Chinese Chemical Letters  2016, Vol.27 Issue (01): 178-184   PDF    
Magnetic nanoparticles used in headspace extraction coupled with DSI-GC-IT/MS for analysis of VOCs in dry Traditional Chinese Medicine
Jing Niea, Yuan-Jie Tenga, Zu-Guang Lia , Wen-Han Liua , Maw-Rong Leeb    
a College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China;
b Department of Chemistry, National Chung-Hsing University, Taichung 40227
Abstract: A novel magnetic method using polystyrene modified magnetic nanoparticles to perform thermoheadspace extraction was successfully developed for extraction and preconcentration of volatile organic components in dry Traditional Chinese Medicine(TCM) based on gas chromatography-ion trap/mass spectrometry with a ChromatoProbe direct sample introduction device. The dried fruit of Amomum testaceum Ridl. was used as the object TCM. The optimum parameters of headspace magnetic solid-phase extraction were investigated, in which desorption solvent ethyl acetate played a key role in this method, and the headspace extraction temperature of 90℃ and the headspace extraction time of 15 min finally decided. Headspace solid-phase microextraction method was also used to analyze volatile compounds in the TCM to compare with the proposed method. The results show that 60 components were identified totally by two methods; most of the low boiling point chemical compounds are isolated by this new method. In this work, an environmental-friendly and cheap analytical method was established, and a new approach to analyze volatile compounds in dry Traditional Chinese Medicine was also provided.
Key words: Magnetic nanoparticles     Direct sample introduction     Volatile components     Traditional Chinese Medicine    
1.Introduction

Traditional Chinese Medicine is an integral part of Chinese culture. Nowadays,both TCM and western medicine have been used to provide medical therapy and health care in China. Hitherto, many researchers have studied the volatile components in different TCMs. Amomum testaceum Ridl. has a long medicine history in TCMs and has been discussed by some groups [1, 2]. The fruit has been used in promoting digestion,warming body, activating spirit and preventing bacteria. Therefore,it has been applied to treatment for abdominal pain,diarrhea,etc. Considering the volatile and special flavors,it is also regarded as condiment, fragrance,an ingredient of perfume [2, 3].

Most analytical methods are available for the determination of the volatile constituents from dried fruits’ or seeds’ essential oil in plants and herbal medicines. The determination of volatile constituents has been considered a way of studying effective ingredients in Traditional Chinese Medicine [4]. Various pretreatment methods,such as Soxhlet extraction,steam distillation, hydro-distillation and solvent extraction can be used for that purpose. Those conventional methods are labor-intensive,timeconsuming and requires large amount of samples. Moreover,low extraction efficiency is the most fatal problem. Some new techniques [5, 6, 7] in the identification of volatile constituents have been developed,which typically use less solvent,time and energy. However,microwave-assisted techniques and ultrasound-assisted [5, 6] techniques need professional equipment to pretreat samples before detection using analytical instruments. Solid-phase microextraction (SPME) is a solvent-free technique proposed by Arthur and Pawliszyn in 1990 [8]. Through unremitting efforts,HS-SPME technique has been widespreadly accepted in analyzing of volatile compounds emitted from the matrix and the analytes which are pre-concentrated on solid phase microextraction fiber,and the corresponding device has been realized commercialization. However,there are still many drawbacks in headspace solidphase microextraction,expensive device,sensitive damaged fiber and sample carryover is sometimes difficult to eliminate. Hence,a novel extraction method for extracting volatile constituents in TCMs should be developed.

