Relative response factors and multiple regression models in liquid chromatography to quantify low-dosed components using alternative standards—proof of concept: total Δ9-THC content in cannabis flowers using CBD as reference

Abstract

A classical quantitative analysis in liquid chromatography is performed using either a one-point calibration or a calibration line, prepared using a reference standard of the compound(s) of interest. However, in some cases, adequate reference standards may be very expensive, rare to obtain, or have limited shelf-life properties. Also, in herbal matrices, multiple compounds could be necessary to be quantified, needing a whole series of different (related) reference standards. In these cases, the use of relative response (sometimes called relative correction factors) factors (RRFs) towards reference standards, different of the compound to be quantified, gained attraction. This study performed a comparison of the use of RRFs and linear relative response factor models (LRRFM) for the quantification of targeted low-dosed compounds using an alternative standard, since it is known that classical RRFs often fail in lower concentration ranges. For this purpose, the determination of the total Δ9-tetrahydrocannabinol (Δ9-THC + Δ9-THC-A) content in dried cannabis flowers, using UHPLC-DAD, was used as a case study. A chromatographic method was implemented and validated, and the use of classical calibration lines, classical RRF, and the LRRFM was applied and compared, with special focus on the concentration around 0.2% (w/w) total Δ9-THC, the legal limit (in most European countries) in these products. Results showed that the newly presented and validated LRRFM approach outperformed the classical RRFs, especially in the low-concentration ranges and that concentrations obtained with the LRRFM were in accordance with the interpolation results obtained with a calibration line.

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

“…There are at least four UPLC (or UHPLC) methods for the analysis of cannabinoids in C. sativa flowers available in the literature. [45][46][47][48] Of them, three used UV-based detection, [46][47][48] while one used ESI-MS/ MS detection. 45 Two of these studies used column particle size of 1.6 μm, 46,47 whereas the other two used 1.7 μm particle size.…”

“…[45][46][47][48] Of them, three used UV-based detection, [46][47][48] while one used ESI-MS/ MS detection. 45 Two of these studies used column particle size of 1.6 μm, 46,47 whereas the other two used 1.7 μm particle size. 45,48 All columns were 100 mm in length, and the diameter was 2.1-3.0 mm (Table 1).…”

“…45,48 All columns were 100 mm in length, and the diameter was 2.1-3.0 mm (Table 1). Three of these analyses were UPLC, [45][46][47] that is, the instrument was from Waters, and one UHPLC (an Agilent instrument). 48 Eleven phytocannabinoids, for example, CBC (1), CBD (5), CBDA (6), CBDV (18), CBG (8), CBGA (9), CBN (10), Δ 8 -THC ( 13), Δ 9 -THC ( 14),…”

“…An Acquity Classic UPLC coupled with a Xevo TQ MS detector, an Acquity UPLC BEH C 18 column (100 Â 2.1 mm, 1.7 μm) connected to an Acquity UPLC BEH C 18 Van-Guard precolumn (5 Â 2.1 mm) and a water-ACN (containing 0.1% HCOOH) gradient were employed in this study. Among the three PDA-based identification methods of phytocannabinoids using a UPLC or UHPLC, [46][47][48] two used an isocratic 46,47 and one used a gradient elution method 48 using a mobile phase composed of water and ACN (Table 1). The method described by Mudge and Brown 48 was quite comprehensive, albeit it was a simple isocratic elution, as it successfully identified CBC (1), CBD (5), CBDA (6), CBG (8), CBGA (9), CBN (10), Δ 9 -THC ( 14), Δ 9 -THCA (15), THCV (16), and THCVA (17) in dried flower samples.…”

“…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 .…”