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`Appendix C
`
`RP-HPLC-MS(SQ) method details
`
`
`Vitas as, Oslo Innovation Centre, Norway
`Vitas P308 Page 24
`
`0000053
`
`
`
`Run Time Checklist
`
`Pre—Run Cmd/Macro:
`
`off
`
`Data Acquisition:
`
`on
`
`Standard Data Analysis:
`
`off
`
`Customized Data Analysis:
`
`off
`
`Save GLP Data:
`
`off
`
`Post—Run Cmd/Macro:
`
`off
`
`Save Method with Data:
`
`off
`
`Agilent 1100/1200 Binary Pump 1
`
`Control
`Column Flow
`Stoptime
`Posttime
`
`Solvents
`Solvent A 2
`Solvent B 1
`
`PressureLimits
`Minimum Pressure
`Maximum Pressure
`
`Auxiliary
`Maximal Flow Ramp
`Compressibility A
`Minimal Stroke A
`
`Compressibility B
`Minimal Stroke B
`
`Store Parameters
`Store Ratio A
`Store Ratio B
`Store Flow
`Store Pressure
`
`Timetable
`
`:
`:
`:
`
`:
`:
`
`:
`:
`:
`
`:
`:
`
`:
`:
`:
`:
`
`0.600 ml/min
`18.00 min
`
`Off
`
`25.0 %
`75.0 %
`
`)
`(
`(ipa 1000 myrsyra 1)
`
`0 bar
`400 bar
`
`100.00 ml/min“2
`80*10‘-6/bar
`Auto
`
`115*10”—6/bar
`Auto
`
`Yes
`Yes
`Yes
`Yes
`
`Time
`
`Solv.B
`
`Flow Pressure
`
`0000054
`
`0000054
`
`
`
`0.00
`10.00
`10.10
`15.00
`16.00
`
`75.0
`85.0
`98.0
`98.0
`75.0
`
`Agilent 1100/1200 Diode Array Detector l
`
`Signals
`
`Signal Store Signal,Bw
`A:
`No
`326
`80
`B:
`No
`326
`80
`C:
`No
`325
`30
`D:
`No
`230
`16
`E:
`No
`280
`16
`
`Reference,Bw [nm]
`650 100
`700 100
`430 100
`360 100
`360 100
`
`Spectrum
`Store Spectra
`
`Time
`
`Stoptime
`Posttime
`
`Required Lamps
`UV lamp required
`Vis lamp required
`
`Autobalance
`
`:
`
`2
`:
`
`:
`:
`
`None
`
`As pump
`Off
`
`No
`No
`
`No
`:
`Prerun balancing
`No
`:
`Postrun balancing
`Margin for negative Absorbance: 100 mAU
`
`Peakwidth
`Slit
`
`:
`:
`
`>
`
`0.2 min
`8 nm
`
`Analog Outputs
`Zero offset ana. out. 1:
`Zero offset ana. out. 2:
`Attenuation ana. out. 1:
`Attenuation ana. out. 2:
`
`Timetable is empty
`
`5 %
`5
`%
`1000 mAU
`1000 mAU
`
`Mass Spectrometer Detector
`
`General Information
`
`Use MSD
`Tune File
`
`StopTime
`
`: Enabled
`: atunes.tun
`
`: asPump
`
`0000055
`
`0000055
`
`
`
`Time Filter
`
`: Enabled
`
`: Condensed
`Data Storage
`: 0.20 min
`Peakwidth
`: Disabled
`Fast Scan
`Fast Scan Data Reconstruction: Disabled
`Polarity Switch Delay
`: 300 ms
`Ionization Switch Delay
`: 50 ms
`
`Signals
`
`[Signal 1]
`
`Ionization Mode
`
`Polarity
`Fragmentor Ramp
`Percent Cycle Time
`
`Scan Parameters
`
`: API—3S
`
`: Posi:ive
`: Disabled
`: 95.00 %
`
`IFrag— I GainIThres—I Step-
`Mass Range
`I
`Time
`Imentorl EMV I hold I size
`Low
`I High
`I
`(min)
`————— --I----—-----I---------I------I-----|~-----I-------
`0.00
`500.00
`1000.00
`150
`1.0
`150
`0.10
`
`[Signal 2]
`
`Ionization Mode
`
`: API—ES
`
`Polarity
`Fragmentor Ramp
`Percent Cycle Time
`Sim On Target Mass
`
`Sim Parameters
`
`: Positive
`: Not Applicable
`: 5.00 %
`: Disabled
`
`SIM IFrag— IGain IActual
`I
`I
`Time
`Ion
`Imentorl EMV IDwell
`I
`Group Name
`I
`(min)
`----- *—|--------~——‘*-----|--—*'*—-I---—--I----‘I———---
`0.00 Group 1
`184.10
`350
`1.0
`50
`
`[Signal 3]
`
`Not Active
`
`[Signal 4}
`
`Not Active
`
`Spray Chamber
`
`0000056
`
`0000056
`
`
`
`Gas Temp
`DryingGas
`Neb Pres
`Quad Temp
`
`VCap (Positive)
`VCap (Negative)
`
`[Time Table]
`
`Time Table is empty.
