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DOI 10.1007/s10337-013-2608-2Chromatographia

OrIgInal

Microwave‑Assisted Extraction/Dispersive Liquid–Liquid Microextraction Coupled with DSI‑GC‑IT/MS for Analysis of Essential Oil from Three Species of Cardamom

Zhi‑Yong Ye · Zu‑Guang Li · Dan Wei · Maw‑Rong Lee

received: 6 august 2013 / revised: 11 november 2013 / accepted: 13 november 2013 © Springer-Verlag Berlin Heidelberg 2013

on comparison of the main bioactive compounds of essen-tial oil, a significant difference was found between Semen Alpiniae Katsumadai or Fructus Amomi Rotundus and Semen Myristicae. This study also provides a new approach for quality assessment of traditional Chinese medicines.

Keywords gas chromatography–mass spectrometry · Direct sample introduction · Microwave-assisted extraction · Dispersive liquid–liquid microextraction · low-density organic solvent · Traditional Chinese medicines

Introduction

Cardamom herbs include Semen Alpiniae Katsumadai, Fructus Amomi Rotundus, and Semen Myristicae, which are dried seed of the plants Alpinia hainanensis K. Schum, Amomum testaceum ridl, and Myristica fragrans Houtt, respectively. These are all common traditional Chinese medicines (TCMs) and can promote digestion, warm the spleen, and expel wind. Thus, they have been applied in treatment of abdominal pain, sickness, indigestion, etc. [1]. The dried seed contains essential oil as a volatile aromatic oily liquid which can be used as an ingredient in perfumes, cosmetics, flavoring agents, and pharmaceuticals. Compari-son of the main chemical constituents of essential oil from cardamoms is a simple and efficient alternative to conven-tional quality assessment of TCMs.

Essential oils (EOs) can be isolated using various differ-ent methods, such as hydrodistillation [2–5], steam distilla-tion [6–8], simultaneous distillation extraction [9–11], and organic solvent extraction [8, 12, 13]. However, these tradi-tional methods suffer from various shortcomings, including the need for a large amount of hazardous organic solvent

Abstract a novel method of microwave-assisted extrac-tion coupled with polyethylene Pasteur-pipette-based dis-persive liquid–liquid microextraction applying low-density organic solvent (MaE-lDS-DllME) was successfully developed for extraction and preconcentration of essential oil from three species of cardamom (Semen Alpiniae Kat‑sumadai, Fructus Amomi Rotundus, and Semen Myristicae). The essential oil was analyzed by gas chromatography-ion trap/mass spectrometry (gC-IT/MS) using a Chromato-Probe direct sample introduction (DSI) device. The effects of various parameters affecting the extraction process, such as the type of extraction solvent and dispersive solvent, ionic strength, microwave power, and irradiation time, were investigated thoroughly and optimized. The optimal condi-tions were extraction solvent of toluene, dispersive solvent of methanol, microwave power of 80 W, irradiation time of 4.0 min, plant material amount of 0.1 g, and no addition of salt. Compared with hydrodistillation, MaE-DllME-DSI-gC–MS is a simple, rapid, low-cost, efficient, and envi-ronmentally friendly method, and the essential oil contains higher amounts of oxygenated compounds, which play an important and valuable role in terms of their contribution to the fragrance of the essential oil. In this work, we also stud-ied the main components of the three varieties of cardamom. Qualitative and quantitative differences in the components of the three essential oils were found to be present. Based

Z.-Y. Ye · Z.-g. li (*) · D. Wei College of Chemical Engineering and Materials Science, Zhejiang University of Technology, Hangzhou 310014, People’s republic of Chinae-mail: lzg@zjut.edu.cn

M.-r. lee Department of Chemistry, national Chung-Hsing University, Taichung 40227, Taiwan

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and plant materials, long duration, and high energy require-ments. In addition, thermally sensitive compounds may be lost during thermal extraction and/or distillation. Therefore, extensive efforts have been focused on extraction of essen-tial oils and related fields for the development of novel techniques to overcome these disadvantages. recently, many kinds of extraction technique such as microwave-assisted extraction (MaE) [4, 14, 15], solvent-free micro-wave extraction (SFME) [16–18], supercritical fluid extrac-tion (SFE) [19, 20], ultrasound-assisted extraction (UaE) [21], solid-phase microextraction (SPME) [22–26], and liquid-phase microextraction (lPME) [27–29] have been developed. SPME is a solvent-free extraction technique and a relatively new method of sample preparation, com-bining extraction and concentration in a single step and in one device. However, the coated fiber used is generally expensive and fragile and has limited lifetime. Further-more, sample carryover is sometimes difficult to eliminate [30]. lPME is a solvent microextraction technique which offers many advantages such as wide choice and low con-sumption of extraction solvents, high extraction efficiency, and simplicity of the experimental setup. However, it also suffers from various drawbacks such as microdrop insta-bility (for single drop microextraction) and relatively low precision [31]. Microwave heating involves internal heat-ing based on conduction and dielectric polarization caused by microwave irradiation [32]. Due to the faster start-up, more effective heating, faster energy transfer, shorter dura-tion, and better performance under atmospheric conditions, MaE has played an important role in isolating essential oils for use in laboratories and industry. Moreover, in com-parison with SFE, the equipment is simpler and less expen-sive. However, sometimes the concentration of analytes in the MaE extract is low, and further extraction and concen-tration are necessary prior to analysis.

