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 Flow Injection Analysis 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 IS ihe GS1 TEM &GS2 Orifice Plate Curtain Plate Voltage, Temperature & Gas Parameters CUR

Ion Spray Voltage [IS] 1000 3000 5000 Infusion 2000 4000 5500 FIA

Nebulizer Gas [GS1] 20 10 30 50 70 9 Infusion 50 20 30 90 70 10 Infusion 50 20 30 90 70 10 Increasing the GS1 from 10 to 50 Increases cocaine ion flux ~20% FIA

Heater [TEM] Infusion FIA TEM= 300 - 600 TEM= 600 TEM= 200 TEM= 500 TEM= 0 (no heat) TEM= 200 TEM= 100 TEM= 0 (no heat) Increasing TEM 0 to 500 Increases cocaine flux >350% Infusion FIA

Heater Gas [GS2] 50 30 70 10 20 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 Q0 Q1 Q2 Q3 EP CEP CXP DP CEM CE Most potentials are relative to the entrance potential (EP). CEM is the setting for the Cascade Electron Multiplier

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 Infusion FIA

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 Spread [CES]

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.

Optimize These Fragments DXM : CE =10 to 100 [summed] Optimize These Fragments

Optimizing 128, 147, 171, 215 171 128 215 147 CE Fragment CE Max 128 84 171 51 147 41 215 32 171 128 215 147 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 Dwell Time MRM Parameters

MS Parameters Enhanced Product Ion Global Parameters Collision Energy Spread [CES] Collision Cell Gas Pressure [CAD] LIT Settings Fill Times LIT Scan Speed Q0 Trapping

Dextromethorphan EPI Typical EPI output... CE =20 CES = 30 Q0 trap = Off Scan Speed = 1000 Fill 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 0,25,50 CES = -30 50, 25, 0

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 Mass Range Scan Speed Set to cover the range dictated by compounds covered (e.g.50 to 500 amu) 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 1000 amu/s 4000 amu/s 250 amu/s

LIT Settings The LIT has four important settings 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. 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 Fixed 20ms Fill Time Q0 On Fixed 20ms Fill Time Q0 OFF Dynamic Fill Time

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 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 IDA (Decision Maker) 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. Dynamic Exclusion Time Threshold Product Ion Scans

IDA Method Flowchart IDA On exclusion list ? Any over 1100 cps? YES NO 130 MRM Transitions Do EPI NO YES 3rd EPI? Add to exclusion list for 15 sec. Dynamic Exclusion

Putting it all together… Overlay of 130 MRM transitions XIC of oxycodone MRM MRM Triggered EPI

The payoff…Library Matching