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Journal of Chromatography A, 1406 (2015) 19–26

Contents lists available at ScienceDirect

Journal of Chromatography A

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

atrix solid phase dispersion method for determination of polycyclicromatic hydrocarbons in moss

stefanía Concha-Granaa, Soledad Muniategui-Lorenzoa,∗, Flavia De Nicolab,esús R. Aboalc, Ana Isabel Rey-Asensiod, Simonetta Giordanoe,f, Ralf Reskig,urificación López-Mahíaa, Darío Prada-Rodrígueza

Grupo Química Analítica Aplicada (QANAP), Instituto Universitario de Medio Ambiente (IUMA), Centro de Investigaciones Científicas Avanzadas (CICA),epartamento de Química Analítica, Facultade de Ciencias, Universidade da Coruna, 15071 A Coruna, SpainDepartment of Sciences and Technologies, University of Sannio, 82100 Benevento, ItalyDepartment of Cellular Biology and Ecology, Faculty of Biology, University of Santiago de Compostela, 15782 Santiago de Compostela, SpainBIOVIA Consultor Ambiental, Edificio Emprendia, Campus Vida, 15782 Santiago de Compostela, SpainDipartimento di Biologia, Università di Napoli Federico II, Campus Monte S. Angelo, Via Cintia 4, 80126 Naples, ItalyAMRA S.c.a.r.l., Via Nuova Agnano 11, 80125 Naples, ItalyPlant Biotechnology, Faculty of Biology, University of Freiburg, and BIOSS Centre for Biological Signalling Studies, and FRIAS Freiburg Institute fordvances Studies, 79104 Freiburg, Germany

r t i c l e i n f o

rticle history:eceived 1 April 2015eceived in revised form 5 June 2015ccepted 5 June 2015vailable online 16 June 2015

a b s t r a c t

In this work a matrix solid-phase dispersion extraction method, followed by programmed temperaturevaporization–gas chromatography–tandem mass spectrometry determination is proposed for the anal-ysis of polycyclic aromatic hydrocarbons (PAHs) in moss samples. A devitalized, cultivated Sphagnumpalustre L. moss clone obtained from the “Mossclone” EU-FP7 Project was used for the optimization andvalidation of the proposed method. Good trueness (84–116%), precision (intermediate precision lowerthan 11%) and sensitivity (quantitation limits lower than 1.7 ng g−1) were obtained. The proposed method

eywords:atrix solid phase dispersion

ryophyteolycyclic aromatic hydrocarbonsTV–GC–MS/MSiomonitoring

was compared with other procedures applied for this complex matrix, achieving a considerable reductionof sample amount, solvent volume and time consumption. The procedure was successfully tested for theanalysis of PAHs in exposed moss clone samples for the monitoring of air pollution. Finally, the methodwas also tested for its suitability in the analysis of PAHs in other moss species as well as a lichen species.

© 2015 Elsevier B.V. All rights reserved.

ir-quality

. Introduction

The International Agency for Research on Cancer (IARC) hasecently branded the exposure to outdoor air pollution and toarticulate matter as carcinogenic to humans (Group 1) [1]. Theuropean Council Directive 2004/107/EC about ambient air qualityssessment and management requires from their Member Stateshe periodical availability of information about air quality withinheir territories. There are increasing evidences that persistentrganic pollutants adversely affect human health and so their mon-toring is required. Monitoring of persistent organic pollutants

evels by reference conventional methods is difficult and expensive,equires physical installation and energy supply. As a consequence,ess samplers can be installed to cover a territory [2]. Polycyclic

∗ Corresponding author. Tel.: +34 981 167000; fax: +34 981167065.E-mail address: smuniat@udc.es (S. Muniategui-Lorenzo).

ttp://dx.doi.org/10.1016/j.chroma.2015.06.014021-9673/© 2015 Elsevier B.V. All rights reserved.

aromatic hydrocarbons (PAHs) levels can largely change in thespace, and thus the use of sampling tools able to assess spatialtrends of PAH depositions at a local scale is of great interest [3].

