Quantitative Proteomics presentation from our quantitative proteomics short courses at the UC Davis Genome Center Proteomics Core Facility

1. Practical Aspects of Quantitation with
Triple-Quadrupole Mass
Spectrometers
Ben Moeller, PhD Candidate
University of California – Davis
K.L. Maddy Equine Analytical Chemistry Laboratory
1

2. Triple Quadrupole use in
Quantitation
Absolute Quantitation
of Analyte in Matrix
Testosterone 500 pg/ml
RT: 0.00 - 2.51 SM: 11G
NL: 1.20E5
m/z= 96.50-97.50 F: + c ESI sid=5.00
SRM ms2 289.234 [77.056-77.066,
79.097-79.107, 81.089-81.099,
97.043-97.053, 109.079-109.089] MS
ICIS C7_003
RT: 1.59
AA: 1222176
100
95
90
85
80
C7_003 #379-403 RT: 1.55-1.64 AV: 5 NL: 1.31E5
F: + c ESI sid=5.00 SRM ms2 289.234 [77.056-77.066, 79.097-79.107, 81.089-81.099, 97.043-97.053, 109.079-109.089]
97.05
100
109.08
95
75
90
85
80
60
70
Relative Abundance
65
60
55
50
20
45
79.10
40
18
35
30
77.06
25
16
81.09
20
55
15
10
Area Ratio
Relative Abundance
65
Testosterone
Y = 0.0222582+0.00197076*X R^2 = 0.9982 W: 1/X
75
70
5
0
50
77.06
m/z
79.10
m/z
81.09
m/z
97.05
m/z
109.08
m/z
45
14
12
10
8
6
40
4
2
35
0
0
2000
30
4000
6000
pg/ml
8000
10000
25
20
15
10
5
0
0.0
0.5
1.0
1.5
Time (min)
2.0
2.5
2

3. Quantitative Method
Development
1. Know what you are looking for and a rough idea
of the concentration range.
2. Obtain reference material (drug, protein, peptide,
etc)
3. Develop method.

Determine sample extraction/cleanup, and what
instrumentation to use
Ex) Immuno-depletion, SPE, tryptic digestion, LC-MS.
4. Validate method with real samples.
5. Run samples, calibrators and quality control
samples identically.

This includes sample clean up, extraction, LC-MS
analysis, peak integration, and quantitation.
3

4. Sample Analysis
•
An absolute quantitation method requires:
•
•
•
Unknown samples (what you trying to analyze)
•
Processed matrix + analytes
Known samples – containing known amounts of
targeted analytes in matrix
•
Calibration standards – generate calibration curve
•
Quality control samples (QCs) – evaluate method
performance
•
Standards spiked in solvent without matrix
Blanks – samples without the analytes
•
Matrix blanks – matrix without analytes
•
Solvent blanks – solvent without analytes
4

5. Quantitative Analysis –
Calibration Curves
 External Standard Method
 Construct calibration curve with increasing amounts of analyte.
 Match unknown samples instrument response to curve
 Method of Standard Addition
 Increasing amounts of analyte spiked into unknown sample.
 Response is measured before and after addition of analyte to give a
curve using linear regression. The x-intercept gives concentration
sample concentration
 Internal Standard (IS) Method
 A known, constant amount of internal standard is added to every
sample including calibrators
 Use the ratio of Analyte to IS to construct calibration curve and use
for determination of unknown sample concentration
 Preferred method because it corrects for sample losses in
5
processing and variations in instrument performance

6. Ideal MS Quantitative Method
Isotope Dilution Mass Spectrometry (IDMS) – use of a isotopically
labeled internal standard.
Sample
1. Take
aliquot
2. Add IS
Internal
standard
(IS)
3. Process
Sample
4. Analyze by
LC-MS/MS
Analyte
IS
5. Integrate and calculate
areas of IS and analyte
peaks – Quantitate using
analyte/IS area ratio
6

7. Internal Standard Selection
 SIS – Surrogate Internal Standard
 Stable isotope labeled version of analyte is
preferable – 13C, 2H, 15N
 Minimize isotopic overlap of SIS and analyte
 SIS co-elution with analyte preferable
 Small molecule – synthesize or purchase
 Proteomics
 Purchase heavy peptides from vendors
 Express protein in culture with heavy media
7

