Anal Bioanal Chem (2016) 408:191–201 DOI 10.1007/s00216-015-9092-5

RESEARCH PAPER

Novel determination of polychlorinated naphthalenes in water by liquid chromatography–mass spectrometry with atmospheric pressure photoionization Athanasios I. Moukas 1 & Nikolaos S. Thomaidis 1 & Antony C. Calokerinos 1

Received: 30 July 2015 / Revised: 25 September 2015 / Accepted: 29 September 2015 / Published online: 10 October 2015 # Springer-Verlag Berlin Heidelberg 2015

Abstract This study presents the development, optimization, and validation of a novel method for the determination of polychlorinated naphthalenes (PCNs) by liquid chromatography-atmospheric pressure photoionization (APPI), using toluene as dopant. The mass spectra of PCN 52, 54, 66, 67, 73, and 75 were recorded in negative ionization. The base ions corresponded to [M–Cl+O]−, where M is the analyte molecule. A strategy, which includes designs of experiments, for the development, the evaluation, and the optimization of the LC-APPI-MS/MS methods is also described. Finally, a highly sensitive method with low instrumental limits of detection (LoDs), ranging from 0.8 pg for PCN 75 to 16 pg for PCN 54 on column, was validated. A Thermo Hypersil Green PAH (100 mm × 2.1 mm, 3 μm) column was used with acetonitrile/water/methanol as mobile phase. The method was applied for the determination of the selected PCNs in surface and tap water samples. A simple liquid–liquid extraction method for the extraction of PCNs from water samples was used. Method LoQs ranged from 29 ng L−1, for PCN 73, to 63 ng L−1, for PCN 54, and the recoveries ranged from 97 to 99 %, for all congeners. This is the first LC-APPI-MS/MS method for the determination of PCNs in water samples.

Electronic supplementary material The online version of this article (doi:10.1007/s00216-015-9092-5) contains supplementary material, which is available to authorized users. * Nikolaos S. Thomaidis [email protected] 1

Laboratory of Analytical Chemistry, Department of Chemistry, School of Sciences, National and Kapodistrian University of Athens, Panepistimioupolis Zografou, 157 71 Athens, Greece

Keywords Polychlorinated naphthalenes (PCNs) . Atmospheric pressure photoionization (APPI) . Dopant . LC-APPI-MS/MS . Pseudo-SRM . Experimental design

Introduction Atmospheric pressure photoionization (APPI) was presented as an ionization source for coupling liquid chromatography (LC) to mass spectrometry (MS) in 2000 by Bruins et al. [1] and Syage et al. [2]. Based on these results, two interfaces are currently commercially available under the trademark names PhotoSpray and PhotoMate, respectively. PhotoMate differs by its orthogonal geometry. APPI is a soft ionization technique able to ionize those molecules that are not, or are poorly, amenable to electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI). This technique has been tested and applied for the analysis of a wide variety of analytes, such as drugs, lipids, natural compounds, pesticides, synthetic organics (i.e., polycyclic aromatic hydrocarbons and polybrominated diphenyl ethers), and petroleum derivatives, in several matrixes [3–5]. However, many of APPI’s potentials are unexplored. Polychlorinated naphthalenes are a group of compounds based on the naphthalene ring system, in which one or more hydrogen atoms have been replaced by chlorine. The chemical formula of PCNs is C10H8–nCln, where n ranges from 1 to 8. Theoretically, 75 different congeners are possible and their basic molecular structure, including the conventional numbering of the substituent positions [6], is shown in Fig. S1 in the Electronic Supplementary Material (ESM). PCNs have found numerous applications [7, 8], but due to their global distribution, persistence, bioaccumulation, and toxic effects to humans and wildlife, they were considered and regulated as persistent organic pollutants (POPs). The most significant