Magnetic solid-phase extraction (MSPE) as a promising sample preparation has attracted wide public concern in some fields over the recent years [9, 10, 11]. This method utilizes analytes through hydrophobic effect,p-p conjugated interaction,hydrogen bonding and ion-exchange on modified magnetic sorbents which have good dispersion in liquids,and they can be separated from the matrix or sample under the action of external magnetic field. It will adsorb more target compounds if the material size reaches nano-scale (nanoparticles,nanowires,nanotubes,etc.) because of the huge specific surface area. "Core-shell" structure is a popular model of these sorbents in MSPE,especially,magnetic nanoparticles based on iron oxides (core) coated with appropriate groups or composites (shell) have been applied to analysis of environmental and food samples [11]. In addition,these sorbents can be washed after work by solvents and recycled. Generally,according to the mechanism of the extraction,the key is not on the "core" but on the "shell". Relevant researches mostly focus on synthesis of new sorbents in accord with target compounds’ structures but not on the method itself [12, 13]. Polymers have a bright future in material field and have wide applications in analytical chemistry for the following reasons that magnetism does not decrease obviously like other materials and has adsorptive selectivity to some extent. Polystyrene is such a material in our views that has a long-pstructure. As a result of this sorbent with surfactant which make particles more dispersive in desorption solvent,but the eluate may contains interference which is harmful to gas chromatography instrument and capillary column. Thus,to overcome this drawback,we adopted a ChromatoProbe direct sample introduction (DSI) device proposed by Amirav et al. [14]. Based on dirty sample introduction into a small glass vial and by direct insertion probes into a temperature-programmable GC injector,the analytes can be applied for following analysis. With the temperature rising,the target analytes are volatilized into GC column while nonvolatile compounds remains in vial.

In this present study,our task is to develop a novel, environmental friendly and low-cost method based on polystyrene coated magnetic nanoparticles (PSt/MNPs) to extract the volatile compounds in dry TCM,and the dried fruit of A. testaceum Ridl. as the model. In consideration of the glass transition temperature of the polystyrene,we adopted an approach to make the nanoparticles indirectly contacts the sample,and to make the vaporized volatile compounds transfer to nanoparticles surface area under a condition of heating. Besides,to protect the analytical apparatus, DSI-GC/MS was employed for the determination of volatile constituents. During the experiments,several parameters such as elution solvent,headspace extraction time and headspace extraction temperature were optimized. Finally,we have established a magnetic method coupled with DSI-GC-IT/MS which was successfully performed in the determination of VOCs in the dry Traditional Chinese Medicine samples.

2.Experimental 2.1. Materials and reagents

The dried fruit of A. testaceum Ridl. was bought from Wulin Medicine Shop (Hangzhou,China) and authenticated by the College of Pharmaceutical Science,Zhejiang University of Technology. Analytical-grade ferric chloride (FeCl3⋅6H2O),oleic acid, sodium dodecyl benzene sulfonate (SDBS) and metals basis (99.95%) ferrous chloride (FeCl2⋅4H2O) were purchased from Aladdin (Shanghai,China). Styrene and n-hexane were supplied by Lingfeng Chemical Reagent Co.,Ltd. (Shanghai,China). Potassium peroxydisulfate (KPS) was brought from Jinhui Taiya Chemical Reagent Co.,Ltd. (Tianjin,China). Methacrylic acid (MA) was obtained from Mitsui Chemicals (Shanghai,China). Sodium hydroxide (NaOH) was supplied by Xiaoshan Chemical Reagent Factory (Hangzhou,China). Acetonitrile was obtained from Tjshield (Tianjin,China) and ethyl acetate was brought from Shuanglin Chemical Reagent (Hangzhou,China).

The manual holder and SPMEfiberwere purchased fromSupelco (Bellefonte,PA,USA),and 50/30 mm divinylbenzene/carboxen/ polydimethylsiloxanewe (DVB/CAR/PDMS) was adopted. Purified water was prepared by HUMAN UP900 purification system(Human Techpia,Korea).

2.2. Preparation of polymer coated magnetic nanoparticles

The method referred to previous chemical coprecipitation report [10] which contains two steps,the first step is using coprecipitation to get the magnetic nanoparticles,and the second step is polymerization process. Briefly,1 g FeCl2⋅4H2O and 2.6 g FeCl3⋅6H2O were dissolved in 12.5 mL purified water and 0.4 mL hydrochloric acid (12 mol/L),then the mixture was added into round bottom flask containing 125 mL NaOH (1.5 mol/L) and 1 mL oleic acid with nitrogen gas stirring under 80 ℃for 1 h. Then 100 mL SDBS (0.007 mol/L) was added into the round bottom flask under the same condition for 30 min. After that,the magnetic nanoparticles were introduced into a steady uniform system of 100 mL purified water with 6 mL styrene and 0.6 mL methacrylic acid,0.1 g KPS and then the mixture was treated with nitrogen gas stirring under 70 ℃for 11 h. The finally product was washed and put in vacuum drying oven 24 h.

