American Journal of Anal yt ical Chemistry, 2011, 2, 929-933
doi:10.4236/ajac.2011.28107 Published Online December 2011 (http://www.SciRP.org/journal/ajac)
Rapid and Sensitive Analysis of Eight Polyphenols in
Tobacco by Rapid Resolution Liquid Chromatogarphy
Fuwei Xie1,2, Ajuan Yu1, Dengke Hou1, Huimin Liu2, Li Ding2, Shusheng Zhang1*
1Chemistry Department of Zhengzhou University, Zhengzhou, China
2Zhengzhou Tobacco Research Institute of CNTC, Zhengzho u, China
Received September 5, 2011; revised October 15, 2011; accepted October 29, 2011
Abstract
A rapid resolution liquid chromatographic (RRLC) method for the determination of eight polyphenols in to-
bacco was developed. Polyphenols were extracted from tobacco samples by methanol/ water in an ultrasonic
bath, then subjected to clean up by solid phase extraction. The separation was performed on a 50 × 4.6 mm,
1.8 μm ZORBAX Eclipse XDB-C18 column. Compared with conventional HPLC method, the analysis time
for eight polyphenols by RRLC method was reduced from 20 to 5 min without sacrificing resolution, and the
sensitivity was improved. This method appears simple, accurate and precious. The relative standard devia-
tions (RSD) of overall analysis procedure for eight tobacco polyphenols were less than 2% with the recover-
ies ranging from 94% to 107%. This method could be applied to the rapid determination of major polyphe-
nols in tobacco with satisfactory results.
Keywords: Solid Phase Extraction, RRLC, Tobacco, Polyphenols
1. Introduction
Polyphenols, including tannic, cumarine, flavonid, and
derivatives of simple phenols, are secondary metabolities
in tobacco plant. They play important roles on the growth
of tobacco and the quality of tobacco leaf [1-3]. The re-
search results showed that in flue-cued tobacco leaves
chlorogenic acid, scopoletin and rutin are the major poly-
phenols [4], and their combustion could generate pheno-
lic compounds considered as carcinogens [5]. Therefore,
it can pose serious harm on consumers’ health during
smoking. To understand the polyphenol content in to-
bacco, polyphenol transfer during smoking and the rela-
tionship between polyphenol and smokers’ health, it is
necessary to develop a practical method for the determi-
nations of polyphenols in tobacco.
To date, several analytical methods for the analysis of
polyphenols in tobacco have been reported by means of
spectrophotometry [6], gas chromatography (GC) or GC-
MS [7], high-performance liquid chromatography (HPLC)
with chemiluminescence detection [8], ultraviolet detec-
tion (UVD) [9,10] and MS detection [11], and capillary
electrophoresis (CE) method [12]. Among these methods,
spectrophotometry is only used for the determination of
the total polyphenols, GC and HPLC are the most pow-
erful. However, GC cannot be used directly to determine
polyphenols due to their poor volatility, high polarity
and/or thermal instability. It needs time-consuming deri-
vation. In contrast GC, HPLC is more effective and ap-
propriate for the separation and determination of poly-
phenols. In order to obtain satisfactory separation of
about 5 - 10 polyphenols by the conventional HPLC, the
longer retention or analysis times between 20 - 30 min
are required. Obviously, it cannot satisfy the requirement
for the rapid analysis of high sample throughput. To
solve this problem, various strategies aimed at increasing
the speed and performance of chromatographic separa-
tion can be considered. Currently, the smaller stationary
phase particles (<2.0 μm), new bridging structure of sta-
tionary phase and high-pressure systems are commer-
cially available. Using this new rapid resolution liquid
chromatography (RRLC) technique, higher linear veloc-
ity, faster run time, higher sensitivity and resolution are
achieved [13-15]. Thus, the aims of the present work are
1) to develop a practical extraction and clean-up proce-
dure prior to the analysis of eight polyphenols (Figure 1)
in tobacco; 2) to develop a rapid and sensitive method to
the analysis of eight polyphenols; and 3) to determine the
polyphenols in actual tobacco samples by the developed
RRLC.