`
`: 350 C
`: 12.0 1/min
`: 60 psig
`:
`0 C
`
`: 3000 V
`: 3000 V
`
`maximum 350 C
`maximum 13.0 l/min
`maximum 60 psig
`maximum 0 C
`
`END OF MS ACQUISITION PARAMETERS
`
`FIA Series
`
`FIA Series in this Method
`
`:
`
`Disabled
`
`Time Setting
`:
`Time between Injections
`Injection Loop Flush Time :
`
`0.25 min
`0.17 min
`
`Agilent 1100 Wellplate Autosampler 1
`
`Injection
`Injection Volume
`Injection Mode
`
`Time
`
`Stoptime
`Posttime
`
`:
`:
`
`:
`:
`
`High Throughput
`Autom. Delay Vol. Reduction :
`Overlapped Injection
`:
`Minimized Carry Over
`:
`
`2.0 ul
`Standard
`
`No Limit
`Off
`
`Off
`disabled
`Off
`
`Injector Cleaning
`Injection Valve Cleaning / Valve Switching
`Time 1 Bypass
`:
`off
`Time 2 Mainpass/Bypass
`:
`off
`Time 3 Mainpass/Bypass
`:
`off
`Time 4 Mainpass/Bypass
`:
`off
`Valve Movements
`:
`1
`
`Auxiliary
`Draw Speed
`Eject Speed
`Draw Position
`
`Equilibration Time
`
`:
`:
`:
`
`:
`
`20 ul/min
`20 ul/min
`5.0 mm
`
`15.0 sec
`
`0000057
`
`0000057
`
`
`
`Sample Flush—Out Factor :
`Vial/Well Bottom Sensing:
`Store Temperature
`'
`
`5.0 times Injection Volume
`No
`No
`
`Agilent 1100/1200 Column Thermostat 1
`
`Temperature settings
`Left
`temperature
`Right
`temperature
`Enable analysis
`2.0°C
`Store left temperature :
`Store right temperature:
`
`:
`:
`:
`
`60.0°C
`Same as left
`When Temp.
`is within setpoint +/—
`
`Yes
`No
`
`Time
`
`Stoptime
`Posttime
`
`Column Switching Valve
`
`Timetable is empty
`
`:
`:
`
`:
`
`As pump
`Off
`
`Position 1
`
`Estimated Sample Purity Calculation
`
`General
`
`Estimated Sample Purity Calculation is disabled.
