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Journal of Chromatography A, 1375 (2015) 1–7

Contents lists available at ScienceDirect

Journal of Chromatography A

j o ur na l ho me page: www.elsev ier .com/ locate /chroma

raphene oxide bound silica for solid-phase extraction of 14olycyclic aromatic hydrocarbons in mainstream cigarette smoke

ui Shia,b, Lihong Yanb, Tongguang Xub, Dongye Liub, Yongfa Zhua,∗, Jun Zhoub,∗∗

Department of Chemistry, Tsinghua University, Beijing 100084, ChinaBeijing Third Class Tobacco Supervision Station, Beijing 101121, China

r t i c l e i n f o

rticle history:eceived 10 September 2014eceived in revised form0 November 2014ccepted 21 November 2014vailable online 27 November 2014

eywords:

a b s t r a c t

Polycyclic aromatic hydrocarbons (PAHs) were considered as a source of carcinogenicity in mainstreamcigarette smoke (MSS). Accurate quantification of these components was necessary for assessing publichealth risk. In our study, a solid-phase extraction (SPE) method using graphene oxide (GO) bound sil-ica as adsorbent for purification of 14 PAHs in MSS was developed. During SPE process, large matricesinterferences of MSS were adsorbed on SPE column. The result of FTIR spectra demonstrated that thesematrices interferences were adsorbed on GO mainly through O H and C O groups. The concentrationsof PAHs in MSS extract were determined by gas chromatography–mass spectrometry (GC–MS). The limit

raphene oxideolid phase extractionolycyclic aromatic hydrocarbonsas chromatography–mass spectrometry

of detection (LOD) and limit of quantification (LOQ) of the developed method for 14 PAHs ranged from0.05 to 0.36 ng/cig and 0.17 to 1.19 ng/cig, respectively. The accuracy of the measurement of 14 PAHs wasfrom 73 to 116%. The relative standard deviations of intra- and inter-day analysis were less than 7.8%and 13.9%, respectively. Moreover, the developed method was successfully applied for analysis of realcigarette containing 1R5F reference cigarette and 12 top-selling commercial cigarettes in China.

© 2014 Elsevier B.V. All rights reserved.

. Introduction

Investigation of harmful and carcinogenic compound in MSSas a critical step aiming to the reduction of harmful health effects

n smokers. It is well known that MSS contain many mutagenicnd carcinogenic chemicals [1]. Of these, PAHs are considered toe one of most toxic and carcinogenic compounds [2]. Their most

mportant source is the incomplete combustion of organic materi-ls during smoking [3,4].

Till now, about 5000 compounds had been identified in MSS [5].raditional methods for PAHs analysis in MSS had been faced chal-enges in obtaining high method precision, accuracy, and sensitivityue to numerous matrices interferences. Therefore, enrichmentnd separation of the PAHs molecules from sample matrices wereery important. Nowadays, various purification methods had beensed in MSS pretreatment, such as solid-phase extraction (SPE),

oxhlet extraction, microwave-assisted techniques, acceleratedolvent extraction and other process [6–12]. Among these meth-ds, SPE was a superior and widely used method because of its

∗ Corresponding author. Tel.: +86 010 62783586; fax: +86 10 62787601.∗∗ Corresponding author. Tel.: +86 010 59028201.

E-mail addresses: zhuyf@mail.tsinghua.edu.cn (Y. Zhu), zhoujun100@sohu.comJ. Zhou).

ttp://dx.doi.org/10.1016/j.chroma.2014.11.057021-9673/© 2014 Elsevier B.V. All rights reserved.

comparatively low cost, ease of operation and automation. The coreof SPE was the adsorbent material that determined the selectivityand sensitivity of the method. In recent years, carbonaceous materi-als such as activated carbon, carbon nanotubes, porous carbon andfullerence had been adopted as SPE adsorbents for pretreatmentin term of their chemical stability, large surface area and excellentmechanical properties [13–18]. However, there was still an increas-ing for the development of excellent and cost-effective adsorbentfor SPE pretreatment.

Graphene, a novel two-dimensional carbon nanomaterial hadattracted a great deal of attentions in recent years. Graphene oxide(GO), as a precursor for graphene, could be obtained by the strongoxidation of graphite [19]. Importantly, GO possessed layered struc-ture and negatively charged surface [20]. NMR and FTIR studiesindicated that carboxyl groups existed at the edges of carbon layers,epoxy and hydroxyl groups were incorporated in the layers [21,22].These copious oxygen-containing groups on GO could significantlyaffect the van der Waals interaction between the graphene lay-ers and also render it strongly hydrophilic. As a result of this,hydrophilic and polar substance, such as pigments, heavy metalions, phenols, aldehydes, esters, basic species and so on, could be

adsorbed on the surface of GO through hydrogen bonds, anion-�and electrostatic interactions [23–28]. Therefore, GO was expectedto be a promising pretreatment adsorbent for the removal of polarmatrices. However, to the best of our knowledge, no relevant work

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R. Shi et al. / J. Chrom

egarding the application of GO on MSS purification had beeneported.

