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Journal of Chromatography A, 1217 (2010) 4587–4600

Contents lists available at ScienceDirect

Journal of Chromatography A

journa l homepage: www.e lsev ier .com/ locate /chroma

practical strategy for the characterization of coumarins in Radix Glehniae byiquid chromatography coupled with triple quadrupole-linear ion trap masspectrometry

ei Yanga, Min Yeb, Man Liua, Dezhi Konga, Rui Shia, Xiaowei Shia, Kerong Zhangc,iao Wanga,∗, Zhang Lantonga

Department of Pharmaceutical Analysis, School of Pharmacy, Hebei Medical University, 361 East Zhongshan Road, Shijiazhuang 050017, ChinaThe State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100038, ChinaApplied Biosystems Trading Co., Ltd., Beijing Branch Office, Beijing 100027, China

r t i c l e i n f o

rticle history:eceived 20 January 2010eceived in revised form 16 April 2010ccepted 28 April 2010vailable online 6 May 2010

eywords:

a b s t r a c t

The aim of the present study was to develop a practical method for the characterization of coumarinsin Radix Glehniae by liquid chromatography–mass spectrometry (LC–MS). First, 10 coumarin standards(including two pairs of isomers) were studied, and mass spectrometry fragmentation patterns and elutiontime rules for the coumarins were found. Then, an extract of Radix Glehniae was analyzed by the com-bination of two scan modes, i.e., multiple ion monitoring-information-dependent acquisition-enhancedproduct ion mode (MIM-IDA-EPI) and precursor scan information-dependent acquisition-enhanced prod-

iquid chromatography–mass spectrometryultiple iononitoring-information-dependent

cquisition-enhanced product ionrecursor scan information-dependentcquisition-enhanced product ion

uct ion mode (PREC-IDA-EPI) on a hybrid triple quadrupole-linear ion trap mass spectrometer. A total of 41coumarins were identified on the basis of their mass spectrometry fragmentation patterns. This is the firsttime that these two scan modes have been combined to characterize chemical constituents in traditionalChinese medicine. This new method allowed the identification of coumarins in Radix Glehniae in traceamounts. The methodology proposed in this study could be valuable for the structural characterizationof coumarins from complex natural and synthetic sources.

oumarinadix Glehniae

. Introduction

Radix Glehniae (Beishashen), from the roots of Glehnia littoralisG. littoralis) Fr. Schmidt ex Miq. (Umbelliferae), is one of the mostommonly used traditional Chinese medicines (TCM). It has beenecorded in Chinese Pharmacopoeia and is used to treat respiratorynd gastrointestinal disorders in China [1]. Coumarins are the mainioactive constituents of Radix Glehniae. Recently, coumarins havettracted increasing interest due to their various activities, includ-ng anticancer [2], anti-inflammation [3], antibacterial [4], hepaticrug-metabolizing enzyme-inducing [5], and antidermatosis func-ions [6]. Until now, only two major types of coumarins, simpleoumarins and linear-type furocoumarins, have been reportedrom Radix Glehniae [7,8]. During our previous study [9], we found

hat Radix Glehniae contained a large number of coumarins, with

ost of them at low levels. It is well-known that traditional Chi-ese medicine (TCM) is commonly believed to operate due to theynergistic effects of all of the major and minor components in the

∗ Corresponding author. Tel.: +86 311 86265625; fax: +86 311 86266419.E-mail address: qiaowang88@hotmail.com (Q. Wang).

021-9673/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.chroma.2010.04.076

© 2010 Elsevier B.V. All rights reserved.

medicines [10]. Therefore, the detection and identification of minorcomponents may be equally important for understanding the phar-macological basis of coumarins and enhancing the product qualitycontrol of Radix Glehniae.

Electron ionization-mass spectrometry (EI-MS) has been usedto analyze coumarins [11,12], but it is more suitable for singlecompound. Liquid chromatography–mass spectrometry (LC–MS)technology has demonstrated its value in analyzing complex mix-tures and has become a powerful tool in the online structuralcharacterization of various natural compounds owing to its highsensitivity, good separation efficacy and our considerable knowl-edge regarding its structure [13–17]. Recently, fragmentationbehaviors and pathways of coumarins in electrospray ionization-mass spectrometry (ESI-MS) have been studied [18], and coumarinswere analyzed with LC–MS in Radix Angelicae Dahuricae [19,20],Korean Angelica [21] and dietary supplements such as lemon peel[22], citrus essential oils [23] and marmalade [24]. However, the

identification of coumarins from Radix Glehniae by LC–MS has notbeen reported.

The traditional way to identify the structures of unknown com-pounds is full scan mass analysis with triggered MS/MS acquisitionbased on intensity. Yet, signals of trace compounds may not be

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588 W. Yang et al. / J. Chroma

etected because of high background noises. Quadrupole-linearon trap mass spectrometry not only has the functions of tripleuadrupole (QqQ) including multiple ion monitoring (MIM), prod-ct ion scan (PI), neutral loss scan (NL), and precursor ion scanPREC), but also has the functions of line ion trap (LIT) referring

series of enhanced sensitive scans, such as enhanced producton scan (EPI). When the information-dependent data acquisi-ion (IDA) is performed, the functions of quadrupole and ion trapan be combined, such as precursor scan information-dependentcquisition-enhanced product ion mode (PREC-IDA-EPI) and multi-le ion monitoring-information-dependent acquisition-enhancedroduct ion mode (MIM-IDA-EPI). Recently, these two IDA meth-ds have been reported and separately applied to the study of drugetabolites and protein modifications [25–28]. They are employed

o trigger EPI scans by analyzing PREC and MIM, respectively. TheREC-IDA-EPI method provides information about parent ions andragment ions of specified ions, while the MIM-IDA-EPI methodrovides information about fragment ions from expected ions.eanwhile, MIM retains the same sensitivity as multiple reactiononitoring (MRM), and no pre-acquisition of MS/MS spectra is

eeded due to the same predicted ion in both Q1 and Q3. On theasis of these considerations, the combination of the two modesould be even more powerful in identifying unknown trace com-ounds in TCM.

