Qualitative and quantitative determinations of pyridalyl and metabolites in excrement of two representative Lepidoptera pests

Abstract

Qualitative and quantitative SPE followed by HPLC-TOF/MS determination of pyridalyl and its potential metabolites in the excrement of Helicoverpa armigera (H. armigera) and Spodoptera exigua (S. exigua) was developed. Two potential metabolites, MP1 and MP2, have been confirmed, and a reported metabolite in mammals, MP3, was indicated to be a product of the hydrolysis of pyridalyl. In addition, the content of pyridalyl, MP1 and MP3 in excrement were similar in both pests, while the content of MP2 was 13% greater compared with its counterpart from S. exigua. This may be due to different metabolic processes in mammals and two Lepidoptera pests. The metabolic pathway of pyridalyl in both pests was then proposed based on the preceding results. Compared with reported isotope-labeled strategies, the proposed method cut down experimental time from several days to 2 hours, and reduced sample consumption from several grams to 30 mg.

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1. Introduction

Pyridalyl is a new pesticide,^1–7 and its target enzyme, which was confirmed to be different from reported ones, is regarded as the source of its specific insecticidal activity for Lepidoptera pests.^3,8–12 Generally, pesticides interact with the target enzyme in the metabolic organ, and certain physiological functions of pests are thus inhibited.^13,14 In this sense, metabolites of pyridalyl in Lepidoptera pests, as well as their metabolic pathways, would be helpful for finding out the target enzyme.^15,16

Related research has focused more on the physiological and biological safety of pyridalyl.^9,17–20 For instance, mammals and insects, e.g. rats and Chironomus yoshimatsui are usually employed as experimental organisms.^2,7,9,17,21 Although the insecticidal performance of pyridalyl is directly dependent on its interaction with its specific target enzyme in pests, its metabolism in practical Lepidoptera pests has never been discussed to our best knowledge.

In related reports, thin layer chromatography as well as two-dimensional gel electrophoresis has been widely employed to prepare samples,^22,23 and metabolites are conventionally determined by isotope-labeled strategies.^7,18,21,24 Both strategies in sample preparation would generally be accompanied with a lot of sample consumption, experimental time and labor work. Moreover, strict safety regulations and chemical treatments were conventionally required in isotope-labeled methods. On the contrary, high performance liquid chromatography coupled with mass spectrometry (HPLC-MS) is capable of efficient separations as well as qualitative and quantitative determinations of analytes in complex samples, and it requires less experimental time and sample consumption.^25–27 Therefore, a rapid and sensitive method, HPLC-MS coupled with solid phase extraction (SPE),^28–32 would be of potential value to study the metabolism of pyridalyl in Lepidoptera pests.

In this study, pyridalyl and its potential metabolites in the excrement of H. armigera and S. exigua, both of which are representative Lepidoptera pests, have been determined based on SPE followed by HPLC-MS. Under optimal conditions, qualitative and quantitative determinations for pyridalyl and its metabolites in the collected excrement were achieved. Moreover, each potential metabolite was carefully evaluated to be from the metabolism of pyridalyl or other resources. At last, its metabolic pathway in both pests was proposed.

2. Materials and methods

2.1. Chemicals

The chemical structures of pyridalyl and three potential metabolites (MP1, MP2 and MP3) are indicated in Fig. 1. The standard chemical pyridalyl was purchased from Dr Ehrenstorfer GmbH (Augusburg, Germany). The pyridalyl used in the pest experiments was synthesized in accordance with ref. 33, and its chemical structure was confirmed by TOF-MS and ¹H NMR (as shown in Fig. S1†). The NMR spectrum is summarized as: ¹H NMR (400 MHz, CDCl₃) δ: 8.45 (s, 1H), 7.77 (d, J = 8.8 Hz, 1H), 6.90–6.81 (m, 3H), 6.11 (t, J = 6.0 Hz, 1H), 4.65 (t, J = 6.4 Hz, 2H), 4.58 (d, J = 6.4 Hz, 2H), 4.14 (t, J = 6.4 Hz, 2H), 2.33–2.27 (m, 2H). Acetonitrile and methanol in HPLC grade were obtained from Merck (Damstadt, Germany). Acetone, tri-chloromethane, formic acid and other reagents were from Sinopharm Chemical Reagent (Shanghai, China). Double distilled water was purified by Simplicity 185 purity system (Millipore Corp., Bedford, MA, USA), and its conductivity was 18.2 MΩ cm.

