Anal Bioanal Chem (2005) 383: 1003–1008
DOI 10.1007/s00216-005-0078-6
ORIGINA L PA PER
Jinchao Shen . Xueguang Shao
A comparison of accelerated solvent extraction, Soxhlet
extraction, and ultrasonic-assisted extraction for analysis
of terpenoids and sterols in tobacco
Received: 30 April 2005 / Revised: 17 July 2005 / Accepted: 16 August 2005 / Published online: 18 October 2005
# Springer-Verlag 2005
Abstract The performance of accelerated solvent extrac-
tion in the analysis of terpenoids and sterols in tobacco
samples was investigated and compared with those of
Soxhlet extraction and ultrasonically assisted extraction
with respect to yield, extraction time, reproducibility and
solvent consumption. The results indicate that although the
highest yield was achieved by Soxhlet extraction, ASE
appears to be a promising alternative to classical methods
since it is faster and uses less solvent, especially when
applied to the investigation of large batch tobacco samples.
However, Soxhlet extraction is still the preferred method
for analyzing sterols since it gives a higher extraction
efficiency than other methods.
Keywords Accelerated solvent extraction . Terpenoids .
Sterols . Tobacco
Introduction
Tobacco is a special product that has attracted widespread
attention, not just because of its organoleptic attraction to
smokers, but also because of the harm it causes to human
health. The smoke obtained when it is burned harms not
only the smoker but also others who breathe in the smoke
“passively”. Among various harmful components, PAHs
and N-nitrosamine are the most notorious carcinogens in
tobacco smoke. Some of them are inherent to tobacco, but
most, especially the PAHs, are derived from the pyrolysis
J. Shen . X. Shao (*)
Department of Chemistry,
University of Science and Technology of China,
Hefei, Anhui 230026, P. R. China
e-mail: xshao@ustc.edu.cn
Tel.: +86-551-3606160
Fax: +86-551-3601592
X. Shao
Department of Chemistry, Nankai University,
Tianjin 300071, P. R. China
or pyrosynthesis of high molecular weight compounds
during burning [1–3]. Therefore, it is very important to
analyze the precursors, including terponoids and sterols,
and especially the terpenoids, which are not only harmful
components but are also precursors to aroma components.
Traditionally, terpenoids and sterols are extracted from
plant materials using solvent extraction [4, 5]. Different
methods, including mixing extraction, ultrasonically-
assisted extraction and Soxhlet extraction have been used.
Unfortunately, these techniques are time-consuming and
require large volumes of expensive and toxic organic sol-
vents that are expensive to dispose of.
In recent years, new extraction techniques have been
introduced in order to reduce the amount of solvent
required, reduce operation time, improve the precision of
analysis and to reduce the cost of sample preparation.
Accelerated solvent extraction (ASE) is one of these new
technologies [6], which requires only small volumes of
solvents, and allows faster extractions than classical
methods. ASE is performed at temperatures ranging from
50 to 200 °C, extraction timesof 5–15 min and pressures of
500–3,000 psi to keep the solvents liquid at high
temperatures. ASE has been found to be a suitable alter-
native to Soxhlet extraction when extracting polycyclic
aromatic hydrocarbons (PAHs), lipids, pesticides, food
additives and medicinal components. from fly ash [7],
environmental sediment samples [8–16], biological materi-
als [17], plant materials [18–21], dietary composites [22],
feeds [23], aerosols [24], food [25] and animals [26].
Surprisingly however, no data have been published on
the possible application of ASE to such a complex plant
like tobacco. We therefore carried out an investigation of
the ASE conditions needed to simultaneously determine 13
terpenoids, seven sterols and the total content of petroleum
ether extracts (PEE), as well as a comparative study to
evaluate ASE as a potential alternative to classical methods
like Soxhlet extraction and ultrasonically-assisted extrac-
tion in terms of extraction time, extraction yields, solvent
consumption and reproducibility during the analysis of
terpenoids, sterols and the total composition of PEE in
tobacco.
1004
Experimental
Reagents and materials
Petroleum ether with BPs from 60 to 90 °C, hydrochloric
acid, NaOH, NaCl and Na2SO4 (Analytica, Shanghai
Reagent Company, China) were used. The terpenoid and
sterol standards (Analytica, Fluka) were dissolved in
alcohol and stored in the dark at 4 °C. Heptadecane was
dissolved in alcohol at a concentration of 7.38 mg ml−1.
