Simultaneous Determination of Oleanolic Acid and Ursolic Acid by in

Mar 21, 2018 - Simultaneous Determination of Oleanolic Acid and Ursolic Acid by in Vivo Microdialysis via UHPLC-MS/MS Using Magnetic Dispersive Solid ...
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Simultaneous determination of oleanolic acid and ursolic acid by in vivo microdialysis via UHPLC-MS/MS using magnetic dispersive solid phase extraction coupling with microwave-assisted derivatization and its application to a pharmacokinetic study of Arctiumlappa L. root extract in rats Zhenjia Zheng, Xian-En Zhao, Shuyun Zhu, Jun Dang, Xuguang Qiao, Zhichang Qiu, and Yanduo Tao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b06015 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 21, 2018

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Journal of Agricultural and Food Chemistry

Simultaneous determination of oleanolic acid and ursolic acid by in vivo microdialysis via UHPLC-MS/MS using magnetic dispersive solid phase extraction coupling with microwave-assisted derivatization and its application to a pharmacokinetic study of Arctiumlappa L. root extract in rats Zhenjia Zheng1, Xian-En Zhao3*, Shuyun Zhu3*, Jun Dang2, Xuguang Qiao1**, Zhichang Qiu1, Yanduo Tao2 1

College of Food Science and Engineering, Shandong Agricultural University, 61 Daizong Street,

Taian 271018, Shandong, P.R. China; 2

Qinghai Provincial Key Laboratory of Tibetan Medicine Research & Key Laboratory of Tibetan

Medicine Research, Northwest Institute of Plateau Biology, Chinese Academy of Science, Xining 810001, Qinghai, P.R. China; 3

College of Chemistry and Chemical Engineering, Qufu Normal University, Qufu 273165,

Shandong, P.R. China

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ABSTRACT

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Simultaneous detection of oleanolic acid and ursolic acid in rat blood by in vivo microdialysis can

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provide important pharmacokinetics information. Microwave-assisted derivatization coupled with

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magnetic dispersive solid phase extraction was established for the determination of oleanolic acid

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and ursolic acid by liquid chromatography tandem mass spectrometry. 2’-Carbonyl-piperazine

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rhodamine B was firstly designed and synthesized as the derivatization reagent, which was easily

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adsorbed onto the surface of Fe3O4/graphene oxide. Simultaneous derivatization and extraction of

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oleanolic acid and ursolic acid were performed on Fe3O4/graphene oxide. The permanent positive

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charge of the derivatization reagent significantly improved the ionization efficiencies. The limits

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of detection were 0.025 and 0.020 ng/mL for oleanolic acid and ursolic acid, respectively. The

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validated method was shown to be promising for sensitive, accurate and simultaneous

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determination of oleanolic acid and ursolic acid. It was used for their pharmacokinetics study in

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rat blood after oral administration of Arctiumlappa L. root extract.

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KEYWORDS: In vivo microdialysis, triterpenic acid, microwave-assisted derivatization,

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magnetic graphene oxide, pharmacokinetics, Arctiumlappa L. root.

16 17 18 19 20 21 22 23 24 25

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INTRODUCTION

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Oleanolic acid (OA) and ursolic acid (UA) belong to triterpenoid compounds that widely exist in

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herbs and fruits. They have been reported to have important pharmacological properties, such as

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anti-inflammatory,

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antihyperlipidemic activities and so on.1,2 Therefore, a sensitive and rapid analytical method is

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necessary and helpful for their pharmacokinetic study to better understand the pharmacological

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activity of related foods and herbs.

hepatoprotective,

antiulcer,

antimicrobial,

antitumour,

anti-HIV,

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OA and UA are triterpine isomers with exactly the same chemical structures, with the only

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difference found in the position of a methyl group in the E ring (Figure 1), thus they are difficult

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to separate and detect rapidly.3-5 A lot of methods have been reported for the determination of OA

