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Analytica Chimica Acta 681 (2010) 33–40

Contents lists available at ScienceDirect

Analytica Chimica Acta

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

new approach for simultaneous screening and quantification of toxicyrrolizidine alkaloids in some potential pyrrolizidine alkaloid-containinglants by using ultra performance liquid chromatography–tandem quadrupoleass spectrometry

an Zhoua, Na Lib, Franky Fung-Kei Choia, Chun-Feng Qiaoa, Jing-Zheng Songa, Song-Lin Lia,in Liua, Zong-Wei Caic, Peter P. Fud, Ge Linb,∗, Hong-Xi Xua,∗

Hong Kong Jockey Club Institute of Chinese Medicine, Shatin, New Territories, Hong Kong, ChinaSchool of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong, ChinaDepartment of Chemistry, Hong Kong Baptist University, Hong Kong, ChinaNational Center for Toxicological Research, Jefferson, AR 72079, USA

r t i c l e i n f o

rticle history:eceived 2 July 2010eceived in revised form 6 September 2010ccepted 8 September 2010vailable online 16 September 2010

eywords:

a b s t r a c t

A rapid, but sensitive and selective method for simultaneous screening and quantification of toxicpyrrolizidine alkaloids (PAs) by ultra performance liquid-chromatography (UPLC) coupled with tandemmass spectrometry (MS/MS) on a tandem quadrupole mass spectrometer (TQ-MS) is described. This wasaccomplished by incorporating the precursor ion scan (PIS) acquisition and multiple reaction monitor-ing (MRM) acquisition in the same UPLC–MS/MS run. Notably, the developed PIS approach for detectingtwo pairs of characteristic product ions at m/z 120/138 or 168/150, allowed specific identification oftoxic retronecine and otonecine types PAs. This PIS method is highly sensitive with over 10-fold sensi-

yrrolizidine alkaloids (PAs)PLC–TQ-MSrecursor ion scan (PIS)ultiple reaction monitoring (MRM)

arasenecio speciesenecio species

tivity improvement upon previously published LC–MS method. Moreover, this new approach is suitablefor high sample throughput and was applied to the screening and quantifying toxic PAs in 22 samplescollected from seven Parasenecio species and four Senecio species. In addition, coupling the MRM withPIS approach generated quantitative results equivalent to those obtained by conventional MRM-onlyapproach. This coupled MRM with PIS approach could provide both qualitative and quantitative results

titive

without the need of repe

. Introduction

With the worldwide rapidly growing interests in the use ofatural products as medical remedies, dietary supplements and

unctional foods, more and more cases of natural products poi-onings have been reported. It is now becoming more generallyecognized that some natural products are toxic and may causeevere adverse effects including fetal problems in human. Amonghe natural toxins, pyrrolizidine alkaloids (PAs) are probably one ofhe most important groups, since most of the naturally occurringAs are known to be hepatotoxic and tumorigenic in humans [1–6].

As are found in a wide variety of plant species, which accountor about 3% of the world’s flowering plants. PAs are esters ofhree types of necines: retronecine (including its 7-� enantiomer),tonecine, and platynecine. The former two types contain unsatu-

∗ Corresponding authors.E-mail addresses: linge@cuhk.edu.hk (G. Lin), xuhongxi@hkjcicm.org (H.-X. Xu).

003-2670/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2010.09.011

analyses.© 2010 Elsevier B.V. All rights reserved.

rated necine bases and are highly hepatotoxic with tendencies tocause hemorrhagic necrosis and veno-occlusion in the liver, whilethe platynecine-type PAs with a saturated necine base are gener-ally suspected to be less or non-toxic [5–7]. The majority of thenaturally occurring PAs belong to the unsaturated toxic PAs andare mainly from three families: Asteraceae (Compositeae), Boragi-naceae and Fabaceae (Leguminosae) [2,3,6–8]. PA poisonings havebeen reported in several countries. The largest known disaster ofhuman PA poisoning occurred in 1975 in Northwest Afghanistan,and was associated with the consumption of bread made fromwheat flour contaminated with PAs. Examination of 7200 inhab-itants from the affected villages showed evidence of liver diseasein 22.6% [9].

