HPLC Analysis of Barbiturates in Serum on Discovery® C18 after SPE using Discovery® DSC-18Lt

HPLC Analysis of Barbiturates in Serum on Discovery® C18 after SPE using Discovery® DSC-18Lt


sample preparation SPE (Solid Phase Extraction)
sample/matrix 0.5 mL porcine serum spiked with 0.5 µg/mL or 1.0 µg/mL each analyte then diluted with 0.5 mL water
SPE tube/cartridge Discovery DSC-18Lt, 500 mg/3 mL (52613-U)
condition 2 mL methanol; 2 mL DI water
sample addition 1 mL at 0.75 mL/min
washing 2 mL 5% methanol, then vacuum or air dry for 5-10 min
elution 1-2 mL methanol
eluate post-treatment dry eluate with nitrogen purge (40 °C; 15-20 min), reconstitute in 20 µL mobile phase
column Discovery C18, 15 cm x 4.6 mm, 5 µm preceded by a 2 cm C18 guard column and 0.5 µm frit filter (504955)
column temp. 30 °C
mobile phase [A] methanol; [B] water (40:60, A:B)
flow rate 1 mL/min
injection 30 µL, diluted porcine serum extract
detector UV, 214 nm


Analysis Note Barbiturates are commonly abused and among the most widely tested compounds in clinical, forensic, or therapeutic drug monitoring applications. Shown here is the baseline separation of a set of barbiturates on a Discovery C18 HPLC column after extraction from spikd serum with Discovery DSC-18Lt SPE. Highest grade HPLC solvents were used to supply low background interference and low particulate contamination for robust, trouble-free operation. Cerilliant and Sigma-Aldrich reference standards provided reliable identification and quantification. A Zymark® RapidTrace® SPE Workstation was used in this study.
Categories Analytical Chromatography, SPE Applications, Clinical, Drugs of abuse, Pharmaceutical & Clinical applications, drugs of abuse
Featured Industry Forensics and Toxicology
Legal Information RapidTrace is a registered trademark of Zymark Corp., Discovery is a registered trademark of Sigma-Aldrich Co. LLC, Zymark is a registered trademark of Sotax Corporation
suitability application for SPE, application for HPLC


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