Extraction of THC and Metabolites from Plasma Using ISOLUTE SLE+ Columns and 96-Well Plates with LC–MS-MS Analysis

February 11, 2011

The Application Notebook

The Application Notebook, The Application Notebook-02-11-2011, Volume 0, Issue 0

A simple and efficient supported liquid extraction (SLE) procedure for the extraction of THC and metabolites from human plasma at low levels has been developed.

A simple and efficient supported liquid extraction (SLE) procedure for the extraction of THC and metabolites from human plasma at low levels has been developed. ISOLUTE SLE+ columns and plates offer a simple, fast, and effective alternative to traditional liquid-liquid extraction providing high analyte recoveries, eliminating emulsion formation, and minimizing matrix effects in LC–MS-MS analysis.

Globally, cannabis is one of the most widely used illicit drugs. The naturally occurring cannabinoids found in hemp species bind to receptors in the brain and cause sensations of relaxation and calm. Widespread legislation against the use of cannabis has led to the necessity for rapid and reliable methods for the analysis and quantitation of cannabinoids and metabolites. The most prevalent markers in biological samples taken from cannabis abusers are Δ9 tetrahydrocannanbinol (THC), cannabidiol, cannabinol in addition to the major THC metabolites;11-hydoxy-Δ9-THC, and 11-nor-9-carboxy-Δ9-THC.

This application note describes the extraction of THC and metabolites from a 200 µL plasma sample using the ISOLUTE SLE+ 200 96-well plate (part #820-0200-P01). Method parameters have been optimized to maximize recoveries and minimize ion suppression in LC–MS analysis.

Experimental

Supported Liquid Extraction

Sample pretreatment: Dilute 100 µL plasma sample 1:1(v/v) with 1% aqueous formic acid.

Sample loading: Load pretreated plasma (200 µL) samples onto the plate and apply a pulse of vacuum to initiate flow. Leave the samples to absorb for 5 min.

Analyte Elution: Elution 1: Apply 500 µL dichloromethane, then a short pulse of vacuum and wait 5 min.

Elution 2: Apply a further 500 µL aliquot of dichloromethane, wait 5 min then apply a short pulse of vacuum to ensure complete elution.

Post Extraction: Evaporate eluate to dryness and reconstitute in 500 µL of 0.1% formic acid in water/acetonitrile (50:50, v/v).

LC–MS-MS

Analyze extracted samples using a Waters Acquity UPLC with an Acquity UPLC BEH C18 column (1.7 µm, 50 × 2.1 mm id) at 35 °C. Mobile phase A was 0.1% formic acid aq and mobile phase B was 0.1% formic acid in methanol, run isocratically at a flow rate of 0.5 mL/min, 10% A and 90% B. Sample injection volumes were 5 µL. The detector was a Premier XE triple quadrupole mass spectrometer (Waters) equipped with an electrospray interface, run in positive ion mode.

Results

Recoveries above 80% with RSDs below 10% were demonstrated as shown in Figure 1. THC-OH, cannabidiol, and THC-COOH exhibit recoveries of 95% or greater.

Figure 1: % recoveries of THC and metabolites from plasma and example chromatogram of THC and metabolites.

Conclusions

This method demonstrates robust and effective extraction of THC and metabolites using ISOLUTE SLE+ 200 96 well-plates. The method could easily be scaled up and optimised for larger sample volumes (up to 1 mL) using ISOLUTE SLE+ columns.

References

(1) L. Williams, "Extraction of THC and metabolites from Plasma using ISOLUTE SLE+." Application Note AN 723 (available from www.biotage.com ) (2010).

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