In this work, a novel SPE method was demonstrated to purify4 PAHs in MSS. To avoid GO escaping from the SPE columns, newdsorbents by binding GO to silica was adopted [29]. 14 PAHs in MSSxtract were analyzed by GC–MS. The performance of the methodas demonstrated by analyzing the Kentucky reference cigarette

R5F and 12 top-selling Chinese cigarette brands in market. Theevelopment of this method aimed to remove matrices interfer-nces and preserve method precision, accuracy, and sensitivity.

. Experimental

.1. Chemicals and standards

The Kentucky reference cigarettes 1R5F were obtained fromhe Kentucky Tobacco Research and Development Center (Lex-ngton, KY). Co-operation Centre for Scientific Research Relativeo Tobacco (CORESTA) Monitor CM6 reference cigarettes wereeceived from CORESTA (Paris, France). 12 top-selling Chi-ese cigarette brands were collected from the market inhina for this study. Graphite powder (−325 mesh, 99.9995%)nd N,N′-dicyclohexylcarbodiimide (DCC) was bought from Alfaesar. Hydrazine hydrate (85%), KMnO4, NaNO3, H2SO4 HClere bought from Sinopharm Chemical Reagent (Shanghai,hina). Analytical-grade methanol (MeOH) and cyclohexane, N,N-imethylformamide (DMF) were bought from J&K Scientific Ltd.minosilica were from Agilent (Santa Clara, CA). 14 PAHs:aphthalene (NP), acenaphtylene (ANP), acenaphthene (ANE),uorine (FLR), phenanthrene (PHE), anthracene (ANT), fluoran-hene (FLT), pyrene (PY), benzo[a]anthracene (BaA), chryseneCHR), 5-methylchrysene (MCHR), benzo[b]fluoranthene (BbF),enzo[k]fluoranthene, (BkF) and benzo[a]pyrene (BaP) was boughtrom J&K Scientific Ltd. All working solution of 14 PAHs were pre-ared in cyclohexane and stored at 4 ◦C in the dark. All chemicalssed were analytic grade reagents, without further purification.

.2. Synthesis of sample and characterization

Graphene oxide (GO) was synthesized by a modified Hummers’ethod [19]. Comparing with Hummer’s method, the reaction timeas prolonged in order to ensuring oxidation reaction fully in this

tudy. Briefly, graphite (10 g) and NaNO3 (5 g) were mixed with30 mL of H2SO4 (98% (w)) in a 2 L beaker and stirred for 30 minelow 0 ◦C. After pre-oxidation process, KMnO4 (30 g) was added

nto the suspension slowly. Then the mixture was stirred at roomemperature until it gradually became a brownish slurry. Afterbout 120 min, brownish slurry was diluted slowly with 500 mLf water, causing violent effervescence and an increase in tem-erature. The diluted suspension, now dark yellow in color, wastirred at this temperature for 120 min. After that, the suspensionas further diluted with 1 L of warm water and treated with 100 mL

f H2O2 (30% (w)) to reduce the residual permanganate and man-anese dioxide. For purification, the mixture was centrifuged andashed with 10% HCl and then deionized water several times to

emove the residual metal ions and acid. After filtration and dryingnder vacuum at room temperature, GO was obtained as powder.

GO was immobilized on aminosilica (designated SiO2/GO) byoupling the amino groups of aminosilica and the carboxyl groupsf GO. The starting materials of 200 mg of GO was dispersed in

00 mL of DMF and ultrasonicated for about 60 min. In what fol-

owed, 5 g of aminosilica and 200 mg of DCC were added. Theuspension was refluxed on a mantle heater at 50 ◦C for 30 h. Theroducts were washed with water and MeOH for several times, and

. A 1375 (2015) 1–7

finally dried at room temperature. The obtained brown powder wasSiO2/GO.

The purity and crystallinity of the as-prepared sample werecharacterized by XRD on a Bruker D8-advance diffractometerusing Cu K� radiation (� = 1.5418 A). The XRD data for index-ing and cell-parameter calculation were collected in a scanningmode with a step length of 0.02◦ and a preset time of 5.6 s/step.The Brunauer–Emmett–Teller (BET) surface area was measured byASAP 2010 V5.02H. The absorbed gas was nitrogen. FTIR spectrawere recorded for KBr disks containing the powdered sample withan FTIR spectrometer (Perkin-Elmer 1600). Raman measurementswere obtained using a Renishaw RM2000 spectrometer equippedwith a notch filter and a CCD detector. AFM images were acquiredin phase mode in air using Digital Instrument Shimadzu SPM-9600.Morphologies of the as-prepared samples were characterized bytransmission electron microscopy (HRTEM JEOL JEM 2010).

2.3. Smoke collection

Before smoking, Cigarettes and glass fiber filters (Whatman, UK)used for this study were conditioned at 22 ◦C and 60% humidityfor at least 48 h. Each glass fiber filter was then placed inside thefilter holder to collect mainstream smoke. Cigarettes were smokedusing a Cerulean (Milton Keynes) SM 450 20-port smoking machineaccording to ISO 3308:2000. Briefly, all cigarettes were smoked in apuff volume of 35 mL with a 2 s puff duration every 60 s. Typically,five cigarettes were smoked per port. All samples were smoked toa butt mark of 3 mm past the tipping paper overwrap.

