5 Chapter Five Investigation of Propoxur and Its Transformation Products in Aquatic Plants and Animals Using Microwave-assisted Extraction Followed by LC-APCI-ITMS 5.1 INTRODUCTION Propoxur, one of the important pesticides of the carbamate group, is widely used against household insects It is a potent inhibitor of cholinesterase and is moderately toxic to humans and animals, having an LD50 of 100 mg/kg in rats [1] It is classified as moderately hazardous by the World Health Organization (WHO) [2] and the acceptable daily intake for humans has been set at 0.02 mg/kg by WHO [3] Due to its high solubility and instability in water, propoxur and metabolites (N-methylformamide and 2isopropoxyphenol) are potential contaminants of the aquatic environment and food resources, such as fish, tortoises and alga, which play significant roles as a carrier of propoxur and its TPs Therefore, evaluation and estimation of the potential risk of propoxur and its transformation products (TPs) for plants and animals in the aquatic environment are necessary As already emphasized previously, LC-MS is one of the most powerful techniques for the analysis of polar pesticide residues In the past few years, much work has been done by LC-MS to analyze carbamate pesticides contained in various types of matrices, such as aquatic samples [4-6], and fruits and vegetables [7-11] To our knowledge, however, little effort has been made to analyze carbamates residues in biological samples with LC-APIMS, except in one study done by Kawasaki et al wherein they used LC-APCI-MS to 120 analyze eight carbamate pesticides (isoprocarb, metocarb, fenobucarb, xylylcarb, XMC, ethiofencarb, propoxur, and carbaryl) in blood from patients suffering from acute poisoning [12] In addition, liquid-liquid extraction (LLE) has been commonly used to extract carbamates (for example, carbofuran, furathiocarb, benfuracarb, carbaryl, and propoxur) from various kinds of biological samples [13-15] However, LLE has many disadvantages - namely, it requires large amounts of toxic organic solvents and is timeconsuming As described above, MAE has become a very attractive sample preparation technique as it overcomes the aforementioned disadvantages Yet, extraction from biological samples by MAE has been limited to the determination of methylmercury [1619], arsenic speciation [20-22], and fatty acids [23] Currently, there are very few MAE methods available for the analysis of pesticide residues in biological samples, except chlorinated pesticides, which can be detected in a freeze-dried mussel tissues using focused MAE [24] Based on the above considerations, in this study, LC-APCI-MS technique coupled to MAE, was selected to monitor trace residues of propoxur and its TPs found in biological materials in the aquatic environment Our work is firstly concentrated on the MAE optimization by using orthogonal array design (OAD) procedure Secondly, the application of optimized techniques for the extraction of target pesticides from spiked biological materials [goldfish, tortoises (Trachemys scripta elegans) and sea lettuce (Ulva lactuca)] is described Finally, the stability studies of propoxur under microwave irradiation and in tested biological extracts at their respective optimum conditions are discussed 121 5.2 EXPERIMENTAL 5.2.1 Reagents and Standards Propoxur was purchased from ChemService (See chapter 2) Both 2-isopropoxyphenol and N-methylformamide were obtained from Aldrich (Steinheim, Germany) Stock standard solutions were prepared in methanol at concentrations of 1000 µg/ml for each compound and stored at - 4°C Working solutions were prepared by diluting the stock solutions with methanol 5.2.2 Sample Preparations The preparation of these biological samples is described in detail in Chapter 5.2.3 Sample Extraction 5.2.3.1 Optimization strategy for MAE In our previous study, very good results (above 95% of recovery) had been obtained for propoxur extraction from soil with 30 ml of methanol, an 80 °C extraction temperature, and 6-min microwave heating The differences between the distribution of propoxur and its transformation products in biota and soils, and additionally the difference in the 122 sample preparation processes (soils were spiked at ambient conditions and air-dried, whereas biota tissues in the present study were freeze-dried), required that there be optimization of extraction step For this, a two-level orthogonal array design (OAD) was