The aim of our study was to establish a practical strategy forhe qualitative analysis of coumarins in Radix Glehniae. First, wetudied the mass fragmentation patterns of 10 coumarin stan-ards in the positive ion mode and their elution properties ineversed-phase high-pressure liquid chromatography (RP-HPLC).n the basis of the summarized new rules, coumarins in a crudextract of Radix Glehniae were characterized by the combined usef the MIM-IDA-EPI and PREC-IDA-EPI modes on a hybrid tripleuadrupole-linear ion trap mass spectrometer, for the first time.orty-one coumarins were identified, and their structures are listedn Fig. 1. It is believed that this novel method could be valuable forhe structural characterization of simple coumarins, furocoumarinsnd their glycosides from natural and synthetic sources.

. Experimental

.1. Standards and reagents

Scopoletin (8), psoralen (21), imperatorin (36) and isoimpera-orin (40) were purchased from the China Institute for Control ofharmaceutical and Biological Products. Bergapten (28), xantho-oxin (22), xanthotoxol (12) and isoimpinellin (26) were obtainedrom Shanghai Tauto Biotech Co., Ltd., PR China. Cnidilin (38) andxypeucedanin (32) from Radix Angelicae Dahuricae were isolatednd purified in our lab. The two compounds were identified by com-arison of their 1H NMR, 13C NMR and MS data with the literatureata. The purities of all ingredients were above 98% according toPLC analysis.

HPLC grade methanol (Fisher, USA) was used for HPLC analy-is. Deionized water was produced by a Heal Force-PWVF Reagent

ater System (Shanghai CanRex Analyses Instrument Corporationimited, PR China). Analytical-grade methanol (Tianjin Chemicalorporation, PR China) was used for sample preparation. Ammo-ium acetate was HPLC grade purchased from Diamond Technology

ncorporated (USA).

.2. Plant materials and sample preparation

A Radix Glehniae sample was collected from Anguo, Hebeirovince of China. The sample was all unpeeled crude herb. Aoucher specimen was deposited in the herbarium of the Schoolf Pharmacy, Hebei Medical University.

1217 (2010) 4587–4600

The unpeeled crude Radix Glehniae were powdered to a homo-geneous size by a mill and sieved through a No. 40 mesh sieve. Anamount of 6 g of Radix Glehnia was extracted with 30.0 mL of 75%methanol in an ultrasonic ice-water bath for 30 min and then fil-tered. The filtrate was evaporated to dryness and the residue wasdissolved in 2 mL of water–methanol (v/v, 1:1). The solution wasfiltered through a 0.45 �m microporous membrane before HPLCinjection of 20 �L.

2.3. Preparation of standard solutions

The appropriate amount of each standard was weighed and dis-solved in methanol to make 10 individual stock solutions. Then,each stock solution was mixed with water–methanol (v/v, 1:1) toprepare a final mixed standard solution. For direct infusion, ESI-MSn

study standard solutions were taken from the stock solutions anddiluted to 5 �g mL−1 with 1 mmol/L ammonium acetate–methanol(50:50).

2.4. Instrumentation

Detections were performed using a 3200 QTRAPTM system fromApplied Biosystems/MDS Sciex (Applied Biosystems, Foster City,CA, USA), a hybrid triple quadrupole-linear ion trap mass spec-trometer equipped with Turbo V sources and a TurboIonsprayinterface. An Agilent 1200 liquid chromatography system (Agi-lent, USA) equipped with a quaternary solvent delivery system,an autosampler and a column compartment were used. All instru-mentations were controlled and synchronized by Analyst software(versions 1.4.2) from Applied Biosystems/MDS Sciex.

2.5. Liquid chromatographic and mass spectrometric methods

The chromatographic separations were performed on an Agi-lent Zorbax SB-C18 column (150 mm × 4.6 mm, 5 �m), with thecolumn temperature set at 25 ◦C. The mobile phase consisted ofmethanol (A) and 1 mmol/L ammonium acetate (B), with a lineargradient elution at a flow rate of 1 mL/min. The gradient programwas as follows: 20–55% A (0–25 min); 55–95% A (25–60 min); 95%A (60–70 min). The LC effluent was introduced into the ESI sourcein a post-column splitting ratio of 3:1.

The instrument was operated using an electrospray ionizationsource in positive mode. The ion spray voltage was set to 5.5 kV,and the turbo spray temperature was maintained at 400 ◦C. Bothnebulizer gas (gas 1) and heater gas (gas 2) were set at 50 psi,respectively. The curtain gas was kept at 25 psi and the interfaceheater was on. Nitrogen was used as nebulizer and auxiliary gas.

MIM-IDA-EPI mode was carried out using the MRM-EPI modewith minimal collision energy in Q2, and the same ions were mon-itored in Q1 and Q3. The declustering potential (DP) and collisionenergy (CE) of MIM were set at 25 V and 5 eV, respectively. Thedwell time of each ion pair was 20 ms. In the IDA criteria, the formertarget ions were excluded for 15 s, and the 3 most intense fragmentions of each analyte that exceed 1000 cps counts were selected todo a product ion scan. An EPI scan was performed at a fill time of50 ms, step size of 0.12 amu and ion scan range from 50 to 400 amuwith a scan rate of 4000 amu/s. The CE of EPI was set at 25 eV (col-lision energy spread 15 eV) or three discontinuous energy levels of10, 25 and 40 eV.

For the PREC-IDA-EPI analysis, the PREC scan was run in positivemode at a scan range from m/z 50 to 800 with a 0.1 amu step size,

5 ms pause between mass ranges and 2 s scan time. The precursorsof PREC scans corresponded to the specified ions monitored in theMIM-IDA-EPI mode. The declustering potential (DP) and collisionenergy (CE) of PREC were set at 25 V and 25 eV, respectively. IDAwas used to trigger the acquisition of EPI spectra for ions exceeding

togr. A

5ow0o

W. Yang et al. / J. Chroma

00 cps, with exclusion in the target for 15 s. The EPI scan was also

perated ranging from m/z 100 to 800 at a scan rate of 4000 amu/sith a fill time of 50 ms in the linear ion trap and a step size of

.12 amu. In the mode, the CE of EPI was set at 25 eV with a spreadf 15 eV.

Fig. 1. Structures of the 41 compounds

1217 (2010) 4587–4600 4589

3. Results and discussion

3.1. Optimization of HPLC–MS/MS conditions

The 10 standards were characterized according to their massspectra using syringe pump continuous infusion analysis to find

identified from Radix Glehniae.

