==== Front Food Chem X Food Chem X Food Chemistry: X 2590-1575 Elsevier S2590-1575(23)00096-2 10.1016/j.fochx.2023.100654 100654 Short Communication Metabolite of chiral cycloxaprid in solvent and in the raw of Puer tea Tian Hen ab Zhang Jiao b Lin Tao a Li Qiwan a Huang Xiangzhong b Liu Hongcheng liuorg@163.com a⁎ a Institute of Quality Standard and Testing Technology, Yunnan Academy of Agricultural Science, Supervision and Testing Center for Farm Product Quality, Ministry of Agriculture, Kunming 650223, PR China b Key Laboratory of Chemistry in Ethnic Medicinal Resources, Yunnan Minzu University, Kunming 650500, PR China ⁎ Corresponding author at: Yunnan Academy of Agricultural Science, Beijing Road 2238 Number, Kunming, PR China. liuorg@163.com 29 3 2023 30 6 2023 29 3 2023 18 10065424 10 2022 16 3 2023 18 3 2023 © 2023 The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). Highlights • The present study was performed with enantioselective degradation, transformation and metabolite of CYC in different solvents under light and raw Puer tea processing. • This degradation pathway in acetone under and Puer tea processing was firstly reported. Cycloxaprid (CYC) with a chiral oxabridged cis- structure contains a pair of enantiomers. Enantioselective degradation, transformation and metabolite of CYC was performed in different solvents under light and raw Puer tea processing. The results showed that cycloxaprid enantiomers in acetonitrile and acetone was stable over 17 day, however the transformation of 1S, 2R-(–)-cycloxaprid or 1R, 2S-(–)-cycloxaprid was founded in methanol. The fastest degradation of cycloxaprid occurred in acetone under light, the metabolites were founded with retention times (TR) at 34.83, 15.78 min, which mainly was via the reduce reaction of NO2 to NO, and rearrange reaction to tetrahydropyran. Degradation pathways were via the cleavage of the oxabridge seven member ring and the whole C ring. However, the degradation pathway under raw Puer tea processing was via the cleavage of whole C ring and the cleavage of oxabridge seven member ring and reducing NO2, then it underwent an elimination of nitromethylene and rearrange reaction. This pathway of Puer tea processing was firstly founded. Keywords Metabolite Cycloxaprid Raw Puer tea processing ==== Body pmcIntroduction Neonicotinoids is a valuable synthetic insecticides piercing-sucking pests of for tea protection (Tomizawa & Casida, 2003). They are used increasingly and occupied 24% of insecticides in world market (Jeschke et al., 2011). In 2013, the European Commission prohibited the use of three neonicotinoide insecticides (Thiamethoxam, imidacloprid and thiamethoxam) that might affect the life cycle of bees, which is urgent to assess other neonicotinoid’s food safety risk (Chen et al., 2017). Li et al. 2011 reported that a new neonicotinoid insecticide of cycloxaprid (CYC) has been synthesized and applied in China. It is different from traditional neonicotinoids, which act as agonists of native and recombinant nicotinic acetylcholine receptors (nAChRs) (Liu and Casida, 1993, Matsuda et al., 1998, Nishimura et al., 1994, Tomizawa and Casida, 2003). Cycloxaprid is chiral compounds, which contains a chiral oxabridged cis-configuration (C ring) leading to a pair of enantiomers, 1R,2S-cycloxaprid and 1S,2R-cycloxaprid. In recent years, chiral pesticides is the main focus of great attention at stereoselective biological activities and environmental processes of isomer selectivity. Chiral pesticides are commonly used as racemic mixtures and their stereoisomers are often degraded stereoselectively in food. Previous studies shown that the stereomiosmer had been found in soil or food. Liu et al. 2022 found the enantioselectivity of cycloxaprid in raw or ripen Puer tea processing. Zhang et al. 2013 observed stereoselective uptake and translocation of