Determination of Terpenoids in Plant Leaves by GC-MS: Development of the Method and Application to Ocimum basilicum and Nicotiana langsdorffii

Abstract

In this work a simple and sensitive analytical method was developed for the determination of terpenoids in plants using gas chromatography coupled to mass spectrometry (GC-MS). Three extraction methods were tested on Ocimum basilicum leaves to maximize terpene extraction: Steam Distillation, Ultrasound-Assisted Extraction, and Microwave-Assisted Extraction (MAE). Best results, both in number of identified compounds and in their concentration, were obtained with MAE, which was used in all subsequent analyses. In particular five terpenoids (α-pinene, β-pinene, limonene, linalool, and α-terpineol) were chosen for the quantitative determination, performed in Selected Ion Monitoring mode. Calibration curves using dodecane as internal standard were drawn, showing good correlation coefficients (0.9981–0.9993). All figures of merit were satisfactory; limits of detection were in the range 14–27 ng for all analytes. The method was also applied to some specimens of wild-type and transgenic (GR and rolC) Nicotiana langsdorffii. Plants were exposed to water and chemical stresses to evaluate possible effects on terpene content. Linalool was present in higher quantities in the plants GR and rolC; both stresses produce a decrease of its concentration if compared to the non-stressed plants. The concentration of α-terpineol, detected only in WT and GR plants, was higher for the genetically modified plant, substantially confirming the trend observed for linalool.

Keywords:

• GC-MS
• Genetically modified plants
• Nicotiana langsdorffii
• Ocimum basilicum
• Terpenes

Acknowledgments

This study was partially funded by the Italian Ministry of University and Research (MIUR) within the framework of the PRIN-2007 project.

Notes

Note: Analytes were identified by the NIST mass spectra library (quality match > 97%); terpenoids confirmed by the comparison with retention times of standards are reported in bold. All compounds are terpenoids except those indicated with*.

Note: Retention times (RT), SIM ions for the quantitative analyses (quantifier ions are reported in bold type), calibration curve parameters (*referred to the curve equation y = ax + b) and limits of detection and quantitation of the analytes expressed in nanograms (calculated as the concentration that would give three and ten times, respectively, the standard deviation of the blank of the whole procedure).

Note: NL WT refers to the wild-type specimens; NL GR to the plants transformed using a binary Agrobacterium vector bearing the gene for the rat glucocorticoid receptor; and NL rolC transgenic for the Agrobacterium rhizogenes rolC gene. CS refers to the plants subjected to the chemical stress and WS to the plants exposed to the water stress.