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

2. 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)

3. Element Mass Average mass of all natural isotopes
Carbon Abundance Integer Mass
12C = Da % 12
13C = % 13
( )(0.9891) + ( )(.011) =

4. Natural isotopes Abundance
Element Abundance Exact Mass X+1 Factor X+2Factor 1H 2H
10B NB
11B
12C
13C NC (NC*NC)
14N
15N NN
35Cl
37Cl NCl
79Br
81Br NBr
127I

5. Natural Isotopes Abundance
Element Abundance Exact Mass X+1 Factor X+2Factor
16O
17O NO
18O NO
19F
28Si
29Si Nsi
30Si NSi
31P
32S
33S NS
34S Ns

6. 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

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

8. 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.

10. 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

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

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

13. 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

14. 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

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

17. 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).

28. 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

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

30. Resolution = 0.6 amu FWHH

31. Resolution = 0.5 amu FWHH

32. 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

33. 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)

34. 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

35. 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

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

37. API3000 Hardware Overview

38. 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: amu/s

39. API Ion Path

40. 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

41. API3000 Gas Function and Setting

42. 10 port valco valve configuration

43. 10 port valco valve configuration

44. 10 port valco valve configuration

45. 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.

46. 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 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).

47. 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

48. 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 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

49. TurboIonSpray - Ion Evaporation
Rayleigh Limit = 10 cm2/V

50. TurboIonSpray (cont.)

51. 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.

52. TurboIonSpray (cont) Needle voltage (+) ion; 1500 to 6000VDC
(-) ion with air as neb; to -4500VDC
Oxygen reduces corona effect in Neg. Mode
(-) ion with N2 as neb; 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

53. 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)

54. 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

55. 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.

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

57. Heated Nebulizer Inlet
APCI Factors:
APCI is a high flow ( 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)

58. 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

59. 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]+

60. 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.

61. 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)

62. 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

63. Vacuum Interface: Curtain Gas &Differentially Pumped Interfaces

64. 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

65. 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)

66. 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

67. 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)

68. 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
IQ V
ST V
RO 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

69. Gridded exit lens hardwired to IQ3 Stubbies 2 and IQ2 linked as IQ2
API 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)

70. 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

71. 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

72. 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)

73. PE-Sciex’s LINAC Technology

74. 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

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

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

77. Selegiline response with different MRM dwell times

78. Effect of dwell time on resolution precursor ion scan mode

79. 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)

80. 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

81. 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

82. 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

83. 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

84. 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

85. 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

86. 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

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

88. 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

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

90. 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)

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

92. 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

93. 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

94. 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

95. 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

96. 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

97. 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

98. 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

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

100. 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

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

102. 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

103. 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

104. 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

105. 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

106. Gas Controllers

107. 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

109. 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)

110. CEM Gain Check (Q1 SIM)

111. CG Optimization (1 Ion)

112. CG Optimization (2 Ions)