applied In this study, two different experiments were designed to optimize MAE conditions for the extraction of the target According to our mode designed earlier, the four important variables selected for optimizing MAE are: (1) extraction solvents (factor A), (2) extraction temperature (factor B), (3) extraction time (factor C), and (4) solvent volume (factor D) One factor not considered was the power setting for the microwave heating This setting was proportional to the number of samples undergoing extraction during one run Furthermore, according to previous experience with MAE and intuition, one twovariable interactions to be considered was B x C (interaction between different extraction temperature and extraction time) Because four two-level variables and one two-variable interaction were to be considered, a total of five degrees of freedom was required, and the OA (2 7) matrix was, therefore, chosen so as to have sufficient degrees of freedom for the assignment of the variables under consideration The assignment of the main variables (A, B, C and D), two-variable interaction, and the level setting values of the main variables applied to aquatic animals (goldfish and tortoises) and sea lettuce are displayed in Table 5-1 123 Table 5-1 Assignment table of variables and the arrangement of the experiment runs using an OA8 (27) matrix Column No A* B* # C* # B x C* D* I Methanol 60οC 20 ml II Acetone/Hexane (1:1) 80οC 30 ml I Methanol 60οC 20 ml II Dichloromethane 80οC 30 ml Extraction Level species settings Goldfish and Tortoises Sea lettuce #: Dummy factor *A: Extraction solvent; B: Extraction temperature; C: Extraction time; D: Volume of extraction solvent; B x C: Interaction between extraction temperature and time 5.2.3.2 MAE procedures and the treatment of extracts For every sample in each species extracted by MAE, g of a spiked sample was accurately weighed out and quantitatively transferred to the PIFE-lined extraction vessel After adding the needed volume of the extracting solvent (Table 5-1 and Table 5-2), 10min equilibration was allowed before extraction During optimization of the MAE conditions, the extraction conditions of each pre-designed experimental trial (a total of 8) were set according to the two-level OAD procedure given in Table 5-1 and Table 5-2 124 Table 5-2 The OA8 (27) matrix with the experimental results Expt No Column No Average recovery (%) Goldfish Tortoises Sea lettuce 1 1 2 2 1 2 1 2 1 2 2 1 2 2 2 2 1 2 1 2 1 2 1 78.6 81.0 84.8 85.4 74.3 71.5 77.9 81.1 78.4 r1 82.4 76.4 79.7 78.9 Goldfish 79.0 79.9 r2 77.0 82.3 79.0 79.8 79.7 78.8 79.6 79.0 78.5 80.1 83.6 81.3 80.5 81.1 89.2 86.7 80.9 72.6 83.7 84.9 80.3 Tortoises 80.0 80.9 78.6 77.7 79.6 78.7 84.1 86.3 76.1 69.4 78.6 81.3 Sea lettuce 81.8 83.3 80.9 83.1 81.6 84.0 * r1 r2 r1 r2 82.2 76.4 84.4 80.5 76.0 82.6 78.8 86.1 79.6 79.0 82.6 82.3 * Average response of each level After extraction, the vessels were cooled down to room temperature before they were opened, preventing any organic solvent fumes from being detected by the solvent detector within the instrument, which would cause a shut-down Sample extracts were further separated by centrifugation at 419 rod S-1 for 15 The supernatant was then evaporated in a rotary evaporator Finally, ml of methanol was added to dissolve the residue that was directly analyzed by LC-MS 125 5.2.4 LC-MS Measurements The LC-APCI-MS instrument was initially tuned with a tuning solution (a mixture of caffeine, MRFA, and ultromark 1621) in both positive and negative ionization modes In order to ensure optimal tuning conditions, the propoxur standard was delivered into the APCI source through an electronically controlled syringe pump Typical tuning parameters were: vaporizer temp: 450.00 °C, sheath gas flow rate: 80 arb, aux gas flow rate: 20 arb, discharge current: 5.00 µA, capillary temp: 150.00 °C, capillary voltage: 35.00 v, tube lens offset: 5.00 v, and corona voltage: 4.50 kv Selective monitoring (SIM) was performed at m/z 60 ([M+H]+), 151([M-H]-) and 210 ([M+H]+) The dwell time for each channel was 0.1 s, with an interchannel delay of 0.02 s and a mass span of mass unit Quantitation was based on the area under the peak from the mass chromatogram of the above molecular ions at m/z 60, 151, and 210 For the LC separation of propoxur and its two transformation products (N-methyl formamide and 2-isopropoxyphenol), a mixture of ultrapure water-methanol (50:50) was used