determination of main taxoids in taxus species by microwave-assisted extraction combined with...

Hao Luo1*Ying-Kun Nie2*Yu-Jie Fu1

Yuan-Gang Zu1**Shuang-Ming Li1

Wei Liu1

Lin Zhang1

Meng Luo1

Yu Kong1

Zheng-Nan Li1

1Key Laboratory of Forest PlantEcology, Ministry of Education,Northeast Forestry University,Harbin 150040, P. R. China

2The Second Hospital Affiliated toHarbin Medical University,Harbin 150081, P. R. China

Original Paper

Determination of main taxoids in Taxus species bymicrowave-assisted extraction combined with LC-MS/MS analysis

A method based on microwave-assisted extraction (MAE) has been developed for thedetermination of paclitaxel and five related taxoids, namely 10-deacetylbaccatin III(10-DAB III), cephalomannine, 10-deacetylpaclitaxel (10-DAT), 7-xyl-10- deacetylpacli-taxel (7-xyl-10-DAT), and 7-epi-10-deacetylpaclitaxel (7-epi-10-DAT) in Taxus species inthis study. The influential parameters of the MAE procedure were optimized, andthe optimal conditions were as follows: extraction solvent 80% ethanol solution,solid/liquid ratio 1:10 (g/mL), temperature 508C, and three extraction cycles, eachcycle 10 min. The method validation for LC-MS/MS analysis was performed. The LODand LOQ were 3.16–9.20 and 12.20 –30.45 ng/mL, respectively. Repeatability andreproducibility for the six taxiods with RSD ranged from 2.78 to 3.85% and from5.26 to 6.60%. The recoveries of the method for the six taxoids were 92.6–105.6%.The developed MAE-LC-MS/MS method was also successfully applied to determinethe contents of six taxoids in different Taxus species.

Keywords: LC-MS/MS / Microwave-assisted extraction / Paclitaxel / Taxoids /

Received: September 18, 2008; revised: October 20, 2008; accepted: October 21, 2008

DOI 10.1002/jssc.200800527

1 Introduction

Paclitaxel, a complex diterpene amide, was first isolatedfrom the bark of the pacific yew tree (Taxus brevifoliaNutt.) [1]. It has demonstrated a novel anticancer mecha-nism; unlike antimitotic drugs that exert their cytotoxiceffect by inhibiting the polymerization of microtubules,paclitaxel induces the formation of highly stable micro-tubules [2–3]. Paclitaxel has been proved to have remark-able antineoplastic effects against a wide variety ofhuman tumors, such as breast, head and neck, ovarian,small-cell, and nonsmall-cell lung cancers, as well as inthe treatment of AIDS-related Kaposi's sarcoma [4–6].The lower content of paclitaxel in all the species and thehigh demand for it in clinical use have led to the ferventresearch for other forms of production, including fungi

culture [7, 8], cell culture [9], total synthesis [10, 11], andpartial synthesis [12]. Among these methods, commercialsemi-synthesis methods have been developed for the pro-duction of paclitaxel from 10-deacetylbaccatin III (10-DAB III) [13]. Moreover, cephalomannine, 10-deacetylpa-clitaxel (10-DAT), 7-xyl-10-deacetylpaclitaxel (7-xyl-10-DAT), and 7-epi-10-deacetylpaclitaxel (7-epi-10-DAT) arealso a potential source of several useful starting materi-als for semi-synthesis [14]. Therefore, the six taxoids havebecome a hot spot in research. Structures of paclitaxel,10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT are shown in Fig. 1.

Considering the small amounts of some constituentspresent and the complex compositions of the plant mate-rial, appropriate extraction techniques are important inchemical analysis. Microwave technology was firstemployed in a laboratory by Abu-Samara et al. [15], andthe work of extraction of organic compounds by micro-wave irradiation was done by Ganzler et al. in 1986 [16].In recent years, microwave-assisted extraction (MAE) hasbeen used for the extraction of interested componentsfrom a wide variety of sample matrices and has been

Correspondence: Professor Yu-Jie Fu, Key Laboratory of ForestPlant Ecology, Ministry of Education, Northeast Forestry Univer-sity, Harbin 150040, P. R. ChinaE-mail: [email protected]: +86-451-82102082