2.3. Headspace magnetic solid-phase extraction (HS-MSPE)

procedure A sample of 1.0 g powdered dry A. testaceum Ridl. fruit which are sieved by 80 mesh sieve was put in a 10 mL vial and evenly spread out on the wall side. 10 mg PSt-MNPs were placed into a 2 mL vial like the way of sample in the vial. Then,making the 2 mL vial immobilized onto the sample’s vial. Next,these vials were put into a heating and drying oven under 90 ℃for 15 min. After that, the 2 mL vial was taken out and added 0.5 mL ethyl acetate by hand shaking for 5 min. And then,the particles were moved to one side with extra-magnetic field and 1 mL eluent taken from the vial by a 10 mL syringe was injected into glass tubular of the direct sample introduction device. Finally,the glass tubular loaded immediately in the direct sample introduction device and the device was inserted in the injection of gas chromatography for analysis. The whole HS-MSPE whole procedure was shown Fig. 1.

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Fig. 1.Magnetic nanoparticles used in headspace extraction.
2.4. Headspace solid-phase microextraction

A sample of 1.0 g powdered dry fruit of medicine was put in a 10 mL vial. The SPME needle pierced the polymer films of the vial; the fiber was extended through the needle and exposed to the headspace above the sample of TCM by an adapter. The volatiles were extracted under the same conditions with HS-MSPE. After extraction,the fiber was inserted into the injector port of the GC- MS system.

2.5. Gas chromatography-mass spectrometry

GC-MS analysis was performed on a Varian GC 3800 (Varian, Walnut Creek,CA,USA) equipped with a 1079 temperatureprogrammable injector connected to a Varian Saturn 2000 ion-trap mass spectrometer. The GC separation was achieved on a DB-5 fused-silica column (30 m × 0.25 mm × 0.25 mm) purchased from J&W Scientific (Folsom,CA,USA). High-purity (≥99.999%) helium gas from Hangzhou Special gas production Co.,Ltd. (Hangzhou, China). The temperature of the column was held at 40 ℃for 9.6 min,increased at 3 ℃/min to 250 ℃and held for 2.5 min. Electronic flow control (EFC) was used to maintain a constant helium carrier gas flow of 0.8 mL/min. For the volatile compounds from the extraction,sample introduction was performed using a direct sample introduction (DSI) device (ChromatoProbe,Varian, USA) attached to a 1079 programmable injector with injection volume of 1.00 mL. The injector temperature was maintained at 70 ℃for 2.5 min with a 50:1 split to evaporate the solvent, increased at 100 ℃/min to 280 ℃in splitless mode and held for 2.4 min,after which the injector cooled back to 70 ℃and the split ratio was 20:1. Full-scan spectra were acquired in electron ionization (EI,70 eV) in the mass range of 40-650 m/z with scan time of three uscans,solvent delay of 7 min,ion-trap temperature of 200 ℃,manifold temperature of 150 ℃and transfer-line temperature of 280 ℃. Kovats retention indices (RI) were calculated for all volatile components using a homologous series of n-alkanes (C6-C18).

2.6. Components identification

The GC-MS data were processed on the Saturn GC/MS workstation-Saturn Vies version 5.52. Identification of the constituents was based on the comparison of the obtained mass spectra data with those of references compounds in the data system of the Wiley library and NIST Mass Spectral Search Program (NIST 2011 version mass spectral database; National Institute of Standards and Technology,Washington,DC,USA) connected to a Saturn 2000 mass spectrometer and homemade library mass spectra built from pure substances and components of known substances and MS literature. The constituents were confirmed by comparison of their Kovats retention indices relative to C6-C18 nalkanes (Sigma Chemical Co.,St. Louis,MO,USA) with those of authentic standards or by publication literature. Identification was assumed when a good match of mass spectrum and RI were achieved. Quantitative analysis in percent was performed by peak area normalization measurements.

3.Results and discussion 3.1. The mechanism of adsorption

To our best knowledge,the volatile substances of nature product in plant are terpenoids or their derivativeswhichcontainp-por p-p conjugated bonds and polystyrene has a long-p structure. Through the intermolecular forces or electrostatic forces and the volatile compounds are easily adsorbed by polystyrene [15, 16].