cycloxaprid in edible vegetables from roots. However, Chen et al. 2017 demonstrated that there was non stereoselective preference for cycloxaprid enantiomer in aerobic soils, so the enantioselectivity was effected by different matrix and the result was different. The degradation of pesticide is found the metabolite which maybe result in high toxicity in environment or food. Liu et al. 2015 identified and tracked 11 and one unknown transformed product in flooded and anoxic soil. Chen et al. 2017 reported three mainly metabolites included cleavage of the oxabridged seven-member ring and C—N between chloropyridinyl methyl and imidazalidine ring, carboxylation of the alkene group, and hydroxylation of imidazolidine ring in aerobic soil. Hou et al. 2017 studied photodegradation of CYC and analyzed 25 photodegradation products were identified via UPLC-TOF-MS/MS in water. Puer tea is the most famous tea in China. Because its special processing, the Puer tea is fermented by traditional technology in long-distance transport with consign for horse. Previous our studied that one mainly metabolite was reported in Puer tea processing (Liu et al., 2022) but, the relevant information of degradation pathway and metabolite of CYC was still incomplete (Fig. 1). Therefore, it is important to obtain the degradation productions and pathway of CYC. The present study was to use racemate CYC to characterize the degradation in raw Puer tea processing, analyzed its metabolites and proposed degradation pathways, which is also significant for the fate of other new pesticides in the food.Fig. 1 The degradation pathway and metabolite of cycloxaprid in Puer tea processing. Materials and methods Reagent A racemic cycloxaprid was provided by Beilinwei technology Ltd. (Beijing, China). Cycloxaprid powder 25 % was supplied by shanghai shengnong pesticide Co Ltd (Shanghai, China). The purified materials of C18, PSA, Carb were supplied by Dima technology co. Ld. (Beijing, China). The stock solutions were produced by dissolving the racemic cycloxaprid in acetonitrile. All solutions were stored in a refrigerator at − 18 °C. HPLC-grade acetonitrile and methanol were provided by Tedia Company Inc. (OH, USA). The initial dose of cycloxaprid 0.15 g/L was used with 0.6 g of cycloxaprid power completely dissolved into 1 L water. High seed centrifuge of TGL-10B was supplied by Anting co. Ld. (shanghai, China); The voter mixer of QL-866 was from Linbei intrument co. Ld. (Jiangsu, China); The high speed of DFT-100A 100was from Linda machine co. Ld (Zhejiang China). Methods Incubation experiments were performed to investigate the enantioselective racemic cycloxaprid and the metabolism in raw Puer tea processing. Five grams of raw Puer tea are sprayed with 0.15 g/L the formulation product solution. A nontreated control was also included. The enantioselective degradation is express as enantiomer fraction (EF) and determined by LC-MS/MS. The metabolomics are analyzed by LC-HRMS. To research stability and transform, 1R,2S -cycloxaprid or 1S,2R-cycloxaprid (1 mg/L otpical pure) are respectively tested with different solutions (methanol, acentrinole, acentone), with light or dark. To research dissipation during light or dark, three hundred grams of raw Puer tea are sprayed with 0.15 g/L the formulation product solution. One sample is placed with black plastic bag on dark room, the other sample is not protected under air temperature. The samples are collected at intervals time on 0 (2 hr), 1, 3, 5, and 14 day. The residues amount expressed as dry sample. Obtaining enantiopure standards: 1R,2S -cycloxaprid and 1S,2R-cycloxaprid The enantiopure of 1R,2S -cycloxaprid and 1S,2R-cycloxaprid is separated by semi-preparative HPLC. The 4.8 mg/mL of cycloxaprid in acetonitrile solution was separated by using a Chiralpak AG (amylose tris (3-chloro-5-methylphenylcarbamate) as stationary