Journal of Chromatography B, 928 (2013) 78– 82
Contents lists available at SciVerse ScienceDirect
Journal of Chromatography B
L
Tc
KPAAHT
1
ttt(asaoD
srsapoPta
lhp
1h
j ourna l h o mepa ge: www.elsev ier .com/ locate /chromb
etter to the Editor
issue distribution profiles of three antiparkinsonian alkaloids from Piper longum L. in rats determined by liquidhromatography–tandem mass spectrometry
a r t i c l e i n f o
eywords:iper longum L.lkaloidntiparkinsonianPLC–ESI-MS/MSissue distribution
a b s t r a c t
The alkaloids of Piper longum L. (PLA) improved motor dysfunction and dopamine depletion in a rat modelof Parkinson’s disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. A rapid, accurate, sim-ple, and high-performance liquid chromatography–mass spectrometry method was developed and fullyvalidated to simultaneously detect three P. longum L. antiparkinsonian alkaloids (piperine (PPR), piper-longuminine (PPL), and ��,�-dihydropiperlonguminine (DPPL)) in rat plasma, heart, liver, spleen, lung,kidney, and brain tissues. Rat plasma and tissue homogenates were pretreated with methanol/acetonitrile(1:1, v/v) using a simple protein precipitation method. Chromatographic separation was achieved on aPhenomenex Gemini C18 column (50 mm × 2.00 mm, 5 �m) with a gradient mobile phase containing0.1% (v/v) formic acid in water or acetonitrile. The elution was pumped at a flow rate of 0.4 ml/min, andthe injection volume was 10 �l with a total running time of 4 min. The analysis was performed by selectedreaction monitoring of the transitions m/z 285.9 → 201.1, m/z 274.3 → 209.9, and m/z 276.2 → 134.9 forPPR, PPL, and DPPL, respectively. All three analytes showed good linearity (R > 0.995) in plasma and tissuehomogenates, and the lower limit of quantification was 0.20 ng/ml. The distribution of PPR, PPL and DPPL
in all 7 tissues was examined. The highest concentrations for PPR and PPL were observed in the liver,while the highest DPPL concentration was observed in the kidney. Following oral administration, thehighest levels of PPR, PPL and DPPL in different tissues were found at approximately 2 h. PPR, PPL andDPPL could cross the blood–brain barrier. The present study provides evidences for that PPR, PPL andprov
DPPL may play roles in im. Introduction
Piper longum L. in the family Piperaceae [1], is used as a tradi-ional medicine in China and Southeast Asia. P. longum L. is used toreat stomach ache [2] and nephrolithiasis [4], and has antioxida-ive [3] and analgesic effects [5]. Piperine (PPR), piperlongumininePPL), and ��,�-dihydropiperlonguminine (DPPL) are the mainctive constituents of the alkaloids from P. longum L. Studies havehown that PPR has nerve-protective [6], antihypertensive [7], andntiparkinsonism [8] effects. PPL and DPPL inhibit the productionf �-amyloid and amyloid precursor protein in SK-N-SH cells [9].PPL also has antihyperlipidemia effect [10].
Due to the pharmacological activity of PLA, a new antiparkin-onian drug has been developed in our laboratory. To provideeliable evidences for the treatment of Parkinsonism, it is neces-ary to investigate the pharmacological actions, drug absorption,nd tissue distribution profiles of the main components of PLA. Theharmacokinetic profiles of PPR and PLA were reported in a previ-us study [11], which showed that the absolute bioavailability ofPR was estimated to be 30.5 ± 14.9% with 50 mg/kg administra-ion by oral gavage, and increased to 37.7 ± 11.7% with 100 mg/kgdministration of PLA by oral gavage.