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and UA, such as gas chromatography (GC),6 thin-layer chromatography (TLC),7 capillary

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electrophoresis (CE),8 high-performance liquid chromatography (HPLC) with UV,9 fluorescence

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detection,3 mass spectrometry (MS) 4, 5 or nuclear magnetic resonance (NMR).10 Each method has

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its own feature, but many of them show a limited enhancement on the sensitivity, accuracy and

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specificity. In the past decade, ultra high performance liquid chromatography with tandem mass

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spectrometry (UHPLC-MS/MS) in the multiple reaction monitoring mode (MRM) has aroused

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wide attention in rapid pharmaceutical analysis and bioanalysis in different biological matrices.11

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However, very low concentrations and strong matrix interferences in real samples usually make

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difficult the sensitive and accurate determination of compounds. Therefore, sensitive, accurate,

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rapid and simultaneous determination of OA and UA is still a challenging task. However, OA and

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UA lack a chromophore and cannot easily gain a charge because of their carboxyl group, thus

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they provide a very low sensitivity in relation to UV, fluorescence and MS detection. Under these

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circumstances, sensitivity enhancement by chemical derivatization can solve problems.12, 13 Some

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derivatization reagents have been reported for them by HPLC fluorescence detection

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GC-MS.19 However, there are almost no synthesized derivatization reagents for the enhanced

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UHPLC-MS/MS determination of OA and UA.

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However, the UHPLC-MS/MS detection sensitivity is frequently compromised by the low

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contents of analytes and serious matrix effect from real samples and derivatization procedure.

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Therefore, efficient sample pretreatment procedure is necessary.20,21 Compared to the popular

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liquid liquid extraction (LLE)

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extraction (d-SPE) is time saving, easy to operate and low consumed of toxic organic solvents.

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Especially, the use of magnetic sorbents for magnetic dispersive solid phase extraction (MDSPE)

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has drawn significant attention because of their excellent dispersibility and ease of separation by

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an external magnetic field.25,

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surface area-to-volume ratio, strong π-π stacking interactions and high mechanical strength,25, 27,

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28

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analytes depending on the hydrogen bonding, hydrophobic interactions, van der Waals forces and

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electrostatic forces.29

11,12,22

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and solid phase extraction (SPE),23,24 dispersive solid-phase

Graphene oxide (GO), a carbon-based material with a high

presents enormous advantages in the separation and determination of small amount of organic

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In vivo microdialysis sampling is a preeminent technique for neuroscience, pharmacokinetic

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(PK), pharmacodynamic (PD) and clinical studies.30 It is commonly used for investigation of

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PK/PD since concentration of the unbound drug is more correlated to the pharmacological effects.

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By coupling with powerful analytical methods, in vivo microdialysis is able to overcome several

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disadvantages of conventional pharmacokinetic techniques which include continuous sampling in

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the same animal, minimizing the number of animals used and also minimizing inter-animal

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variation. However, drug quantification in microdialysis technique remains a major challenge

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because of the low concentration and the small volume of microdialysate. Therefore, a highly

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sensitive, accurate and selective analytical method employing UHPLC-MS/MS (MRM) is always

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recommended.31,32

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In this study, a new method based on in vivo microdialysis by microwave-assisted

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derivatization coupling with MDSPE (MAD-MDSPE) in a single step has been developed for the

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simultaneous determination of OA and UA by UHPLC-MS/MS. 2’-Carbonyl-piperazine 4

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rhodamine B (CPR) was designed and synthesized as derivatization reagent for the labeling of

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carboxyl group of OA and UA. This method was used for the simultaneous pharmacokinetic

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study of OA and UA in rat blood.

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MATERIALS AND METHODS

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Chemicals and Reagents. OA, UA and the internal standard (IS) betulinic acid were

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purchased from National institute for the control of pharmaceutical and biological products

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(Beijing, China). 1-Ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC-HCl)

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and the HPLC grade formic acid were purchased from Sigma Co. (St. Louis, MO, USA). HPLC

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grade acetonitrile and methanol were purchased from Fisher Scientific Co. (Fair Lawn, NJ, USA).