Abbreviations: 10-DAB III, 10-deacetylbaccatin III; 10-DAT, 10-deacetylpaclitaxel; 7-epi-10-DAT, 7-epi-10-deacetylpaclitaxel;HRE, heat reflux extraction; MAE, microwave-assisted extrac-tion; MRM, multiple reaction monitoring; SOE, Soxhlet extrac-tion; USE, ultrasonic extraction; 7-xyl-10-DAT, 7-xyl-10-deacetyl-paclitaxel

The first two authors contributed equally to this work.Additional corresponding author: Professor Yuan-Gang Zu, KeyLaboratory of Forest Plant Ecology, Ministry of Education, NorthEast Forestry University, Harbin 150040, P. R. ChinaE-mail: [email protected]

i 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.jss-journal.com

192 H. Luo et al. J. Sep. Sci. 2009, 32, 192 – 201

J. Sep. Sci. 2009, 32, 192 – 201 Liquid Chromatography 193

used as an alternative sample preparation technique fora number of applications [17–22]. Comparing to conven-tional methods, MAE possesses the advantages of shortextraction time, little solvent consumption, and some-times has excellent efficiency. These advantages com-bined with cell bursting which were created duringmicrowave-heating. Cell bursting phenomenon is morefavorable to entry of the extracting solvent to solubilizeout the target compounds, thus leading to faster andmore efficient extraction. MAE method has beenemployed for the extraction of paclitaxel [23]. However,we found that the optimum extraction temperatureused in that study was 958C, which is not suitable for theextraction of taxoids. Some taxoids are thermosensitiveand are easy to degrade in high temperature. Therefore,further research on simultaneous extraction of taxoidswith MAE method is needed.

Up to now, taxoids have been analyzed by many meth-ods, including TLC [24], immunoaffinity chromatogra-phy (IAC) [25], GC, HPLC [26, 27], high speed countercur-rent chromatography (HSCCC) [28], and micellar electro-kinetic chromatography (MEKC) [29]. With the develop-ment of analytical methods, LC-MS/MS has been accepted

as a more accurate and sensitive method for the identifi-cation and determination of compounds [30, 31]. Espe-cially, it is very effective in the analysis of compoundsfrom complex samples because of its low LOD, high sensi-tivity, and the possibility for short run time [32–34]. LC-MS/MS methods were also applied in the determinationof paclitaxel and related taxoids [35–41]. Some of themfocused on qualitative analysis rather than quantitativeanalysis [35, 36], the others were the LC-MS/MS determi-nation of one or two taxoids in biological samples suchas human plasma, rat plasma, blood, urine, rat tissues,etc. [37–41].

To our knowledge, MAE followed by LC-MS/MS methodfor the rapid determination of paclitaxel, 10-DAB III,cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DATfrom needles and stalks of Taxus species was developedfor the first time in the present study. The operationalparameters of the MAE procedure including extractionsolvent, ratio of solid/liquid, extraction time, extractiontemperature, and number of extraction cycles were opti-mized. LC-MS/MS was used to determine paclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT in the extracts. The method was validated interms of LOD, LOQ, repeatability and reproducibility,and recoveries for the six taxoids were also tested.

2 Experimental

2.1 Chemicals and reagents

Reference compounds of paclitaxel, 10-DAB III, cephalo-mannine were purchased from Sigma (St. Louis, MO,USA). The 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT werebought from Shanghai Jin-He Biological Products (Shang-hai, China). ACN was of chromatographic grade (J & KChemical, China) and all the other reagents were of ana-lytical grade. Deionized water was purified by a Milli-Qwater purification system from Millipore (Bedford, MA,USA).

2.2 Plant materials

Fresh needles and stalks of T. chinensis var. mairei, T. cuspi-data Sieb. et Zucc. and T. media were collected from theBotanical Garden of the Key Laboratory of Forest PlantEcology, Ministry of Education, Northeast Forestry Uni-versity, Harbin, China, and identified by Professor Shao-Quan Nie from the same Key Laboratory. The sampleswere dried at 508C, ground in a disintegrator (HX-200A,Yongkang Hardware and Medical Instrument Plant,China), and then sieved (20–40 mesh).