8. Analyte to SIS Area Count Ratio
 Calibrator 6
RT: 1.00 - 2.47 SM: 7G
RT: 1.63
AA: 606660
100
90
NL: 8.82E4
m/z= 96.50-97.50 F: + c ESI sid=5.00
SRM ms2 289.234 [77.056-77.066,
79.097-79.107, 81.089-81.099,
97.043-97.053, 109.079-109.089] MS
ICIS C6_001
 Analyte/SIS ratio = 1.07
 533 pg/ml based on
C6_001 #384-414 RT: 1.58-1.70 AV: 7 NL: 8.38E4
F: + c ESI sid=5.00 SRM ms2 289.234 [77.056-77.066, 79.097-79.107, 81.089-81.099, 97.043-97.053, 109.079-109.089]
97.05
100
80
95
109.08
90
85
70
calibration curve
80
75
70
60
65
60
Relative Abundance
50
 500 pg/ml nominal
55
50
45
40
40
35
79.10
30
Testosterone
Y = 0.0194483+0.00197195*X R^2 = 0.9982 W: 1/X
25
30
77.06
81.09
20
15
20
10
10
Testosterone
289.2 -> 97.1
0
5
0
77.06
m/z
79.10
m/z
81.09
m/z
97.05
m/z
109.08
20
NL: 8.45E4
18
m/z= 96.50-97.50 F: + c ESI sid=5.00
SRM ms2 292.260 [97.071-97.081]
MS ICIS C6_001 16
RT: 1.62
AA: 603473
100
m/z
C6_001 #384-404 RT: 1.58-1.65 AV: 4 NL: 6.06E4
F: + c ESI sid=5.00 SRM ms2 292.260 [97.071-97.081]
90
97.08
100
95
Area Ratio
85
80
70
75
70
65
Relative Abundance
60
50
60
55
50
45
40
40
30
20
10
14
90
2.0
1.8
10
1.6
8
35
SIS
D3-Testosterone
292.2 -> 97.1
Testosterone
Y = 0.0194483+0.00197195*X R^2 = 0.9982 W: 1/X
2.2
12
Area Ratio
80
30
25
6
20
15
1.4
1.2
1.0
0.8
0.6
10
5
0
97.072
97.074
97.076
m/z
97.078
97.080
4
0.4
0.2
2
0
200
400
600
800
1000
pg/ml
0
1.2
1.4
1.6
1.8
Time (min)
2.0
2.2
Extracted Ion Chromatogram
2.4
0
0
2000
4000
6000
pg/ml
8000
10000
8
Moeller et al (2009)

9. Types of MS scan in
Quantitation
 Four MS scan types used in quantitative
analysis
Full scan MS
Select ion monitoring (SIM)
Product ion MS/MS
Select reaction monitoring (SRM)
9

10. Quantitation using MS
 Types of MS commonly used in quantitation
Single Quad
Ion Traps (2D and 3D)
TOF, QqTOF
Orbitrap type MS
Magnetic Sectors
Triple Quadrupole – the “gold standard”
10

11. Why use Select Reaction
Monitoring (SRM)?
Also known as multiple reaction monitoring (MRM)
Fixed m/z
Q1

Fragment
Q2
Advantages



Targeted Analyte
Monitoring
High Duty Cycle
“Simultaneous”
Monitoring of Multiple
Transitions
Fixed m/z
Q3

Disadvantage

No “advanced”
structural information
11

12. Select Reaction Monitoring
 Quadrupole 1 (Q1) selects the ion of interest
(precursor ion) by its m/z ratio
 Quadrupole 2 (Q2) fragments precursor ion
by collision induced dissociation (CID)
Fixed m/z
Fragment
Q1
Q2
Fixed m/z
Q3
 Quadrupole 3 (Q3) selects specific
fragmentation ions (product ions) which are
counted in the detector
12

13. Why use MS/MS in
Quantitation?
 MS/MS provides additional specificity which
increases signal to baseline (S/B) and
sensitivity allowing for:
Less intense sample preparation.
Shorter chromatographic run times
Decreased Limits of Detection (LOD).
13