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N,N-dimethylformamide (DMF) and pyridine was of analytical grade and obtained from Tianjin

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Guangcheng Chemical Reagent Co. (Tianjin, China). Pure water was obtained on a Millipore

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system (Bedford, MA, USA). All other reagents used were of HPLC grade or at least of

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analytical grade obtained commercially.

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Stock solutions of OA (10.0 µmol/L), UA (10.0 µmol/L) and betulinic acid (IS, 10.0

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µmol/L), and derivatization reagent CPR (100.0 µmol/L) were prepared by HPLC grade

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acetonitrile. All working solutions with different concentrations were prepared by diluting

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corresponding stock solutions with acetonitrile. Solution of 0.10 mol/L coupling reagent EDC

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was prepared in HPLC acetonitrile. The quality control samples (QCs) containing OA (0.5, 5.0,

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100 ng/mL) and UA (0.5, 5.0, 100 ng/mL) were prepared at three concentration levels by adding

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appropriate working standard solutions to drug-free rat microdialysates. When not in use, all the

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solutions were stored at 4 °C.

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Instrumentation. The UHPLC-MS/MS system consisted of an Agilent 1290 UHPLC

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system and an Agilent 6460 Triple Quadrupole MS/MS system (Agilent, USA). The

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chromatographic separation was realized on an Agilent SB C18 column (2.1 mm × 50 mm, 1.8 5

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µm) at 30 °C column temperature with 2.0 µL injection volumes. The flow rate was constant at

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0.2 mL/min. Eluent A was 5% acetonitrile/water (0.1% formic acid) and B was acetonitrile (0.1%

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formic acid). The linear binary gradient elution conditions were as follows: 65-82% B from 0 to 2

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min; 82-98% B from 2 to 6 min; 98-100% B from 6 to 8 min. During 0-5.5 min after injection the

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flow was diverted into waste to protect the mass spectrometer from potential contaminations

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because there were no detectable analytes. The column was equilibrated using the initial mobile

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phase for 1.5 min for each injection. The mass spectrometer was run in positive ion MRM mode

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of electrospray source. The optimal MS conditions were the same as our recent report in 2016.11

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The fragmentor voltage (FV) and collision energy (CE) were also optimized for the target

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derivatives. Experimental conditions for the direct MRM detection of OA and UA were set

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according to the literature.4 Transmission electron microscope (TEM) images were obtained

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using the JEM-2100PLUS microscope (JEOL, Tokyo, Japan).

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In vivo microdialysis sampling was accomplished using in vivo microdialysis system from

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Sweden CMA Co., including a CMA 402 syringe pump (CMA, Solna, Sweden), a CMA 120

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system (CMA, Solna, Sweden) for freely moving animals, and a microdialysis MAB6 probe

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(Stockholm, Sweden). ASI stereotaxic flat skull coordinates were purchased from ASI

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Instruments Inc. (MI, USA). The probe was perfused with Ringer’s solution (5 mmol/L) at a flow

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rate of 2.0 µL/min.

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Synthesis of CPR. The synthesis reaction schematic of CPR was shown in Figure 1.

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The synthesis of raw material N-hydroxysuccinimidyl rhodamine B ester (RB-S) was carried out

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as our previous report.33 The synthesis procedure was as follows: RB-S (1.5 g) and 0.5 g

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piperazine were added into 50 mL of acetonitrile and 25 mL sodium bicarbonate buffer (pH 8.5),

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the solution was heated to 45 oC with continued agitation for 2 h. After reduced-pressure

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distillation of the solvent and recrystallization in dichloroethane/absolute ethanol (v:v, 1:1), CPR

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was obtained as dark red crystals with a yield of 55%. 1HNMR (500 MHz, CDCl3/δ, ppm): 7.69 (t, 6

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J = 9.7 Hz, 2H), 7.61 (d, J = 13.5 Hz, 1H), 7.34 (d, J = 6.9 Hz, 1H), 7.19 (d, J = 9.5 Hz, 2H), 6.98

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(d, J = 9.1 Hz, 2H), 6.75 (s, 2H), 3.82 – 3.55 (m, 12H), 3.03 (d, J = 34.4 Hz, 3H), 1.33 (t, J = 7.0

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Hz, 11H). HRMS: [M+H]+ 511.30692.

