Efficacy of an improved method to screen semiochemicals of insect

Abstract

Background A combination of gas chromatography-electroantennographic detection (GC-EAD) and gas chromatography-mass spectrometry (GC-MS) is typically used to screen active compounds that play a role in the regulation of insect behavior. This method uses two kinds of gas chromatography (GC) equipment and needs to compare compounds between the two chromatograms, and it is tedious and costly. To improve detection efficiency, as well as reduce costs and the rate of missed detection, we designed a system connecting gas chromatography (GC), electroantennography (EAG), and mass spectrometry (MS), with MS used instead of the flame ionization detector (FID) as the GC-EAD detector. To verify the feasibility of the improved method, we compare two methods—GC-EAG-MS and GC-EAD—through a series of experiments. Some researchers made similar improvements, but these were not compared with GC-EAD, and their method needed to be improved in the synchronization and split ratio. Our method has been optimized and improved in these aspects. Methods Helicoverpa armigera was the test organism; the improved method and conventional method were used to detect known and unknown compounds, as well as screen out active compounds that could generate responses in H. armigera antennae. Results Screening known single compounds using the two methods, the active compound benzaldehyde was detected in all seven concentrations of solution. By using the two methods, the five same active compounds of Helicoverpa armigera were detected in high concentration solution of the mixed compounds (100 mg L−1, 50 mg L−1); the four same active compounds were detected at 20 mg L−1 concentration; two identical same compounds were detected in low concentration solution (concentrations of 10 mg L−1 and below). By using the two methods, six identical active compounds of Helicoverpa armigera were detected in unknown compounds. Conclusion The improved method was consistent with the conventional method in terms of accuracy and sensitivity. However, compared with the traditional methods, Gas chromatography-electroantennographic-mass spectrometry (GC-EAG-MS) saved the cost of GC and FID equipment, thereby greatly lowering the experimental cost. In the experiment, GC-EAG-MS combined the two experimental operations of screening active substances by GC-EAD and identifying active substances by GC-MS into one, which not only reduced the experimental steps, but also avoided the false positive caused by the comparison of the two chromatograms, and it greatly reduced the difficulty level of the overall experimental analysis. GC-EAG-MS is more convenient, efficient, economical, and practical, and could confidently replace traditional methods. With further optimization, it could be widely applied in the study of plant and insect chemical ecology.

GC-MS/FID/EAD: A method for combining mass spectrometry with gas chromatography-electroantennographic detection

“…In comparison to other published systems utilizing an MS with GC-EAD, the non-linear relationship between retention times on the MS versus the FID chromatograms observed in our runs may relate to our use of columns after the split which have a thickerbore than those utilized by other practitioners (0.25 mm ID on both sides of the split in the case of our previous single Y-splitter configuration, 0.32 ID and 0.15 mm ID leading to the FID/EAD and MS, respectively, in the system described here, as opposed to combinations of 0.1-0.2 mm ID in other systems, Weissbecker et al, 2004;Li et al, 2021). Using thicker-bore columns also requires longer column lengths after the split in order to maintain the vacuum on the MS, which may further exacerbate the problem.…”

“…Previously described GC-EAD systems that incorporated an MS have all replaced the FID with an MS (Weissbecker et al, 2004;Schott et al, 2013;Bohman and Peakall, 2014;Li et al, 2021). Balancing the vacuum at the outlet for the MS with the EAD outlet at atmospheric pressure has usually been achieved by reducing the column bore leading to the MS. Other authors have not reported whether they observed a non-linear relationship between the elution times of individual compounds at the MS outlet relative to the EAD outlet, although Li et al (2021) report using a GC calculator to work out the precise lengths of column after the effluent split that were required to obtain simultaneous elution at the antenna and MS. We found this effect to be extremely challenging to overcome in a previously tested configuration using a single Y-splitter to divide effluent between EAD and an MS, since antennal depolarizations drifted apart from corresponding peaks in the total ion chromatogram (TIC) obtained from the MS as the run progressed. Because the difference between the antennal depolarization and the corresponding peak in the chromatogram changed throughout the run (it was not simply shifted by a constant time), it was very challenging to accurately assign active compounds in complex odor blends with closely eluting peaks.…”

“…The GC-MS/EAD system described by Li et al (2021) includes a pneumatic control module that allows precise control over the effluent split, although altering the split ratio affected the column lengths downstream of the splitter that were required to optimize elution at the MS and the antenna. Our system described here would result in a 1:1 effluent split between the FID and the EAD.…”

“…Previously described GC-EAD systems that have incorporated an MS (Weissbecker et al, 2004;Schott et al, 2013;Bohman and Peakall, 2014;Li et al, 2021) have replaced the FID with an MS. Here, we propose an alternative method of incorporating MS into a GC-EAD system where the FID is maintained in the system.…”

Identification of Bioactive Plant Volatiles for the Carob Moth by Means of GC-EAD and GC-Orbitrap MS

“…In the current proof-of-principle, GC-EAD and GC-Orbitrap MS were used sequentially. However, it is easily possible to combine EAD and MS in parallel [36,37] and omit the need to compare chromatograms [38]. In combination with Orbitrap MS, the method becomes even more powerful.…”