Talanta 80 (2009) 643–650

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Optimisation and validation of programmed temperaturevaporization (PTV) injection in solvent vent mode forthe analysis of the 15 + 1 EU-priority PAHs by GC–MS

José Ángel Gómez-Ruiz1, Fernando Cordeiro, Patricia López, Thomas Wenzl ∗

European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Retieseweg 111, B-2440 Geel, Belgium

a r t i c l e i n f o

Article history:Received 29 April 2009Received in revised form 3 July 2009Accepted 20 July 2009Available online 28 July 2009

Keywords:15 + 1 EU-priority polycyclic aromatichydrocarbons (PAHs)Large volume injection (LVI)

a b s t r a c t

This paper presents the optimisation of a programmed temperature vaporization solvent vent (PTV-SV)injection gas chromatographic mass spectrometric (GC–MS) method for the analysis of the 15 + 1 EU-priority PAHs in food extracts. Three operation parameters (vent time, vent flow and vent pressure) wereoptimised by applying a D-optimal experimental design. Among these variables, vent time showed thehighest effect on the analytical response (signal intensity) of the target PAHs.

The 15 + 1 EU-priority PAHs were analysed in solvent solutions and in extracts of fortified sausage.In addition, blank lamb meat extracts were prepared and spiked with the target PAHs prior to GC–MSanalysis. The performance of the optimised PTV-SV injection GC–MS method was scrutinised for linearity,precision, matrix effects and robustness. All parameters were found satisfactorily. Compared to PTV

Programmed temperature vaporization(PTV)Gas chromatography mass spectrometry(F

injection in splitless mode, the PTV-SV injection method provided an enhancement of sensitivity for alltarget PAHs. Especially significant was the improvement of the S/N ratios of the compounds with thehighest molecular mass.

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. Introduction

Polycyclic aromatic hydrocarbons (PAHs) are a large group ofrganic compounds containing two or more fused aromatic ringsonstituted of carbon and hydrogen atoms. PAHs constitute a largelass of environmental contaminants. They are emitted from dieselngines, but also stationary sources, e.g. different kinds of indus-ries [1]. The main source of exposure to PAHs for non-smokingumans is food. Food might be contaminated by PAHs throughhe environment or via formation during processing. The dietaryntake of PAHs is of concern because some of them can causeancer in humans [2]. The Scientific Committee on Food (SCF)ssessed in 2002 the toxicity of 33 PAHs and concluded that 15f them are potentially genotoxic and carcinogenic to humans [3].n 2005, the Joint FAO/WHO Expert Committee on Food Additives

JECFA) identified an additional PAH (benzo[c]fluorene) as prob-bly carcinogenic [4]. The joint set of PAHs is recognized as the5 + 1 EU-priority PAHs (Table 1). This terminology serves to dis-inguish them from the 16 PAHs highlighted in the 1970s by the

∗ Corresponding author. Tel.: +32 14 571 320; fax: +32 14 571 783.E-mail address: Thomas.Wenzl@ec.europa.eu (T. Wenzl).

1 Current address: Instituto de Fermentaciones Industriales, Consejo Superior denvestigaciones Científicas (CSIC), C/ Juan de la Cierva 3, 28006 Madrid, Spain.

039-9140/$ – see front matter © 2009 Elsevier B.V. All rights reserved.oi:10.1016/j.talanta.2009.07.041

© 2009 Elsevier B.V. All rights reserved.

US Environmental Protection Agency (EPA) [5]. Though eight ofthe 15 + 1 EU-priority PAHs are identical with PAHs of the US-EPA list they are challenging from the analytical point of view.Difficulties are related to both separation efficiency and sensitiv-ity.

High discrimination of the signal intensities of low and highmolecular weight compounds is a common problem in the analysisof PAHs by gas chromatography [6,7]. The lower abundance of theheavier compounds causes also smaller signal-to-noise (S/N) ratioswhich result in the increase of the limits of detections (LOD). Amongthe 15 + 1 EU-priority PAHs are four dibenzopyrenes (Table 1),which are especially susceptible to discrimination of signal inten-sities due to their high molecular mass.

Large-volume injection (LVI) techniques such as programmed-temperature-vaporization (PTV) and cool-on-column (COC) injec-tion can be used to increase method sensitivity through theinjection of larger volumes of the final extract. PTV injection has alsobeen used to reduce discrimination of certain analytes comparedto classical split–splitless injection [8]. In addition, PTV injection ispreferred to on-column injection when the analyses involve com-

plex samples [9]. Several studies have been recently reported usingPTV methods for the determination of trace pollutants, such asphenols [10], pesticides [11] and polychlorinated biphenyls [12].Likewise, PAHs have been also determined in environmental sam-ples [12–17] and foodstuff [18,19] using PTV injection. However, it

644 J.Á. Gómez-Ruiz et al. / Talanta 80 (2009) 643–650

Table 115 + 1 EU-priority PAHs under investigation (with acronyms). The right part of the table (in bold) shows those PAHs that are not included in the 16 EPA PAHs.