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Preparation of Fe3O4/GO. The preparation of graphene oxide was described in

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Supporting information according to the Hummers method with minor modifications.34

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Fe3O4/GO was prepared based on chemical coprecipitation of Fe2+ and Fe3+ in alkaline media in

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presence of GO as described in Supporting information.35

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MDSPE-MAD procedure. Fe3O4/GO (8 mg) was put into a 1.5 mL vial and then 30

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µL of mixed standards or microdialysates, 30 µL of EDC (0.1 mol/L), 200 µL of CPR reagent

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were added. The vial was sealed and immersed in the ultrasound bath for 2.0 min to form a

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homogeneous dispersed solution. This solution was radiated for 25 min in a microwave reactor

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(450 W) at 60 °C to achieve complete MDSPE-MAD procedure. The derivatization reaction

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scheme is shown in Figure 1. And then the magnetic materials were separated rapidly from the

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derivatization solution by an external magnet. After that, CPR derivatives were eluted from the

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magnetic materials by 300 µL of methanol (containing 1.0 % formic acid) under ultrasound for 1

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min, and 2.0 µL of the solution was analyzed by UHPLC-MS/MS.

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Preparation of Arctiumlappa L. root extract. Dried Arctiumlappa L. root was

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purchased from China Beijing Tongrentang (Group) Co., Ltd. (Beijing). To 50 g of powdered raw

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herb (250 µm) in a 500 mL flask, 300 mL of ethanol was added. The mixture was extracted 2

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times by ethanol refluxing with 6 volume equivalents. The combined extracts were added with 20

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mL of 30% β-cyclodextrin to increase the solubility, and then concentrated to 10 mL under

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vacuum to obtain herb solution for rats. The OA and UA contents in the extract were

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quantitatively determined by the developed method in this work. The contents of free OA and UA

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in the extract were 673.8 and 526.4 µg/g. After saponification reaction with 10% potassium

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hydroxide at 90 °C for 3 h, the conjugated OA and UA in the extract were determined and their 7

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concentrations were 1438.5 and 1142.2 µg/g.

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Pharmacokinetics of OA and UA in rats by in vivo microdialysis. Male

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Sprague-Dawley rats (200-220 g, n=6) were purchased from Shandong Lukang Pharmaceutical

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Co. Ltd. The care and use of animals were in accordance with the related principles of China.

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Rats were narcotized with 20% urethane (1.2 g/kg, i.p.) before surgery, and kept anesthesis in the

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whole surgery of MAB probe implantation. After recovering, rats were free drinking and food

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intake for 24 h, and then fasted for 12 h with free drinking water before the pharmacokinetics test.

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Rats were orally administrated with extracts at a dose of 1.0 g/kg body weight. In vivo

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microdialysate samples from rat carotid artery were collected at 15, 30, 45, 60, 80, 100, 120, 180,

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240, 360, 480 and 600 min after oral administration. Each 30 µL of microdialysates were used for

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MAD-MDSPE procedure and UHPLC-MS/MS analysis.

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RESULTS AND DISCUSSION

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Optimization of Chromatography and MS Conditions. UHPLC and MS

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conditions were optimized for obtaining the best chromatographic separation and maximum MS

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sensitivity. The chromatographic conditions optimization was similar to our previous study.11 The

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optimum conditions were described in the experimental section. The representative MRM

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chromatograms of CPR-derivatives for standards and internal standard were shown in Figure 2A.

