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[Apparatus and conditions]
Perform multiple reaction monitoring (MRM) on a liquid
chromatograph/tandem mass spectrometer (LC/MS/MS) typically
under the following conditions.
Column: C18 reverse phase column2
(2.0 mm i.d., 50 mm long, grain size 3 μm)
Column temperature: 50℃
Mobile phase: 0.02% Ammonia water (A), acetonitrile (B)
Gradient elution: (B), 20 -> 60% in 10 min, linear
Flow rate of the mobile phase: 0.2 ml/min
Monitor ions (precursor/product): m/z = 271/145 for E2, m/z =
274/145 for E2-d3
[Preparation of sample solutions]
Mix 100 ng E2-d3 with 10 g of pulverized feed and add 50 ml
methanol. After leaving the mixture standing for 1 h at ambient
temperature, homogenize for 2 min, irradiate with ultrasound for
10 min, centrifuge, and recover the supernatant. Wash the
residue with 10 ml methanol, centrifuge, recover the
supernatant, and merge with the previously recovered supernatant
to 50 ml. Charge the graphite carbon/aminopropyl silica gel
column with 2.0 ml of the extract, wash with 20 ml methanol and
elute with 10 ml dichloromethane/methanol (50:50). Evaporate the
eluent to dryness under a nitrogen flow, and dissolve in 0.20 ml
of the initial eluent of the LC to obtain the sample solution.
[Calibration curve]
Construct the calibration curve using the E2/E2-d3 peak area
ratios.
[Detection and determination limits]
The standard deviation of operation blank results multiplied by
3 gives the detection limit, and by 10 the determination limit.
[Notes]
The present method was developed considering that some animal
feeds contain fish flour.
1 E.g. Supelco Supelclean ENVI-Carb/LC-NH2 (500 mg/500 mg).
2 E.g. Intakt Corp.'s Cadenza CD-C18, 2.0 x 50 mm, 3 μm. |