In 2006, dispersive liquid–liquid microextraction (DllME) was introduced by assadi and coworkers, being based on a ternary solvent system such as homogeneous liquid–liquid extraction and cloud-point extraction [33]. The advantages of DllME are its rapidity, environmental friendliness, simplicity of operation, low consumption of solvents and time, and high enrichment factor and recov-ery. To date, this innovative method has been successfully applied for isolation and preconcentration of polychlorin-ated biphenyls [30], polycyclic aromatic hydrocarbons [33], polybrominated diphenyl ethers [34, 35], phthalate esters [36], pesticides [37–39], and other organic compounds [40–42] in different matrices [30, 34, 35, 39–41]. However, to the best of our knowledge, isolation and preconcentra-tion of essential oils by DllME has received only limited attention. Sereshti et al. [43] applied DllME to extract essential oils from Elettaria cardamomum Maton, Olive‑ria decumbens Vent. [44], and tea [45]. However, the main

disadvantage of DllME is its insufficient selectivity for complicated matrices. The extract from DllME is dirty and contains many interferences such as pigment, polysac-charide, and other nonvolatile compounds, which rapidly and severely deteriorate analytical instruments due to accu-mulation of harmful residue in the gC injector liner and the first portion of the capillary column. Thus, to overcome this drawback, it is necessary to include a clean-up stage and related alternatives after analyte extraction and before the DllME technique [35]. Direct sample introduction (DSI) was developed by amirav et al. [46, 47], being a simple, rapid, and efficient technique for sampling large-volume dirty samples without a further clean-up stage. It is based on the introduction of the sample into a disposable micro-vial, which is then placed into a ChromatoProbe vial holder, which is directly inserted into a temperature-programmable gC injector. Initially, the extraction solvent is evaporated at a temperature value corresponding to the solvent boiling point minus ~5 °C [48]. Then, with elevation of the tem-perature, the target analytes are thermally extracted into the early portion of the gC column, while the impurity and other nonvolatile residues remain in the vial. This technique has been reported in a wide variety of fields [48–55].

The purpose of the present work is to develop a novel alternative based on microwave-assisted extraction coupled with polyethylene Pasteur-pipette-based dispersive liquid–liquid microextraction applying a low-density organic sol-vent to isolate and preconcentrate essential oils from three species of cardamom (Semen Alpiniae Katsumadai, Fructus Amomi Rotundus, and Semen Myristicae). Subsequently, to protect analytical instruments, reduce matrix interference, and further improve sensitivity, DSI-gC–MS was applied for determination of the components of the essential oils. The influences of several experimental parameters (type of extraction and dispersive solvent, ionic strength, microwave power, and irradiation time) on the extraction efficiency of essential oil were evaluated. To our knowledge, this is the first report describing combined application of MaE-lDS-DllME as the sample preparation and DSI-gC–MS as the analysis technique for extraction and determination of essential oils from TCMs.

Experimental

reagents and Materials

Dried Semen Alpiniae Katsumadai, Fructus Amomi Rotun‑dus, and Semen Myristicae were purchased from Jiuzhou Drugstore (Hangzhou, China). Methanol, acetone, acetoni-trile, cyclohexane, and n-hexane were obtained from Hua-dong Medicine Company (Hangzhou, China), while tolu-ene was from Tedia Company (Fairfield, OH, USa).

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The soft polyethylene Pasteur pipette (ca. 5 ml capac-ity) was from Kangtai Medical appliance Company (Jiang-yan, China). a sketch of the modified Pasteur pipette is shown in Fig. 1 (2a).

Instrumentation

gC–MS analysis was carried out using a Varian gC 3800 (Varian, Walnut Creek, Ca, USa) equipped with a 1079 temperature-programmable injector connected to a Varian Saturn 2000 ion-trap mass spectrometer. The chromato-graphic separation was achieved on a 30-m DB-5 fused-silica column (i.d. 0.25 mm, film thickness 0.25 μm) from J&W Scientific (Folsom, Ca, USa). The temperature of the column was held at 40 °C for 5 min and then increased at 3 °C min−1 to 280 °C, at which temperature it was left for 5 min. Electronic flow control (EFC) was used to maintain a constant helium carrier gas flow of 0.8 ml min−1. For the essential oils from MaE-DllME, sample introduction was performed using a Varian ChromatoProbe direct sample introduction (DSI) device attached to a 1079 programmable injector with injection volume of 5 μl. The injector tem-perature was maintained at 110 °C for 2 min with a 50:1 split to evaporate the solvent, then ramped to 250 °C at 100 °C min−1 in splitless mode and held for 1.6 min, after which the injector cooled back to 110 °C and the split ratio was 20:1. However, for the essential oils from hydrodistil-lation, sample introduction was performed using a 10-μl microsyringe (gaoge, Shanghai, China) with injection vol-ume of 1 μl. The temperature of the injector was held at 250 °C, and it was used in split mode. Full-scan spectra were acquired in electron ionization (EI, 70 eV) or chemi-cal ionization (CI) mode in the mass range of 40–650 m/z

with scan time of three uscans, solvent delay of 11 min, ion-trap temperature of 200 °C, manifold temperature of 50 °C, and transfer-line temperature of 280 °C. Kováts retention indices (rI) were calculated for all organic com-ponents using a homologous series of n-alkanes (C6–C18).

a Tgl-16C centrifuge from anting Scientific Instru-ment (Shanghai, China) was used for centrifuging. a WBFY-201 microwave oven from ruijia Precision Scien-tific Instrument Co., ltd. (Hangzhou, China) was used to facilitate extraction.

Microwave-assisted Extraction/Dispersive liquid–liquid Microextraction Procedures

The MaE-DllME procedures are illustrated in Fig. 1. The three species of cardamom (Semen Alpiniae Katsumadai, Fructus Amomi Rotundus, and Semen Myristicae) were ground to fine powder. a portion (~100 mg) of each sam-ple was placed into a 50-ml glass round flask, followed by addition of 10 ml ultrapure water. MaE was performed at 80 W for 4.0 min. after that, 3.0 ml of cooled aque-ous solution was transferred into a modified disposable polyethylene pipette using a 5.0-ml glass syringe (gaoge, Shanghai, China). Then, a mixture of 100 μl toluene (extraction solvent) and 500 μl methanol (dispersive sol-vent) was rapidly injected into the sample solution using a 1.0-ml syringe (gaoge, Shanghai, China). a cloudy solu-tion was formed, which could achieve migration of ana-lytes from the aqueous sample into toluene droplets in a few seconds. Subsequently, the pipette was placed into a 10-ml Eppendorf tube and centrifuged at 8,000 rpm for 5 min to break down the emulsion and separate two clear phases. Finally, the pipette bulb was squeezed slightly, and

Fig. 1 Schematic of MaE-DllME: 1 microwave-assisted extrac-tion; 2a introduction of 3.0 ml cooled aqueous solution; 2b injection of the mixture of extraction solvent and dispersive solvent, forming a

cloudy solution; 2c centrifugation for phase separation; 2d collection of organic extract after squeezing of pipette

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the upper layer aggregated to the upper narrow neck of the pipette, from where it could be easily withdrawn using a 50.0-μl microsyringe (gaoge, Shanghai, China).

