API 3000 Operator Training Course Analyst-NT Foster City CA PAL Lab

What is LC/MS LC: Liquid Chromatography MS: Mass Spectrometer an instrument that produces ions and separates them in the gas phase according to their mass-to-charge ratio (m/z)

Element Mass Average mass of all natural isotopes Carbon Abundance Integer Mass 12C = 12.0000 Da 98.91% 12 13C = 13.0034 1.09% 13 (12.0000)(0.9891) + (13.0034)(.011) = 12.011

Natural isotopes Abundance Element Abundance Exact Mass X+1 Factor X+2Factor 1H 99.99 1.0078 2H 0.01 2.0141 10B 49.9 10.0129 54.03NB 11B 50.1 11.0093 12C 98.91 12.0000 13C 1.09 13.0034 1.1NC 0.0060(NC*NC) 14N 99.63 14.0031 15N 0.37 15.0001 0.37NN 35Cl 75.77 34.9689 37Cl 24.23 36.9659 32.5NCl 79Br 50.7 78.9183 81Br 49.3 80.9163 98.0NBr 127I 100 126.9045

Natural Isotopes Abundance Element Abundance Exact Mass X+1 Factor X+2Factor 16O 99.76 15.9949 17O 0.04 16.9991 0.04NO 18O 0.20 17.9992 0.02NO 19F 100 18.9984 28Si 92.2 27.9769 29Si 4.7 28.9765 5.1Nsi 30Si 2.2 29.9738 3.4NSi 31P 100 30.9738 32S 95.0 31.9721 33S 0.75 32.9715 0.8NS 34S 4.22 33.9679 4.4Ns

Defining the masses Integer Mass - Sum of Protons and Neutrons of an element Nominal Mass - Sum of the integer masses of the most abundant isotopes of the elements in a compound Average Mass - Sum of the atomic weights of the elements in a compound Exact mass - Sum of the most abundant isotopes of elements in a compound -Theoretical calculation only Accurate mass - experimentally determined mass Monoisotopic mass - sum of the exact masses of the most abundant isotopes of elements in a compound

Examples of Masses Haloperidol: C21H23NO2FCl   C21H23NO2FCl Nominal Mass: 375 Average Mass: 375.8737 Exact Mass: 375.1401 Monoisotopic mass 375.1401 m/z 365 376 386 378 100 75 50 25 % Intensity  C21H23NO2FCl 

Multiply Charged Ions Multiply Charged Ions: Signal = (MW+N)/N (N = # of charges) Since the Quad separates ions based on m/z ratio and ions can have multiple charges in Ion Spray. A series of multiply charged ions is usually produced for larger molecules.

Common adducts POSITIVE MODE Na 22 Da. higher than M+H K 38 Da. higher than M+H Li 9 Da. higher than M+H NH4 18 Da. higher than M+H ACN 41 Da. higher than M+H NEGATIVE MODE TFA 114 Da. higher then M-H (113 and 227 background) Acetate 60 Da. higher then M-H Formic 46 Da. higher then M-H

GAS PHASE Acid-Base Scale for Positive Ion & Neutral Molecules

GAS PHASE Acid-Base Scale for Negative Ions & Neutral Molecules

Ionization Suppression Compounds That Cause Sensitivity Suppression Salts can interfere with ionization and can cluster to complicate spectrum (but also aid in identification) Strong bases or quaternary amines can interfere with positive mode analytes , e.g. Triethylamine (TFA) Acid - Sulfuric/Sulfonic acids and TFA interfere in negative mode Phosphate buffer and non-volatile ion pairing agents (e.g. SDS) can cause severe suppression and complex spectrum Dimerization ([2M+H]+) can occur at high concentration, leading to non-linearity during quantitation Dimer Signal at m/z = (MW*2) + 1 Can cause non-linearity at high concentrations