2.4. Sample preparation and solid-phase extraction procedures

MSS was collected in each glass fiber filter, which then wastransferred to conical flask and extracted with 50 mL of cyclohex-ane for 30 min in an ultrasonic machine. Finally, the extract waspurified through a 0.45-�m PTFE syringe filter before being loadedonto the SPE column.

For all SPE experiments, 100 mg of SiO2/GO powder was placedin a 3 mL SPE column with an upper frit and a lower frit to avoidadsorbent loss. Prior to extraction, SPE columns were activated with3 mL of MeOH and 10 mL cyclohexane with a Supelco Visiprep vac-uum manifold (Bellefonte, PA). After that, 10 mL of extract wereloaded on the SPE columns at a flow rate of 1 mL/min. So onecigarette MSS was purified by one SPE column. During this process,large matrix interferences were retained on SPE column. Finally,the SPE column was washed with 10 mL of cyclohexane to removethe adsorbed PAH from the column. The eluant from both the loadand the wash were combined and concentrated to 1 mL using anitrogen evaporation system for subsequent GC–MS analysis.

2.5. GC–MS analysis

GC–MS analysis was performed using Agilent GC-7890 MS-5975equipped with an autosampler-7693 (Santa Clara, CA). The GC wasequipped with a 30 m (250 �m internal diameter, 0.25 �m filmthickness) DB-5MS fused silica capillary column (J & W ScientificFolsom, CA). The injection port, ion source and interface tempera-ture was 280 ◦C, 230 ◦C and 280 ◦C, respectively. The solvent delaytine was 4 min. The injection volume was 1.0 �L and splitless injec-tion mode was used. The constant flow rate was 1.2 mL/min He.The GC temperature program was 70 ◦C for 1 min, then 20 ◦C/min to200 ◦C and held for 2 min, then 10 ◦C/min to 260 ◦C, then 5 ◦C/minto 280 ◦C and held for 20 min. The MS was operated in selected

ion monitoring mode (SIM). The MS program was set as follow:4–7 min, m/z 128, 127; 7–9.5 min, m/z 152, 151; 9.5–10 min, m/z154, 153; 10–12 min, m/z 166, 165; 14–17 min, m/z 202, 200;17–19 min, m/z 228, 226; 19–21 min, m/z 242, 240; 21–40 min, m/z

R. Shi et al. / J. Chromatogr. A 1375 (2015) 1–7 3

Table 1Selected ion monitoring mode setup for GC/MS analysis of 14 PAHs from MSS.

PAH compound Retentiontime (min)

Quantificationion (m/z)

Confirmationion (m/z)

Naphthalene 6.76 128 127Acenaphthylene 9.32 152 151Acenaphthene 9.61 154 153Fluorene 10.55 166 165Phenanthrene 12.47 178 176Anthracene 12.58 178 176Fluoranthene 15.23 202 200Pyrene 15.75 202 200Benz[a]anthracene 18.67 228 226Chrysene 18.75 228 2265-Methylchrysene 19.93 242 240Benzo[b]fluoranthene 21.53 252 250Benzo[k]fluoranthene 21.61 252 250

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In order to reveal the dominant adsorption mechanism of MSSmatrices on GO, XRD patterns was used. According to XRD pat-tern, a comparison of the (0 0 1) diffraction peaks in the rangeof 2� = 8–12◦ (Fig. 2) showed that the peaks position of GO after

Benzo[a]pyrene 22.43 252 250

52, 250. The quantification ion, confirmation ion and retentionime of PAHs were shown in Table 1.

. Results and discussion

.1. Characterization of GO and GO/SiO2

XRD patterns of graphite, GO and SiO2/GO were shown in Sup-orting Information Fig. S1. The graphite showed a very sharpiffraction peak at 26.5◦ corresponding to a d-spacing of 0.336 nmd0 0 2). Oxidation treatment resulted in a decrease of the peak0 0 2) intensity of graphite and the appearance of (0 0 1) diffractioneak of the GO at 2� = 10.6◦. This peak demonstrated the typi-al loose-layer-like structure of GO corresponding to an interlayerpacing of 0.825 nm, which depended on the method of preparationnd the number of layers of water in the gallery space of the mate-ial [30]. But for SiO2/GO, there were no diffraction peaks in XRDatterns, suggesting that aminosilica in the SiO2/GO existed in anmorphous state and GO was immobilized on aminosilica almostn single layer.

Raman spectrum of GO and SiO2/GO in Supporting Informationig. S2 displayed two prominent peaks at 1580 and 1440 cm−1,hich corresponded to the well-documented G and D bands,

espectively. The intensity ratio of D band to G band (r = ID/IG) wassually used as a measure of the disorder [31]. The intensity ratior) of GO and SiO2/GO showed greatly enhanced value comparedo that of graphite, suggesting the formation of localized disorderefects within sp2 carbon network. These results indicated that GOad been successfully prepared.

