Evaluation of comprehensive two-dimensional gas chromatography coupled to rapid scanning quadrupole mass spectrometry for quantitative analysis

Journal of Chromatography A, 1255 (2012) 177–183

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Journal of Chromatography A journal homepage: www.elsevier.com/locate/chroma

Evaluation of comprehensive two-dimensional gas chromatography coupled to rapid scanning quadrupole mass spectrometry for quantitative analysis Bruno José Gonc¸alves Silva a , Peter Quinto Tranchida b , Giorgia Purcaro b , Maria Eugênia Costa Queiroz a , Luigi Mondello b , Fernando Mauro Lanc¸as c,∗ a

Departamento de Química, Faculdade de Filosofia Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, Ribeirão Preto 14040-901, SP, Brazil Dipartimento Farmaco-chimico, Facoltà di Farmacia, Università di Messina, Viale Annunziata, 98168 Messina, Italy c Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos 13560-970, SP, Brazil b

a r t i c l e

i n f o

Article history: Available online 17 May 2012 Keywords: Comprehensive two-dimensional gas chromatography GC × GC qMS Quantification Pesticide

a b s t r a c t Comprehensive two-dimensional gas chromatography (GC × GC) is a powerful technique that provides excellent separation and identification of analytes in highly complex samples with considerable increase in GC peak capacities. However, since second dimension analyses are very fast, detectors with a rapid acquisition rate are required. Over the last years, quite a number of studies have discussed the potential and limitations of the combination GC × GC with a variety of quadrupole mass spectrometers. The present research focuses on the evaluation of qMS effectiveness at a 10,000-amu/s scan speed and 20-Hz scan frequency for the identification (full scan mode acquisition-TIC) and quantification (extracted ion chromatogram) of target pesticide residues in tomato samples. The following MS parameters have been evaluated: number of data points per peak, mass spectrum quality, peak skewing, and sensitivity. The validated proposed GC × GC/qMS method presented satisfactory results in terms of repeatability (coefficient of variation lower than 15%), accuracy (84–117%), and linearity (ranging from 25 to 500 ng/g), while significant enhancement in sensitivity was observed (a factor of around 10) under scan conditions. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Over the past decade, comprehensive two-dimensional gas chromatography (GC × GC) has gained wide interest and has been increasingly accepted due to its ability to separate and identify analytes in highly complex samples [1]. Since its introduction in 1991 [2], this research tool has attracted growing attention, as seen from the several instruments that are commercially available today and from the hundreds of papers that have been published in this area. The principles, instrumental requirements, applications, and developments of GC × GC have been extensively discussed in recent reviews [3–7]. In the case of GC × GC, apart from set columns selection and modulation, detection is also an important consideration, especially because the second-dimension analyses are essentially very fast (4–8 s). In order to properly describe these very narrow seconddimension peaks (typically 100–600 ms at the baseline) and avoid the extra peak broadening caused by the detector, GC × GC has to

∗ Corresponding author at: Institute of Chemistry at São Carlos, University of São Paulo, São Carlos 13560-970, SP, Brazil. Tel.: +55 16 3373 9983; fax: +55 16 3373 9984. E-mail addresses: fl[email protected], fl[email protected] (F.M. Lanc¸as). 0021-9673/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.chroma.2012.05.035

be coupled to detectors with high data acquisition rate and small internal volume [8,9]. Therefore, it does not come as a surprise that virtually almost all the early studies in this area were conducted by using a flame ionization detector (FID), which provides data acquisition rates up to 200 Hz and dead volumes which are effectively zero [10,11]. Other detectors like the micro electron-capture detector (␮-ECD) [12], sulfur chemiluminescence detector (SCD) [13], and nitrogen chemiluminescence detector (NCD) [14] have been adapted for use in GC × GC. They have been shown to present high selectivity and sensitivity, despite some limitations such as the continuous significant extra band broadening when compared with FID. On the other hand, only the mass spectrometry detector (MS) provides structural information about the analytes, which has prompted the use of this system for detection and identification purposes in development as well as in routine laboratories. The main mass analyzer applied for GC × GC is the time-of-flight mass spectrometer (TOF-MS), which easily reaches the required spectrum acquisition rates (50–100 Hz) for reliable GC × GC peak assignment and, more importantly, for quantification [15–17]. Unfortunately, the high cost of such instrumentation is the main reason behind its limited laboratory utilization. Admittedly, the extremely popular and benchtop quadrupole mass spectrometer (qMS) systems are much less expensive and more user-friendly, and