1 Benz[a]anthracene (BaA) (Mw 228) 9 Benzo[j]fluoranthene (BjF) (Mw 252)

2 Benzo[a]pyrene (BaP) (Mw 252) 10 Cyclopenta[cd]pyrene (CPP) (Mw 226)

3 Benzo[b]fluoranthene (BbF) (Mw 252) 11 Dibenzo[a,e]pyrene (DeP) (Mw 302)

4 Benzo[ghi]perylene (BgP) (Mw 276) 12 Dibenzo[a,h]pyrene (DhP) (Mw 302)

5 Benzo[k]fluoranthene (BkF) (Mw 252) 13 Dibenzo[a,i]pyrene (DiP) (Mw 302)

6 Chrysene (CHR) (Mw 228) 14 Dibenzo[a,l]pyrene (DlP) (Mw 302)

7 Dibenz[a,h]anthracene (DhA) (Mw 278) 15 5-Methylchrysene (5MC) (Mw 242)

8 Indeno[1,2,3-cd]pyrene (IcP) (Mw 276) +1 Benzo[c]fluorene (BcL) (Mw 216)

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ust be stressed that none of the cited studies covered the wholeet of 15 + 1 EU-priority PAHs.

PTV injection offers more operating parameters than split/plitless injection. Hence the optimum combination of parameterettings needs to be identified for each analysis task individually.ome authors [11,13] tackled the optimisation of the PTV injectiony univariate procedures, whereas others applied multivariate sta-istical experimental designs for that purpose [12,14,16,20]. Fromhe latter, only Yusà et al. [14] and León et al. [20] focused in theirtudies on the determination of PAHs, which however covered onlypart of the analytes dealt with in this paper.

The use of statistically based experimental designs (two-levelull factorial designs, fractional factorials, central composite orlackett-Burman designs) can simplify the optimisation of PTVnjection through the use of a selected number of experiments thatandomly combines the desired variables. This approach providesmethod for the simultaneous investigation of multiple variables,stimating any interaction among them, and requires fewer exper-ments to complete the optimisation [14].

Only a few papers have been published on the optimisation

f PTV injection for the determination of PAHs by GC–MS. Theyeported a significant increase in sensitivity by the use of PTVnjection if compared to splitless injection [13,14,16]. These studiesowever have focused only on few selected PAHs [14] or on the 16PA PAHs [13,16]. A study has been published recently concerning

the determination of high molecular mass PAHs by application ofPTV injection GC–MS [21]. This paper reported on the measurementof 15 out of the 15 + 1 EU-priority PAHs in solvent solution and edi-ble oil extracts by using PTV injection (1 �L) in splitless mode [21].However PTV injection in splitless mode has the same limitationswith regard to tolerated solvent volume as conventional splitlessinjection. If the solvent volume exceeds a certain level peak dis-tortions have to be taken into account. PTV injection in solventvent mode (PTV-SV) in contrary offers the possibility to performlarge volume injection and gain thereby extra sensitivity. The limi-tation of this injection technique is provided by the larger amountof matrix that is injected into the instrument, which might interferewith the analyte signal. However, no studies are currently availablethat focus on the employment of PTV-SV injection for the GC–MSanalysis of the 15 + 1 EU-priority PAHs in food samples.

The aim of the present study was the optimisation of a PTV-SVinjection method for the GC–MS analysis of the 15 + 1 EU-priorityPAHs in foodstuff. The optimisation of different operating param-eters of the PTV injection was carried out by application of astatistically based experimental design. Sausage samples prepared

with oil fortified with the 15 + 1 EU-priority PAHs and non-contaminated lamb meat samples were used throughout the study.Different performance parameters such as linearity, precision, andmatrix effects were evaluated for the optimised PTV-SV injectionGC–MS analysis method. Sensitivity and discrimination of signal

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ntensities were compared to standard PTV injection in pulsed split-ess injection mode (PTV-PSL).

. Materials and methods

.1. Reagents and reference materials

All solvents were purchased in chromatographic grade fromWR International (Leuven, Belgium). The drying materialoly(acrylic acid), partial sodium salt-graft-poly(ethylene oxide)as obtained from Aldrich (Milwaukee, USA) and Ottawa sand fromcros Organics N.V. (Geel, Belgium).

The analytes benz[a]anthracene (BaA) CAS#56-55-3, chryseneCHR) CAS#218-01-9, 5-methylchrysene (5-MC) CAS#3697-24-3,enzo[b]fluoranthene (BbF) CAS#205-99-2, benzo[j]fluorantheneBjF) CAS#205-82-3, benzo[k]fluoranthene (BkF) CAS#207-08-, benzo[a]pyrene (BaP) CAS#50-32-8, indeno[1,2,3-cd]pyreneIcP) CAS#193-39-5, dibenz[a,h]anthracene (DhA) CAS#53-70-, benzo[ghi]perylene (BgP) CAS#191-24-2, dibenzo[a,l]pyreneDlP) CAS#191-30-0, dibenzo[a,e]pyrene (DeP) CAS#192-65-4,ibenzo[a,i]pyrene (DiP) CAS#189-55-9, dibenzo[a,h]pyrene (DhP)AS#189-64-0, were commercially available BCR certified ref-rence materials (IRMM, Geel, Belgium). Cyclopenta[cd]pyreneCPP) CAS#27208-37-3, purity >99.0% by GC, was manufac-ured on request (Biochemisches Institut für Umweltkarzinogene,roßhansdorf, Germany). Benzo[c]fluorene (BcL) CAS#205-12-, purity 99.99%, was purchased from Dr. Ehrenstorfer GmbHAugsburg, Germany). The isotopically labelled compoundsenz[a]anthracene (13C6, 99%), benzo[a]pyrene (13C6, 99%),enzo[b]fluoranthene (13C6, 99%), benzo[ghi]perylene (13C12,9%), benzo[k]fluoranthene (13C6, 99%), chrysene (13C6, 99%),ibenz[a,h]anthracene (13C6, 99%), dibenzo[a,e]pyrene (13C6, 99%),ibenzo[a,i]pyrene (13C12, 99%), indeno[1,2,3-cd]pyrene (13C16,9%) and pyrene (13C3, 99%), which was used as internal stan-ard for BcL, were obtained from LGC Promochem GmbH (Wesel,ermany). 9-Fluorobenzo[k]fluoranthene, used as injection stan-ard, was purchased from Chiron AS (Trondheim, Norway). Stockolutions of the individual PAHs were gravimetrically prepared inoluene. The different working solutions containing the 15 + 1 EU-riority PAHs were prepared by mixing appropriate volumes fromhe individual stock solution and adding toluene to achieve theesired concentrations (6 different levels between 15 ng/mL and0 ng/mL).