Due to the safety hazards, the World Health Organization issued

the Health and Safety Guide on PAs in 1989 [10]. Some coun-tries, such as Germany, Austraila and South Africa have set upall-round guidelines or regulations to limit the amount of dailyintake or restrict the sale of PAs-containing pharmaceuticals soas to govern PAs exposure and prevent human posioning [11,12].

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4 Y. Zhou et al. / Analytica C

owever, these regulations are all established based on the casetudies and only appropriate to individual PA-containing products,ut cannot be considered as a general rule applied universally toll PA-containing herbal products. On the other hand, in Mainlandhina and Hong Kong, there have been no regulations to date forhe use of PA-containing Chinese medicinal (CM) herbs and CM-ased products, and no systematic studies have been conductedo determine how many commonly used CM herbal plants con-ain toxic PAs. As such, although more than 6000 plants have beendentified worldwide to contain PAs [2–4,13], only 11 of the 49 PA-ontaining CM herbal plants [2,5] were identified by researchers inhina [5]. Therefore, it is important to establish a reliable and sen-itive screening platform for the identification and quantificationf toxic PAs in the CM herbs and their related products.

The genera Senecio and Parasenecio (Cacalia) both belong to theamily Compositae. It is well accepted that plants from the Senecioenus are known sources of hepatotoxic PAs, with Senecio scan-ens being the most well-known species. In China, there are about60 Senecio species in which 10 of them such as S. nemorensis,. laetus, S. hoi, S. kirilowii, and S. campestris are used as medici-al herb for the treatment of enteritis, conjunctivitis, respiratoryract infections and bacterial diarrhea [14]. Besides, in the South-est mountain area in China a total of 51 species of the genus

arasenecio are distributed, from which 26 species are commonlysed as folk medicine for invigorating the circulation of blood,reating rheumatoid arthritis and other diseases [15]. Despite theiridespread usage, little information on the chemical composition

f Parasenecio genus plants is available, except for a phytochemi-al investigation discussing the presence of PAs at trace levels inarasenecio plants [16].

PAs are normally detected by liquid chromatography mass spec-rometry (LC–MS) [17–22], predominantly by targeted detectionf known PAs. The major shortcoming of the existing targeted MSethods for compounds measurement is that although quantita-

ive information can be obtained, no qualitative information cane derived unless separate LC–MS/MS experiments are performed.heoretically, the modern linear ion trap MS (LIT-MS) allows theollection of MS, MRM and MS/MS scan in the same analytical run23,24], the duty cycle of the instruments when switching between

S and MS/MS modes requires about 2–3 s. When coupled to highesolution chromatography such as UPLC, with peaks width typ-cally in the range of several seconds, inadequate data points toccurately define the peak will be occurred resulting in poor quan-itation.

The tandem quadrupole mass spectrometry (Xevo TQ-MS) canrovide enhanced spectral LC–MS/MS data acquisition capabilities,llowing accurate definition of the very narrow peaks producedy UPLC. Typically a 2-wide peak will have more than 10 pointsspecially in multiple-scan functions. As the collision cell is contin-ously filled with collision gas, the instrument can rapidly switchetween MS and MS/MS scan in the same analytical run, allowingoth simultaneous quantitation using MRM and characterizationsing enhanced scans (either full scan of MS and MS/MS or pre-ursor ion/product ion scan). The precursor ion scan (PIS) modenables specific detection of a class of compounds, which generateproduct ion of interest in MS/MS spectra.