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toxic effects in humans due to the exposure to PCNs are skin lesions (chloracne) and liver degeneration (yellow atrophy) [9–11]. Furthermore, in vivo studies have demonstrated that PCNs were able to induce dioxin-like effects, so they have been included in the toxicity equivalency factor (TEF) system [12, 13]. Although the production of PCNs has stopped, unintentional emissions still contribute to PCN levels in the environment, mainly due to waste incineration. The emissions of PCNs in UNECE-Europe for the year 2000 were estimated to be 1.03 tonnes/year [14]. To control pollution, the existence of sensitive and accurate analytical methods for the determination of PCNs is mandatory. Gas chromatography (GC) with electron capture detector (ECD) was used for the determination of PCNs, but a mass spectrometry (MS) detector was also necessary to confirm their identification [15]. GC–ECD application was limited due to interferences from polychlorinated biphenyls and organochlorine pesticides [16]. Some disadvantages of ECD may be overcome by the use of micro-ECD [17]. Other methods used flame ionization detector (FID) coupled with GC [18], but GC-FID had an additional problem, the high detection limits. Methods that use MS coupled with GC [19, 20] can overcome the above limitations, since MS allows the selective determination of PCNs in trace levels, even in complicated matrices, and the identification of co-eluted isomers. In most cases, the difficult separation of PCN congeners was achieved by two dimensional GC (GC × GC) [17]. Furthermore, the development of MS led to the use of analyzers with higher selectivity and sensitivity and lower detection limits, such as time-of-flight mass spectrometry (TOF/ MS) [21], triple quadruple mass spectrometry (QqQ MS/ MS) [22], and magnetic sector high resolution MS (HRMS) [23–25], but these systems are rather expensive and not widely available. High performance liquid chromatography (HPLC) and thin layer chromatography (TLC) coupled with ultraviolet (UV) detector [26] and GC-UV [27] have also been used for PCNs’ determination, but their analytical figures were not satisfactory. Although acceptable GC methods for determining PCNs exist, the development of an alternative analytical method using LC-APPI-MS/MS will expand the applications of LCMS, and may be useful for specific cases, like biodegradation studies or screening controls. In LC-atmospheric pressure ionization (API)-MS, many parameters have to be optimized for the determination of a batch of compounds, and a compromise between the optimized, for each compound, parameters is necessary. This compromise can be realized following an optimization strategy and applying a design of experiments. Various optimization strategies [28] and a tutorial for statistical design of experiments have been published [29]. Recently, an optimization strategy for the development of LC-APPI-MS methods has been proposed for the first time and applied to the determination of PCBs [30]. However, the development of multi-

A.I. Moukas et al.

component LC-APPI-MS/MS methods is described segmentally in bibliography. To the best of our knowledge, there are no methods that use LC-MS/MS for the determination of PCNs, probably because of the inefficiency of the most widely employed API sources, ESI and APCI, to ionize them. For this reason, in this study, six of the most toxic and commercially available PCNs (PCN 52, 54, 66, 67, 73, and 75) were chosen as model compounds to describe a strategy for the development and optimization of a LC-APPI-MS/MS method (see ESM, Fig. S1). Furthermore, the method was validated and applied for the determination of PCNs in surface and tap water samples. This study clearly demonstrates the applicability of LC-APPI-MS/MS for the determination of PCNs in environmental samples expanding the applications of this technique.

Materials and methods Chemicals and reagents Acetonitrile, methanol, toluene, and acetone HPLC grade were purchased from Merk (Darmstadt, Germany), ethyl acetate HPLC grade from Panreac Quimica (Barcelona, Spain), anisole analytical grade from Janssen Chimica (Geel, Belgium), and isooctane HPLC grade and chlorobenzene analytical grade from Sigma-Aldrich (Steinheim and Munich, respectively, Germany). High-purity water (resistivity 18.2± 0.2 MΩ cm−1) was provided by an ultra pure water Milli-Q system (Millipore Corp., Bedford, MA, USA). Five congeners, PCN 52, 54, 66, 67, and 73 (100 μg mL−1 in nonane), were purchased from Cambridge Isotope Laboratories (Andover, MA, USA) and PCN 75 (10 μg mL−1 in isooctane) was obtained from the laboratory of Dr. Ehrenstorfer-Schafers (Augsburg, Germany). After dilution in isooctane, PCN 52, 54, 66, 67, and 73 solutions of 10 μg mL−1 were prepared. Furthermore, standard stock solutions (1 μg mL−1) of all PCNs were prepared in methanol. In addition, a multi-component solution of the six congeners was prepared in methanol to a final concentration of 0.4 μg mL−1 for each congener. Multi-component working solutions in various concentrations were also prepared in methanol. All solutions were stored in dark vials at 4 °C for up to 1 month. Instrumentation The determination of PCNs was performed by a Thermo LCMS/MS system (San Jose, USA) consisting of a Thermo Surveyor LC pump, a Thermo Surveyor AS autosampler, and a TSQ Quantum Discovery MAX triple quadruple mass spectrometer equipped with a PhotoMate atmospheric pressure photoionization interface. This interface consists of the Thermo atmospheric pressure chemical ionization probe and a