.2. Sample preparation

Contaminated sausage samples were prepared at the Max-ubner-Institut (MRI), Bundesforschungsinstitut für Ernährungnd Lebensmittel, Institut für Sicherheit und Qualität bei FleischKulmbach, Germany) by applying spiked olive oil for the produc-ion of sausages. The sausage samples were split into portions of0 g each and packed into aluminium cans. The final analyte con-ents ranged from 3.9 �g kg−1 to 9.9 �g kg−1. Benzo[a]pyrene, thenly PAH regulated by current EU legislation, was present at a levelf 5.3 �g kg−1, a value slightly above the maximum level specifiedn Commission Regulation (EC) No 1881/2006 (5 �g kg−1) [22].

Blank meat extracts were prepared from commercial mincedamb meat. They were fortified with the 15 + 1 EU-priority PAHsrior to the GC–MS analysis in order to evaluate matrix effectsf the optimised PTV-SV method. The blank extracts were forti-ed to a concentration of 50 ng mL−1 for each PAH, a concentrationimilar to that expected in the final extract of the sausage sample

onsidering sample intake and analyte enrichment.

.2.1. Pressurised liquid extraction (PLE)Extraction was carried out in accordance with the method

escribed by Jira et al. [23]. Briefly, 5 g of homogenised sausage

ta 80 (2009) 643–650 645

was mixed with the same amount of poly(acrylic acid), partialsodium salt-graft-poly(ethylene oxide) and 15 g of Ottawa sand.After adding 200 �L of the 13C-labelled standard solution, the mixwas placed into 33 mL extraction cells with cellulose filters at thebottom. The extraction was performed with n-hexane (at 100 ◦Cand 100 bars at a static time of 10 min) in an ASE 300 pressurised liq-uid extraction system (Dionex, Amsterdam, The Netherlands). Theflush volume was 60% and the purge time 120 s. Two static extrac-tion cycles were performed per sample. Afterwards, the extractantwas evaporated in a Turbo Vap® workstation (Zymark, Hopkinton,USA) at 40 ◦C using a stream of nitrogen.

2.2.2. Gel permeation chromatography (GPC)The evaporated PLE-extract was dissolved in approximately

5 mL of cyclohexane:ethyl acetate (1:1 v/v) and filtered throughPTFE filters of 5 �m pore size (Millipore, Bedford, USA). GPC cleanup was carried out on a GPC column (25 mm internal diameter and320 mm length) filled with 50 g Bio-Beads S-X3 (Bio-Rad Labora-tories, Nazareth-Eke, Belgium) applying a GPC Ultra instrument(LCTech, Dorfen, Germany) connected to a FW-20 detector (LCTech)that was operated at 254 nm. Chromatographic separation wasachieved with cyclohexane:ethyl acetate (1:1 v/v) at a flow rate of4 mL min−1. The PAHs containing fraction eluted between 35 minand 80 min. The volume of this fraction was reduced to 4.5 mLby using an automated concentrator (CPC 2000-II Vacuubrand,Wertheim, Germany) that was integrated in the GPC system. Thesensitivity of the UV detector was not sufficient to detect the elu-tion of the PAHs. As an alternative the elution of toluene, whichgot into the extract as a residue of the spiking with internal stan-dards dissolved in toluene, and the elution of the fat fraction weremonitored for quality control purposes. Toluene eluted shortly afterthe fat fraction and directly in front of the first target analyte,benzo[c]fluorene.

2.2.3. Solid phase extraction (SPE)The GPC eluate was further reduced with a stream of nitro-

gen to 200 �L. Then 800 �L of cyclohexane were added and themix was quantitatively loaded onto a preconditioned silica SPEcartridge (500 mg/4 mL, Alltech, Deinze, Belgium). The PAHs wereeluted with 10 mL of cyclohexane.

2.2.4. Preparation for GC–MS analysisToluene (200 �L) was added as a keeper to the SPE eluate, which

was then concentrated to a volume of about 200 �L. Afterwards,300 �L of toluene containing 9-fluorobenzo[k]fluoranthene wasadded, which served for monitoring the variability of injection.