Many methods have been used to detect PPR, including thin-ayer chromatography [12], ultraviolet spectrophotometry [13],igh-performance liquid chromatography (HPLC) [14,15], ultra-erformance liquid chromatography/quadrupole time-of-flight
570-0232/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jchromb.2013.03.021
ing motor dysfunction and dopamine depletion.© 2013 Elsevier B.V. All rights reserved.
mass spectroscopy (UPLC–qTOF-MS) [16], and liquid chromato-graphy–MS/MS (LC–MS/MS) [17]. These methods, however, onlydetect the content of PPR or its metabolites. Thus, we developeda LC–MS/MS method to simultaneously detect PPR and PPL levelsin rat plasma. To our knowledge, there are no published analyticalmethods for the simultaneous quantification of PPR, PPL, and DPPLin biological fluids.
The aim of present study was to develop a rapid, accurate, andsimple HPLC–electron spray ionization (ESI)-MS/MS method for thesimultaneous detection of PPR, PPL, and DPPL in rat plasma, heart,liver, spleen, lung, kidney, and brain tissues after oral administra-tion of PLA.
2. Experimental procedures
2.1. Chemicals and reagents
PLA containing 50.68% PPR, 3.74% PPL, and 0.62% DPPL was pre-pared in our Chemistry of Chinese Material laboratory at CapitalMedical University (Beijing, China). PPR (batch no. 0775-200203,>98% purity) and terfenadine (internal standard [IS]) were pur-chased from the National Institutes for Food and Drug Control
(Beijing, China). Macroporous resin was purchased from TianjinHaiguang Chemical Co., Ltd (Tianjin, China). The PPL and DPPLreference standards were prepared in our laboratory and identi-fied by determining the ultraviolet, ESI-MS, 1H-nuclear magneticroma
ratH(jp
2
wAs(T(gl
2
dtc14bk
2
lTtdfsw
2
J(GuwtaB3i
drtmis6t
Letter to the Editor / J. Ch
esonance (NMR), and 13C-NMR spectra. The purity of PPL (99.2%)nd DPPL (99.1%) was determined by HPLC with diode-array detec-ion (1260 Infinity, Agilent Technologies, Palo Alto, CA, USA).PLC-grade methanol and acetonitrile were purchased from Fisher
Fair Lawn, NJ, USA). Formic acid was purchased from MREDA (Bei-ing, China). Ultrapure water was prepared using a Milli-Q waterurification system (Millipore, Molsheim, France).
.2. Extracts
The dry seed powder of P. longum L. (3 kg) was first extractedith 85% ethanol (8 L × 3) using heat reflux extraction (15 L, 120 ◦C).fter filtration, the filtrate was concentrated. The concentratedolution was then mixed with clean D101 macroporous resin3 kg) and dried using a rotary evaporator (EYELA, Tokyo, Japan).he dried mixture was subjected to the D101 macroporous resin9 cm × 130 cm, 0.3–1.25 mm) and eluted with an ethanol–waterradient (0%, 20%, 40%, 60%, and 85%). The 60% fraction was col-ected and evaporated to dryness, which contains PLA.
.3. Rat treatment and tissue collection
Twenty-five male Sprague-Dawley rats (220 ± 20 g) were ran-omly divided into five groups. The animals were kept in aemperature-controlled environment with a 12 h/12 h light/darkycle and allowed free access to water. After food-restriction for2 h, the rats were orally administered 100 mg/kg of PLA. At 0.5, 2,, 8, and 24 h after oral administration, five rats were killed to collectlood samples and different tissues. All samples were immediatelyept on ice and then stored at −20 ◦C.
.4. Pretreatment of samples
Tissue samples (0.5 or 1.0 g) including brain, liver, heart, spleen,ung, and kidney were homogenized with distilled water (1:4, w/v).he IS solution (200 �l) and 5 �l of methanol were added to 50 �l ofhe plasma and tissue samples. The IS solution consisted of terfena-ine (5 ng/ml) and methanol/acetonitrile (1:1, v/v). After vortexingor 1 min, the samples were centrifuged at 5000 × g for 10 min. Theupernatant was collected and 10 �l aliquots of each supernatantere injected into the LC–MS/MS system.
