# Optimized Extraction Method for Neutral Cannabinoids Quantification Using UHPLC-HRMS/MS

## TL;DR

This paper presents a faster and more cost-effective method to extract and quantify neutral cannabinoids like CBD and THC from cannabis herbal extracts using advanced mass spectrometry.

## Contribution

A novel optimized extraction method using multivariate design and UHPLC-HRMS/MS for accurate and efficient quantification of neutral cannabinoids in commercial cannabis extracts.

## Key findings

- Agitation and sonication times were the most significant factors in the extraction process.
- The optimized method achieved high precision (<15%) and accuracy (69–98%) for CBD, THC, and CBN quantification.
- The method processed 16 commercial samples in three hours, demonstrating practicality and reproducibility.

## Abstract

The Cannabis market is experiencing steady global growth. Cannabis herbal extracts (CHE) are interesting and sought-after products for many clinical conditions. The medical potential of these formulations is mainly attributed to neutral cannabinoids, such as cannabidiol (CBD), tetrahydrocannabinol (THC), and cannabinol (CBN), and their non-standardized content poses a significant fragility in these pharmaceutical inputs. High-resolution mass spectrometry portrays a powerful alternative to their accurate monitoring; however, further analytical steps need to be critically optimized to keep up with instrumental performance. In this study, Full Factorial and Box–Behnken designs were employed to achieve a multivariate optimization of CBD, THC, and CBN extraction from human and veterinary commercial CHE using a minimum methanol/hexane 9:1 volume and short operational times. A predictive model was also constructed using the Response Surface Methodology and its accuracy was validated. Agitation and sonication times were identified as the most significant operational extraction parameters, followed by the extraction mixture volume. The final setup of the optimized method represented a significantly faster and cheaper protocol than those in the literature. The selected neutral cannabinoids quantification was conducted using ultra high-performance liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-HRMS/MS) with a precision of <15%, accuracy of 69–98%, sensitivity of 23–39 ng kg−1, and linearity regarding pharmaceutical requirements. State-of-the-art levels of analytical sensitivity and specificity were achieved in the target quantification due to high-resolution mass spectrometry. The developed method demonstrated reliability and feasibility for routine analytical applications. As a proof-of-concept, it enabled the efficient processing of 16 samples of commercial CHE within a three-hour timeframe, showcasing its practicality and reproducibility, and highlighting its potential for broader adoption in similar scenarios for both human and veterinary medicines.