Analytical and Bioanalytical Chemistry. Electronic Supplementary Material

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1 Analytical and Bioanalytical Chemistry Electronic Supplementary Material Measuring internal azole and pyrethroid pesticide concentrations in D. magna using QuEChERS and GC-ECD method development with a focus on matrix effects Andreas Kretschmann, Nina Cedergreen, Jan H. Christensen 1

2 Additional Information on Material and Methods If not otherwise stated concentrations listed in the following sections are with respect to the final extracts in the GC vials after addition of analyte and/or surrogate standard (320 µl). Chemicals used Hexaconzole (HEX, CAS# ), Propiconazole (PP, CAS# ), Epoxiconazole (EP, CAS# ), Prochloraz (PC, CAS# ), Bifenthrin (BIF, CAS# ), α-cypermethrin (CYM, CAS# ), Esfenvalerate (ES, CAS# ), and Deltamethrin (DEM, CAS# ) were obtained from Sigma-Aldrich (Riedel de Haen, Fluka), Denmark and were all of analytical grade (Pestanal) with a purity of 99.7%, 98.4%, 99.2%, 98.6%, 98.8%, 99.7%, 99.8%, and 99.7%, respectively. Organisms used Daphnia magna (originally collected from Langedammen in Birkerød, Denmark in 1978) were cultured in artificial M7 medium [1] at a temperature of 20 ± 2 C and a light:dark cycle of 16:8 h and fed with the green algea Pseudokirchneriella subcapitata ( cells/daphnid/d). For experiments, neonates with an age of 24 h were separated from the mothers and kept under normal culturing conditions. After 5 days, daphnids were frozen in groups of 20 in liquid nitrogen and stored at -80 C until homogenization. GC-ECD method An inlet temperature of 270 C was used. For experiments on matrix effects during subsequent matrix injections the following oven temperature program was used: 65 C for 1 min, then at 25 C/min to 210 C, then at 10 C/min to 300 C and held for 5 min, and for the remaining experiments: 150 C for 1 min, then increased to 210, 240, 265, and 300 C at 40 C/min and held for 11.5, 5.75, 3, and 5 min, respectively. H 2 and N 2 were used as carrier and make-up gas at a flow rate of 2 and 60 ml/min, respectively. The detector temperature was kept at 300 C. Extraction of pesticides and sample clean-up The detailed work-flow from homogenization till GC measurement is shown in Fig organisms (total approximate wet weight 24 mg) were homogenized in 750 µl acetonitrile in 2 ml screw cap plastic tubes (Greiner Bio-One GmbH, Frickenhausen, Germany, Art-nr ) filled with mg Zirconia/ Silica (Zr/Si) beads (Ø 0.5 mm, BioSpec Products, Bartlesville, OK, USA) using a FastPrep FP120 Bio101 (Savant Instruments, Inc., NY, USA). Before and after 2

3 homogenization samples were kept on ice. After homogenization the liquid phase was transferred with a glass pasteur pipette into a 4 ml amber glass vial with a polytetrafluoroethylen (PTFE) inlet screw cap (Microlab Aarhus, Denmark, ML33136 and ML33144) and the plastic tubes washed with 500 µl acetonitrile. For liquid-liquid partitioning, 1.25 ml of a mixture of MgSO 4 and NaCl dissolved in H 2 O from a Milli-Q system (286 and 71.4 mg/ml, respectively) were spiked to the collected organic phase. Here, an aqueous solution of the salts instead of the pure salts was added to the extract, since the water content of the daphnid sample was too low to enable liquid-liquid partitioning. After centrifugation (4000 rpm for 5 min), 1 ml of the upper organic phase was transferred into 2 ml dispersive solid-phase extraction (dspe) tubes containing either dspe sorbent A: 150 mg MgSO 4, 50 mg primary-secundary amines, and 50 mg octadecylsilan endcapped (C18) or dspe sorbent B: 150 mg MgSO 4, 50 mg primary-secundary amines, 50 mg C18, and 50 mg graphitized carbon black (roq d-spe kits, Phenomenex, KS and KS0-8917). MgSO 4 removes excess water, primary-secundary amines organic acids, fatty acids, sugars, and anthocyanine pigments, C18 fats, sterols, and other non-polar interferences, and graphitized carbon black pigments from the extract [2-4]. After centrifugation the supernatant was transferred into GC vials with a glas insert. During the liquid-liquid partitioning step, we used a lower MgSO 4 and NaCl concentration than used in the original QuEChERS method (286 and 71,4 instead of 400 and 100 mg/ml H 2 O). When the recommended amounts were applied, the salts did not completely dissolve and precipitated during liquid-liquid partitioning. To obtain a salt solution, which can be spiked directly to the acetonitrile extract after homogenization, instead of adding the salts individually to each sample, the salt solution was diluted just till complete dissolution occurred. Spiking the salts dissolved in H 2 O provided a more convenient and faster way to prepare our samples. This salt solution is presumably saturated with MgSO 4, but contains less NaCl than in the original QuEChERS method. The MgSO 4 and NaCl amount in the water phase controls the amount of water in the acetonitrile phase during liquid-liquid partitioning and influences its polarity and therefore the removal efficiency of polar matrix components from the acetonitrile phase (i.e., higher removal of sugars from the acetonitrile phase with higher NaCl amounts) [2]. Although deviating from the original QuEChERS method, we achieved good results concerning analyte response stability and recovery (see Results and Discussion of the main manuscript). Nevertheless, a change in the salt content during liquid-liquid partitioning should be considered with care for the respective sample type used. 3