Basic steps of LC/MS analysis Sample Introduction：in liquid states by syringe pump or LC pump Ionization：IonSpray ，APCI Vacuum system: prevent collisions of ions with residual gas molecules in the analyzer during the flight from ion source to the detector Interface: prevent excessive gas load Mass analysis : Quadrupole Mass filter ,Time of Flight Data process system : computer based

API Analytical Domains Ionic IonSpray Analyte Polarity APCI GC/MS Neutral 101 102 103 104 105 Molecular Weight

Three types of voltages present on a Quadrupole: Quadrupole Theory Three types of voltages present on a Quadrupole: RFp-p – Fixed frequency (1 MHz) oscillating voltage (VAC  Mass); establishes harmonic ion trajectories. Filtering DC (FDC) – DC voltage difference between poles (linear to RFp-p) that defines the resolution. Rod Offset Voltage – Low DC voltage that defines axial ion energy (velocity through quadrupole). “RF only” mode means: No FDC between poles; only RFp-p, Rod Offset present. Quad. is in “Total Ion Mode” (transmitting all ions, not filtering).

Quadrupole Theory (cont.) Quadrupoles analyzers operate in constant peak width mode (Same Resolution over Mass range) API Resolution Specification Unit = 0.7 (±0.1 amu FWHH) High = .5 (±0.1 amu FWHH) Low = ~1.1 (±0.1 amu FWHH) User can set higher resolution depending on problem to be solved (i.e., setting singly-charged resolution to 0.5–0.6 amu FWHH aids in detecting doubly-charged ions) Resolution offsets set the DC/RF ratio at various points along mass axis Resolution At Unit, High and Low automatically tuned It is always recommended to calibrate mass axis after resolution is adjusted for a quadrupole

Resolution = 0.7 amu FWHH LC2Tune 1.4b1 1.pict LC2Tune 1.4b1 1.pict

Resolution = 0.6 amu FWHH

Resolution = 0.5 amu FWHH

Quadrupole Theory & Ion Energy Effects Resolution and Peak Shape Lower energy (slower) = more cycles, better resolution Higher energy (faster) = less cycles, lower resolution (typically 0.5–2 eV) is given by the voltage difference between the ions “starting potential” (Q0) and the Q1 rod offset (RO1) (times the number of charges) Inc. IE also inc. E of ions to distort peak shape. Always a tradeoff between resolution and sensitivity in all quadrupole analyzers

Quadrupole Theory & Ion Energy Q1 Scan = n(Q0 - RO1); Q1 IE = 0.5-2eV Q3 Scan = n(RO2 - RO3); Q3 IE = (1-6eV) Collision Energy for MS/MS: CAD energy = n(Q0 - RO2); n = # charges Product ion energy = n(RO2 - RO3); n = # charges this affects resolution of product ions (lower energy yields better resolution)

Sensitivity is of course lost as resolution increases Ion Energies Examples of an Ion Energy of .5 eV in Q1 Lower Velocity through Quads allows for greater filtering and better resolution Sensitivity is of course lost as resolution increases

Sensitivity is of course gained as resolution decreases Ion Energies Examples of an Ion Energy of 1.5 eV in Q1 Higher velocity through Quads allows for less filtering and worse resolution Sensitivity is of course gained as resolution decreases

The Resolution Re-evolution Resolution definition for: Quadrupole TOF Resolution definition for Magnetic Sector instruments

API3000 Hardware Overview

API3000 PERFORMANCE: Performance Specifications Q1 Mass Range: 5 to 3000 m/z Q3 Mass Range: 5 to 3000 m/z Dynamic Range : 1 to 4e6 cps Mass Accuracy : 0.01% over the entire mass range Scan Speed: 2400 amu/s

API- 3000 Ion Path

System Overview Sample Introduction Methods Sample Inlets and Ionization Types Vacuum Interface Curtain gas interface Differentially pumped interface Vacuum Chamber & Analyzer Region Quadrupoles, collision cell & ion optics Detector Region

API3000 Gas Function and Setting

10 port valco valve configuration

10 port valco valve configuration

10 port valco valve configuration

Sample Introduction Methods Continuous Infusion - Provides accurate, low flow rates. Allows continuous sample introduction for tuning and optimization. Flow Injection Analysis (FIA) - CombiChem. and Source Optimization Liquid Chromatograph - Allows separations of mixtures with microbore columns at low flows or conventional columns at high flows.

