Novel fluorescence spectroscopy method coupled with N‐PLS‐R and PLS‐DA models for the quantification of cannabinoids and the classification of cannabis cultivars

Abstract

Introduction: Cannabis sativa L. inflorescences are rich in secondary metabolites, particularly cannabinoids. The most common techniques for elucidating cannabinoid composition are expensive technologies, such as high-pressure liquid chromatography (HPLC).Objectives: We aimed to develop and evaluate the performance of a novel fluorescence spectroscopy-based method coupled with N-way partial least squares regression (N-PLS-R) and partial least squares discriminant analysis (PLS-DA) models to replace the expensive chromatographic methods for preharvest cannabinoid quantification.Methodology: Fresh medicinal cannabis inflorescences were collected and ethanol extracts were prepared. Their excitation-emission spectra were measured using fluorescence spectroscopy and their cannabinoid contents were determined by HPLC-PDA. Subsequently, N-PLS-R and PLS-DA models were applied to the excitation-emission matrices (EEMs) for cannabinoid concentration prediction and cultivar classification, respectively. Results:The N-PLS-R model was based on a set of EEMs (n = 82) and provided good to excellent quantification of (À)-Δ9-trans-tetrahydrocannabinolic acid, cannabidiolic acid, cannabigerolic acid, cannabichromenic acid, and (À)-Δ9-transtetrahydrocannabinol (R 2 CV and R 2 pred > 0.75; RPD > 2.3 and RPIQ > 3.5; RMSECV/ RMSEC ratio < 1.4). The PLS-DA model enabled a clear distinction between the four major classes studied (sensitivity, specificity, and accuracy of the prediction sets were all ≥0.9). Conclusions:The fluorescence spectral region (excitation 220-400 nm, emission 280-550 nm) harbors sufficient information for accurate prediction of cannabinoid contents and accurate classification using a relatively small data set.

Progress in the analysis of phytocannabinoids by HPLC and UPLC (or UHPLC) during 2020–2023

“…Several UPLC or UHPLC-based methods have been presented in the literature for the analysis of cannabinoids in inflorescences of C. sativa. [77][78][79][80][81][82][83][84] Like the HPLC analyses outlined above, both PDA or DAD and MS detection technologies have been used with UPLC or UHPLC systems. However, the use of MS detection appears to be more popular with UPLC or UHPLC systems for the analysis of inflorescences for phytocannabinoids.…”

“…CBCA (2), CBDA (6), CBGA (9), Δ 9 -THC ( 14), and Δ 9 -THCA (15) were identified with an isocratic run with 30% HCOOH and 20 mM ammonium formate buffer at pH 2.9 and 70% ACN for a 16 min run at a flow rate of 0.3 ml/min and PDA detection at 228 nm. 77,78 An Acquity Arc FTN-R UPLC system and a Kinetex 1.7-μm XB-C 18 column (150 Â 2.1 mm, 1.7 μm) were used in this analysis. Two other UHPLC-PDA methods were quite similar, 82,84 which employed a gradient elution with water/ACN, both containing 0.1% TFA to identify CBC (1), CBD (5), CBDA (6), CBDV (18), CBG (8), CBN (10), Δ 9 -THC ( 14), and Δ 9 -THCA (15).…”

“…Since the publication of the review article 2 on the application of HPLC and UPLC (or UHPLC) methods in the analysis of phytocannabinoids covering the period of 2010–2019, several publications have appeared in the literature on this topic 12–185 . All these publications highlight the importance of HPLC and UPLC (or UHPLC) techniques as well as showcase remarkable advancements in sensitivity and versatility associated with these techniques in relation to phytocannabinoid analysis 12 .…”

“…A large volume of HPLC and UPLC (or UHPLC) methods that have been reported for the analysis of cannabinoids in C. sativa L. plant samples 13–94 and cannabis consumer products, for example, hashish, marijuana, and cannabis oils, 95–137 since 2020 are presented in Tables 1 and 2 and discussed in the following subsections.…”

“…Both HPLC and UPLC (or UHPLC) methods have been applied for the separation, identification and quantitation of cannabinoids in C. sativa L. samples, mainly including aerial parts, biomass, flowers, inflorescences, and leaves 13–93 (Table 1). However, simple HPLC‐based methods have remained more dominant than UPLC (or UHPLC)‐based methods for the analysis of C. sativa plant samples during 2020–23.…”