4 Short and long term matrix effects and analyte recovery Extracts from D. magna prepared with dspe sorbent A or B were measured in separate experiments. Extracts spiked with analytes were sub-divided in two. Samples were distributed in 3 blocks, each block consisting of 3 replicate samples each alternately injected 3 times. The total number of matrix sample injections was 28 for each sorbent type. In the Pearson correlation analysis the analyte response of CYM and ES of matrix injection #7 (see main manuscript Figure 1) was considered as outlier and not included. Test recoveries for dspe sorbent A and B To determine the loss of analytes during extract preparation with dspe sorbent mixture A or B, the analytes PP, EP, PC, BIF, CYM, and ES were spiked to the daphnid sample prior to homogenization (recovery samples). The analytes spiked to the final extract in the GC vials after sample preparation served as reference. HEX and DEM served as surrogate standards for the azoles and pyrethroids, respectively, and were spiked to the final extracts. Nominal concentrations of azoles and pyrethroids were 94 and 9.4 ng ml -1, respectively. Each sample was prepared in quadruplicate. For dspe sorbent mixture A, the liquid-liquid partitioning step was repeated in order to test potential losses due to partitioning of pesticides in the water phase. In addition to the concentration level 94 and 9.4 ng ml -1 for azoles and pyrethroids, respectively, a lower concentration level was tested with dspe sorbent A by spiking the analytes prior to homogenization and liquid-liquid partitioning resulting in a nominal concentration of 22.5 and 2.3 ng ml -1 in the final extracts for azoles and pyrethroids, respectively (number of matrix blanks n = 3). Recovery samples were measured in four batches each consisting of a recovery, a reference sample and a matrix blank (no analytes spiked) in order to minimize the influence of long-term matrix effects. Recoveries of the repeated liquid-liquid partitioning step were not corrected via the matrix blank response. The single steps of the recovery experiment are depicted in Table SI-2. Data evaluation and statistics In case of stereoisomeric peaks (PP, CYM, ES, DEM), the sum of the peak areas was used, if the minor (diastereomeric) peak (retention times see Table 1) was high enough in intensity and/or not overlayed by a contaminating peak. Assumptions made during the correlation analysis with the Pearson calculation were: The variation in the measured peak area is normal distributed and that measured analyte peak areas are independent from each other. Calibration curves for quantification were obtained via linear least-square regression through the calibration data. 4