phase, 250 × 4.6 mm i.d., 5 μm, Daicel Ltd. JP). Mobile phases are H2O and acetonitrile (55: 45), respectively. The flow rate was 1.0 mL/min. the injection volume is 40 μL. Samples preparation Two gram of tea sample were exactly weighed, then 10 mL water, 10 mL acetonitrile were added. After the mixture was vortexed, and added 4 g NaCl. The tube with vortex mixer was shaken vigorously at 1 min. The mixture was centrifuged at 5000 rpm for 5 min. The upper layer solution was separated and added by 100 mg PSA, 100 mg C18, 50 mg carb and 300 mg anhydrous MgSO4. After shaking and centrifugation at 5000 rpm for 3 min, 0.5 mL of the upper layer was separated and filtered through 0.22 μm filter for UPLC-HFMS or UPLC-MSMS. UPLC-HRMS analysis Sample analysis was achieved in an Ultra Performance Liquid Chromatography – Q Exactive high resolution Mass Spectrometry (Thermo Fisher Scientific, Rockford, IL, U.S.A.) system. The chromatogram seperation was obtained by Hypersil GOLD (100 × 2.1 mm, 1.9 μm), and the temperature is maintained at 30 °C. The mobile phase is comprised of 80% 0.1% formic acid, 1 mmol ammonium with water/20% acetonitrile at a constant flow of 0.3 mL/min. The gradient mobile phase program (min/%B) was 0–3/3–5, 3–10/35–55, 5–10/55, 10–20/10–15, 20–30/15–18, 30–50/20–40, 50–53/40, 53–60/40–95, 60–61/95–3. The injection volume is 1 μL. The instrument was tuned in the positive ESI mode (3.8 kV of spray voltage, 325 °C of capillary temperature, 350 °C of probe heater temperature and 60 V of SLens). The instrument was calibrated using positive calibration solutions. The FS/DIA mode was used. In FS, the scan range was m/z 50 –650; mass resolution at 140,000 FWHM; AGC target and maximum IT were set at 1.0 e6 and 100 ms, respectively. For DIA, the relevant parameters were set as follows: mass resolution: 140,000 FWHM; AGC target: 2 e5; maximum IT: 30 ms; Loop count: 12; MSX count: 1; Isolation window: 50 Da; stepped normalized collision energy (NCE): 20%, 40% and 60%. The spray voltage in positive and negative modes was set as 3.5 kV and 3.0 kV, respectively. The flow rate of sheath gas and aux gas was 45 and 10 (in arbitrary units), respectively. The software was used with TraceFinder 4.1 EFS and Compound Discoverer 3.3. UPLC-MSMS analysis UPLC-MS/MS was used with tandem mass spectrometer AB 4500 (AB Sciex) consisted of a 1290 ultra-high-performance liquid chromatograph (Agilent technology). A Chiral amylose tris (3- chloro- 5-methylphenylcarbamate), 250 × 4.6 mm i.d., 5 μm was used and the column temperature was set at 30 °C. The mobile phase is comprised of 80% 0.1% formic acid, 1 mmol ammonium acetate with water/20% acetonitrile at a constant flow of 0.3 mL/min with 1 μL injection. The gradient mobile phase program (min/%B) was 0–10/35, 10–11/35–55, 11–20/55, and 21–25/95. Anion electrospray ionization mode was used, and multiple reactions monitoring (MRM) was utilized: Precursor ion was set at 323.1 (m/z), Product ion was set at 126.0*/138.1, 90.1 (m/z), Delustering/Focusing potential was set at 77 (V), and Collision energy was set at 40/35, 76 (V). The temperature and flow rate of drying gas (N2) was set at 550 °C and 8.0 L/min, respectively. The nebulizer pressure was set at 20 Pa. Quality control Cycloxaprid at three concentration levels (1, 10, 100 µg/kg) with matrix solution is used for limit of detection limit and recovery test. The chromatogram of spiked sample and elution order of racemic cycloxaprid was determined by online optical rotation. The more separation resolution was obtained by the method than Zhang et al. 2013 reported. No cycloxaprid is residue in the blank sample. The mean recoveries are in the range of 76–108 % for racemate of cycloxaprid, respectively. The corresponding relative standard deviation was 4.6–9.2%. The linearity is the range of 1–100 µg/L with coefficient, R2 > 0.999. The