2.3. Instrumentation and analytical conditions

A gas chromatograph HP 6890N from Agilent Technologies(Waldbronn, Germany) with a programmable-temperature vapor-ization (PTV) injection port (septumless head) was used for theanalysis of the target PAHs. The GC was coupled to an Agilent 5975Bsingle quadrupole mass spectrometer (Agilent Technologies) oper-ated in electron ionization (EI) mode at 70 eV. The analyses wereperformed in selected ion monitoring (SIM) mode recording themolecular ion of each compound (Table 1). Transfer line temper-ature and ion source temperature were maintained at 325 ◦C and300 ◦C, respectively. The mass spectrometer was equipped with a6 mm ultra large aperture draw out lens (Agilent Technologies).Injection was carried out using an automated GC PAL injection

system (CTC Analytics, Zwingen, Switzerland).

PTV-PSL injection was performed to set reference values onwhich basis the performance characteristics of the PTV-SV injectionmethod were evaluated. The parameters of the PTV-PSL injectionmethod were: injection volume 1 �L, injection pulse pressure 30 psi

6 Talanta 80 (2009) 643–650

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or 0.3 min, and injection port temperature 300 ◦C. The purge valveas opened after 1 min (purge flow 30.5 mL min−1).

The parameters of the PTV-SV injection were subject of the opti-isation process and are discussed later in this paper.All analyses were carried out on a DB-17MS column

0 m × 0.25 mm internal diameter, 0.25 �m film thickness (Agilentechnologies, Diegem, Belgium). The oven temperature pro-ramme was 80 ◦C (hold 1 min), to 250 ◦C at 40 ◦C min−1, to 305 ◦Ct 25 ◦C min−1, to 315 ◦C at 2 ◦C min−1, to 330 ◦C at 40 ◦C min−1andold for 35 min. Helium was used as carrier gas at 1.5 mL min−1

ow rate.

.4. Statistical software

Dedicated software for the design of experiments and optimi-ation, MODDE version 8.0 (Umetrics, Umeå, Sweden), was usedor building up the parameter matrix and further evaluation of theesults.

. Results and discussion

.1. Optimisation of PTV-SV injection

All standards and sample extracts were prepared in toluene.lthough toluene is not the preferred solvent for large volume

njection due to its rather high boiling point, it is the most suitableolvent to dissolve PAHs with high molecular mass (i.e. diben-opyrenes). Its application was also recommended to prevent thedsorption of micro-contaminants to glassware [24].

Large volume injection into a PTV injector can be done in differ-nt modes: at-once (i), or using multiple injection (ii) [25]. In modei), the sample is introduced at relatively high speed, whereas in

ode (ii) the sample is introduced at a rate that is theoreticallyqual to that of evaporation.

At-once injection was applied for this study. Different speedsere tested and the best results were obtained by injecting the

ample at a speed of 5 �L s−1.

.1.1. Selection of the inlet linerThe most appropriate PTV inlet liner has to be identified prior

o the optimisation of operating parameters, as this device has arucial role in the trapping and transfer of the analytes. The opti-isation was carried out using a step-wise approach. Four different

TV inlet liners were tested in this study: the single-baffle, theingle-baffle packed with glass wool, the multi-baffle, and the sin-ered glass inlet liner. Another type of liner, which is filled withenax was not considered in this study since it has been reportedhat their use can provoke problems during the transfer of highoiling compounds to the column, leading to peak distortion and

osses among other negative effects [9].Standard solutions containing 100 ng mL−1 of each target ana-

yte were injected ten times into each liner applying PTV-SVnjection, and repeatability of injection was evaluated. An injec-ion volume of only 1 �L was selected to avoid chromatographicroblems stemming from the, at that point of time, non-optimisedTV-SV injection.

The sintered glass inlet liner showed the worst performance inerms of mass transfer and was therefore excluded from furthertudies. Fig. 1 shows the respective chromatograms for the baf-e inlet liner (A) and the sintered glass inlet liner (B). As can be

een strong discrimination in terms of peak height occurred espe-ially for the compounds with the highest molecular weight, theibenzopyrenes.

Table 2 shows the relative standard deviations (RSDs) obtainednder repeatability conditions for the other three inlet liners. The

Fig. 1. Chromatograms of the 15 + 1 EU-priority PAHs on a DB-17MS column60 m × 0.25 mm internal diameter, 0.25 �m film thickness applying a PTV baffle liner(A) and a PTV sintered glass liner (B).

multi-baffle inlet liner provided the best results in terms of preci-sion for all compounds, although a similar level of precision wasobtained with the single-baffle inlet liner for the compounds withlow and medium molecular mass (from BcL to BaP). However theperformance of the multi-baffle liner was superior for analytes withhigh molecular mass.

The results obtained with the single-baffle inlet liner packedwith glass wool were generally worse than those obtained with theother two baffle inserts. Noticeably high RSDs were found for BcLand for high molecular weight compound like DiP and DhP. Thismight be caused by losses of BcL during the solvent evaporationphase, and irreversible adsorption of the heavy PAHs on active sitesof the glass wool.

When increasing the injection volume from 1 �L up to 5 �L, peaksymmetry was maintained only with the multi-baffle inlet liner.

Therefore, the multi-baffle inlet liner was applied for furtheroptimisation of the PTV-SV injection.