14. Determining SRM transitions
Progesterone #1-38 RT: 0.00-0.32 AV: 38 NL: 3.11E6
F: + c APCI Q1MS [170.000-400.000]
315.22
100
95
 Optimize precursor ion formation in Q1
 Source conditions, tube lens, etc
 Optimize several SRM transitions (5 if
possible) and run standards in matrix to
check for interferences
85
80
75
Precursor Ion
Optimization
70
65
60
Relative Abundance
Infusion of pure substance –
Preferably commercially obtained with
certificates of analysis (traceability).
 In Silico - predicted product ions from
software (Proteomics).
Optimization of SRM Transitions
90
55
Progesterone –APCI
50
45
40
35
30
25
20
356.25
15
211.13
225.12
10
181.11
214.11
193.11
202.13
5
313.21
297.20
245.17 255.20
271.19
311.21
279.18
326.22
338.33 353.28
370.62 3
0
180
200
220
240
260
280
300
320
340
m/z
SRM Optimization
14
360
3

15. Common SRM Settings
RT: 0.00 - 2.50 SM: 5G
100
NL:
TIC
289
79.0
97.0
MS
95
 Number of SRM Transitions
90
85
80
75
70
• 3 minimum per analyte, 5 recommended.
• 20% deviation in relative intensities allowed
65
Relative Abundance
60
55
50
45
40
35
30
25
20
15
10
5
0
SS_QC3_003 #384-403 RT: 1.59-1.65 AV: 4 NL: 2.59E5
0.0
F: + c ESI sid=5.00 SRM ms2 289.234 [77.056-77.066, 79.097-79.107, 81.089-81.099, 97.043-97.053, 109.079-109.089]
97.05
100
0.5
1.0
1.5
Time (min)
95
Testosterone SRM
109.08
90
85
80
Product
77.05
79.1
81.09
97.05
109.08
Relative Abundance
Precursor
289.2
289.2
289.2
289.2
289.2
Collision Relative
Energy Abundance
52
18
39
29
35
17
21
100
23
91
75
70
65
60
55
50
45
40
35
30
79.10
25
20
77.06
81.09
15
10
15
5
Moeller et al (2009)
0
77.06
m/z
79.10
m/z
81.09
m/z
97.05
m/z
109.08
m/z
2.0
2.5

16. Selectivity of SRM
Extracted Ion Chromatograms (EIC)
RT: 0.00 - 2.50 SM: 5G
100
NL: 4.81E4
m/z= 76.54-77.54 F: + c ESI sid=5.00 SRM ms2
287.213 [77.043-77.053, 91.061-91.071,
93.077-93.087, 121.060-121.070,
269.175-269.185] MS ICIS C7_003
80
60
40
20
0
100
NL: 5.03E4
m/z= 90.57-91.57 F: + c ESI sid=5.00 SRM ms2
287.213 [77.043-77.053, 91.061-91.071,
93.077-93.087, 121.060-121.070,
269.175-269.185] MS ICIS C7_003
80
60
40
20
0
100
NL: 2.09E4
m/z= 92.58-93.58 F: + c ESI sid=5.00 SRM ms2
287.213 [77.043-77.053, 91.061-91.071,
93.077-93.087, 121.060-121.070,
269.175-269.185] MS ICIS C7_003
80
60
40
20
0
100
EIC used for
quantitation
80
60
40
20
NL: 1.07E5
m/z= 120.50-121.50 F: + c ESI sid=5.00 SRM
ms2 287.213 [77.043-77.053, 91.061-91.071,
93.077-93.087, 121.060-121.070,
269.175-269.185] MS ICIS C7_003
C7_003 #269-291 RT: 1.13-1.19 AV: 4 NL: 1.10E5
F: + c ESI sid=5.00 SRM ms2 287.213 [77.043-77.053, 91.061-91.071, 93.077-93.087, 121.060-121.070, 269.175-269.185]
269.18
100
95
121.07
90
85
80
75
NL: 3.52E5
m/z= 268.50-269.50 F: + c ESI sid=5.00 SRM
ms2 287.213 [77.043-77.053, 91.061-91.071,
93.077-93.087, 121.060-121.070,
269.175-269.185] MS ICIS C7_003
80
60
40
70
65
Relative Abundance
0
100
60
55
50
45
77.05
91.07
40
20
0
0.0
•Choosing the right SRM
transition is key for quantitative
analysis
• Look at EIC to determine
optimal quantitation ion
• Use samples spiked in matrix
to evaluate interferences.
35
30
0.5
1.0
1.5
Time (min)
2.0
2.5
25
20
93.08
15
10
16
5
0
77.05
m/z
91.07
m/z
93.08
121.07
m/z
m/z
269.18
m/z

