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J Sci Food Agric 1998, 76, 617È627
Analysis of Sterols: a Novel Approach for
Detecting Juices of Pineapple, Passionfruit, Orange
and Grapefruit in Compounded Beverages*
Lay-Keow Ng” and Michel Hupe
Research & Development Division, Laboratory & ScientiÐc Services Directorate, Revenue Canada,
79 Bentley Avenue, Ottawa, Ontario, Canada, K1A OL5
(Received 10 June 1996 ; revised version received 31 July 1997 ; accepted 23 September 1997)
Abstract : Direct GC/MS analysis of the hexane extracts of fruit juices provides
an efficient means for demonstrating that very di†erent sterol patterns exist in
the juices of pineapple, passionfruit and the two citrus fruits, orange and grapefruit. Ergostanol and stigmastanol were found to be the sterol markers for pineapple juice, while passionfruit juice was characterised by the presence of an
unidentiÐed but unique sterol referred to as compound C. Juices of orange and
grapefruit yielded very similar sterol proÐles. They were readily distinguished
from pineapple and passionfruit juices by a higher stigmasterol/campesterol
ratio. Valencene/nootkatone response ratio in the hexane extracts was employed
to aid in the di†erentiation of the two citrus juices. Matrix e†ects on the determination of sterol and sesquiterpenoid distributions were found to be insigniÐcant.
Although natural variation and absolute uniqueness of the sterol proÐle for each
of the four fruit juices were not established due to the relatively small number of
fruit samples examined, the results of several compounded beverages clearly
point to the potential usefulness of sterol proÐles for detecting juices of orange,
grapefruit, pineapple and passionfruit in mixed drinks. ( 1998 SCI.
J Sci Food Agric 76, 617È627 (1998)
Key words : juice ; sterols ; sesquiterpenoids ; detection ; compounded beverages ;
gas chromatography/mass spectrometry
INTRODUCTION
materials such as water, sugars, organic acids, Ñavourings, colorants, preservatives, other juices, etc, are added
so that the composition of the original juice is altered.
Therefore, for the purpose of determining the presence
of a juice in beverages, the markers for the juice should
be so chosen such that they remain unique to the target
juice within the matrix of the compounded beverages.
Sterols, in free or conjugated forms, have been identiÐed in parts of various citrus fruits, including grapefruit
peel (Williams et al 1967), orange and tangor juice sacs
(Nagy and Nordby 1971), and peel of Rangpur lime
(Yokoyama and White 1968). Quantitative composition
of free sterols has been used to characterise di†erent
varieties of orange and tangor. Rangpur lime was found
to be the only citrus fruit containing ergosterol. Free
sterols speciÐc to strawberry fruit (Couture et al 1989)
and leaves of pineapple (Pakrachi et al 1975) have also
It is often necessary to conÐrm the declared presence of
a particular juice in compounded beverages for the
purpose of imposing Customs or Excise duty. One
plausible approach would be to detect characteristic
components, or markers, of the juice in the beverages.
Fruit juices are complex mixtures of sugars, organic
acids, volatile Ñavours, fatty acids, sterols, amino acids,
Ñavonoids, pigments, etc in water. Each juice is characterised by a composition which is a unique combination
of these ingredients. In a formulated juice drink,
* Most of the Ðndings from this study were presented at the
21st Annual Conference of the Federation of Analytical
Chemistry and Spectroscopy Societies, 2È7 October 1994, St
Louis, MO, USA.
” To whom correspondence should be addressed.
617
( 1998 SCI.
J Sci Food Agric 0022È5142/98/$17.50.
Printed in Great Britain
L -K Ng, M Hupe
618
been reported. These Ðndings point to the potential of
using free sterols as fruit indices. Unlike volatile Ñavours, sugars and acids, sterols are not added ingredients used in beverage formulation since they are not
Ñavouring agents and are rather expensive chemicals.
Sterols are, therefore, not likely to be masked by added
substances in the juice drinks. They are also not easily
removed from the juice matrices during concentration,
which is important given that most commercial juice
drinks are prepared from juice concentrates. Based on
these considerations, sterols could constitute a useful
group of chemical markers for the identiÐcation of
juices in compounded beverages.
Numerous reports on the detection of fruit juices in
beverages have resulted from e†orts to combat juice
adulteration where a juice has been diluted by a cheaper
juice. Various chemical indices for fruit have been used.
For example, adulteration of orange juice by grapefruit
juice was conÐrmed by the detection of the Ñavonoid
naringin (Greiner and Wallrauch 1984). The high concentration of proline in apple juice was taken as an indication of the addition of pear juice (Wallrauch and
Faethe 1988). Sterols have not been reported to be
employed as index components of fruit juices.