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MS conditions were also optimized similar to our previous study.11 All the CPR derivatives

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showed intense [M+H]+ ions. They were set as precursor ions. The most abundant product ions

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for three CPR derivatives were m/z 398.8 and m/z 443.2. In ESI-MS/MS conditions, these two

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specific product ions contained a permanent intramolecular positive charge. They brought

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enhanced sensitivity by increasing the ionization efficiency in the electrospray ionization. The

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proposed collision-induced dissociation pathways for the precursor ion of CPR-UA were shown

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in Figure 2B. In this study, m/z 398.8 was used for quantitative analysis, and m/z 443.2 was used 8

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for confirmation analysis. The optimal FVs and CEs for two transitions of OA, UA and betulinic

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acid (IS) were shown in Table 1.

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Optimization of MAD-MDSPE. Optimization of the volume of EDC solution.

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Using EDC as the condensing agent, the carboxylic acid group of OA and UA can be selectively

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labeled. A great advantage of this derivatization reaction was that a small amount of water was

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allowed to be present in the EDC condensation derivatization system. Therefore, CPR coupling

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with EDC has the great advantage for the determination of triterpenic acids in aqueous sample

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(such as microdialyaste) and thus was selected in this work. The volumes of CPR solution were

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optimized in the range of 10-60 µL as shown in Figure 3A. The peak area increased with the

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volumes of EDC from 10 to 30 µL and then decreased. Thus, 30 µL of EDC (0.1 mol/L) was

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selected.

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Optimization of the volume of CPR solution. To study the effect of CPR amount, the

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volume of CPR solution was optimized in the range of 50-350 µL as shown in Figure 3B. No less

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than 200 µL of CPR solution was significantly excess and insured the thorough derivatization of

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analytes. The excess CPR was purified by the MDSPE procedure. 200 µL of CPR was chosen for

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the MAD-MDSPE procedure.

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Optimization of derivatization time and temperature. The derivatization time was

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optimized from 10 to 40 min in the MAD-MDSPE procedure (microwave 450 W). As shown in

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Figure 3C, the derivatization reaction was completed rapidly under the microwave assistance.

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There were no remarkable increases of peak areas when the derivatization reaction time was more

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than 25 min. Therefore, 25 min was used to perform the derivatization. The derivatization

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temperature was optimized from 40 to 70 °C while other conditions were kept constant as shown

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in Figure 3D. Optimum peak areas were obtained when OA and UA standards were derivatized at

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60 °C.

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Optimization of sorbent amount. To evaluate the effect of sorbent amount on 9

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extraction efficiency and derivatization efficiency of CPR derivatives, the amount of Fe3O4/GO

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was optimized in the range of 4-16 mg. The incremental amounts of sorbent up to 8 mg possibly

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helped the derivatization reaction by providing a sufficient surface for derivatives adsorption, but

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in higher amounts of it, lower extraction efficiency was obtained. Therefore, the MAD-MDSPE

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procedure was carried out with 8 mg of Fe3O4/GO.

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Optimization of desorption conditions. To achieve good desorption efficiency of

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CPR derivatives, many desorption solutions including acetonitrile, acetone and methanol (each

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containing 1.0 % formic acid) were estimated. The results indicated that methanol had the

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strongest desorption power of CPR derivatives (Figure 4A). Methanol was a stronger polar

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organic solvent than acetonitrile and acetone. Moreover, GO can be easily dispersed in polar

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solvents because of its polar groups on the surface. Therefore, methanol was selected to ensure

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sufficient desorption of the derivatives.

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The effect of the volumes of desorption solution on the desorption efficiency was also

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evaluated. When the methanol volume was increased to 300 µL, desorption efficiency was

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increased because of the high rate of Fe3O4/GO dispersion in methanol. The results showed that

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300 µL of methanol (1.0% formic acid) was enough for the efficient desorption of the derivatives

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(Figure 4B).