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several authors have reported their use with GC × GC under appropriate operating conditions [18–20]. However, in these studies the authors described the difficulty to perform quantification due to the slow quadrupole duty cycle and the need to scan individual masses in the scan range, which results in slow data acquisition. Hence, for many years the qMS system has been employed in combination with GC × GC for identification purposes only. To overcome the limiting scan frequency of the conventional qMS, recent studies have shown how effective (identification and quantitation) GC × GC coupled to qMS can be when operating in the fast scanning mode [21,22], through which satisfaction data acquisition rates required for GC × GC (at least 20–30 Hz) are achieved. This fact opened the possibility of extending the range of applications of GC × GC–qMS methods to the identification and quantification of target analytes in complex samples with results of a good level of confidence. Over the last years, quite a number of studies have discussed the potential and the limitations of the combination of GC × GC with a variety of qMS detectors. According to previous well-established studies, and because of the low data acquisition rate limited by the scan speed and the interscan delay, it became clear that the use of a conventional qMS system coupled to GC × GC is not the best choice when quantitative analysis is required. For example, HP 5973 has been used for mutually divergent applications, but invariably for restricted mass ranges (i.e., 180–200 Da) and at a maximum scan speed of 4 kDa/s, in order to achieve data-acquisition rates close to 20 Hz. Under such conditions, there are only 3–5 data points across the fastest second-dimension peaks, which seriously compromises quantification [23–25]. Although some authors have stated that at least 15–20 data points-peak are necessary for reliable quantification [26], it is generally agreed within the chromatography community that 10 data points/peak are sufficient for reliable peak reconstruction [27]. On the other hand, Debonneville and Chaintreau [28] reported the first example of quantification in a comprehensive GC application using a qMS system, and these authors affirmed that a detection frequency of 30.7 Hz was reached in the study of fragrance allergens analyzed by selected ion monitoring (SIM). According to the authors, this value was sufficient for peak quantitation. It is obvious that the use of the SIM mode is only applicable to known target analytes. These results contributed to a new focus in the GC × GC–MS research field: the development of faster scanning qMS systems. In 2005, Korytár et al. [9] were the first to use a rapid-scanning qMS instrument in a GC × GC experiment (with chemical ionization [electron-capture negative ion (ECNI) mode]). The performance of the MS instrument was studied, and it was found to be capable of producing 23 spectra/s, using a 300-amu mass range. Also in 2005, Adahchour et al. [29] published a study on the principles, practicability, and potential of rapid-scanning qMS instrumentation. The detector was characterized by a maximum scan speed of 10,000 amu/s and was able to reach a frequency of 50 spectra/s within an excessively restricted mass range (95 amu). Moreover, the authors affirmed that only 7 data points/peak (above the baseline) are necessary for accurate peak reconstruction. More recently, in 2010, Purcaro et al. [22] evaluated a very fast scanning qMS instrument, herein used as a GC × GC detector. The novel mass spectrometer was able to operate at a 20,000-amu/s scan speed and generated 50 spectra/s by using a 290-amu mass range (40–330 m/z). GC × GC/qMS applications were directed to the analysis of perfume allergens, and more than 15 data points/peak were attained, meeting the requirements related to reliable peak reconstruction. However, despite these good results, just a few works on the use of a qMS instrument coupled to GC × GC for quantitative purpose were published in the following the next years, and GC × GC/TOF-MS became increasingly established.

