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Analytica Chimica Acta 581 (2007) 53–62

Determination of low level methyl tert-butyl ether, ethyl tert-butyl etherand methyl tert-amyl ether in human urine by HS-SPME gas

chromatography/mass spectrometry

Licia Scibetta, Laura Campo, Rosa Mercadante, Vito Foa, Silvia Fustinoni ∗Department of Occupational and Environmental Medicine, University of Milano and Fondazione IRCCS Ospedale Maggiore Policlinico,

Mangiagalli e Regina Elena, Via S. Barnaba, 8-20122 Milano, Italy

Received 6 June 2006; received in revised form 29 July 2006; accepted 31 July 2006Available online 7 August 2006

bstract

Methyl tert-butyl ether (MTBE), ethyl tert-butyl ether (ETBE) and tert-amyl methyl ether (TAME) are oxygenated compounds added to gasolineo enhance octane rating and to improve combustion. They may be found as pollutants of living and working environments. In this work a robotized

ethod for the quantification of low level MTBE, ETBE and TAME in human urine was developed and validated. The analytes were sampled in theeadspace of urine by SPME in the presence of MTBE-d12 as internal standard. Different fibers were compared for their linearity and extractionfficiency: carboxen/polydimethylsiloxane, polydimethylsiloxane/divinylbenzene, and polydimethylsiloxane. The first, although highly efficient,as discarded due to deviation of linearity for competitive displacement, and the polydimethylsiloxane/divinylbenzene fiber was chosen instead.he analysis was performed by GC/MS operating in the electron impact mode. The method is very specific, with range of linearity 30–4600 ng L−1,

ithin- and between-run precision, as coefficient of variation, <22 and <16%, accuracy within 20% the theoretical level, and limit of detectionf 6 ng L−1 for all the analytes. The influence of the matrix on the quantification of these ethers was evaluated analysing the specimens of sevenraffic policemen exposed to autovehicular emissions: using the calibration curve and the method of standard additions comparable levels of MTBE68–528 ng L−1), ETBE (<6 ng L−1), and TAME (<6 ng L−1) were obtained.

2006 Elsevier B.V. All rights reserved.

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eywords: Methyl tert-butyl ether; Ethyl tert-butyl ether; tert-Amyl methyl eth

. Introduction

Methyl tert-butyl ether (MTBE), ethyl tert-butyl etherETBE) and tert-amyl methyl ether (TAME) are oxygenatedompounds added to gasoline to enhance octane rating, ineplacement of alkyl-lead compounds, and to improve the com-ustion process, in order to reduce emissions of partially oxi-ated compounds as carbon monoxide from motor vehicles [1].

European regulations state that the level of ethers containingve or more carbon atoms in gasoline cannot be higher than 15%v/v) [2]. Data from the European Fuel Oxygenates Association

EFOA) reports that in Italian fuels an average amount of 3%v/v) of ethers is present [1].

∗ Corresponding author. Tel.: +39 02 503 20116; fax: +39 02 503 20111.E-mail address: silvia.fustinoni@unimi.it (S. Fustinoni).

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003-2670/$ – see front matter © 2006 Elsevier B.V. All rights reserved.oi:10.1016/j.aca.2006.07.083

rine; Solid phase microextraction; Biomarkers; Occupational exposure

MTBE, ETBE and TAME are largely produced in petro-hemical plants all over Europe [1]. Beside MTBE, the first andtill principally used ether, great attention is recently devotedo ETBE, due to its production from agriculture sources, andherefore used to prepared the so called biofuel [1].

Since MTBE, ETBE, and TAME are highly volatile and veryoluble in water, they can be easily found both as airborne pol-utants of living and working environments and as contaminantsf drinking water: as a consequence humans may be exposed tohese ethers via both inhalation and ingestion (reviewed in Ref.3]).

Acute and chronic toxicity of MTBE were the object ofeveral investigations (reviewed in Refs. [4,5]). Toxicology ofTBE and TAME was also, even if less extensively, investigated

6–12]. In 1999 the International Agency for Research on CancerIARC) has classified MTBE as “not carcinogenic to humans”group 3) [3], while the American Conference of Governmentalndustrial Hygienists (ACGIH) has classified it as a “confirmed

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4 L. Scibetta et al. / Analytica

nimal carcinogen with unknown relevance to humans” (group3) [13].To regulate airborne exposure to MTBE, ETBE and TAME

n working settings, ACGIH recommends threshold limit val-es (TLV) of 50, 5 and 20 ppm, respectively, as time-weightedverage (TWA), during an 8-h workshift [13].