2.3 Standard solutions

Stock solutions were obtained by dissolution of appropri-ate amounts of the six taxoids standards in ACN and


Figure 1. Chemical structures of paclitaxel, 10-DAB III,cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT.


stored in a refrigerator (48C). Work solutions were pre-pared by diluting the standard solution with ACN. All sol-utions prepared for LC-MS/MS analysis were filteredthrough 0.45 lm nylon membranes before use.

2.4 Chromatographic and mass spectrometricconditions

Chromatographic analysis was performed using an Agi-lent 1100 series HPLC system (Agilent Technologies, SanJose, CA, USA) consisting of a G1312A binary pump, a7725i manual injector, and a G1379A degasser. Separa-tion of the analytes was achieved on an Agilent EclipseXDB-C18 column (5 lm, 150 mm64.6 mm id). Themobile phase was prepared by mixing ACN and water(40:60 v/v). The flow rate was 1 mL/min and the columntemperature was 358C.

The column effluent was monitored by an API 3000(Applied Biosystems, Canada) triple-stage quadrupolemass spectrometer equipped with an ESI source. Ioniza-tion of paclitaxel, 10-DAB III, cephalomannine, 10-DAT,7-xyl-10-DAT, and 7-epi-10-DAT was achieved in the posi-tive ion mode. Multiple reaction monitoring (MRM) wasperformed with 200 ms dwell time. Nitrogen was used asnebulizing gas, curtain gas, collision gas, and auxiliarygas. The collision energy were optimized for the six tax-oids. It is 40 V for paclitaxel and 10-DAB III, 30 V for ceph-alomannine, 15 V for 10-DAT, 20 V for 7-xyl-10-DAT, and35 V for 7-epi-10-DAT. The mass transitions monitoredwere m/z 854.4 fi 286.1 for paclitaxel, m/z 567.4 fi 445.4for 10-DAB III, m/z 832.8 fi 264.1 for cephalomannine,m/z 812.6 fi 286.1 for 10-DAT, m/z 944.9 fi 286.4 for 7-xyl-10-DAT, and m/z 812.6 fi 286.1 for 7-epi-10-DAT. Quanti-tative analysis was carried out using peak areas obtainedfrom the MRM. The optimized MS parameters for thedetection of the six taxoids are listed in Table 1.

2.5 Extraction procedures

2.5.1 MAE

MAE was performed on an MARS-II (1000 W, 2450 MHz)microwave accelerated reaction system from SINEO

Microwave Chemistry Technology (China) equipped witha TFT multicolor liquid crystal screen, a power sensor(the power range 0–1000 W), an infrared temperaturesensor, a temperature controller, a special three necksround-bottomed flask, and electromagnetic stirrers. T.chinensis var. mairei needles and stalks powder (2 g) wasaccurately weighed and introduced into a 50 mL flask.According to the experimental design, the extractionprocess was performed under different conditions. Theeffects of extraction solvent, ratio of solid/liquid, extrac-tion time, extraction temperature, and number of extrac-tion cycles on the extraction yields of paclitaxel, 10-DABIII, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT were investigated.

2.5.2 Ultrasonic extraction (USE)

USE was performed in an ultrasonic bath (Kunshan Ultra-sonic Instrument, China). T. chinensis var. mairei needlesand stalks powder (5 g) was placed into a 100 mL flaskwith 50 mL 80% ethanol solution and extracted for30 min in the ultrasonic bath set at 250 W.

2.5.3 Heat reflux extraction (HRE)

T. chinensis var. mairei needles and stalks powder (10 g)was added into a 250 mL round bottom flask with150 mL 80% ethanol solution, the flask was then placedinto a water bath at 708C and allowed to extract for 10 h.

2.5.4 Soxhlet extraction (SOE)

T. chinensis var. mairei needles and stalks powder (10 g)was placed in a Soxhlet apparatus with 150 mL 80% etha-nol solution at 708C and extracted for 10 h.

After the extraction, the extraction solution obtainedfrom the above experiments was filtered through a 0.22Durapore filter and concentrated to dryness. Dry resi-dues were dissolved in 4 mL methanol and 20 lL wasinjected into the LC-MS/MS system.

