1. Method Translations: Cross-Platform Development & Implementation of Selected Pyrethroid Metabolites and TCPy
The WEBS (Washington Environmental Biomonitoring Survey) Laboratory Team:
Blaine Rhodes, PI
Caroline E. West, Chemist 3
Lynn Skidmore, Chemist 1
Michel Lundy, Chemist 1
Tiffany Firestone, HSC 2
Hello, my name is Caroline and I am the lead chemist for the WEBS group. I would like to talk to you about method translation and development today because this is a VERY important and time consuming part of starting up a biomonitoring program or adding new methods to an existing program.
This slide shows our lab team but I would like to mention there is an epidemiology/toxicology side to the grant as well that works closely with us on deciding what analytes are important to test for and what the clinical significant levels might be.

2. Method Translation: Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS)
Many Different LC/MS/MS Systems Available
Agilent
Waters
Shimadzu
ThermoScientific
AB Sciex
Plus more!
Pyrethroid/TCPy Method Development
CDC: ThermoFinnigan TSQ Quantum MS/MS coupled to an Agilent 1100 HPLC System
WA PHL: Agilent 6410 coupled to an Agilent 1200 HPLC System
METHOD TRANSLATION is an important part of developing and implementing new methods in your laboratory. Many different labs use many different analytical systems which do not have the same working parameters: Some have parameters which can be manually manipulated while others leave it under automation control. DIFFERENT terminology is used to refer to the same parameters and some have different working mechanisms and therefore different parameters all together.
I have listed many different choices of LC/MS/MS manufacturers on the left
We were trained on the CDC’s Universal Pesticide Method at CDC using the Thermo and then came home and had to translate the method to work on our Agilent system

3. Different LC/MS/MS Instruments
Here are some different triple quads: showing a couple Thermo and Shimadzu systems

4. LC = Liquid Chromatograph MS/MS = Mass Spectrometer/Mass Spectrometer
HPLC Stack with LC column
Quad 1
Quad 2
Collision Cell
Basically they all work the same in theory even if not in design
1st the solution is pumped through the HPLC stack carried by the mobile phase through the column to separate the molecules,
2nd The solution is then carried from the column to the ion source where ions are generated,
3rd The ions go through the first mass filter to produce the precursor ions,
4th they get bombarded in the collision cell producing more fragments which then, go through the third cell, another mass filter, to produce the product ions.
Agilent design actually quadrupole/hexapole/quadrupole
LC Column Nebulizer Tandem Mass Spectrometer
Separates Makes Molecular Separates and Counts Ions Molecules Ions by Molecular Mass Twice

5. Main Parameter Differences b/w Agilent and Thermo Systems
WA State PHL Parameters (Agilent)
MS Parameter
Setting
Ionization type
H-ESI (MMI source)
Ion polarity mode
Negative mode
Gas Temperature (same as “Heated capillary”)
150° C
Vaporizer temperature
Capillary Voltage
(-) 3500
Corona Current (same as “discharge current”)
(-) 5 µA
Charging Voltage
2000
Collision gas
Nitrogen
Nebulizer Pressure
60 psi
Gas Flow
5 L/min
CDC Parameters (ThermoFinnigan)
MS Parameter
Setting
Ionization type
H-ESI (APCI/H-ESI source)
Ion polarity mode
Negative mode
Heated capillary
250° C
Vaporizer temperature
150° C
Sheath gas pressure 37
Discharge current
4.5
Aux Gas Pressure 10
Collision gas
Argon
Black Parameters are the same and named the same Red Parameters are not the same or interchangeable
The Blue Parameters are similar but named differently
NOW LET’S TALK ABOUT SOME OF THE DIFFERENCES BETWEEN THE AGILENT AND THERMO QUANTUM SYSTEMS WE DISCOVERED DURING METHOD TRANSLATION AND A LITTLE ABOUT DEVELOPMENT STRATEGIES AND HOW THAT CAN BE DEPENDENT ON TYPE OF SOURCES: MMI VS ESI
Agilent’s MMI source: “If running a cold vaporizer temperature (<175°C) then set the drying gas temperature to vapor temp or lower)/ If running hot vaporizer (>175°C) then set drying gas to 350°C”
Also the MMI source says to ALWAYS have the neb at 60 and the drying gas flow at 5 L/min but the ESI source has you optimize these for the methods.
BASICALLY WHEN YOU HAVE A DIFFERENT INSTRUMENT SOME DEVELOPMENT IS NECESSARY DURING TRANSLATION AND IMPLEMENTATION

