Journal of Food Composition and Analysis 24 (2011) 1069–1072

Contents lists available at ScienceDirect

Journal of Food Composition and Analysis

journal homepage: www.elsevier.com/locate/jfca

Short Communication

Rapid identification and determination of 11 polyphenols in Herba lycopi by

HPLC–MS/MS with multiple reactions monitoring mode (MRM)
Junbo Xie a,b,*, Yanqing Zhang a,b,**, Deqiang Kong a, Marhaba Rexit a
a
Department of Pharmaceutical Engineering, Tianjin University of Commerce, Tianjin 300134, China
b
Tianjin Key Laboratory of Food Biotechnology, The East of Jinba Road, Tianjin 300134, China

A R T I C L E I N F O A B S T R A C T

Article history: A new and rapid method was developed for simultaneous identification and determination of 11
Received 16 July 2010 polyphenols in Herba lycopi, i.e. rosmarinic acid, protocatechuic aldehyde, protocatechuic acid, caffeic
Received in revised form 20 December 2010 acid, ferulic acid, apigenin, luteolin, quercetin, isorhamnetin, chlorogenic acid and rutin, using high
Accepted 29 December 2010
performance liquid chromatography (HPLC) coupled with triple quadrupole mass spectrometry (MS/
Available online 19 February 2011
MS). Quantitation was based on multiple reactions monitoring (MRM) using the precursor ! production
combination for the determination of 11 analytes. The analysis was performed on an Eclipse Plus C18
Keywords:
column (I.D. 4.6 mm  100 mm, 3.5 mm particle size). An electrospray ionization (ESI)-tandem interface
Lycopus lucidus Turcz
High performance liquid chromatography
in the negative mode was employed prior to mass spectrometric detection. With the optimized
Triple quadrupole mass spectrometry conditions, the 11 biomarkers were detected properly within 3 min. Limits of detection (LOD) ranged
Polyphenols from 0.6 to 2.6 ng/ml. The average recoveries ranged from 95.42 to 101.11% with RSDs  2.23%. The
Multiple reactions monitoring applicability of this analytical approach was confirmed by the successful analysis of 3 samples. The
Food analysis established method was validated and found to be sensitive and reliable for the determination of 11
Food composition analytes in Herba lycopi.
ß 2011 Elsevier Inc. All rights reserved.

1. Introduction contribute to the antioxidant activity (Slusarczyk et al., 2009; Woo

Herba lycopi, which is the aerial part of Lycopus lucidus Turcz,
has been used in China as a traditional herbal tea for over one
thousand years (Yang et al., 2010). Due to its anti-inflammatory
(Lee et al., 2008; Yang et al., 2010), thyroid regulatory (Yarnell and
Abascal, 2006), anti-allergic (Shin et al., 2005), estrogenic (Kim et
al., 2008) and hyaluronidase inhibitory activities (Murata et al.,
2010), Herba lycopi is gradually being used more and more in
modern functional foods such as infusions and beverages for
improving health. In addition, it has been more and more widely
used as botanical food supplement, especially as a source of
antioxidants (Slusarczyk et al., 2009).
Chemical constituents from the Herba lycopi that have been
studied include mainly phenols such as rosmarinic acid, proto-
catechuic acid, protocatechuic aldehyde, rutin, luteolin and caffeic
acid (Malik et al., 2002; Sun et al., 2004; Takahashi et al., 1999).
The published studies on L. lucidus have proved that the phenols
* Corresponding author at: Tianjin Key Laboratory of Food Biotechnology, The
East of Jinba Road, Tianjin 300134, China. Tel.: +86 2226667633;
fax: +86 2226686254.
** Corresponding author at: Tianjin Key Laboratory of Food Biotechnology, The
East of Jinba Road, Tianjin 300134, China.
E-mail addresses: junboxie@yahoo.com.cn (J. Xie), zhyanqing@yahoo.com.cn
(Y. Zhang).
and Piao, 2004). It has also been shown that the activity of the
single phenolic constituent proved to be much smaller than that
shown by the crude plant extracts, which illustrated that the
bioactivity came from all of the constituents (Wojciechowski et al.,
1995). Given the insufficient published data on Herba lycopi, it is
necessary to study its potential constituents that help to develop a
dietary source of antioxidants.
Few analytical methods have been reported on the quality
assessment of Herba lycopi (Slusarczyk et al., 2009; Nie et al.,
2006), and only a few phenolics have been assayed. Due to the
complex constituents in the herbal medicine, a complicated
sample preparation process has usually been applied before
analysis in the common HPLC, which consumed large amounts of
organic solvents and time. Recently, the introduction of tandem
mass spectrometric detection (MS/MS) has led to a notable
improvement in detectability and selectivity by employing
multiple reaction monitoring (MRM) data acquisition (Lurie and
Toske, 2008; Gruz et al., 2008). Due to the high selectivity of MRM
mode, sample pre-processing and optimization of chromatograph-
ic separation is greatly simplified. Furthermore, precursor and
production ion monitoring can be used to increase specificity of
detection and identification of the known molecules.
In this study, a new validated and simple HPLC–MS/MS with a
multiple-reaction monitoring mode method was developed for
simultaneous identification and determination of rosmarinic acid,