Fig. 1 Chemical structures of pyridalyl and three potential metabolites.

2.2. Pest experiments

Larvae of H. armigera and S. exigua were collected from cotton plants in Wuhan, China. All pests were fed with standard diets containing wheat germ and soybean powder, and cultured at 26 ± 1 °C with a 16 h-light–8 h-dark period. When both pests formed a sufficient population, as shown in Fig. S2(a) and (b),† the adults were incubated at 60% relative humility and supplied with 10% sugar solution.

Pyridalyl was diluted to 30 mg L⁻¹ with double distilled water. Corn leaves were cut and immersed in the solution for 15 s, and clean leaves in double distilled water were used as the control. Then, the leaves were dried in the air and placed in Petri dishes with a 1% agar layer as shown in Fig. S2(c).† Every 4^th instar larva, starved for 4 h, was placed in a Petri dish as shown in Fig. S2(d).† Four hundred healthy larvae were selected for the pest experiments, and divided into an experimental group and a controlled group. Their excrement in three days were collected as samples.

2.3. Sample pretreatments

Thirty milligram excrement samples from the pests were completely ground in liquid nitrogen. Then, the sample was immediately mixed with three fold volumes of acetone, and extracted using a KQ2200DE ultrasonic generator (Shumei ultrasonic instrument, Kunshan, China). After that, it was centrifuged at 7000 rpm for 10 min, and then, the supernatant was collected. Prior to the combination of all supernatants, the residue was extracted another two times. Then, all supernatants were concentrated to 1.5 mL using a DN-12A nitrogen concentrator (Do-Chrom, Tianjin, China).

Shaking was also used to extract the excrement to compare its performance with ultrasonic treatment. When extracted by slowly shaking with a whist shaker (CAT, Germany), the sample was processed with 400 rpm at 26 °C for 10 min and 30 min, respectively. When extracted using ultrasound, the sample was treated with 60% output power for 5 min, 10 min, 15 min, and 20 min.

A SPE procedure was applied to pretreat concentrated fractions, as shown in Fig. 2. Specifically, 4 mL of aqueous acetonitrile (50 : 50, v/v) was used to activate the SPE column, which was then rinsed by 10 mL methanol, 5 mL aqueous acetonitrile (50 : 50, v/v), 2 mL water, 4 mL aqueous acetonitrile (50 : 50, v/v) and 4 mL methanol to remove the residues. After that, all concentrated fractions (around 1 mL) were loaded to the SPE column, which was eluted by 1 mL 0.1% formic acid aqueous solution (v/v), 2 mL aqueous acetonitrile (50 : 50, v/v), 4 mL methanol and 3 mL acetone. All eluants were collected and concentrated to 150 μL.

Fig. 2 SPE extraction procedures in the proposed method.

In order to optimize the SPE columns for pyridalyl and its metabolites, SPE 1: CEPHY123 (UCT, Bristol, PA, USA), SPE 2: CEC18153 (UCT, Bristol, PA, USA), SPE 3: CLEANERT SC18 (Agela Technologies, Tianjin, China), and SPE 4: SUPELCLEAN LC-18 (Supelco, Bellefonte, PA, USA) were compared in the extraction.

2.4. HPLC-MS determinations

A HPLC 1260 system (Agilent Technologies, USA) was used at a 0.2 mL min⁻¹ flow rate with a ZORBAX Extend-C18 column (2.1 × 50 mm, 1.7 μm, Agilent). The column temperature was set at 25 °C, and a 2 μL sample solution was injected for each separation. Acetonitrile (A) and the 0.1% (v/v) formic acid aqueous solution (B) were the mobile phases. The gradient elution was set as: 0–1 min 45% A, 1–10 min 45–90% A, 10–23 min 90% A.