Biological monitoring is a useful tool to assess dose ofoxic compounds entering the human body. For the assessmentf occupational exposure to MTBE, ETBE and/or TAME theetermination of the ethers per se in blood and in urine wasroposed [14–20]. However, due to the similar significance ofnmetabolised chemicals in blood and urine as biomarkers ofxposure [21], urine is preferable to blood, as sample collec-ion does not require an invasive blood drawing, not always wellccepted by study subjects.

From a search in the literature we found many assaysvailable for the quantification of MTBE in water (reviewedn Refs. [22–24]). These procedures usually perform gashromatographic separation coupled with photo ionizationetector (PID), flame ionization detector (FID) or mass detectorMS). Nevertheless, it has been noted that the use of aspecificetectors as FID or PID to detect MTBE in complex matrixan lead to mistakes and/or false positive results, whereas MSnsures high specificity in the identification of the analytes. Thentroduction of analytes in the chromatographic apparatus iserformed either via direct injection of water samples (DAI)25,26], or using sampling techniques as dynamic headspaceP&T), static headspace [27], solid phase microextractionSPME) [28–36], and solvent microextraction [37,38]. TheAI technique presents some difficulties to be coupled withapillary GC, due to the large expansion volume of water. Directater injections are prone to backflush in the injection port,hich can cause loss of analyte response as well as injectionort contamination. Among the different sampling techniques,PME seems to be the most suitable for the analysis of alkylthers in aqueous matrix, for its characteristics of sensitivitynd specificity. In fact, P&T is complex, compared to otherampling techniques [22,24] and prone to carry over impuritiesnd analytes when highly contaminated samples are introducednto the apparatus. On the other hand static headspace extractionnd solvent microextraction are characterized by low sensitivity,nd present difficulties to be robotized.

Several papers reporting the determination of MTBE andther ethers in human blood [8,14,15,39–48], and urine16–20,41,49,50] were also found. Part of them describe assayspplied in toxicokinetic studies where human volunteers werexposed to relatively high concentrations of ethers [8,45–48],thers deal with biomonitoring of occupational or environmen-al exposure [14–20], while some focus on analytical issues39–44,50]. The majority of these analytical papers describe

method for the determination of MTBE, but also of otherolatile organic compounds, in blood. After this method wasrst proposed, about fifteen years ago, several improvements

nd up-grades have been introduced along the years [39–42,44].n its latest versions MTBE is sampled by headspace SPME andnalyzed by GC/MS [42,44]. This method has very good ana-ytical features: uses labelled internal standards to account for

a

me

ica Acta 581 (2007) 53–62

asual errors and matrix effects, is very specific, applies strictrocedures and precautions to eliminate or reduce possible con-aminations. Accordingly to the use of high- or low-resolution

S [44] or [42], limits of detections below 1 or about 50 ng L−1

re achieved, so that the assay may be applied in epidemiolog-cal surveys in population environmentally or occupationallyxposed to low levels MTBE. Recently a related paper furtherdentifies and eliminates interferences in the quantification oflood MTBE that arise from laboratory materials and that pre-ent MTBE quantification in the range of ng L−1 [43]. The onlyssay focused on the determination of urinary MTBE does noteet the sensitivity required for its application to biomonitoring

ow exposures [50].Aim of this study was the development of a validated and

obotized assay for the quantification of low level MTBE, ETBEnd TAME in human urine. Starting from the above-mentionedublished experiences we decided to base our method on cap-llary gas chromatography coupled with low-resolution masspectrometry, using SPME to sample these volatile analytesrom the headspace of urine and introduce them in the sepa-ation system. We focused our efforts on some aspects that aref major importance in the field of biomonitoring human expo-ure, that are: the development of an assay that, besides urinary

TBE, includes also the determination of ETBE and TAME,s fuels additives whose use is expected to increased in the nearuture; the achievement of good sensitivity, taking into consider-tion the occupational scenario in the developed countries, witharticular attention to low exposures such as those observed inrban environment; the validation of the assay, to insure qual-ty to analytical data; the optimization of the conditions to setn automatic, simple and cost-effective assay, useful for routinepplication on large sample set. The novelties introduced by thisssay in comparison with the previous experiences in the fieldf analytical chemistry applied to biomonitoring low exposuresre: the assay is focused on urine as biological matrix; a compar-son of efficiency of different fibers in sampling MTBE, ETBE,nd TAME, considering their possible interference and compe-ition for fiber absorption sites, is performed; the quantificationf ethers is based on calibration curves obtained using spikedrine, instead of purified water; an evaluation of the effects ofhe complex and variable biological matrix on the quantificationf ethers is attempted.