In the present investigation, the performance of a modern qMS system for use in GC × GC has been studied for both qualitative and quantitative purposes. The principles of coupling a qMS to a GC × GC system are discussed in terms of data acquisition rate, mass range and mass spectral quality. GC × GC/qMS application in a complex sample has been directed to the analysis of pesticides residues in tomatoes samples. The validity of the quantitative results obtained by GC × GC/qMS is demonstrated via several method performance parameters such as selectivity, accuracy, and precision. 2. Experimental 2.1. Chemicals and reagents Certified reference standards of all the pesticides were >98% purity and were purchased from Sigma–Aldrich (Milan, Italy). Ethyl acetate and cyclohexane (HPLC grade) were acquired from Carlo Erba (Milan, Italy). Stock solutions for the pesticides with concentrations of 1 mg mL−1 were prepared by dissolving 10 mg in 10 mL cyclohexane/ethyl acetate (1:1). Working standard solutions were prepared by mixing the 39 pesticides in appropriate proportions, so that a working standard mixture of 100 ␮g/mL would be achieved. The calibration standards of concentrations 25, 50, 100, 200 and 500 ng/mL were obtained by successive dilutions of the above working standards with cyclohexane/ethyl acetate (1:1). All these pesticides solutions were stored at −18 ◦ C during the study. 2.2. GC × GC/qMS analyses The GC × GC/qMS applications were carried out on a Shimadzu GC × GC/qMS system consisting of a GC2010 gas chromatograph and a QP2010 Plus quadrupole mass spectrometer (Shimadzu, Kyoto, Japan). The GC was equipped with an AOC-20i auto-injector and a split–splitless injector (300 ◦ C). The first column was connected by using an SGE SilTite mini-union (Ringwood, Victoria, Australia) to an uncoated tubing (1.4 m × 0.25 mm I.D. internal diameter), which was used to create the modulation loop. The uncoated tubing was connected to the secondary column by using an SGE SilTite mini-union. The first column consisted of an SLB-5 ms 30 m × 0.25 mm I.D. × 0.25 ␮m column [silphenylene polymer virtually equivalent in polarity to poly(5% diphenyl/95% methylsiloxane)], while the second column was a SLB-IL59 1.0 m × 0.10 mm I.D. × 0.08 ␮m film thickness (Supelco, Milan, Italy), which is a polar ionic liquid column with polarity/selectivity similar to that of polyethylene glycol (PEG) columns. An injection volume of 4 ␮L in the split mode (1:10) was set. Modulation was carried out every 4 s by using a loop-type modulator (under the license of Zoex Corp., Houston, TX). The duration of the hot pulse (325 ◦ C) was 375 ms. The oven temperature was as follows: from 150 to 300 ◦ C at ◦ 4 C min−1 , held for 5 min. The carrier gas (helium) was delivered at an initial pressure of 229 kPa (constant linear velocity mode). As for MS parameters, the sample was analyzed in the full scan mode, at a scan speed of 10,000 amu/s, mass range of 50–460 m/z, and sampling frequency of 20 spectra/s. The interface and ion source temperatures were 250 and 220 ◦ C, respectively. The MS ionization mode was electron ionization, and the detector voltage was 1.0 kV. Data were collected by the GCMS Solution software (Shimadzu); bidimensional visualization was carried out by using the Comprehensive Chromatography Manager v.1.0 software (Chromaleont, Messina, Italy). An in-house spectral database as the MS library for spectral matching. Extracted ion chromatogram was used for quantification (see Table 1 ).

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Table 1 Absolute retention times (min), target ion (T), and qualifier ions (Q1 and Q2); peak width at the baseline; number of points per peak for the main modulated peak, and total number of points per peak for each 2D peak for analyses of pesticide residues in tomato samples. No.

Pesticide

tR (1D – min)

tR (2D – s)

T/Q1,Q2

Points per peaka

Total number of points per 2D peak

Peak width (ms)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39

Phorate Hexachlorobenzene Carbofuran Dimethoate Quintozen Terbufos Lindane Disulfoton Etrimfos Prometryn Heptachlor Paraoxon Fenitrothion Malathion Fenthion Parathion Aldrin Bioallethrin (isomer 1) Bioallethrin (isomer 2) Endosulfan p,p-DDE Oxyfluorfen Myclobutanil o,p-DDD Dieldrin Cyproconazole Chlorbenzilate Ethion p,p-DDD 2,4-DDT Carbophenothion 4,4-DDT Propargite Epoxiconazol Fenpropatrin Dicofol ␭-Cyhalothrin Fenvalerate (isomer 1) Fenvalerate (isomer 2)

12.838 13.493 13.858 13.945 14.499 14.637 14.702 15.373 15.574 17.845 17.895 18.127 18.545 18.813 19.389 19.562 19.603 21.045 21.245 23.036 23.835 24.002 24.211 24.241 24.306 24.932 25.317 25.655 25.846 25.903 26.915 27.507 28.048 28.763 29.920 30.165 31.792 38.006 38.545

1.165 0.506 2.430 3.741 0.861 1.114 1.063 1.316 1.367 1.570 0.658 2.481 3.787 3.696 2.278 0.658 3.190 1.620 1.620 1.114 1.013 3.038 1.570 1.367 1.316 2.886 1.975 2.127 1.671 1.114 1.873 1.367 1.823 0.709 2.127 0.810 2.481 3.342 3.797

75/97,121 284/249,142 164/122,142 87/93,125 237/214,295 57/103,231 109/111,219 88/60,142 181/292,153 184/54,241 100/65,272 109/81,149 125/109,277 127/93,173 278/109,125 109/97,291 66/91,263 123/79,136 123/79,136 195/159,241 246/318,176 252/300,361 179/82,150 235/165,199 79/108,263 222/83,139 139/251,111 231/97,153 235/165,199 235/165,199 157/97,121 235/165,199 135/81,173 192/138,165 97/55,181 139/111,250 181/197,208 125/167,225 125/167,225

9 9 13 10 11 11 8 9 9 9 8 12 10 13 12 10 11 8 8 7 8 9 13 9 10 13 11 8 8 7 9 8 10 7 10 7 11 9 9

22 23 31 26 29 20 19 23 22 17 20 31 19 33 24 26 22 15 16 15 18 18 29 17 24 27 24 18 19 17 21 20 27 18 22 17 24 22 23

360 360 660 540 660 600 420 480 420 360 300 600 540 660 600 480 540 360 360 300 300 480 660 420 480 600 540 360 420 300 540 480 600 300 540 300 600 420 420

tR : retention time; 1D: first dimension; 2D: second dimension. a Values for the main modulated peak.