3.1.2. Injection volumeThe maximum tolerable injection volume of toluene was deter-

mined for the multi-baffle inlet liner by injecting different volumesof toluene into the PTV injector without connecting the column.The carrier gas flow was turned on in that experiment. Accordingto Godula et al. [26] the injection volume can be increased until asolvent drop is observed at the exit of the inlet liner. A volume of10 �L of toluene was injected into the multi-baffle inlet liner with-out visible overflow of the liquid and hence further-on used in thisstudy.

3.1.3. Initial inlet temperatureThe initial injector temperature plays an important role in large

volume injection in solvent vent mode. It has to be set to allow

fast removal of the solvent without losses of the analytes. Thetemperature of the injection port was set in the described experi-ments to 55 ◦C because higher temperatures caused losses of themost volatile target analyte, benzo[c]fluorene. This temperaturereflects also the minimum temperature that can be reached in a

J.Á. Gómez-Ruiz et al. / Talanta 80 (2009) 643–650 647

Table 2Repeatability expressed as RSD % obtained for the injection of 1 �L of a standard solution containing the 15 + 1 EU-priority PAHs in toluene in a DB-17MS column 60 m × 0.25 mminternal diameter, 0.25 �m film thickness (100 ng/mL, n = 10). The RSD (%) of three different liners is shown.

Single-baffle liner Single-baffle packed with glass wool liner Multi-baffle liner

RSD (%)Benzo[c]fluorene 1.32 37.84 3.13Benz[a]anthracene 1.73 2.43 1.81Cyclopenta[c,d]pyrene 1.37 5.07 1.51Chrysene 2.38 8.45 2.105-Methylchrysene 1.54 1.62 1.46Benzo[b]fluoranthene 1.10 4.57 0.61Benzo[k]fluoranthene 1.43 1.58 0.85Benzo[j]fluoranthene 1.63 5.01 1.13Benzo[a]pyrene 1.27 0.88 0.39Indeno[1,2,3-cd]pyrene 3.01 3.09 0.90Dibenz[a,h]anthracene 5.05 9.38 0.99Benzo[ghi]perylene 2.98 3.98 0.77

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flow. Contour surface plots were used to visualise the modelledregion and to assist in finding the optimal experimental condi-tions. Fig. 2 shows the contour plots of the response variable forthe most volatile compound within the set of target analytes (BcL),

Table 3Experimental conditions employed for the optimisation of the programmed tem-perature vaporization (PTV) injection in solvent vent mode by applying a D-optimalexperimental design.

Parameter Level

Dibenzo[a,l]pyrene 3.95Dibenzo[a,e]pyrene 6.01Dibenzo[a,i]pyrene 6.61Dibenzo[a,h]pyrene 6.20

easonable period of time without applying cryogenic inlet cool-ng. Compressed air was used as replacement of cryogenic gasesor facilitating the cooling process of the PTV injector. Consequentlyess than 5 min were necessary after each run to reach the initialnlet temperature.

.1.4. Optimisation of PTV operating parametersThe PTV-SV injection can be divided into four different phases:

he injection, the solvent vaporization, the transfer of analytes andhe cleaning phase. During the injection the split valve is open (sol-ent vent mode) and the sample is introduced into the injectionort set at a temperature below the boiling point of the carrier sol-ent. The following step, the solvent vaporization, is critical withegard to the analyte transfer onto the column and its effect onhe quality of separation. The aim of the solvent vaporization iso eliminate the excess of solvent without loosing the analytes.f the residual solvent volume is too high a long flooded zone isormed in the front part of the column leading to peak distortiona phenomenon known as “peak band broadening in space”) [27].oo excessive elimination of solvent will lead to losses of the mostolatile compounds. Hence a small quantity of remaining solventhat is later-on transferred onto the column is desirable, not onlyo prevent analyte losses, but also to form a film at the front part ofhe column that traps and thereby focuses early eluting compounds26].

The solvent elimination is influenced by different parametershat were considered in the optimisation process. They were inarticular vent flow, vent time, vent pressure, and initial inlet tem-erature. The initial inlet temperature was set to 55 ◦C, as detailedefore. Hence the focus was put onto the other three variables. Pre-ious reported studies [28] and own investigations revealed that aigh final temperature of the injection port enhanced the transfer ofhe analytes with high molecular mass onto the column, while thelope of the temperature increase in the PTV injection port did notave any significant influence. Accordingly, the final temperaturef the injection port was maintained throughout the study at 400 ◦Cnd the temperature ramp at the maximum value of 600 ◦C min−1.egardless the conditions selected during optimisation, the splitalve was opened after 3 min (purge time) and kept in this positionor 15 min at 400 ◦C (cleaning step).

Vent time, vent flow and vent pressure were the variables thatad to be optimised in order to maximise the analytical responsepeak area) of the different PAHs.

The type of experimental design was selected taking into consid-ration the number of experiments to be performed and hence the

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time needed for measurements and the information level gained,which is expressed by the efficiency of the design. A full facto-rial experimental design including the three variables at two levelswould consist of eight analyses, but would only allow the set up oflinear models. Any non-linear relationships cannot be identified attwo levels. A full factorial experimental design at three levels wouldconsist without replication of 27 (33), and at four levels even of64 (43) experiments. The advantage of full factorial designs is thatall main effects and all multi-factor interactions can be described.However, if higher order factor interactions are not consideredimportant fractional factorial experimental designs such as the D-optimal design may be applied. This is a computer generated designwhich consists of N runs out of all runs of the full factorial design,select in a way that maximum information can be gained with aminimum number of analyses [29]. The D-optimal design applied inthe current work comprised 21 randomized chromatographic runs(10 corner experiments to which 11 axial points were added) outof 64 possible runs that span maximally the experimental region.

