Introduction
This application note describes the extraction of acidic, basic, and neutral drugs from urine for screening purposes using ISOLUTE® SLE+ supported liquid extraction plates prior to LC-MS/MS analysis. ISOLUTE SLE+ Supported Liquid Extraction products 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. In this application note, ISOLUTE SLE+ is used to quickly screen a panel of drugs from a small amount of urine (100 μL). The limit of detection was determined for each analyte and was found to range from 5 ng/mL to 1.25 ng/mL. The application note describes this quick robust screening assay for the extraction of 32 different drugs from a hydrolyzed urine matrix.
Analytes
Codeine, hydrocodone, oxycodone, norcodeine, oxymorphone, 6-acetyl codeine, alprazolam, clonazepam, diazepam, flunitrazepam, nitrazepam, oxazepam, temazepam, dextromethorphan, buprenorphine, norbuprenorphine, fentanyl, EDDP, benzoylecgonine, tetrahydrocannabinol, normeperidine, naltrexone, hydromorphone, propoxyphene, pentazocine, amphetamine, norfentanyl, MDEA, butalbital, pentobarbital, phenobarbital, secobarbital, 11-nor-9 –carboxy- Δ9-tetrahydrocannabinol, and d3-11-hydroxy-tetrahydrocannabinol.
Sample Preparation Procedure
Format: ISOLUTE® SLE+ 400 μL Supported Liquid Extraction plate, part number 820-0400-P01
Sample Pre-treatment
To 100 μL of sample (calibrator, QC or patient sample), add 100 μL of 100 mM ammonium acetate, pH 5 (hydrolysis buffer). Then add a pre-determined amount of β-glucuronidase (enzyme) to solution to achieve a target concentration of 5000 U/mL. Incubate the sample at optimal temperature for enzymatic hydrolysis (follow vendor guidelines for optimal activity). Post-hydrolysis, add water (200 μL) to the hydrolyzed solution to pre-treat sample.
Sample Loading
Load up to 400 μL total volume of the pre-treated sample into each well and apply a pulse (3–5 seconds) of vacuum (using a vacuum manifold) or positive pressure (using a Biotage® PRESSURE+ 96 Positive Pressure Manifold).
Analyte Extraction
Elute analytes with two successive aliquots of dichloromethane: isopropanol (90:10 [v:v], 700 μL) per well. Elute solution slowly at a rate of 1 mL/min (~10-12 drops/min) under gravity or using minimal pressure/vacuum. Apply a pulse (5–10 seconds) of vacuum or positive pressure to pull through any remaining extraction solvent.
Post Extraction
Dry the extract in a stream of air or nitrogen using a Biotage®SPE Dry 96 (40 °C at 60 L/min) or TurboVap® 96 (40 °C at 1.0 bar/14.5 psi).
Reconstitution
Reconstitute the sample with of methanol:water with 0.1% formic acid (60:40 [v:v], 100 μL) in each well and let sample equilibrate for 15 minutes.
>> Download the full Application Note as PDF
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