Extraction of benzodiazepines from whole blood using ISOLUTE® SLE+

Figure 1. Structure of Diazepam

Introduction

This application note describes the extraction of benzodiazepine compounds from whole blood, prior to GC/MS analysis. This protocol also allows the simultaneous extraction of various other drugs of abuse classes: amphetamines, barbiturates, cocaine 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 10 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

Diazepam-D5, Diazepam, Nordiazepam, Midazolam, Flunitrazepam, 7-amino-flunitrazepam, Bromazepam, Oxazepam, Nitrazepam, Flurazepam, Temazepam,
7-amino-clonazepam, Lorazepam, Clonazepam, 2-hydroxy- ethyl-flurazepam, Estazolam, Alprazolam, Triazolam, Alpha- hydroxy-alprazolam, Alpha-hydroxy-triazolam
 

Sample preparation procedure

Format:

ISOLUTE® SLE+ 1mL 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.

* Note that MTBE can be used as an alternative extraction solvent if a non-chlorinated option is required. MTBE also suitable for extraction of other analyte classes (amphetamines, barbiturates, opiates). If simultaneous extraction of cocaine and metabolites is required, DCM should be used as extraction solvent.

Post-elution and reconstitution

Evaporate the extract in a stream of air or nitrogen using a TurboVap® LV (40 °C, 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 (50 µL) and MTBSTFA (with 1% t-BDMCS) (50 µL), vortex and heat on a block 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

1 μL

Wash solvents

Acetone & ethyl acetate

Oven

Initial temperature 60 °C, hold for 1 minute, ramp 12 °C/min to
330 °C, hold for 0.5 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%.

Figure 2. Typical benzodiazepine recoveries using MTBE or DCM as extraction solvent.

Figure 3. Total Ion Chromatogram of application analytes at 100 ng/mL using the DCM 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 calibration curves. Diazepam-D5 was spiked at 100 ng/mL for each level. The curves are shown in Figure 4.

Figure 4. Chart demonstrating coefficient of determination (r2) values between 0.9922 and
0.9999 for benzodiazepines using the DCM 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).

Cartridge loading: ISOLUTE® SLE+ cartridges are underloaded (750 µL sample on a 1 mL capacity cartridge) to avoid breakthrough of whole blood matrix.
Non-chlorinated extraction solvent alternative: MTBE can be used as an alternative extraction solvent if a non-chlorinated option is required. MTBE also suitable for extraction of other analyte classes (amphetamines, barbiturates, opiates). If simulta- neous extraction of cocaine and metabolites is required, DCM should be used as extraction solvent.

Ordering information

Literature number: AN854