Ionization Sources Turbo IonSpray - IonSpray option which utilizes a heated auxiliary gas flow to provide better sensitivity at higher flow rates (2µL/min—1mL/min) Heated Nebulizer - Sample inlet for flow rates of 0.2-2.0 mL/min. Uses heat (400–500 °C) and nebulizer gas to vaporize HPLC eluent and “steam distill” sample into the gas phase for APCI. Corona Discharge -High voltage needle to ionize compounds in the gas phase by Atmospheric Pressure Chemical Ionization (APCI).

Ionization Sources: TurboIonSpray 1st picture shows a turboionspray with and “hairdryer” portion on the bottom. 2nd picture shows the source from the rear with adjustment options 3rd picture shows the gas inputs, heater cables and interlocks

TurboIonSpray Source: Vacuum Interface IonSpray inlet charged droplets High Voltage + – + + + – + To Q0 1 TOR -3 Sample + + Ions Nebulizer Gas Turbo Gas Ion Source (atmosphere) ~10,000,000 ions on column 4,000,000 - 40,000 ions Operator Impact Area ~1000 ions IS Voltage - Declusters and generates ions IS - Very pH dependent for charging of molecule Nebulizer & Turbo Gas - Declusters and de-solvates Ions

TurboIonSpray - Ion Evaporation Rayleigh Limit = 10 cm2/V

TurboIonSpray (cont.)

Optimal Conditions TIS: TurboIonSpray (cont) Optimal Conditions TIS: Curtain gas (CG) SET AS HIGH AS POSSIBLE (Min. = 8) Can inc. CG for low MW without sensitivity loss. Higher sprayer pressure may require higher cur. gas Sprayer Alignment DO NOT SPRAY DOWN ORIFICE! Generally not compound dependent Spray further away from orifice for higher flow rate.

TurboIonSpray (cont) Needle voltage (+) ion; 1500 to 6000VDC (-) ion with air as neb; -3000 to -4500VDC Oxygen reduces corona effect in Neg. Mode (-) ion with N2 as neb; -2000 to -3500VD Nebulizer Flow Higher liq. flowrate may require higher neb. flow Flowrate and solvent composition Best sensitivity at lower flowrate (2-400 µL/min.) Can operate at up to 100% water, but better sensitivity with some organic content

TurboIonSpray (cont) Modifiers Organic acids (e.g. formic, acetic) promote ionization of basic compounds (sp3 N- containing) Adduct Formation - Neutral compounds containing nucleophilic lone pairs (sp2 N, sp3 O) can be ionized by cationization with alkali metal or ammonium ions. Recommended Buffers - Ammonium formate or acetate are at ( 2-10 mM optimum, can see suppression effects over 20 mM), .1% Formic Acid, (.1% TFA OK)

TurboIonSpray (cont) Compounds That Cause Sensitivity Suppression Salts can interfere with ionization and can cluster to complicate spectrum (but also aid in identification) Strong bases or quaternary amines can interfere with positive mode analytes Acids - Sulfuric and TFA interfere in negative mode experiments. DO NOT USE PHOSPHATE BUFFERS Non-Covalent Dimmers in Ion Spray Dimmer Signal = (MW*2)+1 one cause of non-linearity at high concentrations

Heated Nebulizer (cont.) 1st picture inside of APCI/HN, with corona needle offset between orifice and quartz tube 2nd picture back of APCI/HN gun gas1 and aux connections, heater control and heater power connection, back latter adjustment.