6. Different LC/MS/MS Parameters
Agilent 6410A Triple Quad System
Different Scan Modes used
in Method Development:
MS 2 Scan
MS 2 SIM
MRM (Multiple Reaction Monitoring)
Precursor Ion
Product Ion
LET’S DISCUSS SOME OF THE DIFFERENT PARAMETERS THAT THE AGILENT SYSTEM USES DURING METHOD DEVELOPMENT
FOR EXAMPLE THERE ARE DIFFERENT SCAN MODES THAT CAN BE SET UP DEPENDING ON WHAT PART OF DEVELOPMENT YOU ARE AT:
MS 2 Scan: You select a Start Mass and End Mass for each Scan segment (up to 4), and all masses in these ranges are acquired.
MS 2 SIM: You type the m/z to acquire data at. In selected ion monitoring, SIM, only preselected ions are monitored.
MRM: MOST COMMON TYPE FOR ANALYSIS AND ALSO IMPORTANT IN METHOD DEVELOPMENT
*This mode allows up to 500 transitions in a single Time Segment. These transitions abundances are acquired from high mass to low mass sequentially and repeated until the Time Segment runtime has expired.
* You can have multiple time segments with completely different sets of transitions in a Run.
*Important to note that the more transitions you add to a method (more analytes) the dwell time will also need to be adjusted since adding transitions reduces the time (and hence data points collected) for each peak.
Precursor Ion: A scan type used during method development specifically to help determine the best precursor ions.
Product Ion: A scan type used during method development that helps determine the best product ions.

7. Agilent Supplied Methods for Development
Agilent Method Name
Function of Method
0-FullScan Flow Injection
0-Find the precursor, parent, or quasi-molecular ion
1-Fragmentor Optimization
1-Focus the precursor into the MS, without breaking it into pieces
2-Capillary Optimization
2-Force the ion at a right angle into the MS capillary
3-Product Ion Scan
3-Determine what fragments are formed
4-Collision Energy Optimization
4-Determine the optimum voltage to create each fragment
FOUR MOST IMPORTANT SUPPLIED METHODS FOR HELP WITH TRANSLATION/DEVELOPMENT:
To make the setup easier, methods 0,1,2 are full scan methods. Otherwise: customize the method for each analyte.
Method 3 and 4 must be customized for each analyte for they need to isolate on the precursor ion for that analyte.
All of the methods can be used on mixtures, but best to start with individual compounds. Find optimal for each then add all together afterwards and try out the method
Typical values might be, but yes they vary by analyte:
Fragmentor                       
Capillary voltage               3500
Collision energy                5-30

8. Method Development Steps Taken During Translation Process to the Agilent 6410
1st: Set Up the Initial Source Parameters
Polarity
Drying Gas Temp
Drying Gas Flow
Scan Type/Range of Scan
Nebulizer Pressure
Capillary/Charging Voltages
2nd: Optimize Source and Method Parameters
Determine Precursor Ions
Find Optimum Fragmentor Voltage
Determine Product Ions
Determine Optimum Collision Energy for each MRM acquisition
LEFT SIDE: SOME OF THESE PARAMETERS YOU WILL START WITH YOUR BEST GUESS BASED OFF THE ORIGINAL METHOD/PAPER YOU ARE TRANSLATING FROM AND THEN WILL END UP TWEAKING THEM AS THE METHOD DEVELOPMENT CONTINUES (consider starting with a similar mobile phase and start without the column doing isocratic solvent shots)
RIGHT SIDE: LUCKILY AGILENT HAS SOME PRE-ESTABLISHED METHOD DEVELOPMENT METHODS TO USE WHICH ARE ALREADY PROGRAMMED TO DO THE DIFFENT TYPES OF SCANS AND EVERYTHING ALREADY SET UP—SAW IN LAST SLIDE.
MANY OF THESE METHODS WILL BE CHANGED AND RE-RUN AS YOU GO THROUGH THEM AND TRY DIFFERENT SETTINGS…FOR INSTANCE YOU HAVE TO PROGRAM THE EXACT MASSES OF THE COMPOUNDS TO FIND THE PRECURSORS / YOU WILL HAVE TO PROGRAM DIFFERENT STARTING POINTS FOR VOLTAGES AND FLOWS, ETC.
FOR THE FRAGMENTOR AND COLLISION ENERGIES MULTIPLE RUNS ARE USUALLY NECESSARY BEFORE YOU FIND THE BEST RESPONSE. THEN OF COURSE WHEN YOU HAVE MULTIPLE ANALYTES THE FRAGMENTOR VOLTAGE AND CAPILLARY VOLTAGES HAVE TO BE OPTIMIZED FOR GIVING THE BEST RESPONSES FOR ALL ANALYTES WHICH TAKES SOME GIVE AND TAKE.