3 Results and discussion

3.1 Comparison of different extraction methods

MAE, USE, HRE, and SOE were conducted for comparingtheir performances of extracting taxoids from T. chinensisvar. mairei needles and stalks. The conditions of USE,HRE, and SOE were described in Section 2.5, and for MAE,2 g of T. chinensis var. mairei needles and stalks wasextracted with 20 mL 80% ethanol solution in the 50 mLflask placed symmetrically in the microwave field, andoutput power of 300 W was used. The preheating timeand extraction time were 1 and 10 min, respectively. Theresults are shown in Table 2.

From Table 2, we can observe that the MAE and USEmethods extracted more paclitaxel, 10-DAB III, cephalo-mannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT than


Table 1. Parameters of mass spectrometric conditions

Parameter Value

Declustering potential (V) 80.0Focusing potential (V) 400.0Entrance potential (V) 10.0Nebulizing gas (a.u.) 12Curtain gas (a.u.) 10Collision gas (a.u.) 6Ion spray voltage (V) 5500.0Ion source temperature (8C) 250.0


the HRE and SOE methods. Furthermore, MAE for 10 mingave a little higher yields than those of USE for 30 min.The reason for the above phenomenon may be thatmicrowave possesses unique heating mechanism. Micro-wave heating is a process within a family of electro-heattechniques, which utilize specific parts of electromag-netic energy. As a polar solvent, free water in the plantcells, glands, and other microstructures can efficientlyabsorb microwave energy and leads to efficient heating,which eventually makes the walls rupture. As a result,the soluble compounds within the cells are extractedinto the surrounding solvent.

To sum up, the results indicated that MAE is a rapidand efficient method compared to other test methods.Therefore, MAE was selected for the optimization of theextraction process in the following experiments.

3.2 Optimization of MAE conditions

3.2.1 Extraction solvent

The extraction solvent seriously influences the extrac-tion yields of analytes, so choosing the most appropriate

extraction solvent is necessary for the whole extractionprocess. Taking into account the polarities and solubilityof paclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT and 7-epi-10-DAT, water, methanol, ethanol, 80%ethanol solution, 50% ethanol solution were chosen asthe extraction solvents. T. chinensis var. mairei needles andstalks powder was extracted with chosen solvent forthree cycles, the extraction time for each cycle was10 min and the power was 300 W. The results aredepicted in Fig. 2.

Figure 2 shows that with the decrease in polarities ofthe chosen solvents, the extraction yields of paclitaxel,10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT first increased and then decreased gradually.With 80% ethanol solution, almost all the six taxoidsreached the maximum yields. The possible reason is thatthe solubilities of the six taxoids in 80% ethanol solutionwere higher compared to the other test solvents and thatthe appropriate water content in 80% ethanol solutionwill improve the extraction efficiency of the taxoids con-tained within plant cells, based on the mechanism ofMAE. Hence, 80% ethanol solution was used for the


Table 2. Comparison of the extraction yields of paclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT from T. chinensis var. mairei needles and stalks by different extraction methods (n = 3)

Analyte Extraction yield (average l SD, mg/g)

MAE (10 min) USE (30 min) HRE (600 min) SOE (600 min)

Paclitaxel 0.048 l 0.002 0.043 l 0.003 0.028 l 0.001 0.032 l 0.00110-DAB III 0.426 l 0.019 0.417 l 0.019 0.306 l 0.013 0.318 l 0.014Cephalomannine 0.040 l 0.002 0.035 l 0.002 0.022 l 0.001 0.026 l 0.00110-DAT 0.087 l 0.005 0.078 l 0.004 0.048 l 0.003 0.053 l 0.0047-Xyl-10-DAT 0.956 l 0.039 0.894 l 0.041 0.535 l 0.025 0.582 l 0.0267-Epi-10-DAT 0.160 l 0.008 0.155 l 0.009 0.115 l 0.006 0.128 l 0.007

Figure 2. Effect of different solventson the extraction yields of paclitaxel,10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT from T. chinensis var. maireineedles and stalks by MAE (n = 3).



Figure 3. Effect of solid/liquid ratioon the extraction yields of paclitaxel,10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT from T. chinensis var. maireineedles and stalks by MAE (n = 3).

Figure 4. Effect of temperature onthe extraction yields of paclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT fromT. chinensis var. mairei needles andstalks by MAE (n = 3).