F. W. XIE ET AL. 930
1
2 3
456OH
OH
OH
COOH
H
O
O
OH
OH
O
H3CO
OHO
3-O-caffeoyl-d-quinic acid Scopoletin
Chlorogenic acid (3-O-caffeoyl-d-quinic acid)
Neo Chlorogenic acid (5-O-caffeoyl-d-quinic acid)
O
OH
OH
O
OR'' R'
OH
OH
OH
O
HO
Rutin R’ = OH R” = Rutinosyl Caffeic acid
Quercitrin R’ = OH R” = d-Glucosyl
Kaempferol-3-rutinoside R’ = H R” = l-Rhamnosido-d-Glucosyl R
Figure 1. Structures for the eight polyphenols.
2. Experimental
2.1. Reagents and Solution
All solvent (HPLC grade) were purchased from J&T
Baker (Phillipsburg, NJ, USA), include methanol, ace-
tonitrile and Formic acid. The certified standards used in
this study were 5-O-caffeoylquinic acid (NG, 98%),
chlorogenic acid (CG, 98%), 4-o-caffeoyl-quinic acid
(YG, 98%), caffeic acid (CA, 98%), scopoletin (SP,
98%), rutin(RT, 98%), kaempferol-3-rutinoside (KR,
98%) and quercitrin(QT, 98%), respectively. They are
obtained from Sigma-Aldrich, USA.
Stock solutions of polyphenols were prepared at 1000
µg/ml in methanol and stored under refrigeration at 4˚C
in the dark. Quantification of samples was made using
calibration curves of the eight polyphenols at the final
concentration of 0.5, 1.0, 10, 30, 60 and 120 µg/mL in
the solution of methanol-water (9:1, v/v). Each determi-
nation was performed in triplicate.
2.2. Apparatus and RRLC Conditions
Agilent 1200 RRLC (Agilent, USA) with a binary pump,
a degasser, autosampler and a DAD UV detector; Agilent
1200 High Performance Liquid Chromatographiy (HPLC)
(Agilent, USA) with a binary pump, a degasser, Auto-
sampler and a DAD detector, All the operations and the
data acquiring were controlled by a Agilent chemstation
software.
The separation of eight polyphenols was optimized
and performed on an Agilent Eclipse XDB-C18 RRLC
column, 50 × 4.6 mm id (1.8 µm pore size, Agilent,
USA). The mobile phase consisted of (A) 0.1% formic
acid aqueous solution and (B) acetonitrile. The solvent
program was initially 2 min isocratic with 90% A and
10% B, then from 2 to 5 min linear gradient to 30% B,
finally at 6.5 min linear gradient to 10% B and re-equi-
librium for additional 1.5 min for subsequent analysis.
The flow rate was 2.0 mL/min. The detection wavelength
was set at 340 nm. The injection volume was 5 µL. The
column temperature was set at 30˚C. Thus，the total time
for one-run chromatographic separation was not more
than 8 min.
2.3. Tobacco Sample Pretrement
The tobacco samples were prepared in our laboratory as
follow. One hundred grams of the fluecured tobacco
leaves (without peduncle) were chopped and crushed to
produce the tobacco powder at 40 mesh. 0.25 g of to-
bacco powder was extracted with ultrasonic extraction
for 30 min in 40 mL of methanol-water (70:30, v/v) so-
lution and centrifuged at 10,000 rpm for 10 min. After-
wards, 5 mL solution of centrifuge was loaded onto a
Waters Sep-Park-C18 cartridge (500 mg) previously con-
ditioned with 10 mL methanol and 10 mL deionized wa-
ter, respectively. The first 3 mL eluates were discarded,
the following 2 mL eluates were collected and filtered
through a 0.22 µm membrane. The tobacco extract was
directly analyzed by RRLC.