In the last decades, mosses have gained in popularity as passiveaccumulators of organic compounds because of their usefulnessfor large scale monitoring [2,4–7]. The morphological features ofmosses, and their high efficiency in the uptake of both particulateand gaseous forms of organic pollutants make them excellent toolsfor the monitoring of PAHs and other airborne pollutants [5,8–11].Directive 2004/107/EC admits the use of alternative samplingmethods that prove to give results equivalent to the referencemethod to assess spatial trends of PAH deposition [3], and recently,EN 16414-2014 European standard establishes some guidelines forbiomonitoring of ambient air with mosses [12].

Until now mosses have been used as biomonitors of air quality,but recently several investigators have realized the advantages ofusing devitalized mosses, because it allows disposing transplantsin standard conditions [13]. Nowadays, some moss species (for

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xample several Sphagnum species) as well as their habitats areargely protected, and therefore, the use of in vitro cultivation pro-ides a sustainable way to obtain moss for biomonitoring purposes14]. In view of all this, the “Mossclone” EU-FP7 Project proposeshe use of a devitalized cultivated moss clone as a tool for air pollu-ion monitoring [15]. The use of a moss clone for the biomonitoringf contaminants presents some advantages over the use of wildoss, mainly the reproducibility in the physical characteristics.

he obtained material is homogeneous, standardized and with ini-ial concentrations of PAHs lower than in field-grown moss, whichllows its use for the monitoring of unpolluted areas. Moreover,ome studies developed in the Mossclone project showed that thephagnum palustre L. grown in vitro exhibits, in general, morpholog-cal features similar to the material collected in the field, although amaller size. This fact would suggest a higher surface-to-mass ratiof the moss clone material as a possible benefit for biomonitoringurposes [14].

Biomonitoring programmes, in general, and the monitoringroposed in the Mossclone project in particular, involves largeumbers of samples to analyze, and for this reason the devel-pment of quick, cheap, rugged and safe procedures of analysiss essential. After a review of the state of the art in the analy-is of PAHs in moss [16] we noticed that the extraction is oftenarried out using classical procedures, such as Soxhlet extrac-ion, agitation and sonication that are laborious and not agreedith the Green Analytical Chemistry principles, or more modern

echniques, such as accelerated solvent extraction and microwavessisted extraction, that are more expensive and also time-onsuming.

Among the analytical techniques for solid samples, the matrixolid-phase dispersion (MSPD), developed by Barker et al. in 198917], fits with most of the requirements mentioned before. This pro-ess combines principles of several techniques, performing sampleisruption while dispersing the components of the sample on and

nto a solid support. MSPD involves mixing or blending (dependingn the sample state) a sample with an appropriate sorbent until aomogeneous mixture is obtained [18]. MSPD is simple, fast, usesmall sample sizes and small solvent volumes and does not requireomplex specialized equipment. The main drawback is that MSPDs fairly laborious regarding sample/dispersant mixing and columnacking.

As far as we know, this is the first paper about a determinationf PAHs in moss samples using matrix solid phase dispersion asxtraction procedure. Few papers were found in the literature aboutSPD of PAHs in other matrices like mussels [19–21], soils [22],

ewage sludge [23,24], honey [25], asphalt binder [26], householdust [27], cereals [28], micro-algae [29], fish and seafood [30,31].evertheless, some of the cited papers [25,28,31] should be con-

idered papers on sonication extraction, using sonication to assisthe MSPD. The most common dispersant used in the analysis ofAHs by MSPD is octadecylsilyl silica (C18) [19,29–31]. Others dis-ersants used are alumina [24], diatomaceous earth [32], silica [28],r Florisil [22,26].

MSPD can be coupled easily with a solid phase extraction (SPE)lean-up procedure, by packing a SPE sorbent in the bottom part ofhe MSPD columns. By this way the extraction and clean-up stepsre performed simultaneously, with significant reduction in solventonsumption and without requiring special expensive instrumen-ation [33]. Sorbents used for the clean-up depend on the matrix.

hen moss samples are analyzed, Florisil is the most popular sor-ent [11,34–39].