2.3. Extraction procedure Organic tomato samples were purchased from retail markets. Preliminary analysis showed that they were analyte-free. These blank tomato samples were then spiked with target pesticides and used for the preparation of the calibration curve and analytical validation of the proposed method. For the extraction procedure, 10 mL ethyl acetate were added to 20 g blended tomato. Ethyl acetate was selected as the extraction solvent because provides high recoveries in the analysis of pesticides over a wide range of polarity [30]. After spiking with the pesticides and homogenization, the samples were magnetically stirred for 15 min at 900 rpm, and centrifuged at 4000 rpm for 10 min. The supernatant layer was introduced into a glass vial, evaporated to dryness under a gentle nitrogen stream, and redissolved with 200 ␮L ethyl acetate. No further purification was performed. Subsequently, the vials were closed and placed in the auto-sampler tray for GC × GC/qMS analysis. 2.4. Method validation In order to evaluate the qMS performance in terms of peak reconstruction, mass spectral quality, and analytical repeatability, the method validation was carried out at a 20 Hz acquisition rate in the scan mode, using the same 50–460 mass range. Calibration curves were built at five concentration levels, and two replicates were performed at each level. The linearity was evaluated by

calibration curves constructed using linear regression of the pesticide peak area (Y) versus the pesticide nominal tomato concentration (X, ng/g). The concentrations of the samples ranged from 25 to 500 ng/g. Accuracy and inter-day precision (coefficient of variation CV (%)) values were determined by calibration curves, by means of triplicate assays of the blank tomato samples with a known pesticide concentration. 3. Results and discussion 3.1. GC × GC separation The temperature program and flow rate of the carrier gas were optimized for monodimensional GC (1D-GC) separation before attempting GC × GC. A simple and linear temperature program between 150 and 300 ◦ C as well as an initial pressure of 229 kPa (in a constant linear velocity mode) were setup, to provide the best possible resolution in the first dimension for analysis of standard solutions of pesticides. Thus, to preserve the separation achieved in the 1D-GC, the same inlet pressure was selected for the 2D-GC analysis. As the diameters of the two columns are different, the above cited pressure was maintained constant for the first column and modulation loop only, while the pressure in the second column was much higher. This fact was supported by the calculation of approximate values of the gas linear velocities. We found that when an

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Fig. 1. Sections of the chromatograms obtained by analysis of tomato sample spiked with pesticides at 200 ng/g. (A) 1D GC/qMS, (B) GC × GC/qMS. (1) Interfering compound: m/z = 74, (2) prometryn: m/z = 184 and (3) heptachlor: m/z = 100.

inlet pressure of 229 kPa was applied in the first column, the average linear velocity values were 17.54 (H = 240 ␮m) and 195.15 cm/s (H = 172 ␮m) for the first and second column, respectively. However, in fact it is not possible to assert whether the linear velocity in the second column is maintained constant. On the other hand, as the length of the second column is reduced (1 m), we believe that only small variations in the linear velocity take place. However, the essentially non-selective sample preparation used in the present study yielded extracts that contained rather high concentrations of matrix compounds. As expected, a 1D-GC separation could not separate all the target analytes from the matrix and/or from each other. An example of this problem is given in Fig. 1a for the separation of the pesticides heptachlor and prometryn, from a matrix component. In 1D analysis, prometryn coelutes with both heptachlor and an interfering compound from the matrix. Upon GC × GC application, prometryn is completely separated from the other two compounds, and a pure mass spectrum with high matching values can be obtained (higher than 92%, data not shown). After three modulations, it should be noted that the peaks of the analyte are considerably narrower and around nine times higher as compared to conventional GC (Fig. 1b). This is a result of cryofocusing in the modulator region and the subsequent very fast separation in a second column. Each analyte was identified on the contour plot by combining the retention times (1D (min) and 2D (s)) obtained from analyses of standard solutions. Confirmation was possible by comparing of the mass spectrum of each analyte with those of authentic samples for the GC × GC/qMS method. Regarding the separation of pesticides from the co-extracts, a conventional 30 m × 0.25 mm I.D. nonpolar first column combined with a polarity-based separation on 2D SLB-IL59 column led to significant improvement in resolution, as compared to the 1D setup. Fig. 2 shows the total ion GC × GC/qMS chromatogram from the analyses of a tomato sample spiked with 37 pesticides (resulting in 200 ng/g) after extraction. The total separation was achieved within 26 min, with good peak distribution along the second dimension. A partial coelution between terbufos and lindane (peaks 6 and 7, in Fig. 2) did not compromised the qualification and/or quantification procedure, since integration was conducted by means of an extracted ion chromatogram (in this example, ion m/z = 57 for terbufos, and ion m/z = 109 for lindane). For the retention times and the employed quantifying and qualifying ions, refer to Table 1. In this work, the retention time repeatability was evaluated, so as to consider the possible matrix effects as well as method precision;

