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Tackling the Techniques Tandem Mass Spectrometry
Marie Appleton
Paediatric Metabolic Section
Royal Victoria Infirmary
Newcastle-upon-Tyne
Introduction
Background and history of MS/MS
How MS/MS works
Common errors and troubleshooting
Examples
Mass Spectrometry - History
“I feel sure that there are many problems in Chemistry which could be solved with far greater ease by this than
by any other method. The method is surprisingly sensitive – more so even than that of spectrum analysis – requires an infinitesimal amount of material and does
not require this to be specially purified”…
(Sir J.J Thomson 1921)
Tandem Mass Spectrometry - History
Most significant advance in metabolic disease/NBS screening
Exponential increase in use to biosciences during 1980s
• Development of ionisation methods (ESI, MALDI)
First used in clinical setting in 1982
Inherited metabolic diseases in early 1990s
Short analysis time (2-3 mins)
Broad spectrum – 1 test/ many disorders
Basic Principles
Sample ionisation
(Parent/Precursor ions)
Mass Filtration (m/z)
Fragmentation into smaller ions
(Daughter/Product ions)
Mass Filtration (m/z)
Detection (Mass spectrum)
Types of Mass Spectrometers
Mass Spectrometer
Sample
Solid
Liquid
Gas
Ionisation
ESI, APCI, CI, EI, FAB, MALDI
Mass Analyser
TOF
Quadrupole
Ion trap
FT-ICR
Detector
Electron multiplier
Photo multiplier
Faraday cup
ESI triple quadrupole tandem mass spectrometer LC system
Acetonitrile/methanol/water
Autosampler
Mass spectrometer
ESI triple quadrupole tandem mass spectrometry
Argon gas
(CID)
sample
Mass spectra MS1 MS2
Vacuum
Ion Source – Electrospray Ionisation
Quadrupoles – MS1
Collision-induced Dissociation
+ + +
Ar
Quadrupoles – MS2
Collison cell
Detection – Mass spectra
Molecular ion
Base peak ion
ESI +ve triple quadrupole mass spectrometer
Modes of MS/MS operation
Q1 Q2 Q3
A, B Scan
A-n B-n Scan
A→(A-n) +n B→(B-n) + n
A Select
A A1 A2 A3
A1 A2 A3 Scan
A, B Scan
A→(A-x) + x B→(B-x) + x
X Select
A Select
A1 select
A A1
A2
A3
Product (daughter) ion scan
Precursor (parent) ion scan
Neutral loss scan
Selected ion monitoring (SIM)
ESI +ve triple quadrupole mass spectrometer
Modes of MS/MS operation
Q1 Q2 Q3
A, B Scan
A-n B-n Scan
A→(A-n) +n B→(B-n) + n
A Select
A A1 A2 A3
A1 A2 A3 Scan
A, B Scan
A→(A-x) + x B→(B-x) + x
X Select
A Select
A1 select
A A1
A2
A3
Product (daughter) ion scan
Precursor (parent) ion scan
Neutral loss scan
Selected ion monitoring (SIM)
Acylcarnitine m/z 85 α-Amino acids NL m/z 46 (102 deriv) NBS - MRM
Precursor of m/z 85 scan C:\Xcalibur\...\2015\20150416std2 16/04/2015 17:14:13 CN Std - 020415
RT: 0.00 - 2.98
0.0 0.5 1.0 1.5 2.0 2.5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
0.260.36
1.22 1.971.62 1.82 2.23 2.33 2.58 2.88
NL:1.31E9
TIC MS 20150416std2
20150416std3 #6-15 RT: 0.26-0.59 AV: 5 SM: 7B NL: 3.52E6F: + p ESI Full pr 85.000 [200.000-550.000]
250 300 350 400 450 500 550
m/z
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
260.13
263.14
221.14
456.34
277.21 482.52353.30374.30
288.20 318.30 428.41
Papers
Common Errors & Troubleshooting
Sample Preparation
• Appropriate extraction, internal standard and derivatisation performed
• Eluent compatible with mobile phase
• Good laboratory practice - accuracy
UP TO YOU!