In this work we propose a MSPD procedure for the analysis of 19

AHs from mosses. Both, optimization and validation of the methodere performed using a clone of S. palustre L., and subsequently

he method was applied to analyze exposed moss clone samples.inally, the method was successfully tested for the analysis of PAHs

togr. A 1406 (2015) 19–26

in different moss (Sphagnum sp. and Hypnum cupressiforme) andlichen (Pseudevernia furfuracea) species.

2. Standards and reagents

PAH-Mix 45 (10 �g mL−1 in cyclohexane containing acenaph-thene, acenaphthylene, anthracene, benz[a]anthracene, benzo[b]-fluoranthene, benzo[k]fluoranthene, benzo[g,h,i]perylene, benzo-[a]pyrene, benzo[e]pyrene, chrysene, dibenz[a,h] anthracene,fluoranthene, fluorene, indeno[1,2,3-c,d]pyrene, naphtha-lene, perylene, phenanthrene and pyrene was supplied byDr. Ehrenstorfer GmbH (Augsburg, Germany). Individual stan-dards of benzo[j]fluorathene (10 �g mL−1 in cyclohexane), retene(10 �g mL−1 in cyclohexane) were also supplied by Dr. EhrenstorferGmbH.

As surrogate standards were used: D-labelled PAH surro-gate Cocktail 200 �g mL−1 in 50% methylene chloride (D2, 99.9%)and 50% methanol (D2, 99%) containing [2H8] acenaphthylene(acenaphthylene-d8), [2H12] benzo[a]pyrene (benzo[a]pyrene-d12), [2H12] benzo[g,h,i]perylene (benzo[g,h,i]perylene-d12), [2H10]fluoranthene (fluoranthene-d10), [2H8] naphthalene (naphthalene-d8), [2H10] phenanthrene (phenanthrene-d10), and [2H10] pyrene(pyrene-d10) and supplied by Cambridge Isotope Laboratories, Inc.(Andover, MA, USA) and individual standard of [2H12] chrysene(chrysene-d12, 10 �g mL−1 in cyclohexane) supplied by Dr. Ehren-storfer GmbH.

The internal standards [2H14] dibenz[a,h]anthacene(dibenz[a,h]anthacene-d14, 10 �g mL−1 in cyclohexane) and[2H10] anthracene (anthracene-d10, 100 �g mL−1 in cyclohexane)were supplied by Dr. Ehrenstorfer GmbH.

The working standards were prepared as follows: 0.5 �g mL−1 ofPAHs in hexane, 0.2 �g mL−1 of surrogate labelled standards (sur-rogate cocktail + chrysene-d12) in hexane, 0.5 �g mL−1 of internalstandards (anthracene-d10 and dibenz[a,h]anthacene-d14) in hex-ane.

Dichloromethane (DCM) Super Purity grade was supplied byRomil (Cambridge, UK), hexane (H) Unisolv®, for organic trace anal-ysis, was purchased from Merck (Darmstadt, Germany).

Sorbents used for the dispersion were: diatomaceous earthacid washed not further calcined and silica neutral (Sigma–AldrichChemie Gmbh, Germany), octadecyl functionalized silicaSupelclean-Envi 18 (Supelco, Bellefonte, USA), and Sea sandpro analysi (Merck). All the sorbents were washed with a H andDCM:H (20:80) mixture before use. Silica was activated at 135 ◦C,12 h, and then partially deactivated with milli-Q water.

Clean-up was performed using Envi-Florisil SPE glass tube (1 g)supplied by Supelco.

3. Experimental

3.1. Sampling

S. palustre L. clone samples were obtained as previouslydescribed [14] within the frame of the Mossclone EU-FP7 Project.The moss clone sample obtained in the bioreactor was washed with10 mM ethylendiaminetetracetic acid (EDTA) (using 1 L for each12.5 g d.w. of moss) and 3 times with pure water (1 L for each 10 gd.w.). The devitalization was performed by treatment in an oven asfollows: 8 h at 50 ◦C, 8 h at 80 ◦C and 8 h at 100 ◦C.