Hydrodistillation (HD)

Fifty grams of finely powdered Semen Alpiniae Katsuma‑dai was immersed in a 500-ml round flask with 250 ml ultrapure water and hydrodistilled for 6 h in an all-glass Clevenger apparatus as recommended in the Chinese Phar-macopoeia. The essential oils were collected from the con-denser, dissolved in 5 ml n-hexane, and dried over anhy-drous sodium sulfate prior to analysis by gC–MS.

Identification of Components

The gC–MS data were processed using the Saturn Software package 5.2.1. Identification of the constituents was based on comparison of the obtained mass spectra with those of reference compounds in the data system of the Wiley library and nIST Mass Spectral Search Program (nIST 2011 ver-sion mass spectral database; national Institute of Standards and Technology, Washington, DC, USa) connected to a Sat-urn 2000 mass spectrometer and homemade library mass spectra built from pure substances and components of known oils and MS literature. The constituents were confirmed by comparing the Kováts retention index or gC retention data with those of authentic standards or by published literature. Moreover, the molecular weights of the identified substances were confirmed by chemical ionization using CH3Cn as a liquid CI reagent. Quantitative analysis in percent was per-formed by peak area normalization measurements.

Results and Discussion

Optimization of MaE-DllME Method

In this study, the experimental parameters of MaE (micro-wave power and irradiation time) and lDS-DllME (type of extraction solvent and dispersive solvent, ionic strength) were optimized using the “single-factor-at-a-time” method. Semen Alpiniae Katsumadai was used as the sample, and the number of replicates was three. The peak area of the main representative compounds was employed as the response in the optimization procedure.

DllME Parameters

Selection of Extraction Solvent

The type of extraction solvent is a critical experimen-tal parameter that governs the extraction efficiency of the

DllME process. For lDS-DllME, an appropriate extrac-tion solvent should offer the following physicochemical properties: (1) density lower than water, (2) immiscible with water, (3) high extraction capability for target ana-lytes, (4) fine formation of cloudy state in the presence of a dispersive solvent when injected into aqueous solu-tion, (5) low vapor pressure to prevent evaporation during extraction, and (6) good chromatographic compatibility. On the basis of these considerations, n-hexane (density 0.66 g ml−1, boiling point 68.7 °C), cyclohexane (density 0.81 g ml−1, boiling point 80.7 °C), and toluene (den-sity 0.87 g ml−1, boiling point 110.6 °C) were investi-gated as potential extraction solvents for this purpose. a series of experiments were performed by using 100 μl of the selected extraction solvents and 0.5 ml methanol as dispersive solvent after Semen Alpiniae Katsumadai was extracted under microwave irradiation at 400 W for 4.0 min. Figure 2 shows the peak areas of six representative compounds with the three different solvents. The results reveal that higher extraction efficiency of most target com-pounds was obtained when using toluene compared with the other solvents. Therefore, toluene was employed as extraction solvent in further experiments.

Selection of Dispersive Solvent

The dispersive solvent should be miscible with both water and the extraction solvent in order to form very fine drop-lets and increase the contact surface area of the selected extraction solvent when the mixture of extraction and dis-persive solvents is rapidly injected into an aqueous sample. Based on these criteria, methanol, acetone, and acetoni-trile were evaluated in the following study. The relative amounts of six major compounds obtained using 0.5 ml of the different dispersive solvents and 100 μl toluene as extraction solvent after microwave irradiation at 400 W for 4.0 min are shown in Fig. 3, indicating that the peak area of most target compounds was higher when using methanol

Fig. 2 Effect of extraction solvent on extraction efficiency of 3-phe-nyl-2-butanone (PB), trans-cinnamaldehyde (CI), 2-acetyl-1-tetralone (AT), farnesol (FA), pinocembrin (PI), and 1,7-diphenyl-4,6-hepta-dien-3-one (DHO) from Semen Alpiniae Katsumadai (n = 3)

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or acetone as dispersive solvent compared with acetoni-trile for the same extraction conditions, and the sum of the peaks was highest when using methanol. Hence, methanol was selected as dispersive solvent for the following studies.

Effect of Ionic Strength

In general, adding a salt could adjust the ionic strength and decrease the solubility of analytes in the aqueous phase in order to enhance their extraction into the organic phase. To investigate the influence of ionic strength on the extraction efficiency of the proposed method, a collection of experi-ments were performed with increasing concentrations of sodium chloride (0–30 %, w/v) in the sample solution. The results showed that analytical signals slightly decreased due to increased collected volume of organic phase and viscos-ity of aqueous phase at high salt concentration. Therefore, no salt addition was chosen for subsequent experiments.

MaE Parameters

To fully understand how the important MaE variables affect the EO extraction efficiency, two parameters were considered: microwave power and irradiation time. First, MaE of Semen Alpiniae Katsumadai was performed using different microwave powers [P10 (80 W), P30 (240 W), and P50 (400 W)] for the same irradiation time (4.0 min) prior to DllME. as can be seen from Fig. 4a, with increasing microwave power, the peak areas of most target compounds increased. However, when the microwave power was more than or equal to 240 W, eucalyptol, which is a bioactive compound in Semen Alpiniae Katsumadai, disappeared due to degradation or loss at high temperature. To our knowl-edge, the parameters of microwave power and irradiation time are relevant. Therefore, we also investigated sam-ple extraction under microwave irradiation at 240 W for 1.0 min. The results demonstrated that eucalyptol signifi-cantly decreased. Finally, 80 W was used as the microwave

power in the proposed method for eucalyptol. next, to evaluate the effect of irradiation time, various experiments were performed using microwave irradiation for different times at the same microwave power (80 W). as seen from Fig. 4b, the analytical signals first increased as the irradia-tion time was increased from 1.0 to 4.0 min, then dramati-cally decreased for eucalyptol or slightly increased for most target compounds when the time was further increased to 6.0 min. Therefore, irradiation time of 4.0 min was pre-ferred for this work.