. Experimental

.1. Chemicals and standards preparation

MTBE (99%), ETBE (99%+), TAME (97%+) (Sigma–ldrich, Milano) were used for the preparation of standard solu-

ions; MTBE-d12 (99%) (CDN ISOTOPES, Chemical Research000, Roma) was used for the preparation of the internal stan-ard solutions. Sodium chloride (NaCl) and methanol (MeOH)ere purchased from Carlo Erba Reagenti (Rodano, Milano)

nd were of analytical grade.For the preparation of the calibration curve and the develop-

ent of the assay, standard solutions containing the three ethers,ach at concentrations ranging from 0.036 to 5.4 �g mL−1 in

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L. Scibetta et al. / Analytica

eOH, were prepared. From the pure deuterated compound, annternal standard solution containing MTBE-d12, at the concen-ration of 0.47 �g mL−1 in MeOH (IS), was prepared.

For the development of the assay 0.5 �L of the standard solu-ion containing 2.4 �g mL−1 of each ether was added to 600 �Lf urine to obtain working samples containing MTBE, ETBEnd TAME, each at the concentration of about 2000 ng L−1.

For the calibration curve 0.5 �L of the different standard solu-ions were added to 600 �L of urine to obtain seven calibrationamples, containing the ethers, each at the concentrations ofbout 30, 60, 115, 230, 460, 1960, and 4570 ng L−1. An unspikedample of the same urine was kept as a blank.

Urine used for the preparation of the calibration and workingamples solutions was a pool of urine from non-smoking donorsithout occupational exposure to ethers.Before analysis 0.5 �L of IS solution was added to the calibra-

ion samples or to the blank or to the unknown samples to obtainfinal concentration of MTBE-d12 in urine of 392 ng L−1.

.2. Equipment

For sample analysis 2 mL autosampler glass vials sealed withcrew open-top closure and silicone-polyperfluoroethylene gas-ets were used (Kimble, Superchrom, Milano).

For sampling analytes from the headspace of urine, the fiberf the SPME device was coated with the following materials:olydimethylsiloxane/divinylbenzene with 60 �m film thick-ess (PDMS/DVB), polydimethylsiloxane with 100 �m filmhickness (PDMS), carboxen/polydimethylsiloxane with 85 �mlm thickness (CAR/PDMS) (Supelco, Milano).

A 6890 gas chromatography (Agilent, Cernusco sul Naviglio,ilano), equipped with a 5973N mass spectrometric detector

perating in the electron impact mode (EI, 70 eV) was usedo separate and identify the ethers. Injection was performed,sing the SPME device, by a MPS2 autosampler (Gerstel, RSA,ernusco sul Naviglio, Milano) with the thermostatic unit oper-ting at 30 ◦C. The split/splitless injection port, operating in theplitless mode at 260 ◦C, was equipped with an inlet liner forPME (i.d. 0.75 mm, Supelco, Milano). Chromatographic sep-ration was performed on a capillary column DB1-MS (J&W,0 m length, 0.25 mm internal diameter, 1 �m film thickness,PS Analitica, Milano).

In the field study, storage vials used for both sample col-ection and storage, were prepared according to the followingteps: vials (clear glass, 7.9 mL effective volume, Kimble, Super-hrom, Milano) and septa (butyl rubber/PTFE, 20 mm diameter)ere thermally cleaned overnight at 200 and 90 ◦C, respectively;ials and septa were assembled and sealed using an aluminiumeptum cap and a crimper; sealed vials were evacuated using aater pump connected to the vial via a needle piercing the septa

approximate pressure in the evacuated vial was 30 mmHg).

.3. HS-SPME GC/MS analysis

Analytes are adsorbed for 5 min at 30 ◦C, in the sample’seadspace by SPME using the PDMS/DVB fiber, and ther-ally desorbed for 3 min by insertion of the fiber onto the

t

2s

ica Acta 581 (2007) 53–62 55

hromatographic injection port kept at 260 ◦C. The GC anal-sis is performed at the following conditions: helium carrieras at a constant flow rate of 1 mL min−1; gas chromatographyven temperature programmed from 40 ◦C (5 min initial hold) to2 ◦C at 3 ◦C min−1, then to 250 ◦C at 30 ◦C min−1 (3 min finalold). Under these conditions, the approximate retention timesre: 9.98 min for MTBE, 9.73 min for MTBE-d12, 12.38 min forTBE and 15.51 min for TAME. The overall time required for

he analysis, determined by the total GC run is 24.4 min. TheS detection is performed in the following conditions: trans-

er line temperature 280 ◦C; ion source temperature 230 ◦C;or mass spectra acquisition, the ion range m/z 40–200 wascanned. Quantification is performed in the selected ion moni-oring mode (SIM). The dwell time is 100 ms. From 9 to 11 minhe following ions at mass to charge ratio (m/z) are acquired: 73M•+ − CH3