Devitalized moss samples were exposed for 6 weeks in five dif-ferent locations: an urban site, in a city (ca. 250,000 inhabitants) in

a point affected by road traffic and close to the marine coast; a sub-urban site in an open green area, at 5 km away from the previouslymentioned city and also close to the sea (ca. 500 m); an industrialsite at 1 km away from a metallurgical industry in an industrial area,

E. Concha-Grana et al. / J. Chroma

cwapp

a

3

0g3oa

(f0npt1wgm

3

d

Fig. 1. Scheme of the selected analysis procedure.

lose to a thermal-power station; an agricultural site, in an areaith meadows, crop and pasture fields; and a background site in

remote area selected as background atmospheric contaminationoint by the EMEP–VAG–CAMP Spanish Network for atmosphericollution control.

After the exposure, the samples were collected, oven-dried (24 ht 40 ◦C) and then ground in an ultracentrifuge mill (RetschZM200).

.2. Extraction and clean-up

A summary of the optimized procedure is described in Fig. 1..25 g of moss sample (with 50 �L of working solution of surro-ates) was blended with 0.5 g of C18 in an agate mortar, during

min. Then the homogeneous dispersion was deposited in the topf a Florisil SPE tube (1 g) with 0.5 g of anhydrous sodium sulphate,ll previously washed with 4 mL H and 4 mL DCM:H (20:80).

The PAHs were eluted with 10 mL of H and 10 mL of DCM:H20:80) mixture using a Visiprep vacuum distribution manifoldrom Supelco. The eluate was concentrated to approximately.3 mL in a Syncore® Analyst evaporator from Büchi Labortech-ik AG (Flawil, Switzerland), using the conditions described in arevious work [16] with pressure programme 640 mbar (1 min)o 540 mbar (in 1 min), hold 10 min, 540 mbar to 210 mbar in

min, hold 210 mbar 25 min. Finally 20 �L of internal standardas added. All was transferred to a vial and analyzed by pro-

rammed temperature vaporization–gas chromatography–tandemass spectrometry (PTV–GC–MS/MS) for PAH determination.

.3. Gas chromatography–tandem mass spectrometry

The PTV–GC–MS/MS conditions used were the same asescribed in a previous work [16]. The parent and product ions and

togr. A 1406 (2015) 19–26 21

retention time of PAHs studied are shown in Table 1. The com-pounds in the table are separated by groups, with the labelledsurrogates used for the quantitation of each group in italics.

3.4. Quantification and quality control

Chromatographic control was performed by the injection of twostandard solutions containing all target PAHs, and an instrumen-tal blank for each batch. Procedural blanks were systematicallyevaluated carrying out the whole procedure without samplematrix (surrogates were spiked on the C18). These proceduralblanks were maintained at minimum. Dibenz[a,h]anthracene-d14 (to correct dibenz[a,h]anthracene, indeno[1,2,3-cd]pyrene,benzo[ghi]perylene-d12 and benzo[ghi]perylene) and anthracene-d10 (for the other PAHs) were used as internal standards. Qualitycontrol of the complete procedure and quantification were per-formed using labelled PAHs as surrogates.

4. Results and discussion

4.1. Selection of the initial conditions

Some of the conditions used to perform the experiments wereselected according to our experience in previous works, or accord-ing to the literature. Thus, the dispersion time was set to 3 min,because this time is adequate to achieve a homogeneous dispersion[32].

For the clean-up, Florisil (1 g) was selected, because it is themost popular sorbent in the literature, and together with silica hasprovided good results in a previous work [16].

4.2. Selection of the dispersing agent

For the optimization, an unexposed moss clone sample wasused. The sample was spiked with 50 �L of 0.2 �g mL−1 surrogatestandards and 20 �L of 0.5 �g mL−1 PAHs standard, and then it wasblended with the pestle until all the standards were absorbed andthe sample appears homogenous.

Preliminary assays were performed in order to test different dis-persants. It is important that the mixture of the dispersing agentand the sample become homogeneous. In that sense, Sea sand wastested and discarded because a non homogeneous mixture wasobtained when added to the moss. Moreover, diatomaceous earthwas also rejected because a compact mixture was obtained makingthe contact between sample and eluent solvent difficult.