In the present study, we proposed a new approach to achieveimultaneous screening, identification and quantification of PAs in2 samples of seven Parasenecio species and four Senecio speciesy UPLC coupled with a TQ-MS. Instead of using MRM + MS/MS orRM + MS, we combined the conventional MRM with PIS exper-

ments. In this approach, the toxic unsaturated necine PAs werecreened out by PIS with MS/MS, where the characteristic fragmentons belonging to the retronecine and otonecine type of PAs at spe-ific m/z 120/138 or 168/150 (Fig. 1) were produced after collisionnduced dissociation (CID). Meanwhile, the amount of seven PAs

a Acta 681 (2010) 33–40

including monocrotaline (1), senkirkine (2), riddelliine (3), retror-sine (4), seneciphylline (5), clivorine (6) and senecionine (7) werequantified in the 22 potential PAs-containing plant samples byMRM in the same UPLC run.

2. Experimental

2.1. Chemicals and reagents

Acetonitrile (ACN, HPLC–MS grade) was purchased from FishierScientific UK (Loughborough, UK) and formic acid (spectroscopygrade) was purchased from Sigma–Aldrich (St. Louis, MO, USA).Pure water was prepared from a Milli-Q SP Reagent Water sys-tem (Millipore, Bedford, MA, USA). Hydrochloric acid (AnalyticalReagent grade) was purchased from Merck (Darmstadt, Ger-many), and n-hexane (AR grade), dichloromethane (AR grade) andammonia solution (AR grade) were purchased from BDH-VWR(LeicestersHire, UK). The sulfonic acid Oasis MCX 60 �m (LP) waspurchased from Waters (Milford, MA, USA).

2.2. Sample preparation

2.2.1. Reference standardsThe reference compounds monocrotaline (1) and retrorsine

(4) were purchased from Sigma–Aldrich Chemical Co. (USA).Senkirkine (2), seneciphylline (5) and senecionine (7) were pur-chased from ChromaDex (Irvine, CA, USA). Riddelliine (3) was a giftfrom Dr. Po-Chuen Chan and clivorine (6) (purity >98%) was iso-lated from Ligularia hodgsonii in our laboratory using the previouslyestablished isolation method [25]. The purity of these compoundswas determined to be higher than 98% by HPLC–MS analysis.

2.2.2. Sample preparationThe potential PA-containing plants, including samples of seven

Parasenecio species and four Senecio species were collected fromdifferent localities in China (as shown in Table 1). All herbal mate-rials were identified by Dr. Qiao Chun Feng (Chinese MedicineLaboratory of Hong Kong Jockey Club of Institute of ChineseMedicine, HKJCICM), and all voucher specimens were deposited atthe Chinese Medicine Laboratory of HKJCICM. The dry plant sam-ples were grounded to fine powder by a pulverizer, and 0.2 g ofpowder of each sample was soaked in a 10 mL capped conical flaskwith 6.0 mL 0.05 M hydrochloric acid for 30 min. After soaking, thesample was extracted under supersonic washer (50 Hz) for 1 h, fol-lowed by filtration and extraction of the residue twice with 6.0 mL0.05 M hydrochloric acid. The three extracts were combined andapplied to MCX SPE cartridges. The cartridges were first condi-tioned with 6 mL methanol and 12 mL double-distilled water. Theflow rate was adjusted to 1 drop in 3 s. The sample solution wastransferred into the cartridge and then washed by 6 mL 0.05 Mhydrochloric acid followed by 6 mL methanol. Finally, the cartridgeswere eluted with 12 mL of methanol–10% ammonia (3:1, v/v). Theeluents were combined and evaporated to dryness under reducedpressure at 40 ◦C by rotary evaporator. The residue was dissolvedin 1 mL methanol and filtered with 0.2 �m PTFE membrane filter toform the test solution. An aliquot of each filtrate (2 �L) was injectedinto the UPLC/MS instrument for analysis. Seven reference com-pounds were dissolved in methanol to give stock solutions withconcentration of 500 ng mL−1.

2.3. Chromatography

UPLC analysis was performed on a Waters ACQUITY ultra perfor-mance LC system (Waters Corp., Milford, MA, USA), equipped witha binary solvent delivery system, an auto-sampler, and a PDA detec-tor. The chromatography was performed on a Waters Acquity UPLC

Y. Zhou et al. / Analytica Chimica Acta 681 (2010) 33–40 35

otone

Bwtl

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Fig. 1. Seven PAs standards belonging to the retronecine type and

EH C18 column (100 mm × 2.1 mm, 1.7 �m). The mobile phaseas composed of (A) 0.2% ammonia solution in water and (B) ace-

onitrile. The UPLC eluting conditions were optimized as follows:inear gradient from 5% to 40% B (0–8 min), linear gradient from 40%

able 1ollected crude materials of possible PAs containing plants.