5 Detection limit (DL) and limit of quantification (LOQ) were determined via the mean analyte response (response in the matrix blanks at the retention time of the respective analyte) and the standard deviation of the analyte responses in the samples spiked with analytes:, (SI-I) The DL was determined according to the IUPAC definition as the minimum single result which, with a stated probability, can be distinguished from a suitable blank value [5]. With a sample size of 8 and a chance of 99% that an observed response at the DL is due to the analyte and not to a blank value, the t value accounts to For the calculation of the LOQ a t value of 10 was applied. Precision (repeatability) was determined as the relative standard deviation (% RSD) of the analyte response in the spiked samples: % % 100 (SI-II) Accuracy was calculated as the mean value Rec of the recoveries Rec i of N individual spiked samples (quantified concentration relative to the nominal concentration in ng ml -1 ). % , / 100 (SI-III), Additional information to Results and Discussion Short and long term matrix effects Short term matrix effects: When daphnid matrix samples prepared with sorbent A were injected in the GC system the peak area of CYM was increased by 13% compared to CYM spiked to pure ACN (see Fig. 2). This might have partly been caused by a contamination present in matrix samples co-eluting with CYM. This is discussed in detail in the section Method validation below. When daphnid extracts were cleaned up with sorbent B temporal effects were comparable to extracts prepared with sorbent A except for minor differences (see Fig.SI-1). In contrast to sorbent A, PP and CYM spiked to matrix extracts did not show substantial changes in peak area in case of sorbent B at the beginning of the sequence (below 5 % with respect to first quality control injection). On the other hand, EP decreased by approx. 9 %. Furthermore, HEX spiked to daphnid extracts exhibited a slight gradual increase. The response decrease of EP and PC spiked to matrix prepared with sorbent A was eliminated in case of sorbent B (see Fig. SI-1). 5

6 Long-term matrix effects: When samples without matrix (quality control in pure acetonitrile) were injected between the daphnid extracts CYM, ES, and DEM exhibited a strong matrix-induced response diminishment. CYM, ES, and DEM are type-ii pyrethroids. A feature of type-ii pyrethroids is the α-cyano group, which is responsible for the higher insecticidal activity compared to type-i pyrethroids lacking this group [6]. As key role in the insecticidal activity of pyrethroids the hydrogen bond interaction of the ester carbonyl group with a threonine hydroxyl group in the sodium channels of insect nerve cells was suggested. In type II pyrethroids this interaction is extended by an additional hydrogen bond interaction of the weakly acidic hydrogen atom in α- position [7]. Furthermore, the nitrile group itself might act as hydrogen bond acceptor [8], although rather weakly [9]. The type II pyrethroids CYM, ES, and DEM might therefore indeed form stronger hydrogen bonds with free silanol groups in the GC inlet compared to BIF, which behaved the opposite way (increase in analyte response). The extent of signal reduction increased hereby with increasing retention time (CYM < ES < DEM). This trend might be due to an increased residence time in the GC inlet and therefore to a longer contact time with active sites the later the compounds elutes. Acid-base dissociation constants, which serve as a rough estimation of the hydrogen bond basicity [9], where estimated for the azoles using MarvinSketch (ChemAxon, Dissociation constants were highest for PC. Furthermore, imidazoles (like PC) have been suggested to coordinate in general stronger to the heme iron atom of cytochrome P450 through their imine nitrogen atome compared to triazoles [10]. This supports the finding of a high susceptibility of particularly the imidazole PC to active sites in the GC system compared to the triazole fungicides HEX, PP, and EP. 6

7 7

8 Fig. S1 Matrix effects shown as relative change of a) azole and b) pyrethroid peak area with number of injections of extracts from D. magna prepared with QuEChERS using dspe sorbent B (C18/ primary-secondary amines/ graphitized carbon black). Symbols represent analytes spiked to pure acetonitrile (open) and to daphnid extracts (filled). Changes are expressed with respect to the first injection. Arrows indicate the insertion of a new liner into the GC inlet Matrix effects are in general dependent on the chemical composition of the sample and the amount of sample components extracted during sample preparation [2]. D. magna possess a dry weight, a total lipid, and a total protein content of approx. 10, 1.7, and 3.4 % of the wet weight, respectively [11,12]. In our study, 20 daphnids (approx. 24 mg wet weight) have been used per sample. The dry weight of biological matter was therefore approx. 2.4 mg, the protein and lipid amount approx. 0.4 and 0.8 mg per sample. The QuEChERS method applying C18 and primarysecondary amines as dspe sorbents was shown to remove the majority of co-extractives from nonfatty and fatty food samples. An additional removal of co-extractives with planar ring structures like sterols was achieved with graphitized carbon black [3,4,2]. Differences in daphnid matrix effects (temporary and long-term effects) for dspe sorbent A and B can therefore be explained by an 8