Experiments were carried out using neutral silica (1 g) with dif-ferent degrees of deactivation (5%, 10% and 20%) or C18 (0.5 g) asdispersants. In all the experiment Florisil was used for the clean-upstep. Once the moss sample (0.25 g) was dispersed with the sil-ica or the C18 during 3 min, the dispersion was located in the topof the previously washed SPE clean-up cartridge. The elution of thePAHs was performed using 5 mL of H and 10 mL of a DCM:H (20:80)mixture (Fig. 1).

The eluates obtained for the three assays using silica partiallydeactivated were coloured, and the colour increase in intensity withdecreasing the deactivation degree of silica. The assay performedwith the Supelclean-Envi 18 gave a colourless eluate, which sup-poses less co-extracting matrix compounds. Regarding the recoveryresults, very small differences were observed between the experi-ments (Fig. 2A). For these reasons Supelclean-Envi 18 was selectedas dispersant.

4.3. Study of sample–dispersant proportion

The relation between the amount of sample and the amount ofdispersing agent was also assayed. 0.5 g, 1 g and 1.5 g (relations 1:2,

22 E. Concha-Grana et al. / J. Chromatogr. A 1406 (2015) 19–26

Table 1Analytical parameters: detection limits (LOD), quantitation limits (LOQ), trueness (n = 7), repeatability, intermediate precision (IP) and uncertainty.

Abbreviation Retention time(min)

Parent ion Production

LOD(ng g−1)

LOQ(ng g−1)

Trueness(%)

IP (%)(n = 6)

Uncertainty(%)

Naphthalene d8 25.79 136 108Naphthalene Naph 25.94 128 102 0.72 1.42 101 4.3 8

Acenaphthylene d8 35.60 161 157Acenaphthylene Acy 35.63 152 125 0.57 1.26 98 9.8 10Acenaphthene Ace 36.73 153 150 0.52 1.11 81 5.7 8.0Fluorene Fluo 40.14 166 163 0.10 0.20 92 7.8 18

Phenanthrene d10 46.16 188 160Phenanthrene Phe 46.32 178 152 0.95 1.72 102 4.4 8IS anthracene d10 46.56 188 160Anthracene Ant 46.69 178 152 0.90 1.22 106 3.7 16

Fluoranthene d10 53.61 212 208Fluoranthene Fla 53.74 202 198 0.49 1.00 94 3.7 15

Pyrene d10 55.02 212 208Pyrene Pyr 55.14 202 198 0.54 1.16 89 4.0 20Retene Ret 56.37 219 204 0.18 0.38 104 4.2 8

Benz[a]anthracene BaA 62.67 228 224 1.10 0.21 112 3.4 17Chrysene d12 62.74 240 236Chrysene Chry 62.93 228 224 0.10 0.21 100 4.4 8

Benzo[b + j]fluoranthene B(b + j)F 69.08 252 248 0.25 0.37 83 5.6 22Benzo[k]fluoranthene B(k)F 69.18 252 248 0.27 0.41 91 4.8 17Benzo[e]pyrene B(e)P 70.58 252 248 0.23 0.30 77 6.3 29Benzo[a]pyrene d12 70.74 264 260Benzo[a]pyrene B(a)P 70.9 252 248 0.36 0.54 86 6.7 21

IS dibenz[a,h]anthracene d14 76.90 288 274Dibenz[a,h]anthracene D(ah)A 77.12 278 274 0.42 0.96 116 4.8 22Indeno[1,2,3-cd]pyrene IP 77.24 276 272 0.12 0.09 95 11 22

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Benzo[ghi]perylene d12 78.82 288

Benzo[ghi]perylene B(ghi)Per 79.05 276

:4 and 1:6, respectively) of Supleclean-Envi 18 were assayed forhe 0.25 g of moss.

These assays showed small differences between the three rela-ions tested (Fig. 2B). The proportion 1:2 was selected because itrovides good results minimizing the sorbent and solvent con-umption. These results were obtained using the solvent finalonditions selected in Section 4.4.

