Headspace-Solid Phase Micro Extraction-Gas Chromatography-Quadrupole Mass Spectrometry-based metabolomics for kinetics tracking of natural herbicides’ volatile residues: a simple non-destructive method

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Headspace-Solid Phase Micro Extraction-Gas

Chromatography-Quadrupole Mass Spectrometry-based metabolomics for kinetics tracking of natural herbicides’

volatile residues: a simple non-destructive method

Hikmat Ghosson, Delphine Raviglione, Marie-Virginie Salvia, Cedric Bertrand

To cite this version:

(2)

Headspace-Solid Phase Micro Extraction-Gas Chromatography-Quadrupole Mass Spectrometry

-based metabolomics for kinetics tracking of natural herbicides’ volatile residues:

a simple non-destructive method

Hikmat Ghosson

1,2,*

_{, Delphine Raviglione}

1,2

_{, Marie-Virginie Salvia}

1,2,3

_{, Cédric Bertrand}

1,2,3,4

*: hikmat.ghosson@univ-perp.fr

1: PSL Université Paris: EPHE-UPVD-CNRS, USR 3278 CRIOBE, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France 2: UFR Sciences Exactes et Expérimentales, Université de Perpignan Via Domitia, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

3: Laboratoire d’Excellence « CORAIL », Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France 4: S.A.S. AkiNaO, Université de Perpignan, 52 Avenue Paul Alduy, 66860 Perpignan Cedex, France

Despite their known risks, herbicides are still essential for agriculture. Thus, natural herbicides are increasingly recommended to replace synthetic ones. However, there are still limitations in studying the environmental fate of several of them. The main reason is the lack of methods dedicated for this type of complex bio-herbicides. Hence, in the framework of the Environmental Metabolic Footprinting (EMF) approach, the current work presents a method dedicated to analyze the volatile residues of herbicides applied on soil. The aim is to track the evolution of the volatile compounds issued from the herbicide by an untargeted metabolomics-based kinetics, in order to determine the “resilience time” of the gaseous phase above the soil. The approach aims to explore the environmental fate of these herbicides. In fact, it will supplement other methods based on the EMF approach recently developed. Moreover, it can estimate the exposure of farmers, insects and plants to potential toxic volatile substances.

The method is based on Headspace-Solid Phase Micro Extraction-Gas Chromatography-Electronic Impact Ionization-Quadrupole Mass Spectrometry (HS-SPME-GC-EI-Q MS). The HS-SPME provides a non-destructive extraction. This allows reducing the number of samples. The GC separation technique provides high reproducible analysis for volatile compounds. In addition, it allows the calculation of the Retention Index (RI) as a tool for the molecular characterization. On the other hand, despite the low resolution of the Quadrupole mass analyzer, the Electronic Impact Ionization provides reproducible MS fragmentation patterns used for spectral library search and fast putative identification of unknown compounds.

Context

Giacomoni et al. Bioinformatics (2015), 31(9):1493–1495. doi:10.1093/bioinformatics/btu813

Guitton et al. Int. J. Biochem. Cell Biol. (2017), 93:89–101. doi:10.1016/j.biocel.2017.07.002

Patil et al. Sci. Total. Environ. (2016), 566-567:552–558. doi:10.1016/j.scitotenv.2016.05.071

Popovici et al. Appl. Environ. Microbiol. (2010), 76(8):2451–2460. doi:10.1128/AEM.02667-09

Salvia et al. Environ. Sci. Pollut. Res. Int. (2017), 25(30):29841–29847. doi:10.1007/s11356-017-9600-6

Wehrens et al. J. Chromatogr. B. (2014), 966:109–116. doi:10.1016/j.jchromb.2014.02.051

References

Acknowledgments

1. Introduction

3. Results

2. Material and Methods

4. Conclusion

1.1. Environmental-Analytical concept

1.2. Myrica gale extract



β-Triketone natural herbicide



Complex mixture of metabolites



Active compound: Myrigalone A



Several unknown compounds

Demethoxymatteucinol Uvangoletin

Myrigalone E Myrigalone A

Myrica gale flower

(Popovici et al. 2010)

 6 g of soil incubated in a 20 mL crimped vial

 Two 18Gx1.5" needles to assure aerobic conditions

 Control (Ctrl) vs. Pesticide-spiked (MyrN) soil:

• 8 kinetics points (Day 1 to 38), 3 replicates/group

 24h day/night cycle in climatic chamber:

• 28°C/18°C, 40%/65% RH, light/dark 2.1. Sample preparation © CRIO BE USR 3 27 8 – EPHE -CN RS -UP V D

 Automated HS-SPME extraction (Thermo Triplus):

• Fiber type: DVB/CAR/PDMS

• Extraction time/temperature: 30 min/40°C

 Thermo Focus GC, DB-5MS column, 35 min/run

 Thermo DSQ II EI-Quad MS: m/z 40-400 scan range

BE USR 3 27 8 – EPHE -CN RS -UP V D

Project co-financed by the European Regional Development Fund (ERDF)

 Detection of a wide profile of volatile

xenometabolome

 No volatile endometabolites were detected

0 5 10 15 20 25 30 35 Time [min] 0.0 0.5 1.0 1.5 8 x10 Intens.

 Low matrix effect  Low interferences

 Putative identification of several volatile

xenometabolites C:\Users\...\Data\20190219_009 02/20/19 01:05:23 RT:0.00 - 36.29 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 0 20000000 40000000 60000000 80000000 100000000 120000000 140000000 NL: 1.48E8 TIC F: + c Full ms [40.00-400.00] MS 20190219_009 NL: 1.42E8 TIC F: + c Full ms [40.00-400.00] MS 20190219_006 NL: 1.40E8 TIC F: + c Full ms [40.00-400.00] MS 20190219_011 RT:0.00 - 36.30 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 0 50000000 100000000 150000000 200000000 250000000 300000000 NL: 2.79E8 TIC F: + c Full ms [40.00-400.00] MS 20190219_008 NL: 2.92E8 TIC F: + c Full ms [40.00-400.00] MS 20190219_010 NL: 3.24E8 TIC F: + c Full ms [40.00-400.00] MS 20190219_007

Overlaid TICs of 3 replicates

Control (Ctrl) – Day 1

Overlaid TICs of 3 replicates Spiked (MyrN) – Day 1

 Non-destructive, simple, cheap and green extraction method

 Detection and characterization of volatile xenometabolites

 Reliable performance for kinetics tracking on the same samples

R

Gate

_Profile

A B C D E F G B: L-4-Terpineol Prediction: 56.1% (NIST) C: L-a-Terpineol Prediction: 38.3% (NIST) D: a-Terpinyl acetate Prediction: 43.4% (NIST) A: Eucalyptol Prediction: 83.8% (NIST) F: Aristolene epoxide Prediction: 15.7% (NIST) G: Germacrone Prediction: 69.4% (NIST) E: 3,5,11-Eudesmatriene Prediction: 28.5% (NIST) 3.1. Extracted profile 3.2. Kinetics tracking

 38 days kinetics tracking: application on the same samples

 Degradation of xenometabolites: resilience was almost reached after 38 days

PC3 : 8. 21 % PC1: 27.18% Kinetic tracking and evolution of xenometabolome profile

EICs of fragments of major xenometabolites

Kinetic tracking and evolution of xenometabolome profile

PCA of Control vs. Spiked samples according to time points

Dissipation of xenometabolites - T01 - T02 - T03 - T04 - T08 - T17 - T24 - T38

Similarity of metabolic profiles is almost reached on day 38

(Ctrl vs. MyrN)