Different grades of aged flue-cured tobacco leaves
harvested in different regions in 2002 were dried at
40 °C for 2 hrs, ground to 40 mesh and mixed thoroughly.
The moisture contents of the samples (5.6 wt.%) were
determined according to the standard method.
Soxhlet extraction
A 5 g portion of ground tobacco was accurately weighed
and extracted with 150 ml of petroleum ether for 5 hrs in a
water bath at 90 °C. Finally, the petroleum ether was
concentrated to 60 ml in a rotary evaporator at less than
80 °C under reduced pressure.
Ultrasonically assisted extraction
Extractions were performed with an 870036 ultrasonic
instrument (Shanghai Ultrasonic Instruments, China), the
power of which is 80 W. A 5 g portion of the ground
tobacco was accurately weighed and extracted with 60 ml
Fig. 1 a Mass spectra of
cholesterol in extracts, b stan-
dard mass spectra of choles‐
terol in NIST98
0 100
0 100
of petroleum ether at ambient temperature for 1 hr.
Afterwards, the extract was filtered using Bucher funnel.
Accelerated solvent extraction
Extractions were carried out using a Dionex ASE 300
(Sunnyvale, CA, USA) accelerated solvent extractor at
different temperatures. A 5 g portion of the ground tobacco
was placed in a 33 ml stainless steel vessel and extracted
using petroleum ether as extracting solvent. The extractions
were performed at 1,500 psi, with 5 min equilibration, 5 min
static time, and a 60 s purge for a total of two cycles. The
extractions were done at three temperatures, 100, 125 and
150 °C. About 60 ml of solvent was collected in each
extraction.
Preparation of the samples
The petroleum solutions obtained using the three extraction
methods were washed twice with 20 ml of a 5 wt.% aqueous
solution of hydrochloric acid (20 ml×2) to remove the basic
fraction, and the aqueous solution was re-extracted twice
with 10 ml petroleum ether (10 ml×2). Then the combined
petroleum ether solution was washed twice with 20 ml of a
5 wt.% aqueous solution of sodium hydroxide (20 ml×2) to
remove the acidic fraction and the aqueous solution was
reextracted twice with 10 ml of petroleum ether (10 ml×2).
The remaining petroleum ether solution was dried using
anhydrous sodium sulfate. Finally, 10 μl of internal
standard (7.38 mg ml−1 heptadecane in cyclohexane) was
200 300 400
200 300 400
m/z
 1005

added and the petroleum ether solution was concentrated to
1 ml in a rotary evaporator at less than 80 °C under reduced
pressure; 1 μl was introduced for GC–MS analysis.
GC–MS analysis
A GC–MS system was used for both qualitative and
quantitative analyses. It consisted of a Trace GC 2000
system and a Trace MS detector in electron impact
ionization mode. Chromatographic separations were per-
formed on a CP-Sil 8CB column (30 m×0.25 mm, 0.25 μm
film thickness). The temperatures of the injector, the GC–
MS transfer line and the ion source were 280, 250 and
200 °C, respectively. The ionization voltage was 70 eV.
Ultrahigh purity helium (99.999%) was used as carrier gas
at a constant flow of 1.0 ml min−1. The following GC
temperature program was used for separation: 40 °C held
for 3 min, 4 °C min−1 to 110 °C, held for 5 min, 2 °C min−1
to 180 °C, held for 15 min, 2 °C min−1 to 270 °C, held for
15 min. The split ratio was set at 20:1.
sterols from plant material. Among these, limonene,
3,7,11,15-tetramethyl-2-hexadecen-1-ol and vitamin E
were identified using their mass spectra and by comparison
with the retention times of the standards; others compounds
were identified by comparing the mass spectra with those in
the National Institute of Standards and Technology
(NIST98, Gaithersburg, MD) mass spectral library. Let’s
take cholesterol as an example. After comparing the
unknown mass spectrum with those in NIST98 (Fig. 1), a
match factor of 846 and a reverse match factor of 906 were
obtained, respectively, which strongly suggested that the
component was just cholesterol. By comparing with the
retention times of authentic standards, the back-column
pressure could be adjusted to ensure reproducibility of the
retention time. Quantitative results were obtained by the
internal standard method using heptadecane as the internal
standard without considering calibration factors (i.e.