differences lower than 0.06 min and 0.11 s (data not shown) were observed in the first and second dimensions, respectively. The major result of using nitrogen gas as the cryogen is the fact that peaks elute from the first dimension as very narrow peaks, which have, in view of the mass conservation, enhanced signal intensities. While the peak of the phorate residue presented bandwidth of 11.5 s and intensity of approximately 1.3 × 106 in GC/qMS analyses, after GC × GC/qMS the major phorate peak (3 peaks in total), presented bandwidth of 420 ms (a range of 300–660 ms for the other pesticide residues, see Table 1), which is 25 times narrower than that obtained in 1D-GC, and intensity around 1.25 × 107 . This example of enlarged peak amplitudes is given in Fig. 3. This peak amplitude enhancement lay in the order of 7–16 for the pesticide residues (10 times for phorate). To what extent this enhancement affects the sensitivity also dependents on the data acquisition rate and will be further discussed in next section. 3.2. qMS detection As reported previously, the performance of the qMS instrument as a detector in the GC × GC field has demonstrated that the qMS systems, mainly the conventional ones, could achieve, or could at least closely meet the requirements for analyte quantification by

Fig. 2. Contour plot of pesticides in tomato (at 500 ng/g) after extraction analyzed with GC × GC/qMS. The name of pesticides for each number (from 1 to 39) is in Table 1.

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Fig. 3. Sections of the 1D-GC (above) and raw GC × GC (below) chromatograms showing the increase of detectability (decrease of bandwidth of the modulated peak) in the multidimensional analyses for the pesticide phorate (in red are present the respective values of bandwidths). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

using some limitations, such as SIM acquisition or the application of a reduced mass range. In the present investigation, no restrictions were applied to the MS parameters; i.e., at a scan speed of 10,000 amu/s for a 50–460 m/z scan range corresponding to a scan time of 0.05 s was applied. The extent of peak reconstruction was assessed by counting the number of data points per peak (number of scans) above the baseline for the main modulated chromatographic GC × GC peak, the total number of scans per 2D peak, and the peak width at the baseline for a extracted tomato sample, enriched with the pesticides residues at 500 ng/g, under the experimental conditions reported above (Table 1). The resolution is higher at a faster acquisition rate because of the larger number of data points across a peak, which affords better precision data when quantification analysis is required. In this work, employing a qMS detector working in a frequency of 20 Hz and a peak-base width around 0.4 s, peaks in the GC × GC chromatogram were constructed with around 9 points (ranging from 7 to 13) for each modulated peak. Although other higher acquisition rate detectors (≥50 Hz), such as the flame ionization detector (FID) and the time-of-flight mass spectrometer (TOF/MS), have been preferred for this purpose [8], the number of points per peak found in this study did not seriously compromise quantification (see validation parameters in Table 2). Moreover, in general, there was no significant sensitivity variability at the different acquisition rates, and a satisfactory sensitivity was observed in the case of the scan mode, which was about 16-fold in some situations, with an average increase of a factor of 10 (as shown in Section 3.1, Fig. 3). This last result is very interesting, since a great improvement in sensitivity is achieved, without loss of full-scan information.