3.2. Optimization of parameters in HS-MSPE

The VOCs in dried A. testaceum Ridl. fruit vapored to the headspace in a period of time by heating,then the analytes in the headspace were transferred to the surface of magnetic nanoparticles coated PSt,and then the extraction and concentration were simultaneously accomplished. After heating,the nanoparticles with the analytes were eluted by desorption solvent. In this study, the type of desorption solvent,and the experimental parameters of extraction temperature and time were optimized using the "single-factor-at-a-time" method. Each experimental run was performed in triplicate and peak area of the main representative compounds was employed as the response in the optimization procedure.

An appropriate desorption solvent is essential for the determination of the volatile compounds from medicine. Firstly,the solvent should have a good solubility with them. Secondly,it would be better that PSt/MNPs disperse well in the selected solvent. Thirdly,ChromatoProbe direct sample introduction requires the solvent with lower boiling point so that the compounds can enter the column rapidly. Based on these criterions,we chose acetonitrile,ethyl acetate and n-hexane with different polarity as candidates for the following study. The headspace extraction condition was 90 ℃for 15 min,and the relative amounts of six major compounds ((1S)-6,6-dimethyl-2- methylene-bicyclo[3.1.1]heptane (HE),eucalyptol (EU),L-fenchone (FCh),4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol (CO),pcymen- 8-ol (PC),(-)-1,7-dimethyl-7-(4-methyl-3-pentenyl)-tricyclo[ 2.2.1.0(2,6)] heptanes (TRI)) obtained using 0.5 mL of the desorption solvent to elute particles are shown in Fig. 2a,which indicated that ethyl acetate possessed the best extraction efficiency and was chosen as the desorption solvent for eluting the magnetic nanoparticles. It was found that the peaks obtained by using ethyl acetate were much more obvious than the others,of which the possible reason is that ethyl acetate has p-p structure that can elute analytes from the PSt more easily.

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Fig. 2.Effects of each parameter on extraction efficiency of HE, EU, Ch, CO, PC, TRI from A. testaceum Ridl. (n = 3). a) Desorption solvent, with the same headspace extraction temperature 90 ℃, extraction time 15 min and desorption time 5 min. b) Headspace extractiom temperature, with the same desorption solvent ethyl acetate, extraction time 15 min and desorption time 5 min. c) Headspace extraction time, with the same desorption solvent ethyl acetate, extraction temperature 90 ℃and desorption time 5 min. HE, EU, Ch, CO, PC, TRI are short for 6,6-dimethyl-2-methylene-bicyclo[3.1.1]heptanes, eucalyptol, L-fenchone, 4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol, p-cymen-8-ol, 1,7-dimethyl-7-(4-methyl-3-pentenyl)-tricyclo[2.2.1.0(2,6)] heptanes, respectively.

Furthermore,the headspace extraction temperatures (70 ℃, 90 ℃and 110 ℃) were studied. It was found that higher temperature resulted in a decreased analytical response. Moreover, higher temperature may leads the constituents desorption from the surface of nanoparticles. However,the lower temperature cannot entirely make analytes out of the sample. Thus,90 ℃was selected to ensure the formation of stable and reproducible results, albeit with little loss of polymer material. The relevant chromatogram is shown in Fig. 2b.

As can be seen from Fig. 2c,the analytical signals firstly increased when headspace extraction time was increased from 5.0 to 15.0 min,and then dramatically decreased for HE,EU,FCh TRI and slightly increased for the PC,CO when the headspace extraction time further increased to 20.0 min,with the same headspace extraction temperature of 90 ℃and 0.5 mL of the desorption solvent ethyl acetate to elute particles. Therefore, headspace extraction time of 15.0 min was preferred in this work. We also researched desorption time of eluting particles,and the results shows little difference when desorption time is 5 min or 10 min with shaking. Therefore,desorption time of 5.0 min was preferred in this work.