4590 W. Yang et al. / J. Chromatogr. A 1217 (2010) 4587–4600

(Conti

ttg

Fig. 1.

heir mass spectrometric behaviors and optimize the MS condi-ions. When the electrospray ionization (ESI) source was operated,ood sensitivity and sufficient numbers of fragment ions were

nued ).

obtained. With regard to the 10 standards, positive ion modeoffered better sensitivity than negative ion mode and gave [M+H]+

ions as the base peaks. Therefore, ESI in the positive ion mode was

W. Yang et al. / J. Chromatogr. A 1217 (2010) 4587–4600 4591

n, im

sqv

houpAtc1ammotiP

3

fth

Fig. 2. The PREC spectra of psorale

elected for follow-up analyses. In the following mass data, theuoted m/z values were expressed in integers for presentation con-enience and were obtained by rounding off to the nearest integer.

When ammonium acetate was added into the mobile phase, theigher abundance of [M+NH4]+ could be monitored in the spectraf PREC, which could be used to identify the molecular weight ofnknown coumarins combined with [M+H]+. The spectra of PREC ofsoralen, imperatorin, cnidilin and scopoletin are shown in Fig. 2.mmonium acetate (0.2, 1 and 2 mmol/L) were tested to identify

he optimal mobile phase that produced the best sensitivity, effi-iency and peak shape. The results indicated that the intensity of the0 standard peaks in spectra of MIM was highest when 1 mmol/Lmmonium acetate was added into the mobile phase. Therefore, aobile phase of 1 mmol/L ammonium and methanol was the opti-al choice in both HPLC and ESI-MS analyses. The limit of detection

f MIM-IDA-EPI is 0.08 ng for scopoletin and 0.06 ng for impera-orin, respectively, with which the MS2 spectra of scopoletin andmperatorin can be shown accurately. The limit of detection ofREC-IDA-EPI is 0.08 ng for scopoletin and 0.29 ng for imperatorin.

.2. Analysis of 10 pure standards with ESI-MSn

The standards included two types of coumarins, 9 linear-typeurocoumarins and 1 simple coumarin (scopoletin). The linear-ype furocoumarins are usually substituted at C-5 and C-8 with aydroxyl group [29].

peratorin, cnidilin and scopoletin.

All of the 10 standards were characterized by using positive ESI-MSn, and their fragmentation patterns were studied at three CEvalues of 10, 25 and 40 eV using the [M+H]+ as a precursor ion.Meanwhile, some prominent fragment ions of the 10 standardswere all analyzed by positive PREC mode at CE 25 eV. Fig. 3 andFig. S1 presents the proposed fragmentation pathways of the 10standards.

3.2.1. Fragmentation of xanthotoxin and bergaptenXanthotoxin and bergapten are a pair of isomers and are linear-

type furocoumarins substituted with a methoxyl group at C-8 andC-5, respectively, with [M+H]+ at m/z 217. A characteristic seriesof fragment ions, [M+H-CH3-CO]+ m/z 174, [M+H-CH3-2CO]+ m/z146, [M+H-CH3-3CO]+ m/z 118 and [M+H-CH3-4CO]+ m/z 90, werefound in the MS/MS spectra of xanthotoxin and bergapten. Twofragmentation pathways are possible with an EI source for the twocompounds, the loss of methyl or the loss of CO; both of these havebeen reported in the literature [30]. The pathway of losing CO firstfor bergapten was not observed with an ESI source in the presentstudy. The fragment ion m/z 202 of the two standards was derivedfrom the loss of methyl from their [M+H]+ m/z 217. Comparing the

intensity of m/z 202 and m/z 217 at a CE level of 25 eV, the inten-sity of m/z 217 was larger than m/z 202 for xanthotoxin, and theopposite was observed for bergapten. The reason why the amountof lost CH3 in position 5 is higher than position 8 was probablybecause loss of CH3 at position 5 makes the structure more stable.

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592 W. Yang et al. / J. Chroma

he MS/MS spectra of xanthotoxin and bergapten at CE 25 eV arehown in Fig. 4. It is noteworthy that the fragment ion of [M+H-H3OH]+ m/z 185 was present in xanthotoxin alone, which waslso seen in Fig. 4. In the PREC spectra of each product ion gener-ted from xanthotoxin and bergapten, both [M+H]+ and [M+NH4]+

ere simultaneously found.

.2.2. Fragmentation of imperatorin and isoimperatorinImperatorin and isoimperatorin, with a common molecular

eight of 270 Da, are also a pair of isomers that are linear-typeurocoumarins substituted with an isopentenoxy group at C-8 and-5, respectively. The m/z 203 of imperatorin and isoimperatorinas derived from the neutral loss of C5H8, possibly resulting from

he formation of an intermediate ion with an eight-membered ring19]. The fragmentation of m/z 203 was followed by first loss of COr CO2 and then successive loss of CO, corresponding to the frag-ent ions at m/z 175, 159, 147, 131, 119 and 91. Some fragment ions

rom the cleavage of isopentenyl were also found, such as [M+H-3H6]+ m/z 229, [M+H-C4H8]+ m/z 215 and [C5H9]+ m/z 69, but theragment ions of m/z 229 and m/z 215 were weak. The m/z 185 ionormed from loss of the isopentenoxy group could only be observedn imperatorin. In the PREC spectra, both [M+H]+ and [M+NH4]+ of

ig. 3. The main proposed fragmentation pathway of the 10 standards: xanthotoxin,soimpinellin, cnidilin and scopoletin.

1217 (2010) 4587–4600

imperatorin and isoimperatorin could been accurately monitored(Fig. 2). Occasionally, the parents of [2M+H]+ and [2M+NH4]+ werealso found.

3.2.3. Fragmentation of oxypeucedaninOxypeucedanin is a linear-type furocoumarin substituted with

a deformed isopentenoxy group at C-5, with [M+H]+ at m/z 287.From Fig. 3, it is apparent that the m/z 203 of oxypeucedanin wasproduced from the neutral loss of C5H8O. A characteristic series ofm/z 175, 147, 119 and 91 was produced by the successive loss ofCO from the fragment ion of m/z 203. The m/z 185 formed fromthe loss of the deformed isopentenoxy group could not be foundin the MS2 spectrum of oxypeucedanin. The fragment ions of thedeformed isopentenyl group from oxypeucedanin were [C3H7O]+

m/z 59 and [C5H9O]+ m/z 85. In the PREC spectra of each product ion,the [M+H]+, [M+NH4]+, [2M+H]+ and [2M+NH4]+ of oxypeucedaninwere observed.

