HPLC Analysis of Barbiturates in Serum on Discovery® C18 after SPE using Discovery® DSC-18Lt
Materiales
Analytical column
SPE tube or plate
Standard
Secobarbital solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Phenobarbital solution
1 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Butabarbital solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®Barbiturate Mix-5 solution
250 μg/mL each component in methanol, ampule of 1.0 mL, certified reference material, Cerilliant®Pentobarbital solution
1.0 mg/mL in methanol, ampule of 1 mL, certified reference material, Cerilliant®CONDITIONS
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 × 4.6 mm, 5 μm preceded by a 2 cm C18 guard column and 0.5 μm frit filter (504955)
mobile phase
[A] methanol; [B] water (40:60, A:B)
flow rate
1 mL/min
column temp.
30 °C
detector
UV, 214 nm
injection
30 μL, diluted porcine serum extract
Descripción
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.
Legal Information
Discovery is a registered trademark of Merck KGaA, Darmstadt, Germany
RapidTrace is a registered trademark of Zymark Corp.
Zymark is a registered trademark of Sotax Corporation