0889-1575/$ – see front matter ß 2011 Elsevier Inc. All rights reserved.
doi:10.1016/j.jfca.2010.12.016
1070 J. Xie et al. / Journal of Food Composition and Analysis 24 (2011) 1069–1072

protocatechuic aldehyde, protocatechuic acid, caffeic acid, ferulic Table 1

MS/MS detection parameters for 11 analytes in Herba lycopi (negative ionization
acid, apigenin, luteolin, quercetin, isorhamnetin, chlorogenic acid
mode).
and rutin in Herba lycopi.
Compounds Transition Dwell The fragmentation The collision
(m/z) time energies energies
2. Materials and methods
(ms) of Q1 (V) (eV)

2.1. Reagents and solutions Rosmarinic acid 359.1 ! 161.0 50 100 10

Protocatechuic 137.0 ! 108.0 50 120 35
aldehyde
The substances of 11 polyphenols were purchased from the Protocatechuic 153.0 ! 108.9 50 100 15
Shanghai Touto Biotech Co. Ltd. (Shanghai, China). Three samples acid
of Herba lycopi (Hebei 1#, Henan 2#, Shandong 3#) were obtained Caffeic acid 178.9 ! 134.9 50 100 10
Ferulic acid 193.0 ! 133.9 50 100 10
from different areas in China. The amount of Herba lycopi
Apigenin 269.0 ! 116.9 50 160 30
harvested was 1 kg each, and three independent replicates of Luteolin 285.0 ! 133.0 50 160 30
each sample were gathered. They were all identified by Dr. Junbo Quercetin 301.0 ! 150.9 50 140 20
Xie. The voucher specimens were deposited in the department of Isorhamnetin 315.0 ! 300.0 50 140 25
pharmaceutical engineering, Tianjin University of Commerce, Chlorogenic acid 353.0 ! 191.0 50 100 5
Rutin 609.0 ! 299.9 50 220 35
China. Chromatographic grade methanol was purchased from
Dima Technology Inc. (Richmond Hill, USA), and other chemicals
were of analytical reagent grade. All aqueous solutions were made dissociation (CID). Quantification was performed using multiple
up in deionized water. reaction (MRM) modes. The delta potential of the electron
multiplier (EMV) was set to 200 V for data acquisition. The
2.2. Preparation of sample solutions summary of MS/MS detection parameters is shown in Table 1.
Data acquisition and processing were performed by Agilent
The crude material of Herba lycopi were milled into powder MassHunter Workstation (Zhang et al., 2010).
with a FW100 Universal High-speed Smashing Machine (Taisite
Instrument Co., Ltd., Tianjin, China) and dried at 80 8C for 4 h before 2.5. Validation of HPLC method
determination. The content in samples was established on dry
weight basis. A total of 10 ml of methanol was added accurately to 2.5.1. Limits of detection and quantification
50 mg powder (passed through a 60-mesh screen) in a 25 ml The limits of detection (LOD) and quantification (LOQ) were
volumetric flask. Then it was extracted by sonication in SK3200LH determined at a signal-to-noise (S/N) ratio of 3 and 10,
ultrasonic cleaning instrument (Shanghai Kudos Ultrasonic In- respectively. They were performed using serial diluted reference
strument Co., Shanghai, China) for 40 min at ambient temperature. solution of each compound under the present chromatographic
Three replicates of the extraction process were carried out on the conditions.
independent samples. Before being injected into the HPLC system,
all solutions were diluted to proper concentration and filtered 2.5.2. Precision, reproducibility and accuracy
through 0.45 mm membrane filters. Intra- and inter-day variations were determined at certain
concentration of sample for precision. Five different working
2.3. Preparation of standard solutions solutions prepared from the 1# sample were investigated to
confirm the reproducibility, and variations were expressed by
Stock solution of the mixture of the reference compounds was relative standard deviations (R.S.D.). Recovery test was performed
prepared by dissolving accurately the weighted standards in with the method of standard addition. Accurate amounts of
methanol, transferring it to a 5 ml volumetric flask, and then reference compounds were added to 1# sample, then extracted
adding methanol to make up the volume (Zhang et al., 2010). The and analyzed as described (n = 6).
stock solution was further diluted to make different concentration
ranges. The calibration curve was performed with at least six 3. Results and discussion
appropriate concentrations. Three independent replicates of each
standard were analyzed. 3.1. Optimization of MS/MS conditions