Method Precision of MAE‑DLLME

The method precision can be represented by the relative standard deviation (rSD) value. Under optimum condi-tions, the peak areas of seven representative compounds obtained in three replicate analyses of essential oil of Semen Alpiniae Katsumadai were used to calculate the rSD values. The rSD values of EU, PB, CI, aT, Fa, PI, and DHO were 7.4, 5.3, 7.5, 9.8, 4.9, 9.2, and 8.1 %, which are relatively satisfactory. The results show that MaE-DllME coupled with DSI-gC–MS has good precision.

Chemical Composition of the Essential Oils

gC–MS total ion chromatograms (TICs) of the EOs iso-lated from each of the three species of cardamom are shown

Fig. 3 Effect of dispersive solvent on extraction efficiency of 3-phe-nyl-2-butanone (PB), trans-cinnamaldehyde (CI), 2-acetyl-1-tetralone (AT), farnesol (FA), pinocembrin (PI), and 1,7-diphenyl-4,6-hepta-dien-3-one (DHO) from Semen Alpiniae Katsumadai (n = 3)

Fig. 4 Effect of a microwave power and b irradiation time on extraction efficiency of eucalyptol (EU), 3-phenyl-2-butanone (PB), trans-cinnamaldehyde (CI), 2-acetyl-1-tetralone (AT), farnesol (FA), pinocembrin (PI), and 1,7-diphenyl-4,6-heptadien-3-one (DHO) from Semen Alpiniae Katsumadai (n = 3)

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in Fig. 5. The chemical composition of the EOs was iden-tified by the mass fragmentation patterns and/or retention indices, and the relative content was calculated by the peak area ratio. Based on computer and manual analyses, the qualitative and quantitative differences in the components of the four EOs extracted by HD and MaE-DllME from the three species of cardamom are presented in Table 1. The number of replicates was three.

Comparison of Constituents of EOs Isolated by HD and MaE-DllME from Semen Alpiniae Katsumadai

as can be seen from Table 1, gC–MS and DSI-gC–MS analysis of the EOs extracted from Semen Alpiniae Kat‑sumadai by HD and MaE-DllME resulted in characteri-zation of 50 and 49 different organic compounds, account-ing for 97.08 and 95.72 % of the total oil composition of the two EOs, respectively. The most abundant chemi-cal compounds in the EO isolated by HD were farnesol,

α-caryophyllene, eucalyptol, caryophyllene, carotol, β-cubebene, α-phellandrene, and δ-cadinene, whereas the major aroma components in the EO isolated by MaE-DllME were 3-phenyl-2-butanone, farnesol, 1,7-diphenyl-4,6-heptadien-3-one, pinocembrin, trans-cinnamaldehyde, eucalyptol, methyl cinnamate, 2-acetyl-1-tetralone, and α-caryophyllene. The same 33 compounds including the main bioactive compounds in Semen Alpiniae Katsuma‑dai were obtained by the two methods of HD and MaE-DllME, although the relative concentration of the identi-fied compounds varied. additionally, higher amounts and numbers of oxygenated compounds were present in the EO extracted by MaE-DllME in comparison with HD. These oxygenated compounds are highly odoriferous and play an important and valuable role in terms of their contribution to the fragrance of the essential oil. The greater proportion of oxygenated compounds in the EO extracted by MaE-DllME is probably due to the diminution of thermal and hydrolytic effects, compared with hydrodistillation which

Fig. 5 TICs of essential oils (EOs) extracted by HD and MaE-DllME from three species of cardamom: a EO extracted by HD from Semen Alpiniae Katsumadai, b EO extracted by MaE-DllME from Semen Alpiniae Katsuma‑dai, c EO extracted by MaE-DllME from Fructus Amomi Rotundus, and d EO extracted by MaE-DllME from Semen Myristicae

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Table 1 Main chemical components identified in essential oils from three species of cardamom

no. Compound Calculated rI (literature rI)

Formula Mw Mass spectra (m/z %) Identification method

relative content (%)

a B C D

1 α-Pinene 945 (942) C10H16 136 93 (100), 92 (99), 91 (74) rI, MS 0.67 – – 0.16

2 Camphene 949 (946) C10H16 136 93 (100), 121 (71), 91 (45) rI, MS 0.67 – – –

3 β-Pinene 983 (983) C10H16 136 93 (100), 91 (71), 77 (55) rI, MS 0.42 – 0.08 0.27

4 α-Phellandrene 1,006 (1,003) C10H16 136 91 (100), 93 (99), 77 (49) rI, MS 2.43 – – –

5 α-Terpinene 1,023 (1,023) C10H16 136 121 (100), 93 (89), 136 (75) rI, MS – – – 0.22

6 m-Cymene 1,024 (1,023) C10H14 134 119 (100), 134 (38), 91 (33) rI, MS 1.43 – – –

7 limonene 1,029 (1,028) C10H16 136 67 (100), 93 (71), 94 (51) rI, MS 0.67 – – –

8 Eucalyptol 1,032 (1,034) C10H18O 154 93 (100), 81 (96), 139 (93) rI, MS 8.04 6.30 64.26 –

9 β-Phellandrene 1,035 (1,033) C10H16 136 93 (100), 91 (71), 77 (52) rI, MS – – – 0.06

10 τ-Terpinene 1,062 (1,060) C10H16 136 93 (100), 91 (85), 136 (51) rI, MS 0.11 – 0.04 0.23

11 cis-β-Terpineol 1,071 C10H18O 154 93 (100), 71 (80), 121 (76) MS – – 1.43 0.17

12 Fenchone 1,089 (1,087) C10H16O 152 81 (100), 69 (31), 79 (18) rI, MS – – 0.39 –

13 linalool 1,101 (1,101) C10H18O 154 43 (100), 93 (99), 71 (79) rI, MS 0.54 1.03 1.79 –