Most of the fruit sterols investigated were derived
from juice sacs or other parts of the plants. Few studies
on the composition of free sterols in fruit juices have
been undertaken except for orange juice (Stack et al
1986). This method consisted of gas chromatography
(GC) of a fraction eluted from a C-18 disposable minicolumn. The sample preparation procedure involved
tedious conditioning of the column, solvent elution of
sterols, evaporation of the solvent and derivatisation.
GC analysis of sterols from plant materials is difficult,
due to structural similarity of the compounds which
leads to overlapping of peaks. Gas chromatography/
mass spectrometry (GC/MS) is a more powerful technique for the analysis of co-eluting free sterols.
Components can be extracted from the total ion chromatogram (TIC) using speciÐc ions for qualitative and
quantitative analysis. They can be identiÐed by retention times (RT), as well as by their distinct mass spectral
patterns.
In this study, the potential usefulness of sterols as
diagnostic components for identifying juices of pineapple, passionfruit, orange and grapefruit has been
investigated. The analysis was accomplished by a simple
procedure in which sterols and other components were
extracted into hexane by a one-step liquidÈliquid
extraction, followed by GC/MS analysis.
MATERIALS AND METHODS
Samples
Three oranges from California, Florida and Uruguay,
two pineapples from Costa Rica, one pink-Ñeshed and
two white-Ñeshed grapefruits from Florida, one yellowskinned and two purple-skinned passionfruits, and different brands of commercial beverages composed of
single or mixed juices were purchased from local stores
or supplied by the Excise Department of Thailand.
Solvents and standards
b-Caryophyllene and valencene were obtained from
Fluka Chem Corp (Ronkonkoma, NY, USA) and
Janssen Chimica (Beerse, Belgium), respectively. Cholesterol [99%, stigmasterol 96%, stigmastanol 98% and
b-sitosterol 98É3%, were supplied by Sigma (St Louis,
IL, USA). Campesterol 99% was purchased from
Applied Science Laboratories Inc (State College, PA,
USA). Specially denatured alcohol, formula 3A and
hexane were obtained from JT Baker (Toronto,
Ontario, Canada).
Extraction procedure
Eight millilitres of denatured alcohol formula 3A, 10 ml
of a single-strength juice sample and 1É5 ml of hexane
were pipetted into a centrifuge tube. The tube was
screw-capped and vortexed for 2 min. After centrifuging
the mixture at 8000 rpm for 5 min, 1 ll of the hexane
layer (top) was injected in splitless mode into the
GC/MS system. For compounded beverages, the same
procedure was used except that the hexane extracts
were concentrated to 0É25 ml at room temperature
under a slow stream of nitrogen before 1 ll was injected
into the gas chromatograph.
Gas chromatography/mass spectrometry (GC/MS)
The extracts were analysed using a HewlettÈPackard
gas chromatograph 5890 series II equipped with a 5970
mass selective detector, and a 5990A MS Chemstation
(HP-UX) (HewlettÈPackard, North Hollywood, CA,
USA). The column used was a 12É5 m DB5-MS (5%
diphenyl and 95% dimethyl polysiloxane) fused silica (J
& W ScientiÐc, Falsom, USA), 0É33 lm Ðlm thickness,
0É20 mm id. The carrier gas was helium with a Ñow rate
of 1É0 ml min~1 at 50¡C. The split/splitless injection
port and the interface were at 250¡C and 310¡C, respectively. The oven temperature was programmed as
follows : 50¡C for 1 min, then ramped at 10¡C min~1 to
300¡C and held for 40 min. All mass spectra were
acquired in the electron impact (EI) mode at 70 eV. The
mass spectrometer was scanned in the range of 39È
500 Da at a rate of 1É24 scans s~1, starting 1 min after
injection of the sample. The spectrometer was tuned
using perÑuorotributylamine (PFTBA) as the calibrant
and the autotune procedure incorporated in the Chemstation software package.
Analysis of sterols : detecting fruit juices in compounded beverages
Analysis of overlapping peaks
The purity of TIC peaks was determined by examining
the mass spectrum at di†erent time slices. Co-elution of
components was indicated by appreciable changes of
the mass spectrum as the TIC peak was scanned. A
mass spectrum of each component was taken at a time
slice where overlapping was absent, or by subtracting
the residual spectrum contributed by the co-eluting
component. Computerised library matching, using the
NBS library, was carried out on the mass spectrum to
identify the component. The ions identiÐed as being
characteristic of a component were used to generate
extracted ion chromatograms (EIC). The ion peaks in
these EIC, within the time window where the component eluted, should give the same maxima since they
all belong to the same component. The retention time
(RT) of the component was determined from the
maxima of the extracted ion peaks.
