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Analysis by LC/MS/MS
(i) LC/MS/MS conditions (typical)
Column for analysis: C18 Reverse phase column, 2.1 mm i.d., 150
mm long, grain size 5 μm8
Column temperature: 40℃
Mobile phase for analysis: Water:methanol (0.5 mM ammonium
acetate9)
Flow rate of mobile phase: 0.2 ml/min
Quantity injected: 20 μl
Ionization: Electrospray ionization, negative mode
MS monitor ions: NP: m/z = 219 (precursor ion), 133 (product
ion); m-OP-d5: m/z = 224 (precursor ion), 123 (product ion)
Ionization spray voltage: -4500 V
Ion source temperature: 500℃
(ii) Qualitative and quantitative analysis
Confirm that the MRM peak (m/z = 219 -> 133) permits separation
of the base line, and use the area of the peak for quantitation
using the internal standard. Construct a calibration curve over
a concentration range of 1 to 100 ppb. This range may be
expanded if samples of higher concentrations are to be analyzed.
[Detection and determination limits]
Perform at least five measurements on the standard solution of
the lowest concentration used for construction of the
calibration curve. The standard deviation of the results
multiplied by 3 gives the detection limit, and by 10 the
determination limit. If operation blank tests are performed, use
the standard deviation for the standard solution or for the
blanks, whichever the greater10.
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