Residues of anilinopyrimidine fungicides and suspected metabolites in wine samples

1 Residues of anilinopyrimidine fungicides and suspected metabolites in wine samples G. Castro, L. Pérez-Mayán, I. Carpinteiro, M. Ramil, R. Cela, I. Rodríguez* Department of Analytical Chemistry, Nutrition and Food Sciences. Institute of Research on Chemical and Biological Analysis (IAQBUS). Universidade de Santiago de Compostela, 15782-Santiago de Compostela, Spain. Artículo aceptado para publicación en Journal of Chromatography A, https://doi.org/10.1016/j.chroma.2020.461104 © 2020 Elsevier B.V. This manuscript version is made available under the CC-BY-NC- ND 4.0 license (http://creativecommons.org/licenses/by-nc-nd/4.0/) 2 Residues of anilinopyrimidine fungicides and suspected metabolites in wine samples G. Castro, L. Pérez-Mayán, I. Carpinteiro, M. Ramil, R. Cela, I. Rodríguez* Department of Analytical Chemistry, Nutrition and Food Sciences. Institute of Research on Chemical and Biological Analysis (IAQBUS). Universidade de Santiago de Compostela, 15782-Santiago de Compostela, Spain. Abstract The coexistence of the anilinopyrimidine fungicides pyrimethanil (PYR) and cyprodinil (CYP), and suspected metabolites in wine samples was investigated by liquid chromatography (LC) with tandem mass spectrometry (MS/MS), based on triple quadrupole (QqQ) and quadrupole time-of-flight (QTOF) MS instruments. For the first time, quantitative data obtained after solid-phase extraction (SPE) of wine samples have demonstrated the systematic presence of 4-hydroxyanilino derivatives of PYR and CYP in wines containing residues of parent fungicides, at concentrations from 0.2 to 58 ng mL-1. Higher concentration ratios (hydroxylated derivative/active fungicide) were measured in red than in white wines, particularly in case of PYR. On average, the concentrations of PYR-4OH were twice those measured for PYR in red wines. A targeted search of hydroxyl derivatives in wine extracts by LC-QTOF-MS showed the existence of additional hydroxylation positions in the pyrimidine ring and/or in the alkyl substituents bond to this cycle in the structure of both anti-botrytis fungicides. Moreover, free and glycosylated forms of the hydroxylated metabolites for both fungicides coexist in wine samples. In case of CYP, it is proved that hydroxylated and glycosylated metabolites are already present in grapes before vinification. Keywords: wine; pyrimethanil; cyprodinil; metabolites; liquid chromatography-mass spectrometry *corresponding author e-mail: [email protected] 3 1. Introduction Fungicides are used under conventional agriculture practices to guarantee the productivity and the organoleptic quality of vinification grapes, particularly in those regions with atmospheric conditions promoting the development of fungi. However, the misuse of these compounds and the presence of fungicide residues in elaborated wines are matters of concern for environmentalists, authorities and wine consumers. Thus, the maximum residue limits (MRLs) of approved fungicides in vinification grapes have been regulated [1], studies on transfer factors from grapes to wine are available for most of the marketed fungicides [2–5] and new vine varieties resistant to relevant fungal infections, such as mildew and oidium, are under evaluation in different field trials [6,7]. The presence of fungicide residues in commercial wines has been investigated by several authors [8–10]; however, little is known about the presence of potential transformation products (TPs), either generated by the vines metabolism, or through reactions catalysed by enzymes existing in fermentation yeasts, in wine. As