9 additional removal of daphnid matrix components by graphitized carbon black. GC-ECD chromatograms of extracts from D. magna prepared with sorbent A and B exhibited in general a low number and low intensity of contaminant peaks. Selection of surrogate standards Results of the Pearson Correlation test between analyte reponses for d-spe sorbent A and B are presented in a correlation matrix in Table SI-1a and SI-1b, respectively. By normalizing the peak area to a correlating analyte it was possible to (partly) correct for the gradual matrix induced response enhancement or diminishment and to reduce the variation in the data. This is demonstrated in Fig. SI-2 for DEM and daphnid extracts prepared with dspe sorbent A, where the relative deviation from the average analyte peak area is shown. Normalization of the peak area of DEM to CYM improved the stability of the data and reduced % RSD from 2.7 to 1.6. In contrast, normalization of DEM to BIF did not lead to an improvement, although both are pyrethroids. No clearly correlating analytes could be identified for HEX, PP, and BIF. As surrogate standard for the later method validation the closest eluting azole PP was chosen for HEX instead. PP and BIF were used as surrogate standard for each other, since they exhibited in general similar behavior and correlated well in pure acetonitrile, when injected between daphnid extracts prepared with dspe sorbent A, as well as in daphnid matrix in samples prepared with sorbent B. 9

10 Table S1a and b Analysis of correlation (Pearson correlation) between analytes in the relative change in peak area with subsequent injections of extracts from D. magna. Shown are correlation coefficients and P values. White cells represent analytes spiked to daphnid extracts (matrix samples) and grey cells analytes spiked to pure acetonitrile (quality control samples). Extracts were prepared with a) dspe sorbent A (C18/ primary-secondary amines) and b) dspe sorbent B (C18/ primarysecondary amines/ graphitized carbon black). Number of sample injections: n (matrix) = 28, n (acetonitrile) = 5. The analyte response of CYM and ES of matrix injection #7 (see main manuscript Figure 1) was considered as outlier and not included in the correlation analysis. a) HEX PP EP PC BIF CYM ES DEM HEX , Corr. Coefficient 6.26E E E E E E E-05 P Value PP , E E E E E E E-02 EP , E E E E E E E-03 PC , E E E E E E E-05 BIF , E E E E E E E-01 CYM E E E E E E E-06 ES E E E E E E E-03 DEM E E E E E E E-02 10

11 b) HEX PP EP PC BIF CYM ES DEM HEX PP EP PC BIF CYM ES DEM E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E E-03 Corr. Coefficient P Value Fig. S2 Change of analyte response relative to average response of DEM, with the number of injections. Analytes were spiked to daphnid extracts prepared using dspe sorbent A (C18, primarysecondary amines). The figures on the left show the relative change in peak area, the ones in the middle and on the right the relative change in peak area normalized (response ratio RR) to CYM and BIF, respectively 11

12 Stereoisomers All pesticides used in our study, apart from PC, are chiral compounds. HEX, PP, and EP, possess 1, 2, and 2 chiral centers, BIF, CYM, ES, and DEM 2,3,2, and 3, respectively (see structures in Table 1 in the manuscript). HEX, PP, and BIF are mixtures of all possible stereoisomers (2, 4, and 4, respectively). EP and CYM are racemic mixture of two enantiomers, ES and DEM are enantiomeric pure products. Although consisting of a mixture of stereoisomers, only one peak could be identified in the GC chromatogram for HEX, EP, and BIF. For PP two peaks with an average peak area ratio of 40:60 were present, presumably corresponding to its diastereoisomers (relative t R of the earlier eluting peak = 0.992). The peak area ratio remained stable in samples with and without daphnid matrix during subsequent injections in the experiment on matrix effects (see Fig. SI-3). For CYM, ES, and DEM, an additional peak with lower intensity and at earlier retention times than the main peak was observed (relative t R = 0.989, 0.988, and 0.988, respectively). Although an initial increase during subsequent injections, the peak area ratio of the two stereoisomeric peaks remained rather stable as long as matrix was present in the samples (maximum %RSD of the main peak area = 4 %) with an average value of 8:92, 12:88, and 7:93, respectively. In case of CYM, ES, and DEM spiked to pure acetonitrile and injected between matrix samples in the experiment on matrix effects, a trend towards gradually increasing peak area of the earlier eluting stereoisomeric peak was observed. DEM was most affected with a change in peak area ratio from 5:95 to 14:86. Stereoisomerization of pyrethroids during GC injection was also observed by Mastovska et al. [13]. Here, DEM gradually converted into its diastereomers with subsequent injections of fruit and vegetable extracts into a GC system. The authors suggested an isomerization process at the chiral C atom in α-position during GC injection. The degree of conversion was hereby dependent on the matrix present in the sample. When samples were cleaned up with dspe using primary-secondary amines as sorbent, isomerization up to a 50:50 ratio of stereoisomeric and main peak in case of DEM was observed [13]. In our study, stereoisomerization played a rather minor role, particularly when pyrethroids were injected in daphnid matrix. 12