Novel determination of polychlorinated naphthalenes

vacuum ultra violet (VUV) krypton discharge lamp (Syagen Technology, CA, USA) that generates photons of 10 eV, and it can also operate as an APCI/APPI dual source. For the separation of PCNs, a Thermo Hypersil Green PAH (100 mm×2.1 mm, 3 μm) column was finally used at a constant flow rate of 0.080 mL min−1. Mobile phase consisted of water (solvent A), acetonitrile (solvent B), and methanol (solvent C). The gradient that was used was 0–15 min linear gradient from 75 % B, 20 % A and 5 % C to 95 % B and 5 % C, 15–49 min held at 95 % B and 5 % C, 49–50 min linear gradient from 95 % B and 5 % C to 75 % B, 20 % A and 5 % C, and 50–75 min held at 75 % B, 20 % A and 5 % C in order to equilibrate the column before the next injection. The column oven was set at 25 °C, and the full loop injection volume was set at 5 μL. The ionization source operated in negative ion mode under the following conditions: discharge current 0 μA, vaporizer temperature 260 °C, sheath gas pressure 24 psi, ion sweep gas pressure 0 arbitrary units (a.u.), auxiliary gas 5 a.u., capillary temperature 200 °C, skimmer offset −5 V, and collision pressure 1.5 mTorr. High-purity nitrogen was used as sheath and auxiliary gas and argon was used as collision gas. Collision energy was set at 10 eV and tube lens offset was optimized separately for each analyte. Toluene was used as dopant at a constant flow rate of 16 μL min−1. An external pump from Thermo (model Fusion 100, USA) connected with a Hamilton syringe (USA) delivered the dopant through a T-connection into the mobile phase before the APPI probe. Mass spectrometer parameters were as follows: scan width was set at 0.5 m/z, scan time at 0.1 s, full width half maximum (FWHM) for Q1 (first quadrupole) and Q3 (third quadrupole) at 0.7, and ChromFilter peak width at 10 s. For the ion monitoring, the pseudo-selective reaction monitoring (SRM) technique was used, in which the precursor ion [M–Cl+O]− was left to pass in Q3 trying to avoid its breaking down in Q2 (second quadrupole) by using an optimum collision energy (Table 1). Pseudo-SRM technique is used for compounds with inefficient fragmentation in the collision cell, and it takes advantage of the selectivity of the triple quadrupole mass spectrometer. Q1 and Q3 are at the same m/z, while a small amount of collision energy is applied at Q2. The objective of pseudo-SRM is to reduce the response of interferences [31]. For the integration of peaks, ISIS algorithm was used processed by the smoothing algorithm Boxcar that was set at 15 points. Optimization procedure The development of a LC-APPI-MS/MS method for the simultaneous determination of many compounds is complicate because there are various parameters that are involved and affect the response. To solve this analytical problem, a strategy for the development and optimization of a LC-APPI-MS/MS method is proposed [30].