Principal component analysis
Data based on the presence or absence of compound C,
and area ratios of the extracted ion peaks of selected
components other than compound C/isofucosterol (see
Table 5 below) were subjected to principal component
analysis. Area ratios for which the denominator component, or both the numerator and denominator components, were not detected were assigned a value of
[ 1, and zeros were used to indicate that only the
numerator component was not detected. Compound C
was assigned a value of 1 when detected and zero when
619
not detected. The data were Ðrst autoscaled to zero
mean and unit variance before carrying out principal
component analysis. The commercial software package,
Ein*Sight (InfoMetrix, Seattle, WA, USA) was used for
calculating principal components.
RESULTS AND DISCUSSION
All juice samples were subjected to a simple liquidÈ
liquid extraction procedure to separate sterols from the
matrices of the beverages. Hexane was used as the
extraction solvent. Generally, liquidÈliquid extraction of
fruit juices by an organic solvent is inefficient due to the
formation of an emulsion. In our procedure, ethanol
was added to the juice to precipitate pectins and other
emulsifying agents. The emulsion was destabilised, and
complete separation of the organic and aqueous phases
was achieved.
The extracts were analysed directly by capillary
GC/MS without further treatment. In addition to
sterols, fatty acids were also extracted. Volatile Ñavours
were only detected in the two citrus juices. As shown in
Fig 1, which is a representative TIC of a hexane extract,
volatile Ñavours eluted in the Ðrst 14 min, followed by
fatty acids, with the semivolatile sterols appearing in the
range of 26È29 min. Several unidentiÐed high boiling
components were detected after the sterols.
Free sterols were present in all of the samples of
single juices of orange, grapefruit, pineapple and passionfruit. The common plant sterols, b-sitosterol, campesterol and stigmasterol, were among the major
Fig 1. Total ion chromatogram of the hexane extract of orange juice.
L -K Ng, M Hupe
620
components. Some of the compounds detected in the
sterol fraction were not identiÐed, they may be sterols,
sterol derivatives, triterpene alcohols, etc. For simplicity, they are referred to as sterols in the following discussion. The sterols, along with several terpenic
compounds, were characterised by RT and speciÐc ions
as shown in Table 1. Cholesterol, campesterol, stigmasterol, b-sitosterol and stigmastanol were identiÐed by
computerised library matching and by comparison with
authentic chemicals. Components which partially overlapped with other peaks, such as 24-ethylidene cholesterol (also known as isofucosterol), stigmastanol,
ergostanol, compounds A, B and C, etc, were analysed
as described in the Experimental section.
Single juices
For each juice, samples of freshly squeezed juice were
analysed along with several commercial samples to
determine the nature and the relative abundances of the
sterol components, except for passionfruit juice for
which samples of the commercial counterpart were not
available. The same components were found in the fresh
juices and the commercial counterparts, and their sterol
patterns were comparable as discussed later.
Three fresh and Ðve commercial juices of orange were
analysed. In all of these samples, the major sterols
found were, in order of decreasing prevalence, bsitosterol [ campesterol [ stigmasterol,
isofucosterol
TABLE 1
Chromatographic and mass spectral characteristics of diagnostic components
RT a
(min)
Component
(tentative identity)
MW
Base peak
m/z
Characteristic ions
m/z (relative intensity, %)
11É27
Valencene
204
161b
14É61
Nootkatone
218
147b
26É43
Campesterol
400
400b
26É52
Compound C
424
165
26É54
Ergostanol
402
233b
26É65
Stigmasterol
412
412b
27É24
b-Sitosterol
414
414b
27É31
Isofucosterol
412
314b
27É32
Stigmastanol
416
233b
28É21
Compound B
(stigmast-4-en-3-one)
412
124b
28É28
Compound A
(citrostadienol)
426
285b
91, 93 (65)
105, 107 (60)
133, 204 (50)
147, 189 (37)
79, 121 (84)
91, 133 (65)
105, 175, 203 (56)
218 (21)
315 (60)
382 (50)
367, 289 (50)
145, 213, 255 (30)
424b (99)
203, 205 (20)
215 (80)
234, 402 (70)
397 (30)
255, 271, 300 (50)
351 (33)
329, 396 (50)
303, 381 (40)
281 (34)
229, 299 (26)
416 (70)
215 (65)
234, 401 (55)
412 (80)
229 (60)
288, 289 (50)
370 (30)
328 (40)
411, 313 (10)
426 (5)
a Retention times may change over time, they are listed only to show the relative order
of elution.
b Ions used to extract the corresponding components from the total ion chromatograms.