.4. Study of eluents

The elution of the analytes from the cartridge was carried out inwo fractions collected together (3 mL of H and a 10 mL of DCM:H20:80) mixture). This elution was selected based on the results ofrevious studies [16]. Using only 3 mL of H and 10 mL of DCM:Hixture, a low extraction efficiency was obtained for the elution of

he more apolar PAHs, and for this reason some assays were per-ormed maintaining the volume of the DCM:H mixture and usingigher volume of hexane. As can be seen in Fig. 2C, the recoveryf dibenz[a,h]anthracene and the 6-rings PAHs increases as theolume of hexane increases. For this reason 10 mL of hexane fol-owed by 10 mL of DCM:H (20:80) was selected for the elutiontep.

. Analytical performance characteristics

The scheme in Fig. 1 summarizes the proposed analysis proce-ure.

Table 2 shows a comparison among the proposedSPD–PTV–GC–MS/MS method and other procedures described in

iterature for the determination of PAHs in moss. The simultaneousxtraction and clean-up steps in MSPD allows a significant reduc-ion in solvent and time consumption in comparison with otherrocedures used for the analysis of PAHs in moss samples. Only

284272 0.36 0.75 84 3.6 22

20 mL of solvent per sample are necessary in the whole process(extraction and clean-up), also due to the low amount of mossneeded for the analysis. Additionally, the proposed method doesnot require expensive instrumentation to perform the extractionand clean-up of the sample. Also, the MSPD procedure used in thiswork is fast, the extraction time is lower than in other procedures,and also the concentration step is briefer due to the lower solventvolume. The total time for the preparation of 6 samples by thisprocedure is about 4 h.

The analytical performance characteristics of the proposedmethod were evaluated analysing spiked S. palustre L. clone sam-ples since there are no certificated reference materials available forPAHs in moss, or similar matrices.

Detection limits (LOD) and quantitation limits (LOQ) were calcu-lated as Xb + 3Sl and Xb + 10Sl respectively, where Xb is the averagevalue of blank sample containing no PAHs, and Sl the standarddeviation of a low concentration moss sample [40]. The sensitivityachieved (experimentally verified) in this work is better or compa-rable to other methods in the literature, with LOD values rangedbetween 0.10 and 1.10 ng g−1 and LOQ values lower than 2 ng g−1

for all the PAHs, analysing only 0.25 g of sample (Table 1). Thegreat sensitivity achieved with the proposed procedure using avery small amount of moss is very useful because sometimes itis difficult to obtain enough quantity of moss sample to performthe analysis by other procedures (in general about 5 g of sampleare necessary for Soxhlet or sonication extraction). Thus, using thismethod, a higher number of sample replicates can be performedin order to minimize the variability intrinsic to biological matri-ces.

Regarding the trueness, this was determined using the ana-lytical recoveries of spiked samples at 0.15 �g g−1 level. Truenesswas calculated (n = 7) quantifying using the labelled surrogates. Theoptimized analytical procedure showed good recoveries (77–116%)

E. Concha-Grana et al. / J. Chromatogr. A 1406 (2015) 19–26 23

F ctadee

fl9

a

ig. 2. (A) Study of the dispersing agent: silica deactivated a 5%, 10%, and 20%, and olution solvent volume (n = 3).

or all the native PAHs (Table 1). The recoveries obtained for the

abelled standards used as surrogates were ranged between 62 and4%.

Linearity of the method was determined by the analysis ofn unexposed moss clone sample spiked with target PAHs at 8

cylsilica (C18); (B) study of the relation sample: dispersant amount; (C) study of the

concentration levels, between 0.005 �g g−1 and 1.5 �g g−1, to

obtain the standard addition graphs. A good linearity, with cor-relation coefficients higher than 0.998, was obtained for almost allPAHs between LOQ and 1.5 �g g−1, and between LOQ and 1 �g g−1

for pyrene and fluorene.

24 E. Concha-Grana et al. / J. Chromatogr. A 1406 (2015) 19–26

Table 2Comparison of the proposed matrix solid phase dispersion (MSPD) extraction method, with other methods used for PAHs analysis in moss: Soxhlet, sonication, acceleratedsolvent extraction (ASE), dynamic sonication-assisted solvent extraction (DSASE) and microwave assisted extraction (MAE).