limit of detections (LODs) with three times signal-to-noise (S/N) ratio, and the limit of quantify (LOQ) with ten times S/N ratio is 0.5 μg/L, 0.5 μg/kg respectively. Data analysis The data was obtained by triplicate samples. The results showed as the means with standard errors (means ± STD) using statistical analysis software. The significant difference (p = 0.05) among treatments was determined by one-way analysis of variance. Results and discussion The stability of optical pure compounds in different solvent The stability of optical 1R, 2S-cycloxaprid and 1S, 2R-(–)-cycloxaprid was tested in three solvents. The results showed that 1R, 2S-cycloxaprid or 1S, 2R-(–)-cycloxaprid in acetonitrile and acetone was stable over 17 day. However, optical pure of 1R, 2S-cycloxaprid was not stable in methanol, which was quickly decreased in methanol. But the racemate CYC was stable, the result showed that the transformation of 1S, 2R-(–)-cycloxaprid was formed in Fig. 2. A similar result is observed with other optical pure compounds (Zhang et al., 2013).Fig. 2 The stability of optical pure compounds (1S, 2R-cycloxaprid or 1R, 2S-cycloxaprid) in three solvents. The parent compound of racemate CYC in solvent was more unstable under light than under dark. Hou et al. 2017 reported the pathways of the photoreaction process of CYC was hydroxyl radicals reaction. The degradation of CYC in three solvents was distinctly decreased under light. The CYC in acetone was not detected over 9 day, but an amount of residues in methanol, acetonitrile were founded. This suggest that the degradation of cycloxaprid was catalyzed by light radicals and speed by acetone solvent. Formation of metabolites in acetone under light The metabolite research is major limited with the relatively small number of metabolites commercially available as pure standards and structure determination by NMR or X-ray crystallography, therefore the large number of metabolites with unknown chemical structures that remain to be identified and characterized by High resolution mass spectrum (Ivana et al. 2019). Novel computational tools can predict MS fragmentation patterns in databases (Cajka et al., 2017, Kind et al., 2018), which has been shown that fragmentation spectra can be simulated with quantum chemical and molecular dynamics methods (Wishart et al., 2018). Fig. 3 shows the chromatogram of main metabolites under light with acetone at 13 day after cycloxaprid spiking. There were two large peaks with the retention times (TR) at 34.83(g), 15.78(f) min, and four main peaks were named as a, b, d, e. We were achieved metabolite information using a combination of different tools. The metabolite compounds were identified based on accurate mass and retention time using the NIST17, HMDB 20, GNPS 21, GMD and the Lipid MAPS libraries, which obtained the molecular structure. Then for MSI annotations, we used mzCloud (online second mass spectrometry),ChemSpider (online first mass spectrometry) accurate mass search services for validating and putatively annotating the metabolite feature using mass spectrometry (Table 1).Fig. 3 The total ion chromatogram of cycloxaprid in acetone under light. Table 1 Mass spectrometry data for the identification of cycloxaprid and its metabolites in acetone under light. Product tR (min) Formula Mw Chemical structure Mw (calculated) ESC(+) MS, m/z ESI(+)MS2, m/z (relative abundance %, loss) Parent 17.72 C14H15ClN4O3 322.0833 323.09054 323.09033 323.09033 (60, M + H); 325.08752 (20,M + H + 2); 289.08527 (14,M-O2H2); 277.09681 (36, M-NO2); 276.08992 (36);248.09483 (27, M- NO2 –CO); 234.07930 (22); 220.06371 (12);182.09239 (20);151.08655 (40, M- NO2 + ClC5H3NCH); 138.06631 (16);126.01065 (100, M-OC3H6-N2C3H2O); 128.00773 (30);123.09184 (22, M- CO - ClC5H3NCH); g 34.83 C12 H16 O2 N3 Cl 269.09256 270.10038 270.10032 270.10029 (2, M + H); 272.09685 (1, M + H + 2); 212.05838 (6, M-OC3H6); 197.04748 (10, M-OC3H6-NH); 183.03189 (16, M-OC3H6-NH-CH2); 126.01067 (100, M-OC3H6-N2C3H2O); 128.00766 (24) l 16.66 C14 H15 Cl N4 O2 306.08780 307.09563 307.09518 307.09518 (100,M + H); 309.09239 (22,M + H + 2); 289.08478 (4, M-OH); 276.08971 (4); 261.09000 (6);248.09475 (3); 220.06317 (6, M- OH-NO -C3H4); 208.06335 (4); 169.05252 (4); 164.08178 (4);149.07100 (2);136.08681 (6, M-OC3H6-NC3H2O); 126.01054 (52, M-OC3H6- N2C3H2O); 121.07638 (4); j 45.66 C14H16ClN3O 277.09764 278.10547 278.10544 278.10544 (14,M + H); 280.10185 (6,M + H + 2); 236.09562 (12); 211.06319 (42); 149.02374 (100, M-C6H7NCl); 136.07554 (24);126.01062 (80); k 38.27 C14 H13 Cl N4 O2 304.07215 305.07998 305.07922 305.07922 (36, M + H); 307.07768 (12, M + H + 2); 288.07707 (42, M-OH); 261.08387 (22);259.08668 (100, M-O-NO);258.07942 (50,M-NO2);243.05620 (4);232.06386 (12);224.11781 (14);216.04722 (4);167.03752 (4);147.09109 (4);133.07571 (4);126.01064 (48); b 6.81 C14H16O3N3Cl 309.08747 310.09530 310.09482 310.09482 (38, M + H); 312.09258 (10, M + H + 2); 196.06344(100, M-C5H6O3); 198.06034(24); 160.08690(8); 126.01055(55, M-C8H9O2N3); f 15.78 C14H14ClN3O2 291.07691 324.11095 324.11063 324.11063(29, M + H + MeOH); 306.10770(8, M-H2O + MeOH);196.06357 (100, M-C5H4O2); 126.01058 (48, M-C8H9O2N3); 101.06007 (20); i 17.51 C14 H14 Cl N3 O 275.08199 276.08982 276.08973 276.08974 (100, M + H); 278.08365 (16, M + 2); 248.09497 (28, M-CO); 234.07896 (10,M-O-C2H2); 220.06340 (12,);208.06361 (16);193.03998 (4,M-C5H7O);151.08654 (40);126.01065 (87, M-C8H10N2O);123.09184 (18); r 12.09 C14 H16 Cl N3 O2 293.09256 294.10038 294.10000 294.10000(100,M + H);296.09686(23,M + H + 2);264.05134(1,M-CH2O);238.07403(10,M-CH2O-C2H2);235.02437(4);196.06344(1, M-C5H6O2);169.05281(3); 149.02268(1); 126.01063(38, M-C8H10N2O); 128.00760(10); a 4.70 C9H10N3Cl 195.05578 196.06360 196.06368 196.06371(72, M + H); 198.06058 (15, M + H + 2); 160.08694 (8, M-Cl); 126.01075(100, M-N2C3H6); 128.00772(22);90.03409 (2) h 19.61 C9H10ClN3O 211.05069 212.05852 212.05819 212.05821 (99,M + H); 214.05520 (20,M + 2); 176.08186 (2,M-Cl); 130.02320 (21);128.02615 (100,M-C3H4N2O); 126.01055 (20);99.05565 (32); O 4.02 C10 H12 Cl N3 O 225.06634 226.07417 226.07370 226.07426(96,M + H); 228.07117(24,M + 2); 196.06368(28, M-CH2O); 198.06073(8,M-CH2O + 2); 160.08684(8,M- M-CH3O-Cl); 130.02317(4); 128.00775(22); 126.01075(100,M-C4H8N2O); d 10.02 C10H10 Cl N3 O2 239.04561 240.05343 240.05316 240.05316(52,M + H);242.05013(12,M + H + 2);126.01063(100, M-C4H6O2N2); 128.00759 (22) P 12.25 C10 H12 Cl N3 O2 241.06126 242.06908 242.06847 242.06849(38,M + H); 244.06528(10,M + 2); 224.05838(6,M-H2O); 214.07361(14); 199.04403(8); 197.04720(28, M-COOH); 169.05256(2);143.03676(2); 128.00752(22); 126.01056(100, M-C4H8O2N2); e 14.36 C11H14 Cl N3 O2 255.07691 256.08473 256.08439 256.08439(22,M + H); 258.08192(6,M + H + 2); 238.07397(10, M-H2O); 228.08956 (12, M-CO); 211.06319(8, M-H2O-CHN); 196.06353(42, M-C2H4O2); 126.01063(100, M-C5H10N2O2); The fragment pathways of CYC were three in Fig. 4, One possible pathway was via the cleavage of the oxabridged ring (C ring). The second possible route was oxidated the C ring. The third possible route was removed of chloropyridinyl (A ring). The pathway was similar with Hou et al. 2017 reported the pathway of photodegradation in water.Fig. 4 The fragment pathway of cycloxaprid. The peak at the retention time of 17.72 min with the molecule ion at m/z 323.09033 [M + H]+ and chlorine isotopic ion at m/z 325.08752 [M + H + 2]+ was CYC. The base peak of characteristic ion at m/z 126.01065 and chlorine isotopic ion at m/z 128.00773 corresponding to chloropyridinylmethyl, which could be found in other metabolite. The other major fragments at m/z 276.09681, 248.09485, 151.08655, 123.09183 exhibited the loss of NO2, NO2 + CO, NO2 + ClC5H3NCH, and CO + ClC5H3NCH, respectively. The Fig. 5 showed MS/MS spectra of CYC and main metabolite.Fig. 5 The ms/ms spectra of cyc and main metabolite under light. Metabolite g was the major metabolite in total ion chromatogram. The product ions 270.10032 [M + H]+ containing odd nitrogen and chlorine isotopic ion 37Cl, corresponding to the formula of C12 H16 O2 N3 Cl. The product ions 212.05803, 197.04741 produced via the loss of OC3H6 and OC3H6-NH, which was identified as 1-[(6-chloropyrid-3-yl) methyl]-2-hydroxyl-imidazoline-tetrahydropyran, which probably is a rearrange reaction from metabolism j. Metabolite k, l, j was probably a leading compounds of g. The l of product ion 307.09518 and the k of 305.07922 was reduced oxygen from the NO2 of CYC, and the three metabolites containing product ions m/z 126.01054 resulted from the cleavage of the C—N bond linking the A + B rings. The retention time of metabolite j and k was at 45.66, 38.27 min respectively, but metabolite l was at 16.66 min, which indicated that metabolite l was polar compound with alcohols and metabolitea j, k were low polar compound with oxo-bridge structure in Fig. 4. The mass spectrum of a that appeared at 4.70 min in the chromatogram yielded mass peaks of m/z 196.06368 [M + H]+; the base peak with m/z 126.01074 [M-N2C3H6]+; Metabolite f that appeared at 15.78 min in the total ion chromatogram exhibited an [M + H + MeOH]+ ion at m/z 324.11063, which corresponds to the formula of C14H14ClN3O2, the ion m/z 306.10770 with cleavage of H2O. The base peak of m/z 196.06357 (a) and the ion of m/z 126.01058 (a) which suggested the A and B rings were completed. Characteristic ion 196.06357 and 126.01058 also appeared in the fragments of metabolite b, f, and r. Metabolites of b, f, r also had been detected by photodegradation products of CYC (Hou et al. 2017). Metabolite e, with m/z 238.07397 (10, M-H2O) and m/z 228.08956 (12, M-CO), which suggested that the structure contained simultaneously hydroxyl and aldehyde group. Metabolite p, with an m/z 242.06847, which corresponds to the formula of C10H1°ClN3O2. Characteristic ion 126.01056 appeared in the fragment of metabolite d, o, a. Those compounds had been reported by Hou et al. 2017. Proposed degradation pathway under light Based on the identified degradation products, the possible photodegradation pathways in aqueous solution under light irradiation were proposed. The tentative photodegradation pathways of CYC were shown in Fig. 6. The degradation of CYC in acetone solution under light via three possible pathways.Fig. 6 The hypothetical degradation pathway of cycloxaprid in acetone under light. One main degradation pathway was via the reduce reaction of NO2 to NO, and rearrange reaction to tetrahydropyran, which was not previously reported (Fig. 6) Two degradation pathways were via the cleavage of the oxabridge seven member ring and the whole C ring, resulting in the formation of primary compound. These pathway was very similar to that reported in the photodegradation of CYC under environment. (Hou et al. 2017). Formation of metabolites in raw Puer tea To obtain the transformation, the conversion of cycloxaprid is quickly generated by sun light and the normal fermentation in air temperature. Fig. 7 shows the main metabolites under raw Puer tea processing at 13 day after cycloxaprid spiking and blank. There were three obvious peaks with the retention times (TR) at 13.97 (x), 19.61(h), 39.09 (y) min, and the diversity peaks were similar with these peak on metabolites in acetone under light (metabolite of l, k, j, i).Fig. 7 The total ion chromatogram of raw Puer tea and cycloxaprid spiked. Metabolite × was the major metabolite reported by our previous results (Liu et al., 2022). The metabolite and standard were simultaneously analyzed by LC-HFMS, and their retention time, molecular ion peak, and fragmentation pattern were in agreement