3.3. Method precision of HS-MSPE

The method precision was expressed by the relative standard deviation (RSD) value. The peak areas of six representative volatile compounds in three replicated analytes were used for calculating the RSD values in the optimum condition. The RSD values of 6,6- dimethyl-2-methylene-bicyclo[3.1.1]heptane (HE),eucalyptol (EU), L-fenchone (FCh),4-methyl-1-(1-methylethyl)-3-cyclohexen-1-ol (CO),p-cymen-8-ol (PC),1,7-dimethyl-7-(4-methyl-3-pentenyl)- tricyclo[2.2.1.0(2,6)] heptanes (TRI) were 8.5,9.6,7.2,5.5,6.2,and 8.8%,respectively,which are relatively satisfactory. Theresults show that HS-MSPE coupled with DSI-GC/MS has good precision.

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Fig. 3.TIC of volatile organic components (VOCs) of A. testaceum Ridl. by HS-MSPE. The extraction conditions are desorption solvent ethyl acetate, headspace extraction temperature 90 ℃, extraction time 15 min and desorption time 5 min.
3.4. Determination of volatile constituents in A. testaceum Ridl. by HS-MSPE and HS-SPME

The optimized HS-MSPE parameters were applied for the extraction of VOCs from A. testaceum Ridl. followed by DSI-GC/MS. Total ion chromatogram (TIC) of the volatile components separated from A. testaceum Ridl. is presented in Fig. 3 and the relevant main chemical compositions are listed in Table 1. The chemical compositions were identified by mass fragment peaks and Kovats index,and the relative content of each peak was calculated by the normalization method. From Table 1,we can find the following results: (1) analysis of the VOCs extracted from the medicine by HS-MSPE and HS-SPME resulted in characterization of 45 and 37 organic compounds,accounting for 93.45% and 95.49% of the total composition,respectively. (2) The main constituents identified by HS-MSPE were cis-1-methyl-4-(1- methylethyl)-2-cyclohexen-1-ol (35.22%),eucalyptol (27.33%), benzaldehyde (11.43%),p-cymen-8-ol (2.72%),1-methyl-4-(1- methylethylidene)-cyclohexene (1.73%),1-methyl-4-(1-methylethyl)- 1,4-cyclohexadiene (1.73%). The main constituents identified by HS-SPME were eucalyptol (27.11%),a-terpineol (13.33%),2,4a,5,6,7,8,9,9a-octahydro-3,5,5-trimethyl-9-methylene- 1H-Benzocycloheptene (11.33%),calamenene (5.89%),bselinene (4.88%),[s-(E,E)]-1-methyl-5-methylene-8-(1-methylethyl)- 1,6-cyclodecadiene (3.62%),(-)-1,7-dimethyl-7-(4- methyl-3-pentenyl)-tricyclo[2.2.1.0(2,6)]heptanes (2.59%).

Table 1
Main volatile organic components identified in A. testaceum Ridl.
3.5. Comparison of HS-MSPE and HS-SPME for the analysis of VOCs in A. testaceum Ridl

As can be seen from Table 1,several components are different between two methods,most of the chemical compounds with low boiling point were isolated by HS-MSPE and chemical compounds with high boiling point were extracted by HS-SPME. The results show that the same 22 compounds including the main bioactive compounds in A. testaceum Ridl. were obtained by both of the two methods,although the relative concentrations of the identified compounds were different.

4.Conclusion

In this work,polystyrene modified magnetic nanoparticles being used under thermo-headspace extraction combined with gas chromatography-ion trap/mass spectrometry (GC-IT/MS) with a ChromatoProbe direct sample introduction (DSI) device was successfully developed and applied for determination of volatile compounds in TCMs for the first time. A new pretreatment method was applied to extract volatile organic components in dry TCM,of which the experimental results prove that HS-MSPE is a novel,environmental friendly and cheap method for analyzing volatile constituents in dry TCMs,and the constituents were composed of high olefin compounds. Therefore,the HS-MSPE-GC/ MS-DSI can be employed to other target analytes in dry or dirty matrix.

Acknowledgments

Support of this work by the Department of Education of Zhejiang Province (No. Pd2013016),Hangzhou Qianjiang Distinguished Experts Project (2014),the Science and Technology Department of Zhejiang Province (No. 2015C32006),Key Laboratory of Detection for Pesticide Residues of Ministry of Agriculture Project,and the Sprout Talented Project Program (No. 2011443) is gratefully acknowledged.

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