17. Common SRM Settings
 Scan Time/Dwell Time
• 0.01 – 0.2 seconds
• Need > 9 scans per peak
RT: 1.40 - 1.90 SM: 5G
100
RT: 1.40 - 1.90 SM: 5G
NL: 2.82E5
100
m/z= 96.50-97.50 F: + c ESI sid=5.00
SRM ms2 289.234 [77.056-77.066,
95
79.097-79.107, 81.089-81.099,
95
97.043-97.053, 109.079-109.089]
MS SS_QC3_003
90
•15 scans per peak
•50 msec dwell time
• 21 SRM transitions
monitored for 6 analytes
90
85
80
80
75
75
70
70
65
65
60
60
Relative Abundance
85
Relative Abundance
NL: 7.02E5
TIC F: + c ESI sid=5.00 SRM ms2
289.234 [77.056-77.066,
79.097-79.107, 81.089-81.099,
97.043-97.053, 109.079-109.089]
MS SS_QC3_003
55
50
45
40
55
50
45
40
35
35
30
30
25
25
20
20
15
15
10
10
5
5
0
1.4
1.5
1.6
1.7
Time (min)
1.8
0
1.4
17
1.5
1.6
1.7
Time (min)
1.8

18. SRM Method Setup
•Segments for
Multiple Analytes
•Scan Width
• 1 dalton
•Peak Width
• 0.7 daltons
• Enhanced
Resolution QqQ
0.1 – 0.2 daltons
35 analytes using
Thermo TSQ
Vantage using
Xcalibur software
18

19. Method Development and
Validation
 Limit of Detection
(LOD)
 Limit of Quantitation
(LOQ)
 Linearity of Calibration
 Calibration Range
 Precision
 Accuracy
 Selectivity
 Robustness and
Reproducibility
19

20. Limit of Detection
Signal
RT: 0.00 - 2.51 SM: 7G
100
RT: 2.28
MA: 17172
NL: 2.39E3
m/z= 80.50-81.50 F: + c
ESI SRM ms2 329.281
[81.066-81.076,
95.057-95.067,
121.042-121.052] MS
C25
95
90
85
80
75
70
65
Relative Abundance
60
55
50
1.84
45
1.95
Background
40
35
30
1.10
1.28
25
1.63
20
15
0.28
0.19
10
0.09
1.39
0.43 0.48
0.65
5
0
0.0
0.2
0.4
0.6
0.84
0.8
1.0
1.2
Time (min)
1.4
1.6
1.8
LOD of Stanozolol 25 pg/ml with S/B = 3:1
2.0
2.2
2.4
 Smallest response that is
able to differentiate
between background noise
and your analyte
 Usually defined as a signal
to background (S/B) = 3
 Determined by 1:1
dilutions from a
concentration with a S/B=
50:1
Boyd R, Basic C, Bethem R (2008) Trace 20
Quantitative Analysis by Mass Spectrometry.

21. Limits of Quantitation
 Lower and upper
concentrations that can be
accurately quantitated.
 Lower limit of
quantitation (LLOQ)
usually defined as a
signal to background
(S/B) = 10
 The upper limit of
quantitation (ULOQ) is
usually the highest
calibrator giving a linear
response
RT: 0.00 - 2.51 SM: 5G
RT: 2.27
MA: 51558
100
95
NL: 9.49E3
m/z= 80.50-81.50 F: + c
ESI SRM ms2 329.281
[81.066-81.076,
95.057-95.067,
121.042-121.052] MS
C1_001
S = 9,500 counts
90
85
80
75
LOQ of
Stanozolol150 pg/ml
70
65
Relative Abundance
60
55
50
1.47
45
40
35
30
25
0.58
20
1.95
15
B=665 counts
0.37
10
5
0.21
0
0.0
0.2
0.30
1.10
0.47
0.4
0.69 0.76
0.6
0.8
0.87
1.65
1.21
1.02
1.0
1.78
1.36
1.2
Time (min)
1.4
1.6
1.8
2.0
2.2
Boyd R, Basic C, Bethem R (2008) Trace
Quantitative Analysis by Mass Spectrometry.
2.4
21