Analysis of sterols : detecting fruit juices in compounded beverages
and compound A [ cholesterol and compound B. All of
the identiÐed components have been reported to be
present in the juice sacs of orange fruit (Nagy and
Nordby 1971). These components, with the exception of
isofucosterol, have also been quantiÐed in orange juice
(Stack et al 1986). A typical TIC of sterols in orange
juice is illustrated in the top trace of Fig 2. Isofucosterol
appears as a shoulder on the predominant b-sitosterol
peak, and compound A overlaps with compound B.
Using ions m/z 414, 314, 124 and 285, b-sitosterol, isofucosterol, compounds B and A, respectively, were
extracted from the TIC. The EIC are presented in Figs
2(B)È(E), which show that the RT of isofucosterol was
signiÐcantly di†erent from that of b-sitosterol, and the
co-eluting components, compounds A and B, yielded
slightly, yet noticeably, di†erent RT.
The mass spectra of compounds A and B are shown
in Figs 3 and 4, respectively. These chemicals were ten-
Fig 2. Selective detection of coeluting sterols of orange juice.
1, Cholesterol ; 2, campesterol ; 3, stigmasterol ; 4, b-sitosterol ;
5, isofucosterol ; 6, compound B ; and 7, compound A. The
individual plots are (A) the total ion chromatogram and (BÈE)
the extracted ion chromatograms generated from the theoretical mass of the respective quantitation ion with a limited mass
window of 1 Da.
621
tatively identiÐed by library search as (3b, 4a, 5a, 24Z)4-methylstigmasta-7,24(28)-dien-3-ol, commonly known
as citrostadienol, and stigmast-4-en-3-one, respectively.
The probability based matches (PBM) (McLa†erty et al
1974) with the library mass spectra were 93 and 91%,
respectively. Their identity could not, however, be ascertained because of the unavailability of authentic specimens for direct comparison. These compounds could be
among the unidentiÐed sterols in orange pulp reported
in a previous study (Tushishvili et al 1982).
The relative abundances of several sterols were also
used to characterise orange juice. The results obtained
for the commercial juice samples fall within, or close to,
the range for the fresh juices (Table 2).
Three fresh juices, prepared from one pink-Ñeshed
and two white-Ñeshed grapefruits, and two commercial
grapefruit juices were analysed. They all yielded a sterol
proÐle similar to that reported for grapefruit peel
(Williams et al 1967). The proÐle is also very similar to
that of orange juice.
The sterol proÐles of Ðve commercial and two fresh
juice samples of pineapple can be readily distinguished
from those of the citrus juices. They were all characterised by the presence of signiÐcant amounts of two saturated sterols, namely, ergostanol and stigmastanol.
Stigmastanol and ergostanol have been reported to be
present in the leaves of the pineapple plant (Pakrachi et
al 1975). Their mass spectra displayed the same base
peak at m/z 233. Integration of the ion peak at m/z 233
showed that they were present in almost equal amounts.
All sterols found in orange juice were also present in
pineapple juice. However, the stigmasterol/campesterol,
isofucosterol/campesterol,
cholesterol/campesterol,
compound A/campesterol and compound A/compound
B ratios in pineapple juice are smaller than those found
in orange and grapefruit juices (Table 2).
There are many known species of passionfruit. The
best known are the purple-skinned PassiÑora edulis
Sims and its closely related yellow-skinned mutant
PassiÑora edulis f Ñavicarpa Degener. The sterols
detected are b-sitosterol, compound A, compound C,
stigmasterol, isofucosterol and cholesterol. The
unknown component, compound C, is likely to be a
methyl sterol. Its unique mass spectrum is presented in
Fig. 5. The relative abundance of compound C to isofucosterol was much higher in the yellow-skinned
variety. The ion ratios of stigmasterol/campesterol,
isofucosterol/campesterol and cholesterol/campesterol
were much smaller than those of the citrus juices, but
were of comparable magnitude to those found for pineapple juice (Table 2).
The results for the samples of single juices show that
there are sterol components speciÐc to juices of passionfruit and pineapple. They are present in easily detectable quantities and would be good candidates for
serving as indicators of individual juices in blended beverages. Thus, compound C was chosen as a marker for
L -K Ng, M Hupe
622
Fig 3. Mass spectrum of compound A.
passionfruit juice, and the saturated sterols, ergostanol
and stigmastanol, were employed as index compounds
for pineapple juice. Individual juices were further characterised by quantitative measurements based on
response ratios of selected components shown in Table
2. Although speciÐc sterol markers could not be identiÐed for the two citrus juices, they were characterised by
relatively
high
stigmasterol/campesterol
and
isofucosterol/campesterol ratios.
It is noted that di†erentiation of orange and grapefruit juices cannot be accomplished by the use of sterol
patterns alone. Volatile Ñavours, detected only in the
two citrus juices, may be used to serve this purpose.