13 Fig. S3 Peak area of stereoisomers of PP, CYM, ES, and DEM as function of number of sample injections during the experiment on short and long term matrix effects. Squares represent the main diastereomeric peak, triangles the one with lower intensity (eluting earlier than the main peak). Filled symbols represent analytes spiked to daphnid matrix prepared with dspe sorbent A (C18/ primary-secondary amines) and empty symbols analytes spiked to pure acetonitrile Analyte recoveries Results from the pre-validation experiment on analyte recovery during the sample preparation procedure can be found in Table SI-2. Recovery values fulfill the method validation criteria mentioned in the SANCO guideline for pesticide residue analysis in food and feed in the European Union (required recovery: %) [14]. Low recoveries when using graphitized carbon black during dspe (sorbent B) were observed in our study (see Table SI-2). How strong compounds are retained by GCB depends in general on their structure. A study with cyclic hydrocarbons demonstrated that the more planar the structure is the stronger it is retained on GCB. Interactions with GCB decreased from polyaromatic compounds (planar) to their perhydro-analogues (bended) 13

14 [15]. All of our analyzed pesticides possess at least one (planar) aromatic ring moiety, which might explain their low recoveries by strong sorption to GCB. The liquid-liquid partitioning step was shown to be quantitative: When the water phase was extracted a second time with 1.25 ml of acetonitrile, analytes were not detected or only in very low amounts, apart from PP (23 % extracted). Since recoveries for PP during the whole procedure were around 100 % and since PP was also present in the matrix blanks in that experiment, this was probably due to a contamination problem. Furthermore, CYM contamination was present in the matrix blanks of sorbent A at high concentrations levels. Since peak areas were corrected for the matrix blank values and results are consistent for the two concentration levels tested, we believe that the contamination didn t affect the results. 14

15 Table S2 Recovery of analytes during sample preparation (sample homogenization, extraction, and clean-up with QuEChERS according to Fig.1). Recoveries were tested at two concentration levels (low: 23 and 2.3 ng/ml and high: 94 and 9.4 ng/ml for azoles and pyrethroids, respectively) and the two d-spe sorbent mixtures A (C18/ primary-secondary amines, PSA) and B (C18/PSA/graphitized carbon black, GCB). Analytes were spiked before homogenization (recovery of whole method) and before liquid-liquid partitioning (LLP) (recovery of the QuEChERS procedure only). LLP was repeated in order to test completeness of the first partitioning step. Values are given as mean recoveries ± standard deviation (n = 4) Step Conc level d-spe sorbent mixture Recovery analytes (%) Analyte PP EP PC BIF CYM ES Spiking analytes before homogenization Low A (C18/PSA) ± ± ± ± ± ± 1.4 High A (C18/PSA) ± ± ± ± ± ± 1.6 B (C18/PSA/ GCB) 81.3 ± ± ± ± ± ± 2.1 Spiking analytes before LLP Low A (C18/PSA) ± ± ± ± ± ± 4.3 Repetition of LLP High A (C18/PSA) 23.1 ± ± ± ± (n = 1) n.d. (n = 3) n.d. Methods validation Only the main peaks of PP, CYM, ES, and DEM (retention time written in italics in Table 1) and not the diastereomeric peaks were used for evaluation, since the earlier eluting diastereomeric peak of PP was overlayed by a contaminant peak and for low analyte concentrations, the diastereomeric peaks of the pyrethroids were too low in intensity to be quantifiable. In matrix blanks, a peak eluting at the same time as the main peak of CYM was present, which accounted to approx. 60 % of the peak area in samples spiked with CYM (0.9 ng/ml). When matrix blanks were remeasured with a column of higher polarity (50 % phenyl-50 % dimethylpolysiloxane) this peak still coeluted with CYM. Samples might therefore have been contaminated with CYM through the glass syringes used 15