example, hydroxylation of the fungicide fenhexamide, as well as the conjugation of the parent compound and the hydroxylated derivative with saccarides, has been reported by Polgar and co-workers [11]. However, the extent of compound hydroxylation was less important in vinification grapes in comparison with other fruits and vegetables¡Error! Marcador no definido. [11]. In case of the insecticide imidacloprid (IMI), authorized for open applications until 2018, an oxidized derivative (imidacloprid olefin) was found in wine samples at higher concentrations than the active ingredient [12]. Anilinopyrimidines are a group of fungicides employed to control and/or to prevent diseases caused by Botrytis fungi in several crops, including vines. Pyrimethanil (PYR) and cyprodinil (CYP) are the most often used congeners of the family. In EU, their current MRLs for vinification grapes are 6000 and 3000 ng g-1 for PYR and CYP, respectively. PYR is transferred from grapes to wine with a yield above 90 % [13]. In case of CYP, 4 literature transfer factors vary in the range from 1 to 20 % [13,14]. Available information regarding the metabolization of PYR by animals, soil microorganisms and plant cultures suggests hydroxylation in both aromatic rings (benzene and pyrimidine cycles) and in the alkyl substituents bonded to the pyrimidine ring, as relevant transformation paths [15]. In fact, aryl hydroxylation is recognized as a significant route in the metabolism phase I of many aromatic pesticides ( particularly herbicides) in the vegetable kingdom [16]. Hydroxylation and glycosylation processes have been also reported during in-vitro incubation of CYP with wheat cells and microbial cultures [17,18]. EU regulations dictate that residues of PYR in farm animals must be calculated as the sum of the parent fungicide and the 4-hydroxyanilino (PYR-4OH) derivative. For CYP, the sum of compounds (CYP and 4-hydroxyanilino metabolite, CYP-4OH) must be considered when analysing milk and honey [1]. However, for vegetable food commodities, MRLs are just defined for the free form of both fungicides and, as far as we could trace, the existence of hydroxylated forms of anilinopyrimidine fungicides in wine has not been previously reported. Thus, so far, residues of PYR and CYP in this food matrix have been estimated from concentrations measured for the commercial parent fungicides and no analytical methodology has been proposed for the simultaneous determination of their possible hydroxylated metabolites. Even more, the occurrence of these latter compounds has not been previously reported in vegetable origin food commodities. The aim of this research is to investigate and quantify the potential co-existence of PYR, CYP, PYR-4OH and CYP-4OH in commercial wines. To this end, an analytical approach previously developed for the parent compounds was extended and validated to the above transformation products, using ultra-performance liquid chromatography (UPLC) with tandem mass spectrometry (MS/MS) as determination technique. In addition, a hybrid quadrupole time-of-flight (QTOF) MS instrument was used for retrospective search of additional transformation products of both fungicides, including phase II metabolites generated through glycosylation reactions. Differences between 5 concentration ratios of both fungicides and their phase-I metabolites in red and white wine are discussed. 