Volatile Ñavours are not considered as good index compounds of juices in cases where the beverages are prepared from juice concentrates. In the concentration
process, the majority of the volatiles are removed, and
the recovered essence may not be added back to the
Fig 4. Mass spectrum of compound B.
Analysis of sterols : detecting fruit juices in compounded beverages
623
TABLE 2
Composition of diagnostic components in single juicesa
Orange
Stigmasterol/campesterol
Isofucosterol/campesterol
Cholesterol/campesterol
Compound A/campesterol
Compound C/isofucosterol
Ergostanol/stigmastanol
Valencene/nootkatone
Compound A/compound B
Grapefruit
Pineapple
Fresh passionfruit
Fresh (3)b
Comm (5)
Fresh (3)
Comm (2)
Fresh (2)
Comm (5)
Purple (2)
Y ellow (1)
0É35È0É41
1É02È2É02
0É09È0É19
0É45È0É86
Èc
È
29É1È45É6
2É2È99É1
0É36È0É43
0É32È1É03
0É08È0É13
0É19È0É41
È
È
19É6È44É1
2É0È5É2
0É22È0É30
1É16É2É01
0É06È0É09
0É04È0É43
È
È
0É06È0É16
2É8È50É7
0É26È0É35
0É84È0É93
0É07È0É09
0É04È0É46
È
È
0É24È0É29
9É9È20É7
0É06È0É07
0É11È0É14
0É004È0É011
0É05È0É06
È
0É75È0É96
È
0É05È0É06
0É05È0É12
0É06È0É21
0É004È0É032
0É03È0É08
È
0É73È1É2
È
0É03È0É08
0É05È0É06
0É04È0É10
0É001È0É004
0É11È0É18
0É2È1É3
È
È
3É4È8É0
0É04
0É16
0É04
0É51
50É3
È
È
9É4
a Ratios of components were determined from the relative areas of the component peaks extracted from the total ion chromatograms using speciÐc
ions shown in Table 1.
b Numbers in parentheses are number of samples.
c È indicates a ratio in which the numerator, the denominator, or both the numerator and the denominator components were not detected
(area \ 3000).
concentrates. However, the semivolatile Ñavours, such
as valencene and nootkatone, are less likely to be lost
during the concentration process due to their relatively
high boiling points. Valencene and nootkatone are
characteristic components of juices of orange and
grapefruit (Shaw 1991), respectively. They were selected
to serve as secondary markers for the respective juices.
However, nootkatone is also found in orange juice in
small amount ; similarly valencene is present as a trace
component in grapefruit juice. To increase the speciÐcity of these markers, the response ratio of valencene/
nootkatone was used to di†erentiate the citrus juices. As
shown in Table 2, the valencene/nootkatone ratio was
at least ten times higher in orange juice than in grapefruit juice.
Matrix e†ects on sterol and sesquiterpenoid distributions
ProÐle analysis based on response ratios of components
could be useful for the detection of individual juices in
compounded beverages if the ratios are consistent in the
single juices and in the compounded beverages. Matrix
e†ects on the ratios of selected components of each fruit
juice were studied in the following matrices :
Fig 5. Mass spectrum of compound C.
L -K Ng, M Hupe
624
(1)
(2)
(3)
a 25% juice medium having the same ¡Brix as
the single juice, prepared by diluting the fresh
juice with water and then making up to the ¡Brix
by addition of sucrose ;
a diluted juice of 5 ¡Brix, prepared by diluting
the fresh juice with water until the desired ¡Brix
was obtained ; and
a compounded juice of 20 ¡Brix, prepared by
adding sucrose to the fresh juice until the desired
¡Brix was obtained.
compound C/isofucosterol ratios in all mixtures containing passionfruit juice were smaller than that found
for the single juice. This is because isofucosterol was
found in all four juices and compound C was detected
only in passionfruit juice. In general, it is expected that
in a mixed juice, a response ratio of two components,
which are both present in some or all of the individual
juices in the mixture, should yield a value which lies
between the highest and the lowest values of the same
component ratio of those individual juices. For
example, in the mixtures containing both grapefruit and
orange juices such as GP-OJ, OJ-GP-PN, OJ-GP-PF
and OJ-GP-PN-PF, the valencene/nootkatone ratios
were found to be between 0É15 and 35. Other component ratios, listed in Table 4, also indicate that matrix
e†ects were insigniÐcant.
As shown in Table 3, across the di†erent matrices in
which the same juice was present, all of the response
ratios varied with relative standard deviations (RSD)
ranging 4È16%, indicating that they were essentially
insensitive to changes in ¡Brix or juice concentration.
The only exception was the ratio of compound A/
compound B, which changed by as much as 46È75%
RSD, possibly due to di†erences in the chemical nature
of the two components. Therefore, this component ratio
will not be used to identify the individual juices in mixtures.
