Automated extraction of 11-Nor-9-Carboxy-THC from human urine with ISOLUTE® HAX using Biotage® Extrahera™ HV-5000

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

Figure 1. Structures of analyte and internal standard.

Introduction

This application note describes the extraction of 11-Nor-9- carboxy-THC from human urine using ISOLUTE® HAX SPE cartridges and Biotage® Extrahera™ HV-5000 prior to GC/MS analysis.

The simple sample preparation procedure, based on a mixed- mode/strong anion exchange extraction mechanism, delivers clean extracts and analyte recoveries greater than 90% with RSDs lower than 10%. Linearity of greater than 0.999 is achieved with a urinary concentration range of 1–100 ng/mL.

This application note includes optimized conditions for automated processing of the HAX cartridges (using Biotage® Extrahera™ HV-5000) and manual processing (using the Biotage® PRESSURE+ 48 positive pressure manifold). Data generated using both processing systems is shown.

Analytes

11-Nor-9-carboxy-Δ-9-tetrahydrocannabinol

Internal standards

11-Nor-9-carboxy-Δ-9-tetrahydrocannabinol-D3

Sample preparation procedure

Format:

ISOLUTE® HAX 200 mg/3 mL cartridge, p/n 903-0020-B

Processing conditions

Samples were processed manually using a Biotage® PRESSURE+ 48 positive pressure manifold. Each step below was processed at 2 to 4 psi using the adjustable flow setting. Drying steps were processed at 40 psi using the maximum flow setting.
Automated sample processing was performed using the Biotage® Extrahera™ HV-5000 system. Detailed processing conditions are included in the appendix.

Sample pre-treatment:

Spike urine sample, QC or calibrator (2 mL) with internal standard solution, add 100 µL 10M NaOH and mix thoroughly in a suitable vial. Seal. Hydrolyze at 60 °C for 25 minutes, allow to cool and adjust to pH3 by adding 1 mL glacial acetic acid.

Transfer into 16 x 100 mm test tubes for extraction.

Note: Internal standard solution consists of a 10 ng/µL methanolic solution. 3 µL of this was added to 2 mL of urine to give a 15 ng/mL spike concentration.

Conditioning:

Condition each column with methanol (3 mL).

Equilibration:

Equilibrate each column with deionised (DI) water (3 mL) followed by sodium acetate buffer pH3 (aq) (1 mL).

Sample loading:

Load the full volume of the pre-treated (hydrolyzed) urine sample to each column.

Wash 1:

Elute interferences with DI water (2 mL).

Wash 2:

Elute interferences with 100 mM HCl (aq):ACN (70:30, v/v, 2 mL).

Dry:

Dry columns for 10 mins under positive pressure (40 psi)

Wash 3:

Elute interferences with hexane (100 µL)

Elution:

Elute analytes with hexane:ethyl acetate (50:50, v/v,) with 2% formic acid (3 mL). Elute into a 12 x 75mm test tube.

As THC-COOH contains an acid group, derivatisation of the extracts is required in order to obtain a stabilized analyte that can be analysed via GC/MS.

Post elution & Reconstitution:

Transfer elution solvent tubes to the evaporation system. Dry the extract in a stream of air using a TurboVap® LV at 40 °C, 1.0 L/min, for approximately 14 minutes.

Reconstitute evaporated samples with ethyl acetate (20 µL) and BSTFA:TMCS (99:1, 20 µL). Vortex mix, transfer to high recovery GC/MS vial and derivatise on a heat block at 70° C for 25 minutes. Transfer to GC-MS for analysis.

Note: This extraction method can also be used for THC, Δ8-THC and THC-OH if required.

GC conditions

Instrument

Agilent 7890A GC with purged Ultimate Union

Column

Agilent J&W DB-5ms 30 m, 0.25 mm, 0.25 µm

Mobile phase

GC/MS grade helium

Inlet temperature

260 °C Splitless

Flow rate

1.5 mL/min

Injection volume

1.5 μL

Table 1. GC oven gradient.

MS conditions

Instrument:

Agilent 5975C

Transfer line temp:

280 °C

Source temp:

230 °C

Quad temp:

150 °C

Table 2. MS conditions for target analytes in positive mode.

Results

Recovery and reproducibility

High (> 90%) and reproducible (RSD < 10%) recoveries were achieved for THC-COOH using the method described in this application note using the ISOLUTE® HAX cartridge format processed using the Biotage® Extrahera™ HV-5000 or Biotage® PRESSURE+ 48.
Figure 2. shows analyte % recoveries with RSDs (n=7) using the optimized ISOLUTE® HAX protocol described in this application note.

Linearity

Calibration curve performance was investigated from urine spiked between 1–100 ng/mL. Good linearity was observed for THC-COOH delivering r2 values greater than 0.999. Table 3. details linearity performance for each method of extraction.

Table 3. Analyte calibration curve r2 and LOQ performance.

Calibrator preparation

Calibration standards were prepared by spiking human urine with a 10 ng/µL THC-COOH stock solution to create a 100 ng/mL standard. This was then serially diluted in order to obtain further standards at concentrations of 50, 20, 10, 5, 2, 1 ng/mL.

Internal standard was added to 2 mL of each prepared urine standard and the extraction was performed as decribed.
Figure 3. Calibration curves for THC-COOH extraction on the Biotage® Extrahera™ HV-5000 (a) and Biotage® PRESSURE+ 48 (b) using the ISOLUTE® HAX cartridges to extract hydrolyzed human urine.

Sample preparation time

For a batch size of 24 samples, processing times were:

Conclusion

ISOLUTE® HAX solid phase extraction cartridges provided robust extraction of THC-COOH from hydrolyzed urine samples.

Good, reproducible recoveries were achieved, with an overall processing time of 56 and 66 minutes using the Biotage® PRESSURE+ 48 or the Biotage® Extrahera™ HV-5000 respectively (excluding the common evaporation and derivatisation steps).

Chemicals and reagents

• Methanol (LC-MS grade), hexane, ethyl acetate and acetonitrile were purchased from Rathburn Chemicals Ltd (Walkerburn, UK).
• All analyte standards, deuterated internal standards, sodium hydroxide, hydrochloric acid, formic acid and acetic acid were purchased from Sigma- Aldrich Company Ltd. (Gillingham, UK).
• DI Water used was 18.2 MOhm-cm, drawn daily from a Direct-Q5 water purifier.
• Internal standard (10 ng/mL) was prepared from a 1 mg/mL stock solution by adding 10 µL to 990 µL of MeOH. 3 µL of this solution was then added to each calibration solution.
• Wash 2 solvent (100mM HCl (aq):ACN (70:30, v/v)) was made up by measuring out 70 mL of water (18.2 MOhm-cm) and 30 mL of acetonitrile and adding both to a bottle, then adding 833 µL of concentrated hydrochloric acid.
• Elution solvent (hexane:ethyl acetate (50:50, v/v with 2% formic acid)) was made up by measuring out 50 mL of hexane and 50 mL of ethyl acetate and adding both to a bottle with 2 mL formic acid.

Additional information

All data shown in this application note was generated using human urine donated by healthy human volunteers.

Ordering information

Appendix: Biotage® Extrahera™ HV-5000 settings

The method described in this application note was automated on the Biotage® Extrahera™ High Volume 5000 using ISOLUTE® HAX 200 mg/3 mL cartridges. This appendix contains the software settings required to configure Extrahera™ to run this method. Screenshots may or may not match those here depending upon the instrument software version.

Screenshot                                                                      Settings

Solvent properties

Literature Number: AN970