`
`H
`
`Integration Events
`
`Event
`
`Value
`
`I —————————————————————————————————————————————————————— --I ——————————— ~—s
`Tangent Skim Mode
`Standard
`Tail Peak Skim {eight Ratio
`0.000
`Front Peak Skim Height Ratio
`0.000
`Skim Valley Ratio
`20.000
`Baseline Correction
`Classical
`
`Peak to Valley Ratio
`
`500.000
`
`0000058
`
`0000058
`
`
`
`Initial Slope Sensitivity
`Initial Peak Width
`
`Initial Area Reject
`Initial Height Reject
`Initial Shoulders
`
`1.000
`0.040
`
`1.000
`1.700
`OFF
`
`Initial
`Initial
`
`Initial
`Initial
`Initial
`
`Event
`
`Value
`
`Time
`
`I ——————————————————————————————————————————————————————————— --I ————— ——1
`Initial Slope Sensitivity
`1.000
`Initial
`Initial Peak Width
`0.040
`Initial
`
`Initial Area Reject
`Initial Height Reject
`Initial Shoulders
`
`1.000
`1.700
`OFF
`
`Initial
`Initial
`Initial
`
`Event
`
`Value
`
`Time
`
`I ---------------------------------------------------------- "—| ----- --I
`Initial Slope Sensitivity
`1.000
`Initial
`Initial Peak Width
`0.040
`Initial
`
`Initial Area Reject
`Initial Height Reject
`Initial Shoulders
`
`1.000
`1.700
`OFF
`
`Initial
`Initial
`Initial
`
`Event
`
`Value
`
`Time
`
`I ——————————————————————————————————————————————————————————— --I ————— --I
`Initial Slope Sensitivity
`1.000
`Initial
`Initial Peak Width
`0.040
`Initial
`
`Initial Area Reject
`Initial Height Reject
`Initial Shoulders
`
`0.500
`0.100
`OFF
`
`Initial
`Initial
`Initial
`
`Event
`
`Value
`
`Time
`
`I ——————————————————————————————————————————————————————————— --I ————— --I
`Initial Slope Sensitivity
`20.000
`Initial
`Initial Peak Width
`0.040
`Initial
`
`Initial Area Reject
`Initial Height Reject
`Initial Shoulders
`
`Integration
`Integration
`Integration
`
`0.500
`0.100
`OFF
`
`OFF
`ON
`OFF
`
`Initial
`Initial
`Initial
`
`0.000
`0.400
`0.600
`
`0000059
`
`0000059
`
`
`
`Event
`
`Value
`
`Time
`
`I ——————————————————————————————————————————————————————————— ——I ————— --I
`Initial Slope Sensitivity
`l0.000
`Initial
`Initial Peak Width
`0.030
`Initial
`Initial Area Reject
`0.010
`Initial
`Initial Height Reject
`0.010
`Initial
`Initial Shoulders
`OFF
`Initial
`
`Integration
`Fixed Peak Width
`
`Integration
`Baseline Now
`
`Integration
`
`OFF
`0.030
`
`ON
`
`OFF
`
`0.000
`0.477
`
`0.550
`0.600
`
`1.000
`
`Initial Slope Sensitivity(Full Scan)
`Initial Peak Width(Full Scan)
`Initial Slope Sensitivity(Cond. Scan/SIM)
`Initial Peak Width(Cond. Scan/SIM)
`Initial Area Reject
`Initial Height Reject
`Initial Shoulders
`
`1
`0
`0.
`0.050
`0
`5
`0
`
`l
`Initial
`'
`
`Event
`
`Value
`
`Time
`
`I ———————————————————————————————————————————————————— ——| ————— —-|
`Initial Slope Sensitivity
`5.000
`Initial
`Initial Peak Width
`0.050
`Initial
`Initial Area Reject
`1.000
`Initial
`Initial Height Reject
`0.l00
`Initial
`Initial Shoulders
`OFF
`Initial
`
`Event
`
`Value
`
`Time
`
`I ——————————————————————————————————————————————————————— ——| ----- --I
`Initial Slope Sensitivity
`5.000
`Initial
`Initial Peak Width
`0.050
`Initial
`Initial Area Reject
`5.000
`Initial
`Initial Height Reject
`1.000
`Initial
`Initial Shoulders
`OFF
`Initial
`
`Apply Manual Integration Events: No
`
`0000060
`
`0000060
`
`
`
`Specify Report
`
`No destination selected
`Destination:
`Quantitative Results sorted by: Signal
`Report Style:
`Short
`Sample info on each page:
`No
`Add Chromatogram Output:
`Yes
`Chromatogram Output:
`Portrait
`Size in Time direction:
`100 % of Page
`Size in Response direction:
`40 % of Page
`
`Signal Options
`
`Include: Axes, Compound Names, Retention Times, Baselines, Tick Marks
