Ua

Wa

b

a

ARAA

KAURP

1

gl[phpiptatatO

o

h1

Journal of Chromatography B, 965 (2014) 107–111

Contents lists available at ScienceDirect

Journal of Chromatography B

jou rn al hom ep age: www.elsev ier .com/ locate /chromb

PLC–MS/MS method for determination of avicularin in rat plasmand its application to a pharmacokinetic study

ei-min Zhanga,∗, Rui-fang Lib, Ming Sunb, Da-ming Hub, Jian-fei Qiub, Yun-hao Yanb

The First Affiliated Hospital of Henan University of Science and Technology, Luoyang, Henan 471003, PR ChinaMedical College of Henan University of Science and Technology, Luoyang, Henan 471003, PR China

r t i c l e i n f o

rticle history:eceived 31 January 2014ccepted 14 June 2014vailable online 20 June 2014

eywords:vicularinPLC–MS/MSat plasma

a b s t r a c t

A rapid, sensitive and selective ultra-performance liquid chromatography tandem mass spectrometry(UPLC–MS/MS) was developed and validated for the determination and pharmacokinetic investigationof avicularin in rat plasma. Sample preparation was accomplished through a simple one-step depro-teinization procedure with 0.2 mL of acetonitrile–methanol (9:1, v/v) to a 0.1 mL plasma sample. Plasmasamples were separated by UPLC on an Acquity UPLC BEH C18 column using a mobile phase consist-ing of acetonitrile–0.1% formic acid in water with gradient elution. The total run time was 1.60 min andthe elution of avicularin was at 1.20 min. The detection was performed on a triple quadrupole tandemmass spectrometer in the multiple reaction-monitoring (MRM) mode using the respective transitions m/z

harmacokinetics 434.1 → 301.3 for avicularin and m/z 237.2 → 194.3 for carbamazepine (IS), respectively. The calibrationcurve was linear over the range of 10–3000 ng/mL with a lower limit of quantitation (LLOQ) of 10 ng/mL.Mean recovery of avicularin in plasma was in the range of 84.2–89.5%. Intra-day and inter-day preci-sion were both <12%. This method was successfully applied in pharmacokinetic study after intravenousadministration of 5.0 mg/kg avicularin in rats.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Avicularin (Fig. 1), quercetin 3-O-�-l-arabinofuranoside, is alycoside of quercetin [1] and is known to be included in Taxil-us kaempferi [2], Polygonum aviculare [3], and fruits such as mango4]. Avicularin has been reported to possess a variety of biologicalroperties such as anti-inflammatory, anti-allergic, anti-oxidant,epatoprotective, and anti-tumor activities [5,6]. Moreover, itrotects cardiomyocytes and hepatocytes against oxidative stress-

nduced apoptosis [7,8]. In addition, avicularin has been reported tolay a protective role by inhibiting ureases, which are virulene fac-ors implicated in the pathogenesis of many clinical conditions suchs pyelonephritis and hepatic coma [9]. Recently, it was reportedhat avicularin was anaerobic metabolized to its aglycone quercetinnd two methoxylated avicularin, then quercetin was convertedo quercetin glycosides: quercetin-3-O-rhamnoside, quercetin-3-

-glucoside and quercetin-7-O-glucoside by Bacillus sp. 46 [10].

There have several analytical methods for the determinationf avicularin in different kinds of plants, including TLC [11], HPLC

∗ Corresponding author.E-mail address: 13513792468@139.com (W.-m. Zhang).

ttp://dx.doi.org/10.1016/j.jchromb.2014.06.015570-0232/© 2014 Elsevier B.V. All rights reserved.

[12–17], HPLC–MS/MS [18,19] and UPLC/Q-TOF-MS [20]. However,to our best knowledge, there is no bioanalytical method for deter-mination of avicularin in biological fluid. The evaluation of thepharmacokinetics and bioavailability of botanical drugs in plasmacan link data from pharmacological assays to clinical effects andalso help the design of rational dosage regimens [21]. Thus, a selec-tive, sensitive and reliable analytical method for the determinationof avicularin in rat plasma is required in order to evaluate the phar-macokinetics of avicularin in rats.

