LCMSMS

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LC-MS/MS
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Structure of Melamine and Related Compound
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• ?????????????
• Determination of Melamine and Cyanuric Acid
  using Liquid Chromatography Electrospray
  Ionization Mass Spectrometry (LC/ESI-MS/MS),
  Calgary Laboratory, Canadian Food Inspection
  Agency, version 1.0

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• ??????????????????????????????????????????????
  ????????? LC-MS/MS

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• ?????????????????????????????????? Acetonitrile
  ??????
• ????????????????????????????????????????????????
  ??? LC-MS/MS

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• ?????????????????
• ???????? (18 M? cm)
• Acetonitrile
• Ammonium formate

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• ????????????????????????????? (centrifuge)
• Centrifuge tubes ?????????? ???? 50 mL
• LC vials
• Vol. flask ???? 50 mL
• Mechanical shaker
• Vortex mixer
• ?????????? ???? 5-40 ?l, 40-200 ?l, 100-1000 ?l
• ???????????
• ??????? LC-MS/MS

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• ?????????????????????

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???????????????????????????????????? LC-MS/MS
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LC-System

• LC Column
• Analytical column Atlantis HILIC Silica 3
  ?m 3.0 x 50 mm column
• Column Temperature 35 ? C
• Injection volume 5 ?l

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Mobile Phases
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MS/MS

• Mass Spectrometer API 4000 LC-MS/MS system
• (Applied Biosystems)
• Ion Source Turbo Spray ( ESI )

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MS/MS Parameters

• MS Parameters
• Collision gas (CAD) 7
• Curtain gas (CUR) 20
• Ion source gas 1 (GS1) 65
• Ion source gas 2 (GS2) 60
• Temperature (TEM) 500 ? C
• Ionspray voltage (IS)
  5500.00
• Entrance Potential (EP)
  10

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MS/MS Parameters

• Multiple Reaction Monitoring ( MRM )
• Positive mode

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Melamine
MRM chromatogram of standard melamine 0.5 ng/mL,
column Atlantis HILIC Silica 3 ?m 3.0 x 50 mm
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ammelide
Melamine
cyanuric acid
ammeline
MRM chromatogram of standard cyanuric acid,
ammelide, ammiline 25 ng/mL, column Atlantis
HILIC Silica 3 ?m 3.0 x 50 mm
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Method validation
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• Column ZIC-HILIC

????????? Melamine
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LC-System

• LC Column
• Analytical column ZIC-HILIC 5 um, 200 0A 150
  x 2.1 mm. with ZIC-HILIC Guard 5 um 20 x 2.1 mm,
  SeQuant
• Column Temperature 40 ? C
• Injection volume 5 ?l

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Mobile Phases
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MS/MS Parameters

• Multiple Reaction Monitoring ( MRM )
• Positive mode

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MRM chromatogram of standard melamine 2
ng/mLISTD 5 ng/mL column ZIC-HILIC 5 um, 200
0A 150 x 2.1 mm. with ZIC-HILIC Guard
5 um 20 x 2.1 mm
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Recovery
?????????
????? spiked rec
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????????? ??????? ??????????? ???????????? ???????
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0.2 ppm 1 ppm 2 ppm 1 ppm 1 ppm 1 ppm 2 ppm 0.4
ppm 1 ppm 0.2 ppm
90-96 80-105
97 81-106 80-90
96 -105 95 97
90-104 86
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• Column ZIC-HILIC

????????? Melamine and Cyanuric acid
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Mobile Phases
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MS/MS Parameters

• Multiple Reaction Monitoring ( MRM )
• Negative mode

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MS/MS Parameters

• Multiple Reaction Monitoring ( MRM )
• Positive mode

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MRM chromatogram of standard cyanuric acid,
melamine 20 ng/mL column ZIC-HILIC 5 um, 200
0A 150 x 2.1 mm. with ZIC-HILIC Guard
5 um 20 x 2.1 mm
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MRM chromatogram of standard melamine 0.5 ng/mL,
column ZIC-HILIC ?m 3.0 x 50 mm
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Mobile Phases
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Method Validation of Cyanuric acid
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Method Validation of Melamine
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Calculation

• Melamine (?g/g) (ng/ml blank) x dilution
  factor
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  Sample weight x 1000

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Spike Recovery Calculation

• Recovery (S U) x W x 100
• Cws

• S melamine in spiked sample, ug/g (ppm)
• U melamine in unspiked sample, ug/g (ppm)
• W amount of sample used in spike (g)
• Cws microgram of spiked standard

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Confirmation

• LC Retention Time retention time should not
  exceed 5 tolerance relative to the retention
  time of standards.
• Ions Ratios the relative intensities of the
  detected ions, expressed as a percentage of the
  intensity of the most intense ion or transition,
  must correspond to those of the standards, either
  from calibration standards or from spiked
  samples, at comparable concentrations and
  measured under the same conditions, within the
  tolerances given in the table.

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• a relative intensity ( of base peak)
• b tolerance relative to relative intensity ( of
  peak intensity)

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Control Sample and Spike

• If results for the control or spike sample
  are outside of
• control limits, results are invalid and
  analysis is repeated.
• - Blank must be free of interfering
  peaks.
• - Spike Recovery as per control charts
• - Correlation Coefficients R2 gt 0.98

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Clitical Control Points

• -Sample extracts shall be analysed
  immediately after the sample preparation.

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Reporting

• - at least two chosen MRM transitions are
  detected with correct daughter ratios and
  retention times from the LC column

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• If the results
• lt LOD reports not detected
• gt LOD - lt LOQ reports less than LOQ
• gt LOQ reports actual result
• Reporting Level
• mg/kg (ppm)

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Thank you for your attention