Figure 5. Effect of MAE time on theextraction yields of paclitaxel, 10-DAB III, cephalomannine, 10-DAT,7-xyl-10-DAT, and 7-epi-10-DATfrom T. chinensis var. mairei nee-dles and stalks by MAE (n = 3).


simultaneous extraction of paclitaxel, 10-DAB III, cepha-lomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT.

3.2.2 Ratio of solid/liquid

The ratio of solid/liquid was optimized for increasing theextraction efficiency and decreasing the solvent con-sumption. T. chinensis var. mairei needles and stalks pow-der was extracted for three cycles, each cycle 10 minwith 80% ethanol solution at the solid/liquid ratio of 1:5,1:7.5, 1:10, 1:15, and 1:20 (g/mL). The results are pre-sented in Fig. 3.

As can be seen in Fig. 3, when the ratio was less than1:10, the extraction yields of paclitaxel, 10-DAB III, cepha-lomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DATincreased obviously, however, it did not significantlyincrease when the ratio was higher than 1:10. This sug-gested that the solid/liquid ratio of 1:10 is optimal forMAE of taxoids from T. chinensis var. mairei needles andstalks.

3.2.3 Extraction temperature

In general, higher extraction temperature is beneficialfor extraction due to the improved solubility. However,controlling the temperature is of significance in thestudy because the taxoids are sensitive to temperature.The effect of temperature on the extraction yields ofpaclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT was studied under the tempera-ture controlled from 30 to 708C. The results are shown inFig. 4.

From Fig. 4, we can observe that at lower temperaturethe extraction yields of paclitaxel, 10-DAB III, cephalo-mannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DATincreased with the temperature. When the temperature

was higher than 508C, the extraction yields decreased.The results may be explained that the structure of the sixtaxoids are destroyed when the temperature exceeds acertain level. Hence, the proper temperature was con-trolled at 508C.

3.2.4 Extraction time

A proper extraction time is important for ensuring thedistribution equilibrium of analytes between solidphases and extraction solvents. To investigate the effectof time on the extraction yields of paclitaxel, 10-DAB III,cephalomannine, 10-DAT, 7-xyl-10-DAT and 7-epi-10-DAT,T. chinensis var. mairei needles and stalks powder wasextracted with 80% ethanol solution at the solid/liquidratio of 1:10 for 1, 5, 10, 20, and 30 min for three cycles,respectively. The results are described in Fig. 5.

Figure 5 shows that at the first 10 min, the yields ofpaclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT dramatically increased with theextraction time going. After 10 min the extraction yieldsof the six taxoids stayed at steady levels, indicating thatthey almost reached distribution equilibrium at around10 min. Hence, 10 min was selected as the proper extrac-tion time and this was used in the subsequent tests.

3.2.5 Extraction power

Generally, the MAE power has great influence on theextraction efficiency. In this study, the effect of power onthe extraction yields of paclitaxel, 10-DAB III, cephalo-mannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT wasstudied under the power set between 300 and 700 W. Theresults are described in Fig. 6.

From Fig. 6, we can see that extraction yields of the sixtaxoids at 500 W were the highest. The higher power


Figure 6. Effect of MAE power on theextraction yields of paclitaxel, 10-DABIII, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT from T.chinensis var. mairei needles andstalks by MAE (n = 3).


gave higher yields, but exceeding 500 W the yieldsdecreased. The reason for the above phenomena may bethat excessive MAE power makes molecular internalmotion aggravation, which lead the chemical structureof the six taxoids destroyed.

3.2.6 Number of extraction cycles

An increase in the number of extraction cycles couldimprove matrix partitioning into the extraction solvent,which enhances the extraction yield. Therefore, extrac-tion cycle was tested in this study. T. chinensis var. maireineedles and stalks powder (2 g) was extracted with 20 mLof 80% ethanol solution for 10 min, the extraction powerwas 500 W and the extraction temperature was 508C. Theextraction solution was filtered, and the extraction proc-ess was repeated four times in succession. Because theextraction yields for all the six taxoids were little in thefourth cycle, the data was not shown. The results are pre-sented in Fig. 7.