3.2.4. Fragmentation of psoralenPsoralen is an unsubstituted linear-type furocoumarin. Its frag-

mentation was through two major pathways: the importantpathway was the successive loss of CO, while the other was the

bergapten, imperatorin, isoimperatorin, oxypeucedanin, psoralen, xanthotoxol,

W. Yang et al. / J. Chromatogr. A 1217 (2010) 4587–4600 4593

Fig. 3. (Continued ).

4594 W. Yang et al. / J. Chromatogr. A 1217 (2010) 4587–4600

thotox

c1mopHt

3

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3

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3

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Fig. 4. The MS2 spectra of xan

leavage pathway of M+H-CO2-CO. Therefore, its [M+H]+ of m/z87 yielded the fragment ions [M+H-CO2]+ m/z 143, [M+H-2CO]+

/z 131, [M+H-CO2-CO]+ m/z 115 and [M+H-3CO]+ m/z 103. More-ver, the PREC spectra of its fragment ions exhibited two commonrominent parent ions, [M+H]+ and [M+NH4]+, at m/z 187 and 204.owever, in the PREC spectra of psoralen (Fig. 2), we could not find

he parent ions of [2M+H]+ and [2M+NH4]+.

.2.5. Fragmentation of xanthotoxolThe MS/MS data of xanthotoxol (8-hydroxycoumarin) revealed

he main fragmentation pathway of xanthotoxol, which is the suc-essive loss of 4 CO units until the oxygen atoms are completely lost,ielding prominent ions at m/z 175, 147, 119 and 91. In the MS/MSpectrum of xanthotoxol, m/z 185 formed through the loss of H2Ohrough cleavage at the hydroxy substituent at C-8 was found. Itsragment ions at m/z 159, 131 and 103 were formed by the suc-essive elimination of CO2, CO and CO. The cleavage pathway ofanthotoxol is shown in Fig. S1.

.2.6. Fragmentation of isoimpinellinIsoimpinellin has a molecular weight of 246 Da and is a linear-

ype furocoumarin substituted with two methoxy groups. Theragmentation pattern of isoimpinellin was the continuous loss ofH3, yielding the predominant ions [M+H-CH3]+ m/z 232 and [M+H-CH3]+ m/z 217. After completely losing the substituent groups, aeries of fragment ions including m/z 189, 161, 133 and 105 wasroduced through the successive loss of CO. The ions [M+H]+ m/z47, [M+NH4]+ m/z 264, [2M+H]+ m/z 493 and [2M+NH4]+ m/z 510ere the common parent ions of each predominant product ion.

.2.7. Fragmentation of cnidilinCnidilin is a linear-type furocoumarin with [M+H]+ m/z 301 that

s substituted with an isopentenoxy group at C-5 and a methoxyroup at C-8. It produced [M+H-C5H8]+ m/z 233 through the neu-ral loss of a rearranged isopentenyl moiety, followed by loss ofhe methyl group, leading to the formation of characteristic ionM+H-C5H8-CH3]+ m/z 218. The isopentenoxy group of cnidilin

in and bergapten at CE 25 eV.

fragmented and produced [C5H9]+ m/z 69, which was also be foundin the MS/MS spectrum of imperatorin and isoimperatorin. The par-ent ions of two standards, [M+H]+ and [M+NH4]+, were monitoredand were simultaneously evident in the PREC spectra (Fig. 2). Theparents of [2M+H]+ m/z 601 and [2M+NH4]+ m/z 618 were alsosometimes observed.

3.2.8. Fragmentation of scopoletinScopoletin (6-methoxy-7-hydroxy-coumarin) was the only sim-

ple coumarin among the 10 standards. When the collision energywas 10 and 25 eV, the product ion spectra of [M+H]+ m/z 193 wascharacterized by the first loss of CH3 and CO. However, when thecollision energy was increased to 40 eV, the [M+H]+ ion at m/z 193lost CH3OH to generate a product ion at m/z 161. Therefore, therepresentative fragment ions of scopoletin were [M-CH3]+ m/z 178,[M-CO]+ m/z 165, [M-CH3OH]+ m/z 161, [M-CH3-CO]+ m/z 150, [M-2CO]+ m/z 137, [M-CO-CH3OH]+ m/z 133, [M-CH3-2CO]+ m/z 122,[M-2CO-CH3OH]+ m/z 105, [M-CH3-3CO]+ m/z 94 and [M-CH3-4CO]m/z 66. The fragmentation pathways of scopoletin are presented inFig. 3. In the PREC spectra of each product ion, [M+H]+ m/z 193and [M+NH4]+ m/z 210 were monitored, but [2M+H]+ m/z 385 and[2M+NH4]+ m/z 402 were only found occasionally. The PREC spec-trum with a precursor ion at m/z 193 is shown in Fig. 2.

3.3. Fragmentation rules deduced from pure standards

(1) The distinctive fragmentation pattern of simple coumarins andlinear-type furocoumarins was the successive loss of CO. More-over, both the nature of the substituent and the position havean effect on the fragmentation patterns [30].

(2) Most of the linear-type furocoumarins substituted with isopen-tenoxy and deformed isopentenoxy groups easily lost theirside chains through the formation of an intermediate ion with

an eight-membered ring [19]. In the case of the linear-typefurocoumarins substituted with a single isopentenoxy groupor deformed isopentenoxy groups, most of them fragmentedto generate 8-hydroxypsoralen or 5-hydroxypsoralen with m/z203 [30].

W. Yang et al. / J. Chromatogr. A

Fig. 5. The total ion chromatograms (TIC) of the 10 standards (A) and the extract ionchromatograms (XIC) of multiple ion monitoring (MIM) chromatograms of the 10specified ions (B). The 41 compounds have been presented in these chromatograms,which is consistent with Table 1.

1217 (2010) 4587–4600 4595

(3) A series of distinctive fragment ions at m/z 203, 175, 147,119 and 91 could be used to identify the skeleton of 8-hydroxypsoralen or 5-hydroxypsoralen. It is worth noting thatthe relative abundances of m/z 175 and 119 were generallyweaker, so these two fragment ions could not be found.

(4) With the linear-type furocoumarins substituted with a singleoxysubstituent group, the fragment ion at m/z 185 was diag-nostic for identification of the substituent group at C-8.

(5) Based on the MS/MS spectra of imperatorin, isoimperatorinand cnidilin, the fragment ion of an isopentenyl group was atm/z 69. The product ions of the deformed isopentenyl group ofoxypeucedanin were observed at m/z 59 and 85.