MSPD Soxhlet Sonication ASE DSASE MAE

Sample amount 0.25 g 5 g 3–5 g 1–5 g 0.2 g 0.5 gSolvent consumption 20 mL 200–300 mL 150–300 mL 20–200 mL 4 mL 40 mLExtraction time 3 min 8–24 h 10–30 min 10–30 min 10 min 16 minClean-up On-line Off-line Off-line On-line/off-line Off-line Off-lineCost Low Low Low High High MediumSensitivity LOQ: 0.09–1.72 ng g−1 LOQ: 0.3–1 ng g−1 LOD: 1–3 ng mL−1 MQL: 3.3–7.8 ng g−1 7−350 ng g−1 a LOQ: 0.1–1.7 ng g−1

Precision (RSD) 3.4–11% 10–25% 10–15% 1–22% 1.8–17% 2.7–12%Reference This work [34,37,39,42–46] [4,47,48] [5,38,49,50] [51] [16]

a Instrumental detection limits.

btaine

tirpefpcio

mETc(u√

p

TD

Fig. 3. PAH profile and concentration o

The precision of the method was evaluated by determininghe repeatability and the intermediate precision. The repeatabil-ty was calculated as within-day RSD of concentrations, using foureplicates of spiked moss samples (0.15 �g g−1) analyzed with theroposed method during the same day and the same analyst andquipment. The repeatability obtained expressed as RSD was satis-actory for all the PAHs, with values lower than 8%. The intermediaterecision (IP) of the method was calculated as between-day RSD ofoncentrations over the course of 4 weeks (6 replicates). A goodntermediate precision, with %RSD lower than 11% in all cases wasbtained (Table 1).

The uncertainty (U) of the analytical method was also esti-ated on the basis of in-house validation data according to the

URACHEM/CITAC guide for all compounds at 0.15 �g g−1 level.he main sources of uncertainty were identified and quantified andombined uncertainty was calculated as U = k

√u2

1 + u22 + |� − x|,

coverage factor k = 2, for a 95% of confidence) where thencertainties associated with the spiked sample (u1 = Csample ∗

(Sstandard/Cstandard)2 + (Spipette/Vpipette)2 + (Sflask/Vflask)2 + (Sbalance/mstandard)2),recision (u2 = Sprec/

√N) and trueness |� − x| were taken into

able 3iagnostic ratios calculated for exposed moss clone samples.

Agricultural Urban

Fla/Fla + Pyr 0.57 0.56

Ant/Ant + Phe 0.12 0.08

BaA/BaA + Chry 0.22 0.18

BaP/BaP + BeP 0.11 0.21

IP/IP + BghiPer 0.56 0.52

Ret/Ret + Chry 1.00 1.07

BaP/BghiP 0.32 0.60

d for the exposed moss clone samples.

account. The relative expanded uncertainty calculated for thewhole method at 0.15 �g g−1 level was lower than 30% in all cases(Table 1).

6. Analysis of samples

The proposed method was tested analysing 5 moss cloneexposed samples from agricultural, urban, sub-urban, backgroundand industrial locations in order to verify the suitability of theextraction procedure for the different levels of contamination. Thesum of the 20 PAHs analyzed (expressed as �g g−1 d.w.) was 0.84for the Industrial sample, 0.76 for the urban sample, 0.53 for thesub-urban, 0.15 for the background sample, and 0.09 for the agricul-tural sample. As can be seen in Fig. 3, both, the light PAHs associatedto gas phase and the heavy PAHs associated to the particle phasewere extracted from samples, which demonstrates the suitabil-

ity of the proposed procedure for the analysis of both fractions.Similar PAH profiles and levels were obtained for the industrialand urban samples, whereas lower levels with very low concen-trations of the heavier PAHs were obtained for the background and

Sub-urban Background Industrial

0.48 0.50 0.560.27 0.17 0.110.26 0.25 0.320.28 0.19 0.370.53 0.52 0.551.05 1.01 1.090.73 0.88 0.77

E. Concha-Grana et al. / J. Chromatogr. A 1406 (2015) 19–26 25

Table 4Analysis of two natural moss species (Sphagnum sp. and Hypnum cupresiforme) and one lichen species (Pseudevernia furfuracea). Concentration of native PAHs (ng g−1) anddeuterated surrogate recoveries (%R).