Sample code Species

CMED-487-01 Parasenecio sinicusCMED-487-02 P. sinicusCMED-488-01 Parasenecio hastatusCMED-488-02 P. hastatusCMED-489-01 Parasenecio otopteryxCMED-490-01 Parasenecio ainsliiflorusCMED-491-01 Parasenecio ambiguusCMED-491-02 P. ambiguusCMED-492-01 Parasenecio phyllolepisCMED-493-01 Parasenecio bulbiferoidesCMED-495-01 Senecio vulgarisCMED-496-01 Senecio nemorensisCMED-496-02 S. nemorensisCMED-496-03 S. nemorensisCMED-497-01 Senecio scandensCMED-497-02 S. scandensCMED-497-03 S. scandensCMED-497-04 S. scandensCMED-497-05 S. scandensCMED-497-06 S. scandensCMED-497-07 S. scandensCMED-500-01 Senecio argunensis

cine type PAs obtained based on their characteristic product ions.

to 90% B (8–10 min), linear gradient from 90% to 5% B (10–11 min).The flow rate was 0.5 mL min−1. The column and the auto-samplerwere maintained at 35 ◦C and 10 ◦C, respectively. Each wash cycleconsisted of 200 �L strong solvent (80% acetonitrile) and 600 �L

Parts Location of collection

Whole plant Hubei, ShenlongjiaWhole plant HenanWhole plant HenanWhole plant Shanxi, Qinshui-LishanWhole plant Hubei, ShenlongjiaWhole plant Hubei, ShenlongjiaWhole plant HenanWhole plant Shanxi, Qinshui-LishanWhole plant Hubei, ShenlongjiaWhole plant Hubei, LichuanWhole plant Hubei, ShenlongjiaWhole plant Hubei, BadongWhole plant Shanxi, Qinshui-LishanWhole plant NeimengWhole plant Hubei, ShenlongjiaWhole plant Shanxi, Qinshui-LishanWhole plant GuangdongWhole plant AnhuiWhole plant Guangxi NanningWhole plant Hong Kong, Tai Mo ShanWhole plant Hong Kong, Tai PoWhole plant Shanxi, Qinshuilishan

36 Y. Zhou et al. / Analytica Chimic

Table 2The retention time (Rt.), MS, MS/MS fragment ion, cone voltage (CV) and collisionenergy (CE) of seven reference PAs.

No. Compounds Rt. (min) MS, m/z MS/MS, m/z CV (V) CE (eV)

1 Monocrotaline 3.10 326 120 40 252 Senkirkine 3.97 366 168 40 253 Riddelliine 4.65 350 120 45 26

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4 Retrorsine 5.22 352 120 53 285 Seneciphylline 5.71 334 120 40 256 Clivorine 5.96 406 168 37 257 Senecionine 6.42 336 120 40 25

f weak solvent (30% acetonitrile) and the injection volume of thetandards and sample was 2 �L.

.4. Mass spectrometry

Mass spectrometry was operated on a Waters Xevo TQ tan-em quadrupole mass spectrometer (Micromass MS Technologies,anchester, UK) using an ESI operated in positive ion mode. The

esolvation gas was set to 1000 L h−1 at temperature of 500 ◦C, andhe source temperature was set to 150 ◦C. The capillary voltage andxtractor voltage were set to 0.75 kV and 3 V, respectively. The col-ision gas flow was set at 0.15 mL min−1. Six MS/MS functions wereerformed in a single UPLC run; function 1 was the MRM mode, andhe transition channel was switched from the precursor ion to theroduct ion, and corresponding cone voltage and collision energyf each PA standard were clearly defined (as shown in Table 2) withhe dwell time at 0.025 s; function 2 was the PIS of ion at m/z 120,unction 3 was the PIS of ion at m/z 138, function 4 was the PIS ofon at m/z 168, function 5 was the PIS of ion at m/z 150 and func-ion 6 was the MS scan; the scan duration of function 2–6 was set at.18 s, and collision energy ramping from 25 to 40 V for PIS scans.