•]+ (quantifying ion) and 57 [C(CH3)3]+ (qualify-ng ion) for MTBE; 82 [M•+ − CH3

•]+ (quantifying ion) and 66C(CD3)3]+ (qualifying ion) for MTBE-d12. From 11 to 16 minhe following ions at mass to charge ratio (m/z) are acquired: 87M•+ − CH3

•]+ (quantifying ion) and 59 [(CH3)2COH]+ (qual-fying ion) for ETBE; 73 [C(CH3)2OCH3]+ (quantifying ion)nd 87 [M•+ − CH3

•]+ (qualifying ion) for TAME.

.4. HS-SPME

To optimise the HS-SPME experimental conditions, the fol-owing parameters were evaluated: different fiber coatings, addi-ion of a saturating amount of salt to the sample, samplingime, and desorption time. All trials were performed in triplicateampling ethers from working samples prepared as previouslyescribed (see Section 2.1).

Other parameters affecting sampling efficiency, as samplegitation and sampling by soaking fiber in urine, were not con-idered. This was based on our previous experience of littledvantages deriving from the adoption of these additional pro-edures and/or increase of stress for the fiber with significantife shortening [51].

.4.1. Comparative evaluation of different fiber coatings

.4.1.1. Relative extraction efficiency. Three different fiberoatings were initially evaluated for their relative efficiency ofxtraction: PDMS, PDMS/DVB and CAR/PDMS. Before these, these fibers were conditioned as suggested by the supplier.or this experiment, the analytes were sampled from workingamples applying a sampling time of 5 min and a desorption timef 3 min.

.4.1.2. Linearity of response. A successive trial was per-ormed with PDMS/DVB and CAR/PDMS fibers to evaluatehe linearity of the response of the calibration samples.

.4.2. Optimisation of sampling parametersThe following parameters were optimised only for the

DMS/DVB fiber, since this was found to be the best fiber in

he previous experiments.

.4.2.1. Effect of salt addition. Since the ionic strength of aolution has an influence on the partition coefficient of volatile

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nalytes between liquid and headspace [51,52], the effect ofdding saturating amount of salt (NaCl) was studied. This trialas performed with working samples added with 300 mg ofaCl.

.4.2.2. Sampling and desorption times. Aim of this experi-ent was to establish adsorption and desorption times for SPME

ampling. The kinetic of adsorption of ethers, sampled by theDMS/DVB fiber, was evaluated in the headspace of urine keptt 30 ◦C, ranging adsorption time from 1 to 120 min, and keep-ng desorption time constant at 3 min. The kinetic of thermalesorption of ethers from the fiber, in the inlet of the GC injec-ion port kept at 260◦, was evaluated ranging desorption timesrom 1 to 10 min, and keeping adsorption time constant at 3 min.hese experiments were performed on urine working samplesdded with a saturating amount of NaCl.

.5. Calibration curves, limits of detection, within- andetween-run precision, accuracy

Calibration samples, prepared as described in Section 2.1,ere analysed using the procedure outlined in Section 2.3 tobtain a calibration curve. Least squares linear regression anal-sis was applied to estimate the slope (m) and the interceptq) of the function y = mx + q, where y is the ratio between thehromatographic peak area of each ether versus IS, and x is theoncentration (ng L−1) of MTBE, ETBE or TAME in the sample.he limit of detection (LOD) of the assay, for each component,as calculated according to the expression:

OD = 3S.E.q + q

m

here S.E.q is the standard error of the intercept [53].The within- and between-run precision and accuracy of the

ssay were determined by analysing four pools of urine spikedith known amount of each ether to obtain solutions contain-

ng the ethers at a theoretical concentration of about 30, 115,60 and 1960 ng L−1 each, with slight differences depending onhe analyte. The same analyst analysed three replicates of eachool on three different days. Precision was expressed as coeffi-ient of variation (CV). Accuracy was estimated as percent ratioetween the value calculated from the calibration curve and theheoretical value [54].