(6) In the spectra of PREC, both [M+H]+ and [M+NH4]+ of the10 standards could be monitored simultaneously regardlessof what fragment ions were used as the precursor. In thissense, with the great relevance of the ammonium adduct[M+NH4]+ and the proposed protonated molecular [M+H]+ ion,the molecular weight of unknown compounds could be accu-rately identified. The [2M+H]+ and [2M+NH4]+ could assist usin further determining the molecular weight.

3.4. RP-HPLC elution order of isomers

All of the peaks of standards in the mixed standard solutionwere unambiguously identified by comparison of retention timeand parent and fragment ions with standards in the MIM-IDA-EPIspectra. The chromatograms of the mixed standard solutions areshown in Fig. 5(A). From the chromatograms, certain tendencies ofthe isomers regarding the elution order were observed.

Xanthotoxin and bergapten, and imperatorin and isoimpera-torin are two pairs of isomers that are all substituted with a singleoxysubstituent group. The substituent groups of xanthotoxin andimperatorin were at C-8, and those of bergapten and isoimperatorinwere at C-5. The retention times of xanthotoxol and bergapten were23.7 and 28.3 min, respectively, which suggested that the polarityof xanthotoxol was higher than that of bergapten. Meanwhile, theretention time of imperatorin was less than that of isoimperatorin.It was deduced that with the linear-type furocoumarins substitutedwith the same single oxysubstituent group, the retention timesof linear-type furocoumarins substituted with an oxysubstituentgroup at C-8 were less than those with substitution at C-5. Thisspecific rule could be reliably and effectively used to identify theposition of the substituent group when a pair of linear-type furo-coumarin isomers substituted with the same single oxysubstituentgroup were found.

3.5. Analysis of the extract of Radix Glehniae

3.5.1. Analytical strategyThus far, the furanocoumarin biosynthetic pathway has not been

well characterized [31]. However, according to known informa-tion on coumarins and the hypothesis that plants in one familycould have the same compounds, coumarins possibly existing inRadix Glehniae was assumed. By analyzing the structures of thosecoumarins, we defined the predicted analytical ions. A combina-tion of MIM-IDA-EPI and PREC-IDA-EPI was chosen to identify theunknown coumarins with the monitored ions.

First, the MIM-IDA-EPI mode was used to find fragment ionsand retention times of coumarins from the predicted ions. Accord-ing to the spectrum of each transition, some predicted ions wereprecluded when the compounds found with the transitions did not

have the distinctive fragmentation pattern of successive losses ofCO. After screening the predicted ions, 10 transitions were keptand analyzed by the PREC-IDA-EPI mode. The extracted ion chro-matograms (XIC) of the 10 MIM transitions are shown in Fig. 5(B).Comparing the fragment ions of unknown coumarins from one

4596 W. Yang et al. / J. Chromatogr. A 1217 (2010) 4587–4600

ombin

tcfTpicMcmcct

3e

obxttaaiasda

3

s

glucoside.

Fig. 6. The process of identification with the c

ransition with each other, it was easy to find some unknownoumarins with the same structure of fragment ions, which laid theoundation for further identification of the unknown coumarins.he PREC-IDA-EPI mode was applied to provide information on thearent ions, some fragment ions and the retention times of 10 spec-

fied ions, so that the molecular weight of the unknown coumarinsould be confirmed, the information of fragment ions from theIM-IDA-EPI mode could be supplemented and the retention times

ould be further verified. Adequate data including retention time,olecular weight and fragment ions from the 10 monitored ions

ould be used to accurately identify the structure of unknownoumarins. The process of identification with the combined use ofhe two modes is presented in Fig. 6.

.5.2. Structural characterization of coumarins in Radix Glehniaextract

In the chromatograms of Radix Glehniae samples, the peaksf compounds 8, 12, 21, 22, 26, 28, 32, 36, 38 and 40 coulde unambiguously identified as scopoletin, xanthotoxol, psoralen,anthotoxin, isoimpinellin, bergapten, oxypeucedanin, impera-orin, cnidilin and isoimperatorin by comparing their retentionimes, molecular weights and fragment ions with standards. Bynalyzing the ions detected in the two scan modes of MIM-IDA-EPInd PREC-IDA-EPI, the other unknown coumarins were tentativelydentified. The retention times, molecular weights, fragment ionsnd parent ions of the 41 compounds plausibly identified are pre-ented in Table 1, and their structures are shown in Fig. 1. For a clearescription, we narrated the process of structural characterizationccording to the 10 monitored ions as follows.

.6. Analysis of precursor ion of m/z 163

With the monitored ion of m/z 163 used in the two modes, thetructure of compounds 1 and 7 were detected. In the PREC spec-

ation use of MIM-EPI-IDA and PREC-EPI-IDA.

trum of [M+H]+ m/z 163, [M+NH4]+ m/z 180 and [2M+H]+ m/z 325showed compound 7 have a molecular weight of 162 Da. Combinedwith the fragment ions, such as [M+H-CO]+ m/z 135, [M+H-CO2]+

m/z 119, [M+H-2CO]+ m/z 107, [M+H-CO2-CO]+ m/z 91 and [M+H-3CO]+ m/z 79, compound 7 was identified as umbelliferone, whichis the precursor of bergapten and is known to be produced in violetand white cell cultures of G. littoralis [29]. The proposed MS frag-mentation pathway of compound 7 is shown in Fig. 7. The fragmentions of compound 1 were similar to umbelliferone. The molec-ular weight of compound 1 of 324 Da was identified by [M+H]+

m/z 325 and [M+NH4]+ m/z 342, corresponding to the direct addi-tion of Glu (162 Da) to umbelliferone. In previous reports, the mostprobable sugars of known coumarin-O-glycosides are glucose andapiose [32]. According to the literature, 162 Da is related to theloss of a hexose and 132 Da is associated with the loss of a pentose[33]. Therefore, the compound 1 was identified as umbelliferone-

Fig. 7. The proposed MS fragmentation pathway of representative compounds.

W.Yang

etal./J.Chrom

atogr.A1217 (2010) 4587–4600

4597

Table 1Characterization of coumarins by HPLC–MS from Radix Glehniae.