.5. Instruments and analytical conditions
The samples were analyzed using an HPLC system (Shimadzu,apan) and tandem Sciex API 4000 Qtrap mass spectrometerApplied Biosystems, Concord, Ontario, Canada). A Phenomenexemini C18 column was used (50 mm × 2.00 mm, 5 �m). The col-mn was run at room temperature. The samples were separatedith a mobile phase that consisted of water (solvent A) and ace-
onitrile (solvent B). 0.1% formic acid was added to both solvent And solvent B. The solvent gradients were 0.01–0.60 min with 10%, 0.60–2.00 min with 10–98% B, 2.00–3.00 min with 98% B, and.01–4.50 min with 10% B. The flow rate was 0.4 ml/min, and the
njection volume was 10 �l.Tandem MS analyses were performed using a triple quadrupole
etector in positive ESI mode. The data were acquired by selectedeaction monitoring (SRM). The precursor-to-product ion transi-ions were m/z 285.9 → 201.1 for PPR, m/z 274.3 → 209.9 for PPL,/z 276.2 → 134.9 for DPPL, and m/z 472.3 → 436.3 for the IS. The
on spray needle voltage was 5000 V. The cone voltage and colli-ion energy were 71 V and 27 eV for PPR, 56 V and 25 eV for PPL,6 V and 29 eV for DPPL, and 104 V and 35 eV for IS. The capillaryemperature was 500 ◦C.
togr. B 928 (2013) 78– 82 79
2.6. Calibration standards and quality control sample preparation
The stock solutions of PPR, PPL, and DPPL were separatelyprepared in methanol (1.00 mg/ml). The standard solutions wereprepared with serial dilutions of the stock solution with methanol(2000, 1000, 500, 200, 50, 20, 5.0, and 2.0 ng/ml). The quality con-trol (QC) solutions were prepared with serial dilutions of the stocksolution with methanol (0.5, 50, 160 ng/ml). The IS stock solutionwas prepared in methanol (1.00 mg/ml) and diluted to a final con-centration of 10 ng/ml with methanol/acetonitrile (1:1, v/v).
2.7. Method validation
2.7.1. SpecificitySelectivity was assessed by comparing blank plasma, blank
plasma with standards (PPR, PPL, DPPL, and IS; 0.2 and 50 ng/ml),and rat tissue homogenate samples. The samples were obtained 2 hafter oral administration of PLA.
2.7.2. Linearity of calibration curves and lower limit ofquantification
The standard working solution (5 �l) was spiked into blankplasma and tissue homogenate samples (50 �l). The concentrationsof serial dilutions were 0.2, 0.5, 2, 5, 20, 50, 100, and 200 ng/ml forplasma and tissue homogenate samples. The sample solutions wereprepared and analyzed as described in Section 2.4. The results werefitted using linear regression analysis to calculate the calibrationequation with a weighted factor (1/x2). The lower limit of quantifi-cation (LLOQ) was determined by calculating the signal-to-noiseratio (S/N) of 10:1 with acceptable accuracy and precision.
2.7.3. Recovery and matrix effectThe recovery was evaluated by comparing the peak areas of
blank homogenates spiked at concentrations of QC samples withthe areas of methanol spiked at corresponding QC samples. The con-centrations of QC samples were 160 ng/ml (maximum), 50 ng/ml(medium), and 0.5 ng/ml (minimum). The recovery of the sampleswas greater than 80% (RSD < 15%), indicating that few method liabil-ities are caused by matrix effect and other interferences. Therefore,we did not need to determine the matrix effect separately.
