| |
[Apparatus]
Column switching-liquid chromatograph/mass spectrometer (LC/MS):
LC/MS equipped with a column switching valve and capable of
selective ion monitoring (SIM) (m/z = 219 [M-H]-) using an
electrospraying ion source. While the LC/MS/MS measurements are
possible, monitoring the deprotonated ion of m/z = 219 is
adopted here.
[Sample preparation]6
Procedure A: Place 1 ml serum in a test tube and treat with
glucuronidase. After the enzyme reaction is completed, add
purified water to 1.5 ml. Add the internal standard to the
temporary concentration. Inject the solution directly into the
column switching-LC/MS.
Procedure B: Place 1 ml serum in a test tube, treat with
glucuronidase, and add the internal standard to the temporary
concentration. Add the same amount of acetonitrile as the serum,
mix and centrifuge (3000 rpm, 30 min, 4℃). Transfer the
supernatant to a vial through a filter7
[Operation]
(i) LC/MS conditions (typical)
Column for analysis: C18 Reverse phase column, 2.0 mm i.d., 100
mm long, grain size 5μm8
Column temperature: 40℃
Mobile phase for analysis: Water:acetonitrile (gradient elution
with 0.02% ammonium acetate9:
30:70 (8 min) -> 5:95 (10 min) -> 5:95 (20 min))
Flow rate of mobile phase: 0.5 ml/min
Column for extraction: Inner face reverse phase column, 4.6 mm
i.d., 10 mm long, grain size 5μm4
Mobile phase for extraction: Purified water (100%10
Flow rate of mobile phase: 0.5 ml/min
Valve switching time: 5 min
Quantity injected: 30μl
Ionization: Electrospray ionization, negative mode
MS monitor ions: m/z = 219 (NP), m/z = 224 (m-OP-d5)
|