One of the main advantages of a target analysis is the relative facility of confirming the identity of the investigated components with a certain level of confidence. The quality of the mass spectra, relative to the selected pesticide residues, was assessed at the acquisition frequency applied: the similarity match (MS%) with the MS library-contained compounds was evaluated at each data point of the modulated peak. However, an important issue in rapid scanning qMS detection and/or fast eluting peaks is the distortion of the mass spectrum across a peak, known as “mass spectral skewing”, which causes fluctuations in the relative ion abundances (from the high- to low-mass) [21,32,33]. This phenomenon, and obviously the consistency of the mass spectral profiles, was evaluated by plotting m/z ratios between the target ion (T) and the qualifier ions (Q1 and Q2) abundances across the eluting time-profile of the chromatographic peak for some pesticide residues. As an example, two graphs containing the three aforementioned spectral parameters, relative to those of ethion and 2,4-DDT (MS%, T/Q1, T/Q2) are reported in Fig. 4. Among the several investigated compounds, these two pesticide residues were chosen because they are characterized by a poor peak shape and low number of points per peak (8 and 7 points per peak, respectively, for the higher modulated peak). According to the results, both compounds showed high MS (%) values (ranging from 89% to 98%) and very stable m/z ratio behavior over the elution profile, with no skewing. This attests to the mass spectral quality even in considerably low acquisition frequency (20 Hz). The relatively low MS (%) values (around 89%) for the peak extremities were probably due to an increase in the background noise relative to the signal. Nevertheless, it was possible to confirm the identity of compounds and/or identify unknown compounds in the matrix. Interferents of the 1D-GC analyses as

Fig. 4. Spectral quality evaluation of ethion and 2,4-DDT at 20 Hz in terms of mass spectrum similarity (MS%) at each data point acquired and ion ratios across the eluting time-profile of the chromatographic peak. Ethion m/z ratios: 231/97 (T/Q1), 231/153 (T/Q2); 2,4-DDT m/z ratios: 235/165 (T/Q1), 235/199 (T/Q2).

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Table 2 GC × GC/qMS linear regression analysis parameters (slope, intercept, and correlation coefficient, R2 ), precision, and accuracy. Pesticide

Phorate HCB Carbofuran Dimethoate Quintozen Terbufos Lindane Disulfoton Etrimfos Prometryn Heptachlor Paraoxon Fenitrothion Malathion Fenthion Aldrin Parathion Bioallethrin I Bioallethrin II Endosulfan p,p-DDE Oxyfluorfen Myclobutanil o,p-DDD Dieldrin Cyproconazole Chlorbenzilate Ethion p,p-DDD 2,4-DDT Carbophenothion 4,4-DDT Propargite Epoxiconazol Fenpropatrin Dicofol ␭-Cyhalothrin Fenvalerate I Fenvalerate II

Linear regression (linear range: 25–500 ng/g)

CV (%), n = 3

Accuracy (%)

Slope (×105 )

Intercept (×106 )

R2

25 ng/g

500 ng/g

25 ng/g

500 ng/g

0.63111 0.13161 0.32608 0.56458 0.04526 0.42397 0.06392 0.37956 0.19764 0.14951 0.16460 0.55398 0.17519 0.15611 0.19005 0.27008 0.14471 0.15619 0.39650 0.01234 0.29815 0.13474 0.17923 0.17465 0.19471 0.26955 0.46557 0.32265 0.30365 0.16212 0.22023 0.15455 0.30189 0.24026 0.03600 0.14407 0.16027 0.19738 0.19385

2.012089 0.867101 1.133817 0.880176 0.361067 3.719091 0.314823 4.501380 2.481811 0.930009 0.195980 2.150252 0.965869 1.109489 0.800374 1.721802 0.769245 0.127245 2.489683 0.076404 0.812377 0.641027 −0.004878 0.616685 0.490553 −0.132291 2.857108 0.778993 0.426964 0.364819 0.915986 0.319561 0.974384 1.136653 0.136856 0.242771 0.154006 0.282611 0.244356

0.9949 0.9945 0.9826 0.9982 0.9935 0.9845 0.9960 0.9860 0.9879 0.9982 0.9920 0.9976 0.9924 0.9837 0.9962 0.9948 0.9962 0.9888 0.9924 0.9917 0.9973 0.9929 0.9964 0.9982 0.9982 0.9897 0.9919 0.9901 0.9894 0.9917 0.9879 0.9943 0.9894 0.9884 0.9983 0.9972 0.9972 0.9983 0.9966

4.7 4.6 7.1 7.6 1.3 3.4 2.6 13.2 4.9 13.9 4.4 9.4 8.4 8.1 1.6 4.2 5.8 6.3 8.5 4.2 8.8 11.2 3.9 6.3 1.0 4.2 2.5 3.7 13.8 11.5 9.1 11.0 2.6 11.6 3.5 14.0 12.5 2.9 2.4

2.5 2.3 4.0 6.3 2.7 2.5 3.7 5.7 4.0 10.2 4.5 6.4 2.8 3.4 3.7 3.4 1.7 3.3 1.4 4.5 4.8 5.8 6.5 3.6 2.4 3.2 5.4 6.3 8.8 2.1 6.0 7.9 3.5 8.0 5.2 11.6 7.8 1.3 1.2