Ultra-performance liquid chromatography coupled with tan-dem mass spectrometry (UPLC–MS/MS) is recognized as a powerfultool for the quantitative determination of the active compoundof a herbal drug in biological samples due to the selectivity,sensitivity, robustness and sample throughput [22,23]. There-fore, it is worthwhile to develop a simple, precise, and accurateUPLC–MS/MS method for the determination of avicularin in ratplasma. In this study, the pharmacokinetic data of avicularin inrats as well as the bioanalytical method for the determination ofavicularin in biological fluids were developed. The sample prepa-

ration of this method was simple one-step protein precipitationby acetonitrile–methanol (9:1, v/v), which was time- and effort-saving. The analysis time and the sensitivity could also meet therequirements of high-throughput bioanalysis. Once developed and

108 W.-m. Zhang et al. / J. Chromatogr. B 965 (2014) 107–111

he pre

vn

2

2

I(aU(

2

pM1(wi(Abtm

eCtmTMfp4ta

2(

i

Fig. 1. The chemical structures of avicularin and IS in t

alidated, this method was successfully applied to a pharmacoki-etic study in rats.

. Materials and methods

.1. Chemicals materials

Avicularin (purity >98%) and carbamazepine (internal standard,S, purity >98%) were purchased from Aladdin Chemistry CompanyShanghai, China). Acetonitrile and methanol were of HPLC gradend were purchased from Merck Company (Darmstadt, Germany).ltra-pure water was obtained using a Millipore Milli-Q system

Millipore, Bedford, USA).

.2. UPLC–MS/MS conditions

Liquid chromatography was performed on an Acquity ultraerformance liquid chromatography (UPLC) unit (Waters Corp.,ilford, MA) with an Acquity BEH C18 column (2.1 mm × 50 mm,

.7 �m particle size) and inline 0.2 �m stainless steel frit filterWaters Corp., Milford, USA). A gradient program was employedith the mobile phase combining solvent A (0.1% formic acid

n water) and solvent B (acetonitrile) as follows: 30–80% B0–0.50 min), 80% B (0.50–1.40 min), 80–30% B (1.40–1.60 min).

subsequent re-equilibration time (1 min) should be performedefore next injection. The flow rate was 0.40 mL/min and the injec-ion volume was 10 �L. The column and sample temperature were

aintained at 40 ◦C and 4 ◦C, respectively.An AB Sciex QTRAP 5500 triple quadruple mass spectrometer

quipped with an electro-spray ionization (ESI) source (Toronto,anada) was used for mass spectrometric detection. The detec-ion was operated in the multiple reaction monitoring (MRM)

ode under unit mass resolution (0.7 amu) in the mass analyzers.he dwell time was set to 250 ms for each MRM transition. TheRM transitions were m/z 434.1 → 301.3 and m/z 237.2 → 194.3

or avicularin and IS, respectively. After optimization, the sourcearameters were set as follows: curtain gas, 30 psig; nebulizer gas,0 psig; turbo gas, 60 psig; ion spray voltage, 3.5 kV; and tempera-ure, 400 ◦C. Data acquiring and processing were performed usingnalyst software (version 1.5, AB Sciex).

.3. Standard solutions, calibration standards and quality control

QC) sample

The stock solution of avicularin that was used to make the cal-bration standards and quality control (QC) samples was prepared

sent study: (A) avicularin and (B) carbamazepine (IS).

by dissolving 10 mg in 10 mL methanol to obtain a concentration of1.00 mg/mL. The stock solution was further diluted with methanolto obtain working solutions at several concentration levels. Calibra-tion standards and QC samples in plasma were prepared by dilutingthe corresponding working solutions with blank rat plasma. Finalconcentrations of the calibration standards were 10, 20, 50, 100,200, 500, 1000 and 3000 ng/mL for avicularin in rat plasma. Theconcentrations of QC samples in plasma were 20, 400, 2400 ng/mLfor avicularin. IS stock solution was made at an initial concen-tration of 1.00 mg/mL. The IS working solution (2000 ng/mL) wasmade from the stock solution using methanol for dilution. All stocksolutions, working solutions, calibration standards and QCs wereimmediately stored at −20 ◦C.