Heated Nebulizer (cont.) A position setting of 2-4 on the housing unit is usually sufficient to avoid spraying down the orifice.

Heated Nebulizer Inlet APCI Factors: APCI is a high flow (0.2-2.0 mL/min.) inlet Suitable for polar, thermally stable cmpds Usually, MW < 1300 amu Probe is heated to facilitate vaporization Requires nebulizing and auxiliary gas Requires corona discharge needle to produce ionization (APCI)

Atmospheric Pressure Chemical Ionization (APCI) APCI utilizes corona discharge APCI is a “three” step process: 1) Needle at high voltage ionizes nebulizing gas (air or nitrogen) forming primary ions 2) Primary ions react immediately with solvent molecules forming reagent ions 3) Reagent ions react (by proton transfer) with analyte molecules forming (M+H)+ in positive ion mode or (M-H)- in negative ion mode

Atmospheric Pressure Chemical Ionization (APCI) Corona discharge example - positive ion 1) EI on atmosphere cause e- removal from N2, O2 forming N2+•,O2+• 2) In a complex series of reactions N2+•,O2+• react with H2O, CH3OH forming H3O+ and CH3OH2+ as reagent ions for CI. 3) H3O+, CH3OH2+ donate protons to analyte forming [M+H]+

Heated Nebulizer - APCI APCI Optimal Conditions: Probe Alignment DO NOT SPRAY DOWN ORIFICE! Not compound dependent Optimize on tuning or test compound Probe Temperature Distillation Limits actual temperature experienced by Analyte ~ 150 Degrees C Minimum temperature set by solvent and flow rate. May cause thermal degradation of analyte. Flowrate and solvent composition Works from 0.2 to 2.0 mL/min.

Heated Nebulizer - APCI Buffers: Buffers/modifiers not required for ionization Volatile buffers tolerated up to 50 mM Very polar modifiers may reduce sensitivity to less polar analyte Corona discharge needle position: Slightly off-axis to probe; align with orifice Curtain Gas: SET AS HIGH AS POSSIBLE (with no sens. loss) Minimum value is 9 (MW < 1000)

Vacuum Interface: Curtain Gas&Differentially Pumped Interfaces Skimmer diameter is 2.6 mm, Larger then other instruments for better sensitivity Orifice diameter is unchanged (254 µm diameter) Orifice-ring-skimmer spacing is unchanged Orifice-skimmer pressure is 1 Torr

Vacuum Interface: Curtain Gas &Differentially Pumped Interfaces

System controller monitors vacuum system “transparently” Safety overrides disable electronics and shut down turbos if: BAG pressure higher than 1 x 10-4 torr CG pressure less than 20 psi Rotary pumps are never disabled by system, they are turned on/off manually

Vacuum Interface: Curtain Gas &Differentially Pumped Interfaces Curtain Gas (CG) - Ultra-High Purity nitrogen keeps non-ionized species from being sucked in to vacuum chamber CG aids in ion declustering (with CID potentials) Two stage transition from atmosphere to low pressure region of analyzer (1 x 10-5 torr) Ions are drawn in due to: Pressure differential (both ions & CG) Electric field gradients (ions only)

Analyzer - Q0 Region Atmos. to vac. flow is a free-jet expansion OR–SK region is “high pressure” (1 torr), CID fragmentation may occur here (also in SK–Q0 region, 6 x 10-3 torr) Ions are focused into SK by the RNG, confined by Q0, and attracted toward IQ1 Neutral species are pumped away Q0 uses “collisional focusing” of ions