F. W. XIE ET AL.931
3. Results and Discussion
3.1. Optimization of RRLC Conditions
The separation of eight polyphenols was initially per-
formed on a ZORBAX Eclipse XDB-C18 column (150 ×
4.6 mm id, 5 μm) by conventional HPLC. Although they
were achieved the batter separation under the optimized
HPLC conditions, the analysis time was more than 20
min (Figure 2(a)). It is not suitable for the throughput
analysis. Thus, a new RRLC separation system was cho-
sen. Using the Eclipse XDB-C18 RRLC column (50 × 4.6
mm i.d., 1.8 µm), different mobile phase, elution pro-
gram, flow rate and column temperature were investi-
gated. For example, at flow rate 1.0 mL/min and column
temperature 20˚C, The longest retention time was less
than 6 min, however SP and RT can not be resolved
(Figure 2(b)). By increasing the flow rate from 1 to 2
Figure 2. Separations of eight poly phenols by (a) HPLC and
(b) RRLC at 20˚C. (a) HPLC column: Eclipse XDB-C18
150 × 4.6 mm i.d., 5 µm; Mobile A: 0.1% formic acid /H2O;
B: cetonitrile; Flow rate: 1.0 mL/min; Temperature: 20˚C.
(b) RRLC column: Eclipse XDB-C18 50 × 4.6 mm i.d., 1.8
µm; Mobile A: 0.1% formic acid/H2O; B: acetonitrile; Flow
rate: 1.0 mL/min; Temperature: 20˚C; Peaks: 1, 5-O-caf-
fioylquinic acid; 2, chlorogenic acid; 3, 4-o-caffioylquinic
acid; 4, caffeic acid; 5, scopoletin; 6, rutin; 7, kaempferol-
3-rutinoside; 8, quercitrin.
mL/min and column temperature from 20˚C to 30˚C,
eight polyphenols were separated in baseline within 5
min (Figure 3(a)). More importantly, the analysis was
faster 3 times than conventional HPLC without sacrific-
ing resolution. micellar liquid chromatography (MLC)
also can separate and quantify 3 tobacco polyphenols
(chlorogenic acid, rutin and scopoletin) in less than 10
min, but the theoretical plate of chlorogenic acid in to-
bacco sample using RRLC (2116 plates) was much
higher than that using MLC (576 plates).
3.2. Choice of Extraction and Cleanup Method
To extract polyphenols from tobacco leaf powder, the
following three methods were compared using 40 mL
methanol/ water (70:30, v/v) as extraction solvent for
0.2500 g tobacco leaves: 1) refluxed at 60˚C for 40 min;
2) ultrasonic extraction under 50 Hz for 40 min; and 3)
Figure 3. Chromatograms for (a) polyphe nol standards and
(b) tobacco samples by RRLC RRLC column: Eclipse
XDB-C18 50 × 4.6 mm i.d., 1.8 µm; Mobile A: 0.1% formic
acid/ H2O; B: acetonitrile; Gradient: 0 min (10% B) ~ 2.0
min (15% B ~ 5.0 min (30% B) ~ 6.5 min (50% B) ~ 8.0 min
(10% B); Flow rate: 2.0 mL/min; Temperature: 30˚C.
Peaks: 1, 5-o-caffioylquinic acid; 2, chlorogenic acid; 3,
4-O-caf-fioylquinic acid; 4, caffeic acid; 5, scopoletin; 6,
rutin; 7, kaempferol-3-rutinoside; 8, quercitrin.

F. W. XIE ET AL. 932
mechanical shaking extraction under 15 cycles per min-
ute for 40 min. The results indicated that the extraction
efficiency of refluxed extraction and ultrasonic extrac-
tion (Recoveries of eight polyphenols >90%) were better
than mechanical shaking extraction (Recovery of Sco-
poletin <80%). Because ultrasonic extraction is simpler
than two others, it was chosen for the polyphenols ex-
traction in this paper.
SPE has been proven to be an effective tool for selec-
tively removing interferences, enabling sensitive, selec-
tive and robust analysis. There are some weak polar ma-
terials in tobacco extract which can not be removed
completely from C18 column by mobile phase, such as
fatty substance, wax, pigment, and so on. Moreover, the
smaller stationary phase particles make it easier plug for
RRLC than HPLC. So it is necessary to clean up the ex-
tract before RRLC analysis. In this paper, the solution of
extraction was clean up with a Waters Sep-Park C18 car-
tridge, by which the weak polar material was retained,
and the ployphenol fractions were collected for RRLC
analysis.