2. Experimental 2.1. Solvents, standards and sorbents Methanol (MeOH) and acetonitrile (ACN), both HPLC grade, as well as, formic acid (FA), ethanol and tartaric acid were obtained from Merck (Darmstadt, Germany). Ultrapure water was obtained from a Milli-Q Gradient A-10 system (Millipore, Bedford, MA, USA). OASIS HLB solid-phase extraction (SPE) cartridges, containing 200 mg of sorbent, were purchased from Waters (Mildford, MA, USA) and employed for extraction and concentration of wine samples. Standards of PYR, CYP and their deuterated analogues (PYR-d5 and CYP-d5, deuterium atoms are attached to the phenyl ring) were acquired from Sigma-Aldrich (St. Louis, MO, USA). PYR-4OH was supplied by Toronto Research Chemicals (North York, Canada) and CYP-4OH by HPC Standards GmbH (Borsdorf, Germany). Individual standard solutions of each compound were prepared in MeOH and stored at -20 ºC. Mixtures of PYR, CYP, PYR-4OH and CYP-4OH in MeOH were employed to prepare spiked wine samples used during method optimization and validation. Calibration standards were prepared in ACN:MeOH (80:20). The mixture of deuterated fungicides, employed as internal surrogates (ISs) through the analytical procedure, was also made in MeOH. Diluted calibration standards were stored at 4 ºC and used for a maximum of 2 weeks after preparation. 2.2. Samples and sample preparation Samples employed in the current study correspond to commercial wines. Most of them were acquired from retail markets and the rest kindly provided by local associations of wine producers. Wine bottles were stored in the dark, at a temperature of 16 ºC, for a maximum of one month before opening. The extraction and concentration of fungicides 6 was performed immediately after opening each bottle of wine. SPE extracts were stored at -20 ºC. Sample collection and analysis, was performed during a period of two years. Only those wines containing concentrations of PYR and CYP above the LOQs of the method were considered in this research. SPE conditions were adapted from a previous multianalyte study dealing with the determination of pesticide residues in wine[9]. That methodology was re-validated for the two commercially available hydroxylated derivatives of PYR and CYP. Under final working conditions, 10 mL of wine were diluted with ultrapure water and concentrated using a SPE cartridge previously conditioned with 5 mL of MeOH and the same volume of an ethanol:water (12:88) solution. After sample concentration, the cartridges were rinsed with 5 mL of ultrapure water and dried under a gentle stream of nitrogen.Compounds were finally recovered with 2 mL of the ACN:MeOH (80:20) mixture. The extract was homogenized, filtered (0.22 µm) and injected in the LC-MS system without any additional treatment. Procedural blanks were carried out using aliquots of synthetic wine (ethanol:water, 12:88, containing 4 g L-1 of tartaric acid) spiked only with the mixture of ISs. SPE recoveries, independent of matrix effects, were calculated as the ratio between responses obtained for spiked aliquots of wine samples and SPE extracts, obtained from same wines, fortified at the end of the extraction step. Matrix effects during electrospray ionization (ESI) were estimated as the ratio between the slopes obtained from calibration curves prepared using spiked SPE extracts (from red and white wines), and those corresponding to solvent-based standards, multiplied by 100. The range of concentrations varied from 2 to 400 ng mL-1, with 9 different concentration levels. Global recoveries of the method were calculated as the normalized ratio between concentrations measured for spiked and non-spiked aliquots of different wines and added values. Concentrations in sample extracts were determined using solvent-based calibration standards, after correcting the responses measured for each compound with 7 those of ISs (PYR-d5 for PYR and PYR-4OH, and CYP-d5 for CYP and CYP-4OH, respectively). When