2.4. Sample preparation

Before analysis, the plasma sample was thawed to room tem-perature. In a 1.5 mL centrifuge tube, an aliquot of 10 �L ofthe IS working solution (2000 ng/mL) was added to 100 �L ofcollected plasma sample followed by the addition of 200 �Lacetonitrile–methanol (9:1, v/v). The tubes were vortex mixedfor 1.0 min and spun in a centrifuge at 9000 × g for 8 min. Thesupernatant (10 �L) was injected into the UPLC–MS/MS system foranalysis.

2.5. Method validation

The method was validated for selectivity, linearity, accu-racy, precision, recovery and stability according to the literatures[24,25] for validation of bioanalytical methods (US Food and DrugAdministration, 2001). Validation runs were conducted on threeconsecutive days. Each validation run consisted of one set of cali-bration standards and six replicates of QC plasma samples.

The selectivity of the method was evaluated by analyzing blankrat plasma, blank plasma spiked with avicularin and IS and a ratplasma sample.

Calibration curves were constructed by analyzing spiked cal-ibration samples on three separate days. Peak area ratios ofavicularin to IS were plotted against analyte concentrations, andstandard curves were fitted to the equations by linear regressionwith a weighting factor of the reciprocal of the concentration (1/x)in the concentration range of 10–3000 ng/mL. The lower limit of

quantitation (LLOQ) was defined as the lowest concentration onthe calibration curves.

To evaluate the matrix effect, blank rat plasma were extractedand then spiked with the analyte at 20, 400 and 2400 ng/mL. The

W.-m. Zhang et al. / J. Chromatogr. B 965 (2014) 107–111 109

Fig. 2. Representative chromatograms of avicularin and IS in rat plasma samples. (A) A blank plasma sample; (B) a blank plasma sample spiked with avicularin and IS; (C) arat plasma sample 45 min after intravenous administration of single dosage 5.0 mg/kg avicularin.

Table 1Precision, accuracy and recovery for avicularin of quality control sample in rat plasma (n = 6).

Analyte Concentration (ng/mL) CV (%) Accuracy (%) Recovery (%)

Intra-day Inter-day Intra-day Inter-day

1187

csrem

o4e

att

a

TS

Avicularin 20 10.3

400 7.6

2400 5.4

orresponding peak areas were then compared with those of neattandard solutions at equivalent concentrations, and this peak areaatio is defined as the matrix effect (ME). The matrix effect of IS wasvaluated at the working concentration (2000 ng/mL) in the sameanner.Accuracy and precision were assessed by the determination

f QC samples at three concentration levels in six replicates (20,00 and 2400 ng/mL) in three validation days. The precision wasxpressed by the coefficient of variation (CV).

The recovery of avicularin was evaluated by comparing peakrea ratios of extracted QC samples with those of reference QC solu-

ions reconstituted in blank plasma extracts (n = 6). The recovery ofhe IS was determined in a similar way.

The stabilities of avicularin in rat plasma were evaluated bynalyzing five replicates of plasma samples at the concentrations

able 2ummary of stability of avicularin under various storage conditions (n = 5).

Condition Concentration (ng/mL)

Added Fou

Ambient, 2 h 20 2400 38

2400 258Autosampler, ambient, 24 h 20 1

400 412400 230

Three freeze–thaw 20 2400 37

2400 222−20 ◦C, 42 days 20 1

400 402400 238

.2 94.6 92.9 84.2 ± 3.5

.9 104.5 96.7 89.5 ± 4.4

.7 99.4 107.8 86.6 ± 3.9

of 20, 400 and 2400 ng/mL. These results were compared withthose obtained for freshly prepared plasma samples. The short-term stability was determined after the exposure of the spikedsamples at room temperature for 2 h, and the ready-to-injectsamples (after protein precipitation) in the UPLC autosampler atroom temperature for 24 h. The freeze–thaw stability was eval-uated after three complete freeze–thaw cycles (−20 to 25 ◦C)on consecutive days. The long-term stability was assessed afterstorage of the standard spiked plasma samples at −20 ◦C for42 days.