.5. Validation of quantitative method

.5.1. Calibration curvesThe calibration curves (7-point) were obtained using an exter-

al standard calibration for the seven analytes by injecting eacholution twice. The curves were then constructed by plotting theeak area versus the concentration of each analyte (Table 3).

.5.2. LOD and LOQThe stock solution containing seven reference compounds was

iluted to a range from 0.26 to 36 pg mL−1 of the reference PAs,nd the injection volume was 5 �L. The limits of detection (LOD)nd quantification (LOQ) under the present chromatographic con-itions were determined at a signal-to-noise ratio (S/N) of about 3nd 10, respectively (Table 3).

.5.3. Precision and accuracy

Intra- and inter-day variations were chosen to determine the

recision of the developed method. For the intra-day variabilityest, three concentration levels of the seven mixed standards wereeparately spiked into sample CMED-497-01 (Table 1) solutionsnd were extracted and analyzed in triplicates within one day. For

able 3egression equation, correlation coefficients, linearity ranges and limit of detection (LOD

No. Compounds Regression equation Linear range (

1 Monocrotaline Y = 675.4x − 607.3 0.1–502 Senkirkine Y = 1530.5x + 996.7 0.1–503 Riddelliine Y = 49259x + 5952 0.1–504 Retrorsine Y = 1536.1x + 1820 0.1–505 Seneciphylline Y = 407.1x + 7914 0.1–506 Clivorine Y = 501.5x − 139.4 0.1–507 Senecionine Y = 896.9x + 7820 0.1–50

a Acta 681 (2010) 33–40

the inter-day variability test, the solutions were examined in dupli-cates for three consecutive days (Table 4). Precision was expressedby relative standard deviation (RSD).

In the absence of certified reference materials, the recovery wasused to evaluate the accuracy of the method. Three concentra-tion levels of the seven mixed standards were spiked into 0.2 g ofsample CMED-497-01, and then extracted and analyzed under thisproposed method (Table 4).

2.5.4. Matrix effectThe matrix effect (ME) was defined as the ion suppression or

enhancement of the ionization of analytes, which was evaluated bycomparing the corresponding peak areas of matrix sample (CMED-497-01) after sample preparation with that of a neat standard. Theexperiments were performed at three concentration levels of theseven mixed standards in triplicate.

3. Results and discussion

3.1. Establishment of the coupled PIS and MRM approach

PAs are conventionly quantified by targeted detection of knownPAs during MRM MS/MS experiments, thus allowed sensitive deter-mination but would also miss detecting the PAs derivatives with thesame product ions. To identify analogs with similar fragmentationpattern, the PIS approach can be used to selectively detect the pre-cursors with the same product ions. As such, the combination ofthe MRM with PIS approach could achieve simultaneous quantifi-cation and screening of structurally similar analogs with commonfragments.

The basic idea of the method rests on the fact that the toxicretronecine-type and otonecine-type PAs contain specific fragmentions, e.g., m/z 120 and 138 for retronecine type; and m/z 150 and 168for otonecine type PAs. Therefore, we developed the PIS method forthe detection of these two types of toxic PAs present in the crudeextracts using their characteristic fragment ions.

In the MS/MS spectral analysis of the seven PAs standards, theTQ-MS experiments were optimized and developed through raisingthe cone voltage and collision energy to induce abundant charac-teristic fragment ions. Under optimal MS/MS conditions (detailedin Section 2.4), the intensity and sensitivity of m/z 120 were higherthan those of m/z 138; similarly, the intensity and sensitivity of m/z168 were higher than those of m/z 150. Therefore, four PIS togetherwith the MRM scan functions were simultaneously used for search-ing the presence of the predefined product ions at m/z 120/138 orm/z 168/150. The molecular mass range of the PIS was set between70 and 500 Da with reference to the typical structure of PAs.