A 6224 TOF-MS (Agilent Technologies, USA) was coupled with the HPLC system to achieve the proposed determination. The MS parameters were set as the following: scanning range 100–1100 amu, gas temperature 325 °C, drying gas 11 L min⁻¹, nebulizer 45 psig, voltage of capillary 4000 V. The formula of the analyte was extracted and simulated by the software Qualitative Analysis (version B.05.00). Pyridalyl solutions at 6.96 mg L⁻¹, 5.22 mg L⁻¹, 3.48 mg L⁻¹, 1.74 mg L⁻¹, 0.87 mg L⁻¹, 0.65 mg L⁻¹ and 0.435 mg L⁻¹ were obtained by adding acetonitrile as the diluent and determined for its liner calibration curve.

3. Results and discussion

3.1. Determination of pyridalyl

Under optimal conditions, the MS determination of pyridalyl is indicated in Fig. 3A. [M + H]⁺ (m/z 489.9745), isotopic [M + H]⁺ (m/z 491.9718, m/z 493.9691, m/z 495.9665) and [M + Na]⁺ (m/z 513.9474) were all observed. Considering the inorganic residues in the ESI source as well as the chemical components in the reference standard, the presence of [M + Na]⁺ seemed reasonable. It is worthy to emphasize the additives in the mobile phase, e.g., TFA, HFBA and formic acid, which were indispensable for a satisfactory ionization efficiency of pyridalyl. As shown in Fig. 3B, the EIC ([M + H]⁺, m/z 489.9745) suggested zero abundance for the pyridalyl, when the mobile phase was without any additives. This may be because the additives improved the volatility of the mobile phase as well as the ionization of pyridalyl.

Fig. 3 MS spectra of pyridalyl with and without formic acid. ((A) MS spectrum of pyridalyl by acetonitrile/formic acid aqueous solution (90 : 10, v/v); (B) EIC of pyridalyl by acetonitrile/water (90 : 10, v/v)).

Then, pyridalyl solutions at different concentrations were determined, and a linear calibration curve corresponding to the peak areas in the EICs was obtained as shown in Fig. S3.† It can be observed that its linear range is from 0.4 mg L⁻¹ to 7 mg L⁻¹.

3.2. Optimization of sample pretreatments

A mixture of 10 μL of a 6.96 mg L⁻¹ pyridalyl solution and 30 mg of excrement of H. armigera in the control group was used as the spiked sample to optimize the solvents in the sample pretreatments. Isopropanol, toluene, methanol, acetonitrile, chloroform, n-hexane, ethyl acetate, benzene, and acetone were used to pretreat the spiked sample, and their performances are compared in Fig. 4.

Fig. 4 The recovery of pyridalyl by different solvents.

It can be observed that n-hexane, ethyl acetate, benzene, and acetone indicated better recovery compared with the other solvents. Because of their weak polarity, solvents with no or weak polarity indicate a satisfactory recovery for pyridalyl. In addition, the volatility of the solvents also influenced their recovery. This was because the more volatile the solvent was, the faster the concentration would be, during the concentration of all solvents with various analytes. In this sense, a fast concentration would lose comparably less analyte, which thus improved the recovery. Isopropanol possesses a better recovery compared with toluene in Fig. 4, and this can probably be attributed to its stronger volatility. Overall, acetone was finally applied for its good recovery, strong volatility and low toxicity.

In addition, different extraction strategies are compared in Fig. 5, and it can be observed that 5 min ultrasonic extraction showed the best performance. And its recovery became worse with a longer extraction. This may be because of the high energy of the ultrasonic treatment, which would result in greater decomposition of pyridalyl with a longer time. On the contrary, the recovery with shaking was improved with a longer time. This may be related with the lower energy of shaking compared with ultrasonic treatments. Compared with the other treatments, 5 min ultrasonic treatment showed the best recovery, and it was chosen as the optimal sample pretreatment.

Fig. 5 The recovery of pyridalyl using different extractions.