F=1.00 for all compounds), and the values were corrected
for moisture content to give dry-basis qualitative data.
Results and discussion

Compound identification and quantification Effect of temperature on ASE

In total, 13 representative terpenoids and seven sterols were During ASE, it is important to optimize different param-
qualitatively and quantitatively analyzed in order to assess eters, including the extraction solvent used, the extraction
the performance of the ASE at extracting terpenoids and temperature, the number of static cycles and the static time,
Table 1 ASE, Soxhlet extraction and ultrasonically assisted extraction yields at different temperatures (results in μg g−1)
tR (min) Compound ASE (100 °C) ASE (125 °C) ASE (150 °C) Soxhlet Sonic
(RSD%) (RSD%) (RSD%) (RSD%) (RSD%)

14.15 Limonene (t1) 14.49(3.6) 10.90(1.2) 11.48(9.9) 68.19(5.7) 62.02(2.7)

31.35 Geranyl isovalerate (t2) 5.02(0.3) 3.96(7.8) 3.83(14.3) 0.88(8.2) 1.48(4.5)
34.26 (E)-6, 10-Dimethyl-5, 2.49(2.3) 1.93(2.7) 1.96(1.3) 3.96(5.4) 4.16(0.3)
9-undecadien-3-one (t3)
34.91 5,9,13-Trimethyl-4,8,12-tetradecatriene (t4) 2.60(3.9) 2.43(7.3) 2.75(16.2) 6.06(3.8) 2.97(11.7)
41.90 2-Methyl-4-[2,6,6-Trimethyl-cyclohex-1-enyl] 0.74(8.0) 0.69(1.2) 0.83(12.8) 1.53(12.8) 1.10(2.9)
but-2-en-1-ol (t5)
56.25 3,7,11,15-Tetramethyl-2-hexadecen-1-ol (t6) 157.2(3.9) 181.56(5.5) 179.3(3.3) 289.6(9.8) 263.8(4.5)
56.42 6,10,14-Trimethyl-2-pentadecanone (t7) 2.99(6.0) 3.38(0.7) 3.92(16.1) 7.06(12.9) 8.75(5.1)
59.73 [E,E]-6,10,14-Trimethyl-5,9,13- 2.49(5.7) 3.01(4.9) 3.03(2.5) 5.13(8.0) 4.85(3.3)
pentadecatriene-2-one (t8)
59.95 Squalene (t9) 1.38(2.3) 1.11(5.7) 1.34(4.4) 6.05(0.3) 5.97(6.6)
73.13 Phytol (t10) 3.92(5.0) 3.92(5.1) 4.41(7.1) 8.54(10.0) 9.93(5.9)
91.97 2,6,10,14,18-Pentamethyl-2,6,10,14, 5.00(2.9) 4.53(0.3) 4.87(0.5) 20.02(0.5) 19.03(2.3)
18-eicosapentene (t11)
118.98 γ-Tocopherol (t12) 4.18(3.3) 2.33(2.9) 2.89(11.4) 18.20(2.1) 17.98(7.5)
121.65 Vitamin E (t13) 17.29(4.7) 14.53(2.5) 14.32(18.6) 100.7(8.2) 75.4(12.4)
108.49 Stigmasterol (s1) ND ND ND 0.31(15.4) 0.34(20.9)
113.90 β-Sitosterol (s2) ND 9.32(3.4) 9.50(4.5) 0.92(17.6) ND
120.64 Cholesterol (s3) 6.16(7.2) 23.92(0.8) 25.43(5.0) 28.38(6.1) 27.61(3.3)
122.51 Ethyliso-allocholate (s4) 3.00(2.5) 2.87(1.8) 2.67(8.5) 20.20(0.6) 13.0(11.0)
124.61 Campesterol (s5) 4.13(1.0) 11.77(1.1) 13.26(9.5) 52.96(2.3) 54.19(3.7)
125.83 Stigmasterol-5,22-dien-3-ol (s6) 19.46 (7.5) 18.54(1.5) 20.86(11.1) 90.54(1.5) 84.01(6.6)
129.25 [3α,24Z]-Stigmasta-5,24[28]-dien-3-ol (s7) 12.09(5.3) 10.51(1.9) 11.47(8.5) 50.96(3.2) 51.51(5.4)
ND: not detected
1006
in order to achieve a satisfactory extraction efficiency.