with the synthesized structure standard of 2 -chloro-5 -[[-2 -(nitromethylidenc) imidazodin-1 -yl] methyl] pyridine (CAS 10336–63-4). A similar result was also observed by Chen et al. 2017. The result indicated that most CYC might transform to the active intermediate x. Furthermore, Shao et al. 2010 showed that CYC may be a slow-release reservoir for (nitromethylene) imidazole (x), which was considered as the final active ingredient. The mass spectrum of h that appeared at 19.61 min in the chromatogram yielded mass peaks of m/z 212.05819 [M + H]+, which corresponded to the formula of C9H10ON3Cl. The Fig. 8 showed MS/MS spectra of main metabolite. The base peak was more different with m/z = 128.02614 than with characteristic ion m/z = 126.01055. The result suggested that the pyridine ring was added hydroxyl group and oxygen rearrange reaction.Fig. 8 The ms/ms spectra of main metabolite in puer tea processing. Metabolite y that appeared at 38.85 min in the total ion chromatogram exhibited an [M + H]+ at m/z 262.07383, which corresponds to the formula of C13H12ClN3O, m/z 244.06299 [M-H2O]+,which showed that the compound contain hydroxyl or carbonyl group. The chromatogram condition exhibited the low polarity compound with long retention time (38.85 min), so the chemical structure was deducted in Tab 2. Metabolite t that appeared at 14.41 min in the total ion chromatogram exhibited an [M + H]+ at m/z 239.0691 and chlorine isotopic ion m/z 241.06527, which corresponds to the formula of C10H11ClN4O. The fragment m/z 208.06329 [M-NO]+ showed that the B ring contain NO group, the even nitrogen rule indicated the complete A ring at m/z 191.96248 [M-NO-NH2]+. Metabolite z was overlapped by tea matrix at 17.51 min exhibited an [M + H]+ at m/z 182.09239, which corresponds to the formula of C8H11N3O2. The fragment m/z 164.0817 remove H2O, and the fragment m/z 136.08684, 124.09956 removed C2H4, C3H4 which showed still the whole frame of B ring and C ring. Proposed degradation pathway under Puer tea processing One degradation pathway was via the cleavage of whole C ring, resulting in the formation of mainly degradation compound X, which had been reported as the final active ingredient (Chen et al., 2017, Hou et al., 2017,; Liu et al. 2022). Metabolism X also underwent an elimination of nitromethylene and methylation of the imidazolidine ring, which produced h and w. The other degradation pathway was via the cleavage of oxabridge seven member ring and reducing NO2, resulting in the formation of metabolism l, which was similar to degradation in acetone under light. Metabolism l also underwent an elimination of nitromethylene and rearrange reaction, which produced y. This pathway in Fig. 9 was very different to photodegradation in water (Hou et al., 2017) or our previously reported results (Liu et al., 2022). Compared to the photolysis, the metabolite of CYC was more complex in Puer tea processing. This study can provide insights to the fate of pesticide in the Puer tea processing.Fig. 9 The hypothetical degradation pathway of cycloxaprid in raw Puer tea processing. Conclusion The enantioselective degradation, transformation and metabolite of CYC in different solvents under light and raw Puer tea processing was detected. Chiral cycloxaprid in acetonitrile and acetone was stable, however the transformation of 1S, 2R-(–)-cycloxaprid or 1R, 2S-(–)-cycloxaprid was founded in methanol. The fastest degradation of cycloxaprid occurred in acetone under light, the degradation pathway was via the reduce reaction of NO2 to NO, and rearrange reaction to tetrahydropyran. The degradation pathway under raw Puer tea processing was via the cleavage of whole C ring, and via the cleavage of oxabridge seven member ring and reducing NO2, then it underwent an elimination of nitromethylene and rearrange reaction (Table 2).Table 2 Mass spectrometry data