Method Building Essentials

Technology Transfer Workshop

Method Building Essentials• Source Parameter Optimization

– Extracting the solution ion from the mobile phase

• Compound Optimization for MRM– Getting parent ion to the collision cell and

smashing it to bits

• MS optimization for EPI– Producing reproducible full scan MS with lots of

detail.

• Linking MRM with EPI using IDA


Method Parameters• TurboSpray Parameters

– The goal is to maximize ion formation based on the liquid matrix.

– Set globally in the method.– In multi-compound methods should be generalized

• Compound Parameters– The goal is two fold

• 1st maximize parent ion reaching the collision cell• 2nd Optimize fragmentation

– MS experiment determines if generalized and set globally• MRM = drug specific• EPI = generalized and set globally.

• MS Parameters– The goal is optimized resolution and sensitivity.– Minimize scan time


Infusion and FIA• Infusion

– Syringe pump driven

– 10 mcL/min

– Compounds ~ 5mg/L in H20 mixed with a small amount of 50:50 A:B mobile phase.

• Flow Injection Analysis– Syringe pump still used

– No chromatography

– LC mobile phase added at mixing-T on source. • Syringe pump +A + B = 200 mcl/min• Mobile phase A & B: 0.95 mL/min each• Syringe Pump 10 mcl/min.


TurboSpray Settings

• Ion Spray Voltage [IS]• Nebulizer Gas [GS1]• The “hairdryers”

– Heater [TEM]

– Heater Gas [GS2]

• Curtain Gas [CUR]• Interface Heater [ihe]

– On or Off…we leave it on.


3200 Qtrap Overview: The Source

Curtain Plate

CUR

GS1 IS

TEM &GS2

ihe

Orifice Plate

Voltage, Temperature & Gas Parameters


Ion Spray Voltage [IS]

1000 3000 50002000 4000 5500

Infusion

FIA


Nebulizer Gas [GS1]

1020

30 5070 9

0

1020 30 50 70 90

Increasing the GS1 from 10 to 50 Increases cocaine ion flux ~20%

Infusion

FIA


Heater [TEM]

TEM= 0 (no heat)

TEM= 100

TEM= 200

TEM= 300

TEM= 400TEM= 500

TEM= 600

TEM= 0 (no heat)

TEM= 100

TEM= 200

TEM= 300 - 600

Infusion FIAIncreasing TEM 0 to 500Increases cocaine flux >350%


Heater Gas [GS2]

3010 20

5070 90

[LC Baseline: GS1 =10, GS2 = 0]


TurboSpray Parameters• The curtain gas [CUR] should be maximized to repel

neutrals and droplets without dramatically impacting sensitivity.– The geometry of the 3200 source makes it less

important than on older sources.• Mobile phase composition and flow affects source

parameters– Increasing water content will require higher temperature

and gas pressures– Increasing flow rate will require increased temperature

and gas pressures.• Using FIA reproduces mobile phase conditions at

compound elution and can help to maximize sensitivity.


Compound Optimization Overview• Select Mass [M+H]+

– Needs to be the exact mass of the most abundant isotope

• Optimize compound voltages – maximize parent ion abundance– DP,EP, & CEP

• Optimize compound fragmentation– Select and optimize transition masses– CE

• Quantitative Optimization– Instrument’s automatic routine


Compound Optimization Parameters

• Getting ions to the collision cell– Declustering Potential [DP]

– Entrance Potential [EP]

– Collision Cell Entrance Potential [CEP]

• Controlling Fragmentation– Collision Energy [CE]

– Collision Gas Setting [CAD]

– Collision Cell Exit Potential [CXP]• Usually 4V

– Collision Energy Spread [CES]


3200 Qtrap: Potentials

• Most potentials are relative to the entrance potential (EP).

• CEM is the setting for the Cascade Electron Multiplier

Q0 Q1 Q2 Q3

EP CEP CXPDP

CE CEM


Declustering Potential• “The potential applied to the orifice plate (OR)

has the greatest effect on the amount of declustering in the orifice region of the interface.”