2.4. HPLC–MS/MS instrumentation and conditions
The HPLC analyses were performed on an Agilent 1200 series
(Agilent, Santa Clara, USA) equipped with a G1312B Binary pump,
G1367D-HiPALS SL autosampler and a G1316B-TCCSL column
oven. The sample was separated using Agilent Eclipse Plus C18
column (I.D. 4.6 mm  100 mm, 3.5 mm particle size; Agilent) and
the column temperature was 25 8C. Isocratic elution was applied
with 0.1% acetic acid and methanol (20:80, v/v). The flow rate was
0.30 ml/min, and the injection volume was 20 ml.
Determination was performed using a Agilent Technologies
6410 Triple Quad LC/MS equipped with electrospray ionization
(ESI). The compounds were ionized in the negative ion polarity
mode. The ionization source conditions were as follows: spray
voltage of 4000 V, source temperature of 100 8C and desolvation
temperature of 350 8C. Nitrogen was used as nebulizer gas and
pressure was set at 35 p.s.i at a flow rate of 8 L/min. The pressure
of high purity nitrogen was 0.15 MPa for collision-induced
The MS/MS parameters were optimized to improve the overall
sensitivity and minimize the matrix effects according to the peak
areas of the analytes, such as capillary voltage, ion mode, collision
energy, cone voltage and dwell times. For example, when the
fragmentation energies of Q1 for rosmarinic acid was optimized to
be 100 V, the maximum response of the compound fragment ion
peaks was achieved. Matrix effects result from co-eluting matrix
components that affect the ionization of the target analyte,
resulting in either ion suppression or ion enhancement (Chambers
et al., 2007). In the study, it was also found that matrix effects
influenced the assay results greatly. The result showed that the ESI
ion mode and the assayed solutions concentration were the main
effective factors. ESI in both negative and positive ion modes were
tried and the results showed that ESI in negative ion mode was
more sensitive for phenolic acids in the present study. In addition,
the assayed solutions must be diluted to the proper concentration.
The optimized MS/MS parameters and MRM transitions were
found to be consistent with those previously described research
 J. Xie et al. / Journal of Food Composition and Analysis 24 (2011) 1069–1072
Fig. 1. LC/MS/MS chromatographic profiles of Herba lycopi sample. 1—rosmarinic acid, 2—protocatechuic aldehyde, 3—protocatechuic acid, 4—caffeic acid, 5—ferulic acid, 6—
apigenin, 7—luteolin, 8—quercetin, 9—isorhamnetin, 10—chlorogenic acid, and 11—rutin.
overall (Liu et al., 2010; Wan et al., 2007; Bastos et al., 2007). The
fragmentation mechanism of precursor ! production was pre-
sumed as shown as followed, i.e. m/z 359.1 [MH] ! 161.0
[MH–C9H10O3] for rosmarinic acid, m/z 153.0 [MH] ! 108.9
[MH–COO] for protocatechuic acid, m/z 137.0 [MH] ! 108.0
[MH–CHO] for protocatechuic aldehyde, m/z 179.0
[MH] ! 135.0 [MH–COO] for caffeic acid, m/z 353.0
[MH] ! 191.0 [MH–caffeoyl] for chlorogenic acid and m/z
193.0 [MH] ! 133.9 [MH–COO–CH3–] for ferulic acid. Due
to the identical flavone aglycone structure, quercetin, luteolin and
apigenin had the similar fragmentation pattern. Some neutral
diagnostic losses and specific retro Diels–Alder reaction contrib-
uted to the product ions spectra of deprotonated molecule ions
[MH]. As a result, quantification was performed using multiple
reaction (MRM) mode at m/z 301.0 ! 150.9 for quercetin, m/z
285.0 ! 133.0 for luteolin and m/z 269.0 ! 117.0 for apigenin. In
addition, after collision-induced dissociation, one rutinose group
fell off from deprotonated rutin [MH] (m/z 609.0) producing a
distinctive radical of [MH-309] (m/z 299.9). Deprotonated
isorhamnetin [MH] (m/z 315.0) lost a methyl and produced
[MH–CH3] (m/z 300.0).
3.2. Method validation for quantitative determination of the bioactive
markers
With the optimized conditions, the 11 biomarkers were
detected properly within 3 min (Fig. 1). The biomarkers in Herba
lycopi were identified and confirmed by comparing the retention
time and MS spectrum of the reference standards. A calibration
1071
curve for each marker was constructed and tested for linearity. The
calibration curve was prepared for each polyphenolic compound
by measuring six different concentrations of the standard solution
in the concentration range of 2.3–2000 ng/ml. All injections were
repeated three times (n = 3). For calibration, the plots for the
concentration (X) versus mean peak area (Y) were drawn to obtain
linearity. Table 2 shows the results of linearity, LOD and LOQ for
each compound. All calibration curves were linear with good
correlation coefficients (R2  0.998). The results showed that the
precision and reproducibility of the 11 biomarkers were good. The
average recoveries ranged from 95.42% (luteolin) to 101.11%
(ferulic acid) with RSDs  2.23% (ferulic acid).
3.3. Sample analysis
Polyphenols are the group of substances, including phenolic
acids and flavonoids, which are widely distributed in the plants.
Due to multiple health value of the natural products, it is necessary
to develop more analytical techniques to find them from the
natural resources (Boros et al., 2010).
Compared with the common HPLC method, the novelty of the
method was mainly in its application to a wide group of
polyphenolic compounds assayed in a fairly shorter time. It was
subsequently applied to a simultaneous determination of the 11
bioactive markers in Herba lycopi. The assay results are listed in
Table 3.
It was found that there were remarkable differences between
the samples, in terms of concentrations of the 11 bioactive
markers. The abundant and various phenolics contained in the
1072 J. Xie et al. / Journal of Food Composition and Analysis 24 (2011) 1069–1072