assessing the recoveries at low addition levels, ecological production wines were selected in order to minimise the content of native compounds in non-spiked samples. Grapes (Mencía variety) were provided by a local farmer who declared a single treatment with a commercial mixture of CYP and Fludioxonil (Switch, Syngenta), at the maximum recommended dose, 33 days before harvest. Grapes (around 5 kg of bunches from 4 different vines) were de-stemmed, homogenized and extracted using the same mixture of solvents employed during elution of SPE cartridges. Around 2 g of the homogenized slurry was spiked with the ISs and shaken with 10 mL of solvent for 10 min. An aliquot of the upper phase was filtered (0.22 µm pore size filters) and injected in the LC-MS systems employed in the study. Grape extracts were considered only for semiquantitative purposes; thus, the extraction yield of the above approach was not evaluated. 2.3. Determination conditions Two different LC-MS systems were employed in this study. Target determination of anilinopyrimidine fungicides and their 4-hydroxyanilino derivatives was performed using a LC-MS/MS XEVO TQD, triple quadrupole mass spectrometer, from Waters (Milford, MA, USA). Search of additional metabolites of PYR and CYP in wine extracts was carried out using a hybrid QTOF-MS (6550 model) acquired from Agilent (Wilmington, DE, USA). Both MS instruments were furnished with an ESI ionization source working in positive mode and coupled to UPLC systems from same supplier as the MS instrument (Acquity from Waters and Agilent 1290 models). LC separations were carried out under identical conditions in both instruments. To this end, an UPLC Zorbax Eclipse Plus C18, Rapid Resolution column (50 mm x 2.1 mm, 1.8 µm particle size), connected to a C18 2.1 mm i.d. guard cartridge from Phenomenex (Torrance, CA, USA) was employed. The column was maintained at 40 ºC, using ACN (B) and ultrapure water (A), both 0.1 % in FA, as 8 mobile phases at 0.4 mL min-1. The injected volume was 1 µL and the composition of the mobile phase was programmed as follows: 2 % B (0 min), 30 % B (5 min), 50 % B (7-8 min), 100 % B (9-10.5 min), 2 % B (11-14 min). Using the LC-QqQ-MS instrument, two transitions were selected per compound and ISs. These transitions were considered for quantification (Q1) and qualification (Q2) purposes attending to their relative intensities. Identification of target species in wine extracts is based on retention time and Q2/Q1 ratio match with solvent-based standards. Maximum differences were set at 0.1 min and ± 30 % of the average Q2/Q1 ratio in calibration standards. The LC-QTOF-MS system operated in the 2 GHz acquisition mode, offering a mass resolution of 17000 (FWHM) for ions with m/z ratios in the range from 200 to 300. The m/z axis was continuously recalibrated using reference ions at m/z of 121.05087 and 922.00979. MS spectra were acquired in the range of m/z values from 50 to 1700, at a frequency of 1 Hz. Product ion scan spectra were recorded in the range from 40 to 700, at a frequency of 4 Hz, considering different collision energies. This system was employed to search additional hydroxylated derivatives of CYP and PYR, as well as their glycosylated forms, produced during phase II of metabolism. Sample (wines or grapes) extracts were first injected in the MS mode to identify the retention time of the potential derivatives of parent fungicides. Selective chromatograms for their pseudo-molecular ([M+H]+) ions were extracted with a mass window of 10 ppm. Thereafter, the product ion scan spectra of candidate peaks were acquired in a 2nd injection. Fragmentation patterns observed in these spectra were compared to those recorded for known compounds in order to propose the chemical structure of the additional metabolites. 