14 cis-1-Methyl-4- (1-methylethyl) -2-cyclohexen-1-ol

1,101 (1,103) C10H18O 154 93 (100), 81 (88), 71 (80) rI, MS – – – 0.36

15 nonanal 1,107 (1,104) C9H18O 142 57 (100), 70 (91), 67 (85) rI, MS – 0.04 – –

16 trans-1-Methyl-4- (1-methylethyl) -2-cyclohexen-1-ol

1,125 (1,125) C10H18O 154 43 (100), 121 (75), 93 (75) rI, MS 0.08 – – 0.06

17 Camphor 1,146 (1,146) C10H16O 152 95 (100), 108 (73), 81 (56) rI, MS 0.26 0.58 0.29 –

18 Dialin 1,154 (1,158) C10H10 130 130 (100), 129 (82), 115 (47) rI, MS – 1.41 – –

19 Umbellulone 1,163 (1,170) C10H14O 150 53 (100), 135 (69), 107 (64) rI, MS – – 0.17 –

20 Hydrocinnamic aldehyde 1,164 (1,160) C9H10O 134 91 (100), 134 (80), 92 (65) rI, MS – 1.53 – –

21 Borneol 1,171 (1,171) C10H18O 154 95 (100), 67 (20), 93 (10) rI, MS 0.92 1.64 1.92 –

22 Dihydrocarvone 1,176 (1,186) C10H16O 152 95 (100), 67 (31), 110 (26) rI, MS – – 0.07 –

23 4-Terpineol 1,180 (1,180) C10H18O 154 71 (100), 93 (99), 111 (73) rI, MS 0.52 1.00 0.98 1.63

24 p-Cymen-8-ol 1,188 (1,187) C10H14O 150 135 (100), 91 (21), 65 (14) rI, MS – – – 0.04

25 α-Terpineol 1,195 (1,197) C10H18O 154 121 (100), 136 (93), 93 (82) rI, MS 1.08 2.18 15.54 0.40

26 Myrtenol 1,202 (1,201) C10H16O 152 91 (100), 92 (76), 79 (55) rI, MS 0.51 1.14 0.22 –

27 trans-Piperitol 1,208 (1,206) C10H18O 154 83 (100), 84 (83), 139 (62) rI, MS – 0.06 – 0.07

28 cis-Carveol 1,220 (1,220) C10H16O 152 109 (100), 73 (37), 91 (35) rI, MS – – 0.16 –

29 cis-geraniol 1,224 (1,225) C10H18O 154 69 (100), 93 (44), 67 (42) rI, MS – 0.09 – –

30 β-Citronellol 1,228 (1,228) C10H20O 156 67 (100), 81 (64), 69 (59) rI, MS 0.17 0.28 – –

31 Carvone 1,244 (1,243) C10H14O 150 82 (100), 93 (66), 54 (66) rI, MS – – 0.23 –

32 3-Phenyl-2-butanone 1,245 C10H12O 148 148 (100), 105 (81), 91 (49) MS 1.92 15.34 – –

33 Thymoquinone 1,250 (1,249) C10H12O2 164 164 (100), 93 (95), 121 (82) rI, MS – – 0.34 –

34 trans-geraniol 1,251 (1,251) C10H18O 154 69 (100), 41 (84), 67 (40) rI, MS 0.28 0.75 – 0.06

35 Piperitone 1,254 (1,253) C10H16O 152 110 (100), 82 (85), 95 (78) rI, MS – 0.14 0.08 –

36 4-Phenyl-2-butanol 1,259 C10H14O 150 117 (100), 132 (89), 91 (82) MS – 0.47 – –

37 trans-Cinnamaldehyde 1,275 (1,268) C9H8O 132 131 (100), 103 (57), 132 (41) rI, MS – 7.30 – –

38 Bornyl acetate 1,284 (1,283) C12H20O2 196 95 (100), 93 (66), 136 (61) rI, MS 0.25 0.10 – –

39 Safrole 1,290 (1,287) C10H10O2 162 162 (100), 131 (40), 104 (39) rI, MS – – – 0.74

40 Carvacrol 1,290 (1,291) C10H14O 150 135 (100), 150 (37), 107 (22) rI, MS – 0.08 0.44 –

41 Cuminol 1,292 (1,291) C10H14O 150 135 (100), 150 (39), 107 (26) rI, MS – – 0.24 –

42 Thymol 1,298 (1,296) C10H14O 150 135 (100), 150 (48), 107 (22) rI, MS 0.22 0.52 – 0.04

43 2,3-Pinanediol 1,321 C10H18O2 170 111 (100), 126 (99), 108 (69) MS – – 0.23 –

Z. Ye et al.

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Table 1 continued

no. Compound Calculated rI (literature rI)

Formula Mw Mass spectra (m/z %) Identification method

relative content (%)

a B C D

44 Fumaric acid, di (2-methylallyl) ester

1,326 C12H16O4 224 55 (100), 70 (77), 98 (56) MS – – 0.40 –

45 α-limonene diepoxide 1,345 C10H16O2 168 97 (100), 55 (53), 107 (47) MS – – 1.04 –

46 δ-Elemene 1,347 (1,344) C15H24 204 121 (100), 93 (76), 136 (55) rI, MS – – – 0.05

47 α-Terpineol acetate 1,347 (1,348) C12H20O2 196 121 (100), 93 (64), 136 (60) rI, MS – – 0.70 –

48 5-Isopropenyl-2-methyl -7-oxabicyclo[4.1.0] heptan-2-ol

1,352 C10H16O2 168 97 (100), 55 (65), 107 (52) MS – – 0.95 –

49 Eugenol 1,352 (1,353) C10H12O2 164 164 (100), 149 (32), 103 (27) rI, MS – – – 1.41

50 trans-Benzalacetone 1,356 C10H10O 146 103 (100), 131 (99), 145 (61) MS 0.34 1.37 – –

51 neryl acetate 1,360 (1,360) C12H20O2 196 69 (100), 93 (81), 67 (75) rI, MS – – 0.13 0.06

52 α-gurjunene 1,371 (1,373) C15H24 204 161 (100), 105 (48), 119 (34) rI, MS 1.29 0.12 – –