The nitrogen adsorption–desorption isotherms were shown inupporting Information Fig. S3. The BET surface area of GO was87 m2 g−1. The discrepancy on experimental data and theoreti-al value of surface area of GO was attributed to the incompletexfoliation and aggregation during reaction process. After GOas immobilized on aminosilica, the surface area of SiO2/GO was

36 m2 g−1, which was sufficiently high for a SPE adsorbent.The morphological and structure of GO was further examined

y TEM and AFM. The TEM image of GO (Supporting Informationig. S4a) clearly exhibited a typically wrinkled, sheet-like structureith the size of several micrometers. Similar results were observed

n the AFM image (Supporting Information Fig. S4b). The size ofheets ranged from dozens of nanometers to several micrometers.n addition, the corresponding section analysis of GO sheets indi-

ated that the average height was about 0.9 nm which meant theeneration of single layer of GO sheets. These results were consis-ent with the other literature references [32].

Fig. 1. Selected ion chromatogram of the PAHs in MSS extract preconditioned with-out (a) and with (b) SPE.

3.2. Mechanism of adsorption and purification

As a result of the existing of oxygen-containing functionalgroups, such as hydroxyl, epoxy, and carboxyl groups, GO couldeasily adsorb polar molecules or polymers via the functional groupsas anchors. The intrinsic properties could render it excellent qualityas adsorbent for normal-phase SPE. Then, we tested the adsorptionand purification capacity of GO in normal-phase SPE. After apply-ing this SPE procedure to MSS extract of CM6 reference cigarette,a visually cleaner extract was obtained. The color of MSS extractwas changed from yellow to colorless after being loaded onto theSPE column. During this process, large matrices interferences, suchas pigments, heavy metal ions, phenols, aldehydes, esters, basicspecies and so on, were retained on SPE column. Elimination of thematrices interferences was further confirmed by visual examina-tion of the selected ion chromatograms of PAHs (Fig. 1). Comparingwith untreated MSS extract, the chromatogram of pretreatmentextract showed more simplify because of reduced background andmatrices interferences. Due to thousands of polar compounds exist-ing in MSS extract, a complete study of potential interferences fromthe matrices was not possible. Selected ion chromatograms demon-strated excellent separation for most of the PAHs analytes. Only apair of compounds were not well-resolved: benzo[b]fluoranthenecoeluted with benzo[k]fluoranthene (see the insert in Fig. 1).

Fig. 2. XRD patterns of GO and GO after adsorption.

4 R. Shi et al. / J. Chromatogr. A 1375 (2015) 1–7

Table 2The linear range, regression data, limits of detection (LODs), limits of quantification (LOQs) for SPE of 14 PAHs from MSS.

PAH compound Concentration range (ng mL−1) R2 Slope Intercept LOD (ng/cig) LOQ (ng/cig)

Naphthalene 1–200 0.9996 17.14 18.65 0.24 0.79Acenaphthylene 1–200 0.9998 20.44 19.51 0.06 0.20Acenaphthene 1–200 0.9998 11.76 20.33 0.12 0.40Fluorene 1–200 0.9997 14.51 25.87 0.22 0.73Phenanthrene 1–200 0.9991 21.81 49.09 0.36 1.19Anthracene 1–200 0.9994 20.78 −7.59 0.08 0.26Fluoranthene 1–200 0.9991 24.30 13.51 0.14 0.46Pyrene 1–200 0.9992 25.88 11.22 0.09 0.30Benz[a]anthracene 1–200 0.9976 19.74 −69.84 0.05 0.17Chrysene 1–200 0.9984 23.73 −46.22 0.07 0.235-Methylchrysene 1–200 0.9965 21.36 −70.9 0.17 0.57

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dsorption shifted slightly toward a lower 2� value. According toragg’ s law, the decrease in 2� value resulted in the increase in

attice parameters (d(0 0 1) value). Therefore, the observed shift ofiffraction peak toward lower angle could be due to the disturbancef MSS matrices. The adsorbed MSS matrices might intercalate theattice of GO and broaden its interlayer spacing, which was similaro result of other literature references [24,33].

FTIR spectroscopy was a useful tool in identifying the variation ofertain functional groups on the surface of a solid. The interactionsetween MSS matrices and GO were also investigated using theTIR spectra to explore the position MSS matrices can be adsorbedn. As can be seen from Fig. 3, the strong peak at 3380 cm−1 coulde attributed to the stretching vibration of O H groups. The peakt 1725 cm−1 indicated the existence of C O bonds in carboxyliccid and carbonyl moieties. The peak at 1625 cm−1 was associ-ted with skeletal vibration of aromatic C C bonds. The peak atround 1416 cm−1 belonged to carboxyl O C O bonds. The peakt 1222 cm−1 referred to epoxy bonds. The peak at 1044 cm−1 wasrom alkoxy C O bonds [28,34]. Compared with GO, it could beound that almost all the intensity of absorption peaks decreasedfter MSS adsorption, especially, for the peaks of O H and C Oroups, which meant the lower population of O H and C O func-ional groups. Presumably, MSS matrices could attack these twounctional groups. Moreover, the stretching vibration of O H groupad a large shift from 3380 to 3184 cm−1, which could be ascribedo the formation of hydrogen bonding between GO and MSS matri-es. The peak corresponding to C O group also shifted from 1725 to

714 cm−1, indicating that there might be interaction between C Oroup of GO and MSS matrices. Therefore, the result of FTIR spectra

Fig. 3. FTIR spectra of GO (a) and GO after adsorption (b).

