HPLC Analysis of 3-Methylpyrazole and 4-Methylpyrazole in Urine on Discovery^® C18 after SPE using Discovery^® DSC-SCX

sample preparation

SPE (Solid Phase Extraction)

sample/matrix

urine spiked with 3- and 4-methylpyrazole at 1 mg/mL

SPE tube/cartridge

Discovery DSC-SCX, 500 mg/3 mL (52686-U)

condition

2 mL methanol; 2 mL DI water

sample addition

1 mL

washing

2 mL DI water

elution

2 mL 5% methanol in 250 mM potassium phosphate buffer, pH 7.4

column

Discovery C18, 15 cm × 4.6 mm, 5 μm preceded by a 2 cm guard column and 0.5 μm frit filter (504955)

mobile phase

MeOH:5 mM phosphate buffer, pH 6 (20:80)

flow rate

1 mL/min

column temp.

30 °C

detector

UV, 220 nm

injection

25 μL, dilute urine extract