From Fig. 7, we can observe that with the increase inextraction cycle, the extraction yields of paclitaxel, 10-DAB III, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT obviously decreased. Furthermore, little sixtaxoids were extracted in the fourth extraction. There-fore, three extraction cycles were selected to assure an

efficient extraction of the six taxoids considering theconsumption of solvent and time.

3.3 Method validation

For linearity test of the chromatographic determination,standard solutions of paclitaxel, 10-DAB III, cephaloman-nine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT at eight lev-els over the concentration range of 0.25–35.2 lg/mLwere injected in triplicate. The linearity was wellexpressed, and the regression equations, concentrationranges, and correlation coefficients are shown in Table 3.

LODs and LOQs of the six taxoids were studied by grad-ually diluting the individual standard solution withACN; LOD and LOQ were calculated for ratio S/N of 3 and10, respectively. The results are shown in Table 4.

The repeatability and reproducibility were studiedwith RSD for the extraction yields of the six taxoids. Inrepeatability tests, the MAE-LC-MS/MS process wasrepeated for five times on the same day under the opti-mal MAE conditions. The obtained RSD varied between2.78 and 3.85%. The reproducibility was assessed by theseries of five independent extractions on five differentdays, and the RSD ranged from 5.26 to 6.60% (Table 4).


Figure 7. Effect of number of extrac-tion cycles on the extraction yields ofpaclitaxel, 10-DAB III, cephaloman-nine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT from T. chinensis var.mairei needles and stalks by MAE(n = 3).

Table 3. Regression equations, concentration ranges, and correlation coefficients of the six taxoids in the extracts of T. chinensisvar. mairei needles and stalks (n = 3)

Analyte Regression equation Concentration range (lg/mL) Correlation coefficient

Paclitaxel y = 19338281x – 1605571 0.25–35.2 0.992510-DAB III y = 10846953x – 84188 0.60–15.7 0.9978Cephalomannine y = 11393158x – 102538 0.40–18.6 0.994610-DAT y = 10452680x + 13989 0.80–20.5 0.99667-Xyl-10-DAT y = 7965026x – 26020 0.50–16.5 0.99857-Epi-10-DAT y = 12438981x + 43354 0.40–22.3 0.9993

y: peak area, x: concentration of standards.


The recovery experiments of the six taxoids were car-ried out by adding two concentration levels of standardto known amounts of T. chinensis var. mairei samples. Theresultant samples were then extracted and determinedwith the optimal MAE-LC-MS/MS method. The recoveriesfor the six taxoids ranged from 92.6 to 105.6% (Table 5).

3.4 Application of the MAE-LC-MS/MS method

The contents of paclitaxel, 10-DAB III, cephalomannine,10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT in T. cuspidataSieb. et Zucc., T. media, and T. chinensis var. mairei needlesand stalks were determined using the developed MAE-LC-

MS/MS method. The representative LC-MS/MS chromato-grams of the six taxoids in samples with MRM are shownin Fig. 8. The contents of the six taxoids in three Taxus spe-cies are summarized in Table 6.

From Table 6, we can observe that contents of the sixtaxoids vary in different species. The contents of pacli-taxel, 10-DAB III, and 10-DAT in T. media are the highest inthe three species. The other taxoids in T. cuspidata Sieb. etZucc., and T. media needles and stalks are less than in T.chinensis var. mairei. The results indicated that we couldselect appropriate species so as to get more target tax-oids.


Table 4. LOD, LOQ, repeatability, reproducibility of the six taxoids in the extracts of T. chinensis var. mairei needles and stalks(n = 5)

Analyte LOD(ng/mL)

LOQ(ng/mL)

Repeatability Reproducibility

Average l SD,mg/g

RSD% Average l SD,mg/g

RSD%

Paclitaxel 7.11 24.46 0.078 l 0.003 3.85 0.076 l 0.004 5.2610-DAB III 9.20 28.38 0.554 l 0.018 3.25 0.565 l 0.032 5.66Cephalomannine 6.34 18.19 0.056 l 0.002 3.57 0.053 l 0.003 5.6610-DAT 3.20 12.20 0.130 l 0.004 3.08 0.125 l 0.007 5.607-Xyl-10-DAT 5.25 20.85 1.358 l 0.040 2.95 1.382 l 0.084 6.087-Epi-10-DAT 3.16 30.45 0.216 l 0.006 2.78 0.212 l 0.014 6.60