20.79 −167.07 0.21 0.6923.96 −176.08 0.24 0.8021.16 −183.72 0.27 0.9

demonstrated that MSS matrices were effectively adsorbed on GOmainly through O H and C O functional groups as anchors.

3.3. Validation of the method

All the standard solutions underwent the same sample prepa-ration and SPE procedure. The linear regression, the limit ofdetection (LOD) and limit of quantification (LOQ) data were listedin Table 2. Excellent linear relationships were obtained in all cases(R2 between 0.9948 and 0.9998). The LOD and LOQ were calculatedas the concentration corresponding to the signals of 3 and 10 timesthe standard deviation of the baseline noise. The LOD and LOQ for14 PAHs were found to be 0.05–0.36 ng/cig and 0.17–1.19 ng/cig,respectively. The LOD were much lower than normal level of PAHsin most commercial cigarette [35,36].

The accuracy of this method was established with PAHs spikedat low, medium, and high concentration levels on glass fiber filterscontaining MSS from CM6 cigarettes. The accuracy was determinedby comparing the calculated amounts of PAHs from the spiked sam-ple with the total spiking amount. As listed in Table 3, the accuracyof the 14 PAHs were in the range from 73 to 116%. The precisionof this method was investigated by calculating intra- and inter-dayrelative standard deviations (RSD). The RSDs data were summa-rized in Table 4. The intra-and inter-day RSD of 14 PAHs were lessthan 7.8% and 13.9%, respectively. The acceptable accuracy and pre-cision indicated matrices had been widely eliminated through thepretreatment of SiO2/GO SPE procedure. Moreover, we expectedthe accuracy and precision of this method could be improved ifan isotopically labeled analog were used as internal standard. Thereproducibility of the SPE material was investigated by run-to-runtest. Encouragingly, good run-to-run RSD were obtained based onsix extractions on a single SiO2/GO SPE (RSD < 9.1%, Table 4), whichconfirmed that SiO2/GO was reusable SPE adsorption.

The stability to reagents of SiO2/GO was also explored. Priorto SPE process, SiO2/GO adsorbent was dispersed in methanol, n-hexane, HCl solution (0.1 mol/L) and NaOH solution (0.1 mol/L),respectively. Then all bottles were placed in a shaking machineat a speed of 200 rpm for 10 h. After filtration and drying, treatedSiO2/GO adsorbent was placed in SPE column with an upper fritand lower frit. Under same conditions, all adsorbent were appliedto extract PAHs from spiked MSS extract. These results were shownin Fig. 4. It can be seen that the performance of SiO2/GO was verystable in most reagents, except for NaOH solution. GO could beescaped from SiO2 because of hydrolysis of acid amide in alkalineenvironment.

In order to evaluate the cleanup capability of SiO2/GO, its perfor-mance was compared with the other adsorbent materials, includingsilica and cyclohexyl. 100 mg of different adsorbents was packed in3 mL SPE cartridges. Under optimal conditions, all adsorbent were

R. Shi et al. / J. Chromatogr. A 1375 (2015) 1–7 5

Table 3Method accuracy at three different concentrations for SPE of 14 PAHs from MSS.

PAHs Concentration (ng/cig) Added (ng/cig) Found (ng/cig) Accuracy (% ± RSDs, n = 3)

Naphthalene 710500 1175.4 93.1 ± 7.2

1000 1653.3 94.3 ± 5.81500 2134.8 95.0 ± 6.9

Acenaphthylene 116.250 158.8 85.2 ± 3.5

100 201.8 85.6 ± 4.4200 295.3 89.6 ± 8.7

Acenaphthene 62.850 105.6 85.6 ± 4.7

100 154.5 91.7 ± 5.9200 247.1 92.2 ± 6.6

Fluorene 321.6200 528.7 103.6 ± 6.7300 603.5 94.0 ± 9.1400 701.5 95.0 ± 6.2

Phenanthrene 249.8200 412.5 81.4 ± 7.1300 526.9 92.4 ± 1.9400 630.3 95.1 ± 3.4

Anthracene 78.150 121.7 87.2 ± 5.1

100 171.6 93.5 ± 5.7200 265.6 93.8 ± 6.3

Fluoranthene 107.050 150.9 87.8 ± 7.1

100 194.1 87.1 ± 3.2200 285.8 89.4 ± 1.8

Pyrene 118.150 159.4 82.6 ± 7.0

100 209.3 91.2 ± 3.4200 300.7 91.3 ± 2.5

Benz[a]anthracene 25.710 37.3 116.0 ± 10.120 44.9 96.0 ± 9.630 53.8 93.7 ± 5.8

Chrysene 18.510 29.3 108.0 ± 6.220 41.6 115.5 ± 8.830 49.7 104.0 ± 7.4

5-Methylchrysene

N.D.a50 44.6 89.2 ± 7.9

100 90.6 90.6 ± 8.8200 205.0 102.5 ± 8.5

Benzo[b]fluoranthene 17.210 24.9 77.0 ± 7.620 31.8 73.0 ± 7.930 42.4 84.0 ± 9.0

Benzo[k]fluoranthene 1.710 10.3 86.0 ± 6.320 18.6 84.5 ± 9.930 29.1 91.3 ± 5.6

Benzo[a]pyrene 15.210 24.6 94.0 ± 3.720

30

a 5-Methylchrysene was not detected in MSS of CM6 cigarette.