53 Copaene 1,375 (1,375) C15H24 204 161 (100), 119 (70), 105 (67) rI, MS 0.55 – – 0.03

54 β-Cubebene 1,377 (1,379) C15H24 204 161 (100), 121 (39), 162 (25) rI, MS 3.62 0.33 – –

55 geranyl acetate 1,379 (1,377) C12H20O2 196 69 (100), 67 (57), 93 (43) rI, MS – – – 0.06

56 Methyl cinnamate 1,384 (1,389) C10H10O2 162 131 (100), 103 (66), 162 (62) rI, MS 1.11 3.54 – –

57 Unknown 1,394 71 (100), 111 (85), 100 (72) – – 0.49 –

58 3,4,5-Trimethoxytoluene 1,397 (1,407) C10H14O3 182 167 (100), 182 (91), 139 (44) rI, MS – 0.20 – –

59 Methyleugenol 1,408 (1,407) C11H14O2 178 178 (100), 163 (31), 107 (27) rI, MS – 0.20 – 19.10

60 Diosphenol 1,411 C10H16O2 168 69 (100), 98 (94), 126 (82) MS – – 0.37 –

61 Caryophyllene 1,417 (1,418) C15H24 204 133 (100), 91 (98), 105 (69) rI, MS 5.85 0.72 – 0.02

62 cis-Bicyclo[4.4.0] decan-1-ol-3-one

1,419 C10H16O2 168 69 (100), 98 (90), 111 (80) MS – – 0.48 –

63 4-Hydroxy-6-isopropyl -3-methyl-2-cyclohexen -1-one

1,426 C10H16O2 168 98 (100), 69 (83), 126 (61) MS – – 0.14 –

64 α-Bergamotene 1,431 (1,430) C15H24 204 119 (100), 93 (80), 91 (72) rI, MS 0.23 – – –

65 Isoeugenol 1,450 (1,451) C10H12O2 164 164 (100), 149 (20), 103 (20) rI, MS – – – 2.42

66 α-Caryophyllene 1,454 (1,454) C15H24 204 93 (100), 147 (55), 91 (46) rI, MS 19.43 2.77 – –

67 τ-Muurolene 1,473 (1,473) C15H24 204 161 (100), 105 (43), 119 (42) rI, MS 1.00 0.11 – –

68 α-amorphene 1,476 (1,475) C15H24 204 105 (100), 161 (89), 93 (54) rI, MS 0.36 – – –

69 Veratraldehyde 1,483 C9H10O3 166 166 (100), 165 (88), 77 (37) rI, MS – – – 0.05

70 β-Selinene 1,488 (1,490) C15H24 204 93 (100), 107 (94), 79 (90) rI, MS – – 0.25 –

71 Valencene 1,495 (1,496) C15H24 204 93 (100), 91 (99), 161 (80) rI, MS – – 0.41 –

72 α-Muurolene 1,496 (1,496) C15H24 204 161 (100), 105 (99), 91 (42) rI, MS 0.44 – – –

73 Methylisoeugenol 1,501 (1,491) C11H14O2 178 178 (100), 107 (39), 163 (37) MS – – – 6.82

74 β-Bisabolene 1,507 (1,508) C15H24 204 93 (100), 41 (71), 91 (70) rI, MS 0.29 0.04 0.34 –

75 τ-Cadinene 1,510 (1,513) C15H24 204 161 (100), 105 (40), 119 (34) rI, MS 0.64 – – –

76 δ-Cadinene 1,516 (1,519) C15H24 204 161 (100), 204 (74), 119 (68) rI, MS 2.29 0.35 – 0.02

77 Unknown 1,520 161 (100), 204 (72), 159 (65) 1.38 – – –

78 Myristicin 1,529 (1,528) C11H12O3 192 192 (100), 91 (20), 119 (18) rI, MS – – – 16.69

79 1,2,3,4,4a,7-Hexahydro-1,6-dimethyl-4-(1-methylethyl)-naphthalene

1,530 (1,528) C15H24 204 161 (100), 119 (96), 105 (94) rI, MS 0.52 0.26 – –

80 Selina-3,7(11)-diene 1,533 (1,527) C15H24 204 161 (100), 105 (82), 204 (73) rI, MS 0.41 – – –

81 Unknown 1,538 157 (100), 161 (59), 200 (36) MS 0.45 – – –

82 Elemol 1,555 (1,554) C15H26O 222 67 (100), 93 (90), 69 (73) rI, MS – – 0.32 –

83 Elemicin 1,559 (1,558) C12H16O3 208 208 (100), 193 (55), 209 (21) rI, MS – – – 33.22

84 nerolidol 1,562 (1,564) C15H26O 222 93 (100), 69 (97), 41 (93) rI, MS 0.69 – 0.39 –

Microwave-Assisted Extraction/Dispersive Liquid–Liquid Microextraction

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Table 1 continued

no. Compound Calculated rI (literature rI)

Formula Mw Mass spectra (m/z %) Identification method

relative content (%)

a B C D

85 Dodecanoic acid 1,570 (1,567) C12H24O2 200 73 (100), 129 (86), 55 (83) rI, MS – – – 0.04

86 3-Methoxypiperonal 1,574 (1,570) C9H8O4 180 179 (100), 180 (65), 57 (29) rI, MS – – – 0.04

87 Caryophyllene oxide 1,579 (1,581) C15H24O 220 79 (100), 91 (95), 93 (95) rI, MS 0.56 0.31 – –

88 Viridiflorol 1,590 (1,593) C15H26O 222 67 (100), 69 (75), 119 (69) rI, MS – – 0.47 –

89 Carotol 1,599 (1,596) C15H26O 222 161 (100), 105 (34), 67 (29) rI, MS 4.33 1.80 – –

90 Methoxyeugenol 1,600 (1,609) C11H14O3 194 194 (100), 91 (22), 119 (20) rI, MS – – – 5.30

91 p-Ethyldiphenylmethane 1,603 C15H16 196 167 (100), 196 (51), 168 (12) MS – – – 0.06