Table 5. Recovery of the six taxoids in the extracts of T. chinensis var. mairei needles and stalks (n = 5)

Analyte Original amount(ng)

Amount added(ng)

Amount found(ng)

Recovery

Average l SD% RSD%

Paclitaxel 15.2 10.1 25.2 99.0 l 4.4 4.4420.2 35.0 98.0 l 4.6 4.69

10-DAB III 108.5 20.1 128.0 97.0 l 3.2 3.3040.3 146.9 95.3 l 3.2 3.36

Cephalomannine 11.2 15.2 26.3 99.3 l 4.6 4.6330.4 41.2 98.7 l 4.4 4.46

10-DAT 26.5 30.2 58.4 105.6 l 4.2 3.9860.3 88.9 103.5 l 3.6 3.48

7-Xyl-10-DAT 272.3 40.5 309.8 92.6 l 3.1 3.3580.1 347.4 93.8 l 3.5 3.73

7-Epi-10-DAT 43.6 35.5 80.4 103.7 l 4.5 4.3470.2 115.3 102.1 l 5.6 5.48

Figure 8. LC-MS/MS chromato-gram (MRM mode) of six taxoids inT. chinensis var. mairei needlesand stalks. Peaks: 1, (10-DAB III);2, (7-xyl-10-DAT); 3, (10-DAT); 4,(cephalomannine); 5, (7-epi-10-DAT); and 6, (paclitaxel).


4 Concluding remarks

A MAE method followed by LC-MS/MS for the determina-tion of paclitaxel, 10-DAB III, cephalomannine, 10-DAT,7-xyl-10-DAT, and 7-epi-10-DAT in Taxus species was devel-oped in the present study. After the optimization steps,the optimal conditions for MAE were as follows: extrac-tion solvent 80% ethanol solution, ratio of solid/liquid1:10 (g/mL), temperature 508C, and three extractioncycles, each cycle 10 min. The MAE-LC-MS/MS methodshowed acceptable repeatability (RSD f 3.85%), reprodu-cibility (RSD f 6.60%), and recovery (92.6–105.6%). Goodlinearity and low LOD and LOQ were obtained by the LC-MS/MS determination. From all these results, the MAE-LC-MS/MS method was simple, efficient, and reliable forthe extraction and determination of paclitaxel, 10-DABIII, cephalomannine, 10-DAT, 7-xyl-10-DAT, and 7-epi-10-DAT. We may conclude that it could be applied on sam-ples from other Taxus species.

The authors gratefully acknowledge the financial supports byInnovative Program for Importation of International AdvancedAgricultural Science and Technology, National Forestry Bureau(2006-4-75), National Key Technology R & D Program(2006BAD18B0405), Heilongjiang Province Science Foundationfor Excellent Youths (JC200704), National Natural Science Founda-tion of China (30770231), Key Project of Chinese Ministry of Educa-tion (108049), and Research Foundation for Science and Technol-ogy Innovation Talents of Harbin (2006RFXXS001).

The authors declared no conflict of interest.

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Table 6. Contents of the six taxoids in the extracts of T. cuspidata Sieb. et Zucc., T. media and T. chinensis var. mairei needlesand stalks (n = 5)

Analyte T. cuspidata Sieb. et Zucc. T. media T. chinensis var. mairei

Average l SD, mg/g RSD% Average l SD, mg/g RSD% Average l SD, mg/g RSD%

Paclitaxel 0.083 l 0.004 4.82 0.267 l 0.014 5.24 0.077 l 0.003 3.9010-DAB III 0.329 l 0.014 4.26 1.607 l 0.053 3.30 0.552 l 0.025 4.53Cephalomannine 0.022 l 0.001 4.55 0.038 l 0.002 5.26 0.055 l 0.003 5.4510-DAT 0.078 l 0.004 5.13 0.265 l 0.013 4.91 0.129 l 0.007 5.437-Xyl-10-DAT 0.066 l 0.003 4.55 0.071 l 0.003 4.23 1.367 l 0.055 4.027-Epi-10-DAT 0.086 l 0.004 4.65 0.079 l 0.004 5.06 0.218 l 0.010 4.59


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determination of main taxoids in taxus species by microwave-assisted extraction combined with...

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