2.6. Application to a pharmacokinetic study

Male Sprague-Dawley rats (180–220 g) were obtained from Lab-oratory Animal Center of Wenzhou Medical University (Wenzhou,

CV (%) Accuracy (%)

nd

2.2 ± 1.9 8.7 111.34.2 ± 26.5 6.9 96.10.1 ± 158.4 6.1 107.58.7 ± 1.6 8.7 93.54.4 ± 24.9 6.0 103.69.7 ± 134.7 5.8 96.20.2 ± 1.5 7.5 101.18.9 ± 25.3 6.7 94.76.6 ± 122.5 5.5 92.89.0 ± 1.8 9.5 95.19.9 ± 23.3 5.7 102.52.1 ± 134.7 5.7 99.3

1 matogr. B 965 (2014) 107–111

CwRrWf1s14TTPlM

3

3

oimicthiwatatcadt

maoTscausIwtpr

3

ppa

219t

in concentration was within ±15% of nominal values, and theestablished method was suitable for the pharmacokinetic study(Table 2).

Table 3The main pharmacokinetic parameters after intravenous administration of5.0 mg/kg avicularin in six rats.

Parameters Avicularin

t1/2 (h) 0.75 ± 0.09C (ng/mL) 2323.8 ± 423.7

10 W.-m. Zhang et al. / J. Chro

hina) used to study the pharmacokinetics of avicularin. All six ratsere housed at Wenzhou Medical University Laboratory Animalesearch Center. All experimental procedures and protocols wereeviewed and approved by the Animal Care and Use Committee of

enzhou Medical College and were in accordance with the Guideor the Care and Use of Laboratory Animals. Diet was prohibited for2 h before the experiment but water was freely available. Bloodamples (0.3 mL) were collected from the tail vein into heparinized.5 mL polythene tubes at 5, 10, 20, 30, 45, 60, 90, 120, 180, 240, 360,80 min after intravenous administration of avicularin (5.0 mg/kg).he samples were immediately centrifuged at 3000 × g for 8 min.he plasma obtained (100 �L) was stored at −20 ◦C until analysis.lasma avicularin concentration vs. time data for each rat was ana-yzed by DAS (Drug and statistics) software (Version 2.0, Wenzhou

edical University, China).

. Results and discussion

.1. Method development and optimization

Liquid–liquid extraction (LLE) and solid-phase extraction areften used to prepare biological samples due to their ability tomprove the sensitivity and robustness of assays. However, these

ethods are both time-consuming and expensive, and may resultn environmental pollution. In this study, a simple protein pre-ipitation method was used to reduce the sample preparationime. No further concentration procedure was needed. To obtainigh levels of extraction efficiency, three different protein precip-

tation agents (acetonitrile, methanol and acetonitrile–methanol)ere investigated. Higher extraction efficiency was obtained using

cetonitrile–methanol (9:1, v/v) as the precipitation solvent ratherhan using acetonitrile or methanol. High extraction efficiency waslso achieved when acetonitrile–methanol (9:1, v/v) was appliedo the IS. acetonitrile–methanol (9:1, v/v) was chosen as the pre-ipitation solvent, and the mean recoveries of avicularin were allbove 80.0%. The protein precipitation sample preparation proce-ure was much simpler and less expensive. Thus, this method methe requirements of high sample throughput in bioanalysis.

