Research article

Received 9 November 2010, Accepted 11 December 2010 Published online in Wiley Online Library: 10 February 2011

(wileyonlinelibrary.com) DOI 10.1002/bmc.1609

1360

Application of LC‐MS/MS method for thein vivometabolite determination of oleuropeinafter intravenous administration to ratTing Zhoua,b, Tianxiu Qiana*, Xiaoying Wanga, Xianen Lia,Li Caoa and Shuangying Guib*

ABSTRACT: A highly sensitive, specific and simple LC‐MS/MS method was developed to investigate in vivo bio‐transformationof oleuropein in rat. Rat urine samples collected after the intravenous administrations were determined using liquidchromatography coupled to tandem mass spectrometry with electrospray ionization in the negative‐ion mode. The assayprocedure involves a simple liquid–liquid extraction of parent oleuropein and the metabolite from rat urine with ethylacetate. Chromatographic separation was operated with 0.1% formic acid aqueous and methanol in gradient program at aflow rate of 0.80 mL/min on an RP‐C18 column with a total run time of 30 min. This method has been successfully applied tosimultaneous determination of oleuropein and its metabolite in rat urine. Oxygenation was found to be the major metabolicpathway of the oleuropein in rat after intravenous administration. Copyright © 2011 John Wiley & Sons, Ltd.

Keywords: liquid chromatography; tandem mass spectrometry; oleuropein; in vivo metabolite; urine

* Correspondence to: Tianxiu Qian, Institute of Medicinal Plant Develop-ment, Chinese Academy of Medical Sciences and Peking Union MedicalCollege, 151 Malianwa North Road, Haidian District, Beijing 100193,People’s Republic of China. E-mail: txqian88@yahoo.com

Shuangying Gui, School of Pharmacy, Anhui University of Traditional ChineseMedicine, Hefei, People’s Republic of China. E-mail: guishy0520@yahoo.com.cn

a Institute of Medicinal Plant Development, Chinese Academy of MedicalSciences and Peking Union Medical College, 151 Malianwa North Road,Haidian District, Beijing 100193, People’s Republic of China

b School of Pharmacy, Anhui University of Traditional Chinese Medicine,Hefei, People’s Republic of China

Abbreviations used: MDQ, Mao‐Dong‐Qing; OE, oleuropein.

IntroductionMao‐Dong‐Qing (MDQ), the dried roots of Ilex pubescens Hook.et Arn., is widely used for the treatment of cardiovascular diseasesand hypercholesterolemia in China. Pharmacological investiga-tion has demonstrated that the extracts of Mao‐Dong‐Qing notonly dilate blood vessels but also improve minicirculation, lowerblood pressure, inhibit platelet aggregation, prevent thrombosis,reduce cardiac ischemia, decrease the excitation of the cardiacconduction system and enhance anoxia resistance (Yang andPang, 1986). Recently, the new drug Mao‐Dong‐Qing capsule,made from extract of Ilex pubescens, has been approved by theState Food and Drug Administration of China to treatcardiovascular diseases. In our laboratory, the chemical constit-uents of Mao‐Dong‐Qing capsule were investigated, whichresulted in oleuropein (OE) and other glycosides being isolatedand identified. We are interested in OE as it is one of the majorcompounds in Mao‐Dong‐Qing capsule.

OE is a nontoxic natural iridoid glycoside. It has also been foundin many Chinese herbal medicines, such as flowers of Jasminumofficinale (Zhao et al., 2008), Syringa pubescens (Wu et al., 2003),Syringaoblata (Zhang et al., 2006) and Ilex pubescens (Yang et al.,2007). It has many important biological activities, includingantioxidant (Mannaa et al., 2004; Ruíz‐Gutiérrez et al., 1995;Al‐Azzawie and Alhamdani, 2006), anti‐atherogenic (Visioli andGalli, 2001; Carluccio et al., 2003), anti‐HIV (Lee‐Huang et al., 2007)and other virus (Micol et al., 2005; Zhao et al., 2009), anti-cancer (Owen et al., 2000; Trichopoulou et al., 1995; Hamdi andCastellon, 2005; Visioli and Galli, 1994) and hypoglycemic(Al‐Azzawie and Alhamdani, 2006) properties. Aforementionedpharmacological research reports led us to study its in vivobio‐transformation.