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Firstly, the appropriate ionization mode and polarity should be selected as well as a dopant and its flow rate for the beginning of the experiments. By infusing a standard solution (10 μg mL−1) of each congener into the source, the ionization mode and its polarity were selected. During this experiment, a candidate dopant was mixed with the standard solution though a T-connection, and at this step, a full scan MS (50–600 m/z) was recorded. As the basic information about target congeners were extracted, the selection of mobile phase and column’s stationary phase was the next step, aiming at a sufficient chromatographic separation of isomers. Various mobile phases were tested in order to evaluate the following columns: Waters XSELECT HSS T3 (2.5 μm, 100×2.1 mm), Thermo Hypersil Gold (3 μm, 100×2.1 mm), Zorbax SB C18 Rapid Resolution HT (1.8 μm, 30×2.1 mm), KINETEX PFP (1.7 μm, 50×2.1 mm), XSELECT CSH Phenyl-Hexyl (2.5 μm, 100×2.1 mm), and Thermo Hypersil Green PAH (3 μm, 100×2.1 mm). A selection was made regarding the basic composition of mobile phase and the column, recording the selected monitoring MS of the chosen precursor ions. Then, preliminary screening experiments were conducted with loop injections (5 μL) of standard solution (1 μg mL−1) of each congener in order to assess the effect of the following parameters on each congener ionization and its signal response (in that order): dopant, dopant percentage, mobile phase flow, probe position, y-distance, skimmer offset, sheath gas, auxiliary gas, vaporizer temperature, and capillary temperature. The univariate optimization was carried out by recording the SIM signal of the chosen precursor ions. A decision about the appropriate gradient of mobile phase was made at that step. The selected parameters were kept constant, and based on the univariate results, a full factorial experimental design was performed for the optimization of sheath gas, auxiliary gas, vaporizer temperature, and capillary temperature, in order to test which ones were critical and if joint effects exist. For the non-critical parameter, the capillary temperature, a definite value was selected. As for the critical parameters—sheath gas, auxiliary gas, and vaporizer temperature—additional multi-level experiments were conducted using a central composite design (CCD) so that their optimal values could be located. All these experiments were performed with loop injections (5 μL) of standard solution (1 μg mL−1) of each congener at the chosen mobile phase and flow conditions (95 % acetonitrile/5 % methanol, 80 μL min−1). Experiments with loop injections were realized using the final conditions so as to select the precursor ion of each congener and its product ions, while collision energies were also optimized. Through injections of multi-component working solutions of PCNs into the column, SRM experiments were compared with pseudo-SRM experiments and the final scan mode was chosen. Finally, selections were made for the appropriate scan time and ChromFilter.

194 Table 1 Congener

A.I. Moukas et al. Pseudo-SRM conditions and retention times of the LC-APPI-MS/MS determination of selected PCNs Observed ions (negative mode)

Collision energy (eV)

Tube lens voltage (V)

Retention time (min)±SD (n=21)

Q1

Q3

PCN 52

281

281

10

−109

34.01±0.03

PCN 54 PCN 66

281 315

281 315

10 10

−64 −152

28.88±0.10 39.54±0.15

PCN 67 PCN 73

315 349

315 349

10 10

−53 −87

40.17±0.14 44.70±0.15

PCN 75

383

383

10

−91

39.36±0.14

For the experimental design and the statistical treatment of data, the Statgraphics Centurion XV software (Stat Point, Inc., Warrenton, VA, USA, ver. 2002) was used. Determination of PCNs in water samples Liquid–liquid extraction For the isolation of the selected PCNs from water samples, a simple and efficient liquid–liquid extraction technique was used. Initially, the samples were filtered through a Millipore vacuum filtration device using glass fiber filters (type A/E, 47 mm, Pall Corporation, USA). Then, 200 mL of the filtered sample was extracted twice with 40 mL of ethyl acetate in a separatory funnel. Both ethyl acetate layers were collected in a round bottom flask. The ethyl acetate extracts were percolated through a glass tube with sodium sulfate for the removal of water residues. Subsequently, the combine extracts were condensed at 35 °C with a vacuum rotary evaporator until a final volume of 5 mL. The ethyl acetate aliquot was transferred into a test tube, and the flask was washed out three times with small aliquots of ethyl acetate which were combined with the initial extract. Finally, the ethyl acetate extract was further evaporated under a steam of nitrogen to a volume of 0.5 mL. One milliliter of methanol was added and, after stirring with Vortex, the solvent’s volume was reduced again with nitrogen to 0.5 mL. An additional 1 mL of methanol was added and the previous step was repeated. The remaining solvent was transferred in a volumetric flask of 1 mL and filled up with methanol. Then, the sample was transferred in an autosampler vial for the analysis.

