HPLC Analysis of Chloramphenicol in Milk on Ascentis^® C18 after SPE using SupelMIP^® SPE-Chloramphenicol

sample preparation

SPE (Solid Phase Extraction)

sample/matrix

whole pasteurized milk spiked with chloramphenicol at 15 ng/mL and 38 ng/mL

SPE tube/cartridge

SupelMIP SPE - Chloramphenicol, 25 mg/10mL (LRC) (53210-U)

condition

1 mL methanol;1 mL DI water

sample addition

1 mL

washing

2 x 1 mL MS-grade water, 1 mL 5% acetonitrile in 0.5% acetic acid, 2 x 1 mL MS-grade water, 1 mL 20% acetonitrile in 1% ammonium hydroxide. (dry SPE tubes for 15 min. under gentle vacuum; 3 x 1 mL dichloromethane; dry SPE tubes for 1 min. under gentle vacuum)

elution

elute chloramphenicol with 2 x 1 mL methanol:acetic acid:MS-grade water (89:1:10, v/v/v)

eluate post-treatment

evaporate combined eluate to dryness at 50 °C under nitrogen. reconstitute in 150 mL LC mobile phase prior to LC-MS analysis

column

Ascentis C18, 2.1 mm x 10 cm I.D., 3 μm particles (581301-U)

mobile phase

[A] 100 mM ammonium acetate: [B] MS-grade water: [C] acetonitrile (10:60:30, A:B:C)

flow rate

0.2 mL/min

column temp.

35 °C

detector

MS, ESI(-), 320-323 m/z range

injection

5 μL