Recently, Fotini et al. (2010) reported the quantification of OEand its metabolites in rat plasma by LC‐MS after oral

Biomed. Chromatogr. 2011; 25: 1360–1363 Copyright © 2011 John

administration for 80 days, and another report appeared aboutthe LC‐MS analysis of OE and its metabolite hydroxytyrosol in ratplasma and urine after oral administration (Del Boccio et al.,2002). However, OE was poorly absorbed from perfused ratintestine (Edgecombe et al., 2000). The metabolites may be traceamounts in biological samples. Therefore, a highly sensitive andspecific LC‐MS/MS method is much needed. Furthermore, nobio‐transformation of OE after intravenous administration hasbeen reported. To better understand the pharmacologicalmechanism of Mao‐Dong‐Qing capsule and further develop anew therapeutic formula or pure compound, study of themetabolism of OE after intravenous dosing is of importance.Therefore, this study focused on the development andapplication of LC‐MS/MS method to in vivo metaboliteidentification of oleuropein.

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LC‐MS/MS for the metabolite determination of oleuropein

Experimental

Chemicals

OE (purity >99%) was isolated and purified in our laboratory fromMao‐Dong Qing. Its structure (Fig. 1) was identified by MS and NMRanalyses and phytochemistry research results will be published in aseparate paper. HPLC‐grade methanol was purchased from FisherScientific (Somerville, NJ, USA). The water used in the experiments wascollected from a Milli‐Q Ultra‐pure water system (Millipore, Billerica, MA,USA). Other chemicals (analytical grade) were purchased from SinopharmChemical Reagent Beijing Co. Ltd (Beijing, China).

In Vivo study with intravenous administrations of OE

OE was dissolved in saline (0.9% NaCl in water). An i.v. dose (5 mg/kg)with 1 ml dosing solution was given to each male Sprague–Dawleyrat (body weight 180–200 g). The rat urine was collected from 0 to24 h.

HO O

O O

O

O

α

β

HO

OCH3

O-glc

4

5’

3’ 1’ 3

1

6 5 7

9

8

10

11

Oleuropein, MW 540

Figure 1. The molecular structure of oleuropein.

2 4 6 8 10 12 14

Tim

0.02000.04000.06000.08000.0

1.0e41.2e41.4e41.6e41.8e42.0e42.2e42.4e42.6e42.8e43.0e4

Intensity, cps

11.00

2 4 6 8 10 12 140.0

1.0e4

2.0e4

3.0e4

4.0e4

5.0e4

6.0e4

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8.0e4

Intensity, cps

14

Figure 2. Extracted mass chromatograms obtained from the negative LC‐Mm/z 539(A) and its mono‐oxygenated metabolite at m/z 555 (B).

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Urine sample preparation

A urine sample of each rat was extracted with an equal volume of ethylacetate three times. The extract was dried and the residue was dissolvedin 1 ml methanol. After the centrifugation at 8000g for 20 min, 5 μl ofthe supernatant was analyzed using LC‐MS and LC‐MS‐MS for bothparent OE and metabolites.

LC‐ESI‐MS analysis

HPLC separation was carried out on an LC system equipped with anautosampler and a normal mode pump (HP1200, Agilent Technologies,Germany). A reversed‐phase column (Merck Lichrospher RP‐C18,4 × 250 mm, 5 μm) was used for separating the OE and the metabolites.The temperature of the auto‐sampler was set at 15°C. The mobile phaseconsisted of two eluents. Eluent A was water with 0.1% formic acid andeluent B was methanol. The gradient program was 20% B held for 5 minthen changed to 50% B within 1 min and held at 50% B for 15 min, thenback to 20% B within 1 min and held until 31 min at a flow rate of0.8 mL/min. The effluent from the LC column was diverted to waste forthe first 4 min following the injection, then 30% effluent from the LCcolumn diverted to the MS ion source.