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Desorption time was optimized in the range of 10-100 s (ultrasound 120 W, 40 KHz).

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Significantly increased peak areas of the CPR-derivatives were detected in 10-60 s and no

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significant increase was obtained with the enhance of desorption time in 60-100 s. In the end, 1.0

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min was employed as the optimal desorption time.

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Method validation. To investigate the applicability of the developed MAD-MDSPE

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coupled to UHPLC-MS/MS, linearity ranges, limits of detection (LODs), quantification (LOQs),

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repeatability, recovery, precision, accuracy and matrix effect (ME) were determined. The linearity

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of this method was established using internal standard spiked calibration solutions at 7 10

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concentration levels. To evaluate the dynamic ranges of this method, six batches of calibration

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microdialysate samples were prepared and determined. The peak-area ratio between the analyte

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and IS of each spiked microdialysate sample was determined. The calibration curves were then

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constructed by plotting the peak-area ratio with the spiked concentrations using linear regression

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for each analyte, respectively. The regression equations were y=9.836x+0.082 (R=0.991) for OA

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and y=9.923x−0.051 (R=0.995) for UA, where y was the peak-area ratio of the analyte and IS

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and x was the concentration of analyte (ng/mL). The calibration curves covering the

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concentration range of 0.050-100 ng/mL showed good linearity with correlation coefficient R >

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0.99. The LODs were 0.025 and 0.020 ng/mL for OA and UA (S/N > 3). The LOQs for OA and

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UA in microdialysates were 0.090 and 0.080 ng/mL (S/N > 10).

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As shown in Table 2, the precision was in the range of 2.20-6.01 %, and the accuracy was in

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the range of 85.1-112.3% from the actual QCs. The intra- and inter-day accuracy and precision

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were all within 15% by FDA. The matrix effect of the analytes ranged from 92.6-111.3% at 3

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concentration levels. The repeatability of the method was in the range of 2.34-7.35%. The

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recoveries of the OA and UA were in the range of 97.6-109.8% at 3 concentration levels. These

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results indicated that the developed method could be well used for the sensitive, specific and

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accurate determination of OA and UA in rat microdialysate samples.

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Pharmacokinetics of OA and UA from in vivo rat blood microdialysates.

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The developed MAD-MDSPE coupled to UHPLC-MS/MS method has been used to analyzing

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microdialysate samples from rat blood after oral administration of Arctiumlappa L. root extract.

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Typical MRM chromatograms of internal standard (IS) betulinic acid, OA and UA derivatives in

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a rat blood microdialysate sample were shown in Figure 5. The mean plasma drug

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concentration-time profiles were shown in Figure 6. Table 3 presented the pharmacokinetic

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parameters including the maximum plasma concentration (Cmax), the time for reaching the

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maximum concentration (Tmax), terminal half-life (t1/2), the area under the concentration-time 11

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curve (AUC), mean residence time (MRT), the apparent volume of distribution (Vz/F) and

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time-averaged total body clearance (CL/F). After oral administration, both OA and UA could be

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absorbed into the blood. However, their systemic exposures were quite different. OA appeared to

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be absorbed slightly quickly into the plasma with a Tmax of 45 min, while UA relatively absorbed

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slowly with a Tmax of 50 min. However, the absolute bioavailability of UA was obviously better

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than OA with Cmax of 40 vs 10 ng/mL. This finding is consistent with previous study,5 which will

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be helpful for future pharmacology, pharmacodynamics and drug development.

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In conclusion, we developed a rapid, selective and sensitive strategy based on MAD-MDSPE

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coupled to UHPLC-MS/MS (MRM) for the simultaneous determination of OA and UA in the rat

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blood microdialysates. The derivatization, extraction and purification of OA and UA occurred on

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the surface of Fe3O4/GO and were integrated into one step. Furthermore, the validated method

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was successfully used to the pharmacokinetics study.

