Extraction of benzodiazepines from human urine using ISOLUTE® SLE+

This application note describes the extraction of a range of benzodiazepines from human urine using ISOLUTE® SLE+ supported liquid extraction cartridges prior to derivatization and GC-MS analysis.

Introduction

Benzodiazepines are psychoactive drugs used to treat various conditions including anxiety, insomnia, seizures and alcohol withdrawal. Long-term use can cause physical dependence and withdrawal upon cessation of use, due to this their application as medication has to be monitored and measured. This application note details an optimized extraction protocol for the use of ISOLUTE® SLE+ supported liquid 1 mL sample volume cartridges to extract a range of benzodiazepines from human urine. This simplified and efficient extraction method has analyte recoveries from 90% with LOQs as low as 40 ng/mL. The application has also been performed with halogenated and non-halogenated extraction solvents in the interests of providing for green chemistry.

Figure 1: Structure of diazepam

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 time.

Analytes

Diazepam, nordiazepam, midazolam, flunitrazepam, 7-aminoflunitrazepam, bromazepam, oxazepam, nitrazepam, flurazepam, temazepam, 7-aminoclonazepam, lorazepam, hydroxyethylflurazepam, estazolam, alprazolam, triazolam, α-hydroxyalprazolam, α-hydroxytriazolam.

ISOLUTE® SLE+ procedure

Format: ISOLUTE® SLE+ 1 mL Sample Volume cartridges, part number 820-0140-C

Sample pre-treatment: Dilute human urine (500 µL) with sodium acetate (100 mM, pH 5 (500 µL ). Perform enzymatic hydrolysis with α-glucuronidase (H3) ex. H.pomatia (Sigma-Aldrich G8885) 50 µL/mL urine (approx. 4500 U/mL urine) at 60 °C ± 2 °C for 2 hours. Adjustment to pH 7 using 25 % ammonium hydroxide (conc.), 10 µL/mL urine.

Sample loading: Load pre treated urine (1 mL in total) onto the ISOLUTE® SLE+ cartridge followed by a pulse of vacuum to initiate flow (VacMaster-10 Sample Processing Manifold 121-1016), allow the sample to absorb for 5 minutes.

Analyte elution: Apply dichloromethane (2.5 mL), wait five minutes to allow the solvent to adsorb, apply a short pulse of vacuum if solvent not fully adsorbed. Apply a second aliquot of dichloromethane (2.5 mL), allow to adsorb for a further 5 minutes and then apply a final pulse of vacuum.

Post Extraction: Evaporate the eluate to dryness with 80 L/min air at 35˚C and reconstitute in 50 µL of ethyl acetate. Sample then derivatized 1:1 with with TBDMSTFA, N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide with 1% tert-butyldimethylchlorosilane (Sigma-Aldrich 375934) at 70 °C ± 2 °C for 20 minutes.

GC conditions

Instrument: 7890A GC / 5975A MSD (Agilent Technologies Inc., Santa Clara CA, USA).

Column: Zebron ZB-5Msi, 30 m x 0.25 mm, 0.25 µm df. Deactivated fused silica, 5 m x 0.25 mm pre-cartridge. )

Carrier gas: Helium 1.2mL min-1 (constant flow)

Inlet: Pulsed Splitless, 9.8 psi at 250 °C Pressure pulse 20 psi to 0.75 min Purge flow 50 mL/min at 1.5 min

Injection: 2 µL

Oven: 50 °C for 1.5 min, 25 °C/min to 150 °C then 10 °C/min to 320 °C, hold 6 min

Transfer Line: 280 °C

MS conditions

Source: 230 °C

Quadrupole: 150 °C

Solvent Delay: 6 min

MSD mode: SIM

SIM Groups:  1 – 6.1 min to 18.4 min / 2 – 18.4 min to 19.1 min
                     3 – 19.1 min to 20.2 min / 4 – 20.2 min to 22.6 min
                     5 – 22.6 min to 23.9 min / 6 – 23.9 min to 25.3 min
                     7 – 25.3 min to 28 min

Figure 2: Overlaid target ion chromatograms sim groups 1-7

Figure 2 shows a collection of the overlaid target ion chromatograms for all benzodiazepine analytes spiked at 500 pg/μL. Ethyl acetate was determined to be an acceptable non-halogenated alternative to dichloromethane as an extraction solvent, this is demonstrated alongside the dichloromethane recoveries shown in figure 3.

However, recoveries are lower due to decreased signal: noise in proportion to extract cleanliness.

An LOQ of 40 ng/mL was reached (500 μL matrix) for all analytes. Peak-to-peak signal/noise 14:1 or greater with RSDs < 10% (n=3).

Figure 3: Analyte % recoveries for the full range of extracted benzodiazepines, comparing two different extraction solvents, halogenated and non-halogenated

Literature number: AN756