HPLC Analysis of Bronchodilators Theophylline and Other Caffeine Metabolites in Serum on Discovery^® RP-AmideC16 after SPE using Discovery^® DSC-18

sample preparation

SPE (Solid Phase Extraction)

SPE tube/cartridge

Discovery^® DSC-18, 500 mg/3 mL (52603-U)

condition

2 mL methanol, then 2 mL water

sample preparation

1 mL porcine serum spiked with 0.1 μg/mL, 0.50 μg/mL, or 1.0 μg/mL each analyte

sample preparation

1 mL methanol; evaporate to dryness with nitrogen stream at room temperature; reconstitute in 200 μL mobile phase containing 0.2 μg/mL or 7-methylxanthine (IS)

sample preparation

2 mL 5% methanol in water; dry tube 10 min with nitrogen stream

column

Discovery RP-AmideC16, 15 cm × 4.6 mm, 5 μm particles, preceded by a 2 cm RP-AmideC16 guard column and 0.5 μm frit filter (505013)

mobile phase

[A] Methanol; [B] 1% acetic acid (17:38, A:B)

flow rate

1 mL/min

column temp.

30 °C

detector

UV, 272 nm

injection

20 μL reconstituted porcine serum extract