Table 2
Summary of regression data, LODs, LOQs for 11 analytes in Herba lycopi.

Analyte Calibration curve R2 Linear range (ng/ml) LODa (ng/ml) LOQb (ng/ml)

Rosmarinic acid Y = 101.6X + 153.8 0.9993 10–2000 2.1 6.5

Protocatechuic aldehyde Y = 5.035X + 109.39 0.9998 6.5–650 1.4 2.9
Protocatechuic acid Y = 6.4601X + 29.778 0.9999 15–900 2.2 4.7
Caffeic acid Y = 19.165X  129.17 0.9998 15.5–1550 2.1 5.1
Ferulic acid Y = 31.53X  87.13 0.9999 2.3–230 0.6 1.6
Apigenin Y = 24.569X + 58.389 1 3.5–350 0.7 1.8
Luteolin Y = 21.701X  48.722 0.9999 15–750 0.9 3.1
Quercetin Y = 9.714X  53.833 0.9999 15–750 2.6 5.5
Isorhamnetin Y = 15.763X + 18.5 1 10–500 1.6 4.2
Chlorogenic acid Y = 3.6307X + 26.83 0.9981 10–500 2.0 5.7
Rutin Y = 14.119X  46.278 0.9999 6.1–610 1.3 2.8
a
Limit of detection (3 S/N).
b
Limit of quantitation (10 S/N).