The proÐles of mixed juices were also investigated.
The samples were prepared by mixing two, three or all
four fruit juices. In the absence of matrix e†ects, the
ratio of components speciÐc to a particular juice, such
as the ergostanol/stigmastanol ratio for pineapple juice,
should remain the same in any mixtures containing that
juice as in the single juice. As shown in Table 4, the
ergostanol/stigmastanol ratios of all mixtures containing pineapple juice, ie GP-PN, OJ-PN, PN-PF, OJ-GPPN, GP-PN-PF, OJ-PN-PF and OJ-GP-PN-PF, were
found to be between 0É87 and 0É96, which was very
close to that (0É96) of the pineapple juice used to
prepare the mixed juices, indicating that matrix e†ects
were not signiÐcant. Similarly, for mixed juices containing either orange or grapefruit juice, the valencene/
nootkatone ratios were the same as that of the
corresponding citrus juice present in the mixture, ie
D35 and D0É15, respectively, for the orange and the
grapefruit juices used in this study. On the other hand,
Compound beverages
The results shown in Tables 3 and 4 demonstrate the
consistency of the sterol and the sesquiterpenoid distributions of the individual juices in various matrices. In
this study, these proÐles were used to conÐrm the
alleged presence of the juices in various commercial
juice beverages. The results are presented in Table 5.
The data on single juices should not be taken as deÐnitive standards of the component distributions due to the
small number of samples investigated ; they are presented only to serve as a guide to the characteristic composition of the individual juices.
Samples of orangeade, OJ-A1 and OJ-A2, which
contain 25% and 40% of orange juice, respectively, with
added water, sugars, acids and Ñavors, were found to
yield sterol proÐles in accordance with orange juices.
The citrus components in OJ-A1 and OJ-A2 were
further conÐrmed to be derived from orange juices by
the measurements of the valencene/nootkatone ratio.
Three commercial samples of passionfruit drinks
analysed in this study, PF-A1, PF-A2 and PF-A3, contained 25% juice with added sugar and water. Com-
TABLE 3a
Matrix e†ects on response ratios of selected components
Orange
Stigmasterol/campesterol
Isofucosterol/campesterol
Cholesterol/campesterol
Compound A/campesterol
Compound C/isofucosterol
Ergostanol/stigmastanol
Valencene/nootkatone
Compound A/compound B
Grapefruit
Pineapple
Passionfruit
SJ
M1
M2
M3
SJ
M1
M2
M3
SJ
M1
M2
M3
SJ
M1
M2
M3
0É37
1É03
0É11
0É45
Èb
È
34É56
2É20
0É38
1É03
0É11
0É46
È
È
32É29
1É28
0É35
0É95
0É12
0É43
È
È
34É76
0É71
0É36
0É90
0É12
0É41
È
È
30É66
0É52
0É22
1É16
0É07
0É25
È
È
0É16
17É45
0É22
1É17
0É07
0É28
È
È
0É16
11É30
0É20
1É06
0É09
0É31
È
È
0É18
2É87
0É21
1É18
0É09
0É31
È
È
0É18
4É30
0É06
0É14
0É005
0É06
È
0É96
È
0É37
0É07
0É13
0É003
0É06
È
0É86
È
0É28
0É08
0É18
0É002
0É08
È
1É21
È
0É10
0É07
0É14
0É008
0É06
È
0É93
È
0É14
0É05
0É04
0É003
0É11
1É26
È
È
8É01
0É06
0É04
0É004
0É12
1É41
È
È
7É64
0É06
0É04
0É005
0É15
1É60
È
È
3É23
0É05
0É04
0É004
0É14
1É31
È
È
3É54
a SJ, single juice ; ¡Brix, 10É8 (orange) ; 10É4 (grapefruit) ; 14É3 (pineapple) ; and 15É1 (passionfruit) ; M1, 25% juice having the same ¡Brix as the corresponding single juice ;
M2, diluted juice having ¡Brix 5 ; M3, compounded juice having ¡Brix 20.
b È, see footnotes of Table 2.
