HPLC Analysis of NNAL in Urine on Ascentis^® Express C18 after SPE using SupelMIP^® SPE-NNAL

sample preparation

SPE (Solid Phase Extraction)

sample/matrix

human urine spiked with 60 or 100 pg/mL NNAL

SPE tube/cartridge

SupelMIP SPE - NNAL, 25 mg/10 mL (LRC) (53206-U)

condition

1 mL dichloromethane; 1 mL methanol; 1 mL DI water

sample addition

2 mL (60 pg/mL sample), 5 mL (100 pg/mL sample)

washing

2 x 1 mL DI water, 10 min vacuum; mL toluene; 1 mL toluene:dichloromethane (9:1, v/v); 1 mL toluene:dichloromethane (4:1, v/v), 2 min. vacuum

elution

2 x 1 mL 10% methanol in dichloromethane, apply gentle vacuum between each fraction

eluate post-treatment

evaporate under nitrogen and reconstitute in 0.15 - 0.25 mL mobile phase

column

Ascentis Express C18, 5 cm x 2.1 mm I.D., 2.7 μm particles (581307-U)

mobile phase

[A] 10 mM ammonium acetate; [B] 10 mM ammonium acetate in acetonitrile

flow rate

0.3 mL/min

column temp.

35 °C

detector

MS/MS, MRM Transitions, ESI(+) (210.2/180 and 210.2/93.2 m/z)

injection

20 μL