88 104 97 92 113 107 89 106 105 94 87 88 99 105 89 84 98 86 116 85 90 103 117 96 96 114 106 109 97 115 103 118 105 92 97 111 114 91 84

101 103 109 104 102 100 102 104 100 103 98 102 103 111 106 99 102 103 105 107 94 104 107 104 101 100 101 103 97 109 99 107 101 103 104 106 106 105 107

All data calculated by using the scan mode and integration by means of extracted ion chromatograms.

such hexadecanoic, stearic, oleic, and ascorbic (vitamin C) acids could be identified with similarity matches higher than 93% in GC × GC. Because of these results and for quantitation purposes, each 2D peak is the sum of each single modulation of each 1D-eluted peak. The above experimental setting was adopted for validation of the pesticide residues GC × GC/qMS analysis method. 3.3. Validation procedure and quantitative analyses of the GC × GC/qMS method The curve calibration of the GC × GC/MS method was carried out with blank tomato samples spiked with pesticide standard solutions with concentrations that included the maximum residue limits (MRL) established by the Codex Alimentarius (from 50 to 500 ng/g [34]) for most of the investigated pesticides. Only three of the investigated pesticides presented MRL above the higher concentration in the calibration curve: propargite (2000 ng/g), and fenpropatrin and dicofol (1000 ng/g). Moreover, quintozen was characterized by a MRL of 20 ng/g, slightly outside the calibration range. The linearity was evaluated by calibration curves constructed using linear regression. These sample concentrations ranged from 25 to 500 ng/g, and each point of the curve was made in triplicate. Very good linearity was achieved in the concentration range available (R2 > 0.9826), and the coefficient of variation (CV) ranged from 1.0% to 14.0% in the lower concentration level. The developed method showed adequate accuracy,

with values ranging between 84% and 117%. Both precision and accuracy were determined in two levels of concentration, low (25 ng/g) and high (500 ng/g). Table 2 reports these quantitative results. It is known that by the both the limits of detection and quantification attained by GC × GC/MS are considerably lower than those obtained by 1D GC/MS analysis. Some authors have described enhancement factors (expressed as the ratio of the signals from the GC × GC system and those from the 1D-GC system) around 1.50 to 50 [35,36]. As mentioned in Section 3.1, by application of the GC × GC/qMS technique, the calculated enhancement factors range between 7 and 16, compared with 1D GC/MS analysis. These results are especially important for pesticides known for their poor detectability by conventional GC–MS, and even for the low concentration level that the pesticides can be reliably determined in tomato matrices and be unambiguously identified by means of their full mass spectra. In addition, they are eluted as sharp and symmetrical peaks from the secondary column. To ascertain its applicability, this method was employed for determination of pesticide residues in several tomato samples cultivated according to conventional agricultural procedures. The samples were purchased from different retailers in the city of Messina, Italy, and immediately processed following the above described procedure (Section 2.3). No residues of the studied pesticides were detected in these samples using the proposed method, for concentrations covered by the calibration curve, which included MRL for all the pesticides.

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4. Conclusions The results of this study demonstrate the potential use of the rapid-scanning quadrupole mass spectrometer, with a 20Hz scan frequency and scan speeds of 10,000 amu/s, as detector for the GC × GC technique for both qualitative (high similarity matches values and no spectral skewing) and quantitative analyses. Although a poor number of points per peak were acquired (around 9 scans for each of the main modulated peak), compared with faster acquisition rate detectors, like the TOF-MS system (at least 50 Hz), the validation parameters such as accuracy, precision, and linearity proved that the qMS detector was satisfactory for quantification in a properly optimized GC × GC experiment. The GC × GC/qMS method was successfully applied to the analysis of selected pesticide residues in tomato samples. Obviously, the use of GC × GC enable separation of the target analytes from each other, but, most importantly it also allowed for their separation from matrix compounds, which tend to seriously interfere in 1D-GC/MS procedures. In summary, this work once again showed the use of a quadrupole MS instrument for quantification in comprehensive GC separations, even when a large mass range (50–460 m/z) is utilized in the scan mode. The results presented here confirm the present interest in the development of faster scanning qMS systems (scan frequency of 50 Hz, or more), for hyphenation with the GC × GC technique. The advantages are that it is a much less expensive instrument, compared with the well-established TOF-MS instrumentation. References [1] [2] [3] [4]

J. Dallüge, J. Beens, U.A.Th. Brinkman, J. Chromatogr. A 1000 (2003) 69. Z. Liu, J.B. Phillips, J. Chromatogr. Sci. 29 (1991) 227. J.B. Phillips, J. Beens, J. Chromatogr. A 856 (1999) 331. P.J. Marriott, R. Shellie, Trends Anal. Chem. 21 (2002) 573.