.6. Stability of standards, internal standard solutions, andalibration samples

The stability of the standard solutions of the ethers, of theS solution, of calibration samples (four calibration levels andhe blank), and of unknown samples stored in storage vials inhe dark at −20 ◦C, was evaluated at various time points overmonths.

.7. Application of the assay to the biomonitoring and

nfluence of matrix on the quantification

The assay was applied to urine samples collected from sevenraffic policemen, exposed to autovehicular emissions in a large

pc

ica Acta 581 (2007) 53–62

ity located in Northern Italy, Milan, characterized by intenseraffic. The subjects were informed about the aims of the researchnd signed an informed consent.

Urine samples were collected at the end of 7 h-workshiftn disposable polyurethane bottles. From each sample a 7 mLliquot was immediately transferred into a pre-evacuated closedtorage vial, using a disposable syringe and stored at 0 ◦C.re-evacuated vials were used to improve and facilitate sam-le collection: in fact the use of open vials requires imme-iately seal by the operator and could be prone to pollutionnd/or loss of analytes, while the use of non-evacuated closedial requires a double needle system to allow sample intro-uction.

Within 16 h samples were delivered to the laboratory, wherehey were kept at −20 ◦C and analyzed, according to their sta-ility.

For analysis, urine samples, kept in storage vials, were leftt room temperature until completely thawed. After shakingnd waiting for few minutes, an aliquot of 600 �L of urineupernatant was transferred into a 2 mL autosampler glass vialontaining 300 mg of NaCl, immediately spiked with 0.5 �L ofS and readily sealed. To reduce the possible loss of volatile ana-ytes from the specimen, this operation was performed withoutecapping the storage vial, but piercing the septa with a dis-osable syringe equipped with a 26-gauge needle. Analysis waserformed accordingly to the procedure described in Section.3.

The quantification of the analytes was performed applyingwo different approaches: the calibration curve and the methodf standard additions. This was done to evaluate the influencef urine matrix on the quantification of samples.

In the first approach all samples were quantified using theame calibration curve. This curve was obtained analysing thealibration solutions prepared as described in Section 2.1, andpplying to results least squares linear regression analysis, asescribed in Section 2.5.

In the second approach the samples were quantified usinghe method of the standard additions. For each subject a specificalibration curve, with three calibration levels prepared spik-ng each subject’s urine with calibration solutions of MTBE,TBE and TAME (additional levels at about 115, 460, and960 �g L−1 of each compound), and an unspiked sample withhe unknown concentration of ethers were analysed. Applyinghe least squares linear regression analysis to estimate the slopemi) and the intercept (qi) of the function y = mix + qi, the con-entration of the ethers in the unknown sample (xunknown) wasbtained as intercept on axes x of the straight line, that is for= 0, xunknown = −qi/mi.

. Results and discussion

.1. Chromatographic separation and mass spectracquisition

The single ion chromatograms of a urine sample of an unex-osed volunteer, spiked with MTBE, ETBE, TAME, at a con-entration of about 1950 ng L−1 each, and MTBE-d12 at the

L. Scibetta et al. / Analytica Chimica Acta 581 (2007) 53–62 57

Fig. 1. Single ion mass-chromatograms obtained registering the ions m/z 73 for MTBE and TAME, and m/z 87 for ETBE, in a urine working sample containing theanalytes at the concentration of about 1950 ng L−1 each (a), and in a urine sample of a traffic policeman, containing MTBE, ETBE, TAME, at the concentration of318, <6, and <6 ng L−1 (b). For both samples is reported also the single ion mass-chromatogram obtained registering the ion m/z 82 for internal standard MTBE-d12a

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oncentration of 392 ng L−1 are shown in Fig. 1a; the singleon chromatograms of a urine sample of a traffic policeman,ontaining MTBE, ETBE, TAME, at the concentration of 318,6, and <6 ng L−1, and MTBE-d12 at the concentration of92 ng L−1 are shown in Fig. 1b. The analytes were identi-ed based on their retention times and different mass to chargeatio.

The introduction of MTBE-d12 as internal standard, insteadhat of MTBE-d3, used in some analytical procedures previously

eported for the quantification of MTBE in water [18–20,23],as necessary because in the ion chromatogram obtained regis-

ering m/z = 76, corresponding to quantifier ion [M•+ − CH3•]+

f MTBE-d3, there was an interfering peak that obscured the cor-

crai

ect quantification of the internal standard. This peak, derivingrom an unknown compound, was tentatively attributed to CS2molecular weight 76), a volatile chemical ubiquitously presentn human urine [55].