LC Pressure System
RT: 0.00 - 1.70 SM: 7G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
0
20
40
60
80
1000.43
1.11 1.31 1.48
1.20
0.15
0.86 0.970.49 0.71
0.40
1.08 1.311.19 1.53
1.00
1.620.710.18
0.40
0.07
NL: 3.76E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkA140513
NL: 7.49E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkB140513
NL: 4.35E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkC140513
NL: 3.83E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkD140513
NL: 4.53E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkE140513
RT: 0.00 - 1.70 SM: 7G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
0
20
40
60
80
1000.43
1.11 1.31 1.48
1.20
0.15
0.86 0.970.49 0.71
0.40
1.08 1.311.19 1.53
1.00
1.620.710.18
0.40
0.07
NL: 3.76E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkA140513
NL: 7.49E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkB140513
NL: 4.35E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkC140513
NL: 3.83E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkD140513
NL: 4.53E6
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 119.50-120.50] MS NSSsampchkE140513
Autosampler
• Common symptom is Low intensity
• Syringe
• Blockages - sample port/stata valve
• Leaks – rotor seal
• Wrong sample injection – user error!
Contamination
• Highly sensitive instrument
• Contamination may results from
• Sample preparation – other reagents and handcreams
• Bad laboratory practice
• Mobile phase – microbe growth in water
• Dirt build up in source
• Improper cleaning
Mass spectrometer • Physical
• Clean cone shield and ion transfer tube – reduced signal intensity
• Probe correct position from the cone – proper nebulization
Mass spectrometer • Physical
• Acquisition Parameters
• ESI Probe/source settings – temperature and gas flow optimised per compound (polar
mobile phases need high temperatures)
• Collision energy too high or too low –
no product ions formed
• Incorrect transitions or scan mode
• Incorrect flow rates
Mass spectrometer • Physical
• Acquisition Parameters
• Ion suppression
• Sample matrix and mobile phase additives (acids, salts) alter signal of target analyte.
Mass spectrometer • Physical
• Acquisition Parameters
• Ion suppression
• Data Processing
ARE YOU LOOKING AT THE RIGHT THING??
RT: 0.00 - 2.98
0.0 0.5 1.0 1.5 2.0 2.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
0.26
0.36
1.22 1.971.62 1.82 2.23 2.33 2.58 2.88
NL:1.31E9
TIC MS 20150416std2
RT: 0.00 - 2.98
0.0 0.5 1.0 1.5 2.0 2.5
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Rel
ativ
e Ab
unda
nce
0.26
0.36
1.22 1.971.62 1.82 2.23 2.33 2.58 2.88
NL:1.31E9
TIC MS 20150416std2
20150416std2 #31-41 RT: 1.52-2.02 AV: 11 SM: 7B NL: 1.74E5T: + p ESI Full pr 85.000 [200.000-550.000]
250 300 350 400 450 500 550
m/z
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
260.1
456.4
291.1
218.1323.3
237.2
20150416std3 #6-17 RT: 0.26-0.68 AV: 6 SM: 7B NL: 3.23E6T: + p ESI Full pr 85.000 [200.000-550.000]
250 300 350 400 450 500 550
m/z
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
260.1
221.1
456.3
277.2 482.5
353.3
374.3288.2 318.3
428.4
Examples
Example 1
Courtesy of Liverpool
Misaligned microtitre plate
Example 2
• Plate positioned correctly
• Sample in well
• LC-solvents correct
• No Leaks/blocks
• Correct acquisition
parameters
RT: 0.00 - 1.70
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
0.911.410.21 0.88 1.400.23
1.431.070.80 1.560.18 0.980.02 0.36 0.750.600.56 1.331.291.651.150.33
NL:4.35E1
TIC F: MS NSSsampchkA141028
Example 2
Power supply 3 assembly failure Call an engineer
RT: 0.00 - 1.70
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
0.911.410.21 0.88 1.400.23
1.431.070.80 1.560.18 0.980.02 0.36 0.750.600.56 1.331.291.651.150.33
NL:4.35E1
TIC F: MS NSSsampchkA141028
Example 3 RT: 0.00 - 1.69 SM: 5B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0.40
0.40
1.14
NL:3.45E7
TIC F: MS SPA140108RC1
NL:1.14E7
TIC F: MS SPAR601759
RT: 0.00 - 1.69 SM: 5B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0.40
0.40
1.14
NL:3.45E7
TIC F: MS SPA140108RC1
NL:1.14E7
TIC F: MS SPAR601759
RT: 0.00 - 1.69 SM: 5B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
0.40
1.14
NL:3.45E7
TIC F: MS SPA140108RC1
NL:1.14E7
TIC F: MS SPAR601759
Example 3 RT: 0.00 - 1.69 SM: 5B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
NL:1.14E7
TIC F: MS SPAR601759
NL:1.14E7
TIC F: MS ICIS SPAR601759
Area 159777277
Area 188572354
Incorrect seating of ferrule causing dead volume
Example 4 RT: 0.00 - 1.69 SM: 7B
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative
Ab
un
da
nce
0.64
0.11
NL:1.09E8
TIC F: MS SPAR596134A
Sample overload
Example 5
Courtesy of Birmingham
Improper nebulization due to ESI capillary misaligned following maintenance.