for the identification of cycloxaprid and its metabolites in raw Puer tea processing. Product tR (min) Formula Mw Chemical structure Mw (calculated) ESC(+) MS, m/z ESI(+)MS2, m/z (relative abundance %, loss) Parent 17.72 C14H15ClN4O3 322.08272 323.09054 323.09033 323.09033 (60, M + H); 325.08752 (20,M + H + 2); 289.08527 (14,M-O2H2); 277.09681 (36, M-NO2); 276.08992 (36);248.09483 (27, M- NO2 –CO); 234.07930 (22); 220.06371 (12);182.09239 (20);151.08655 (40, M- NO2 + ClC5H3NCH); 138.06631 (16); 126.01065 (100, M-OC3H6-N2C3H2O); 128.00773 (30); 123.09184 (22, M- CO - ClC5H3NCH); x 14.04 C10H11ClN4O2 254.05650 255.06433 255.06411 255.06399 (91,M + H); 257.06104 (32,M + H + 2); 238.06126 (28,M-OH); 221.05871 (68,M-O2H2); 208.06350 (100,M-HNO2); 174.10261 (8,M-NO2-Cl); 167.03701(14);140.02584 (8); 126.01052 (36); 114.06659 (30); 98.07171(86); 96.05608 (20); 83.06102 (24); 70.05330 (10); T 14.41 C10H11 Cl N4 O 238.06159 239.06942 239.06915 239.06915 (58,M + H); 241.06527 (14,M + 2); 221.05863 (22);208.06329 (12,M-HNO); 191.96248(10, M-NO-NH2);173.09434 (3); 126.01048 (1 0 0); 98.07162 (26); 83.06103 (6); Y 38.85 C13H12ClN3O 261.06634 262.07417 262.07383 262.07383 (100,M + H); 264.07060 (20,M + H + 2); 244.06299 (12);226.09721 (4,M-Cl);195.03209 (8,M-C4H5N);167.03708 (3,M-C5H5ON); 135.05511 (7);126.01055 (90,M-C7H8ON2); 90.03410 (2) K 38.27 C14 H13 Cl N4 O2 304.07215 305.07998 305.07922 305.07922 (36, M + H); 307.07768 (12, M + H + 2); 288.07707 (42, M-OH); 261.08387 (22);259.08668 (100, M-O-NO);258.07942 (50,M-NO2);243.05620 (4);232.06386 (12);224.11781 (14);216.04722 (4);167.03752 (4);147.09109 (4);133.07571 (4);126.01064 (48); Q 17.51 C14 H14 Cl N3 O 275.08199 276.08982 276.08973 276.08973 (100,M + H); 277.09464(24); 278.08497 (14,M + 2); 262.07416 (2, M-H2O); 248.09478 (28, M-H2O-CH2); 234.07907 (18, M-H2O-C2H4); 220.06322 (12, M-H2O-C3H6); 208.06347 (16,M-H2O-C4H6); 193.03991 (4); 151.08648 (28); 126.01063 (16); 123.09178 (14); J 45.66 C14H16ClN3O 277.09764 278.10547 278.10544 278.10544 (14,M + H); 280.10306 (6,M + 2); 236.09562 (12); 211.06319 (42); 149.02374 (1 0 0); 126.01062 (80);80.06086 (8);57.07081 (8); L 16.66 C14 H15 Cl N4 O2 306.08780 307.09563 307.09518 307.09518 (100,M + H); 309.09239 (22,M + 2); 289.08478 (4, M-H2O); 276.08971(4, M-NOH); 261.09000 (6); 248.09475 (3); 220.06317 (6); 208.06335 (4);169.05252 (4); 164.08178 (4); 149.07100 (2); 136.08681 (6); 126.01054 (52); 121.07638 (4); H 19.61 C9H10ClN3O 211.05069 212.05852 212.05851 212.05821 (99,M + H); 214.05520 (20,M + H + 2); 176.08186 (2,M-Cl); 130.02320 (21); 128.02615 (100,M-C3H4N2O); 126.01055 (20); 99.05565 (32); W 14.29 C10H11 N3 173.09475 174.10257 174.10264 174.10264(100,M + H);172.08679(29); 158.08382(3); 145.07596 (8, M-C2H3N); 133.07619 (6, M-C2H3N-CH); 118.06555 (6); z 17.51 C8H11 N3 O2 181.08458 182.09240 182.09239 182.09239(100, M + H); 164.08173(14, M-H2O); 154.09725(8);136.08684(20, M-H2O-C2H4); 124.09956 (10); 123.09186(7); 121.07616 (6) CRediT authorship contribution statement Hen Tian: Methodology. Jiao Zhang: Software. Tao Lin: . Qiwan Li: Conceptualization. Xiangzhong Huang: Validation. Hongcheng Liu: Writing – review & editing. Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Liu hongcheng reports article publishing charges was provided by Yunnan Academy of Agricultural Science. Liu hongcheng reports a relationship with Yunnan Academy of Agricultural Sciences that includes: employment. Liu hongcheng has patent No pending to Yes. employee. Data availability Data will be made available on request. Acknowledgements We are grateful for supporting funded 10.13039/501100001809 National Natural Science Foundation of China (31760490), Yunnan province Science and Technology Major Project (Nos. 202102AE090021, 202002AE3200053). ==== Refs References Cajka T. Smilowitz J.T. Fiehn O. 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