9 3. Results and discussion 3.1. Optimization of determination conditions Table 1 summarizes retention times, ionization and quantification parameters corresponding to parent fungicides, their isotopically labelled analogues and the commercially available hydroxylated derivatives. Compounds were separated in less than 8 min, with a total LC analysis time of 14 min. The relative intensity of the 2nd transition for each compound stayed above 0.39, and linear responses were attained for standards in the range of concentrations from 1 to 400 ng mL-1. The instrumental limits of quantification (LOQs) of the UPLC-ESI-MS/MS (QqQ) system varied between 0.4 to 0.9 ng mL-1. These values were calculated as the lowest concentration providing a peak with a signal to noise (S/N) ratio of 10 for the qualifying (Q2) transition. SPE was selected as sample preparation technique to extract and concentrate compounds from wine. ACN and ACN:MeOH (80:20) mixtures were investigated for compounds elution. The first solvent permitted the effective elution of the two parent fungicides, leading to free-pigment extracts even from red wines; however, PYR-4OH and CYP-4OH required up to 10 mL of ACN for their complete desorption. Addition of a 20 % of MeOH to the elution solvent permitted to recover all the compounds from the SPE sorbent using just 2 mL. Breakthrough studies, considering 10 mL of wine diluted with the same volume of ultrapure water, showed that all compounds were quantitative retained in the 200 mg cartridge (data not shown). Recoveries of the SPE process, without considering MEs, were estimated for samples spiked at 20 ng mL-1 (equivalent to 100 ng mL-1 in the SPE extract). Spiked and non-spiked fractions of each wine were processed in triplicate. SPE recoveries were in the range of 92-101 %, with relative standard deviations (RSDs) between 2 and 3 %, Table 2. The MEs, calculated as described in section 2.2, varied between 88 and 112% (Table 2), which points out to 16 samples is developed and validated. Moreover, the co-occurrence of both kinds of compounds (free and 4-hydroxyanilino forms) is reported through analysis of a relevant number of commercial wines, produced in different years. Ratios between 4- hydroxyanilino species and parent compounds were higher in red than in white wines. Moreover, PYR-4OH showed higher concentration levels than PYR in red wines. Thus, the sum of concentrations for parent and 4-hydroxyanilino species is recommended to estimate their total residues. Qualitative data obtained in this study demonstrate, again for the first time, that hydroxylation takes place also in different positions of the pyrimidine ring of parent fungicides, and also that these compounds are already present in grapes treated with parent fungicides (proved for CYP). In addition, the hydroxyl derivatives of anilinopyrimidine fungicides coexist in wine with their glycosylated forms. Further research is required to understand those parameters controlling the extent of hydroxylation and glycosylation reactions at vines and during wine elaboration, as well as the relative toxicity of the metabolites described in this research versus that of parent fungicides sprayed in vineyards. Acknowledgements L.P.M acknowledges a FPU grant to the Spanish Ministry of Science. 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Schabacker, Microbial Transformations of the Fungicide Cyprodinil (CGA-219417), J. Agric. Food Chem. 45 (1997) 3647–3651. doi:10.1021/jf970298l. [19] J. Godshaw, A.K. Hjelmeland, J. Zweigenbaum, S.E. Ebeler, Changes in glycosylation patterns of monoterpenes during grape berry maturation in six cultivars of Vitis vinifera, Food Chem. 297 (2019) 124921. doi:10.1016/j.foodchem.2019.05.195. 