Did not extend beyond the probe tip
Example 6 RT: 0.00 - 1.70
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
1000
20
40
60
80
100 NL:1.25E7
TIC F: MS NSSsampchkD140513
NL:1.38E7
TIC F: MS NSSsampchkA140513
NL:2.61E6
TIC F: MS NSSsampchkB140513
NL:1.56E7
TIC F: MS NSSsampchkC140513
NL:1.77E7
TIC F: MS NSSsampchkE140513
Example 6 RT: 0.00 - 1.70
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
1000
20
40
60
80
100 NL:1.25E7
TIC F: MS NSSsampchkD140513
NL:1.38E7
TIC F: MS NSSsampchkA140513
NL:2.61E6
TIC F: MS NSSsampchkB140513
NL:1.56E7
TIC F: MS NSSsampchkC140513
NL:1.77E7
TIC F: MS NSSsampchkE140513
Example 6 RT: 0.00 - 1.70
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
20
40
60
80
1000
20
40
60
80
1000
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
1000
20
40
60
80
100 NL:1.25E7
TIC F: MS NSSsampchkD140513
NL:1.38E7
TIC F: MS NSSsampchkA140513
NL:2.61E6
TIC F: MS NSSsampchkB140513
NL:1.56E7
TIC F: MS NSSsampchkC140513
NL:1.77E7
TIC F: MS NSSsampchkE140513
Pump running at low flow rate 50ul/min. Fix = Back pressure/retention loop (2m length peek) added
Example 7 RT: 0.00 - 8.00 SM: 9B
0 1 2 3 4 5 6 7
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
MMA
SA3.50
4.36
MMA
SA4.28
5.58
NL:1.09E5
TIC F: - c ESI SRM ms2 117.100 [72.850-73.350] MS MMA040714test22
NL:8.66E4
TIC F: - c ESI SRM ms2 117.100 [72.850-73.350] MS MMA040714test01
Example 7 RT: 0.00 - 8.00 SM: 9B
0 1 2 3 4 5 6 7
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Re
lative
Ab
un
da
nce
MMA
SA3.50
4.36
MMA
SA4.28
5.58
NL:1.09E5
TIC F: - c ESI SRM ms2 117.100 [72.850-73.350] MS MMA040714test22
NL:8.66E4
TIC F: - c ESI SRM ms2 117.100 [72.850-73.350] MS MMA040714test01
Mobile phase composition
Example 8
Courtesy of Liverpool
Poor response for Vit D3 but D2 and IS# are OK
Fresh mobile phase....Formic acid in methanol should be SHAKEN not STIRRED!
Example 9 C:\Xcalibur\...\Sept\08\ExtScreenUnk190 09/09/2014 06:35:52 Unk190
RT: 0.01 - 1.75 SM: 7G
0.5 1.0 1.5
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
0
50
100
Re
lative
Ab
un
da
nce
0
50
100
0
50
100
0
50
1000.33
0.33
0.34
0.41
0.35
0.35
0.29
0.29
NL: 1.03E7
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 85.50-86.50] MS
ExtScreenUnk190
NL: 1.48E7
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 88.50-89.50] MS
ExtScreenUnk190
NL: 8.90E4
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 103.50-104.50] MS
ExtScreenUnk190
NL: 2.42E6
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 106.50-107.50] MS
ExtScreenUnk190
NL: 3.68E6
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 119.50-120.50] MS
ExtScreenUnk190
NL: 1.55E7
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 124.50-125.50] MS
ExtScreenUnk190
NL: 1.31E6
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 135.50-136.50] MS
ExtScreenUnk190
NL: 2.80E6
TIC F: + p sid=-10.00
SRM ms2 [email protected]
[ 139.50-140.50] MS
ExtScreenUnk190
RT: 0.00 - 1.75 SM: 7G
0.0 0.5 1.0 1.5
Time (min)
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100
Re
lative
Ab
un
da
nce
0
20
40
60
80
100
0
20
40
60
80
100
0
20
40
60
80
100 1.46
0.30
1.53
1.401.14
1.211.54
0.25
1.601.35
1.02
0.32
1.611.22
0.32
1.16
NL: 4.13E5
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
NL: 3.33E5
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
NL: 2.27E4
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
NL: 1.22E5
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
NL: 2.56E4
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
NL: 6.10E5
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
NL: 4.78E4
TIC F: + p sid=-10.00
SRM ms2
