# HS‐FET‐GC/MS‐Method Development and Validation for Analysis of 45 Terpenes—Creating a Complementary Tool for Comprehensive Profiling of Cannabis Flowers in Forensics

## TL;DR

This paper introduces a validated method to analyze 45 terpenes in cannabis flowers, helping distinguish between medicinal and recreational cannabis in forensic contexts.

## Contribution

A new HS-FET-GC/MS method was developed and validated for forensic analysis of 45 terpenes in cannabis flowers.

## Key findings

- The method successfully validated 45 terpenes, including monoterpenes, monoterpenoids, sesquiterpenes, and sesquiterpenoids.
- Thermal instability was observed in higher-boiling terpenes during FET-sampling.
- Cannabis strains with similar THC content showed distinct terpene profiles.

## Abstract

Cannabis sativa
is one of the oldest and most versatile plants with many facets ranging from intoxicant to medicine. Legalisation of medicinal cannabis leads to an increasing complexity of specific forensic questions to distinguish between recreational and medicinal use, for example, in context with participation in road traffic. Hence, there is a recent interest in finding objective markers that enable the differentiability of cannabis flowers. Terpenes, volatile hydrocarbons with a modular construction principle of isoprene subunits, are currently suggested as a second substance class alongside phytocannabinoids for the classification of cannabis material. A headspace full evaporation technique gas chromatography mass spectrometry (HS‐FET‐GC/MS) methodology was successfully validated according to forensic guidelines for the analysis of 45 terpenes in cannabis flowers including 16 monoterpenes, 16 monoterpenoids, 7 sesquiterpenes and 6 sesquiterpenoids. FET‐sampling was developed in detail experimentally, revealing evidence of thermal instability of higher‐boiling terpenes. Validation included selectivity, linearity of calibration (ranges 10–2000 μg/g), analytical limits (at least 6 μg/g), accuracy (bias) as well as intraday and interday precision. The use of a retention time index mixture as an internal standard and measurement in SIM‐scan mode also allows for the qualitative identification of further terpenes present in cannabis. Application to a set of cannabis strains with similar Δ9‐THC content demonstrated differences and similarities in their terpene profiles.

Legalisation of medicinal cannabis leads to an increasing complexity of specific forensic questions. Terpenes, volatile hydrocarbons with modular construction principle of isoprene subunits, are suggested as second substance class alongside phytocannabinoids for classification of cannabis material. A headspace full evaporation technique gas chromatography mass spectrometry (HS‐FET‐GC/MS) methodology was developed and successfully validated according to forensic guidelines for analysis of 45 terpenes in cannabis flowers. Application to cannabis strains with similar Δ9‐THC‐content demonstrated differences and similarities in their terpene profiles.