2.7.4. Precision and accuracyThe QC samples of heart, liver, spleen, kidney, and lung tissues at
three concentrations (0.5, 50, and 160 ng/ml; n = 6) were tested onthe same day for intra-day precision (% relative standard deviation[RSD]) and accuracy. The inter-day precision (% RSD) and accuracywere determined by testing the QC samples repeatedly for 3 days.The concentrations were calculated from the calibration curve.
2.7.5. StabilityThe stability studies included freeze–thaw stability, short-term
stability, and long-term stability. All stabilities were assayed atthree concentrations (0.5, 50, and 160 ng/ml). Freeze–thaw stabilitywas assessed by three consecutive freeze–thaw cycles. Short-termstability was studied by evaluating the samples at 4 ◦C for 24 h.Long-term stability was evaluated for 30 days at −20 ◦C. Theconcentrations were compared with fresh QC samples, and theconcentration deviation percentage was then calculated.
3. Results and discussion
3.1. Chromatographic and mass spectrometric conditions
The chromatography and ionization response studies of PPR,PPL, DPPL and IS were performed using reversed phase HPLCcolumns and various mobile phases. A Phenomenex Gemini C18
80 Letter to the Editor / J. Chromatogr. B 928 (2013) 78– 82
tation
cla(tt2
Towrfmtm
3
3
tSbais
3
ws
3.2.3. Precision and accuracyAccuracy and precision were assayed by testing QC samples
at different concentrations (0.50, 50 and 160 ng/ml; Table 2). The
Table 1The calibration curves of PPR, PPL and DPPL in rat plasma and tissues (n = 6).
Sample Tissue Standard curves Correlationcoefficient (R)
PPR
Plasma y = 0.1620x + 0.0210 0.9976Liver y = 0.1673x + 0.0337 0.9972Lung y = 0.1573x + 0.0550 0.9975Brain y = 0.1360x + 0.0085 0.9977Heart y = 0.1507x + 0.0236 0.9960Kidney y = 0.1457x + 0.0333 0.9970Spleen y = 0.1113x + 0.0038 0.9982
PPL
Plasma y = 0.0837x + 0.0054 0.9970Liver y = 0.0982x + 0.3113 0.9962Lung y = 0.0831x + 0.0170 0.9975Brain y = 0.0762x + 0.0028 0.9977Heart y = 0.0897x + 0.01150 0.9966Kidney y = 0.0920x + 0.0137 0.9972Spleen y = 0.0712x + 0.0076 0.9973
DPPL
Plasma y = 0.1450x + 0.0234 0.9967Liver y = 0.1620x + 0.0265 0.9975Lung y = 0.1520x + 0.0392 0.9975Brain y = 0.1531x + 0.0002 0.9963
Fig. 1. Full-scan product ion spectra of [M+H]+ ions and fragmen
olumn (50 mm × 2.00 mm, 5 �m) was used to separate three ana-ytes and the IS. We chose terfenadine as the IS, which is a readilyvailable compound. The mobile phase, consisting of solvent A0.1% formic acid in water) and solvent B (0.1% formic acid in ace-onitrile), was used to obtain a good peak shape and short runningime. The retention times were 2.77 min, 2.73 min, 2.71 min, and.21 min for PPR, PPL, DPPL, and terfenadine, respectively.
Three analytes and the IS were scanned in ESI positive ion mode.he ion spray needle voltage was set to 5000 V, and the temperaturef the capillary column was 500 ◦C. The optimal collision energiesere 27 eV, 56 eV, 25 eV, and 29 eV for PPR, PPL, DPPL, and the IS,
espectively. The parent fragmentation ions were m/z 286 [M+H]+
or PPR, m/z 274 [M+H]+ for PPL, m/z 276 [M+H]+ for DPPL, and/z 472 [M+H]+ for the IS. The precursor-to-product ions showed
ransitions of m/z 285.9 → 201.1 for PPR, m/z 274.3 → 209.9 for PPL,/z 276.2 → 134.9 for DPPL, and m/z 472.3 → 436.3 for the IS.