has not been used in calibration curves. For each congener, the instrumental limit of detection (LoD) and the limit of quantification (LoQ) were defined as 3.3×SD/b and 10×SD/b, respectively, SD being the standard deviation of congeners’ response after six replicate injections of the standard working solution at the lowest concentration in the calibration curve and b being the slope of the calibration curve of the corresponding congener. By carrying out six replicate injections of the standard working solution of 100 μg L−1, the injection repeatability was also estimated. For each congener, standard addition curves were constructed by analyzing surface water samples spiked at 0.05, 0.125, 0.25, 0.5, 0.75, and 1 μg L−1, and at each level three spiked samples were extracted. The method LoDs and LoQs were estimated as 3.3×SD /b and 10×SD/b, respectively, with SD being the standard deviation of congeners’ response after the analysis of six surface water samples spiked at 0.05 μg L−1 for each congener and b being the slope of the standard addition curve of the corresponding congener. By analyzing six surface water samples spiked at 0.05 and 0.5 μg L−1 for each congener, the trueness and the intra-day precision of the method were estimated. Similarly, the method’s overall recovery for each congener was calculated by using the following equation: % overall recovery ¼

slope of standard addition curve  100 slope of calibration curve

Results and discussion

Method validation

Optimization of APPI-MS/MS conditions

The linearity of the LC-APPI-MS/MS method was initial evaluated with calibration curves, obtained by triplicate injections of eight standard working solutions, with concentration levels of 1, 5, 10, 25, 50, 100, 150, and 200 μg L−1 for each congener prepared in methanol. Since the response of each congener was different, the level of 1 μg L−1 for PCN 52 and PCN 54

The most important issue for the development of a LC-MS/ MS method is the efficient ionization of target compounds. So, the first choice that should be made was the selection of ionization mode and its polarity. Since ESI and APCI mode could not ionize PCNs efficiently, APPI was the next option. Yet, since in most cases APPI has to be assisted by a dopant,

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195

toluene was selected for the initial experiments. Full scan spectra (m/z 50–600) using APPI mode (discharged current at corona was set at 0 μA) were obtained for each congener, by infusion of a standard solution (10 μg mL−1) of each congener. The ionization of the congeners was insufficient in positive polarity, but it was sufficient in negative mode ionization. In all cases during the infusion of the congeners in negative polarity, an intense peak was recorded at m/z corresponding to [M–Cl+O]− ions and their isotopes, where M is the analyte molecule (Fig. 1). The potential mechanism may be described by the following equations: D þ hv→Dþ þ e− ; where D is the dopant −

O2 þ e →O2 −

−

ð1Þ ð2Þ

−

M þ O2 →½M–Cl þ O þ OCl



ð3Þ

APCI/APPI dual mode was also tested, and different values of discharged current were applied to the corona during the infusion of the congeners. The application of discharged

current resulted in an immediate and significant decrease of the obtained signal and in all cases blocked the ionization. This was also observed with compounds, for which the dual source APCI/APPI was applied, i.e., naphthalene [5]. Then, preliminary experiments for SRM transitions were conducted under these conditions, and for each congener, the four most intense fragments were found (see ESM, Fig. S2). These fragments were derived by removing one or two chlorine atoms from the precursor ion while also taking into account the existence of different isotopes (Table 2). The next step was the selection of a suitable chromatographic column and mobile phase composition (systematic study presented in the BOptimization of the chromatographic parameters^ section). It was observed that the congeners were eluted when the composition of mobile phase was 95 % acetonitrile and 5 % methanol. For this reason, it was decided to use the system 95 % acetonitrile/5 % methanol as mobile phase in all experiments with loop injections.

Fig. 1 Mass spectra of examined PCNs obtained from LC-APPI-MS/MS in negative ion mode during the infusion after the optimization (Q1 FWHM= 0.1). The intense peak in all cases corresponds [M–Cl+O]− ions, where M is the analytes’ molecule

196 Table 2 Observed precursor and product ions for the tested PCNs

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Congener

PCN 52 PCN 54 PCN 66 PCN 67 PCN 73 PCN 75

Observed SRMs (negative mode) Q1 (precursor ion)

Q3 (four most intense product ions)

[M–Cl+O]− (281)

[M–Cl2+O]− (245, 243), [M–Cl3+O]− (217, 215)