Table 4Method precision for SPE of 14 PAHs from MSS.

PAH compound RSD (%)

Intra-day (n = 6) Inter-day (n = 6) Run-to-run (n = 6)

Naphthalene 1.7 4.8 3.5Acenaphthylene 3.9 1.9 4.1Acenaphthene 2.4 6.1 3.9Fluorene 1.7 5.6 3.3Phenanthrene 0.8 2.7 3.1Anthracene 2.1 3.6 5.2Fluoranthene 6.8 7.5 5.4Pyrene 2.4 5.8 6.8Benz[a]anthracene 7.8 9.7 7.2Chrysene 5.0 12.5 9.15-Methylchrysene N.D.a N.D.a N.D.a

Benzo[b]fluoranthene 7.6 12.8 8.8Benzo[k]fluoranthene 5.9 13.9 9.0Benzo[a]pyrene 5.0 11.9 7.9

a 5-Methylchrysene was not detected in MSS of CM6 cigarette.

36.7 107.5 ± 8.848.9 112.3 ± 5.8

applied to extract PAHs from spiked MSS extract. These resultswere shown in Fig. 5. It can be seen that SiO2/GO demonstrated thebest performance toward most target analytes. The performanceof silica was also acceptable, but demonstrated lower recoveriesto SiO2/GO because of insufficient adsorption capacity for matricesinterferences. In order to obtain satisfying recoveries, more silicamust be used to enhance the adsorption capacity. Cyclohexyl wasmedium polar adsorbent, its lowest recoveries indicated that cyclo-hexyl showed a poor adsorption capacity for matrices interferences.The maximum sample matrix interferences can lead to poor analyterecoveries and precision.

3.4. Analysis of reference and commercial cigarette

To demonstrate the consistency of this method compared with

other methods, Kentucky reference cigarette 1R5F were analyzedand compared with previously reported literature data [6,37].Cigarettes were smoked according to ISO 3308:2000 in the all meth-ods. As can be seen from Table 5, most PAHs levels measured in 1R5F

6 R. Shi et al. / J. Chromatogr. A 1375 (2015) 1–7

Fig. 4. Comparison of the stability performance of SiO2/GO in different reagent.

Fig. 5. Comparison of the cleanup capability of SiO2/GO, silica and cyclohexyl SPE.

Table 5Comparison of PAHs concentrations (ng/cig) of 1R5F Reference Cigarette.

PAHs Forhand et al. Tarrant et al. Current study

Naphthalene 57.5 63.6 65.8Acenaphthylene 11.3 12.2Acenaphthene 9.6 9.8Fluorene 33.8 28.1 35.1Phenanthrene 31.0 29.3 30.5Anthracene 13.9 12.0 13.4Fluoranthene 16.2 12.3 13.1Pyrene 10.2 8.9 11.3Benz[a]anthracene 3.0 2.5 3.5Chrysene 4.7 3.6 4.05-Methylchrysene N.D.a

Benzo[b]fluoranthene 1.4 1.5Benzo[k]fluoranthene 0.5 0.2

fPttNcTcL

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Table 6Summary of PAHs concentration (n = 3) in 12 top-selling Chinese cigarette brands.

PAHs Range (ng/cig) Mean (ng/cig) Median (ng/cig)

Naphthalene 467.5–270.6 351.5 359.9Acenaphthylene 74.6–44.9 56.5 55.5Acenaphthene 40.3–25.5 31.8 31.3Fluorene 207.3–146.3 171.8 171.8Phenanthrene 171.9–118.1 146.5 149.2Anthracene 60.6–33.4 44.7 44.9Fluoranthene 80.9–47.9 61.5 60.6Pyrene 96.6–58.8 73.5 73.3Benz[a]anthracene 18.5–9.4 14.3 14.4Chrysene 13.9–6.4 10.6 11.05-Methylchrysene N.D.a N.D.a

Benzo[b]fluoranthene 12.2–7.4 9.6 9.7Benzo[k]fluoranthene 1.05–0.56 0.8 0.8

[3] M.N. Kayali, S. Rubio-Barroso, Determination of benzo(a)pyrene in total par-

Benzo[a]pyrene 1.6 1.3 1.3

a 5-Methylchrysene was not detected in MSS of 1R5F cigarette.

rom these different methods were consistent. Minor differences inAHs profile were most likely due to the batch variability amonghe tobacco itself. However, the situation was less clear with Naph-halene and Benzo[k]fluoranthene. The lowest molecular weightaphthalene could evaporate during the extraction and precon-entration processes, which could lead to the variation of content.he variation of Benzo[k]fluoranthene level among those methodsould be attributed to its low concentration, which was close to its

OD.

It was known that PAHs varied among different tobacco speciesnd could vary among cigarette tobaccos from different countries.

Benzo[a]pyrene 9.5–5.0 7.7 7.9

a 5-Methylchrysene was not detected in MSS of Chinese cigarette.