.2. Method validation
.2.1. SelectivityThe full-scan product ion spectra of [M+H]+ ions and fragmen-
ation schemes for PPR, PPL, DPPL and the IS were shown in Fig. 1.electivity was assessed by comparing blank rat liver homogenates,lank rat liver homogenates spiked with standards (PPR, PPL, DPPL,nd the IS; 50 ng/ml), and rat tissue homogenates after oral admin-stration of PLA (100 mg/kg). The results were shown in Fig. 2,howing no interference between these analytes and the IS.
.2.2. Linearity lower limit of quantificationThe peak area ratio (y) and concentration of each analyte (x)
ere subjected to weighted (1/x2) least squares linear regres-ion analysis. The slope, intercept, correlation coefficient (R), and
schemes for (A) PPR, (B) PPL, (C) DPPL and (D) terfenadine (IS).
linear range were shown in Table 1. The calibration curves of threeanalytes exhibited good linearity (R > 0.995). The LLOQ was0.2 ng/ml for three analytes.
Heart y = 0.1773x + 0.0194 0.9969Kidney y = 0.1697x + 0.0154 0.9977Spleen y = 0.1413x + 0.0056 0.9969
Concentration range of PPR, PPL and DPPL: 0.2–200 ng/ml.
Letter to the Editor / J. Chromatogr. B 928 (2013) 78– 82 81
F udiedw 2 h af
aT
3
ew
3
s
TT
C
ig. 2. Representative chromatograms from rat liver homogenate spiked with the stith 50 ng/ml of PPR, PPL, DPPL and terfenadine; (C) rat liver homogenate obtained
ccuracies of three QC samples were within the range of 80–120%.his indicates that the method is accurate and reproducible.
.2.4. RecoveryThe samples were prepared by precipitating protein. The recov-
ry results were shown in Table 2. The recovery of three QC samplesas greater than 80% and RSD was less than 10%.
.2.5. StabilityThe stability tests included freeze–thaw stability, short-term
tability, and long-term stability. The value of each test was greater
able 2he intra-day (n = 6) precisions and accuracies, inter-day (n = 3) precisions and accuracies
Tissue Alkaloid Intra-day (n = 6) Inter-d
Precision(RSD, %)
Accuracy(mean %)
Precis(RSD,
PlasmaPPR 5.22–6.12 98.3–101.0 5.47–PPL 6.79–8.86 98.6–101.4 6.43–DPPL 3.77–7.53 98.7–100.8 3.78–
LiverPPR 5.92–7.08 97.3–100.5 5.85–PPL 99.6–101.0 4.51–6.27 4.51–DPPL 4.97–6.96 97.3–102.0 4.91–
LungPPR 3.01–7.20 100.5–102.2 5.57–PPL 5.56–6.15 100.0–103.7 5.09–DPPL 4.10–5.80 96.1–101.3 4.71–
BrainPPR 2.51–6.34 96.7–104.1 3.51–PPL 4.38–6.99 98.2–101.8 4.96–DPPL 3.30–5.80 96.9–101.4 4.83–
HeartPPR 2.65–6.08 95.3–98.2 4.17–PPL 3.73–7.03 102.5–104.5 4.85–DPPL 4.49–5.98 98.7–99.1 4.85–
KidneyPPR 4.83–8.53 96.4–102.3 5.65–PPL 4.25–7.34 99.2–102.5 3.98–DPPL 3.99–7.53 99.0–101.5 3.82–
SpleenPPR 4.31–4.60 93.3–100.9 4.97–PPL 5.08–8.01 96.4–101.1 5.14–DPPL 4.10–7.14 99.7–102.0 5.81–
oncentrations of PPR, PPL and DPPL were added to homogenates in 3 levels: 0.50 ng/ml
compounds. (A) Blank rat liver homogenate; (B) blank rat liver homogenate spikedter oral administration of PLA (100 mg/kg).
than 85% and RSD was less than 13.8%. This indicates that the sam-ples are stable in the experiment.