A number of commercially available UPLC columns and variousobile phases were evaluated for its chromatographic behavior

nd the ionization response of avicularin. The best response wasbtained from acetonitrile and water (containing 0.1% formic acid).he addition of 0.1% formic acid to the mobile phase increased theensitivity of avicularin. The gradient elution mode was applied inhromatographic separation and showed a better peak shape andppropriate retention time. A Waters Acquity UPLC BEH C18 col-mn (2.1 mm × 50 mm, 1.7 �m) with isocratic delivery providedatisfactory chromatographic results with minimal matrix effects.n this assay, no significant signal suppression or enhancement

as found using the current conditions. The whole separation ofhe analyte and IS was completed within only 1.60 min per sam-le. Avicularin and IS were eluted at about 1.20 and 1.01 min,espectively.

.2. Selectivity and matrix effect

Fig. 2 shows the typical chromatograms of a blank plasma sam-le, a blank plasma sample spiked with avicularin and IS, and alasma sample. No interfering endogenous substance was observedt the retention time of the analyte and IS.

The ME for avicularin at concentrations of 20, 400 and

400 ng/mL was measured to be 103.1 ± 5.4, 97.8 ± 4.9 and01.4 ± 5.5% (n = 6), respectively. The ME for IS (2000 ng/mL) was9.6 ± 5.2% (n = 6). As a result, ME from plasma was negligible inhis method.

Fig. 3. Mean plasma concentration time profile after intravenous administration of5.0 mg/kg avicularin in six rats.

3.3. Calibration curve and sensitivity

The linear regressions of the peak area ratios vs. concentrationswere fitted over the concentration range 10–3000 ng/mL for avic-ularin in rat plasma. A typical equation of the calibration curve is:y = 0.00312x − 0.00698, r = 0.9995, where y represents the ratios ofavicularin peak area to that of IS and x represents the plasma con-centration. The LLOQ for the determination of avicularin in plasmawas 10 ng/mL. The precision and accuracy at LLOQ were 9.3 and98.2%, respectively.

3.4. Precision, accuracy and recovery

The precision of the method was determined by calculatingCV for QCs at three concentration levels over three validationdays. Intra-day precision was 11% or less and the inter-day pre-cision was 12% or less at each QC level. The accuracy of themethod ranged from 92.9 to 107.8% at each QC level. Mean recov-eries of avicularin were better than 80.0%. The recovery of theIS (2000 ng/mL) was 85.7 ± 4.5%. Assay performance data is pre-sented in Table 1. The results demonstrate that the values arewithin the acceptable range and the method is accurate andprecise.

3.5. Stability

The autosampler, room temperature, freeze–thaw and long-term (42 days) stability results indicated that the analyte wasstable under the storage conditions described above since the bias

max

AUC0→t (ng/mL h) 2823.3 ± 161.7AUC0→∞ (ng/mL h) 2860.1 ± 168.3MRT0→t (h) 0.92 ± 0.10MRT0→∞ (h) 0.93 ± 0.11

mato

3

mipy

4

reltocamsr

R

[

[

[[

[[

[

[[

[

[

[

[

W.-m. Zhang et al. / J. Chro

.6. Application of the method in a pharmacokinetic study

The method was applied to a pharmacokinetic study in rats. Theean plasma concentration–time curve after intravenous admin-

stration of 5.0 mg/kg avicularin was shown in Fig. 3. The mainharmacokinetic parameters from non-compartment model anal-sis were summarized in Table 3.

. Conclusions

A UPLC–MS/MS method for the determination of avicularin inat plasma was developed and validated. To the best of our knowl-dge, this is the first report of the determination of avicularinevel in rat plasma using an UPLC–MS/MS method. Compared withhe analytical methods reported in the literatures, the methodffered superior sample preparation with a simple one-step pre-ipitation of plasma protein by acetonitrile–methanol (9:1, v/v)nd shorter run time of 1.60 min. The method meets the require-ent of high sample throughput in bioanalysis and has been

uccessfully applied to the pharmacokinetic study of avicularin inats.

eferences

[1] T. Konishi, T. Nishio, S. Kiyosawa, Y. Fujiwara, T. Konoshima, Yakugaku zasshi116 (1996) 148–157.

[2] T. Fukunaga, K. Nishiya, I. Kajikawa, K. Takeya, H. Itokawa, Chem. Pharm. Bull.37 (1989) 1543–1546.