• “The working range of DP is typically 0 to 100 V, although it may be set higher.”

• Decluster what?– Example clusters include: [M+H3O]+, [M+Na]+,

[M+H+CH3OH]+

• If set too high DP can actually fragment a compound in the source… BAD for LC/MS/MS.


Cocaine DP Ramp 5.0 to 100v

Infusion (10 mcl/min)

FIA 200 mcl/min 50:50 H2O: MeOH


Cocaine DP= 20, 40, 80

DP=20 DP=40 DP=80


Entrance Potential [EP]

• The EP parameter controls the entrance potential, which guides and focuses the ions through the high-pressure Q0 region.

• It is typically set at 10 V (for positive ions) or

–10 V (for negative ions) and affects the value of all the other instrument voltages.


Cocaine EP = 1 to 12V

FIA

Infusion


Collision Cell Entrance Potential

• CEP (Collision Cell Entrance Potential)– The CEP parameter controls the collision cell

entrance potential, which is the potential difference between Q0 and IQ2.

– It focuses ions into Q2 (collision cell). CEP is used in Q1, MS/MS-type, and LIT scans.

• Generally the most mass dependent.


Cocaine CEP = 1 to 188V

Infusion

FIA


Fragmentation Parameters

• Controlling Fragmentation– Collision Energy [CE]

– Collision Gas Setting [CAD]

– Collision Cell Exit Potential [CXP]• Usually 4V



Collision Energy (CE)• Fragmentation of parent ion is caused by collision with

low pressure N2 in the collision cell.• The more energy generally results in greater

fragmentation.• The energy of that collision is controlled by the

Collision Energy (CE) setting.– CE is a voltage difference between the Q0 and Q2 (EP –

RO2). – CE can be optimized for each drug.

• (Quantitative Optimization)

– Higher CE results in greater fragmentation of the parent molecule.

• Consider dextromethorphan…


DXM: CE =10


DXM: CE =20


DXM: CE =30


DXM: CE =40


DXM: CE =50


DXM: CE =60


DXM: CE =70


DXM: CE =80


DXM: CE =90


DXM: CE =100


Fragment Ion Selection

• In MRM screening, generally a single transition is selected for each drug. – For example dextromethorphan 272 128– The sensitivity of the method is directly

proportional to the intensity of the fragment ion.– Attention should also be given to the

uniqueness or the transition.

• How do you select a transition ion to monitor?


Fragment Ion Selection• Quantitative Optimization

– Starts with compound optimization • DP, EP, & CEP

• Fragment parent mass at many different collision energies.

• Pick the four most intense ions – Avoid loss of H2O

• (fragments δm >19 amu of parent)

• Determine the collision energy that produces the maximum amount of each transition ion.


DXM : CE =10 to 100 [summed]

Optimize These Fragments


Optimizing 128, 147, 171, 215

128

147

171

215

Fragment CE Max

128 84

171 51

147 41

215 32

CE


Dwell Time

• Dwell time is the amount of time (msec) the instrument spends at each transition.

• Effects sensitivity up to a point.– Increasing the DT >50 msec produces little if

any additional sensitivity.

– For most drugs DT> 25 has little effect.

• Also longer dwell times lengthen total MRM scan times.


Method Building: MRM

• Optimization of source parameters

• Optimization of compound parameters– Compound

– Fragmentation

• Selection of drug transitions– Repeated 130 times…..

• Congratulations You’re Ready to Build an MRM Method!


MRM Method for DXM

Parent [M+H]+

Daughter Transition

Ion

DwellTime

MRM Parameters


MS Parameters

• Enhanced Product Ion – Global Parameters


– Collision Cell Gas Pressure [CAD]

– LIT Settings• Fill Times• LIT Scan Speed• Q0 Trapping


Dextromethorphan EPI

Typical EPI output...CE =20

CES = 30Q0 trap = Off

Scan Speed = 1000Fill time fixed 20ms


EPI: Global Parameters• Source Settings (TEM, GS1 etc…) are set

globally and are the same for MRM and EPI.