`Font:
`Arial, Size:
`8
`
`Ranges: Use Ranges
`
`I Min Value I Max Value I
`——————— ——+———————————+~————-———-—+
`
`I
`Time
`Response I
`
`0.000 I
`-0.500 I
`
`1 500 I
`5 000 I
`
`Multi Chromatograms: Overlaid, All the same Scale
`
`Calibration Table
`
`Retinyl palmetate i kapsel
`
`Calib. Data Modified
`
`Calculate
`Based on
`
`:
`
`:
`:
`
`Saturday, April 30, 2005 1:33:11 PM
`
`External Standard
`Peak Height
`
`Rel. Reference Window :
`Abs. Reference Window :
`Rel. Non—ref. Window :
`Abs. Non-ref. Window :
`
`30.000 %
`0.000 min
`30.000 %
`0.000 min
`
`Use Multiplier & Dilution Factor with ISTDS
`Uncalibrated Peaks
`:
`not reported
`Partial Calibration
`:
`Yes,
`identified peaks are recalibrated
`Correct All Ret. Times:
`No, only for identified peaks
`
`Curve Type
`Origin
`Weight
`
`:
`:
`:
`
`linear
`Included
`Equal
`
`Recalibration Settings:
`Average Response
`:
`Average Retention Time:
`
`Average all calibrations
`Floating Average New 75%
`
`Calibration Report Options
`Printout of recalibrations within a sequence:
`Calibration Table after Recalibration
`
`0000061
`
`0000061
`
`
`
`Normal Report after Recalibration
`If the sequence is done with bracketing:
`Results of first cycle (ending previous bracket)
`
`Signal 1: MSDl TIC, MS File
`
`Peak Sum Table
`
`***No Entries in table***
`
`0000062
`
`0000062
`
`
`
`Appendix D
`
`RP-HPLC-MS/MS(3Q) method details
`
`Vitas as, Oslo Innovation Centre, Norway
`
`0000053
`
`Vitas P308 Page 25
`
`0000063
`
`
`
`Acquisition Method RP-
`._|1fl§:__M,S/FMS L§Q),,-.,,
`
`Method Name
`
`M°th°d Path
`
`grad mrm
`110929a.m
`
`D:\Vitas\pad\aker biomarin\p308 1109Z9\grad mrm
`110929a.m
`
`Method Description
`
`was p303
`
`Device List
`h-ALS
`
`Bin Pump
`Column
`
`MS QQQ
`
`QQQ Mass Spectrometer
`
`ESI
`atunes.tune.x
`ml
`BY
`Stoptime
`
`80
`
`n
`
`0,2
`
`Ion Source
`
`Tune File
`
`Stop Mode
`Stop Time
`
`Time Filter
`
`Time Filter Width
`
`Time Segments
`
`Time Seg #
`1
`
`Scan
`Time Type
`0 MRM
`
`Div
`Ion
`Mode Valve
`ESI
`To Ms
`
`Delta Sto
`EMV re
`0
`
`Time Segment
`
`1
`
`Scan Segments
`
`Compound Name
`
`ISTD?
`
`Prec MS1
`Ion
`Res
`
`Prod MS2
`Ion
`Res
`
`Dw
`ell
`
`Frag
`(V)
`
`DHA
`
`DHA
`
`DHA
`
`El
`
`El
`
`D
`
`878,4
`
`Unit
`
`568,2
`
`Unit
`
`878,4
`
`878,4
`
`852,4
`
`Unit
`
`Unit
`
`Unit
`
`550,2
`
`184
`
`568,2
`
`Unit
`
`Unit
`
`Unit
`
`50
`
`50
`
`50
`
`50
`
`200
`
`200
`
`200
`
`200
`
`30
`
`30
`
`40
`
`30
`
`CE Polari
`(V)
`tyPositiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`Positiv
`e
`
`30
`
`30
`
`30
`
`40
`
`30
`
`30
`
`40
`
`DHA EPA
`
`DHA EPA
`
`DHA EPA
`
`DHA EPA
`
`DHA EPA
`
`EPA
`
`EPA
`
`EPA
`Source Parameters
`Valu
`e
`
`Parameter
`
`El
`
`El
`
`El
`
`El
`
`El
`
`El
`
`El
`
`El
`
`852,4
`
`852,4
`
`852,4
`
`852,4
`
`826,4
`
`826,4
`
`826,4
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`550,2
`
`542,2
`
`524,2
`
`184
`
`542,2
`
`524,2
`
`184
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`Unit
`
`50
`
`50
`
`50
`
`50
`
`50
`
`50
`
`50
`
`200
`
`200
`
`200
`
`200
`
`200
`
`200
`
`200
`
`0000064
`
`0000064
`
`
`
`Gas Temp (‘’C)
`Gas Flow
`(I/min)
`Nebulizer (psi)
`Capillary (V)
`
`350
`
`12
`60
`3000
`
`Chrom Type
`
`TIC
`TIC
`Instrument Curves
`Actua
`
`I #
`
`N/A
`
`Chromatagrams
`Y-
`Labe
`I Offset Range
`100000
`00
`
`0
`
`Wellplate Sampler
`
`Name
`Ordinal
`#
`
`h-ALS Model
`Optio
`ns
`
`1
`
`G1367
`B
`
`THM
`
`Stop Time (min)
`
`As
`Pump
`
`Post Time
`(min)
`
`=§O
`
`Injection
`Volume
`Draw
`Position
`
`Draw Speed
`Flush Out
`Factor
`Equilibratio
`n Time
`
`Wash
`Location
`
`Wash
`Cycles
`Temp.
