Semiquantitative Screening of THC Analogues by Silica Gel TLC with an Ag(I) Retention Zone and Chromogenic Smartphone Detection

With the ever-evolving cannabis industry, low-cost and high-throughput analytical methods for cannabinoids are urgently needed. Normally, (potentially) psychoactive cannabinoids, typically represented by Δ9-tetrahydrocannabinol (Δ9-THC), and nonpsychoactive cannabinoids with therapeutic benefits, typically represented by cannabidiol (CBD), are the target analytes. Structurally, the former (tetrahydrocannabinolic acid (THCA), cannabinol (CBN), and THC) have one olefinic double bond and the latter (cannabidiolic acid (CBDA), cannabigerol (CBG), and CBD) have two, which results in different affinities toward Ag(I) ions. Thus, a silica gel thin-layer chromatography (TLC) plate with the lower third impregnated with Ag(I) ions enabled within minutes a digital chromatographic separation of strongly retained CBD analogues and poorly retained THC analogues. The resolution (Rs) between the closest two spots from the two groups was 4.7, which is almost 8 times higher than the resolution on unmodified TLC. After applying Fast Blue BB as a chromogenic reagent, smartphone-based color analysis enabled semiquantification of the total percentage of THC analogues (with a limit of detection (LOD) of 11 ng for THC, 54 ng for CBN, and 50 ng for THCA when the loaded volume is 1.0 μL). The method was validated by analyzing mixed cannabis extracts and cannabis extracts. The results correlated with those of high-performance liquid chromatography with ultraviolet detection (HPLC-UV) (R2 = 0.97), but the TLC approach had the advantages of multi-minute analysis time, high throughput, low solvent consumption, portability, and ease of interpretation. In a desiccator, Ag(I)-TLC plates can be stored for at least 3 months. Therefore, this method would allow rapid distinction between high and low THC varieties of cannabis, with the potential for on-site applicability.


Figure S1
HPLC -UV calibration curves of THC, CBN, THCA, CBD, CBG  and CBDA  Page S3   Table S1 THCA, CBN, THC, CBD, CBG and CBDA content in samples  Page S4  Table S2 Preparation of mixed cannabis extracts Page S5 Figure S2 Design of Ag(I)-TLC plate Page S6 Protocol S1 Optimization of TLC plates Page S7 Figure S3 Images of different storage conditions for Ag(I)-TLC plates Page S8 Figure S4 Glass spray bottle for spraying color reagents Page S8 Figure S5 Images of light box for controlling photographing conditions Page S9 Figure S6 Photographs of Ag(I)-TLC plates for parameter optimization Page S10

Figure S7
Resolution of standards and analysis time under different experimental parameters by Ag(I)-TLC Page S11 Protocol S2 Procedure for extracting cannabinoids from TLC plates Page S13 Table S3 LC-MS/MS acquisitions parameters for the 12 cannabinoids Page S14 Figure      there is a reference sample loading line which is in the middle of this zone, namely 5 mm below the top of the plate. When performing TLC parameter optimization experiments, there was no reference zone so that the TLC plate was 1 cm shorter than the above-described TLC plate.

Optimization of total height of the TLC plate
As shown in Figure S7A, with the increasing total height of the TLC plate, the analysis time increased, while the resolution of THCA-CBD first increased and remained constant above a total height of 7 cm. The resolution of THC-THCA increased a lot from a TLC height of 4 cm to 6 cm, and then remained constant at a height of 6 cm and 7 cm. When the total height was further increased, greater resolution of THC-THCA could be achieved but the CBD spot tended to migrate out of the Ag(I) retention zone due to the longer running time. Therefore, a total TLC height of 7 cm was chosen for all further analyses.

Optimization of AgNO3 solution concentration for modification
Next, the influence of the AgNO3 concentration on the resolution and analysis time was investigated ( Figure S7B). The analysis time was almost not influenced by the AgNO3

Optimization of height of Ag(I) retention zone
With the above parameters fixed, the height of the Ag(I) retention zone was varied from 14 mm to 26 mm and its effect on the resolution observed ( Figure S7C) with the color analysis. Therefore, a height of 20 mm was selected for the Ag(I) retention zone.

Optimization of drying methods for Ag(I)-TLC plates
The AgNO3 for the modification was dissolved in acetonitrile. Passive drying and accelerated drying with a blow dryer were compared for the separation performance of three pairs of standards ( Figure S7D). Accelerated drying leads to better resolution of both THCA-CBD and THC-THCA. This is probably because accelerated drying leads to a more complete evaporation of acetonitrile (boiling point 82 °C) and thus avoids competition of ACN and cannabinoids for binding with Ag(I) considering that acetonitrile can complex with Ag(I). 2 Based on the above, the optimized TLC plate is 7 cm high, has a 20 mm-high Ag(I) retention zone modified with 1.5 M AgNO3 solution and dried by blow dryer. All the above optimized parameters were adopted in later experiments.  Table S3.     RGB color analysis was performed on different cannabis samples according to the method described in Figure S17 and the obtained normalized (B−R) value were labelled in Figure S17.