Summary
This application note describes the solid phase extraction of a variety of basic drugs in urine which are typically screened for forensic toxicology panels using EVOLUTE® EXPRESS CX in a 10 mg 96-well plate format.
Application Note AN806Introduction
The screening of patient urine samples for basic drugs is typically performed following a lengthy sample preparation methodology that requires a time consuming dry down step. A novel elution protocol coupled to the EVOLUTE EXPRESS advanced plate technology affords a fast sample preparation solution that follows a LOAD-WASH-ELUTE-ANALYZE strategy. Basic drugs are fortified into urine and extracted in 3 easy steps using EVOLUTE EXPRESS CX solid phase extraction in a 96-well plate format.
Figure 1. Structure of Diazepam, Fentanyl, and Codeine.Analytes
Alprazolam, Clonazepam, Diazepam, Flunitrazepam, Oxazepam, Temazepam, Nitrazepam, Normeperdine, Naltrexone, Morphine, Codeine, Oxymorphone, Hydromorphone, Oxycodone, Hydrocodone, 6-Acetyl Codeine, 6-Acetyl Morphine, Fentanyl, Buprenorphine, EDDP
Sample Preparation Workflow: Load >> Wash >> Elute >> Analyze
Format: EVOLUTE® EXPRESS CX 10 mg Fixed Well Plate, part number 601-0010-PX01 Sample Pre-treatment: Pipette patient urine (200 μL) into a container and add appropriate amount of internal standard.
Pipette urine calibrators and QC samples (200 μL) and add appropriate amount of internal standard.
Pre-treat each of the samples with HCl (0.01N, 200 μL) and gently vortex the solutions. Sample Loading: Load pre-treated samples onto wells. Allow to flow through sorbent via gravity. Sample Washing: 1) Add water (1000 μL) to each well and allow to flow by gravity.
2) Add 0.01N HCl/isopropanol (80:20, v/v, 1000 μL) to each well and allow to flow by gravity. Collect into waste container. Apply vacuum or positive pressure as needed between each step. Analyte Elution: Add a solution of tetrahydrofuran:methanol:acetonitrile:2% aqueous ammonium hydroxide (60:20:19:1, v/v/v/v, 600 μL) and allow solvent to gravity flow into a sample collection plate. Apply positive pressure or pull slight vacuum as needed during collection process to facilitate a flow rate of 1 mL per minute. Post Extraction Add water (600 μL) to the eluent in each well. Load the collection plate onto the HPLC autosampler. Additional Information: Prepare elution solution at a 50 mL total volume by mixing THF (30 mL): MeOH (10 mL): Acetonitrile (9.5 mL): 2% aq NH4OH (0.5 mL). Mix thoroughly before use.
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