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Note 94: Detection of Nepetalactone in the Nepeta Cataria Plant by Thermal Desorption GC/MS

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By Robert Frey, Ronald E. Shomo II and John J. Manura, Scientific Instrument Services, 1027 Old York Road, Ringoes, NJ 08551
(presented at ASMS2007)

Photo of Nepeta Cateria (catnip) plat

Introduction

Nepetalactone, the active ingredient found in Nepeta Cataria, commonly known as Catnip, makes most cats behave in an intoxicated state. The leaves, stems, and seed pods of Nepeta Cataria are covered with microscopic bulbs called trichomes. Trichomes store the nepetalactone oil until they reach maturity and burst. Nepetalactone is released into the atmosphere as cats rub or chew on the catnip plant, bursting these bulbs containing the active ingredient.1 An olfactory gland detects the nepetalactone and produces a euphoric high for the cat. Previous attempts to characterize and quantify nepetalactone involved labor intensive solvent extractions. Utilizing direct thermal desorption GC/MS on fresh grown catnip, we were able to detect nepetalactone in different parts of the plant and determined which portion of the plant contained the most nepetalactone without the use of solvent extractions. The sample preparation is less than 5 minutes with Direct Thermal Extraction.

Materials and Methods

Nepeta Cataria was grown in an outdoor garden from seedlings and harvested when the plant was mature. The harvested plant was carefully separated into its components (leaves, stems, seeds, flower pods, roots, and base stalk) and placed into individual glass containers and allowed to air dry for two weeks prior to analysis.

Desorption tubes (stainless steel 4 mm id x 4 inch long) were heat conditioned in a 24 port conditioning oven for 4 hours at 320 °C with an ultrapure Nitrogen gas purge at 25 ml/min. Tubes were removed from the heat conditioning oven then allowed to cool for 10 minutes and capped with stainless steel caps with a PTFE seal to avoid adsorption of contaminants.

Nepeta Cataria samples (2.7-13.8 mg) were loaded into a pre-conditioned desorption tube that had a small glass wool plug (2-3 mm) in one end to prevent the sample from falling out of the tube. The desorption tube was connected to an SIS ADS2000 Thermal Desorption System. A 35 mm desorption needle was connected to one end of the desorption tube. The needle acts as the transfer line when desorption takes place insuring the highest transfer of sample to the GC and eliminates any carryover effects observed in many other desorption systems.

The desorption tube was placed in a 12 position autosampler. The Thermal Desorption unit (Figure 1) picked up the desorption tube from the autosampler, performed a 1 minute He purge using the GC's carrier gas, and then injected the needle through the septum of the GC injection port. After the injection port pressure stabilized, all of GC carrier gas flow was diverted through the desorption tube.

Figure 1 - schematic of
SIS AutoDesorb, Automated Short Path Thermal Desorption

Desorption blocks heated to 150°C then closed around the desorption tube and heated the tube for 5 minutes. During the desorption process, a cryofocusing unit (SIS CryoTrap) inside the GC oven cooled a 2" section of a guard column (0.53 mm ID deactivated fused silica) to -45°C with CO2. This trapped the desorbed compounds during the desorption process improving the chromatography by cryofocusing the analytes.

Thirty seconds after the desorption process was completed, the cryogenic cooling was stopped and the CryoTrap ballistically heated to 200°C and held for 3 minutes to elute the trapped components at the head of the capillary column.

The Thermal Desorption controller then sent a remote start signal to the GC to begin the temperature ramp from 40°C to 200°C at a rate of 5°C per minute and then to 280°C at 10°C per minute for a total GC run time of 45.0 minutes. The MS transfer line was held at 280°C. The GC (Agilent 6890) oven contained a capillary column Agilent HP5MS (5% Phenyl Polysilphenylene-siloxane) (0.25 mm ID x 30 m length x 0.250 μm film thickness). The GC injection port was held at 280°C and the GC was run in "split" mode set at a 5:1 ratio. Total helium flow through the desorption tube was 8.8 ml/minute for the length of the 5 minute desorption run. Helium flow through the GC capillary column was 1.0 ml/minute. A Mass Spectrometer (Agilent 5973 MSD) was operated in Electron Impact Mode (EI) and scanned over a mass range between 35 and 500 daltons.

All analytes were identified using Agilent ChemStation software with the NIST AMDIS software using the NIST mass spec library as well as the Wiley NBS mass spec library running in the full scan mode. Quantification was performed utilizing dodecalactone injected onto a 25 mg Tenax® TA adsorbent bed in a preconditioned desorption tube.

Results and Discussion

Thermal Desorption GC/MS proved to be an extremely effective analytical tool for measuring the concentration levels of nepetalactone found in various parts of the Nepeta Cataria plant. The nepetalactone levels in portions of the plant were quite significant, but the ADS2000 Thermal Desorption system's design utilizing a "short-path" transfer line eliminated any carry over problems that have been problematic with other desorption systems. A summation of the levels of nepetalactone found in each portion of the plant is shown in Table 1

Table 1
SampleWeight of Sample (mg)Total Nepetalactone (ng)Nepetalactone per Sample (ng/mg)
Root6.0Not DetectedN/A
Main Stalk5.6Not DetectedN/A
Seed13.8254.518.4
Lead Stems2.7227.984.4
Seed Pods13.83679.7408.9
Outer Leaf7.55859.0781.2
Middle Leaf5.714,728.32,583.9

Figure 2 shows the chromatogram resulting from the analysis of 2.7 mg of a portion of the lead stem of the plant. Figure 3 is the mass spectrum generated from nepetalactone found in the lead stem.

Figure 2 - GC chromatogram of Nepetalactone in lead stem
Figure 3 - MS spectrum of Nepetalactone in lead stem

Figure 4 is the chromatogram of 13.8 mg of catnip seeds analyzed by Thermal Desorption GC/MS. In addition to the nepetalactone that is found in the seeds, there are a whole host of additional components that can be identified. The sensitivity of the ADS2000 allows for full scan mass spectral data acquisition so a single run can yield multiple component identification. As an example two additional components were identified as caryophyllene and its oxide. Caryophyllene is a natural bicyclic sesquiterpene that is a constituent of some essential oils. Caryophyllene is notable for having a cyclobutane ring, a rarity in nature. It is also responsible for the spicyness of black pepper2. The ability to utilize multiple component analysis that is an inherent feature associated with the ADS2000 leads to a more complete analysis of the sample in less time.

Figure 4 - GC chromatogram of catnip seeds Conclusion

Nepetalactone was found to be concentrated in the seed pods and surrounding leafy structures of the Nepeta Cataria plant. This is consistent with the results reported in the literature1. Concentrations of nepetalactone were found to be up to 2,583.9 ng/mg of plant material. This level of analyte could lead to significant carry-over in many thermal desorption systems. The "short-path" design utilized in the ADS2000 eliminated any carry-over issues with this application. Thermal Desorption GC/MS was the method of choice for detection of nepetalactone, it required minimal sample prep (just trim a portion of the plant and place in the desorption tube), and no solvent based extractions. The sensitivity of the thermal desorption system allowed the mass spectrometer to be operated in full scan mode. This yielded a complete analysis of the volatile and semi-volatiles present in the sample in a single run.

References
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