Extraction of cocaine and metabolites from whole blood using ISOLUTE® SLE+

Figure 1. Structure of Benzoylecgonine

Introduction

This application note describes the extraction of cocaine and major metabolites from whole blood, prior to GC/MS analysis. This protocol also allows the simultaneous extraction of various other drugs of abuse classes: amphetamines, barbiturates, benzodiazepines and opiates ISOLUTE® SLE+ cartridges with 1 mL sample capacity are used to extract whole blood samples following a straightforward sample dilution. No protein precipitation or other pre-treatment is required prior to sample loading. The sample preparation procedure delivers clean extracts, good recoveries and RSD values and LLOQs from 20 ng/mL (analyte dependant).

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

Anhydroecgonine methyl ester (AEME), Ecgonine (EME), Cocaine, Cocaethylene, Benzoylecgonine-D3, (BZE-D3), Benzoylegonine (BZE)

Sample preparation procedure

Format

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

Sample pre-treatment

To 1 mL of whole blood, add 10 µL of ISTD (total 100 ng/mL). Allow to equilibrate and add 1 mL of 1% ammonium hydroxide (aq). Vortex.

Sample loading

Load 750 μL of the pre-treated whole blood 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 dichloromethane (DCM, 2.5 mL) and allow to flow under gravity for 5 minutes. Collect in an appropriate glass tube.

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 vessel.

Post-elution and reconstitution

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

Reconstitute the extracts with ethyl acetate (250 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 (40 °C, 20 to 40 L/min).

Reconstitute extracts with ethyl acetate (40 µL) and BSTFA (with 1% t-BDMCS) (40 µL), vortex and heat for 30 minutes at 70 °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

260 °C, Splitless, purge flow: 50 mL/min at 1.0 min

Injection

2 μL

Wash solvents

Acetone & ethyl acetate

Oven

Initial temperature 60 °C, hold for 1 minute, ramp 50 °C/min to 200 °C , hold for 1.5 minutes, ramp 10 °C/min to 250°C.

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

Results

Blank whole blood was spiked at 100 ng/mL for recovery testing. Reproducible data was observed from the typical recovery data with RSD values <10% as shown in Figure 2.


Figure 2. Typical recoveries for cocaine and metabolites.

Figure 3. Total Ion Chromatogram of cocaine and metabolites at 100 ng/mL
 

Calibration curves

Whole blood was spiked prior to extraction, at concentrations of 10, 20, 50, 75, 100, 200 and 500 ng/mL for each analyte to create calibration curves. BZE-D3 was spiked at 100 ng/mL for each level. The curves are shown in Figure 4.

Figure 4. Charts demonstrating coefficient of determination
(r2) values between 0.9953 and 0.9995 for the cocaine and metabolites

Additional notes

Solvents and reagent preparation:

• All solvents were HPLC grade.
• 1% ammonium hydroxide (aq): Add concentrated ammonium hydroxide (28–30%) (1 mL) to HPLC grade water (99 mL).

Cartridge loading: ISOLUTE® SLE+ cartridges are underloaded (750 µL sample on a 1 mL capacity cartridge) to avoid breakthrough of whole blood matrix.
Simultaneous extraction of other drug classes: This protocol allows the simultaneous extraction of amphetamines, barbiturates, benzodiazepines and opiates.

Ordering information

Literature number: AN856