--- title: Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using EVOLUTE® EXPRESS ABN description: This application note describes the fully automated extraction of carboxy-THC from urine, following base hydrolysis prior to GC/MS analysis. --- [Literature ](https://www.biotage.com/literature) # [Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using EVOLUTE® EXPRESS ABN](https://www.biotage.com/literature/application-note/automated-extraction-thc-hydrolyzed-urine-evolute-express-abn) Written by [Biotage](https://www.biotage.com/literature/author/biotage) | Mar 13, 2026 5:29:27 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 EVOLUTE EXPRESS ABN cartridges. The method delivers clean extracts and analyte recovery of 90% with RSD lower than 10% for carboxy-THC and its deuterated internal standard. Using the Biotage® Extrahera, 24 samples were extracted in approximately 38 minutes. The limit of quantitation of 2 ng/mL is below the SAMHSA/ EWDTS confirmation cut off of 15 ng/mL for workplace testing applications. The EVOLUTE® EXPRESS range of SPE cartridges and 96-well plates combine powerful EVOLUTE sorbent chemistry with innovative features that provide robust, reliable SPE, giving ultra-pure extracts and high analyte recoveries. With improved flow-through characteristics and by eliminating the need for conditioning and equilibration, samples can be prepared much faster using the simplified Load-Wash-Elute procedure. ## Analytes Carboxy-THC and carboxy-THC-D9 as internal standard ## Sample preparation procedure ### Format EVOLUTE® EXPRESS ABN 60 mg/3 mL tabless SPE cartridges, part number 610-0006-BXG. ### Sample pre-treatment Apply 40 μL of a 1 ng/μL aqueous internal standard solution to 2 mL of urine, and allow to equilibrate for 1 hr at room temperature. Add 100 µL sodium hydroxide (10N) to this urine sample and heat at 60 °C for 20 minutes. Allow to cool and add 1 mL glacial acetic acid. ### Sample loading Load the full sample volume onto the column and apply positive pressure to achieve approximately 3 mL/minute. Gravity or very low pressure may be sufficient for this step. ### Wash 1 Apply 2 mL of HPLC water and apply pressure to elute. ### Wash 2 Apply a further 2 mL of HPLC water and apply pressure to elute. ### Pre-elution step Dry the column for 10 mins using the Biotage® ExtraheraTM dry plate function, or at 10 psi using other processing systems. ### Elution Elute direct into GC vials with hexane/ethyl acetate (1 mL, 50/50, v/v) ### Post elution and derivatization Dry the extract in a stream of air or nitrogen using a TurboVap (1.2 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 oC, 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 EVOLUTE® EXPRESS ABN protocol demonstrated analyte recoveries of 90% and 92% 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 EVOLUTE® EXPRESS ABN with the optimal protocol. Analyte concentrations visible here are 2, 5, 10, 20, 50 and 100 ng/ mL showing r2 values of greater than 0.999. The carboxy-d9 deuterated internal standard concentrations is at 20 ng/mL. Signal to noise measurement shows LOQ to be 2 ng/mL. ## Additional information - Sodium Hydroxide 10N is prepared with 40 g of pellets in 100 mL deionised 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% ## Ordering information | **Part Number** | **Description** | **Quantity** | | --- | --- | --- | | **610-0006-BXG** | EVOLUTE® EXPRESS ABN 60 mg/3 mL cartridge | 50 | | **414001** | Biotage® ExtraheraTM | 1 | | **415041** | Configuration Kit 24 Positions, Dual Flow | 1 | | **415000** | TurboVap® LV Evaporator | 1 |   Literature number: AN893 [View full post](https://www.biotage.com/literature/application-note/automated-extraction-thc-hydrolyzed-urine-evolute-express-abn) ```json { "@context" : "http://schema.org", "@type" : "BlogPosting", "author" : { "@type" : "Person", "name" : "Biotage" }, "dateModified" : "2026-03-13T17:29:27.320Z", "datePublished" : "2026-03-13T17:29:27Z", "headline" : "Automated extraction of 11-nor-9-Carboxy-Δ9- THC from hydrolyzed urine using EVOLUTE® EXPRESS ABN", "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-evolute-express-abn", "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" } } ```