Extraction of amphetamine and designer amphetamine analogues from whole blood using ISOLUTE® SLE+

Figure 1. Structure of Amphetamine and Ethylone

Introduction

This application note describes the extraction of a range of amphetamine-type compounds from whole blood, prior to GC/MS analysis. A protocol that allows the simultaneous extraction of various other drugs of abuse classes: barbiturates, benzodiazepines, cocaine and opiates, is also evaluated.

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 10 ng/mL (analyte dependent).

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

Amphetamine, Amphetamine-D5 Methamphetamine, Methcathinone, Mephedrone, 5-APB, 6-APB, MDA, pMMA, Methedrone, BZP, TFMPP, MDMA, MDEA, Methylone, Butylone, Ethylone, Ethylone-D5, 2C-B, mCPP, MDPV, Naphyrone

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 MTBE* (2.5 mL) and allow to flow under gravity for 5 minutes. Collect in an appropriate glass tube containing HCl in methanol (0.2 M, 100 µL). This acts to stabilize free-base analytes in the solvent prior to evaporation.

Apply a second aliquot of MTBE* (2.5 mL) and allow to flow under gravity for 5 minutes. Collect as before. Apply vacuum or positive pressure (5–10 seconds) to pull through any remaining extraction solvent.

*Note that dichloromethane (DCM) can be used as an alternative extraction solvent if simultaneous extraction of other analyte classes (barbiturates, benzodiazepines, opiates and cocaine including the BZE metabolite) is required

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 (ambient room temperature, 20 to 40 L/min).

Reconstitute extracts with ethyl acetate (25 µL) and pentafluoropropionic anhydride (PFPA)(25 µL). Vortex mix then heat on a block for 20 minutes at 70 °C to complete derivatization.

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 (50 µL) and vortex.

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

250 °C, Splitless, purge flow: 50 mL/min at 1.0 min

Injection

2 μL

Wash solvents

Acetone & ethyl acetate

Oven

Initial temperature 25 °C/min to 350 °C, hold for 0.4 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

Results

Blank whole blood was spiked at 100 ng/mL for recovery testing; the typical recovery data is shown in Figure 2. Both MTBE and DCM protocols gave reproducible data with RSD values <10% for barbiturates, benzodiazepines and opiates.

Figure 2. Typical amphetamine/analogue recoveries using MTBE or DCM extraction solvent.

Figure 3. Total Ion Chromatogram of amphetamines and amphetamine type analytes at 100 ng/mL using the MTBE extraction protocol.
 

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 calibrators. The internal standards were 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 0.9939 and 0.9999 for the amphetamine analytes using the MTBE extraction protocol.

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).
• 0.2M HCl in methanol: Add concentrated HCl solution (37%) (200 μL) to methanol (11.8 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: MTBE is the optimum solvent for amphetamine extraction. However if simultaneous extraction of cocaine and its BZE metabolite is required, dichloromethane (DCM) should be used. DCM will also allow the simultaneous extraction of barbiturates, benzodiazepines and opiates. Comparable recoveries, RSDs, LLOQs and linearity for amphetamines are observed with both solvents.

Ordering information

Literature number: AN855