• Compound parameters individualized to optimize transition ion formation (MRM) must be set globally for EPI. (DP,EP,CEP,CE,CXP)

• The goal of EPI optimization is to find the best settings to produce good MS data that is reproducible and library searchable.


Collision Energy Spread [CES]• “The CES parameter controls the spread of collision

energies used when filling the LIT. It is used in conjunction with the Collision Energy (CE) parameter.” – “The advantage of using a collision energy spread is

that you do not have to optimize the collision energy.”

• “By specifying the CE and CES parameters, low, medium, and high collision energies are used in a single scan to provide maximum information in the product ion spectra (low and high mass fragments).”

• We have chosen CE 20:CES 30 (20/30) or (25/30)– Provided the best fragmentation across the widest

number of drugs.


Cocaine CE = 25 CES=30/-30

CES = 30

CES = -3050, 25, 0

0,25,50


Collision Gas Pressure [CAD]

• Controls the pressure of the N2 gas in the collision cell

• In simplified mode you get three settings: – Variable by instrument

• Low: 1.9x10-5 Torr• Medium: 2.6x10-5 Torr• High: 3.3x10-5 Torr

• Collision with the gas causes fragmentation, but also helps to “cool” fragment ions and focus them into the LIT.


Collision Gas Pressure [CAD]

CAD = High (3.3x10-5 Torr)

CAD = Medium (2.6x10-5 Torr)

CAD = Low (1.9x10-5 Torr)


LIT Settings• The LIT has four important settings

1. Mass Range • Set to cover the range dictated by compounds

covered (e.g.50 to 500 amu)

2. Scan Speed• Slower Scan Speed give better mass resolution

but cost time and sensitivity.• Qtrap has three 250, 1000 and 4000 amu/s


LIT Scan Speed

4000 amu/s1000 amu/s250 amu/s


LIT Settings• The LIT has four important settings

3. Fill Time– The amount of time the trap remains open to

accept ions. – Can be fixed time (e.g. 50 msec) or Dynamic– Dynamic fill time (DFT)

– Fill time based on presampling of incoming ion flux– Prevents over or under filling of the LIT.

4. Q0 trapping [on or off]– Like the LIT Q0 can store ions coming in from

the source while the LIT is closed.– Can increase sensitivity


Fixed vs. Dynamic Fill Times

Dynamic Fill Time

Fixed 20ms Fill Time Q0 OFF

Fixed 20ms Fill Time Q0 On


Building a Qualitative Method• Use MRM as the survey method

– Each compound setting optimized for sensitivity and specificity

• Use EPI to generate a full scan mass spectrum with a lot of detail to facilitate positive identification.– Use standard parameters for reproducibility and

library searching.

• Use Information Dependent Acquisition (IDA) to switch between MRM and EPI.


Information Dependent Acquisition• Information Dependent Acquisition

– Allows on-the-fly software switching between MS modes.

• IDA examines– The results of the MRM cycle to see if any

drugs are above the a threshold.• IDA determines

– IF any drugs will be selected for EPI– Which drugs will be selected

• IDA switches – The instrument from MRM mode to EPI mode


IDA Example• For Example

– Experiment 1: MRM• Survey Scan of 100+ drugs

– IDA (Decision Maker)• When a transition is above a threshold…• And is the most intense transition…• And is not on an exclusion list… • Then trigger a second experiment

– Experiment 2: Product Ion Scan• Full scan mass spectrum for compound

identification.


IDA: Dynamic Exclusion• A software technique to handle coeluting peaks

especially when one is much less intense.

Threshold

Dynamic Exclusion

Time

Product Ion Scans


IDA Method Flowchart

130 MRM Transitions

Any over 1100

cps?

Add to exclusion list for 15 sec.

On exclusio

n list ?

3rd EPI?

Do EPI

YES

YES

YES

NO

NO

NO

IDAIDA

Dynamic ExclusionDynamic Exclusion


Putting it all together…

XIC of oxycodone MRM

MRM Triggered EPI

Overlay of 130 MRM transitions


The payoff…Library Matching

Date post:	31-Dec-2015
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Method Building Essentials

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