[M–Cl+O] (281) [M–Cl+O]− (315) [M–Cl+O]− (315) [M–Cl+O]− (349) [M–Cl+O]− (383)

Then, a univariate optimization of the most important APPI parameters was performed. Dopant was the first parameter that should be examined and as previously mentioned toluene was selected as dopant for the first experiments. However, the most widely used dopants, namely toluene, chlorobenzene, anisole, and acetone, were also tested and compared for the ionization efficiency of PCNs. Keeping all other conditions constant, loop injections (n=5), at full scan (m/z 50–600) of each congener, were carried out using each dopant. The results (Fig. 2) revealed that toluene was indeed the best choice for all congeners, and it was finally selected and used for the rest of experiments. For the remaining experimental parameters of the univariate optimization, loop injections (n=4 injections) were carried out by recording the SIM of the chosen precursor ions. The flow rate of the dopant is another important parameter, which is usually referred to as the percentage of the mobile phase flow. A percentage between 5 and 20 % is used in most cases, and therefore the percentages 5, 10, and 20 % were examined. The 20 % dopant level produced the highest signal for all the PCNs (see ESM, Fig. S3a) and finally selected and was kept constant for the remaining experiments. Toluene was also the best dopant for PCBs’ ionization, but lower percentages were required for PCBs than for PCNs [30]. The flow rate of mobile phase is also very important in APPI, Fig. 2 Effect of dopant on PCNs’ ionization. Examination of toluene, chlorobenzene, anisole, and acetone

−

[M–Cl2+O]− (245, 243), [M–Cl3+O]− (217, 215) [M–Cl2+O]− (279, 277), [M–Cl3+O]− (251) Cl− (35) [M–Cl2+O]− (279, 277), [M–Cl3+O]− (251, 249) [M–Cl2+O]− (313, 311), [M–Cl3+O]− (285, 283) [M–Cl2+O]− (355, 348), Cl− (37, 35)

and for this reason, flow rates of 50, 80, 100, and 200 μL min−1 were examined with loop injections. The ideal flow rate appeared to be 50 μL min−1, while at 200 μL min−1, the response was almost negligible (Fig. 3). At this point, a compromise between response and chromatographic analysis time should be made. Thus, 80 μL min−1 was finally selected. It was evident that ionization of PCNs in APPI was flow dependent. Vaporization at higher flow rates is probably not as complete as at lower flow rates, especially when using relatively low vaporization temperatures—as in this study. Regarding the probe position in relation to the sampling cone, probe at BA^ position and y-distance (micrometer position) at 0 a.u. were chosen (ESM, Fig. S3b, c). Skimmer offset was a crucial parameter. Values of 0, −5, −10, and −20 V were tested (Fig. 4), and it was observed that a small voltage at skimmer was necessary for solvent declustering and increase of the signal. The optimum value was −5 V. Sheath gas and auxiliary gas were set at 25 psi and 2 a.u., respectively (ESM, Fig. S3d, e). A vaporizer temperature of 300 °C and an ion transfer tube temperature of 200 °C were chosen (ESM, Fig. S3f, g). All these values were lower for PCNs’ ionization than those observed for efficient PCBs’ ionization, denoting that PCNs are transferred in the gas phase and in ionized state easier than PCBs [30].

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197

Fig. 3 Effect of mobile’s phase flow rate on PCNs’ ionization

The selected parameters were kept constant and, according to the above results, a design of screening experiments (factorial in two blocks, 1 center point per block, 0 replicates, 6 error degrees of freedom; ESM, Table S1), was performed for the following factors: sheath gas, auxiliary gas, vaporizer temperature, and capillary temperature. The peak area of the precursor ion of each congener was chosen as the response variable. This was necessary in order to examine which parameters were more critical and if joint effects between the parameters existed. Loop injection experiments were performed, measuring the

Fig. 4 Effect of skimmer offset on PCNs’ ionization

selected monitoring MS of each congener at each combination of conditions. In order to create those combinations, a definition of a low and a high value for each parameter was necessary and that was made from the results of the univariate experiments (ESM, Table S2). Pareto charts for each congener were created after the statistical treatment of the results (ESM, Fig. S4). These charts revealed that capillary temperature was not critical, and thus, it was set at 200 °C. Regarding the critical parameters (ESM, Fig. S4), namely the sheath gas, auxiliary gas, and vaporizer