Table 3 shows the parameter settings employed for each vari-able in the D-optimal experimental design.

The correlation matrix obtained from the experimental dataindicated an extremely high correlation between the responsesobtained for all PAHs, which justifies the application of one sin-gle model for all the 15 + 1 EU-priority PAHs. The data was fittedby means of partial least square regression (PLS-R) providing anindependent multivariate regression curve (model) for each PAH.No significant lack-of-fit was identified for any of the responses.Among the selected factors, vent time showed the highest influ-ence on the analytical responses (negatively correlated with allthe 15 + 1 EU-priority PAHs) followed by vent pressure and vent

Minimum Intermediate Maximum

Vent time (min) 0.5 1 and 1.5 2Vent flow (mL min−1) 25 50 and 75 100Vent pressure (bar) 0.25 0.5 and 1 1.5

648 J.Á. Gómez-Ruiz et al. / Talanta 80 (2009) 643–650

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ig. 2. Contour surface plots of BcL, BaP and DhP for two independent variablesressure = 0.5 bar). Values inside the plots represent analytical responses in arbitrar

or BaP, for which maximum levels in food are specified in EU leg-slation, and for the last eluting PAH (DhP) for the two independentariables vent time and vent flow at a constant value of the ventressure (0.5 bar). The contour plots show that small variations inent time provoke significant changes in the analytical responsef the PAHs (the response decreases as the vent time increases).minimum value of the vent time (0.5 min) combined with vent

ow values ranging between 60 mL min−1 and 100 mL min−1 max-mised the analytical response of all compounds. Long vent timeshowed a negative effect on the response even though the vent flowas kept low.

The optimisation of the response for the individual analytes isot the way to success if the target is performance at the best for thehole set of analytes, because parameter settings to maximise per-

ormance for the volatile target analytes might be improper for theix-ring PAHs, which show low volatility. Therefore, the optimisernd the prediction list (two features provided by MODDE software)ere used to predict the best experimental conditions considering

hat the response variables for all 15 + 1 EU-priority PAHs have theame importance. This is done by optimising an overall desirabil-ty function that is combining the individual desirability for eachesponse. However, a finding that is not reflected in the models

as that broad and fronting chromatographic peaks were obtainedhen short vent times were combined with low vent flows. Similar

esponses were gained for vent flows in the range of 60 mL min−1 to00 mL min−1 when combined with low vent time (0.5 min) (Fig. 2).herefore it was decided to set for the optimisation of the model

able 4erformance characteristics of the optimised PTV injection in solvent vent mode – GC–M

Compound PTV injection in solvent vent mode

Solvent solution

Calibration function Repeatabilitya IntermediatR2 RSDr (%) RSDi (%)

Benzo[c]fluorine 0.997 3.34 3.60Benz[a]anthracene 0.996 1.32 2.45Cyclopenta[cd]pyrene 0.998 1.62 2.15Chrysene 0.998 0.77 2.425-Methylchrysene 0.996 3.34 4.92Benzo[b]fluoranthene 0.996 2.85 3.49Benzo[k]fluoranthene 0.998 1.79 2.50Benzo[j]fluoranthene 0.998 1.36 2.12Benzo[a]pyrene 0.998 0.93 2.57Indeno[1,2,3-cd]pyrene 0.993 2.47 5.56Dibenz[a,h]anthracene 0.998 1.69 2.45Benzo[ghi]perylene 0.998 1.20 2.91Dibenzo[a,l]pyrene 0.998 1.45 1.79Dibenzo[a,e]pyrene 0.999 1.71 3.67Dibenzo[a,i]pyrene 0.992 3.05 3.07Dibenzo[a,h]pyrene 0.996 3.54 4.00

2: coefficient of determination from the analysis of 6 different concentrations in the rana 50 ng mL−1 standard mix of the 15 + 1 EU-priority PAHs.b Sausage meat extract (3.9 �g kg−1 to 9.9 �g kg−1 for the 15 + 1 EU-priority PAHs).c Average recovery (%) plus standard deviation calculated from triplicate analysis of bla

rior to their analysis by GC–MS.

time and vent flow) at a constant value of the other independent variable (vents.

the vent flow to the highest value to ensure adequate solvent elim-ination. Hence the following parameter settings were found mostappropriate for the PTV-SV injection of PAH solutions: vent time0.5 min, vent flow 100 mL min−1 and vent pressure 0.5 bar.

3.2. Method validation

The optimised PTV-SV method was validated to assess its appli-cability for the analysis of the 15 + 1 EU-priority PAHs in differentsamples. Different parameters such as linearity, precision (repeata-bility and intermediate precision), influence of matrix effects, androbustness of the PTV-SV injection method were evaluated.