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Supporting Information

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The Supporting Information is available free of charge on the ACS Publications website.

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Preparation of Fe3O4/GO.

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Corresponding Authors

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*E-mail: [email protected] (Zhao XE), Tel: +86-537-4456301, Fax: +86-537-4456305;

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*E-mail: [email protected] (Zhu SY);

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**E-mail: [email protected] (Qiao XG).

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ORCID

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Xian-En Zhao: 0000-0003-3500-9518;

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Shuyun Zhu: 0000-0002-9632-8187;

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Funding

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This work was supported by the National Natural Science Foundation of China (Nos. 21775088,

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21405094, and 81303179), the Special Fund for Agro-scientific Research in the Public Interest 12

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(Grant No. 201503142), the Innovation Platform for the Development and Construction of

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Special Project of Key Laboratory of Tibetan Medicine Research of Qinghai Province (No.

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2017-ZJ-Y11), and the Open Projects Program of the Key Laboratory of Tibetan Medicine

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Research of Chinese Academy of Sciences.

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Notes

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The authors declare that they have no conflict of interest.

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prior to gas chromatography–mass spectrometry for the characterization of the triterpenic fraction 13

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in olive leaves. J. Chromatogr. A 2007, 1165, 158-165.

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high-performance thin-layer chromatography. JPC-J. Planar Chromat. 2006, 19, 68-72.

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8. Gao, R. B. ; Wang, L. T.; Yang, Y.; Ni, J. M.; Zhao, L.; Dong, S. Q.; Guo, M. Simultaneous

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from plant material before HPLC determination of triterpenicacids. Talanta 2014, 122, 51-57.

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and P-31 NMR determination of pentacyclic triterpenic acids. Anal. Methods 2017, 9, 949-957.

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dispersive liquid-liquid microextraction coupled with microwave-assisted derivatization for

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14. Li, G. L.; You, J. M.; Song, C. H.; Xia, L. A.; Zheng, J.; Suo, Y. R. Development of a new

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HPLC method with precolumn fluorescent derivatization for rapid, selective and sensitive

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detection of triterpenic acids in fruits. J. Agric. Food Chem. 2011, 59, 2972-2979.

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15. Chen, G.; Li, J.; Song, C. H.; Suo, Y. R.; You, J. M. A sensitive and efficient method for

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simultaneous trace detection and identification of triterpene acids and its application to

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pharmacokinetic study. Talanta 2012, 98, 101-111.

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16. Wang, Y. W.; Suo, Y. R.; Sun, Y. N.; You, J. M. Determination of triterpene acids from 37

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different varieties of raspberry using pre-column derivatization and HPLC fluorescence detection.

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Chromatographia 2016, 79, 1515-1525.

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17. Wozniak, L.; Marszalek, K.; Skapska, S.; Jedrzejczak, R. Novel method for hplc analysis of

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triterpenic acids using 9-anthryldiazomethane derivatization and fluorescence detection.

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18. You, J. M.; Wu, D.; Zhao, M.; Li, G. L.; Gong, P. W.; Wu, Y. Y.; Guo, Y.; Chen, G.; Zhao, X.

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E.; Sun, Z. W.; Xia, L.; Wu, Y. N. Development of a facile and sensitive HPLC-FLD method via

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fluorescence labeling for triterpenic acid bioavailability investigation. Biomed. Chromatogr. 2017,

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of pentacyclic triterpenes prior to their gas chromatography-mass spectrometry analysis in plant

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extracts. Talanta 2016, 147, 35-43.

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20. Tang, S.; Zhang, H.; Lee, H. K. Advances in sample extraction. Anal. Chem. 2016, 88,

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Analysis of amino acid and monoamine neurotransmitters and their metabolites in rat urine of 15

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microextraction with UHPLC–MS/MS. J. Pharmaceut. Biomed. 2017, 135, 186-198.