16 for spiking, although careful precautions like several washing steps in different solvents were undertaken. Since the highly lipophilic pyrethroids tend to sorb to experimental equipment [16] a strict separation of the equipment used for high and low concentrations is recommended in order to avoid contamination of samples with analytes, especially when working with very low pyrethroid concentrations as done in our study. Another reason for the contamination overlaying with CYM might be a contamination present in the EP standard. Excluding EP from the analyte mixture eliminated the background contamination. In addition to testing for linearity, the quality of the calibration curves was further assessed through the maximal deviation of individual data points (maximum residual) as well as the average deviation of all calibration standards (mean residual) from the regression line. Maximum residuals were partly very high (41 % for DEM). The application of different weighing schemes for linear regression (1/x or 1/x 2 ) substantially improved the fit (see Table 1). As a measure for the long-term matrix effect on analyte reponse, the relative change in the slope of the calibration curve at the end compared to the beginning of the sequence was calculated. Between calibration standards 19 matrix samples (matrix blanks and spiked matrix samples) were measured. Determined changes in the slopes (see Table 1) are in general in accordance with the gradual increase or decrease observed in the experiments on short and long term matrix effects. Particularly DEM exhibited a rather strong decrease in slope by 8.6 %. The changes in slopes caused by the daphnid matrix were minimized by surrogate standard normalization in case of EP, PC, ES (with DEM as surrogate standard), and DEM. In case of CYM the change in slopes increased from 0.7 to 9.4 % when normalized to ES. This can be explained by a slightly stronger impact of long-term matrix effects on ES peak area leading to a stronger decrease in response than compared to CYM. But normalization of CYM peak area to ES as surrogate standard proved in general to be beneficial as can be seen by reduced mean/maximal residuals of the calibration curves and an improved accuracy and precision (see Table 1 in the main manuscript). When measuring daphnid extracts after in vivo exposure to CYM, where more than 40 matrix samples were measured between calibration curves this effect was not present and ES proved to be a suitable surrogate standard. In general, if long-term matrix effects introduce a systematic error even when normalized to a surrogate we recommend to reduce the number of samples measured within one measurement sequence and/ or to randomly distribute calibration curve samples within one sequence instead of measuring separate curves at the beginning and end of a sequence. 16

17 Internal concentrations during in vivo exposure of D. magna The inter-experimental variation in internal CYM concentrations of the two duplicate experiments calculated as the difference between samples at each sampling time point was 23 ± 17 % for the exposure to CYM alone (mean ± stdev). For the exposure to the mixture (CYM + PP) the variation amounts to 32 ± 12 % for internal CYM and to 17 ± 9 % for internal PP concentrations. Considering that sampling times differed slightly between experiments and that experiments were performed six months apart from each other these results are satisfactory. Daphnid samples taken during the pulse or elimination phase have been measured in separate batches for exposure and elimination phase samples either within the same or two separate measurement sequences (including the renewal of the GC liner before each sequence). Samples taken at the end of the α- cypermethrin pulse were remeasured in the elimination phase sample batch. Although normalization to the surrogate standard (ES) stabilized the analyte response, remeasured internal CYM concentrations differed from the first measurement by 1 19 % (see Fig. 4). These variations might be due to the measurement of exposure and elimination samples in separate sequences. It is therefore recommended not to separate uptake and elimination samples into two separate batches, but to randomize all samples within one sample batch in order to reduce this variation. Peak areas of CYM were corrected for matrix blanks. This has introduced additional variation in the remeasured sample, where 19% difference was observed. In that case, only one matrix blank was available for correction of the recovery samples, which might not have been representative for the average matrix blank value. In the second exposure experiment three matrix blanks were prepared for each sample type (samples taken during exposure or elimination phase) and the mean blank value used for blank correction, which didn t introduce additional variation in the remeasured samples. 17

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