3.2.1. LinearityThe linearity of the optimised PTV-SV injection GC–MS method

was evaluated in the range of 15 ng mL−1 to 80 ng mL−1 fromcalibration standards applying Mandel’s fitting test. All calibra-tion functions were found linear. Table 4 presents performancecharacteristics of the method that were determined from repli-cate injections of six PAH standard solutions, respectively sausageextracts. The coefficients of determination of the calibration func-tions were for all 15 + 1 EU-priority PAHs higher than 0.99.

3.2.2. Precision and matrix effectsThe precision of the PTV-SV method was evaluated both under

repeatability and intermediate precision conditions. Standard solu-tions in solvent (toluene) and sausage extracts containing the 15 + 1

S analysis method.

Meat extracts

e precisiona Repeatabilityb Intermediate precisionb Recoveryc

RSDr (%) RSDi (%) (%)

3.94 9.95 98.6 ± 2.33.09 3.09 99.1 ± 2.22.38 3.77 102.5 ± 2.52.92 3.63 98.1 ± 1.95.30 5.30 98.8 ± 3.63.09 3.44 99.3 ± 3.73.34 3.34 97.5 ± 2.63.98 4.08 98.3 ± 2.62.84 4.01 97.5 ± 2.03.43 3.86 97.5 ± 2.54.38 4.53 98.8 ± 1.91.53 4.74 97.7 ± 1.93.60 4.30 100.0 ± 2.43.39 3.39 98.3 ± 2.04.38 4.94 95.2 ± 2.13.84 13.64 104.6 ± 3.4

ge 15–80 ng mL−1.

nk lamb meat extracts (n = 3) fortified with the 15 + 1 EU-priority PAHs (50 ng mL−1)

J.Á. Gómez-Ruiz et al. / Talanta 80 (2009) 643–650 649

F ined wE ationB

Eaeaopsem

SsDt

bmmatBpsidrsitsee1yp

3

u

ig. 3. SIM chromatograms of BcL (m/z 216), BaP (m/z 252) and DhP (m/z 302) obtaU-priority PAHs (concentration ranging from 3.9 �g kg−1 to 9.9 �g kg−1). ConcentraP and DhP, respectively.

U-priority PAHs were analysed. Repeatability was calculated fromnalysis results of three consecutive injections on the same day ofither one PAH standard solution in toluene (50 ng mL−1 of eachnalyte), or one sausage extract sample (3.9 �g kg−1 to 9.9 �g kg−1

f the different PAHs), while intermediate precision was evaluatederforming one-way ANOVA of three consecutive injections of bothamples on three different days. Hence the calculated precisionstimates reflect only the contribution of the GC–MS measure-ents on the overall precision of the analysis method.The relative repeatability standard deviations (RSDr) of the PTV-

V injection GC–MS measurements were satisfactory for the PAHolution in toluene, ranging between 0.77% for CHR and 3.54% forhP. The respective relative intermediate precision standard devia-

ion (RSDi) ranged between 1.79% for DlP and 5.56% for IcP (Table 4).The influence of matrix effects on the GC–MS measurement can

e deduced from the precision estimates of the sausage sample. Forost analytes the intermediate precision was of the same order ofagnitude as the repeatability, which indicates that the gradual

ccumulation of co-extractives in the injection liner did not nega-ively impact precision. They were significantly different only forcL and DhP (Table 4). The precision estimates for the sausage sam-les were somewhat higher than the respective estimates for theolvent solution, which was to be expected. However the vapor-zation of the analytes from food extracts was not significantlyifferent to that from solvent solutions. This was confirmed by theeplicate injection of blank lamb meat extract samples that werepiked with the target analytes to a level of 50 ng mL−1 just beforenjection and comparison of the results to those for solvent solu-ions. The ratio of the results for the meat sample and of the solventolution would indicate matrix effects, if it was significantly differ-nt from one. The last column of Table 4 contains this information,xpressed as percent recovery. Most of the results were close to00%, which reveals the absence of matrix interferences in the anal-sis of meat samples applying this particular combination of sample

reparation and PTV-SV-injection-GC–MS analysis.

.2.3. RobustnessThe robustness of the optimised PTV-SV method was evaluated

sing a screening fractional factorial design. Robustness measures

ith PTV-SV and PTV-PSL injection of an extract of sausage fortified with the 15 + 1of the presented compounds were 3.9 �g kg−1, 5.3 �g kg−1 and 9.9 �g kg−1 for BcL,

the capacity of the method to remain unaffected by small but delib-erate variations in the method parameters [30]. Assuming that theprecision of the temperature control and of the control of pneu-matics of the PTV injector is better than 5%, this value was selectedfor testing of the robustness of the PTV-SV injection. Therefore,values between 0.475 min and 0.525 min were selected for venttime, between 95 mL min−1 and 105 mL min−1 for vent flow andbetween 0.475 bar and 0.525 bar for vent pressure. A t-test was usedto estimate the significance of each effect relative to the randomvariability (expressed as the observed standard deviation obtainedunder repeatability conditions at the centre of the experimentalregion). The PTV-SV method was considered robust since none ofthe three investigated factors had a statistically significant effect atthe 95% confidence level on the analytical response of the differentPAHs.