198

Fig. 5 Desirability plot of [M–Cl+O]− peak areas in APPI as function of vaporizer temperature and sheath gas flow rate. Auxiliary gas, 5 a.u.

temperature, a surface response design of experiments was done (central composite design, CCD, 23+ star, 2 center points, 1 replicate, 21 error degrees of freedom; ESM, Table S3), aiming at locating their optimal values. Again, the response value was the peak area. A loop injection of each congener was conducted at each combination of conditions and, to create those combinations, it was also necessary to define the low and high values of each parameter. The choice was made from the results of the previous screening experiments (see ESM, Table S4). The Pareto charts and the surface response plots for each congener were created after the statistical treatment of the

A.I. Moukas et al.

obtained results. The most critical parameters were the vaporizer temperature and sheath gas. Using desirability plots for all the compounds [28], a compromise was made, and the optimal values for those parameters were finally selected (Fig. 5). Finally, the vaporizer temperature was set at 260 °C, sheath gas was 24 psi, and the auxiliary gas was 5 a.u. At the final source conditions, loop injections were performed in order to confirm the product ions of the selected precursor ions. At this stage, collision energies were also optimized. It was observed that fragmentation of PCN 75 was not efficient under flow conditions, and the response of its product ions was too low to be useful for quantitative trace analysis purposes. To overcome this problem, the use of pseudo-SRM technique was performed. Pseudo-SRM technique gave higher responses not only for PCN 75 but also for all congeners, so it was finally used for all of them. For comparison purposes, chromatograms of the same multi-congener PCNs standard solution (100 μg L−1) obtained with the same chromatographic method under SRM and pseudo-SRM techniques were recorded and depicted in Figs. S5 (see ESM) and 6, respectively. Pseudo-SRM technique produced the best sensitivity (the best signal-to-noise ratios) than SRM technique for all congeners (see ESM, Table S5). The optimization of collision energy for the pseudo-SRM of each congener was carried out manually (Fig. 7), since it was important to avoid any fragmentation of PCNs and select the optimum collision energy for the pseudo-SRM technique for each congener. For all congeners, 10 eV was the optimum collision energy.

Fig. 6 Chromatogram of multi-congener standard solution 100 μg L−1 (for each congener) obtained using the pseudo-SRM technique

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199

Fig. 7 Effect of collision energy on the response of PCNs at pseudo-SRM mode

Optimization of the chromatographic parameters The first step of the optimization of the chromatographic parameters was the selection of the stationary and an initial mobile phase. Various columns and solvents for reversed phase chromatography, such as methanol, acetonitrile, and mixtures of them with water, in various analogies, were examined. Mobile phases consisting of methanol/water could not separate the isomers PCN 66 and PCN 67, regardless of the stationary phase. On the contrary, mobile phases consisting of acetonitrile/water could separate those two PCNs with the Thermo Hypersil Green PAH column. The acetonitrile/water system was not efficient for the other columns. As mentioned, the compromised flow rate and the gradient of the mobile phase were chosen on both the optimum peak shape and analysis time (the last congener was eluted at

44.60 min and it was PCN 73). It is important to state that the existence of a small amount of methanol (5 %) in acetonitrile enhanced the ionization. This probably occurred because both PCNs and toluene are more soluble in methanol than acetonitrile. Probably, this small amount of methanol in the mobile phase provided better elution of PCNs from the column and better commixture of the eluent with the dopant. Furthermore, water in the column during the elution was obligatory for the separation of the congeners. Validation A wide linearity range was shown in APPI for all congeners, with correlation coefficients being higher than 0.99. Table 3 summarizes the instrumental LoD and LoQ, which where quite low, precision (%RSD),

Table 3 Instrumental LoDs and LoQs, injection repeatability (%RSD), correlation coefficient (R2), and regression coefficients with standard errors of the calibration equations of each congener Congener

PCN 52 PCN 54 PCN 66 PCN 67 PCN 73 PCN 75 a

LoD (pg on column)

5.4 16 2.1 1.5 1.4 0.8

LoQ (pg on column)

16 48 6.4 4.6 4.3 2.4

Repeatability (n=6, % RSD)

6.9 4.0 3.2 3.9 3.7 2.2

Correlation coefficient (R2)

0.991 0.992 0.992 0.990 0.985 0.984

Regression coefficients with standard errors of the calibration equationsa (b±sb)×103

(a±sa)×104

35.6±1.5 32.7±1.3 62.0±2.3 58.7±2.4 65.3±3.3 53.3±2.8

−8±16 −3±14 −11±23 −12±23 −23±32 −15±27

Linear calibration line y=bx+a was used, where y is area and x concentration of the congener in microgram per liter

200

A.I. Moukas et al.

