--- title: Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using ISOLUTE® SLE+ description: This application note describes the fully automated extraction of carboxy-THC from urine. --- [Literature ](https://www.biotage.com/literature) # [Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using ISOLUTE® SLE+](https://www.biotage.com/literature/application-note/automated-extraction-thc-hydrolyzed-urine-isolute-sle) Written by [Biotage](https://www.biotage.com/literature/author/biotage) | Mar 5, 2026 3:01:31 PM 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® ExtraheraTM , 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. | SIM Group | Analyte | Target (Quant) Ion | 1st  Qual Ion  | 2nd Qual Ion | | --- | --- | --- | --- | --- | | 1 | THC-COOH-D9 | 380 | 479 |   | | 1 | THC-COOH | 371 | 488 | 473 | ## 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 | **Part Number** | **Description** | **Quantity** | | --- | --- | --- | | **820-0140-CG** | ISOLUTE® SLE+ 1 mL Sample Volume Cartridge (Tabless) | 30 | | **414001** | Biotage® ExtraheraTM | 1 | | **415041** | Configuration Kit 24 Positions, Dual Flow | 1 | | **415000** | TurboVap® LV Evaporator | 1 |   Literature number: AN892 [View full post](https://www.biotage.com/literature/application-note/automated-extraction-thc-hydrolyzed-urine-isolute-sle) ```json { "@context" : "http://schema.org", "@type" : "BlogPosting", "author" : { "@type" : "Person", "name" : "Biotage" }, "dateModified" : "2026-03-05T15:01:31.161Z", "datePublished" : "2026-03-05T15:01:31Z", "headline" : "Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using ISOLUTE® SLE+", "image" : { "@type" : "ImageObject", "height" : 60, "url" : "/hs/hsstatic/content_shared_assets/static-1.4092/img/default-amp-logo.png", "width" : 60 }, "mainEntityOfPage" : "https://www.biotage.com/literature/application-note/automated-extraction-thc-hydrolyzed-urine-isolute-sle", "publisher" : { "@type" : "Organization", "logo" : { "@type" : "ImageObject", "height" : 60, "url" : "/hs/hsstatic/content_shared_assets/static-1.4092/img/default-amp-logo.png", "width" : 60 }, "name" : "Literature" } } ```