3.2. PAs screening by precursor ion scan (PIS)

The PIS approach was applied to screen and identify toxic

retronecine-type and otonecine-type PAs. In comparison to a Q-TOF-MS, the TQ-MS has significantly higher sensitivity and speedof acquisition when recording precursor ion and neutral loss spec-tra. This is because the duty cycle of the Q-TOF is much longer thanthat of a static TQ-MS.

) and quantitation (LOQ) for the seven reference PAs.

ng mL−1) R2 LOD (pg mL−1) LOQ (pg mL−1)

0.9988 11 360.9999 0.42 1.30.9991 8.7 110.9999 0.33 1.00.9989 1.07 3.20.9996 3.6 110.9985 0.26 0.78

Y. Zhou et al. / Analytica Chimica Acta 681 (2010) 33–40 37

Table 4Precision, recovery and matrix effect of the seven reference PAs .

No. Compounds Spiked standards(ng mL−1, n = 3)

Precision Recovery % ± RSD (n = 3) Matrix effect % (n = 3)

Intra-day RSD% (n = 3) Inter-day RSD% (n = 3)

1 Monocrotaline 0.36 4.9 6.6 92.5 ± 6.8 89.90.53 2.4 3.2 106.6 ± 5.3 95.81.26 3.6 4.8 92.8 ± 4.3 97.2

2 Senkirkine 0.45 5.2 10.5 107.8 ± 5.9 99.60.59 4.1 7.2 104.7 ± 7.5 92.31.07 3.2 9.2 91.4 ± 6.3 95.1

3 Riddelliine 0.13 2.0 7.2 97.1 ± 10.5 98.00.37 3.1 9.2 95.3 ± 7.6 108.81.21 4.1 12.2 102.4 ± 7.9 102.3

4 Retrorsine 0.34 4.6 4.9 92.8 ± 9.8 96.80.44 4.8 9.9 103.9 ± 4.7 86.31.92 9.9 11.8 101.2 ± 12.0 95.1

5 Seneciphylline 0.15 1.3 3.6 95.6 ± 8.7 85.90.36 3.6 5.9 103.4 ± 3.6 93.11.93 2.7 8.7 93.3 ± 4.7 88.6

6 Clivorine 0.32 3.1 6.5 91.5 ± 5.6 106.00.56 4.4 8.5 102.4 ± 6.9 102.42.24 5.2 10.3 94.7 ± 8.9 98.3

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Fig. 2 shows representative total ion chromatograms (TIC) of PISith MRM of seven standard PAs obtained by UPLC–TQ-MS. The TIC

f seven MRM transitions was shown in Fig. 2A. The TIC of the PISo detect the characteristic product ion at m/z 120 (Fig. 2B) clearlyhowed, as expected that only the precursor ion of retronecine typeAs were detected at the retention time of 3.1, 4.6, 5.2, 5.7 and.4 min, corresponding to compounds 1, 3, 4, 5, and 7, respectively.ig. 2D representing the TIC of the PIS for detecting the character-stic product ion at m/z 168 clearly showed only the otonecine typeAs were detected at retention time of 3.9 and 6.0, correspondingo compounds 2 and 6.

The detection specificity of the PIS approach was evaluated bynalyzing seven standards of PAs at different concentration levels.ll analytes were detected below concentration of 0.05 �g mL−1,hich is over 10-fold sensitivity improvement upon previouslyublished LC–MS method recorded in MS full scan using extracted

on chromatograms [22].The PIS approach is also highly selective. Only the PAs frag-