3.3. PTV in solvent vent mode and PTV splitless injection.Sensitivity of the optimised method

The performance of the optimised PTV-SV injection method wascompared to the PTV-PSL injection (injection volume 1 �L) in termsof the signal-to-noise (S/N) ratios of two different samples: a stan-dard solution containing the 15 + 1 EU-priority PAHs (15 ng mL−1)and a sausage sample fortified with the 15 + 1 EU-priority PAHs(concentration ranging from 3.9 �g kg−1 to 9.9 �g kg−1). A PTV-PSL injection method was already published by Bordajandi et al.[21]. The aim of the current experiments was to identify poten-tial improvements of sensitivity of the PTV-SV injection method(10 �L) compared to the pulsed splitless injection method (1 �L).Fig. 3 shows selected SIM chromatograms of both PTV-SV injectionand PTV-PSL injection of the meat extract for the ions with m/z 216,252 and 302 corresponding to the base peak ions of BcL, BaP andDhP, respectively. The S/N ratios were for all analytes much higherin the chromatograms recorded after injection of the meat extract

with the optimised PTV-SV method. Significantly increased was thesensitivity obtained for DhP, with an almost seven-fold higher S/Nratio after PTV-SV injection compared to PTV-PSL injection.

The gain of sensitivity with the PTV-SV injection was noticeablealso for the other six-ring PAHs (Table 5). An increase in S/N ratios

650 J.Á. Gómez-Ruiz et al. / Talanta 80 (2009) 643–650

Table 5Signal-to-noise (S/N) ratio of GC–MS analysis applying the optimised PTV injection in solvent vent mode (PTV-SV) and PTV injection in pulsed splitless mode (PTV-PSL) forthe analysis of both a solution of the 15 + 1 EU-priority PAHs in solvent (15 ng mL−1 in toluene) and an extract of a sausage samples fortified with the15 + 1 EU-priority PAHs(content ranging from 3.9 �g kg−1 to 9.9 �g kg−1).

Compound S/N ratio S/N ratios for the sausage extract normalised to BaP

Solvent solution Sausage extract PTV-SV PTV-PSL

PTV-SV PTV-PSL PTV-SV PTV-PSL

Benzo[c]fluorene 538 246 2484 785 1.75 1.45Benz[a]anthracene 7938 241 1439 590 1.01 1.09Cyclopenta[c,d]pyrene 5918 176 1111 426 0.78 0.79Chrysene 2902 299 2017 578 1.42 1.075-Methylchrysene 975 70 1231 221 0.87 0.41Benzo[b]fluoranthene 5072 265 2013 327 1.42 0.61Benzo[k]fluoranthene 541 191 1722 378 1.21 0.70Benzo[j]fluoranthene 3489 205 2779 550 1.96 1.02Benzo[a]pyrene 3551 175 1420 540 1.00 1.00Indeno[1,2,3-cd]pyrene 926 136 1155 330 0.81 0.61Dibenz[a,h]anthracene 4597 118 1794 368 1.26 0.68Benzo[ghi]perylene 585 127 1202 221 0.85 0.41

8305

rliHtsondalSto(wtPnc

4

1ompmsoEcisrm

[[[

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[[[

[

[

[[[[

[[[

[[[28] B. Veyrand, A. Brosseaud, L. Sarcher, V. Varlet, F. Monteau, P. Marchand, F. Andre,

Dibenzo[a,l]pyrene 779 43 60Dibenzo[a,e]pyrene 811 46 64Dibenzo[a,i]pyrene 483 37 38Dibenzo[a,h]pyrene 442 33 72

anging between 5.1 and 6.7 times was observed. This is particu-arly important with regard to the typical discrimination of signalntensities that is observed in the analysis of PAHs by GC–MS [6,7].owever the effect of the injection technique on the discrimina-

ion of the signal intensities was visualised by determining relativeignal-to-nose ratios in order to demonstrate that the enhancementf signal-to-noise ratios in PTV injection in solvent vent mode wasot only the consequence of the higher amount of analyte intro-uced into the instrument. For that purpose the S/N ratio of thenalytes were normalised to the S/N ratio of BaP (Table 5). For theast eluting PAHs (DiP and DhP) a 2.5-fold increase of the normalised/N ratios was observed when applying PTV-SV injection comparedo PTV-PSL injection. Likewise, the normalised S/N values of thether two dibenzopyrenes (DlP, DeP) as well as the rest of PAHsexcept for BaA and CPP that showed similar values) were improvedhen the PTV-SV injection was used. Therefore, the PTV-SV injec-

ion does not only improve the sensitivity of the 15 + 1 EU-priorityAHs, it is moreover also able to reduce the discrimination of sig-al intensities between the low- and the high-molecular weightompounds.

. Conclusions

A PTV method in solvent mode (PTV-SV) for the analysis of the5 + 1 EU-priority PAHs has been optimised and validated. The usef statistical experimental design simplified the optimisation of theost important parameters governing the PTV injection. As com-

ared to PTV injection in splitless mode, the optimised PTV-SVethod provided higher sensitivity for all studied analytes, irre-

pective of the type of injected sample being it a solvent solutionf the target analytes or an extract of a fortified sausage sample.specially significant was the improvement in the S/N ratio of the

ompounds with the highest molecular weight (more than six-foldncrease for DeP, DiP and DhP). The optimised PTV-SV injectionhowed very good performance in terms of linearity, precision, andobustness. Matrix effects were not experienced in the analysis ofeat extract samples.

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119 0.43 0.22105 0.45 0.19

62 0.27 0.11108 0.51 0.20

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