Mass spectrometric experiments were performed on a 3200 QTRAP®LC/MS/MS (AB Sciex, Totonto, Canada). Negative ESI ion mode was usedto analyze OE and the metabolites in rat urine samples. The followingparameters of the turbo‐ionspray for negative ion mode were used:ionspray voltage −4500 V, declustering potential −70 V, entrancepotential −10 V. The ion source gas 1, gas 2 and curtain gas were 25, 15and 20, respectively, and collision energy for the LC‐MS/MS experimentwas set at −40 for parent OE and −30 for the oxygenation metabolite,respectively. The temperature of GS2 was set at 400°C.

Full‐scanmass spectra at a mass range ofm/z 100–1000 were acquired.Mass chromatograms for the [M − H]− ion of OE and themetabolites were

16 18 20 22 24 26 28

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A

B

S analysis of rat urine sample after i.v. administration of OE: parent OE at

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50 100 150 200 250 300 350 400 450 500 550 600 m/z, amu

2.0e6

4.0e6

6.0e6

8.0e6

1.0e7

1.2e7

1.4e7

1.6e7

Intensity, cps

275.2

139.0

89.1 307.2

119.1

223.2

377.2 539.3

345.2 403.3

100 150 200 250 300 350 400 450 500 550 600 m/z, amu

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151.0

555.5

223.1

291.3 178.9 537.4 118.8 88.7 323.3 361.0

393.4 403.2

[M-H]-

[M-264-H]-

[M-232-H]-

[M-194-H]-

[M-152-H]-

[M-162-H]-

A

B

[M-H]-

[M-264-H]-

[M-232-H]-

[M-194-H]-

[M-136-H]-

[M-162-H]-

Figure 3. LC‐MS/MS spectra of OE (A) and its mono‐oxygenated metabolite (B).

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used for determination. Molecular ion masses of potential metaboliteswere examined and the corresponding extracted mass chromatogramswere recorded.

HO O

O O

O

O

α

β

HO

OCH3

O-glc

HO O

O O

O

O

α

β

HO

OCH3

O-glc

O+

[O]

OE, MW 540

Monooxygenated OE, MW 556

136

152

Figure 4. Proposedmetabolic pathway of OE in rat urine sample after i.v.administration of OE.

Results and discussion

Metabolites in rat urine collected after intravenousadministration of OE

One metabolite was detected in rat urine samples collected inthe period of 0–24 h after the oral administration of OE. Themetabolite detections were achieved from LC‐MS analyses andconfirmed with the MS‐MS experiments in negative ESI mode.Figure 2 shows that one oxygenated metabolite was detected. Itwas identified and confirmed by LC‐MS/MS analyses (Fig. 3).Figure 3(A) shows the fragmentation pattern of the parent OE,and the major daughter ions produced from deprotonatedmolecular ionm/z 539 [M − H]− were m/z 377 (M − 162), m/z 345(M − 194), m/z 307 (M − 232) and m/z 275 (M − 264). Themetabolite demonstrated a similar MS‐MS fragmentationpattern to the parent OE from the analyses of their [M − H]−

ions (Fig. 3). The fragmentation pattern of the metabolite, whichwas produced from the [M − H]− ion at m/z 555, was interpretedand compared with that of OE. The metabolite had the samefragment patterns, i.e. the same fragments were lost from themolecular ion and the corresponding daughter ions at m/z 393(M − 162), m/z 361 (M − 194), m/z 323 (M − 232) and m/z 291(M − 264) (Fig. 3B).

In addition, daughter ions at m/z 403 (M − 152, 3B), m/z 403(M − 136, 3A) and m/z 151 (M − 404, 3B) indicate that the

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oxygenated site may be at α or β of the parent OE (Fig. 4). Theresult shows that the oxygenation is major metabolic pathwayof OE in rat blood circulatory system (Fig. 4).

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LC‐MS/MS for the metabolite determination of oleuropein

ConclusionHPLC coupled with ESI‐MSwas proved to be a powerful analyticaltechnique for the simultaneous determination of OE and itsmetabolites in rat urine samples. Oxygenation was found to bethe major metabolic pathway of the OE in rat blood circulatorysystem after intravenous administration. The obtained resultprovides information for studying the pharmacology of OE withintravenous administration for research and development of newdrugs for treating the cardiovascular disease.

AcknowledgmentsWe acknowledge the Ministry of Science and Technology of thePeople’s Republic of China for financial support of this project(National S&T Major Project, 2009ZX09301‐003).

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