Pressure, which is required to maintain the solvents in their
liquid states, has very little impact on analyte recovery, and
1,500 psi is the standard pressure used for most applica-
tions. Due to the nonpolar character of terpenoids and
sterols, petroleum ether was selected as the extraction
solvent, which is a solvent routinely used in the extraction
of tobacco lipids. The standard ASE method states that the
static cycle is 1 min and the static time 5 min, respectively. It
has been suggested that the first extraction step extracts
most of the compounds of interest, the yield of the second is
much lower but not negligible, and the third extraction yield
is very low [18, 27]. Also, the yield from a 10 minute
extraction was nearly the same as that for a 15 minute
extraction, while the yield from a 10 minute extraction was
higher than that from a 5 minute extraction but lower than
the yield from two successive 5 minute extractions [27].
Therefore, a static extraction time of 2×5 min was chosen
for all further investigations. The results from the extrac-
tions of terpenoids and sterols from tobacco by ASE at
different temperatures are summarized in Table 1. Because
the molecular names are relatively long, different symbols
are used, such as t1, t2 and t3. . . for terpenoids and s1, s2 and
s3 . . . for sterols, respectively.
As shown in Table 1, the effect of temperature on the
extractions of terpenoids with different characteristics
varies. The yields of diterpenes, such as t1, t2 and t3, are
highest at 100 °C. When the temperature is raised from 100
to 125 °C, the yields decrease, otherwise, there are only
minor changes from 125 to 150 °C. This may due to partial
pyrolysis of diterpene up to temperatures as high as 125 °C,
while the pyrolysis approaches equilibrium after this
temperature. A similar phenomenon was observed when
extracting saturated triterpenoids with phenyl, such as t12
and t13. For triterpenes with hydroxy groups, such as t4 and
t5, hexterpene t9, and pentaterpenoid t11, there temperature
did not affect extraction efficiency. For triterpenoids with
carbonyl, such as t7 and t8, and the semi-saturated
tetraterpenoid t6, the extraction yields increased when the
temperature was raised from 100 to 125 °C, but further
increases in extraction yields were not seen when the
temperature was raised. However, the yield of another
tetraterpenoid, t10, which is an isomeric compound of t6 but
has a different stereoconformation, increases from 125 to
150 °C. In Table 1, it was also found that the extraction
yields of most terpenoids show poorer reproducibilities
below 150 °C compared with those at 100 and 125 °C. This
may be because more terpenoid pyrolysis takes place at
higher temperatures.
Unfortunately, when assessing the extraction efficiency
of sterols by ASE, as shown in Table 1, it was found that s1,
which is one of the most important sterols in tobacco, can-
not be extracted by ASE under the temperatures investi-
gated. However, the yields of another three important
sterols, s2, s3 and s5, increase as the temperature is raised
from 100 to 125 °C, although no further increases were seen
beyond this temperature. For others, such as s4, s6 and s7, as
shown in Table 1, we can see that temperature had no effect
on their yields. Although the yields of sterols that are
difficult to extract may increase at temperatures higher than
150 °C, the evocative smell produced at around this tem-
perature suggests that some destructive reactions took
place. Similar to terpenoids (Table 1), the extraction yields
of sterols show poorer reproducibilities below 150 °C
compared with those at 100 and 125 °C.
The total yields of sterols and terpenoids, as shown in
Fig. 2, increase as the temperature is raised from 100 to
125 °C, but not beyond this temperature.
Comparison of ASE with Soxhlet extraction
and ultrasonically assisted extraction
To investigate the relative efficiency of ASE at extracting
terpenoids and sterols from tobacco, experiments were
carried out using ASE, Soxhlet extraction and ultrasoni-
cally assisted extraction. The results are listed in Table 1,
where qualitative ASE data under 100 °C are taken into
account. The corresponding GC–MS total ion chromato-
grams (TIC) are shown in Fig. 3.