22. Generation of Calibration
Curve
 Optimize for an expected concentration range (if
known)
 Develop method around that range
Rosiglitazone
Y = 0.0313243+0.00277886*X R^2 = 0.9949 W: Equal
3.0
Y = mx + b
2.5
Detector saturation
2.0
1.5
1.0
ULOQ
0.5
0.0
0
200
400
600
ng/mL
800
1000
22

23. Generation of Calibration Curve
and Quality Control Samples
Calibrators C1
Sample
C2
1 ng/ml
LOQ
C3
C4
C5
C6
C7
5 ng/ml
10 ng/ml
25 ng/ml
50 ng/ml
75 ng/ml
100 ng/ml
ULOQ
Quality Control Samples
QC2
QC1
QC3
3 ng/ml
15 ng/ml
60 ng/ml
n=6
n=6
Internal
Standard
n=6
23

24. Generation of Calibration
Curve
 6 to 9 calibrators prepared in
matrix blanks
 Must include LLOQ and
ULOQ
 Linear regression commonly
used – R2 ≥ 0.98
 Be aware of deviations from
linearity at higher conc.
 Avoid forcing through zero
 Internal Calibration
 Ratio of analyte area to SIS area
24

25. Quality Control (QC)
Samples
 QC samples:
 Blank matrix containing a known amount of analyte
 Run dispersed thorough out the assay
 At least 3 different levels (n=6)
 One near LLOQ
 One in the middle of linear range
 One at the high end of the linear range
 Determine accuracy and precision of method during validation and
monitor performance during sample runs
 Use QC’s for determination of both inter-assay (between runs) and intraassay (same run) precision and accuracy
25

26. Quality Control Samples –
Accuracy and Precision
 Accuracy: Trueness
 % expected = (Conc of Peak)/(Expected Conc)*100
 mean within 15% of nominal
 Precision: Reproducibility
 % Coefficient of Variation = (Standard Dev)/(Mean)*100
 % CV ≤ 15%
 Also expressed as relative standard deviation (RSD)
26

27. Accuracy and Precision
RT: 0.00 - 2.52 SM: 5G
Testosterone
Y = 0.010179+0.00178017*X R^2 = 0.9991 W: 1/X
RT: 1.62
MA: 37906
100
90
85
80
75
18
70
 210 injections over 19 hours
65
60
Relative Abundance
16
NL: 7.71E3
m/z= 96.50-97.50 F: + c ESI sid=5.00
SRM ms2 289.234 [77.056-77.066,
79.097-79.107, 81.089-81.099,
97.043-97.053, 109.079-109.089]
MS C1_002
LOQ –
25 pg/ml
95
2.03
55
50
45
40
35
14
30
1.89
25
0.98
1.10
20
1.19
0.92
15
12
1.29
0.77
0.55
2.22
1.45
10
0.22
5
0.41 0.47
2.35
2.47
0.63
0.10
0
0.0
10
0.2
0.4
0.6
0.8
1.0
1.2
Time (min)
1.4
1.6
1.8
2.0
2.2
2.4
RT: 0.00 - 2.50 SM: 5G
RT: 1.62
AA: 1018291
100
NL: 1.64E5
m/z= 96.50-97.50 F: + c ESI sid=5.00
SRM ms2 289.234 [77.056-77.066,
79.097-79.107, 81.089-81.099,
97.043-97.053, 109.079-109.089] MS
ICIS Inter_QC2_001
95
90
8
85
80
75
70
6
65
Relative Abundance
60
4
55
QC2 –
750 pg/ml
50
45
40
35
2
30
25
20
15
0
10
0
2000
Testosterone
(n=6 per level)
4000
6000
pg/ml
8000
10000
RT: 2.04
AA: 26482
5
0
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Time (min)
1.4
1.6
1.8
2.0
2.2
2.4
Average
Standard
Deviation
%CV
% of expected
QC1 – 75 pg/ml
74.1
2.21
2.98
98.7
QC2 - 750 pg/ml
742.7
29.32
3.94
99.0
QC3 - 3000 pg/ml
2798.6
57.31
2.05
93.3
27
Moeller et al (2009)