Fig. 8 Chromatogram of a spiked surface water (100 ng L−1 for each congener)

correlation coefficients (R2), and regression coefficients with standard errors of the calibration equations of each congener. Standard addition curves were constructed by measuring surface water samples (Fig. 8, spiked surface water 100 ng L −1 ). Adequate correlation coefficients were found (higher than 0.99). Table 4 presents method LoDs and LoQs, precision (%RSD), correlation coefficients (R2), regression coefficients with standard errors of the calibration equations, and the overall recovery of each congener. The estimated method LoDs were similar to previous GC-MS methods [15, 20, 22, 24], demonstrating the applicability of LC-APPI-MS/MS technique

for the determination of PCNs in environmental samples. Moreover, the overall recoveries were almost quantitative, demonstrating the absence of matrix effects and systematic errors during ionization and extraction, as expected, because APPI is a gas phase ionization technique (like APCI, where no matrix effects were observed in various environmental matrices [28]). Taking into account the low LoDs of the method and the satisfactory precision and recoveries, this method is fit for the trace determination of PCNs in real water samples. Five lake samples from the water reservoirs of Athens were analyzed, and the target PCNs were found below the method LoDs.

Table 4 Method LoDs and LoQs, precision (%RSD), overall recovery, correlation coefficient (R2), and regression coefficients with standard errors of the standard addition calibration equations of each congener Congener

PCN 52 PCN 54 PCN 66 PCN 67 PCN 73 PCN 75

LoD (μg L−1)

0.015 0.021 0.011 0.012 0.009 0.010

LoQ (μg L−1)

0.045 0.063 0.032 0.035 0.029 0.030

Precision (n=6, % RSD)

Trueness (mean recovery±SD, n=6)

0.05 μg L−1

0.5 μg L−1

0.05 μg L−1

0.5 μg L−1

11 15 9.5 10 8.4 8.4

17 12 16 15 13 14

82.7±8.9 82±11 67.5±6.4 69.3±7.1 68.6±5.7 70.6±5.9

77±11 79.6±9.0 81±11 82±12 82.1±9.4 82±10

Overall recovery %

97 99 99 98 97 97

Correlation coefficient (R2, n=6)

0.977 0.982 0.984 0.986 0.984 0.986

Regression coefficients with standard errors of the calibration equationsa (b±sb)×105

(a±sa)×105

69.0±5.3 64.6±4.3 123.2±8.0 115.0±6.9 127.4±7.9 103.7±6.2

−2.7±3.0 −2.1±2.4 −5.3±4.5 −3.9±3.9 −5.6±4.4 −4.1±3.5

a Linear calibration line y=bx+a was used, where y is area and x is concentration of the congener, micrograms per liter, in the sample before the preconcentration

Novel determination of polychlorinated naphthalenes

Conclusions This study presents the ionization of PCNs using APPI for the first time. Mass spectra of PCN 52, 54, 66, 67, 73, and 75 by negative ionization LC-APPI-MS/MS were presented. A detailed optimization strategy is suggested for the development of LC-APPI-MS/MS methods. Compromised conditions were chosen for the sensitive determination of all the congeners using desirability plots. A highly sensitive and accurate method for the determination of the target PCNs was finally proposed and validated for surface and tap water samples. The low instrumental and method LoDs and the adequate recoveries indicate the potential use of LC-APPI-MS/MS for the determination of PCNs in traces in real samples and widen the applications of LC-APPI-MS in matrices where detection of PCNs is more often and usually at higher concentrations. Compliance with ethical standards Conflict of interest The authors declare that they have no competing interests.

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