3.3. Tissue distribution study
The tissue concentrations of PLA at 0–24 h after oral administra-tion were shown in Fig. 3. PLA was widely and rapidly distributedin the body. After administering PLA, the concentration reached to
the maximum level within 2 h in all detected tissues. The highesttissue concentrations of PPR and PPL were detected in the liver(456 ± 291 ng/g for PPR at 2 h; 34.4 ± 40.3 ng/g for PPL at 0.5 h).The highest tissue concentration of DPPL was found in the kidney, total recovery (n = 3) of PPR, PPL and DPPL in rat plasma and tissues.
ay (n = 3) Recovery (n = 3)
ion%)
Accuracy(mean %)
Precision(RSD, %)
Measured(mean ± SD%)
6.42 100.4102.5 1.15–2.84 82.3 ± 3.04–84.7 ± 2.708.78 100.0–101.1 2.40–3.98 82.1 ± 1.97–83.1 ± 3.316.29 98.1–101.5 1.56–2.30 80.3 ± 1.8–83.3 ± 1.306.84 98.1–99.8 1.25–8.17 83.5 ± 6.82–86.9 ± 1.086.27 98.7–100.1 2.51–4.18 83.0 ± 3.47–86.3 ± 2.177.00 97.6–103.7 2.72–3.87 82.1 ± 3.18–85.8 ± 2.606.78 98.5–102.1 1.72–5.44 81.3 ± 4.42–86.3 ± 3.838.50 99.0–101.8 1.60–6.20 81.1 ± 1.30–84.6 ± 1.916.52 98.3–99.4 1.06–5.48 82.1 ± 4.50–86.5 ± 2.536.54 97.9–100.9 1.69–2.22 81.8 ± 1.39–86 ± 1.916.50 99.5–100.1 1.00–6.26 81.7 ± 5.12–87.0 ± 0.876.52 97.6–100.3 1.69–4.88 80.9 ± 1.21–87.5 ± 3.486.24 97.5–101.6 3.074.81 81 ± 3.89–84.8 ± 2.607.39 100.2–101.7 1.85–4.38 80.8 ± 3.54–84.8 ± 1.577.39 100.2–101.7 1.49–3.40 80.9 ± 1.21–85.7 ± 2.917.46 99.6–103.5 1.60–5.06 81.8 ± 1.31–85.6 ± 4.337.18 100.7–101.9 1.89–3.30 81.5 ± 2.69–83.3 ± 1.576.30 98.8–100.7 0.72–7.36 82.6 ± 6.08–85.6 ± 3.317.01 99.8–102.0 1.93–5.04 81.9 ± 4.13–85.6 ± 1.658.04 99.2–102.5 1.25–1.48 80.3 ± 1.01–84.5 ± 1.146.85 101.3–102.1 1.28–5.06 82.6 ± 1.06–84.8 ± 1.91
(minimum), 50.00 ng/ml (medium) and 160.00 ng/ml (maximum).
82 Letter to the Editor / J. Chromatogr. B 928 (2013) 78– 82
d DPP
(ra
4
daawdtfa
A
dHUP(
A
i2
R
[
[[[
[[[
[
Fig. 3. Tissue distribution of PPR (A), PPL (B) an
8.35 ± 5.69 ng/g at 2 h). PLA was also detected in brain tissue. Theseesults indicate that three compounds may have pharmacologicalntiparkinsonian action.