3.3. Linearity of Calibration and Limit of
Detection for Eight Ployphenols
Using the optimized RRLC condition, the linear ranges
of the UV response at 340 nm were observed over the
concentration range from 0.5 to 120 µg/mL for eight
ployphenols. The regressions between peak area (y) and
concentration (x, µg/ml) yielded the linear equations as
decribed in Table 1.
The limit of detection (LOD) was evaluated by calcu-
lating a signal to noise ratio of 3 (S/N = 3). The result
was summarized in Table 1.
From Ta ble 1, it can be seen that the LODs by RRLC
were lower than those by HPLC, which enhanced the
detection sensitivity for eight ployphenols.
3.4. Method Validation
A series of samples analysis were performed to validate
the performance of the method. The accuracy was as-
sessed with recovery assay by adding eight standard
polyphenols to sample at low and high levels. The re-
covery was calculated by comparing the found mount of
standards to those of added. The precision was evaluated
from replicated determinations (n = 5) performed on the
different day for same samples. The recoveries of eight
polyphenols ranged from 94% to 107% with the relative
standard deviation (RSD) less than 2.0%.
3.5. Application to Real Tobacco Samples
The proposed RRLC method was used for routine analy-
sis of four tobacco samples from different areas. A typi-
cal chromatogram was displayed in Figure 3(b) and the
results obtained were summarized in Table 2. The results
are consistent with those determined by Zhang [16] and
Table 1. Regression equation and the LODs.
Compound Regression equation Correlation coefficient LOD/RRLC (ng/mL) LOD/HPLC (ng/mL)
NG y = 1.9951x – 0.5391 R2 = 0.9994 59.4 128.2
CG y = 1.8874x – 0.9993 R2 = 0.9996 105.3 211.2
YG y = 1.7039x – 0.6706 R2 = 0.9999 103.5 178.6
CA y = 3.5595x – 0.9966 R2 = 0.9991 51.3 69.4
SP y = 4.2813x + 0.1804 R2 = 0.9995 30.3 43.0
RT y = 1.2785x + 0.2096 R2 = 0.9995 66.7 93.8
KR y = 1.3913x + 0.3109 R2 = 0.9995 58.8 84.7
QT y = 1.0483x + 0.1352 R2 = 0.9995 84.5 121.0
Table 2. The determination results of eight polyphe nols in tobacco sample s.
Compounds Flue-cured tobacco (Yunnan) Burley tobacco (Hubei) Oriental tobacco (Yunnan) Zimbabwel
NG 0.247% 0.008% 0.156% 0.203%
CG 1.522% 0.024% 0.666% 1.156%
YG 0.361% 0.013% 0.289% 0.263%
CA 0.016% 0.007% 0.016% 0.025%
SP 0.017% 0.005% 0.008% 0.032%
RT 0.779% 0.024% 0.424% 0.075%
KR 0.060% 0.004% 0.080% 0.049%
QT 0.022% unfound 0.014% 0.020%

F. W. XIE ET AL. 933
Li [17]. The method demonstrated that it is usable and
applicable to the rapid and sensitive determination of
polyphenols in tobacco.
4. Conclusions
In this paper, a simple, fast, sensitive and reproducible
RRLC analytical method for for eight polyphenols in the
tobacco samples was developed by coupling with a prac-
tical sample pretreatment. With ultrasonic extraction and
Solid-phase extraction (SPE), higher recoveries were
obtained with lower matrix interfering for RRLC separa-
tion of eight polyphenols. The developed method was
suitable for high throughput analysis of the tobacco sam-
ples.
5. Acknowledgements
The authors gratefully acknowledge for the financial
support of the National Nature Science Foundation of
China (No. 20875083, 21077095), the Scientific Founda-
tion of the State Tobacco Monopoly Administration of
China, and the Innovation Scientists & Technicians Troop
Construction Projects of Zhengzhou City (10LJRC192).
They also thank Dr. Julie Rimes for assistance with edit-
ing the text.
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