as a mobile phase at a constant flow-rate of 0.6 ml/min A Phenomenex ODS 150 x 3.2 mm column was utilized for separation The HPLC system was interfaced with the ion trap through the APCI source Mass spectra collected in full-scan mode were obtained by scanning over a range of m/z 50 to 250 Maximum injection time was set at 150 ms Time scheduled mass conditions were shown as following scheme: 126 15 22 30 LC time (min) Full-scan mass range of m/z 50 to 250 SIM mode (m/z 60) SIM mode (m/z 151 and 210) in the negative and positive ion detection modes, respectively 5.3 RESULTS AND DISCUSSION 5.3.1 LC-MS Analysis The structures of propoxur and its two metabolites (2-isopropoxyphenol and N-methyl formamide) are given in Fig 5-1, together with their typical APCI spectra in full scan mode (m/z 50-250) acquired from direct injections of standard solution of each compound MS2 spectra were recorded by isolating the quasi-molecular ions, followed by a 30% collision-induced dissociation (CID) energy, and are also presented in Fig 5-1 From Fig 5-1(a), it can be seen in the mass spectrum that the quasi-molecular ion of propoxur at m/z 210 is the most intense peak The MS2 spectrum reveals that two fragment ions were detected One is [M+H-42] + at m/z 168, probably because of the loss of a 1-propene (C3H6) molecule, the other is the base peak at m/z 153 [M+H-57]+, which probably resulted from the neutral loss of a CH3NCO group MS studies of other carbamate pesticides have reported an identical product ion [M-CONCH3+H]+ [15] that is believed to be a key characteristic of N-methylcarbamate pesticides Fig 5-1 (b) shows that the most abundant ion in the MS spectrum of 2-isopropoxyphenol is [M-H]- at m/z 127 151 The MS2 analysis of that ion, at m/z 151 with 30% CID energy, shows fragment ions corresponding to cleavage of C-C and C-O bonds A fragment ion at m/z 121 [M-H-30]was detected as two CH3 groups were lost, whereas the m/z 109 [M-H-42]- probably resulted from the neutral loss of 1-propene, similar to the formation of a fragment ion of propoxur at m/z 168 For N-methylformamide, one peak at m/z 60 [M+H]+ was detected in the MS spectrum, whereas no MS2 spectrum was obtained, as m/z values of fragment ions are too small to be recorded (Fig 5-1 (c)) In all the cases, small peaks can be ignored when their relative intensities are less than 20% of the corresponding base peak In this study, SIM of quasi-molecular ions in each tested compound at m/z 210, m/z 151 and m/z 60, were selected for quantitative analysis Fig 5-2 shows the individual SIM chromatogram for all three compounds in a mixture of 0.10 ng/µl of a solvent-based standard +C APCI Full MS (m/z 50-250) (a) +C Full MS2 210.00 @ 30 [50.00-250] 128 -C APCI Full MS (m/z 50-250) (b) -C Full MS2 151.00 @ 30 [m/z 50.00-250] +C APCI Full MS (m/z 50-250) (c) Fig 5-1 APCI full MS (and MS2) spectra of (a) propoxur and (b) 2-isopropoxyphenol and (c) N-methylformamide 129 3.21 (a) 100 % Relative abundance (%) 18.92 100 (b) % 21.01 100 (c) % 12 16 20 24 Retention Time (min) Fig 5-2 SIM chromatogram for (a) N-methylformamide, (b) propoxur and (c) 2isopropoxyphenol in a standard mixture (0.10 ng/µl of each analyte in methanol) 130 5.3.2 Data Analysis Strategy for MAE Optimization After implementing the eight experimental trials, which were pre-designed according to the OA8 (27) matrix, the corresponding average recovery (AR) for each experimental trial was calculated and then tabulated (Table 5-2) The average of the responses (r1 and r2) for each factor at different levels was also calculated and is shown in Table 5-2 Table 5-2 reflects the values for goldfish, tortoises, and sea lettuce Based on methods described in previous work [22, 23], the results of the sums of squares for different variables and so-called interaction columns for each extraction species were calculated (see Table 5-3, 5-4 and 5-6) For each tested species, two-variable interactions were assigned to column Furthermore, columns and were treated as dummies and used for estimating the error variance in this optimization Subsequently, the ANOVA tables were constructed (Table 5-3 to 5-5) Table 5-3 Variance analysis table in the OA (27) matrix for the optimization of MAE from goldfish A B C D Extraction solvent Extraction temperature Extraction time Solvent volume SS 58.3 69.6 1.6 d.f 1 MS 58.3 F-Value Significancea Items BхC Error 7.2 2.4 2.0 1 69.6 1.6 7.2 2.4 1.0 58.3 69.6 1.6 7.2 2.4 P