92 1,5,5,8-Tetramethyl -12-oxabicyclo[9.1.0] dodeca-3,7-diene

1,607 (1,607) C15H24O 220 67 (100), 96 (64), 109 (55) rI, MS 2.04 0.86 – –

93 Unknown 1,607 67 (100), 93 (51), 79 (49) – – 0.39 –

94 Cubenol 1,625 (1,625) C15H26O 222 161 (100), 119 (87), 105 (52) rI, MS 0.68 0.51 0.18 –

95 Unknown 1,630 67 (100), 91 (61), 95 (52) 1.09 0.92 – –

96 τ-Cadinol 1,639 (1,642) C15H26O 222 161 (100), 204 (45), 105 (30) rI, MS 0.57 – – –

97 Isoelemicin 1,648 C12H16O3 208 208 (100), 193 (99), 133 (23) MS – – – 0.95

98 α-Cadinol 1,653 (1,654) C15H26O 222 104 (100), 204 (86), 161 (78) rI, MS 0.81 0.76 – –

99 Propioveratrone 1,654 C11H14O3 194 165 (100), 194 (38), 137 (17) MS – – – 0.11

100 3-(1,3-Benzodioxol -5-yl)-2-propenal

1,661 C10H8O3 176 176 (100), 89 (50), 147 (49) MS – – – 0.02

101 Juniper camphor 1,663 (1,675) C15H26O 222 189 (100), 204 (94), 161 (94) rI, MS – – 0.49 –

102 β-Bisabolol 1,670 (1,673) C15H26O 222 67 (100), 93 (94), 82 (85) rI, MS – – 0.49 –

103 Bulnesol 1,670 (1,666) C15H26O 222 59 (100), 204 (79), 95 (77) rI, MS 1.55 0.89 – –

104 Unknown 1,687 69 (100), 67 (99), 55 (83) – – 0.54 –

105 cis-α-Santalol 1,695 C15H24O 220 93 (100), 94 (75), 91 (75) – – 0.39 –

106 1,3-dimethoxy-5-(1-propenyl)-benzen-2-acetate

1,696 C13H16O4 236 194 (100), 91 (22), 119 (20) MS – – – 0.24

107 2-acetyl-1-tetralone 1,699 C12H12O2 188 131 (100), 155 (88), 170 (72) MS 0.36 3.33 – –

108 Farnesol 1,716 (1,713) C15H26O 222 69 (100), 41 (76), 81 (68) rI, MS 24.35 12.83 – –

109 2,6-Diisopropylnaphthalene 1,717 (1,717) C16H20 212 197 (100), 132 (62), 212 (50) MS – – 0.21 –

110 Unknown 1,736 69 (100), 67 (53), 93 (53) – 0.24 – –

111 2,6-Di-tert-butyl-4-ethyl-phenol

1,753 (1,760) C16H26O 234 85 (100), 57 (94), 71 (80) MS – – 0.42 –

112 Unknown 1,763 195 (100), 224 (47), 196 (11) – – – 0.34

113 Unknown 1,768 67 (100), 109 (57), 95 (56) – 0.33 – –

114 Myristic acid 1,774 (1,773) C14H28O2 228 129 (100), 55 (84), 73 (77) rI, MS – – – 2.26

115 Methyl 3-(3,4,5- trimethoxyphenyl)- 2-oxiranecarboxylate

C13H16O6 268 197 (100), 268 (92), 169 (53) MS – – – 0.12

116 Farnesyl acetate C17H28O2 264 69 (100), 41 (63), 81 (58) MS 0.77 0.24 – –

117 Unknown 93 (100), 95 (43), 107 (41) – 0.29 – –

118 Unknown 165 (100), 222 (57), 166 (14) – – – 0.18

119 Unknown 91 (100), 95 (93), 79 (87) – – 0.15 –

120 Unknown 206 (100), 177 (32), 178 (29) – – – 0.19

121 Methyl 3,4- dimethoxycinnamate

C12H14O4 222 222 (100), 179 (68), 151 (28) MS – – – 0.10

122 7,9-Di-tert-butyl- 1-oxaspiro[4,5]deca -6,9-diene-2,8-dione

C17H24O3 276 205 (100), 148 (94), 82 (86) MS – – 0.19 –

123 Unknown 91 (100), 254 (72), 239 (64) – – 0.15 –

124 Unknown 190 (100), 147 (85), 162 (79) – 0.41 – –

Z. Ye et al.

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uses a large amount of water and requires a long time and high energy [17]. a series of compounds with high boiling point (such as 7-phenyl-6-heptenophenone, 2-benzylidene-1-tetralone, pinocembrin, and 1,7-diphenyl-4,6-heptadien-3-one) were not, or only slightly, extracted by HD, while they were extracted by the proposed method. However, α-phellandrene, m-cymene, α-pinene, camphene, β-pinene, and limonene, which are a series of volatile compounds with low boiling point, were not identified from the EO extracted by MaE-DllME, whereas they were identi-fied from the EO extracted by HD. This is probably due to the process of solvent evaporation of the ChromatoProbe direct sample introduction (DSI) device, in which low-vol-atile compounds evaporate with solvent. Hence, the MaE-DllME method followed by DSI-gC–MS is more suitable for analysis of semivolatile compounds.

as is well known, the conventional HD method requires a long time to isolate the essential oil and a large volume of organic solvent to perform dissolution and dilution. Obvi-ously, the proposed method needs a short time and little solvent and consumes less plant material. SFME is a novel alternative based on the combination of microwave heat-ing and dry distillation under atmospheric pressure. The method [16–18] avoids use of organic solvent or water, but it also takes a longer time and consumes a larger amount of plant material than the proposed method. Therefore, compared with SFME and the conventional HD technique,

MaE-DllME is a simple, rapid, low-cost, efficient, and environmentally friendly method for isolation of essential oils from TCMs.

