Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using ISOLUTE® SLE+

For research use only. NOT for use in diagnostic procedures.

Figure 1. Structure of 11-nor-9-carboxy-Δ⁹-THC.

Introduction

This application note describes the fully automated extraction of carboxy-THC from urine, following base hydrolysis prior to GC/MS analysis. The method was automated using Biotage® Extrahera^TM , configured for use with ISOLUTE SLE+ cartridges.

This application note describes an effective and efficient ISOLUTE SLE+ protocol optimized for extraction of 1 mL of pre-hydrolyzed urine. The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 80% with RSDs lower than 10% for carboxy-THC and its deuterated internal standard. Using Biotage Extrahera, 24 samples are extracted in approximately 35 minutes. Limit of quantitation is below the SAMHSA/EWDTS confirmation cut off of 15 ng/mL for workplace testing applications.

ISOLUTE® SLE+ Supported Liquid Extraction plates and cartridges offer an efficient alternative to traditional liquid- liquid extraction (LLE) for bioanalytical sample preparation, providing high analyte recoveries, no emulsion formation, and significantly reduced sample preparation.

Analytes

Carboxy-THC and carboxy-THC-D₉ as internal standard

Sample preparation procedure

Format

ISOLUTE® SLE+ 1 mL sample volume cartridges (Tabless), part number 820-0140-CG.

Sample pre-treatment

Apply 20 μL of a 1 ng/μL aqueous internal standard solution to 1 mL of urine and allow to equilibrate for 1 hr at room temperature. Add 50 µL sodium hydroxide (10N) to this urine
sample and heat at 60 °C for 20 minutes. Allow to cool and add 60 µL glacial acetic acid.

Sample loading

Load 1 mL of the hydrolyzed sample onto the cartridge and apply a pulse of vacuum or positive pressure (3–5 seconds) to initiate flow. Allow the sample to absorb for 5 minutes.

Analyte extraction

Apply hexane/ethyl acetate (2.5 mL , 50/50, v/v) and allow to flow under gravity for 5 minutes. Apply a further aliquot of hexane/ethyl acetate (2.5 mL, 50/50, v/v) and allow to flow for another 5 minutes under gravity. Apply vacuum or positive pressure to pull through any remaining extraction solvent. (5–10 seconds).

Post elution and derivatization

Dry the extract in a stream of air or nitrogen using a Biotage® TurboVap® (1.2 bar at 40 °C for 25 mins).

Reconstitute the extracts with 250 μL ethyl acetate and vortex for 10 seconds before transferring to high recovery GC vials. Dry the extract in a stream of air or nitrogen using a TurboVap (1.0 bar at 40 °C for 10 mins).

Upon dryness, reconstitute with 20 μL ethyl acetate and 20 μL BSTFA:TMCS 99:1 and vortex for 20 seconds. Place in a heating block set to 70 °C, for 25 minutes. Remove vial from the block and allow to cool.

GC conditions

Instrument

Agilent 7890A with QuickSwap

Column

Restek Rxi-5ms, 30 m x 0.25 mm ID x 0.25 μm

Carrier

Helium 1.2 mL/min (constant flow)

Inlet

280 °C, Splitless, purge flow: 50 mL/min at 1.0 min

Injection volume

2 µL

Wash solvents

Methanol and ethyl acetate

Oven

Initial temperature 125 °C

Ramp 50 °C/min to 300 °C, hold for 2.5 minutes

Ramp 50 °C/min to 330 °C, hold for 1.4 minutes

Post run

Backflush for 1.6 minutes (2 void volumes)

Transfer line

280 °C

MS conditions

Instrument

Agilent 5975C

Source

230 °C

Quadrupole

150 °C

MSD mode

SIM

SIM parameters

Table 1. Ions acquired in the Selected Ion Monitoring (SIM) mode.

Results

This optimized SLE+ protocol demonstrated analyte recoveries of 82% and 85% from urine for the carboxy-THC-D9 and carboxy-THC respectively, as shown in Figure 2. RSDs were lower than 10% (n=7).
Figure 2. Analyte Recoveries.Figure 3. A calibration curve of carboxy-THC constructed following extractions using ISOLUTE SLE+ with the optimal protocol. Analyte concentrations shown here are 5, 10, 20, 50 and 100 ng/mL with r2 values of greater than 0.999. The carboxy-D9 deuterated internal standard concentrations is at 20 ng/mL.

The analyte signal allows an approximate inferred limit of quantitation of between 5 and 10 ng/mL.

Additional information

• Sodium Hydroxide 10N is prepared with 40 g of pellets in 100 mL deionized water. Prepare this solution with extreme care. As an added precaution, place the beaker on ice prior to gradual pellet addition.
• Glacial Acetic Acid was purchased from Sigma-Aldrich at ≥ 99.85%
• The use of MTBE is an acceptable alternative to hexane/ethyl acetate (50/50, v/v)
• For manual extraction protocols, ISOLUTE® SLE+ 1 mL Sample Volume columns are available in tabbed format (p/n 820-0140-C) and can be used as an alternative to the tabless version used in this Biotage® ExtraheraTM automated protocol.

Ordering information

Literature number: AN892