In our study, PAHs levels were measured from 12 top-selling Chi-nese cigarette brands. As can be seen from Table 6, in all cigarettebrands, naphthalene was the dominating compound, followed byfluorine and phenanthrene. The high-molecular-mass PAHs (4–5rings) exhibited small concentrations compared to low-molecular-mass PAHs (2–3 rings). This trend was similar to that of referencecigarette. The level of benzo[a]pyrene was important because itwas leading compound of this group and one of the most poten-tial carcinogenic PAH. In the Chinese cigarette brands analyzed,the mean concentrations of benzo[a]pyrene was 7.7 ng/cig, whichwas roughly five times greater than that of Kentucky referencecigarettes 1R5F.

4. Conclusion

We had developed a reliable method to purify and determine 14PAHs in MSS. The proposed method revealed great potentials of GOas advantageous purification material in pretreatment of MSS. GOshowed high adsorption efficiencies toward most polar matricesinterferences mainly through abundant oxygen-containing groupson GO, especially, O H and C O functional groups. This work notonly proposed a useful method for quantification of PAHs in MSS,but also revealed great potentials of GO as an excellent material inanalytical field.

Acknowledgments

This work was partly supported by National Basic ResearchProgram of China (973 Program) (2014CB632403), NationalHigh Technology Research and Development Program of China(2012AA062701) and Chinese National Science Foundation(20925725 and 21473121).

Appendix A. Supplementary data

Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.chroma.2014.11.057.

References

[1] S.S. Hecht, Tobacco smoke carcinogens and lung cancer, J. Natl. Cancer Inst. 91(1999) 1194–1210.

[2] A. Rodgman, C.R. Green, Toxic chemicals in cigarette mainstreamsmoke–hazard and hoopla, Beitr. Tobakfor. Int. 20 (2003) 481–539.

ticulate matter of Virginia and black tobacco smoke by HPLC with fluorimetricdetection, J. Liq. Chromatogr. 18 (1995) 1617–1623.

[4] J.P. Buchet, M. Ferreira, J.B. Burrion, T. Leroy, M. Kirsch-Volders, P. Van Hum-melen, J. Jacques, L. Cupers, J.P. Delavignette, R. Lauwerys, Tumor markers in

atogr

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

R. Shi et al. / J. Chrom

serum, polyamines and modified nucleosides in urine, and cytogenetic aberra-tion in lymphocytes of workers exposed to polycyclic aromatic hydrocarbons,Am. J. Ind. Med. 27 (1995) 523–543.

[5] C.R. Green, A. Rodgman, The tobacco chemists’ research conference: a half cen-tury of advances in analytical methodology of tobacco and its products, RecentAdv. Tob. Sci. 22 (1996) 141–304.

[6] J.E. Tarrant, K. Mills, C. Williard, Development of an improved method forthe determination of polycyclic aromatic hydrocarbons in mainstream tobaccosmoke, J. Chromatogr. A 1216 (2009) 2227–2234.

[7] J. Haib, I. Hofer, J.M. Renaud, Analysis of multiple pesticide residues in tobaccousing pressurized liquid extraction, automated solid-phase extraction clean-upand gas chromatography–tandem mass spectrometry, J. Chromatogr. A 1020(2003) 173–187.

[8] C. De, O. Van, L. Van, On-line-solid-phase extraction with ultra performanceliquid chromatography and tandem mass spectrometry for the detection ofnicotine, cotinine and trans-3′-hydroxycotinine in urine to strengthen humanbiomonitoring and smoking cessation studies, J. Pharm. Biomed. Anal. 76 (2014)126–143.

[9] L.K. Ng, M. Hupe, Effects of moisture content in cigar tobacco on nicotine extrac-tion: similarity between soxhlet and focused open-vessel microwave-assistedtechniques, J. Chromatogr. A 1011 (2003) 214–219.

10] Z. Li, D. Huang, Z. Tang, C. Deng, X. Zhang, Fast determination of chlorogenicacid tobacco residues using microwave-assisted extraction and capillary zoneelectrophoresis technique, Talanta 82 (2010) 1181–1185.

11] X.L. Zhu, Q.D. Su, J.B. Cai, J. Yang, Optimization of microwave-assisted solventextraction for volatile organic acids in tobacco and its comparison with con-ventional extraction methods, Anal. Chim. Acta 579 (2006) 88–94.

12] Y. Li, T. Pang, Z. Guo, Y. Li, X. Wang, J. Deng, K. Zhong, X. Lu, G. Xu,Accelerated solvent extraction for GC-based tobacco fingerprinting and itscomparison with simultaneous distillation and extraction, Talanta 81 (2010)650–656.

13] R. Vallant, Z. Szabo, S. Bachmann, R. Bakry, M. Najam, M. Rainer, N. Heigl,C. Petter, C. Huck, G. Bonn, Development and application of C60-fullerenebound silica for solid-phase extraction of biomolecules, Anal. Chem. 79 (2007)8144–8153.

14] J. Yu, L. Dong, C. Wu, L. Wu, J. Xing, Hydroxyfullerene as a novel coating forsolid-phase microextraction fiber with sol–gel technology, J. Chromatogr. A978 (2002) 37–48.