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[5] M. Adahchour, J. Beens, R.J.J. Vreuls, U.A.Th. Brinkman, Trends Anal. Chem. 25 (2006) 438. [6] M. Adahchour, J. Beens, U.A.Th. Brinkman, J. Chromatogr. A 1186 (2008) 67. [7] H.J. Cortes, B. Winniford, J. Luong, M. Pursch, J. Sep. Sci. 32 (2009) 883. [8] M. Adahchour, J. Beens, R.J.J. Vreuls, U.A.Th. Brinkman, Trends Anal. Chem. 25 (2006) 540. [9] P. Korytár, J. Parera, P.E.G. Leonards, J. de Boer, U.A.Th. Brinkman, J. Chromatogr. A 1067 (2005) 255. [10] P. Korytár, H.G. Janssen, E. Matisová, U.A.Th. Brinkman, Trends Anal. Chem. 21 (2002) 558. [11] L.C.A. Amorim, J.M. Dimandja, Z.L. Cardeal, J. Chromatogr. A 1216 (2009) 2900. [12] E.M. Kristenson, P. Korytár, C. Danielsson, M. Kallio, M. Brandt, J. Makela, R.J.J. Vreuls, J. Beens, U.A.Th. Brinkman, J. Chromatogr. A 1019 (2003) 65. [13] F.C.Y. Wang, W.K. Robbins, F.P. Di Sanzo, F.C. McElroy, J. Chromatogr. Sci. 41 (2003) 519. [14] F.C.Y. Wang, W.K. Robbins, M.A. Greaney, J. Sep. Sci. 27 (2004) 468. [15] J.A. van der Meer, H.C. Trap, D. Noort, M.J. van der Schans, J. Chromatogr. B 878 (2010) 1320. [16] P. Wojtowicz, J. Zrostlíková, T. Kovalczuk, J. Schurek, T. Adam, J. Chromatogr. A 1217 (2010) 8054. [17] J.-F. Focant, A. Sjodin, D.G. Patterson Jr., J. Chromatogr. A 1019 (2003) 143. [18] G.S. Frysinger, R.B. Gaines, J. High Resolut. Chromatogr. A 22 (1999) 251. [19] S.M. Song, P. Marriott, P. Wynne, J. Chromatogr. A 1058 (2004) 223. [20] P.Q. Tranchida, G. Purcaro, C. Fanali, P. Dugo, G. Dugo, L. Mondello, J. Chromatogr. A 1217 (2010) 4160. [21] C. Cordero, C. Bicchi, D. Joulain, P. Rubiolo, J. Chromatogr. A 1150 (2007) 37. [22] G. Purcaro, P.Q. Tranchida, C. Ragonese, L. Conte, P. Dugo, G. Dugo, L. Mondello, Anal. Chem. 82 (2010) 8583. [23] M. Kallio, T. Hyotylainen, M. Lehtonen, M. Jussila, K. Hartonen, M. Shimmo, M.-L. Riekkola, J. Chromatogr. A 1019 (2003) 251. [24] R.A. Shellie, P.J. Marriott, Analyst (Cambridge, UK) 128 (2003) 879. [25] S.M. Song, P.J. Marriott, P. Wynne, J. Chromatogr. A 1058 (2004) 223. [26] N. Dyson, J. Chromatogr. A 842 (1999) 321. [27] C.F. Poole, The Essence of Chromatography, Elsevier, Amsterdam, 2003, p. 66. [28] C. Debonneville, A. Chaintreau, J. Chromatogr. A 1027 (2004) 109. [29] M. Adahchour, M. Brandt, H.-U. Baier, R.J.J. Vreuls, A.M. Batenburg, U.A.Th. Brinkman, J. Chromatogr. A 1067 (2005) 245. [30] D. Sharma, A. Nagpal, Y.B. Pakade, J.K. Katnoria, Talanta 82 (2010) 1077. [32] M. Kirchner, E. Matisova, S. Hrouzkova, J. de Zeeuw, J. Chromatogr. A 1090 (2005) 126. [33] R.J.J. Vreuls, J. Dallüge, U.A.Th. Brinkman, J. Microcol. Sep. 11 (1999) 663. Alimentarius, Pesticides Residues in Food and Feed, [34] Codex (accessed June 2011). [35] J. Zrostlíková, J. Hajˇslová, T. Cajka, J. Chromatogr. A 1019 (2003) 173. [36] J. Schurek, T. Portolés, J. Hajslova, K. Riddellova, F. Hernandez, Anal. Chem. Acta 611 (2008) 163.