Table 3 Chambers, E., Wagrowski-Diehl, D.M., Lu, Z., Mazzeo, J.R., 2007. Systematic and
The mean contents of 11 analytes in Herba lycopi samples (mean  S.D., n = 3). comprehensive strategy for reducing matrix effects in LC/MS/MS analyses.
Journal of Chromatography B 852 (1–2), 22–34.
Analyte Contents (mg/100 g) Gruz, J., Novák, O., Strnad, M., 2008. Rapid analysis of phenolic acids in beverages by
UPLC–MS/MS. Food Chemistry 111 (3), 789–794.
Hebei Henan Shandong Kim, I.G., Kang, S.C., Kim, K.C., 2008. Screening of estrogenic and antiestrogenic
Rosmarinic acid 90.70  2.11 174.86  3.57 120.22  3.35 activities from medicinal plants. Environmental Toxicology and Pharmacology
Protocatechuic aldehyde 5.38  0.08 10.21  0.07 8.19  0.05 25 (1), 75–82.
Lee, Y.J., Kang, D.G., Kim, J.S., Lee, H.S., 2008. Lycopus lucidus inhibits high glucose-
Protocatechuic acid 23.39  1.72 36.19  1.19 25.63  0.83
induced vascular inflammation in human umbilical vein endothelial cells.
Caffeic acid 84.86  1.90 106.75  2.41 73.58  1.27
Vascular Pharmacology 48 (1), 38–46.
Ferulic acid 1.09  0.10 2.37  0.04 1.87  0.04 Liu, Y., Li, X., Lia, Y., Wang, L., Xue, M., 2010. Simultaneous determination of
Apigenin 4.10  0.11 10.58  0.20 6.46  0.08 danshensu, rosmarinic acid, cryptotanshinone, tanshinone IIA, tanshinone I
Luteolin 28.38  1.20 20.32  1.92 27.81  2.51 and dihydrotanshinone I by liquid chromatographic–mass spectrometry and
Quercetin 27.38  2.17 21.34  3.01 32.84  1.33 the application to pharmacokinetics in rats. Journal of Pharmaceutical and
Isorhamnetin 2.86  0.12 5.15  0.09 3.53  0.17 Biomedical Analysis 53 (3), 698–704.
Chlorogenic acid 5.22  0.29 6.44  0.36 5.43  0.16 Lurie, I.S., Toske, S.G., 2008. Applicability of ultra-performance liquid chromatog-
Rutin 10.80  1.58 14.11  1.99 15.42  0.78 raphy–tandem mass spectrometry for heroin profiling. Journal of Chromatog-
raphy A 1188 (2), 322–326.
Malik, A., Yuldashev, M.P., Obid, A., Ismoil, T., Ping, L.Y., 2002. Flavonoids of the
herb contributed to the antioxidant activity, which showed that it aerial part of Lycopus lucidus. Chemistry of Natural Compounds 38 (6), 612–613.
is a good natural source of dietary antioxidants (Slusarczyk et al., Murata, T., Watahiki, M., Tanaka, Y., Miyase, T., Yoshizaki, F., 2010. Hyaluronidase
Inhibitors from Takuran, Lycopus lucidus. Chemical and Pharmaceutical Bulletin
2009). 58 (3), 394–397.
Nie, B., Liu, Y., Xu, Q., Shi, J.L., Liang, X.M., Xiao, P.G., 2006. HPLC determination of
4. Conclusions caffeic acid in Herba Lycopi. China Journal of Chinese Materia Medica 31 (11)
882–883, 891.
Shin, T.Y., Kim, S.H., Suk, K., Ha, J.H., Kim, I., Lee, M.G., Jun, C.D., Kim, S.Y., Lim, J.P.,
An HPLC–MS/MS method with multiple reactions monitoring Eun, J.S., Shin, H.Y., Kim, H.M., 2005. Anti-allergic effects of Lycopus lucidus on
mode method was developed for simultaneous identification and mast cell-mediated allergy model. Toxicology and Applied Pharmacology 209
(3), 255–262.
