Introduction

The two-dimensional gas chromatography with time-of-flight mass spectrometry detection (GC×GC-TOFMS) is a recently developed instrumental technique that offers substantially better analytical selectivity than a classical GC-MS, and thus should be an ideal tool for analysis of complex mixtures of natural origin. In principle it consists of two GC systems connected by an interface with a cryogenic trap which repeatedly condensates fractions of compounds eluting from the primary column (the fraction width is optional) and releases them periodically as short pulses to the secondary column. Such system preserves the GC separation obtained in the first dimension, allows additional separation in the second dimension, and increases the sensitivity and signal to noise ratio dramatically. Since the second column is quite short (1 - 2 m), the system produces very narrow peaks (down to 50 ms, depending on the frequency of modulation) and only the coupled TOFMS detector with an extraordinarily high acquisition rate (up to 500 spectra/s) allows full-scan screening. TOFMS detector offers not only the acquisition rate sufficient to keep up with fast GC runs of the second dimension, but also its source of ionisation operates at a pulsed nature. The pulsed mode avoids the problem of spectral skewing common in continuous ionisation mode due to the rapidly changing concentration flows into the source. This allows TOFMS detector to operate with a high accuracy independently on concentration. Additionally, sophisticated software algorithms perform automatic data processing, looking for all present compounds in a given chromatogram while mathematically separating spectra of coeluting peaks. From this, analytes masked by matrix components can easily be identified and accurately quantified.

The persimmon bark borer, Euzophera batangensis Caradja, is a small moth from Pyralidae family distributed widely in East Asia (China, Korea, Japan). Originally harmless polyphagous insect became a serious pest of the jujube tree (Ziziphus jujuba) orchards in North China. E. batangensis has an economical importance especially in Hebei province where jujube is the key fruit tree cultivated on area over 100 000 hectares. Harmfulness of E. batangensis is closely associated with girdling (the removal of a ring of phloem on tree trunk) which is a common horticultural practice used to enhance jujube fruit harvest. The E. batangensis overwinters beneath the bark as larva and occurs in several generations per year. Females lay eggs to fresh girdles where the hatched larvae feed on the tree cambium (callus), and as a result of larval feeding, jujube tree vigour is heavily diminished (trees are dying often). It is difficult to control E. batangensis due to its endophytic behaviour. Sex pheromone of the species is not known. Its identification might innovate the integrated pest management strategies and increase their efficiency substantially.

Results

GC-EAD analyses of E. batangensis gland extract showed one major EAD response (Fig. 1A). The compound eliciting the EAD response was not FID-detectable (Fig. 1B). Since Pyralidae usually utilize linear aliphatic C₁₂ - C₁₄ acetates, alcohols oraldehydes with one or more double bonds we determined Kovats' index of EAD activity (I_K,EAD) and compared it with tabular I_K values of pheromone-like compounds. The EAD Kovats' index determined at DB-5 column (I_K,EAD = 1681) didn't correspond with any tabular I_K value suggesting that the prominent EAD activity is not associated with C₁₂ - C₁₄ saturated or monounsaturated alcohol, acetate or aldehyde. This observation indicated the presece of the additional double bonds within the active molecule. Further GC-EAD experiments with available diunsaturated synthetic standards showed a perfect match of EAD response of unknown compound from the pheromone gland extract with EAD response to synthetic 9Z,12E-tetradeca-9,12-dien-1-ol (9Z,12E-14:OH; Figs. 1C and 1D).

Standard one-dimensional GC-MS analysis of the extract didn't provide any relevant mass spectrum to confirm such identification, since MS signal was deeply hidden in the background noise. But the GC×GC-TOFMS analysis of the area corresponding with EAD activity disclosed several compounds (Fig. 2AB) including 9Z,12E-14:OH. Figure 2B also illustrates nicely the high "wall" of the column bleed that would hide the analyte peaks in standard 1D-GC analysis, but is really well separated in the second dimension of the GC×GC experiment. The mass spectrum (Fig. 3) and 2D-retention times of the synthetic 9Z,12E-14:OH were identical with 9Z,12E-14:OH found in the gland extract (Fig. 4). The quantitative analysis showed that the detection threshold of GC×GC-TOFMS to 9Z,12E-14:OH was roughly 10 pg, which is substantially lower than detection limits of most available standard one-dimensional GC-MS systems. Such sensitivity almost approaches the sensitivity of GC-EAD.

The intimate inspection of 3D-surface plots of GC×GC-TOFMS chromatograms in the EAD activity area showed besides 9Z,12E-14:OH (peak 1; Fig. 2B) also traces of another pheromone-like compound eluting slightly earlier than 9Z,12E-14:OH in both dimensions (peak 2; Fig. 5A) as well. Mass spectrum of this compound together with two-dimensional retention parameters was identical with 9Z-tetradec-9-en-1-ol (9Z-14:OH), suggesting a possibility that E. batangensis might have two-component pheromone. The presumable physiological function of 9Z-14:OH was confirmed by separate GC-EAD experiments with synthetic 9Z-14:OH (Fig. 6A; co-injection with 9Z,12E-14:OH) and more concentrated gland extract (Fig. 6B). The amount of 10 ng of this compound elicited obvious but substantially weaker EAD response in comparison with 9Z,12E-14:OH response.

Conclusions

The presented study showed the perfect suitability of GC×GC-TOFMS system for infochemicals analyses. Based on results of GC-EAD and GC×GC-TOFMS analyses we suggest that 9Z,12E-tetradeca-9,12-dien-1-ol and 9Z-tetradec-9-en-1-ol are female sex pheromone components in Euzophera batangensis. Later field tests in China supported our suggestions...

Research team

Michal Hoskovec, Blanka Kalinová, Jan Žďárek, Pavel Jiroš

Published in...

Kalinová B., Jiroš P., Žďárek J., Wen, X., Hoskovec M.:
GC × GC/TOF MS technique - A new tool in identification of insect pheromones: Analysis of the persimmon bark borer sex pheromone gland.
Talanta 69 (3): 542-547, 2006.