15] Y. Cai, G. Jiang, J. Liu, Q. Zhou, Multi-walled carbon nanotubes as superiorsolid phase extraction adsorbent for the determination of bisphenol A, 4-nonylphenol and 4-tert-octylphenol in environmental water samples by highperformance liquid chromatography-fluorimetric detection, Anal. Chem. 75(2003) 2517–2521.

16] Y. Cai, Y. Cai, S. Mou, Y. Lu, Multi-walled carbon nanotubes as a solid-phaseextraction adsorbent for the determination of chlorophenols in environmental

water samples, Chromatogr. A 1081 (2005) 245–247.

17] L. Ravelo, A. Herrera, J. Hernandez, M. Rodriguez, Carbon nanotubes: solid-phase extraction, J. Chromatogr. A 1217 (2010) 2618–2641.

18] M. Shibukawa, A. Unno, T. Miura, A. Nagoya, K. Oguma, On-column deriva-tization using redox activity of porous graphitic carbon stationary phase: an

[

. A 1375 (2015) 1–7 7

approach to enhancement of separation selectivity of liquid chromatography,Anal. Chem. 75 (2003) 2775–2783.

19] W. Hummers, R. Offeman, Preparation of graphitic oxide, J. Am. Chem. Soc. 80(1958) 1439.

20] T. Szabo, E. Tombacz, E. Illes, I. Dekany, Enhanced acidity and pH dependent sur-face charge characterization of successively oxidized graphite oxides, Carbon44 (2006) 537–545.

21] A. Lerf, H. He, M. Forster, J. Klinowski, Structure of graphite oxide revisited, J.Phys. Chem. 102 (1998) 4477–4482.

22] T. Szabo, O. Berkesi, I. Dekany, DRIFT study of deuterium-exchanged graphiteoxide, Carbon 43 (2005) 3186–3189.

23] Y. Gao, Y. Li, L. Zhang, H. Huang, J.J. Hu, S.M. Shah, X.G. Su, Adsorption andremoval of tetracycline antibiotics from aqueous solution by graphene oxide,J. Colloid Interface Sci. 368 (2012) 540–546.

24] T. Wu, X. Cai, S. Tan, H. Li, J. Liu, W. Yang, Adsorption characteristics of acry-lonitrile, p-toluenesulfonic acid, 1-maphthalenesulfonic acid and methyl blueon grapheme in aqueous solutions, Chem. Eng. J. 173 (2011) 144–149.

25] M. Seredych, T. Bandosz, Removal of ammonia by graphite oxide via its inter-calation and reactive adsorption, Carbon 45 (2007) 2140–2142.

26] Y. Matsuo, Y. Nishino, T. Fukutsuka, Y. Sugie, Removal of formaldehyde from gasphase by silylated graphite oxide containing amino groups, Carbon 46 (2008)1162–1163.

27] S.T. Yang, S. Chen, Y.L. Chang, A. Cao, Y.F. Liu, H.F. Wang, Removal of methyleneblue from aqueous solution by grapheme oxide, J. Colloid Interface Sci. 359(2011) 24–29.

28] V. Chandra, J. Park, Y. Chun, J. Lee, I. Hwang, K. Kim, Water-dispersiblemagnetite-reduced graphene oxide composites for arsenic removal, ACS Nano4 (2010) 3979–3986.

29] Q. Liu, J. Shi, J. Sun, T. Wang, L. Zeng, G. Jiang, Graphene and grapheme oxidesheets supported on silica as versatile and high-performance adsorbents forsolid-phase extraction, Angew. Chem. Int. Ed. 50 (2011) 5914–5917.

30] Z.H. Liu, Z.M. Wang, X.J. Yang, K. Ooi, Intercalation of organic ammonium ionsinto layered graphite oxide, Langmuir 18 (2002) 4926–4932.

31] A.C. Ferrari, J. Robertson, Interpretation of Raman spectra of disordered andamorphous carbon, Phys. Rev. B 61 (2000) 14095–14107.

32] J. Xu, Y.F. Zhu, Elimination of Bisphenol A from water via grapheme oxideadsorption, Acta Phys. Chim. Sin. 29 (2014) 829–836.

33] C. Xu, X. Wang, L.C. Yang, Y.P. Wu, Fabrication of a graphene-cuprous oxidecomposite, J. Solid State Chem. 182 (2009) 2486–2490.

34] J. Xu, L. Wang, Y.F. Zhu, Decontamination of bisphenol A from aqueous solutionby graphene adsorption, Langmuir 28 (2012) 8418–8425.

35] R.F. Arrendale, R.F. Severson, M.E. Snook, Quantitative determination of naph-thalene in tobacco smoke by gas chromatography, Beitr. Tabakforshc. 10 (1980)100–105.

36] V. Noman, An overview of the vapor phase, semivolatile and nonvolatile com-

ponents of cigarette smoke, Recent Adv. Tob. Sci. 3 (1977) 28–58.

37] J.B. Forehand, G.L. Dooly, S.C. Moldoveanu, Analysis of polycyclic aromatichydrocarbons, phenols and aromatic amines in particulate phase cigarettesmoke using simultaneous distillation and extraction as a sole sample clean-upstep, J. Chromatogr. A 898 (2000) 111–124.