Extraction of THC and its metabolites from human nail samples using ISOLUTE® SLE+ prior to UPLC-MS/MS analysis

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

Figure 1. Structure of Δ9-tetrahydrocannabinol (THC).

Introduction

The testing of alternative matrices in forensic and/or clinical toxicology is gaining popularity, partly due to less invasive means of collection. Matrices such as hair or nail can provide a more rounded picture of abstinence or abuse and associated timeframes.

This application note describes a procedure for sample pre-treatment and extraction of THC and its metabolites from human nail samples, using the Biotage® Lysera for matrix pulverisation, and pre-concentration of the sample prior to clean up using ISOLUTE® SLE+ supported liquid extraction.

Manual processing protocols were developed using the Biotage® Pressure+ 96 (plate format) or 48 (column format) Positive Pressure Manifolds.

This application note contains procedures optimized for both individual column format and 96-well plate format for higher throughput applications. The methodology delivers clean extracts with analyte recoveries > 70% (plate format) or > 75% (column format) with %RSD < 10% for all analytes and LLOQ as low as 20 fg/mg of nail.

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

Analytes

Tetrahydrocannabinol (THC), 11-Nor-9-carboxy-Δ⁹- tetrahydrocannabinol (THC-COOH), 11-Hydroxy-Δ⁹- tetrahydrocannabinol (THC-OH), Δ9-tetrahydrocannabinolic acid-A (THCAA), cannabidiol (CBD), and cannabinol (CBN)

Internal standards

Tetrahydrocannabinol –D3 (THC- D3), 11-Nor-9-carboxy-Δ⁹- tetrahydrocannabinol-D3 (THC- COOH-D3) and 11-Hydroxy-Δ⁹- tetrahydrocannabinol- D3 (THC-OH- D3)

These were used during the calibration process and recovery work was also done alongside the standards.

Sample preparation procedure

Format

ISOLUTE® SLE+ 400 µL capacity cartridges (p/n 820-0055-B) or
ISOLUTE® SLE+ 400 µL capacity 96-well plate (p/n 820-0400-P01)

Matrix preparation

Weigh 10 mg of nail into 2 mL Biotage® Lysera tubes (p/n 19-620) containing 4 x 2.4 mm stainless steel beads (p/n 19-640).

Micropulverisation procedure

Grind to a fine powder using Biotage® Lysera (Conditions - 8 x 6.95 m/sec for 45 seconds with a 45 second dwell.)

Add 1 mL 0.1% formic acid in methanol to each nail sample. Also add 10 µL of a 10 ng/mL ISTD making a 10 pg/mg spike. Mix.

Centrifuge tubes for 10 minutes at 13,300 rpm (Heraeus Pico 17 Microcentrifuge (Thermo Scientific) with 24 position, 2 mL rotor).

Post micropulverisartion

Transfer a 250 µL aliquot of supernatant into 12 x 75 mm evaporation tubes and evaporate extracts to dryness using a TurboVap® LV at 40 °C. Reconstitute in methanol:water (80:20, v/v, 250 µL).

Supported liquid extraction conditions

UHPLC conditions

Instrument

Shimadzu Nexera X2 UHPLC

Column

Restek Pinnacle DB Biphenyl 1.9um 50 x 2.1 mm combined with a Restek EXP guard holder and biphenyl guard

Mobile phase

A: 0.01% Formic Acid (aq)
B: 0.01% Formic Acid in MeCN

Flow rate

0.7 mL/min

Table 1. UHPLC gradient.

Injection volume

5 µL

Column temperature

50 °C

MS/MS conditions

Instrument

Shimadzu 8060 Triple Quadrupole MS using ES interface

Nebulizing gas flow

3 L/min

Drying gas flow

5 L/min

Heating gas flow

15 L/min

Interface temperature

400 ^oC

DL temperature

300 ^oC

Heat block temperature

500 ^oC

CID gas flow

270 kPa

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

Results

This simple sample preparation method delivers clean extracts and analyte recoveries mostly greater than 75% with RSDs lower than 10% for all analytes (see fig 2). LLOQs are below 10 pg/mg and as low as 20 fg/mg for THCA-A (see table 3) for ISOLUTE® SLE+ cartridge formats used.

Recoveries

Figure 2. Representative analyte recoveries.

Calibration curves

Figure 3. Calibration curves for THC (a), CBN (b) and THCA-A (c) using human nail with 400 µL capacity cartridge format (loading 250 µL of extracted sample as described).

Calibration curve performance was investigated from nails spiked between 0.01–100 pg/mg. Good linearity was observed for all analytes typically delivering r² values greater than 0.999. Table 3. details linearity performance and associated LOQ for each analyte using both ISOLUTE® SLE+ formats.

Table 3. Analyte calibration curve r² and LOQ performance.

Chemicals and reagents

• Methanol (LC-MS grade), Acetonitrile (Gradient MS) were purchased from Honeywell Research Chemicals (Bucharest, Romania).
• All analyte standards and deuterated internal standards, and acetic acid (99.7%) (LC-MS grade) were purchased from Sigma-Aldrich Company Ltd. (Gillingham, UK).
• Water used was 18.2 MOhm-cm, drawn daily from a Direct-Q5 water purifier.
• Mobile phase A (0.01% Formic acid aq) was prepared by adding 50 µL acid to 500 mL of purified water.
• Mobile phase B (0.01% Formic acid in acetonitrile) was prepared by adding 50 µL acid to 500 mL of HPLC grade acetonitrile.
• Internal standards (10 pg/mg) were prepared from a 1 ng/µL stock solution by adding 10 µL of each of to 970 µL of MeOH. This makes a 10 ng/mL stock and then 10 µL of this solution was added to each calibration sample.

Additional information

• All data shown in this application note was generated using various fingernail samples, provided by healthy human volunteers.
• THC and some of its metabolites bind to plastic, so in order to increase recoveries and reduce RSDs the pre-concentration evaporation steps should always be performed in glass tubes.
• Biotage®Lysera hints and tips
  
  • A minimum of four tubes must be loaded in the tube carriage to ensure balance during processing.
  • Ensure vial caps are firmly tightened and Lysera locking mechanism is fully engaged.
  • To minimize sample transfer and manipulation steps, 2 mL Lysera tubes were placed directly into the centrifuge (Heraeus Pico 17 Microcentrifuge (Thermo Scientific) with 24 position, 2 mL rotor).

Conclusion

This application note demonstrates that ISOLUTE®SLE+ cartridges or plates can be used in conjunction with Biotage® Lysera to provide a simple but effective sample preparation procedure for determination of THC and metabolites from human nail samples.

Ordering information

Literature Number: AN929