ented into two pairs of product ions at m/z 120/138 or 168/150ould be identified, thereby reducing the endogenous interferencesf the co-elution for identification of PAs. The representative PIShromatograms of Senecio argunensis (CMED-500-01) were shownn Fig. 3. The total ion chromatograms for the MS full scan datare shown in Fig. 3A. As can be seen from Fig. 3A, the detection ofAs in the crude extracts was more difficult due to the increase inndogenous interferences. Most endogenous interferences elutedetween 1 and 6 min hampering the detection of some PAs. Oppo-itely, the PAs were more easily detected by PIS because of itsigh selectivity. In Fig. 3B, as expected that only tandem masspectrum acquired during the chromatographic separation wasegistered for the product ions at m/z 120 for retronecine-type PAsetection at the retention time of 3.17, 3.67, 3.78, 3.98, 4.28, 5.74,.28 and 6.47 min. Notably, two peaks were detected at retentionime of 3.67 and 3.78 in the PIS chromatogram, which was a large

eak at retention time of 3.75 in the MS full scan chromatogramFig. 3A), indicating the higher selectivity of PIS. Two peaks at Rt of.73 and 6.47 were unambiguously identified as seneciphylline (5)nd senecionine (7) by comparison with those of authentic stan-ards. Five other peaks at retention time of 3.17, 3.67, 3.78, 4.28,

4.9 97.8 ± 6.8 98.65.5 106.5 ± 10.2 96.43.8 110.8 ± 9.4 87.9

and 6.28 could only be tentatively assigned to be seneciphyllineN-oxide [21], senecionine N-oxide [21], 21-hydroxyintegerrimine[25], erucifoline (Z) [26], and integerrimine [26], respectively, bycomparison their MS/MS data with literature.

The screening of PAs by means of the above described PISmethod was then applied to 22 samples collected from sevenParasenecio species and four Senecio species. In general, largeramounts and varieties of PAs were detected in the four Seneciospecies, where S. argunensis was found to be the most abundant PAssource. A few PAs were detected in the seven Parasenecio specieswith the PIS approach, and the distribution of PAs varied consider-ably among the Parasenecio species.

3.3. Validation of quantification method

The linearity, regression, and linear ranges of seven PA standardswere determined using the developed MRM method. The correla-tion coefficient values (r > 0.999) indicated appropriate correlationsbetween the investigated compound concentrations and their peakarea within the test ranges (Table 3). The LOD and LOQ were in therange from 0.26 to 11 and from 0.78 to 36 pg mL−1 (Table 3), and theoverall intra- and inter-day variations (RSD) of the seven PAs werein the range from 1.2% to 9.9% and from 3.2% to 12.2%, respectively(Table 4). The recoveries were between 91.4% and 110.8%, and therepeatability present as RSDs (n = 6) was in the range from 3.6% to12.0% (Table 4). The matrix effect percentage ranged from 85.9 to108.8% (Table 4), indicating that the matrix effect on the ionizationof analytes was not obvious under these conditions. These resultsindicated that the developed UPLC–TQ-MS method was sensitive,repeatable, and accurate for the quantification analysis of PAs inplants.

3.4. Quantification of PAs

The optimized UPLC–TQ-MS method was subsequently appliedfor the simultaneous quantitative analyses of the seven PAs com-pounds in 22 samples from the four Senecio species and sevenParasenecio species. The contents of the marker compounds in eachsample were analyzed by a regression equation, as shown in Table 5.

38 Y. Zhou et al. / Analytica Chimica Acta 681 (2010) 33–40

F RM)b the PIS

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ig. 2. (A) The total ion chromatograms (TIC) from multiple reaction monitoring (My detection of the characteristic fragment ion at m/z 120 and 138, and; (D) and (E)

The analysis revealed that the total content of the targeted PAsn dried herb were higher than 1.6 �g g−1 but varied considerablymong the four Senecio species, except for sample CMED-497-7. Retrosine (4), seneciphylline (5), and senecionine (7) were

transitions of seven standards; (B)–(C) the precursor ion scan (PIS) chromatogramchromatogram by detection of the characteristic fragment ion at m/z 168 and 150.

presented abundantly in Senecio vulgaris; senkirkine (2), seneci-phylline (5), and senecionine (7) were in very high in S. argunensis.High amount of retrosine (4) was found only in S. vulgaris. Thevariation in PAs contents amongst the S. scandens may attribute

Y. Zhou et al. / Analytica Chimica Acta 681 (2010) 33–40 39

Fig. 3. Representative TIC of the precursor ion scans of Senecio argunensis: (A) the TIC of full MS scan; (B) the TIC of PIS by detection of the characteristic fragment ion at m/z120.

