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New Analytical Methods

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

1

ACS Paragon Plus Environment


1

ABSTRACT

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

3

provide important pharmacokinetics information. Microwave-assisted derivatization coupled with

4

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

5

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

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

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

2


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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

29

anti-inflammatory,

30

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

31

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

32

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

34

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

35

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

41

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

3


14-18

and


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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,

61

28

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

63

electrostatic forces.29

11,12,22

26

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

64

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

79

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

83

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

89

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

90

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

92

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

95

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

96

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

98

solutions were stored at 4 °C.

99

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

100

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

101

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



102

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

103

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

105

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

111

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

112

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

117

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

118

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

119

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

123

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

124

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

125

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

129

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

136

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

137

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

139

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

140

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

142

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

146

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

148

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

149

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

150

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

151

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.

153

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

155

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,

161

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

162

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

163 164

RESULTS AND DISCUSSION

165

Optimization of Chromatography and MS Conditions. UHPLC and MS

166

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

167

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

168

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

169

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

170

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

171

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

174

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

176

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

181

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

184

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

185

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

186

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

187

selected.

188

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

195

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

199

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

200

60 °C.

201

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



202

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

206

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

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Optimization of desorption conditions. To achieve good desorption efficiency of

208

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

209

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

210

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

211

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

212

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

213

sufficient desorption of the derivatives.

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

215

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

216

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

217

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

218

(Figure 4B).

219

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

220

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

222

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),

225

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

226

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

231

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

233

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

234

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

235

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

239

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

240

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

241

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

242

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

243

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

246

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

248

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

249

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

250

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

251

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



252

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

253

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

254

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

255

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

256

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

257

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

258

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

260

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

261

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

262

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

263

was successfully used to the pharmacokinetics study.

264

Supporting Information

265

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

266

Preparation of Fe3O4/GO.

267

Corresponding Authors

268

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

269

*E-mail: [email protected] (Zhu SY);

270

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

271

ORCID

272

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

273

Shuyun Zhu: 0000-0002-9632-8187;

274

Funding

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

276

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.

281

Notes

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The authors declare that they have no conflict of interest.

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394 395 396 397 398 399 400 401 402 403 404 405 17



406

FIGURE CAPTIONS

407

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

408

UA.

409

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

410

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

411

fragmentation schematics of CPR-UA derivative.

412

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

413

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

414

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

415

volumes of desorption solution.

416

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

417

derivatives in a rat blood microdialysate sample.

418

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

419

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

420

(n=6).

421

18


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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

19



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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

20


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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

21



Figure 1

22


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Figure 2 (A, B)

23



Figure 3 (A, B, C, D)

24


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Figure 4 (A, B)

25



Figure 5

26


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Figure 6

27



Graphic for Table of Contents

28


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Simultaneous determination of oleanolic acid and ... - ACS Publications

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