Sphagnum sp. Hypnum cupresiforme Pseudevernia furfuracea

ng g−1 %R ng g−1 %R ng g−1 %R

Naphthalene d8 43% 41% 41%Naphthalene 39 58 50Acenaphthylene d8 73% 92% 76%Acenaphthylene <1.26 <1.26 <1.26Acenaphthene 0.6 3.9 1.4Fluorene <0.20 7.3 <0.20Phenanthrene d10 78% 92% 83%Phenanthrene 4.0 22.8 36Anthracene 1.7 4.2 9.2Fluoranthene d10 87% 98% 96%Fluoranthene <1.0 7.9 5.7Pyrene d10 90% 101% 102%Pyrene 0.1 3.7 30Retene 4.5 12.2 13Benz[a]anthracene 0.9 0.6 1.3Chrysene d12 90% 103% 100%Chrysene <0.21 5.8 2.7Benzo[b + j]fluoranthene 4.0 3.1 2.1Benzo[k]fluoranthene 4.3 5.4 2.0Benzo[e]pyrene 3.3 2.7 1.3Benzo[a]pyrene d12 90% 90% 86%Benzo[a]pyrene 2.9 1.6 1.8Dibenz[a,h)anthracene 2.2 1.8 1.3

af

osac(t((c

mccsmm

7

dbiiaasacc

mc

Indeno[1,2,3-cd]pyrene 4.1

Benzo[ghi]perylene d12 89%

Benzo[ghi]perylene 3.7

gricultural samples. The PTV–GC–MS/MS chromatogram obtainedor the industrial sample is included in the supplementary material.

Some PAH diagnostic ratios [41] were determined (Table 3) inrder to investigate if these results agree with the expected PAHources for each kind of sample. The relations Ant/Ant + Phe (>0.1)nd BaA/BaA + Chry (0.2–0.35) show pyrogenic processes, specifi-ally from coal combustion for all samples except the urban onepetrogenic process). In all cases the relation Ret/Ret + Chy (closeo 1) indicates wood burning processes. The relation BaP/BaP + BeP<0.5 in all samples) should indicate photodegradation processesold emission). A grass, wood and coal combustion source is indi-ated for the relations Fla/Fla + Pyr (>0.5) and IP/IP + BghiPer (>0.5).

The method was also tested for the analysis of PAHs in naturaloss of the same genus (Sphagnum sp.), another moss species (H.

upressiforme) and in a lichen (P. furfuracea) applying the same pro-edure. As can be seen in Table 4, good recoveries of the labelledurrogates were obtained in all cases, and therefore the proposedethod can also be applied for the analysis of PAHs in severalosses and lichen species.

. Conclusions

A novel procedure for the analysis of PAHs in moss samples waseveloped, tested and validated. The MSPD extraction, followedy the PTV–GC–MS/MS determination, presents many advantages

n comparison with the existing procedures. The great sensitiv-ty of the method allows the considerable reduction of samplemount and solvent consumption. The simultaneous extractionnd clean-up allow the elimination of one of the concentrationteps typically needed in other procedures, reducing also the totalnalysis time. For all these reasons the proposed method can beonsidered “greener” than the existing ones for the analysis of a

omplex matrix like moss samples.

The analytical performance characteristics of the proposedethod are good, improving the results obtained by other pro-

edures (Table 2). The method was proven to be suitable for the

3.1 2.775% 74%

3.3 2.1

analysis of moss samples and can be very helpful for the fast analysisof hundreds of samples in air quality biomonitoring programmes.

Acknowledgements

Financial support is acknowledged to the Program of Consol-idation and Structuring of Units of Competitive Investigation ofthe University System of Galicia (Xunta de Galicia) potentiallyco-financed by ERDF in the frame of the operative Program of Gali-cia 2007–2013 (reference: GRC2013-047) and “Mossclone” EU-FP7Project (ENV. 2011-Eco-innovation).

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.chroma.2015.06.014

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