Moreover, the performances of four SPE columns were compared as shown in Fig. S4.† It can be observed that SPE 2 and SPE 3 suggested better extractions. SPE 2 showed better extractions for pyridalyl, MP2 and MP3 compared with SPE 3, although its performance for MP1 was slightly worse than the latter. Therefore, SPE 2 was chosen as the optimal SPE column.

3.3. Optimization of ionization sources

Electrospray ionization (ESI) and atmospheric pressure ionization (APCI) were compared to optimize the ionization efficiency in the proposed method. For pyridalyl and all metabolites, ESI showed better performances compared with APCI as shown in Fig. 6. According to the chemical structures of pyridalyl and its metabolites, ESI would be sufficient to achieve their ionization, and APCI may be too strong for these analytes, all of which were in low concentrations. Therefore, ESI was appointed as the optimal ionization source.

Fig. 6 Comparison of ESI and APCI for pyridalyl and metabolites.

3.4. Confirmation of metabolites

The excrement of H. armigera in the experimental group was determined by the proposed method: SPE followed by HPLC-TOF/MS. Pyridalyl and three potential metabolites MP1 ([M + H]⁺, 382.022), MP2 ([M + H]⁺, 440.027) and MP3 ([M + H]⁺, 453.998) were determined as shown in Fig. 7(1–4).

Fig. 7 EICs of pyridalyl and three metabolites in experimental and spiked excrement.

In order to confirm the preceding potential metabolites, pyridalyl was added in the excrement from the control group. Then, this spiked sample was determined by the proposed method, and EICs of pyridalyl and three potential metabolites are presented in Fig. 7(5–8). From the comparison between Fig. 7((2), (3), (6) and (7)), MP1 and MP2 could be confirmed to be metabolites of pyridalyl. However, MP3 was observed in the spiked sample as shown in Fig. 7(8). This suggested that MP3, which was reported to be a metabolite of pyridalyl in other experimental organisms,^7,21 would no longer be its metabolite in H. armigera.

In order to be sure of the source of MP3, its extracted ion chromatograms (EIC) were extracted from all total ion chromatograms (TIC) for the linear calibration curve of pyridalyl. It could be observed that there was an obvious peak in each EIC. Then, all the peak areas for MP3 were collected and calibrated for its linear equation, in which the slope was 0.741. Considering the linear slope of pyridalyl is 0.633 in Fig. S3,† it could be concluded that the content of MP3 increased with the content of pyridalyl, and their growth were approximately synchronous. Based on the chemical structure of MP3, it could be generated from the pyridalyl dehydrochlorination process. Therefore, MP3 was predicted to be from the hydrolysis of pyridalyl by electrospray ionization, which was under high temperature and high voltage.

If the preceding hypothesis was established, the content of MP3 would decrease using the mobile phase without water. Both pyridalyl and MP3 could be observed when the mobile phase included water, and their MS spectra are shown in Fig. 8A and B. When there was no water in the mobile phase, MP3 was absent from its EIC in Fig. 8D, while pyridalyl was well observed in Fig. 8C. The foregoing results revealed that the hydrolysis of pyridalyl should at least be one of the sources of MP3. Therefore, it is subjective to regard all the MP3 in Fig. 7(4) to be the metabolite of pyridalyl.

Fig. 8 MS spectra for pyridalyl and MP3. ((A) MS spectrum of pyridalyl using acetonitrile and water; (B) MS spectrum of MP3 using acetonitrile and water; (C) MS spectrum of pyridalyl using acetonitrile with 0.1% formic acid and without water; (D) EIC of MP3 using acetonitrile with 0.1% formic acid and without water).

In addition, corn leaves in which pyridalyl was present, were fed to H. armigera and S. exigua, and evaluated by the proposed method. EICs corresponding to pyridalyl and three potential metabolites were extracted, and their content was calculated based on their peak areas. It was found that the content of pyridalyl and MP3 were 2.51 mg kg⁻¹ (n = 5, RSD = 1.85%) and 0.90 mg kg⁻¹ (n = 5, RSD = 1.64%), respectively. Moreover, there was no MP1 or MP2 detected from the corn leaves. This indicated that MP1 and MP2 were not generated in leaves, and they should be metabolites of both Lepidoptera insects. MP3 was detected without the presence of any pests, and this result supported the hypothesis that MP3 came from the dehydrochlorination of pyridalyl. To sum up, MP3, a reported metabolite in other experimental organisms,^7,21 should not be regarded as a metabolite of pyridalyl in H. armigera and S. exigua.