Moreover in Fig. 2b, as well as in other chromatogramsbtained analysing urine samples from traffic policemen, itas observed a broad interfering signal at time between 15.2

nd 15.8 min. Such interference is not expected to negativelyffect the quantification of TAME, as it is observed for ion

hromatogram registered at m/z 87. Such mass to changeatio is used for qualifying TAME, while ion chromatogramt m/z 73, used for its quantification, is not affected by suchnterference.

58 L. Scibetta et al. / Analytica Chimica Acta 581 (2007) 53–62

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ig. 2. Linearity of response trial: chromatographic area of MTBE, ETBE, TAMber (c and d), at concentration of each ether ranging from 30 to 5000 and at 392

.2. HS-SPME

.2.1. Comparative evaluation of different fibre coatings

.2.1.1. Relative extraction efficiency and linearity of response.he relative extraction efficiencies of the investigated fiber coat-

ngs for the different analytes are reported in Table 1; these valuesre expressed as percent of the extraction efficiency obtainedor CAR/PDMS fiber. The PDMS fiber had the lower extrac-ion efficiency, with percent ranging from 10 to 15, dependingrom the analyte. The fibers with mixed coating, as CAR/PDMS

nd PDMS/DVB, had higher relative extraction efficiency, andherefore may be regarded as potential tools for sampling ethersrom urine.

able 1xtraction efficiencies for the investigated ethers, using different SPME fiberoatings; the values are expressed as percent of the extraction efficiency obtainedsing the CAR/PDMS fiber

iber coating MTBE ETBE TAME

DMS 10 11 15DMS/DVB 85 98 95AR/PDMS 100 100 100

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MTBE-d12 obtained with the CAR/PDMS fiber (a and b) and the PDMS/DVB1 for the internal standard. (�) MTBE; (�) ETBE; (♦) TAME; (�) MTBE-d12.

This result confirms what was recently reported by othersnvestigators, who proposed these mixed coatings both for sam-ling volatile organic compounds in air [56,57] and MTBE inroundwater [28,29,31–33,36]. However, notwithstanding thedvantage given by their high affinity for the ethers of con-ern, fibers containing carboxen and divinylbenzene may showignificant deviation from linearity when used for quantitativeetermination of MTBE. In fact these component of the fibers,ampling by adsorption, have only a limited number of sitesvailable for the interaction with the substrate; in the co-presencef several chemicals with affinity for the adsorption sites, aompetition and a displacement of the analytes from the sub-trate may occur: in this case calibration curves show significanteviation from linearity, especially at the higher concentrations36,57–59].

Fig. 2a and b report responses, as chromatographic peak areaersus analyte concentration, for MTBE, ETBE, TAME, andTBE-d12 obtained with CAR/PDMS fiber, while Fig. 2c andreport responses obtained with PDMS/DVB fiber. In Fig. 2a

deviation from linearity was clearly observed for MTBE, forhich, at concentration higher than 1250 ng L−1, due to a com-etitive displacement by ETBE and TAME, chromatographicrea is lower than expected; such effect was also very evi-

L. Scibetta et al. / Analytica Chimica Acta 581 (2007) 53–62 59

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ig. 3. Kinetic of adsorption of ethers from urine by HS-SPME using the PDMf thermal desorption of ethers from PDMS/DVB fiber, in the injection port of

ent for MTBE-d12 (see Fig. 2b). At the same time, but to aower extent, chromatographic area higher than expected werebserved for both ETBE and TAME. On the contrary, in Fig. 2clinear increase of the chromatographic response up to about

000 ng L−1 for all the analytes was found; furthermore, thenternal standard was not affected by analytes concentration, ashown in Fig. 2d.

Our results are qualitatively and quantitatively in line withhose of Nakamura et al. [36] who, describing an assayor the determination of several volatile organic compounds,

TBE and musty odours in water, underlined that, extrac-ion efficiency and linearity range were inversely proportionalassing from CAR/PDMS fiber (highest extraction efficiency,nd narrowest linearity range: 0.1–1 �g L−1), to PDMS/DVBber (intermediate extraction efficiency, intermediate linearityange: 0.1–5 �g L−1), and to PDMS fiber (lowest extractionfficiency, widest linearity range: 0.1–100 �g L−1). However,hile these authors focused on the PDMS fiber, according to

apanese requirements for water regulation, we focused on theDMS/DVB fiber, in line with the expected concentration ofTBE, ETBE and TAME in urine of subjects exposed to low

evels of these ethers in polluted working settings.