No.a Rt min Tentatively identificationb M.W.c (Da) Parent ions (m/z) Fragment ions (m/z)

1 5.53 Umbelliferone-glucoside 324 [M+H]+ 325 [M+NH4]+ 342 325, 163, 135, 1072 6.37 Scopoletin-glucoside� 354 [M+H]+ 355 [M+NH4]+ 372 355, 193, 178, 150, 137, 133, 122, 105, 943 7.14 Scopoletin-apiosyl-glucoside� 486 [M+H]+ 487 [M+NH4]+ 504 487, 355, 193, 161, 150, 133, 122, 944 9.98 Xanthotoxol-triglucoside� 688 [M+H]+ 689 [M+NH4]+ 706 689, 527, 365, 203, 175, 147, 131, 129, 119, 101, 915 11.58 Xanthotoxol-diglucoside� 526 [M+H]+ 527 [M+NH4]+ 544 527, 365, 203, 175, 147, 131, 119, 916 12.41 8-Methoxy-5-hydroxypsoralen-glucopyranoside� or

5-methoxy-8-hydroxypsoralen-glucopyranoside�556 [M+H]+ 557 [M+NH4]+ 574 557, 365, 233, 218, 173, 162, 134

7 12.64 Umbelliferone� 162 [M+H]+ 163 [M+NH4]+ 180 [2M+H]+ 325 163, 135, 119, 107, 91, 798* 12.91 Scopoletin 192 [M+H]+ 193 [M+NH4]+ 210 [2M+H]+ 385 [2M+NH4]+ 402 193, 178, 161, 150, 137, 133, 122, 105, 94, 669 13.46 Leptophyllin-2glucoside� 586 [M+H]+ 587 [M+NH4]+ 604 587, 425, 263, 203, 191, 175, 147, 91

10 14.83 Xanthotoxol-glucoside 364 [M+H]+ 365 [M+NH4]+ 382 365, 203, 175, 147, 131, 129, 119, 101, 9111 15.37 Rutaretin� 262 [M+H]+ 263 [M+NH4]+ 280 263, 245, 217, 203, 187, 175, 147, 119

12* 16.34 Xanthotoxol 202 [M+H]+ 203 [M+NH4]+ 220 203, 175, 147, 131, 129, 119, 101, 9113 17.16 6-(3,3-Dimethylallyl)-7-hydroxycoumarin-diglucoside� 554 [M+H]+ 555 [M+NH4]+ 572 555, 393, 231, 175, 147, 119, 9114 17.65 5-Methoxy-8-hydroxypsoralen� 232 [M+H]+ 233 [M+NH4]+ 250 233, 218, 190, 173, 162, 145, 134, 106, 7815 18.08 Leptophyllin� 262 [M+H]+ 263 [M+NH4]+ 280 263, 245, 217, 203, 191, 189, 147, 9116 18.33 Marmesinin 408 [M+H]+ 409 [M+NH4]+ 426 409, 247, 229, 213, 185, 175, 147, 119, 9117 19.75 6-(3,3-Dimethylallyl)-7-hydroxycoumarin–apiosyl-

glucoside�524 [M+H]+ 525 [M+NH4]+ 542 525, 393, 231, 175, 147

18 20.54 6-(3,3-Dimethylallyl)-7-hydroxycoumarin-glucoside� 392 [M+H]+ 393 [M+NH4]+ 410 393, 231, 175, 147, 119, 9119 20.83 8-Methoxy-5-hydroxypsoralen� 232 [M+H]+ 233 [M+NH4]+ 250 233, 218, 190, 173, 162, 134, 10620 22.69 Marmesin 246 [M+H]+ 247 [M+NH4]+ 264 247, 229, 213, 185, 175, 147, 119, 91

21* 23.33 Psoralen 186 [M+H]+ 187 [M+NH4]+ 204 187, 143, 131, 115, 10322* 23.67 Xanthotoxin 216 [M+H]+ 217 [M+NH4]+ 334 217, 202, 189, 185, 174, 161, 146, 131, 118, 9023 24.30 Byak-angelicin� 334 [M+H]+ 335 [M+NH4]+ 352 335, 233, 218, 173, 162, 145, 13424 25.08 Oxypeucedanin hydrate� 304 [M+H]+ 305 [M+NH4]+ 322 [2M+H]+ 609 [2M+NH4]+ 626 305, 203, 175, 159, 147, 131, 119, 103, 91, 5925 25.76 8-(2-Hydroxy-3-methylbut-3-enoxy)-psoralen� 286 [M+H]+ 287 [M+NH4]+ 304 287, 269, 227, 203, 201, 173, 157, 147, 145, 129, 117, 91, 89

26* 27.04 Isopimpinellin 246 [M+H]+ 247 [M+NH4]+ 264 [2M+H]+ 493 [2M+NH4]+ 510 247, 232, 217, 189, 161, 133, 10527 27.99 Pabulenol� 286 [M+H]+ 287 [M+NH4]+ 304 287, 269, 227, 203, 201, 175, 173, 159, 157, 147, 145, 131,

12928* 28.23 Bergapten 216 [M+H]+ 217 [M+NH4] +334 217, 202, 174, 161, 146, 131, 118, 9029 30.11 Prangenin� 286 [M+H]+ 287 [M+NH4]+ 304 287, 203,175, 159, 147, 131, 9130 31.17 Byak-angelicol� 316 [M+H]+ 317 [M+NH4]+ 334 317, 233, 218, 190, 189, 175, 173, 162, 13431 31.58 Isooxypeucedanin� 286 [M+H]+ 287 [M+NH4]+ 304 287, 202, 174, 146,118, 90

32* 31.93 Oxypeucedanin 286 [M+H]+ 287 [M+NH4]+ 304 287, 203, 175, 159, 147, 131, 119, 103, 91, 5933 33.58 4′′-Hydroxyimperatorin� 286 [M+H]+ 287 [M+NH4]+ 304 287, 269, 239, 203 187, 159, 147, 13134 33.79 7-O-(3,3-Dimethylallyl)-scopoletin 260 [M+H]+ 261 [M+NH4]+ 278 261, 193, 178, 161, 150, 137, 133, 122, 9435 36.85 8-Geranyloxypsoralen 338 [M+H]+ 339 [M+NH4]+ 356 339, 203, 175, 159, 147

36* 37.08 Imperatorin 270 [M+H]+ 271 [M+NH4]+ 288 [2M+H]+ 541 [2M+NH4]+ 558 271, 203, 185, 175, 159, 157, 147, 131, 119, 91, 6937 38.01 6-(3,3-Dimethylallyl)-7-hydroxycoumarin 230 [M+H]+ 231 [M+NH4]+248 231, 187, 175, 147, 131, 119, 103, 91, 77, 69

38* 39.30 Cnidilin 300 [M+H]+ 301 [M+NH4]+ 318 [2M+H]+ 601 [2M+NH4]+ 618 301, 233, 218, 190, 173, 162, 145, 134, 106, 78, 6939 41.36 Phellopterin 300 [M+H]+ 301 [M+NH4]+ 318 [2M+H]+ 601 [2M+NH4]+ 618 301, 233, 218, 190, 173, 162, 145, 134, 121, 117, 106, 78

40* 43.36 Isoimperatorin 270 [M+H]+ 271 [M+NH4]+ 288 [2M+H]+ 541 [2M+NH4]+ 558 271, 203, 175, 159, 147, 131, 119, 91, 6941 50.26 Bergaptin 338 [M+H]+ 339 [M+NH4]+ 356 339, 203, 175, 159, 157, 147, 131, 129

a The 10 standards have been marked by *.b The components not reported in Radix Glehniae have been marked by� .c M.W. = molecular weight.