API-3000 Ion Path Differential Pumping Zones (Skimmer, QO & Main DP/OR IQ1 FP/RNG IQ2 CEM EP/Q0 ST RO1 ST3 RO3 DF RO2 (LINAC) 6 mTorr 1 Torr Leybold 361 1e-5 torr Varian 550 S25B Pump Backed by D10E Differential Pumping Zones (Skimmer, QO & Main Q0= first Quad. Skimmer, IQ# = Inter-Quad. Lens, ST = Stubbies Quad., RO# = Quad., RO2 LINAC, DF = Deflector, CEM Ion energies Q1=Q0-RO1, CE=Q0-RO2 (Collision Energy), Q3=RO2-RO3 (eV) Tunable Potentials (Mass Or Cmpd. Dependent) OR, RNG, RO2, (ST3 unlinked) EP fixed (IQ1,ST RO1 Linked EP), IQ2 Fixed, (ST3, R03 linked RO2)

Analyzer - IQ1, ST & RO1 Ions passing through IQ1 are focused by stubbies (pre-filter) into Q1 (mass filter) Q1 separates ions based on mass/charge ratio Stubbies have RF voltage and DC “offset” Typical voltages for (+) ion: EP/Q0 Fixed @ -10 V IQ1 Fixed @ -11 V ST Fixed @ -15-17 V RO1 Fixed @ -10.5 - 11 V (Individual instruments may vary) CEP & CXP - Collision cell potential CEP = Collision cell entrances = Q0-IQ2 Fixed on 3000 CXP = Collision cell exit potential = RO2-ST3 (mass dependent) Mass dependent on 3000

Gridded exit lens hardwired to IQ3 Stubbies 2 and IQ2 linked as IQ2 API- 3000 Ion Path (cont) Gridded exit lens hardwired to IQ3 Stubbies 2 and IQ2 linked as IQ2 IQ3 hardwired 1 volt more attractive than Q2 (RO2)

Stubbies 2 and 3 held in Q2 endcaps API-3000 Mass Filter Rail Stubbies 2 and 3 held in Q2 endcaps Stubbies 2 coupled to Q1 RF and stubbies 3 coupled to Q3 RF

API-3000 Collision Cell - Linac Q2 Linac (linear accelerator) eliminates cross-talk and allows faster MS/MS scanning without sensitivity losses Q2 rods are tilted and separate DC potentials are applied to each pair of rods to create an axial electric field

Collisional Focusing Collisional focusing is a patented technique, owned by PE-Sciex, which improves ion transmission in Q0 and Q2. High pressure (8 mtorr) Low pressure (<8 mtorr)

PE-Sciex’s LINAC Technology

Generation of the LINAC Axial Field Gradient Field gradient established by angling rods, each pair having a different DC potential Entrance of collision cell Exit of collision cell +4v +6v field on center line ≈ 5.75 v field on center line ≈ 4.25 v • entrance exit

Ion Path Linac “Off” Injection of selegiline to determine the level of cross-talk in the amphetamine MRM transition

Ion Path with Linac Injection of selegiline to determine the level of cross-talk in the amphetamine MRM transition with a Linac Q2

Selegiline response with different MRM dwell times

Effect of dwell time on resolution precursor ion scan mode

API Triple Quad Basic Scan Modes Q1 full scan Q3 full scan Selected ion monitoring (SIM) Product ion scan Precursor ion scan Constant neutral loss scan Multiple reaction monitoring (MRM)

Q1 Full Scan (Start – Stop) Single MS Operation Q1 Full Scan (Start – Stop) Q1 always used as single MS analyzer Used primarily for ident. of precursor ion In the API-2000, Q3 operates in RF-only mode API-2000 operates very similar to an API-150EX

Single MS Operation (cont.) Multiple Masses/Center Width Mode Multiple small scan ranges of the same width Resolution tune and calibrate mass axis (with PPG’s) or establish exact mass of molecular ion/fragments Q3 Scan used only to calibrate Q3 mass (can also calibrate Q3 in MS/MS mode): CAD gas is on (=1) to slow ions down Q1 operates in RF-only mode

Single MS Operation (cont.) SIM - Selected Ion Monitoring (Width ~ 0): Used to optimize analyzer for specific ions SIM used for quantitative analyses Q1 SIM used to “optimize” precursor ion Maximize signal in preparation for MS/MS

