8min at 230C (corresponding to com-
mercial roasting). However, the amount (in mg 100/g,
dry base) of cafestol and, notably, kahweol remained
stable even during aggressive thermal processing due
to the relative increase of the lipid concentration during
roasting.
Regarding commercial coffees, which are mainly ara-
bica and canephora blends, kahweol contents between
50 and 800mg 100/g and cafestol contents between
190 and 700mg 100/g were observed20,28 (Table 109.2).
Adding canephora to arabica coffee is expected to sig-
nificantly reduce the diterpene levels, particularly the
kahweol content. This pattern was described by Cam-
panha etal.25 for coffee blends prepared using differ-
ent degrees of roasting. De Souza etal.26 also reported
higher concentration of kahweol in gourmet coffees
(pure arabica).
Interestingly, Campanha etal.25 reported that the
content of defective beans did not influence the diter-
pene concentrations (Table 109.3). Since the diterpenes
are relatively stable in the roasting process and the
presence of defective beans do not affect the kahweol
and cafestol concentrations, the profile of diterpenes
could indicate the presence of canephora coffee in a
commercial blend.
 References
De Souza and Benassi28 described that, in general,
high levels of caffeine and low levels of diterpenes (kah-
weol and cafestol) were related to higher proportions
of canephora in the commercial coffees, which were
reflected in a decreasing kahweol/cafestol ratio and an
increasing caffeine/kahweol ratio.
As mentioned before, several authors also described
the use of Raman techniques, based on the kahweol
content, as a tool for detecting unwanted admixtures of
low-value canephora coffee with the high-quality ara-
bica coffee.3,16,34 In addition, Pacetti etal.5 proposed an
authentication method for roasted coffee (proportion of
canephora in a blend) based on the ratio of kahweol and
16-O-methylcafestol.
The content of diterpenes of different coffee brews has
been investigated, highlighting the interest in cafestol
after it was discovered its cholesterol-enhancing prop-
erties. However, it is very difficult to compare the data
from different studies and brew methods because of the
diversity of the extraction parameters, such as the grind
sizes, the coffee to water ratios, the time and temperature
of the process, and the use of various equipment and cof-
fee pads. Furthermore, there were also different units of
expression, given that there is no definition of a standard
cup of coffee (Table 109.4).
Generally, unfiltered preparation methods such as
the Scandinavian type of boiled coffee and Turkish
coffee exhibited higher content of diterpenes, followed
by French press and mocha coffees, which showed
moderate levels. Kahweol and cafestol are retained
in the grounds and do not pass through paper fil-
ters, which explains the negligible contents observed
in filtered coffees (filter paper, electric drip). Soluble
coffees were almost devoid of diterpenes20,22,24,29,32,37
(Table 109.4).
109.5 SUMMARY POINTS
E xtraction is a critical step in the analysis of
diterpenes due to their low thermostability and
photostability.
Most usual chromatographic conditions: reverse-
phase octadecyl column, mobile phase of
acetonitrile:water, and ultraviolet detection.
Genetic diversity greatly contributes to the diversity
in the profile of diterpenes of different species and
varieties of coffee, particularly their kahweol content.
Despite the formation of degradation products
during roasting, no significant changes were
observed in the contents of cafestol and kahweol.
Authentication methods for roasted coffee are
proposed, which determine the proportion of
canephora in a blend with arabica based on the
kahweol content.
IV. ANALYSIS AND METHODS
1003
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