Analysis of sterols : detecting fruit juices in compounded beverages
625
TABLE 4a
Comparison of found and expected response ratios of diagnostic components in mixed juices
GP-PF
Stigmasterol/campesterol
Isofucosterol/campesterol
Cholesterol/campesterol
Compound A/campesterol
Compound C/isofucosterol
Ergostanol/stigmastanol
Valencene/nootkatone
GP-PN
Expected
Found
Expected
Found
Expected
Found
Expected
0É07
0É17
0É01
0É15
0É26
Èb
0É15
0É05È0É22
0É04È1É16
0É001È0É068
0É11È0É25
0È1É26
È
0É16
0É13
0É55
0É06
0É15
È
0É95
0É14
0É06È0É22
0É14È1É16
0É005È0É068
0É06È0É25
È
0É96
0É16
0É30
1É08
0É08
0É35
È
È
3É7
0É22È0É38
1É03È1É16
0É06È0É09
0É25È0É45
È
È
0É16È34É6
0É09
0É14
0É02
0É17
0É27
È
31É3
0É05È0É38
0É04È1É03
0É001È0É094
0É11È0É45
0È1É26
È
34É6
PN-PF
OJ-GP-PN
GP-PN-PF
Found
Expected
Found
Expected
Found
Expected
Found
Expected
0É18
0É44
0É05
0É20
È
0É92
34É5
0É07È0É38
0É13È1É03
0.005È0É094
0É06È0É45
È
0É96
34É6
0É06
0É05
0É004
0É12
0É77
0É96
È
0É05È0É06
0É04È0É14
0É001È0É004
0É06È0É11
0È1É26
0É96
È
0É19
0É63
0É06
0É21
È
0É91
4É7
0É06È0É38
0É14È1É16
0É004È0É094
0É06È0É45
È
0É96
0É16È34É6
0É07
0É16
0É01
0É14
0É21
0É91
0É16
0É05È0É22
0É04È1É16
0É001È0É063
0É06È0É25
0È1É26
0É96
0É16
OJ-GP-PF
Stigmasterol/campesterol
Isofucosterol/campesterol
Cholesterol/campesterol
Compound A/campesterol
Compound C/isofucosterol
Ergostanol/stigmastanol
Valencene/nootkatone
OJ-PF
Found
OJ-PN
Stigmasterol/campesterol
Isofucosterol/campesterol
Cholesterol/campesterol
Compound A/campesterol
Compound C/isofucosterol
Ergostanol/stigmastanol
Valencene/nootkatone
GP-OJ
OJ-PN-PF
OJ-GP-PN-PF
Found
Expected
Found
Expected
Found
Expected
0É10
0É25
0É02
0É19
0É16
È
4É20
0É05È0É38
0É04È1É16
0É001È0É094
0É11È0É45
0È1É26
È
0É16È34É6
0É09
0É15
0É02
0É16
0É26
0É88
32É2
0É05È0É38
0É04È1É03
0É001È0É094
0É06È0É45
0È1É26
0É96
34É6
0É10
0É23
0É02
0É17
0É15
0É87
4É3
0É05È0É38
0É04È1É16
0É001È0É094
0É06È0É45
0È1É26
0É96
0É16È34É6
a OJ, orange juice ; GP, grapefruit juice ; PN, pineapple juice ; and PF, passionfruit. These were the same single juices used in Table 3. Each mixed
juice sample was composed of equal volumes of the constituent juices.
b È, see footnote c of Table 2.
pound C was found to be present in all three samples,
indicating the presence of passionfruit juice. Relative
abundances of the various components were in comparable magnitudes to the fresh passionfruit juices. The
isofucosterol/campesterol ratios in the beverages were,
however, slightly higher than those of the three fresh
juice samples analysed in this study. In BEV1, a commercial beverage comprised of pineapple and grapefruit
juices in unknown proportion, the markers for pineapple, ergostanol and stigmastanol, were detected in a
TABLE 5a
Composition of diagnostic components in single juices and compounded beverages
Compounded beverages
Stigmasterol/campesterol
Isofucosterol/campesterol
Cholesterol/campesterol
Compound A/campesterol
Compound C/isofucosterol
Ergostanol/stigmastanol
Valencene/nootkatone
Single juicesb
OJ-A1
OJ-A2
PF-A1
PF-A2
PF-A3
BEV 1
BEV 2
BEV 3
Orange
Grapefruit
Pineapple
Passionfruit
0É39
0É84
0É07
0É53
Èc
È
45É2
0É36
0É54
0É06
0É20
È
È
19É2
0É07
0É29
0É05
0É17
4É4
È
È
0É08
0É22
0É06
0É25
5É2
È
È
0É09
0É29
0É04
0É02
3É9
È
È
0É14
0É26
0É04
0É16
È
0É82
0É18
0É18
0É06
0É03
0É15
1É1
1É1
25
0É08
0É23
0É03
0É11
0É54
0É93
È
0É35È0É43
0É32È2É02
0É08È0É19
0É19È0É86
È
È
19É6È45É6
0É22È0É35
0É84È2É01
0É06È0É09
0É04È0É46
È
È
0É06È0É29
0É05È0É12
0É06È0É21
0É004È0É01
0É03È0É08
È
0É73È1É2
È
0É04È0É06
0É04È0É16
0É001È0É04
0É11È0É51
0É2È50É3
È
È
a OJ-A1 and OJ-A2 orangeades ; PF-A1, PF-A2 and PF-A3, passionfruit drinks ; BEV1, a beverage composed of pineapple and grapefruit juices ; BEV2, a beverage
composed of pineapple, orange and passionfruit juices ; and BEV3, a beverage composed of pineapple and passionfruit juices.
b Ranges of response ratios are the combined results of the fresh and commercial juices shown in Table 2.
c È, see footnote c of Table 2.