Q1 MS Scans Q1 Full Scan (Start – Stop) Q1 always used as single MS analyzer Used primarily for ident. of precursor ion Q3 operates in RF-only mode SIM - Selected Ion Monitoring (or multiple ions): Used to optimize analyzer for specific ions for MS/MS SIM used for quantitative analyses

Q3 MS Scans Q3 Full Scan (Start – Stop) Q3 always used as single MS analyzer Used primarily for ident. of precursor ion or for IDA use Q1 operates in RF-only mode SIM - Selected Ion Monitoring (or multiple ions): Used to optimize analyzer for specific ions for MS/MS SIM used for quantitative analyses

Fragmentation When an Ion fragments the following formulas apply: Positive mode (Parent)+  A+ + B Neutral Negative mode (Parent)-  A- + B Neutral Two common types modes of fragmentation: CAD = Collisionally Activated Dissociation in collision cell, gas molecules collide and cause weak bonds to break. CID = Collisionally Induced Dissociation in curtain gas region, OR/DP & EP/Q0, non-selective

MS/MS - Product Ion Scan Product ion scan- common MS/MS mode After identification, the precursor ion is sent into the collision cell and fragmented by CID Q1 is fixed, Q3 sweeps a given mass range Used for structural information gathering and identification of product ions First step to developing quantitative method

MS/MS - Product Ion Scan (cont.) Product ion spectrum of a particular compound m1+ set m2+ scan

MS/MS Product Ion Scan m1+ fixed After identification, the precursor ion is sent into the collision cell and fragmented by CID Q1 is fixed, Q3 sweeps a given mass range Used for structural elucidation First step to developing quantitative method m3+ scanned

Example of Product Ion Spectrum Ephedrine, MW = 165 (M+H)+ 166

MS/MS - Precursor Ion Scan Q1 sweeps a given mass range, Q3 is fixed Used to determine the “origin” of particular product ion(s) created in the collision cell Frequently used for drug metabolite identification (common product ion observed in the metabolites)

MS/MS - Precursor Ion Scan (cont.) A set of compounds with a common product ion m1+ scan m2+ set

MS/MS Precursor Ion Scan m1+ scanned Q1 sweeps a given mass range, Q3 is fixed Used to determine the “origin” of particular product ion(s) created in the collision cell Frequently used for drug metabolite identification (common product ion observed in the metabolites) m3+ fixed

Analysis of peptides that have Lucine or isolucine Example of Precursor Ion Spectrum 600 900 1200 1500 1800 m/z, amu 5 8 7 . 4 6 9 2 3 520.3 1105.0 1704.4 3.0e6 6.0e6 9.0e6 1.2e7 Intensity, cps 802.4 3.0e4 6.0e4 9.0e4 Precursors of 86 (Ile/Leu) Q1 Scan 745.6 Analysis of peptides that have Lucine or isolucine

MS/MS Constant Neutral Loss Neutral loss scan Q1 & Q3 both scan a given mass range but with a constant difference between ranges scanned Spectrum indicates which ions lose a neutral species equal to Q1 - Q3 difference Complement to Precursor Ion Scan Neutral “gain” indicates a multiply charged precursor ion was fragmented

MS/MS Constant Neutral Loss (cont.) Constant Neutral Loss Scan m1+ m2+ A set of compounds with a common neutral fragment m1+ scan m2+ scan m -m

MS/MS Constant Neutral Loss Scan m1+ scanned Δm Q1 & Q3 both scan a given mass range but with a constant difference between ranges scanned Spectrum indicates which ions lose a neutral species equal to Q1 - Q3 difference Complement to Precursor Ion Scan Neutral “gain” indicates a multiply charged precursor ion was fragmented m3+ scanned