4598 W. Yang et al. / J. Chromatogr. A 1217 (2010) 4587–4600

-hydr

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Fig. 8. The EPI spectra of cnidilin, phellopterin, 5-methoxy-8

.7. Analysis of precursor ion of m/z 233

Using m/z 233 as the monitored ion, the structural characteri-ation of compounds 6, 14, 19, 23, 30, 38 and 39 was completed.ompound 38 was identified as cnidilin. Compound 39 had theame molecular weight of 300 Da as compound 38. In addition,imilar fragmentation behavior and the characteristic ions at m/z33, 218, 190, 173, 162, 134 and 106 were also present in thePI spectra of compound 39, so compound 39 was unquestionablydentified as phellopterin. Cnidilin and phellopterin are a pair of iso-

ers substituted with the same isopentenoxy and methoxy groups,ut at different positions, as shown in Fig. 1. The retention timesf cnidilin and phellopterin, at 39.3 and 41.4 min, showed that thetronger hydrophobicity caused by the isopentenoxy group at C-8esulted in a longer retention time. This could be used to identifyhe position of the more strongly hydrophobic group when theinear-type furocoumarins were substituted two oxysubstituentroups. The [M+H]+ of cnidilin and phellopterin produced the

ame fragment ions of [M+H-C5H8]+ m/z 233 and [M+H-C5H8-CH3]+

/z 218. However, the relative abundance of the two fragmentons was different at a CE value of 25 eV: [M+H-C5H8]+ > [M+H-5H8-CH3]+ (phellopterin) and [M+H-C5H8]+ < [M+H-C5H8-CH3]+

cnidilin). This result demonstrated that a methoxyl group at C-

oxypsoralen and 8-methoxy-5-hydroxypsoralen at CE 25 eV.

8 made it easier to lose a methyl group from m/z 233 than amethoxyl group at C-5. The EPI spectra of the two compoundsat CE 25 eV are shown in Fig. 8. Based on the changes of rel-ative abundance of m/z 233 and 218, the position of the morestrongly hydrophobic constituent group also could be tentativelyidentified.

Compounds 14 and 19 had the same molecular weight of 232 Daand characteristic fragment ions at m/z 233, 218, 190, 162, 134and 106. Comparing the EPI spectra of compounds 14 and 19with that of cnidilin, it was found that the EPI spectrum of cni-dilin contained all of the characteristic ions of compounds 14 and19. Therefore, we deduced that the structures of compounds 14and 19 might be 5-methoxy-8-hydroxypsoralen or 8-methoxy-5-hydroxypsoralen, the same structure as the fragment ions ofcnidilin and phellopterin after losing isopentenyl with an inter-mediate ion having an eight-membered ring. According to theretention times of 17.6 and 20.8 min, we believed methyl as ahydrophobicity group should be at C-8 for compound 19. There-

fore, compound 14 might be 5-methoxy-8-hydroxypsoralen andcompound 19 might be 8-methoxy-5-hydroxypsoralen. Moreover,the relative abundance of [M+H]+ m/z 233 and [M+H-CH3]+ m/z 218at CE 25 eV were consistent with the deduction that a methoxylgroup at C-8 made it easier to eliminate methyl from m/z 233 than

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methoxyl group at C-5. The EPI spectra of compounds 14 and 19t CE 25 eV are shown in Fig. 8.

Comparing the fragment ions of compound 6 with compounds4 and 19, similar fragment ions at m/z 233, 218, 173, 162 and34 were found in the five compounds. It was deduced that com-ound 6 could have the skeleton of 5-methoxy-8-hydroxypsoralenr 8-methoxy-5-hydroxypsoralen. Combined with informativeragment ions such as [M+H]+ m/z 557, [M+H-162]+ m/z 365nd [M+H-162-162]+ m/z 203, compound 18 was preliminar-ly identified as 5-methoxy-8-hydroxypsoralen-diglucoside or-methoxy-5-hydroxypsoralen-diglucoside. The structure couldot be further identified due to its limited fragment ions.

Based on the parent ion of compound 23, m/z 335 and 352,he molecular weight was identified as 334 Da. The fragment ionsere insufficient but similar to cnidilin and phellopterin. With theeak fragment ions [M+H-H2O]+ m/z 317 and [M+H-2H2O]+ m/z

99, compound 23 was tentatively identified as byak-angelicin,hich has been isolated from Angelica Apaensis and Radix Angel-

cae Dahuricae. In the PREC spectrum of the ion m/z 233, m/z 317nd 334 demonstrated the molecular weight of compound 30 toe 316 Da. The characteristic fragment ions of compound 30 werehe same as cnidilin and phellopterin, such as m/z 233, 218, 190,89, 173, 162 and 134. The distinctive fragment ion [C5H9O]+ m/z5 implied that compound 30 might possess the same deformed

sopentenyl group as oxypeucedanin. Therefore, compound 30 wasentatively identified as byak-angelicol, which has also been iso-ated from Angelica Apaensis and Radix Angelicae Dahuricae.