9. More Advanced Development after Establishing Basic Conditions
3rd : Next Phase of Settings for Optimizing
Add Column
Add Matrix
Flow Rate/Run Time
Isocratic or Gradient?
Tuning for analytes optimum response
4th: Re-Visit Initial Parameters
Combine all Analytes into a mixed solution
Program Initial Analytical Method using Optimized Parameters
Tweak Initial Settings
Once you have established the original conditions and you have started to dial in all the compounds of interest you often have to go back and look again at some of the initial settings because they may change now that you have more parameters optimized and set up. For instance you started with a certain capillary voltage. See if increasing or decreasing helps the abundances…etc. —YOU WILL FIND YOU MAY NEED TO TWEAK SOME THINGS TO GET THE BEST RESPONSE FOR ALL ANALYTES SO SOME THINGS MAY HELP ONE ANAYLTE BUT MAKE ANOTEHR WORSE, ETC.
There is often a reciprocal relationship between certain parameters (EX: sensitivity and resolution) that also comes into play while finding the optimum settings.

10. Further Method Development/Translation Considerations
Theoretical Considerations
Mobile Phase Ph/Strength of Buffers
Mobile Phase Compositions & Ratios of Organic/Aqueous
Column Temperature
All These Affect RT
(retention time), Peak Shape, Resolution, etc.
Physical Considerations
Cleanliness of Systems
Ion Suppression
Sample Prep/ Stability
SPE Process & Recovery (Solid Phase Extraction)
Guard Column/Pre-Filter
After programming first method based on optimizations these types of things are also to be considered
Some General Rules for Reverse Phase (small molecules mostly <500mw):
RETENTION: affected by solvent strength: retention decreases ~ a factor of 3 for every 10% change (decrease organic means increased RT) ALSO affects peak spacing since different components affected differently
TEMPERATURE: Isocratic RT changes ~2% for each degree C change in temperature (increase temp means decreased RT)
MOBILE PHASE: Organic component in reconstitution solutions should not be higher in organic content than the mobile phase usually
ALSO Solvent blending can improve peak separation sometimes if having issues with current mobile phase conditions. THINGS LIKE THAT.
MANY OF THESE MAY BE INCLUDED IN THE ORIGINAL METHOD OR PAPER BUT I HAVE RUN INTO CASES WHERE THEY WEREN’T SO IT’S IMPORTANT TO LOOK INTO THESE AS WELL.
EXAMPLE: WITH THE MMI SOURCE WE ARE SUPPOSED TO USE MUCH LOWER CONCENTRATIONS OF BUFFER THAN FOR THE ESI SOURCE SO INSTEAD OF RUNNING 0.1% ACETIC ACID WE GET BETTER RESPONSES ON OUR SYSTEM USING 0.05%.
EXAMPLE: WE FOUND THAT INCREASING THE RUNTIME SLIGHTLY IMPROVED THE COLUMN LIFE
Some

11. Quality Management Considerations
Reproducibility
Accuracy
Precision
LOD (Limit of Detection), LOQ (Limit of Quantitation)
AMR (Analytical Measurement Range)
Clinically Significant Ranges
Validation of Method
Quality Controls in Place
Once you have the method in general working condition you now need to start looking into the quality management and control or assurance issues that have to be addressed during method translation/development—You may have to go back if any of these quality conditions are not met.
There are rules for all of the QM conditions listed here and different labs may have slightly different rules or values for some of these items.
Some important things to note is to make sure you list how you determined your values, FOR EXAMPLE: LOD and LOQ: NEED TO TELL what kind of samples these were based on and how you determined the values. Some labs use blanks others use low spikes. They can be determined statistically or empirically. FOR PYRETROIDS WE BASED OUR LOD/LOQ ON LOW SPIKES DURING VALIDATION AND ASSIGNED VALUES STATISTICALLY: LOD IS STD *3 AND LOQ STD*10.
FOR OUR QUALITY CONTROL CHARTING OF QCs WE USE 20 pt rolling LEVEY-JENNINGS CHARTS BASED OFF WESTGARD RULES.
Precision: RSD lower than 3% according to our rules. Accuracy: should be b/w +/-20% of target.
Does your AMR encompass the clinically significant ranges? ETC.

12. FINISHED! AMR Encompasses Clinical Significant Ranges
Validation Passed
Acceptable Accuracy/Precision
READY to join Proficiency Testing Programs!
READY to Analyze Patient Samples!
Once you have ensured all your quality objectives are in place and you have validated you method you are ready to join a proficiency program and start on your patient samples!

13. Questions?
Contact Info:
Caroline E. West, lead chemist WEBS
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