determination of rosmarinic acid, protocatechuic aldehyde, pro- Slusarczyk, S., Hajnos, M., Skalicka-Wozniak, K., Matkowski, A., 2009. Antioxidant
tocatechuic acid, caffeic acid, ferulic acid, apigenin, luteolin, activity of polyphenols from Lycopus lucidus Turcz. Food Chemistry 113 (1),
quercetin, isorhamnetin, chlorogenic acid and rutin to evaluate 134–138.
Sun, L.N., Chen, W.S., Tao, Z.Y., Yuan, Y., Zhang, H.M., 2004. Studies on the chemical
the quality of Herba lycopi. The method has been validated with
constituents of Lycopus Lucidus II. Pharmaceutical Journal of Chinese People s
good sensitivity, precision and reproducibility. The results Liberation Army 20 (3), 172–174.
indicated that the HPLC–MS/MS method provided improved Takahashi, Y., Nagumo, S., Noguchi, M., Nagai, M., 1999. Phenolic constituents of
Lycopus lucidus. Natural Medicines 53 (5), 273–274.
chromatographic conditions to simplify sample throughput with
Wan, L., Guo, C., Yu, Q., Li, Y., Wang, X., Wang, X., Chen, C., 2007. Quantitative
less time and lower limits of quantitation (LOQ) compared with the determination of apigenin and its metabolism in rat plasma after intravenous
usual analytical method. bolus administration by HPLC coupled with tandem mass spectrometry. Journal
of Chromatography B 855 (2), 286–289.
Wojciechowski, H., Gumbinger, H.G., Vahlensieck, U., Winterhoff, H., Nahrstedt, A.,
Acknowledgement Kemper, F.H., 1995. Analysis of the components of Lycopus europaeus L. in body
fluids during metabolism studies comparison of capillary electrophoresis and
This research was financially supported by National Natural high-performance liquid chromatography. Journal of Chromatography A 717
(1–2), 261–270.
Science Foundation of China (No. 31000749). Woo, E.R., Piao, M.S., 2004. Antioxidative constituents from Lycopus lucidus.
Archives of Pharmacal Research 27 (2), 173–176.
References Yang, X.B., Lv, Y., Tian, L.M., Zhao, Y., 2010. Composition and systemic immune
activity of the polysaccharides from an herbal tea (Lycopus lucidus Turcz).
Bastos, D.H., Saldanha, L.A., Catharino, R.R., Sawaya, A.C.H.F., Cunha, I.B.S., Carvalho, Journal of Agricultural and Food Chemistry 58 (10), 6075–6080.
P.O., Eberlin, M.N., 2007. Phenolic antioxidants identified by ESI-MS from Yerba Yarnell, E., Abascal, K., 2006. Botanical medicine for thyroid regulation. Alternative
Maté (Ilex paraguariensis) and green tea (Camelia sinensis) Extracts. Molecules and Complementary Therapies 12 (3), 107–112.
12 (3), 423–432. Zhang, Y.Q., Xie, J.B., Liu, Y., Wang, D.P., 2010. Identification and simultaneous
Boros, B., Jakabová, S., Dörnyei, Á., Horváth, G., Pluhár, Z., Kilár, F., Felinger, A., determination of mangiferin, neomangiferin, timosaponin A-III, and timosapo-
2010. Determination of polyphenolic compounds by liquid chromatography– nin C in Rhizoma anemarrhenae by rapid resolution liquid chromatography
mass spectrometry in Thymus species. Journal of Chromatography A 1217 coupled with triple quadrupole mass spectrometry. Analytical Letters 43 (14),
(51), 7972–7980. 2210–2219.