Extraction of illicit drugs and pesticides from liver tissue using ISOLUTE® SLE+

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

Figure 1. Structures of amphetamine, diazepam, butabarbital and atrazine respectively.

Introduction

This application note describes the extraction of a broad range of analytes from liver tissue matrix prior to GC/MS analysis using ISOLUTE® SLE+ supported liquid extraction cartridges. A protocol has been developed that allows the simultaneous extraction of various drugs of abuse classes: amphetamines, barbiturates, benzodiazepines, cocaine, ketamine, THC. In addition to these drug panels, simultaneous extraction of carbamate, organochlorine, organophosphate, pyrethroid, and triazine pesticide classes is demonstrated.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.

This application note describes an effective and efficient ISOLUTE SLE+ protocol optimized for the 1 mL capacity cartridge format. The simple sample preparation procedure delivers clean extracts, good recoveries and RSD values and LLOQ of 50 ppb, suitable for screening applications.

Analytes

Amphetamine, Bendiocarb, Methamphetamine, Propanil, MDMA, Chlorothalonil, Atrazine, Butabarbital, Secobarbital, Ketamine, Malathion, Phenobarbital, Cocaine, Methadone, THC, Bifenthrin, Diazepam, Nitrazepam, Midazolam, Clonazepam, Estazolam, Alprazolam and Triazolam.

Sample preparation procedure

Format:

ISOLUTE SLE+ 1 mL Sample Volume Cartridge, part number 820-0140-C

Sample pre-treatment:

Weigh 200 mg of liver and place in 7 mL Biotage® Lysera tubes containing 2.8 mm ceramic beads. Add methanol:water (50:50, v/v, 1.8 mL). Add internal standard mix (500 ppb, 10 μL in methanol). Load tubes into the Lysera and homogenize using the following program: 1 cycle for 30 seconds at 5.3 m/s.

Transfer the homogenized liver into 2 mL Eppendorf tubes and place in a micro centrifuge for 10 minutes at 13,300 rpm.

Sample loading:

Load 500 μL of the pre-treated liver supernatant onto the column 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 dichloromethane (DCM) (2.5 mL) and allow to flow under gravity for 5 minutes. Collect in an appropriate glass tube containing 100 µL HCl in methanol (0.2 M). This acts to stabilize free-base analytes in the solvent prior to evaporation.

Apply a second aliquot of DCM (2.5 mL) and allow to flow under gravity for 5 minutes. Apply vacuum or positive pressure (5–10 seconds) to pull through any remaining extraction solvent into the collection tube.

Evaporation and derivatization:

Evaporate the extract in a stream of air or nitrogen (ambient, 20 to 40 L/min).

Reconstitute the extracts with ethyl acetate (200 µL) and vortex for 20 seconds before transferring to high recovery GC vials. Evaporate the extract in a stream of air or nitrogen using a SPE Dry (ambient room temperature, 20 to 40 L/min).

Reconstitute extracts with ethyl acetate (25 µL) and MSTFA (25 µL). Vortex mix, then heat on a block for 30 minutes at 80 °C to complete derivatization.

GC conditions

Instrument

Agilent 7890A with QuickSwap

Column

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

Carrier

Helium 1.2 mL/min (constant flow)

Inlet

300 °C, Split (5:1), Septum purge flow: 3 mL/min

Injection

1 µL

Wash solvents

Methanol and ethyl acetate

Oven

Initial temperature 55 °C

Ramp 25 °C/min to 325 °C, hold for 3.2 minutes

Post run

Backflush for 1.6 minutes (2 void volumes)

Transfer line

300 °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 (5 minute solvent delay)

Results

Blank liver supernatant was spiked at 1000 ppb for recovery testing; typical recovery data is shown in Figure 4. The protocol described offered reproducible recovery with most RSD values <10%.
Figure 2. Chart demonstrating typical analyte recoveries.
Figure 3. Total ion chromatogram of extracted analytes spiked into liver at 250ppb

Calibration curves

Liver supernatant was spiked prior to extraction, at concentrations of 50, 100, 250, 500, 1000 and 2500 ppb for each analyte to create calibration curves. The internal standards were spiked at 500 ppb for each level. The curves are shown in Figure 4.
Figure 4. Coefficient of determination (r²) values between 0.9931 and 0.9995 were achieved for the analytes. 

Table 2. Lower Limits of Quantitation (LLOQ) using the ISOLUTE® SLE+ procedure described in this application note

Additional information

All solvents were HPLC grade.

0.2M HCl in methanol: Add 200 μL concentrated HCl solution (37%) to 11.8 mL methanol. Mix thoroughly.

Ordering information

*Biotage® Lysera is available in North America, Europe and China only.

Literature Number: AN864