Table 5The contents of seven standard PAs in the tested samples (�g g−1).

samples Monocrotaline Senkirkine Riddelliine Retrorsine Seneciphylline Clivorine Senecionine

CMED-487-01 – 0.005 – 0.009 0.065 – 0.11CMED-487-02 – 0.002 – – 0.002 – 0.066CMED-488-01 – 0.003 – 0.007 0.006 – –CMED-488-02 – 0.006 0.003 – 0.009 – 0.003CMED-489-01 0.021 0.036 – – 0.014 – 0.18CMED-490-01 – 0.001 0.005 0.18 0.15 – 0.31CMED-491-01 0.006 0.93 – 0.05 0.37 – 0.015CMED-491-02 – 0.004 0.17 – 0.16 – 0.02CMED-492-01 0.072 0.004 – 0.018 0.023 – 0.25CMED-493-01 – – 0.005 0.054 0.075 – 0.012CMED-495-01 – 0.013 – 65 12.3 – 40CMED-496-01 0.008 0.037 0.007 – 2.1 – 6.3CMED-496-02 0.004 – – – 1.3 – 3.3CMED-496-03 – – – – – – 1.6CMED-497-01 – 4.3 3.9 – 6.1 – 8.1CMED-497-02 – 6.3 0.37 2.7 59 - 44CMED-497-03 0.005 44 – – 0.60 – –CMED-497-04 – 0.006 0.01 – 13 – 15

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CMED-497-05 0.005 23 0.02CMED-497-06 – 22 1.3CMED-497-07 – 0.23 –CMED-500-01 – 39 2.5

o different geographic locations where they were grown. In con-rast, the contents of the targeted PAs were generally low in theeven Parasenecio species.

The superior efficiency of the UPLC produces extremely narroweaks which were separated within 6 s or less from the base. Theevo TQ MS TQ-MS employs traveling-wave technology that can

mprove the MS capabilities by performing simultaneous and mul-ifunctional data acquisition in a single injection, such as MRMnd PIS acquisition, all within a timescale compatible with the.7 �m UPLC column. The rapid data acquisition of the TQ-MSllows MS/MS data of PIS to be acquired while still collecting a

– 0.18 – –0.90 – – –0.041 0.051 – –1.1 105 – 106

sufficient number of points across the designated analyte peak (15scanned points in 6 s) in MRM mode for accurate quantification.

4. Conclusion

A new approach to simultaneously perform PAs screening and

quantititon of toxic retronecine-type and otonecine-type PAs in 22samples from seven Parasenecio species and four Senecio specieson a new TQ-MS was developed. This was achieved by combin-ing the PIS acquisition and the MRM acquisition within the sameUPLC–MS/MS run. As a result, two types of toxic PAs could be

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eparately screened and seven PAs could be quantified simulta-eously, so that it reduces analytical time required for repetitivePLC–MS/MS runs and improves productivity of bioanalyticalethod research and development. The results presented here

llustrate the utility of the PIS in collecting qualitative informationhat would be missed using the targeted analysis approach of MRM

ode only, so that non-targeted PAs could also be screened out withigh selectivity and sensitivity. This strategy also has the potentialo be utilized as the ideal platform for simultaneously acquir-ng useful qualitative and quantitative information from complex

atrixes in the fields of natural products and so on.

cknowledgements

This research was supported by the Hong Kong Jockey Club Char-ties Trust (JCICM-15-07). The authors are grateful to Dr. Karl Lo of

aters Corp. The contents of this article do not necessarily reflecthe views and policies of the U.S. Food & Drug Administration. The

entioning of trade names and commercial products does not con-titute endorsement or recommendation for use.

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.aca.2010.09.011.

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