Constant NL Scan - Phase II Metabolites Buspirone Glucuronide MS3 of 578  402 N O 138 265 222 HO 2 4 6 8 10 12 14 16 18 Time, min 1000 2000 3000 4000 5000 6000 7000 8000 Intensity, cps Buspirone Glucuronide EPI of 578 N O Gluc-O NL 176 MS/MS For Glucuronide, the TIC of the Neutral Loss Experiment clearly identifies where the 3 species eluted; two of them are hydroxy-buspirone glucuronide (1) and one of them is the buspirone glucuronide (2). Ret.Time 6.6 min 2 1 - Hydroxy-Buspirone Glucuronide 1 2 - Buspirone Glucuronide

MS/MS - Multiple reaction Monitoring (MRM) If Q1 and Q3 width=0, then MRM Many precursor to product ion pairs can be monitored (A-B, A’-B’, A”-B”, etc.) MRM analysis is the best way to maximize signal intensity of product ions MRM used primarily for quantitation studies

MS/MS - Multiple Reaction Monitoring (MRM) (cont.) Precursor ion set Fragmentation (CAD) Product ion set

MS/MS Multiple Reaction Monitoring (MRM) Many precursor to product ion pairs can be monitored (A-B, A’-B’, etc.) MRM is the best way to maximize signal intensity of product ions MRM used primarily for quantitation Precursor ion fixed Fragmentation (CAD) Product ion fixed

LC/MS/MS BioAnalysis of Multiple Drugs High Throughput MRM: LC/MS/MS BioAnalysis of Multiple Drugs atenolol, pindolol, acebutolol amoxicillin, dicloxicillin, ampicillin

Selectivity: LC/MS vs. LC/MS/MS To Detector 1.0 2.0 3.0 4.0 10 20 30 40 50 60 70 80 90 100 5.0 6.0 Precursor ion selection Fragmentation (CAD) Product ion selection Crude racehorse urine extract dexamethazone (21 hr post dose) 2ng in sample injected both cases This slide just reiterates why it is preferable to use a triple-quadrupole instrument rather than a single quadrupole instrument for quantitative studies. In this figure, the top TIC or total-ion-chromatogram shows the selectivity that can be afforded using a triple quadrupole instrument. Raw horse urine was injected onto the LC/MS/MS API 300 instrument and the assay performed under MRM or “multiple reaction monitoring” conditions. One peak is observed for the drug of interest, in this case dexamethazone. In the bottom figure, the same sample was analyzed under SIM or “single ion monitoring” conditions, or what would be used on a single-quadrupole MS. One huge broad peak is observed and the analyte is obscured by all of the other components present in the crude horse urine sample. Ion selection To Detector

Why LC/MS/MS for Quantitation ? e.g. how much cocaine is in urine? Retention Time Select by MW Select by Structure Separate on HPLC Increased Sensitivity & Specificity Intensity Retention Time

API-3000 Analyzer- Q3 Operation In all MS/MS modes, Q3 is a mass filter Ions are separated based on mass/charge ratio Q2 is “source” of product ions entering Q3 In single MS scans, Q3 is RF-only mode Q3 transmits all ions toward detector region Q3 acts as “ion guide” for product ions

Gas Controllers Nebulizer (Gas1) operating pressure is 90 psi (max 100 psi) Auxiliary (Gas 2) and curtain gas are unchanged CAD gas uses “clean Nitrogen” Gas manifold moved to back of instrument

Gas Controllers

Detector & Signal Handling Detector consists of CEM, DF & signal handling circuit card (all in one assembly) Ions are attracted to CEM “horn” voltage Deflector (DF) enhances electric field around CEM horn to aid ion attraction Ion striking the horn generates a “pulse” Pulse passes onto computer as a signal

Detector - CEM Voltages Horn voltage is fixed: Positive ion: -6000V Negative ion: +4000V Bias voltage set by operator 2000 to 3000V more positive than horn voltage Deflector voltage set by operator (+400V)

CEM Gain Check (Q1 SIM)

CG Optimization (1 Ion)

CG Optimization (2 Ions)