Inert LC Columns—The Answer to Poor THC Sensitivity?
16 Apr 2025Whole blood toxicology analysis of cannabinoids by LC-MS/MS is difficult for several reasons, but one of the biggest challenges is achieving the low detection limits needed to meet reporting guidelines. Most forensic laboratories strive to achieve a limit of quantitation (LOQ) of at least 0.5 ng/mL for Δ9-tetrahydrocannabinol (Δ9-THC); 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-Δ9-THC); and 11-nor-9-carboxy-Δ-9-THC (Δ9-THC-COOH), which are the analytes typically tested for to determine cannabis use. Achieving these low detection limits can be challenging, especially for instrument platforms that are older and less sensitive. If you’ve already tried all the usual tricks to squeeze a little more sensitivity out of your method, you might consider trying an inert LC column.
Inert LC columns are just like our traditional stainless-steel LC columns, but with an inert coating applied to the surface. This coating helps to prevent nonspecific binding for analytes that chelate, or stick, to metal surfaces. Chelation can cause poor peak shapes or a loss of analyte that negatively impacts sensitivity. Not all compounds will experience nonspecific binding/chelation, though acidic compounds may be more prone to it.
Although not all analytes will see a difference from the use of an inert column, it can lead to an improvement in sensitivity for those that do. In the following experiment, we compared an inert LC column and traditional stainless-steel column for the analysis of cannabinoids in whole blood. The developed method uses a Raptor FluoroPhenyl stationary phase to separate Δ8-THC; Δ9-THC; and their hydroxy and carboxy metabolites. To determine if an inert column would be beneficial for this analysis, a sample containing the analytes of interest was run on the method using both a Raptor Inert FluoroPhenyl column and a standard Raptor FluoroPhenyl column. The results are shown below.
Figure 1: Comparison of Δ9-THC; Δ8-THC; and Metabolites on Raptor FluoroPhenyl vs. Raptor Inert FluoroPhenyl
| Peaks | Conc. (ng/mL) | Precursor | Product 1 | Product 2 | |
|---|---|---|---|---|---|
| 1. | 11-OH-Δ8-THC | 100 | 331.0 | 313.0 | 201.1 |
| 2. | 11-OH-Δ9-THC | 100 | 331.0 | 313.0 | 201.1 |
| 3. | Δ8-THC-COOH | 500 | 345.1 | 327.0 | 299.2 |
| 4. | Δ9-THC-COOH | 500 | 345.1 | 327.0 | 299.2 |
| 5. | Δ8-THC | 100 | 315.0 | 193.0 | 123.2 |
| 6. | Δ9-THC | 100 | 315.0 | 193.0 | 123.2 |
| Column | See notes. | ||||||||||||||||||||||||||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| Dimensions: | 100 mm x 3 mm ID | ||||||||||||||||||||||||||||||||||||
| Particle Size: | 2.7 µm | ||||||||||||||||||||||||||||||||||||
| Pore Size: | 90 Å | ||||||||||||||||||||||||||||||||||||
| Temp.: | 40 °C | ||||||||||||||||||||||||||||||||||||
| Standard/Sample | |||||||||||||||||||||||||||||||||||||
| Δ8-Tetrahydrocannabinol (Δ8-THC) (cat.# 34090) | |||||||||||||||||||||||||||||||||||||
| Δ9-Tetrahydrocannabinol (Δ9-THC) (cat.# 34067) | |||||||||||||||||||||||||||||||||||||
| (±)11-nor-9-carboxy-Δ-9-THC (Δ9-THC-COOH) (cat.# 34068) | |||||||||||||||||||||||||||||||||||||
| Other compounds obtained separately. | |||||||||||||||||||||||||||||||||||||
| Diluent: | 40:60 Water:methanol, both with 0.1% formic acid (v/v) | ||||||||||||||||||||||||||||||||||||
| Inj. Vol.: | 5 µL | ||||||||||||||||||||||||||||||||||||
| Mobile Phase | |||||||||||||||||||||||||||||||||||||
| A: | Water, 0.1% formic acid | ||||||||||||||||||||||||||||||||||||
| B: | Methanol, 0.1% formic acid | ||||||||||||||||||||||||||||||||||||
| |||||||||||||||||||||||||||||||||||||
| Max Pressure: | 390 bar |
| Detector | Shimadzu 8045 MS/MS |
|---|---|
| Ion Mode: | ESI+ |
| Instrument | Shimadzu Nexera X2 |
| Sample Preparation | Five hundred microliters of whole blood was transferred to a 12 mL glass test tube. Fifty microliters of internal standard and 50 µL of control material were transferred to the test tube and vortexed. Five hundred microliters of HPLC grade water was added to each sample and vortexed. One hundred microliters of 10% acetic acid was added to each sample and vortexed. Two and a half milliliters of 80:20 hexanes:ethyl acetate was added to each sample and vortexed until visibly combined. Samples were centrifuged at 2800 rpm for 15 minutes. The top layer was transferred to a new glass test tube and dried down under nitrogen. Samples were reconstituted with 100 µL of 40:60 methanol:water, both containing 0.1% formic acid, and vortexed. Samples were transferred to 2 mL screw-thread vials (cat.# 21143) with glass inserts (cat.# 21776) and capped with short-cap, screw-vial closures (cat.# 24498). |
| Notes | The column was stored in 100% acetonitrile when not in use. Columns are: Raptor Inert FluoroPhenyl (cat.# 9319A1E-T) Raptor FluoroPhenyl (cat.# 9319A1E) |
Looking at these results, we can see that all six analytes had some increase in peak height and area when using the inert column. The parent compounds saw little increase in peak height/areas (2-7%), while the hydroxy metabolites had a more substantial increase (17-27%), and the carboxy metabolites had the most significant change (31-39%) when analyzed on an inert column. The carboxy compounds likely see the largest increase in sensitivity because of their carboxylic acid functional group, which is more prone to nonspecific binding. The hydroxy and parent compounds are more neutral, so they are less affected using an inert column. Nonetheless, we do see increased sensitivity for these cannabinoids when they are analyzed on an inert column.
If you are struggling to meet the low detection limits needed for cannabinoid analysis in whole blood, an inert LC column might be just what you need. If you’re interested in analyzing THC isomers in biological fluids, make sure you check out LC-MS/MS Analysis of THC Isomers & Metabolites in Whole Blood and Urine to learn more about our complete workflow solutions for this topic.