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liquid–liquid microextraction by UHPLC-MS/MS. RSC Adv. 2016, 6, 108635-108644.

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hydroxyproline derivate as template combined with in situ derivatization for the specific

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FIGURE CAPTIONS

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Figure 1. The synthesized of CPR and the derivatization reaction scheme of CPR with OA and

408

UA.

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Figure 2. (A) The representative MRM chromatogram of CPR derivatives of internal standard

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(IS) betulinic acid, OA and UA standards, (B) product ion spectrum and the proposed

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fragmentation schematics of CPR-UA derivative.

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Figure 3. Optimization of MAD conditions (n = 5), (A) volumes of EDC, (B) volumes of CPR

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solution, (C) time (min), and (D) temperature (°C).

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Figure 4. Optimization of desorption conditions (n = 5), (A) types of desorption solution, (B)

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volumes of desorption solution.

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Figure 5. Typical MRM chromatograms of internal standard (IS) betulinic acid, OA and UA

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derivatives in a rat blood microdialysate sample.

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Figure 6. Mean concentration-time curves of OA and UA in rat plasma microdialysates after oral

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administration of Arctiumlappa L. root extract. Each point represents the mean ± standard error

420

(n=6).

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Tables Table 1. MRM Parameters of OA, UA and Betulinic Acid (IS) Analytes

Fragmentor (V)

Quantitation Transition (m/z)

Collision Energy (eV)

Confirmation Transition (m/z)

Collision Energy (eV)

OA

240

949.6 > 398.8

82

949.6 > 443.2

78

UA

240

949.6 > 398.8

81

949.6 > 443.2

78

Betulinic Acid (IS)

250

949.6 > 398.8

79

949.6 > 443.2

76

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Table 2. Results of Recovery, Matrix Effect, Repeatability, Precision, and Accuracy for OA and UA (n = 6)

Analytes

OA

UA

Spiked Levels (ng/mL)

Recovery (%)

Matrix Effect (%)

Repeatability (RSD, %)

Intra-day Precision

Inter-day Precision

(RSD, %)

(RSD, %)

Accuracy (%)

Peak Area

Retention Time

Peak Area

Retention Time

Peak Area

Retention Time

Intra-day

Inter-day

0.50

98.4±4.2

98.6±6.5

7.35

3.14

3.59

3.35

4.80

3.50

93.0

92.8

5.0

97.6±3.1

94.6±6.1

6.62

2.34

5.81

2.57

3.45

3.21

99.1

112.3

50.0

104.1±5.1

103.3±7.0

4.23

4.50

3.87

2.90

2.33

3.89

101.8

87.2

0.50

98.7±5.1

111.3±6.5

4.75

3.70

4.41

4.20

5.58

2.20

101.4

85.1

5.0

109.8±4.3

103.7±6.9

5.52

2.88

5.27

3.77

6.01

5.74

98.0

103.4

50.0

103.5±3.6

92.6±4.5

6.26

2.70

5.70

3.29

5.12

3.10

96.4

110.5

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Table 3. The Plasma Pharmacokinetic Parameters after Oral Administration of Arctiumlappa L. Root Extract to Rats. Parameter

Unit

UA

OA

t1/2

min

294.67±83.66

333.89±107.88

Tmax

min

50±7.75

45±9.49

Cmax

ng/mL

40.55±4.11

10.51±3.26

AUC0−t

ng/mL*min

6897.44±428.33

1249.10±245.91

AUC0−∞

ng/mL*min

9491.76±953.31

1749.03±470.50

MRT

min

457.42±87.94

473.65±110.73

Vz/F

(ng)/(ng/mL)

36.60±7.35

228.31±40.28

CL/F

(ng)/(ng/mL)/min

0.09±0.009

0.50±0.12

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Figure 2 (A, B)

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Figure 3 (A, B, C, D)

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Figure 4 (A, B)

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Figure 5

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Figure 6

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