19 Table 1. LC-ESI-MS/MS determination parameters, including quantification (Q1) and qualification (Q2) ions, linearity assessment and instrumental LOQs values corresponding to the UPLC-QqQ-MS instrument. Compound Retention time (min) [M+H]+ ion Cone Voltage (V) Q1 (CE) Q2(CE) Q2/Q1 ratio Linearity (1-400 ng mL-1, R2) LOQs (ng mL-1) PYR 5.67 200.1 51 107.1 (24) 82.1 (24) 0.76 0.996 0.5 CYP 7.08 226.1 56 93.1 (33) 108.1 (25) 0.72 0.996 0.4 PYR-4OH 3.01 216.1 55 107.1 (25) 82.1 (28) 0.75 0.995 0.8 CYP-4OH 4.24 242.1 60 93.1 (34) 109.1 (30) 0.39 0.998 0.9 PYR-d 5 5.64 205.1 51 107.1 (24) 82.1 (24) 0.82 - - CYP-d 5 7.03 231.2 56 93.1 (33) 108.1 (25) 0.53 - - CE, collision energy. 20 Table 2. Summary of SPE recoveries and matrix effects (MEs, %) of the analytical procedure. Compound Recoveries (%, SDs) MEs (%, SDs) Red wine White wine Red wine White wine PYR CYP PYR-4OH CYP-4OH 101 (3) 99 (3) 98 (3) 92 (2) 98 (3) 99 (2) 99 (3) 98 (2) 106 (4) 112 (6) 95 (6) 88 (2) 109 (4) 108 (4) 95 (6) 94 (6) 21 Table 3. Global recoveries (%) of the method, with standard deviations, for wine samples spiked at 3 different concentration levels. Values obtained using solvent-based calibration standards, after ISs correction. Compound Recoveries (%, SD) LOQs (ng mL-1) 4 ng mL-1 10 ng mL-1 20 ng mL-1 Red wine White wine Red wine White wine Red wine White wine PYR CYP PYR-4OH CYP-4OH 70 (1) 87 (2) 78 (2) 87 (2) 86 (8) 88 (9) 80 (9) 90 (9) 103 (1) 101 (1) 97 (3) 101 (1) 107 (2) 107 (1) 85 (2) 82 (2) 73 (1) 87 (2) 72 (2) 83 (1) 81 (1) 83 (3) 83 (4) 87 (1) 0.1 0.1 0.2 0.2 22 Table 4. Summary of concentrations in commercial wine samples. Data obtained for a set of 60 wines processed in duplicate. Values in ng mL-1. Compound Average Median Maximum Positive samples PYR CYP PYR-4OH CYP-4OH 18.7 7.9 11.4 2.7 8.0 7.6 4.6 1.7 99.6 31.9 58.0 17.3 54 50 54 45 23 Table 5. Summary of potential transformation products of PYR and CYP identified by LC-QTOF-MS. Parent fungicide TP Formula [M+H]+ Calculated mass (Da) Retention time (min) [M+H]+ Experimental Mass (Da) Mass error (ppm) Normalized score (0-100) PYR PYR-4OH C 12 H 13 N 3 O 216.1131 2.55 216.1128 -1.4 95 PYR-TP216A C 12 H 13 N 3 O 216.1131 4.50 216.1122 -4.2 96 PYR-TP216B C 12 H 13 N 3 O 216.1131 4.71 216.1117 -2.8 97 PYR-TP378 C 18 H 23 N 3 O 6 378.1660 2.17 378.1654 -1.6 94 CYP CYP-4OH C 14 H 15 N 3 O 242.1288 3.96 242.1287 -0.4 94 CYP-TP242A C 14 H 15 N 3 O 242.1288 6.30 242.1292 1.7 99 CYP-TP242B C 14 H 15 N 3 O 242.1288 6.60 242.1270 -7.4 97 CYP-TP242C C 14 H 15 N 3 O 242.1288 6.81 242.1290 0.8 75 CYP-TP404A C 20 H 25 N 3 O 6 404.1816 2.97 404.1814 -0.5 76 CYP-TP404B C 20 H 25 N 3 O 6 404.1816 4.94 404.1815 -0.2 94 CYP-TP404C C 20 H 25 N 3 O 6 404.1816 5.03 404.1820 1.0 88 CYP-TP404D C 20 H 25 N 3 O 6 404.1816 5.28 404.1808 -2.0 87 24 Fig. 1. Plots of concentrations for PYR-4OH/PYR and CYP-4OH/CYP in white wine (W.W.) (A) and red wines (R.W.) (B). Slope = 0.1995 R² = 0.8386 0 5 10 15 20 25 020 40 60 80 100 120 PYR-OH (ng mL -1 ) PYR (ng mL -1 ) W.W. Slope = 0.1137 R² = 0.365 0 1 1 2 2 3 3 0 5 10 15 20 CYP-OH (ng mL -1 ) CYP (ng mL -1 ) W.W. Slope = 2.0679 R² = 0.7026 0 10 20 30 40 50 60 70 0 5 10 15 20 25 30 PYR-OH (ng mL -1 ) PYR (ng mL -1 ) R.W. Slope = 0.472 R² = 0.8992 0 2 4 6 8 10 12 14 16 18 20 0 5 10 15 20 25 30 35 CYP-OH (ng mL -1 ) CYP (ng mL -1 ) R.W. A B 25 Fig. 2. Product ion scan spectra of PYR-TP216B (A) and PYR-TP216A (B). 