.2.2. Optimisation of sampling parameters

.2.2.1. Effect of salt addition. Results of this trial showed that,n the presence of saturating amount of NaCl in urine, the extrac-ion efficiency of the ethers improved from 2.6- to 3.1-fold. Forhis reason in all the subsequent steps, HS-SPME sampling waserformed in presence of NaCl.

.2.2.2. Sampling and desorption time. In Fig. 3a the kinetic ofdsorption of ethers from urine at 30 ◦C, is shown. Chromato-raphic area of the various analytes increased with sampling

ime, but the equilibrium was not reached even after 120 min.rom this experiment we concluded that it is not feasible tobtain the maximum signal in a reasonable sampling time, there-ore a sampling time of 5 min was chosen. This is a good com-

ttem

B fiber at 30 C, in the presence of saturating amount of NaCl (a), and kinetic260 ◦C (b). (�) MTBE; (�) ETBE; (♦) TAME.

romise between a significant chromatographic signal, about7–65% the value obtained at 120 min, and a short samplingime; in fact for SPME fiber sampling also in adsorbtion, as inhe case of PDMS/DVB, short sampling may represent an advan-age, due to the reduction of the time available for a competitiveisplacement at the adsorbtion sites to occur [60].

In Fig. 3b the kinetic of thermal desorption of ethers fromDMS/DVB coating onto the injection port of the GC kept at60 ◦C is shown. The chromatographic area of the various ana-ytes was constant for desorption time ranging from 1 to 10 min.ased on this evidence a desorption time of 3 min was arbitraryhosen. No chromatographic peak broadening was observed forong desorption times, as precautions were taken to avoid suchffect: low volume SPME insert liner was used in the injectionort of the gas chromatograph, constant and low oven temper-ture was initially set to focus analytes in a narrow band in therst part of the chromatographic column, and a thick columnlm (1 �m) was chosen to increase the retention capability of

he column. On the other hand, desorption of ethers is indeedery fast at the working temperature, i.e. injection port at 260 ◦C,nd practically independent from time, as shown in Fig. 3b.

Sampling temperature is another important parameter thatffects HS-SPME, in fact, playing a major role in determin-ng the kinetic energy of the molecules, influences the partitiononstants between the phases involved, i.e. urine, air and SPMEber coating. In the present work the effect of temperature onecovery of ethers was not experimentally evaluated. Previoustudies on the determination of volatile organic compounds [61]nd/or MTBE [28,36], reported optimized conditions in whichow or below ambient temperatures, were preferable to enhanceS-SPME recovery. Moreover, in our previous experience with

he determination of urinary benzene, toluene, ethylbenzene andylenes by HS-SMPE using a PDMS fiber, we could verify that

emperatures higher than 40 ◦C played an unfavourable role onhe extraction efficiency of these chemicals [51]. Based on thesevidences we chose to perform sampling at 30 ◦C, as this is theinimum working temperature for the thermostatic unit of the

60 L. Scibetta et al. / Analytica Chimica Acta 581 (2007) 53–62

Table 2Within- and between-run precision and accuracy for the determination of MTBE, ETBE and TAME

Theoretical concentration

MTBE (ng L−1) ETBE (ng L−1) TAME (ng L−1)

29 114 456 1958 29 115 458 1963 30 119 475 2037

Day 1Mean (N = 3) 34 113 418 1994 27 100 430 1964 30 104 442 2043CV%a 1 5 16 3 9 6 4 3 22 10 4 0% Theoretical 116 99 92 102 93 87 94 100 101 88 93 100

Day 2Mean (N = 3) 34 110 456 1991 29 105 449 1955 27 106 444 2050CV%a 1 6 6 3 10 8 0 3 11 1 6 0% Theoretical 116 96 100 102 100 91 98 100 89 98 98 101

Day 3Mean (N = 3) 28 101 421 1951 25 99 417 1991 26 100 434 2056CV%a 16 17 2 3 1 1 10 11 3 7 3 1% Theoretical 97 88 92 100 88 86 91 110 85 84 91 101

OverallMean (N = 9) 32 108 432 1979 27 101 432 1970 27 104 447 2050CV%b 8 4 9 5 8 8 6 8 14 6 5 1% Theoreticalc 110 95 95 101 93 88 94 100 92 87 94 101

attns

3b

T∼tetblAabc[

3c

stgga

3i

ttmiTl

ePpresence of volatile organic compounds in urine, e.g. acetone, inthe order of mg L−1, that, compared to the concentration of theanalytes, in the order of tens or hundreds of ng L−1, tends to levelthe unspecific interactions between matrix and fiber coating,