. Conclusion
Using the UPLC–ESI-MS/MS method, PPR, PPL, and DPPL wereetected simultaneously. The method was simple, rapid, reliable,nd sensitive. The LLOQ of the analytes was 0.2 ng/ml. PLA wasbsorbed rapidly and distributed widely in the body. PPR and PPLere predominantly distributed in the liver, while DPPL was pre-ominantly distributed in the kidney. Furthermore, PLA could crosshe blood–brain barrier. These results provide evidences for theunctions of PPR, PPL, and DPPL in improving motor dysfunctionnd dopamine depletion.
cknowledgements
This work was supported by National Natural Science Foun-ation of China (No. 81073016), Funding Project for Academicuman Resources Development in Institutions of Higher Learningnder the Jurisdiction of Beijing Municipality (KM 201010025013,HR201008402) and Key Laboratory of Brain Diseases in Beijing2011NZDB03).
ppendix A. Supplementary data
Supplementary data associated with this article can be found,n the online version, at http://dx.doi.org/10.1016/j.jchromb.013.03.021.
eferences
[1] S. Kumar, J. Kamboj, Suman, S. Sharma, J. Acupunct. Meridian Stud. 4 (2011)134.
[2] Y. Bi, X. Wu, X.Q. Chen, Chin. Pharm. J. 46 (2011) 1697.[3] A. Mishra, S. Kumar, A. Bhargava, B. Sharma, A.K. Pandey, Cell. Mol. Biol. 57
(2011) 16.
L (C) in rats after an oral administration of PLA.
[4] M.A. Patel, P.K. Patel, A.K. Seth, Pharmacologyonline 2 (2011) 1169.[5] S. Venkatesh, K.D. Durga, Y. Padmavathi, B.M. Reddy, R. Mullangi, Arzneimittel
Forschung. 61 (2011) 506.[6] A. Lublin, F. Isoda, H. Patel, K. Yen, L. Nguyen, D. Hajje, M. Schwartz, C. Mobbs,
PLoS One 6 (2011) e27762 www.plosone.org[7] L. Hlavacková, A. Janegová, O. Ulicná, P. Janega, A. Cerná, P. Babál, Nutr. Metab.
8 (2011) 72.[8] P. Chonpathompikunlert, T. Yoshitomi, J. Han, H. Isoda, Y. Nagasaki, Biomateri-
als 32 (2011) 8605.[9] H.S. Qi, P. Liu, S.Q. Gao, Z.Y. Diao, L.L. Yang, J. Xu, X. Qu, E.J. Han, Chin. J. Physiol.
52 (2009) 160.10] Z. Jin, G. Borjihan, R. Zhao, Z. Sun, G.B. Hammond, T. Uryu, Phytother. Res. 23
(2009) 1194.11] J.H. Liu, Y. Bi, R. Luo, X. Wu, J. Chromatogr. B 879 (2011) 2885.12] B.G. Bhat, N. Chandrasekhara, Toxicology 44 (1987) 99.13] S. Chopra, S.K. Motwani, F.J. Ahmad, R.K. Khar, Spectrochim. Acta. A. Mol.
Biomol. Spectrosc. 68 (2007) 516.14] C. Li, Y. Xiao, H. Yang, Y. Du, L. Wei, Chin. J. Chin. Mater. Med. 36 (2011) 1046.15] A. Itharat, I. Sakpakdeejaroen, J. Med. Assoc. Thai. 93 (2010) 198.16] B.S. Sachin, I.A. Najar, S.C. Sharma, M.K. Verma, M.V. Reddy, R. Anand, R.K.
Khajuria, S. Koul, R.K. Johri, J. Chromatogr. B 878 (2010) 823.17] S. Bajad, R.K. Khajuria, O.P. Suri, K.L. Bedi, J. Sep. Sci. 26 (2003) 943.
Haolong LiuRong Luo
Xiaoqing ChenJunhui Liu
Ying BiLi ZhengXia Wu ∗
School of Traditional Chinese Medicine, CapitalMedical University, 10 Youanmen, Xitoutiao, Beijing
100069, China
∗ Corresponding author. Tel.: +86 10 83911671;fax: +86 10 83911627.
E-mail addresses: [email protected],
[email protected] (X. Wu)5 November 2012Available online 28 March 2013