Comparison of Constituents of EOs Isolated by MaE-lDS-DllME from the Three Species of Cardamom

The optimal MaE-DllME parameters were applied for isolation of EOs from the three species of cardamom, followed by DSI-gC–MS analysis. Figure 5b–d shows that the total ion profiles of the chemical composition of the three EOs were very different. From Table 1, we find the following results: (1) 49 main compounds represent-ing 95.72 % were identified in the EO of Semen Alpiniae Katsumadai, including 3-phenyl-2-butanone, farnesol, 1,7-diphenyl-4,6-heptadien-3-one, pinocembrin, trans-cinnamaldehyde, eucalyptol, methyl cinnamate, 2-acetyl-1-tetralone, and α-caryophyllene; (2) 43 main compounds representing 98.77 % were identified in the EO of Fructus Amomi Rotundus, the major components being eucalyp-tol, α-terpineol, borneol, linalool, and cis-β-terpineol; (3) 40 main compounds representing 94.92 % were identified in the EO of Semen Myristicae, with elemicin, methyl-eugenol, myristicin, methylisoeugenol, methoxyeugenol, isoeugenol, myristic acid, 4-terpineol, eugenol, and safrole being the major compounds. The results also show that only

Table 1 continued

no. Compound Calculated rI (literature rI)

Formula Mw Mass spectra (m/z %) Identification method

relative content (%)

a B C D

125 n-Hexadecanoic acid C16H32O2 256 55 (100), 41 (98), 129 (77) MS 0.49 0.70 – 0.09

126 Unknown 266 (100), 181 (28), 223 (22) – – – 0.12

127 7-Phenyl-6-heptenophenone C19H20O 264 105 (100), 91 (98), 264 (86) MS – 1.40 – –

128 Oleic acid C18H34O2 282 55 (100), 69 (88), 41 (85) MS 0.06 0.90 – –

129 2-Benzylidene-1-tetralone C17H14O 234 233 (100), 91 (31), 234 (19) MS – 0.36 – –

130 Pinocembrin C15H12O4 256 256 (100), 255 (66), 124 (48) MS – 7.36 – –

131 1,7-Diphenyl-4,6-heptadien-3-one

C19H18O 262 262 (100), 157 (66), 171 (49) MS 0.29 10.55 – –

132 Unknown 91 (100), 104 (80), 105 (67) – 0.62 – –

133 Unknown 91 (100), 104 (89), 105 (82) – 2.39 – –

134 Unknown 165 (100), 194 (60), 137 (27) – – – 0.19

135 Unknown 356 (100), 281 (55), 313 (42) – – – 0.33

136 Unknown 326 (100), 324 (97), 325 (27) – – – 0.75

137 Unknown 209 (100), 208 (26), 193 (18) – – – 0.20

138 Unknown 194 (100), 237 (55), 193 (37) – – – 1.95

139 Unknown 194 (100), 193 (18), 195 (14) – – – 0.83

140 Desmethylnomifensine C15H16n2 224 194 (100), 195 (40), 224 (33) MS – – – 1.20

141 Clionasterol C29H50O 414 329 (100), 414 (90), 57 (86) MS – – 0.62 –

a Semen Alpiniae Katsumadai (HD); B Semen Alpiniae Katsumadai (MaE-DllME); C Fructus Amomi Rotundus (MaE-DllME); D Semen Myristicae (MaE-DllME); “–” not detected

Microwave-Assisted Extraction/Dispersive Liquid–Liquid Microextraction

1 3

two compounds (4-terpineol and α-terpineol) were found to be present in the EOs of all three varieties of cardamom. The same 11 compounds were identified in the EOs of Semen Alpiniae Katsumadai and Fructus Amomi Rotun‑dus. Between Alpiniae Katsumadai and Semen Myristicae, nine compounds were the same. The number of common compounds was five among Fructus Amomi Rotundus and Semen Myristicae.

The main bioactive compound in Semen Alpiniae Kat‑sumadai and Fructus Amomi Rotundus is eucalyptol, which has been used as a febrifuge, antibiotic, and antibacterial. as can be seen from Table 1, in Fructus Amomi Rotundus, the relative content of eucalyptol was 64.26 %, which was higher compared with Alpiniae Katsumadai. However, we could not find eucalyptol in Semen Myristicae. Myr-isticin and safrole, which have an antibacterial effect, are the main bioactive compounds in Semen Myristicae. The relative contents of myristicin and safrole were 16.69 and 0.74 %, respectively. However, these two compounds were not identified in the other samples. These qualitative and quantitative differences in the chemical components, espe-cially in the main bioactive compounds, may lead to dif-ferent pharmacologic effects of the three species of carda-mom to some extent. In fact, bioactive compounds play an important role in the treatment of some diseases by TCMs. Therefore, there was a significant difference between Semen Alpiniae Katsumadai or Fructus Amomi Rotundus and Semen Myristicae. The MaE-DllME method cou-pled with DSI-gC–MS is a feasible alternative for quality assessment of traditional Chinese medicines.

Conclusions

In this work, MaE-lDS-DllME combined with DSI-gC–MS was successfully developed and for the first time applied for extraction and determination of essen-tial oils from three species of cardamom. Microwave-assisted extraction supplied sufficient energy to favor release of essential oil from the plant matrix in a short time. For DllME process, a low density solvent (tolu-ene) was selected as extraction solvent instead of chlo-rinated solvents, which could expand the applicability of DllME to a wider range of solvents. a cheap and dis-posable polyethylene Pasteur pipette was employed as a vessel for extraction, preconcentration, and collection of target analytes, offering advantages such as avoid-ance of carryover problems and simplicity of withdraw-ing the low-density extraction solvent. In addition, the whole sample preparation consumed small amounts of plant material (0.1 g) and organic solvents (100 μl tolu-ene and 0.5 ml methanol) and required a much shorter time (~10 min) compared with conventional methods.

Compared with the conventional HD method, MaE-lDS-DllME-DSI-gC–MS is a simple, rapid, low-cost, and environmentally friendly method for analysis of essential oil from TCMs, and the essential oil is more valuable and composed of highly odoriferous aromatic compounds. Comparison of the main chemical constitu-ents of the essential oil showed a significant difference between Semen Alpiniae Katsumadai or Fructus Amomi Rotundus and Semen Myristicae. Therefore, the MaE-DllME method coupled with DSI-gC–MS is also a feasible alternative for quality assessment of traditional Chinese medicines.

Acknowledgments Support of this work by the Ministry of Science and Technology of Zhejiang Province (no. 2010r10044), the Sprout Talented Project Program (2011443), and the Key Innovation Team of Science and Technology in Zhejiang Province (2010r50018) is grate-fully acknowledged.

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