Table 3Levels of MTBE in seven traffic policemen exposed to autovehicular traffic,quantified using the calibration curve and the method of standard additions, andthe percent difference between the two quantifications [Δ% = (MTBE calibrationcurve − MTBE standard additions) × 100/MTBE calibration curve]

Sample ID MTBE (ng L−1) Δ%

Calibration curve Standard additions

1 73 73 02 318 362 143 528 505 −4

a Within-run precision.b Between-run precision.c Accuracy.

utoinjector in use. This choice is supported by the satisfac-ory extraction efficiency obtained: actually 30 ◦C as samplingemperature should be regarded as a compromise between theeed of achieving good sensitivity and of setting a robust assay,uitable for automation.

.3. Calibration curves, limits of detection, within- andetween-run precision, accuracy

The calibration curves obtained for MTBE, ETBE andAME were linear all over the investigated range (up to5000 ng L−1), with correlation coefficient typically higher

hen 0.995. The analytical limit of detection was 6 ng L−1 forach ether. Within- and between-run precision and accuracy ofhe assay are summarised in Table 2. Observed CV was mostlyelow 17% with the exception of the lowest concentrationevel of TAME, for which a CV as high as 22% was observed.ccuracy is in line with the requirement of US-FDA for the bio-

nalytical methods validation, that stated that this value shoulde within 15% of the theoretical, except for concentrationslose to the LOD, that should not deviate by more than 20%54].

.4. Stability of standards, internal standard solutions, andalibration samples

The standard solutions of the ethers and the IS solution weretable at least for 3 months. Calibration samples were stable up

o 5 weeks. As acceptance criteria was considered a chromato-raphic signal of each analyte within ±20% its original values,iven that fact that 20% is the between-run precision of thessay.

4567

.5. Application of the assay to the biomonitoring andnfluence of matrix on the quantification

Table 3 reports concentrations of MTBE in urine of sevenraffic policemen quantified using both the calibration curve andhe method of standard additions. These data were in good agree-

ent, with differences within the precision of the assay (0–14%)n all samples, but one, for which a difference of 37% was found.he levels of ETBE and TAME were not tabled because always

ower than 6 ng L−1, LOD of the assay for both analytes.Based on the result of this comparison we concluded that the

ffect of matrix on the quantification of urinary ethers using theDMS/DVB fiber is negligible. This may be due to the normal

263 258 −270 68 −2

181 189 4113 71 −37

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rrespectively from the difference in the fine composition of thepecimen [55].

The major outcome of this trial is that, for the quantificationf urinary ethers, there is no need to adopt the tedious methodf the standard additions, but rather that the quantification cane conveniently performed using the calibration curve.

The levels of MTBE and other ethers found in the studyolicemen exposed to traffic exhaust fumes, were in the samerder of magnitude than those reported for Italian gasoline atten-ants [19], but lower than the levels found in Finnish workersxposed to gasoline vapours during truck loading and unload-ng [16–18]. Moreover, in the latter studies also the presencef TAME was detected, while such presence was not found atquantifiable level in the present study, even if traces of bothTBE and TAME were seen in the chromatograms (for instanceee Fig. 2b).

.6. Conclusions

In this work, an analytical assay based on HS SPME GC/MSuitable for the determination of a low level of MTBE, ETBEnd TAME in human urine is presented. Although CAR/PDMSber has been previously recommended for sampling MTBE andthers volatile organic compounds from biological matrices, weound that, even at the low concentrations investigated, it showsignificant deviation from linearity in the quantification of uri-ary ethers, and therefore it was discarded; instead a less efficientDMS/DVB fiber was chosen for this application. Nevertheless

he assay is very sensible, uses a small amount of specimen,erforms all steps in one vial, introduces an internal standard,voids the use of solvents and limits the manual work, samplingnd injecting the analytes in the chromatographic system auto-atically. Analysis, performed by GC/MS, ensures good and

ong lasting performance in separation and high specificity inhe identification of the analytes. The determination of MTBEn a group of occupationally exposed subjects showed that theffect of matrix on this quantification is negligible and that thisay be conveniently performed by calibration curve. Based on

hese features we conclude that the present assay is a usefulool for the biological monitoring of exposure to MTBE, ETBEnd TAME through the determination of urinary unmetabolizedompounds.

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