`
`
`
`>\2>\ZOOU1-§'r-I
`
`Standard Injection
`Disable overlapped
`Injection
`
`0 5
`
`0
`
`NO
`
`N/A
`
`N/A
`
`Injection Type
`
`Overlap Time
`
`Draw Position Detection
`
`Eject Speed
`
`Automatic Delay Volume Reduction
`
`Wash Vessel
`
`Wash Time
`
`Ready Temp. Range
`
`Contact
`1
`Contact
`2
`Contact
`3
`Contact
`4
`
`0
`
`0
`
`0
`
`0
`
`Injector Program
`Signals Selected
`Contact: Time Table
`
`Binary Pump
`
`Name
`Ordinal
`#
`
`Bin
`Pump Model
`Optio
`ns
`
`1
`
`G1312
`A
`
`SSV
`
`0000065
`
`0000065
`
`
`
`ipa 1000 1 ml myrsyra 1g/I
`amonium acetat
`
`80
`
`Solvent B
`Solvent
`Ratio B
`
`Solvent Type
`B1
`Solvent Type
`B2
`
`Stop Time (min)
`
`18
`
`Post Time
`(min)
`
`0
`ff
`
`01
`
`0
`0
`
`0.
`6
`
`Pressure Min (bar)
`
`40 Max Flow Gradient
`0
`(ml/min)
`
`Flow (ml/min)
`
`Pressure Max (bar)
`
`Solvent A
`
`vatten 500 ml metanol 500 ml 1 ml myrsyra 1g/I
`amonium acetat
`
`Solvent Ratio A
`
`20
`
`Solvent Type A1
`
`Solvent Type A2
`
`Compress. A (*10-6/ bar)
`
`5 Compress.B(*10-
`0 6/ bar)
`
`1
`5
`
`Stroke A Auto
`
`Stroke
`3
`
`Auto
`
`Stroke Syncronization
`
`Contact
`1
`Contact
`2
`Contact
`3
`Contact
`4
`
`0
`
`0
`
`0
`
`0
`
`Pump Time Table
`
`Time
`
`0
`
`10
`
`10.1
`
`15
`
`Flow
`No
`Change
`No
`Change
`No
`Change
`N0
`Change
`No
`Change
`16
`Signals Selected
`Cantacs Time Table
`
`Pressure
`
`Nochange
`
`No Change
`
`NoChange
`
`Nochange
`
`No Change
`
`Solv
`RatioB
`
`75
`
`85
`
`98
`
`98
`
`75
`
`Thermostated Column
`
`,,9°"!B3;‘!!!!§!.‘ E
`
`lr
`
`Name
`Ordinal
`#
`
`Colum
`n
`
`1
`
`Model
`Optio
`ns
`
`G1316
`A
`
`0000066
`
`0000066
`
`
`
`Stop Time (min)
`
`As
`Pump
`
`Post Time
`(min)
`
`0
`ff
`
`60
`When Temp Within Set Point +/—
`0.8
`
`1
`
`Right
`Temp.
`Right
`Ready
`
`Same as |eft
`When Temp Within Set Point
`+/- 0.8
`
`Left Temp.
`
`Left Ready
`Valve
`Position
`
`Contact
`1
`Contact
`2
`Contact
`3
`Contact
`4
`
`0
`
`0
`
`0
`
`0
`
`Temperature Time Table
`Signals Selected
`
`Description
`Temperature of left heat exchanger
`Contacb Time Table
`
`0000067
`
`0000067
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