3.5. Quantitative determination of pyridalyl and its metabolites in the excrement of H. armigera and S. exigua

The pyridalyl and metabolites in the excrement of S. exigua were determined as shown in Fig. S5.† Because H. armigera and S. exigua are both Lepidoptera, Noctuidae insects, pyridalyl should be metabolized through a similar pathway in both pests. There were few differences between Fig. S5 and 7(1–4), with the data obtained from H. armigera. This result indirectly supports the reliability of the proposed method.

The content of pyridalyl and three potential metabolites in the excrement of both pests are compared in Table 1. The content of MP1 was greater than the content of MP2 in both pests. Moreover, it could be observed that content of pyridalyl, MP1 and MP3 from both pests were similar, while the content of MP2 in the excrement of S. exigua was 13% higher than its counterpart in H. armigera. This may be related to the different content of the unknown target enzyme for pyridalyl in both pests.

Table 1 The content of pyridalyl, MP1, MP2 and MP3 in the excrement of H. armigera and S. exigua

	H. armigera		S. exigua
	W (mg kg^−1)	RSD (%, n = 3)	W (mg kg^−1)	RSD (%, n = 3)
Pyridalyl	1.14	1.23	1.17	0.99
MP1	1.15	5.08	1.12	2.68
MP2	0.92	0.52	1.04	2.55
MP3	0.87	1.38	0.84	0.37

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3.6. Proposed metabolic pathway of pyridalyl in H. armigera and S. exigua

The metabolic pathway of pyridalyl in both pests is proposed in Fig. 9, based on their molecular structures as well as their content in the excrement. To be exact, MP2 was predicted to be formed from the acylation of the olefinic bond in the side chain. Furthermore, MP1 was the product of pyridalyl or MP2 by losing the whole side chain. Moreover, MP3 should not be regarded to be a metabolite of pyridalyl in Lepidoptera insects, and it should be related with the hydrolysis of pyridalyl. Therefore, the metabolism of pyridalyl in Lepidoptera insects was deduced to be different from its reported metabolism in mammals.^7,21

Fig. 9 Proposed metabolic pathway of pyridalyl in H. armigera and S. exigua (×××××× stands for “not happened”).

4. Conclusions

A qualitative and quantitative method based on SPE followed by HPLC-TOF/MS to determine pyridalyl and its potential metabolites in the excrement of H. armigera and S. exigua was developed. Under optimal conditions, qualitative and quantitative determinations for pyridalyl and three potential metabolites were achieved. Moreover, the metabolic pathway of pyridalyl in both pests was proposed, and its metabolism in both Lepidoptera insects was indicated to be different from its metabolism in mammals. Compared with reported strategies^7,21, the proposed method took less time from several days to 2 hours, and reduced the sample consumption from several grams to 30 mg. The proposed method provided the possibility to determine pesticides and their metabolites in complex samples, and it would be of certain value in finding out the target enzyme of pyridalyl in Lepidoptera insects finally.

Acknowledgements

Prof. Guangfu Yang and Dr Jun Li (Central China Normal University) are sincerely appreciated for providing the pyridalyl. Dr Roma Smith and Dr Elaine Cage (Wuhan University, China) are acknowledged for their revisions of English usages. This research was supported by the National Basic Research Program of China (2010CB126103) and the National Key Technology R&D Program (2011BAE06B05). This research was also financially supported by self-determined research funds of CCNU from the colleges’ basic research and operation of MOE (No. CCNU14A05005, CCNU15KFY004) and the National Natural Science Foundation of China (No. 21205044, 21302060).

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Footnote

† Electronic supplementary information (ESI) available: Spectroscopic data with ¹H NMR spectra, EIC spectra, photographs of pests, and the calibration curve. See DOI: 10.1039/c5ra21984a

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