L -K Ng, M Hupe
626
proportion consistent with a pineapple juice. The presence of grapefruit juice was indicated by the signiÐcant
amount of nootkatone and the low valencene/
nootkatone ratio. Other component ratios assumed
values which were intermediate between those of pineapple and grapefruit juices. The results conÐrmed that
the sample contained juices of pineapple and grapefruit.
The sterol proÐle of sample BEV2, which was a composite of juices of pineapple, orange and passionfruit,
was similar to that of BEV1. This beverage was determined to contain orange, rather than grapefruit juice,
based on the relative abundances of the sesquiterpenoid
components. The presence of passionfruit juice was
indicated by the detection of compound C. In the case
of sample BEV3, a mixture of 90% pineapple and 10%
passionfruit juices, the marker compounds for pineapple
and passionfruit juices were detected. Quantitative
aspects of the sterolic proÐle were also consistent with
the alleged composition of the beverage. Several commercial beverages composed of juices of pineapple or
passionfruit blended with other juices not investigated
in this study were also analysed, with a view to determining the presence of markers. Stigmastanol and
ergostanol were detected at approximately equal
amounts in a sample comprised of pineapple and apple
juices, as well as in a beverage containing 10% pineapple juice compounded with banana puree and guava
juice. Similarly, in commercial drinks composed of 10%
passionfruit juice in banana puree and sugar solution,
and 20% passionfruit admixed with mango juice and
added sugar and water, the presence of passionfruit
juice was indicated by the positive detection of compound C.
Principal component analysis
Principal component analysis (PCA) provides a simple
graphical picture of the variability in a set of data. A
sample having n-variables can be represented by a point
in n-dimensional space. Principal component analysis is
a technique of reducing the dimensionality of the data
by constructing a series of new data axes, called principal components, which are linear combinations of the
variables in the data set. The mutually orthogonal principal components are given a prioritisation based on the
amount of variance in the data for which they can
account. In e†ect, a projection of the data onto the new
axes allows one to visualise graphically the relationship
of the samples on a two-dimensional plot if the variance
described by the Ðrst two principal components is sufficient to account for most of the variance of the data.
A data set based on the data of the selected sterol and
terpenoid components of the juice samples was subjected to PCA. It was established and pre-processed
before PCA as described in the Experimental section.
Figure 6 presents PCA results and shows the pattern of
association among the samples. In the two-dimensional
plot, samples of juices of pineapple, passionfruit, orange
Fig 6. Plot of the Ðrst two principal components of juices of pineapple, passionfruit, orange and grapefruit, as well as compounded
beverages. Total variance \ 82%. 1, Orange juice ; 2, grapefruit juice ; 3, pineapple juice ; 4, passionfruit juice ; OJ-A, orangeade ;
PF-A, passionfruit drink ; BEV1, composed of pineapple and grapefruit juices ; BEV2, composed of pineapple, orange and passionfruit juices ; and BEV3, composed of pineapple and passionfruit juices.
Analysis of sterols : detecting fruit juices in compounded beverages
and grapefruit form well-separated groups. The closeness of the orange and grapefruit clusters indicates that
the two citrus juices are very similar in composition.
The two orangeades fall within the cluster of orange
juice as expected since no other juices, which would
alter the ratios of the diagnostic components, have been
added. The same observation was made with the three
passionfruit drinks. Samples of mixed juices, on the
other hand, fall between the clusters of their constituent
juices. The PCA results clearly illustrate the discriminatory power of assays of sterols and sesquiterpenoids.
CONCLUSION
The results of this study indicate that analysis of sterol
patterns is a useful approach for conÐrming the presence of juices of pineapple, passionfruit, orange and
grapefruit in compounded beverages. Sterols such as
ergostanol, stigmastanol, and compounds A and C,
which have not been reported previously to be present
in the juices, were found to be diagnostic components
for the identiÐcation of these juices. The relative abundances of various selected sterols, as well as valencene
and nootkatone, are of particular value for this purpose.
In principle, the scope of the applicability of this
approach could be extended to the detection of adulteration of fruit juices.
ACKNOWLEDGEMENTS
The authors thank the Excise Department of Thailand
for supplying some of the juice samples. Thanks are also
627
due to Dr Jan Kovar for valuable discussions throughout this study. Particular thanks to Dr Andre H Lawrence for helpful suggestions during the preparation of
this paper.
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