84.50-85.50] MS
ExtScreenUnk190
Amino acids Acylcarnitines
Example 9
RT: 0.00 - 1.75 SM: 7G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
Re
lative
Ab
un
da
nce 0
50
100
0
50
100
0
50
100 1.46
0.30
1.53
1.401.14
1.21 1.54
0.25
1.601.35
1.02
0.32
1.611.22
0.32
1.16
NL: 4.13E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
NL: 3.33E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
NL: 2.27E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
NL: 1.22E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
NL: 2.56E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
NL: 6.10E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
NL: 4.78E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS ExtScreenUnk190
RT: 0.00 - 1.72 SM: 7G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6
Time (min)
0
50
100
0
50
100
0
50
100
0
50
100
Re
lative
Ab
un
da
nce 0
50
100
0
50
100
0
50
1000.51
0.35
0.77
0.35
1.00
0.51
0.32
1.321.10
0.32 0.45
0.97
0.32
0.52
0.840.71
0.36
0.39
0.68
NL: 7.55E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
NL: 4.43E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
NL: 5.37E3
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
NL: 7.39E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
NL: 1.33E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
NL: 8.06E5
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
NL: 3.16E4
TIC F: + p sid=-10.00 SRM ms2 [email protected] [ 84.50-85.50] MS NSA15372828
Formic acid in sample NOT mobile phase
Example 10
Courtesy of Birmingham
Contamination of reconstituting reagent (ACn) with Internal standard
m/z 403
Partial derivatisation • Not drying samples properly • Insufficient reagent/heating • Derivatising reagent ‘gone off’
Example 11 20140721002 #6-17 RT: 0.26-0.68 AV: 6 SM: 7B NL: 2.89E6T: + p ESI Full pr 85.000 [200.000-550.000]
250 300 350 400 450 500 550
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lative A
bu
nd
an
ce
263.2
221.1
465.5
353.3277.2
288.3316.3
SM: 7B
275 280 285 290 295
m/z
0
50
100
0
50
100
0
50
100
Re
lative A
bu
nd
an
ce
0
50
100
0
50
100277.1
282.3
288.3284.4280.1
282.3
277.2
284.3280.2 288.1
277.2
284.4
277.2
275.0 284.4
277.3
284.2279.3
NL: 5.46E5
20140722023#6-17 RT: 0.26-0.68 AV: 6 T: + p ESI Full pr 85.000 [200.000-550.000]
NL: 7.20E5
20140724002#6-17 RT: 0.26-0.68 AV: 6 T: + p ESI Full pr 85.000 [200.000-550.000]
NL: 7.25E5
20140722027#6-17 RT: 0.26-0.68 AV: 6 T: + p ESI Full pr 85.000 [200.000-550.000]
NL: 5.75E5
20140722029#6-17 RT: 0.26-0.68 AV: 6 T: + p ESI Full pr 85.000 [200.000-550.000]
NL: 8.44E5
20140722025#6-17 RT: 0.26-0.68 AV: 6 T: + p ESI Full pr 85.000 [200.000-550.000]
Contamination from
Conclusion
MSMS are robust analytical instruments
Sensitive to small changes
Contamination will be detected – Good Lab Practice performed
Common problems with leaks and blocks in peek tubing
Take care with connection during maintenance
Conclusion
Contamination – sample preparation, dirty source
Peak shifts and shape – blocks or leaks, mobile phase
Low intensity- sample injection, clogged transfer capillary
No peaks – acquisition parameters, sample prep/position
Troubleshooting Strategy
• Start from easiest to fix first
• Eliminate the obvious
• Examine your
chromatographic profile
RT: 0.00 - 3.00
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
0
10
20
30
40
50
60
70
80
90
100
Rela
tive A
bundance
0.26
0.36
0.31
0.480.22
0.56
0.64
0.73
0.81
0.90
NL:6.98E8
TIC MS 20150408std1
NL:4.20E8
TIC MS 20150408std3
• Familiarise yourself
with YOUR TICs – tells
you alot!
• If it doesn’t look
right…..Do something
about it!
Troubleshooting Strategy