As shown in Table 1, Soxhlet extraction and ultrason-
ically assisted extraction have similar extraction efficien-
cies, which are several times that of ASE except in the case
of the extraction of geranyl isovalerate. Indeed, when
Soxhlet extraction was employed, stigmasterol and β-
sitosterol (which are not usually extracted by ASE and are
seldom extracted by ultrasonically assisted extraction) were
extracted, although with low recoveries. It is also apparent
from Fig. 2 that the total yields of terpenoids and sterols
were highest when Soxhlet extraction was used for a period
as long as 5 h. On the other hand, due to the automization of
ASE, the reproducibility was better (0.3–8.0%) than that of
Soxhlet extraction (0.5–12.9%) and ultrasonically assisted
extraction (0.3–12.4%). Here, the reproducibility of stig-
masterol and β-sitosterol were not taken into account.
Total content of petroleum ether extracts
The total content of PEE is an important index for eval-
uating the quality of flue-cured tobacco. PEE mainly
sterols
500 terpenoids
400
300
ug g-1
200
100
0
ASE(100°C) ASE(125°C) ASE(150°C) ultrasonic Soxlet
Fig. 2 Total yields of sterols and terpenoids extracted by ASE,
Soxhlet extraction and ultrasonically assisted extraction

Fig. 3 GC–MS total ion chro- 2.0x107
matograms (TICs) of extracts
obtained with different tech-
niques: a ultrasonically assisted
extraction, b ASE extraction
under 100 °C, c Soxhlet
extraction
2.0x107
2.0x107
includes volatile components that directly enter smoke and
precursors of aroma components, which are transferred into
volatile components during the course of ripening, ageing,
and burning of tobacco. To quantify the PEE, after GC–MS
the peaks of all of the fluent components were integrated
and the total yield of PEE was obtained by the internal
standard method using heptadecane as internal standard
without considering calibration factors (i.e. F=1.00 for all
compounds).
It is apparent from Fig. 4 that the yields of PEE were
highest value at 100 °C when ACE was employed. The
yields decreased when the temperature was raised from 100
to 125 °C. Otherwise, there were only minor changes from
125 to 150 °C. The reason may be that the loss of yield
caused by the pyrolysis of some components in PEE
exceeded the increase in yield of other components for
temperatures as high as 125°C. Soxhlet extraction has a
much better yield, almost three times better, than ASE.
4000
Petroleum ether extracts
3000
ug g-1
2000
1000
0 References
ASE (100°C) ASE (125°C) ASE (150°C) ultrasonic
Fig. 4 Total PEE yields for ASE, Soxhlet extraction and
ultrasonically assisted extraction
1007
a
20 40 60 80 100 120
20 40 60 80 100 120
c
20 40 60 80 100 120
Retention time (min)
Ultrasonically assisted extraction gives a similar PEE
extraction yield to Soxhlet extraction.
Conclusions
The extraction of terpenoids and sterols from plant materials
is a matrix- and method-dependent process. The possibility
of extracting terpenoids, sterols and total PEE from tobacco
using ASE was investigated. The results indicate that for
components present at high levels, such as terpenoids, which
are predominant in PEE, ASE is quite suitable for quanti-
tative analysis. When comparative studies were carried out, it
was found that the highest efficiency was obtained by
Soxhlet extraction, and this technique is especially suited to
sterols that cannot usually be extracted by ASE or are seldom
extracted by ultrasonically assisted extraction. On the other
hand, ASE exhibits better reproducibility and allows the
sequential extraction of samples due to automation. Because
of these advantages, together with its higher speed and
reduced solvent consumption, ASE appears to be a
promising alternative to classical methods for extracting
terpenoids and PEE from tobacco, especially when investi-
gating large batches of tobacco samples comparatively.
However, for sterol analysis, the preferred method is still
Soxhlet extraction.
Acknowledgements This study is supported by the outstanding
youth fund (No. 20325517) from National Scientific Foundation of
China (NNSFC), and the Teaching and Research Award Program for
Outstanding Young Teachers (TRAPOYT) in higher education
institutions of the Ministry of Education (MOE), PR China.
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