[3] X.Q. Zhang, L.S. Xu, Chung-kuo i hsueh k’o hsueh yuan, Chung-kuo hsieh ho i k’ota hsueh hsueh pao (Proceedings of the Chinese Academy of Medical Sciencesand the Peking Union Medical College, 4), 1989, pp. 193–195.

[[

[

gr. B 965 (2014) 107–111 111

[4] A. Schieber, N. Berardini, R. Carle, J. Agric. Food Chem. 51 (2003) 5006–5011.[5] R.J. Williams, J.P. Spencer, C. Rice-Evans, Free Radic. Biol. Med. 36 (2004)

838–849.[6] V.A. Vo, J.W. Lee, J.E. Chang, J.Y. Kim, N.H. Kim, H.J. Lee, S.S. Kim, W. Chun, Y.S.

Kwon, Biomol. Ther. 20 (2012) 532–537.[7] S.M. Kim, K. Kang, E.H. Jho, Y.J. Jung, C.W. Nho, B.H. Um, C.H. Pan, Phytother.

Res. 25 (2011) 1011–1017.[8] J.A. Kim, Y.S. Jung, M.Y. Kim, S.Y. Yang, S. Lee, Y.H. Kim, Phytother. Res. 25 (2011)

1612–1617.[9] S. Shabana, A. Kawai, K. Kai, K. Akiyama, H. Hayashi, Biosci. Biotechnol. Biochem.

74 (2010) 878–880.10] M. Zhao, J. Xu, D. Qian, J. Guo, S. Jiang, E.X. Shang, J.A. Duan, J. Yang, L.Y. Du, J.

Chromatogr. B 949–950 (2014) 30–36.11] M.H. Kim, A. Nugroho, J. Choi, J.H. Park, H.J. Park, Arch. Pharmacal Res. 34 (2011)

971–978.12] K. Fujimori, M. Shibano, J. Agric. Food Chem. 61 (2013) 5139–5147.13] A. Nugroho, T.J. Rhim, M.Y. Choi, J.S. Choi, Y.C. Kim, M.S. Kim, H.J. Park, Arch.

Pharmacal Res. (2013).14] A. Nugroho, E.J. Kim, J.S. Choi, H.J. Park, J. Pharm. Biomed. Anal. 89 (2014) 93–98.15] N.H. Le, K.E. Malterud, D. Diallo, B.S. Paulsen, C.S. Nergard, H. Wangensteen, J.

Ethnopharmacol. 139 (2012) 858–862.16] R. Rodriguez Madrera, A. Picinelli Lobo, B. Suarez Valles, J. Agric. Food Chem.

54 (2006) 120–124.17] M. Olszewska, Acta Pol. Pharm. 62 (2005) 435–441.18] A. Lhuillier, N. Fabre, F. Moyano, N. Martins, C. Claparols, I. Fouraste, C. Moulis,

J. Chromatogr. A 1160 (2007) 13–20.19] Y. Zhang, C. Liu, Z. Zhang, J. Wang, G. Wu, S. Li, J. Chromatogr. B 878 (2010)

3149–3155.20] H. Huang, Y. Feng, W. Rui, M. Jiang, L. Han, Zhongguo Zhong yao za zhi 34 (2009)

875–878.21] V.A. Bhattaram, U. Graefe, C. Kohlert, M. Veit, H. Derendorf, Phytomedicine 9

(Suppl. 3) (2002) 1–33.22] T. Toyo’oka, J. Chromatogr. Sci. 46 (2008) 233–247.

23] J. Wang, L. Liang, Q. Zhang, X. Li, Z. Fu, J. Chromatogr. B 942–943 (2013) 70–76.24] J.H. Song, T.H. Kim, J.W. Jung, N. Kim, S.H. Ahn, S.O. Hwang, N.S. Kang, S.E. Yoo,

T.S. Koo, Biomed. Chromatogr. (2014).25] Y. Lu, Z. Sun, Y. Zhang, X. Chen, D. Zhong, J. Chromatogr. B 921–922 (2013)

27–34.