3 x10 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 1.2 +ESI Product Ion (rt: 4.542-4.614 min, 6 scans) Frag=80.0V [email protected] (216.1131[z=1] -> **) 198.1022 156.0681 183.0785 171.0907 119.0602 72.0442 92.0482 131.0601 143.0723 Counts vs. Mass-to-Charge (m/z) 510 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 230 235 240 245 250 186.1021 C12H13N3 Calc. 198.1026 Error -2.02 ppm C11H9N3 Calc. 183.0791 Error -3.28 ppm C10H8N2 Calc. 156.0682 Error -0.64 ppm PYR-TP216A N HN NOH N HN N -CH 3 -CH 3 -HCN -HCN 216.1122 [M+H]+ C12H13N3O Calc. 216.1131 Error -4.16 ppm B 2 x10 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6 6.5 7 7.5 8 8.5 9 9.5 +ESI Product Ion (rt: 4.708-4.776 min, 5 scans) Frag=80.0V [email protected] (216.1131[z=1] -> **) 77.0388 68.0498 115.0539 44.0500 53.0388 130.0648 156.0678 93.0575 143.0728 104.0495 169.0761 83.0364 199.0861 216.1117 Counts vs. Mass-to-Charge (m/z) 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 123.0543 C6H7N2O Calc. 123.0553 Error -8.13 ppm [M+H]+ C12H13N3O Calc. 216.1131 Error -6.48 ppm C6H7N Calc. 93.0573 Error 2.15 ppm C6H5 Calc. 77.0386 Error 2.60 ppm N HN N OH PYR-TP216B -NH 3 -CH 2 O N N OH NH 2 A 32 Fig. S1. Product ion scan spectra of PYR-TP378 and CYP-TP404A. 4 x10 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 +ESI Product Ion (rt: 2.130-2.260 min, 2 scans) Frag=80.0V [email protected] (378.1660[z=1] -> **) 216.1129 378.1654 Counts vs. Mass-to-Charge (m/ z) 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 [M+H] + C 18 H 23 N 3 O 6 Calc. 378.1660 Error -1.6 ppm [M+H] + C 12 H1 3 N 3 O Calc. 216.1131 Error -0.93 ppm HO N HN N O N HN N O OH HO HO HOH 2 C A 4 x10 0 1 2 3 4 5 6 7 8 +ESI Product Ion (rt: 2.969-3.037 min, 3 scans) Frag=80.0V [email protected] (404.1816[z=1] -> **) … 242.1286 Counts vs. Mass-to-Charge (m/ z) 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 404.1814 [M+H] + C 20 H 25 N 3 O 6 Calc. 404.1816 Error -0.49 ppm [M+H] + C 14 H 15 N 3 O Calc. 242.1288 Error -0.83 ppm O N HN N O OH HO HO HOH2C HO N HN N B 33 Fig. S2. Product ion scan spectra for PYR (A) and PYR-4OH (B). 4 x10 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0.6 0.65 0.7 0.75 0.8 0.85 0.9 0.95 1 1.05 1.1 1.15 +ESI Product Ion (rt: 5.528-5.649 min, 5 scans) Frag=80.0V [email protected] (200.1182[z=1] -> **) 42.0345 77.0389 53.0392 168.0684 67.0293 181.0756 107.0602 92.0494 140.0491 115.0545 200.1177 131.0598 156.0680 Counts vs. Mass-to-Charge (m/z) 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 93.0576 82.0654 80.0496 65.0390 183,0917 141.0574 [M+H] + C 12 H 13 N 3 Calc. 200.1182 Error -2.49 ppm PYR C 6 H 7 N Calc. 93.0573 Error 3.22 ppm C 2 H 4 N Calc. 42.0344 Error 2.38 ppm C 6 H 5 Calc. 77.0386 Error 3.89 ppm C 4 H 5 Calc. 53.0391 Error 1.89 ppm C 6 H 7 N 2 Calc. 107.0604 Error -1.87 ppm N HN N C 12 H 11 N 2 Calc. 183.0917 Error 0 ppm N N -NH 3 -CH 3 N N NH 2 3 x10 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 +ESI Product Ion (rt: 2.598-2.628 min, 2 scans) Frag=80.0V [email protected] (216.1131[z=1] -> **) 42.0343 82.0653 109.0516 65.0389 53.0390 197.0709 93.0332 184.0624 154.0647 170.0870 135.0547 216.1127 147.0915 Counts vs. Mass-to-Charge (m/z) 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155 160 165 170 175 180 185 190 195 200 205 210 215 220 225 199.0866 158.0589 107.0595 80.0494 C 6 H 7 NO Calc. 109.0522 Error -5.50 ppm C 6 H 7 N 2 Calc. 107.0604 Error -8.41 ppm C 6 H 5 O Calc. 93.0335 Error -3.22 ppm [M+H] + C 12 H 13 N 3 O Calc. 216.1131 Error -1.85 ppm HO N HN N PYR-4OH -NH 3 -CH 3 NH 2 HO N HN C 5 H 6 N Calc. 80.0495 Error -1.25 ppm C 5 H 8 N Calc. 82.0651 Error -2.44 ppm C 5 H 5 Calc.65.0386 Error -4.61 ppm A B 34 Fig. S3. Product ion scan spectra for CYP-TP242C