.8. Analysis of precursor ion of m/z 263

Using m/z 263 as a precursor, the structures of compounds 9, 11nd 15 were identified. The molecular weight of compounds 11 and5 was 262 Da, which has not been reported in Radix Glehniae. Theragmentation pattern of compound 15 was deduced by the frag-

entation ions produced at different CE values. At a CE value of0 eV, the fragment ions [M+H-H2O]+ m/z 245, [M+H-C3H8O]+ m/z03 and [M+H-C4H8O]+ m/z 191 were produced. In the EPI spec-rum of CE 25 eV, the fragment ion of [M+H-H2O-CO2]+ m/z 217as generated. When the CE value was set at 40 eV, the fragment

ons of [M+H-C3H8O-2CO]+ m/z 147 and [M+H-C3H8O-4CO]+ m/z 91ere observed. The existence of m/z 203, 147 and 91 suggested that

ompound 15 could have the skeleton of 8-hydroxypsoralen or 5-ydroxypsoralen. Moreover, some fragmentation pathways werehe same as marmesin, such as [M+H-H2O]+ and [M+H-C4H8O]+.hus, compound 15 was plausibly identified as rutaretin or lep-ophyllin. The fragment ions of compound 11 were similar toompound 15 but without the characteristic product ion at m/z 191.he variation in the major product ion was possibly due to the dif-erent substituent position of the hydroxyl group. According to thelution order rule of linear-type furocoumarins substituted withhe same single oxysubstituent groups, the retention time of com-ound 11 (15.3 min) and compound 15 (18.1 min) suggested thatompound 11 might be rutaretin and that compound 15 might beeptophyllin. The fragmentation pathway of compound 15 is shownn Fig. 7. Considering the fragment ions of [M+H-162]+ m/z 425,M+H-162-162]+ m/z 263 and [M+H-162-162-C4H8O]+ m/z 191 andhe similar fragmentation pathways to leptophyllin, compound 9as confirmed to leptophyllin-diglucoside.

.9. Analysis of precursor ion of m/z 287

The characterization of compounds 25, 27, 29, 31, 32 and 33as completed when m/z 287 was used as the monitored ion. It

s worth noting that compounds 25, 27, 29, 32 and 33 could alsoe found in the PREC spectrum using m/z 203 as the monitored

on. The molecular weights of the six compounds were all 286 Da.

1217 (2010) 4587–4600 4599

Compounds 29 and 32 had the same characteristic fragmentationpathway. Compound 32 was identified as oxypeucedanin, and com-pound 29 was tentatively identified as prangenin, the isomer ofcompound 32 that is substituted with the same deformed isopen-tenoxy group as oxypeucedanin, but at C-8. The retention time ofcompound 32 was longer than compound 29, which confirmed tothe polarity rule of linear-type furocoumarin isomers substitutedwith a single oxysubstituent group.

With the same fragment ions, compounds 25 and 27 were pre-liminarily identified as a pair of isomers substituted at differentpositions. Based on a series of fragment ions at m/z 203, 175, 147and 91, it was deduced that the skeletons of 8-hydroxypsoralenor 5-hydroxypsoralen were present in compounds 25 and 27. Theexistence of fragment ions of [M+H-C5H10O]+ 201, [M+H-C5H10O-CO]+ 173, [M+H-C5H10O-2CO]+ 145 and [M+H-C5H10O-4CO]+ 89was possibly due to the different type of substituent. The fragmentions of the side chain, including [M+H-H2O]+ 269 and [M+H-H2O-C3H6]+ 227, suggested that the side chain might containone hydroxyl group. According to the retention time of the twocompounds, compounds 25 and 27 were preliminary identifiedas 8-(2-hydroxy-3-methylbut-3-enoxy)-psoralen and pabulenol,which has been isolated from Radix Angelicae Dahuricae.

In the EPI spectrum of compound 33, a series of fragmentions at m/z 203, 159, 147, 131, 119 and 91 was found. Thisresult implied that the skeleton of 8-hydroxypsoralen or 5-hydroxypsoralen was present in compound 33. Fragmentationof the side chain produced [M+H-H2O]+ m/z 269, [M+H-C3H6O]+

m/z 229, [M+H-C4H8O] m/z 215 and [M+H-C5H8O]+ 203. Com-paring the fragment ions of the side chain from compound 33with the isopentenyl group, the structure of the side chain wasidentified as 4-hydroxylisopentenyl. In previous reports [34], 4′′-hydroxyimperatorin-4′′-O-�-d-glucopyranoside has been isolatedfrom G. littoralis, suggesting that this type of substituent group, 4′′-hydroxylisopentenyl, is known. With the fragment ion at m/z 185,compound 33 was tentatively identified as 4′′-hydroxyimperatorin.

Based on the characteristic fragment ions of compound 31,such as [M+H-C5H9O]+ m/z 202, [M+H-C5H9O-CO]+ m/z 174,[M+H-C5H9O-2CO]+ m/z 146, [M+H-C5H9O-3CO]+ m/z 118 and[M+H-C5H9O-4CO]+ m/z 90, it was deduced that compound 31could not produced the structure of hydroxypsoralen that resultedfrom the formation of an intermediate ion with an eight-memberedring. Moreover, considering the molecular weight of 286 Da and thecharacteristic fragment ions without m/z 185, compound 31 wasidentified as isooxypeucedanin [35].

The identification of other analytical ions (m/z 193, 231, 203,247) has been put into supplementary material and the figuresinvolving in these ions have been put into Fig. S2.

Up to now, there were some reports about the fragment ruleof coumarins [18,19,21,29,30,35]. Comparing with these reports,fragmentation rules (4) and (6) in the present study are firstly sum-marized. Meanwhile, by the combination of these two scan modes,some coumarins and coumarin glycosides were found in RadixGlehniae for the first time, which have been marked in Table 1.

4. Conclusions

In this study, a highly sensitive and effective LC–MS method forthe qualitative analysis of trace coumarins in Radix Glehniae hasbeen developed using a combination of the MIM-IDA-EPI and PREC-IDA-EPI scan modes. This is the first report on the chemical analysis

of TCM using these two modes. It is also the first structural identi-fication of the trace coumarins in Radix Glehniae with LC–MS/MS.This study has demonstrated the unprecedented advantage of thecombination of these two scan modes. The MIM-IDA-EPI mode issensitive, and no pre-acquisition of MS/MS spectra of the parent

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on is required due to the same precursor ion and product ion. TheREC-IDA-EPI mode was used to provide information on the par-nt ions, fragment ions and retention times of specified ions sohe molecular weights of unknown coumarins and their glycosidesould be identified. The information on the fragment ions from theIM-IDA-EPI mode could be supplemented, and the retention time

ould be verified. Therefore, the characterization of trace coumarinsas become very easy and accurate by the combined use of the twoodes and may play an important role in controlling the quality ofedicinal herbs.

cknowledgement

We thank the financial support from Department of Science andechnology of Hebei Province of China (09276423D) for this work.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.chroma.2010.04.076.

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