28. Quantitation Software
 Peak Integration
 Determine settings in validation
and use throughout study
 Integration must be consistent for
Calibrators, QC’s, and samples
 Avoid manual integration
 Set up Calibrator and QC levels
Thermo Quan Browser
 Fail runs that fall outside expected
concentration and % CV
 Fail runs with calibration curves R2
<0.98
28

29. Review
 Quantitation Methodology – IDMS preferred
 MS used – Triple Quad is the “Gold
Standard”
 SRM collecting multiple transitions
 Internal standard selection is important
 Defined LOD, LLOQ, ULOQ
 Generation of calibration curve
 Accuracy and precision using QCs
 Integration software
29

30. Validated Quantitative
Multiple Analyte Method
Analyte
 Stanozolol
 Testosterone
 Boldenone
 Nandrolone
 Trenbolone
LOD (pg/mL)
25
20
50
150
150
LOQ (pg/mL)
150
150
250
250
250
RT: 0.00 - 2.51 SM: 7G
RT: 0.00 - 2.51 SM: 5G
NL: 2.39E3
m/z= 80.50-81.50 F: + c
ESI SRM ms2 329.281
[81.066-81.076,
95.057-95.067,
121.042-121.052] MS
NC_a_01
100
95
90
NC Serum
80
95
Stanozolol
25 pg/ml
90
85
80
75
75
70
70
65
65
2.04
60
Relative Abundance
55
2.27
50
2.12
1.87
1.82
40
2.38
1.11
NL: 9.49E3
m/z= 80.50-81.50 F: + c
ESI SRM ms2 329.281
[81.066-81.076,
95.057-95.067,
121.042-121.052] MS
C1_001
95
Stanozolol
150 pg/ml
90
80
75
70
65
60
55
50
1.84
45
1.95
55
50
1.47
45
40
35
RT: 2.27
MA: 51558
100
85
60
2.21
45
NL: 2.39E3
m/z= 80.50-81.50 F: + c
ESI SRM ms2 329.281
[81.066-81.076,
95.057-95.067,
121.042-121.052] MS
C25
Relative Abundance
85
RT: 0.00 - 2.51 SM: 5G
RT: 2.28
MA: 17172
100
40
35
35
1.56
1.52
30
0.61
25
20
0.19
1.10
1.28
25
0.56
0.34
0.67
0.11
15
0.85
10
0.09
0.4
0.6
0.8
1.0
1.2
Time (min)
1.4
1.6
1.8
2.0
2.2
2.4
0
0.0
0.2
0.4
0.37
10
5
0.2
1.95
1.39
0.43 0.48
0.65
5
0.58
20
15
0.28
0.19
10
0
0.0
25
1.63
20
0.22
15
30
30
1.69
1.36
0.91
0.6
0.84
0.8
5
1.0
1.2
Time (min)
1.4
LOD
1.6
1.8
2.0
2.2
2.4
0.21
0
0.0
0.2
1.78
0.30
1.10
0.47
0.4
0.69 0.76
0.6
0.8
0.87
1.02
1.0
1.65
1.21
1.36
1.2
Time (min)
1.4
1.6
LOQ
1.8
2.0
2.2
2.4
30

31. Calibration Curves
Testosterone
Y = -0.0183096+0.00114189*X R^2 = 0.9987 W: Equal
 Calibrator
12
 C1 - 150 pg/ml
Area Ratio
Concentrations
10
8
6
4
 C2 - 250 pg/ml
2
 C3 - 500 pg/ml
0
0
 C4 - 750 pg/ml
2000
4000
6000
pg/ml
8000
10000
Stanozolol
Y = 0.113603+0.00136788*X R^2 = 0.9976 W: Equal
 C5 – 1,000 pg/ml
 C6 – 2,500 pg/ml
14
 C7 – 5,000 pg/ml
10
Area Ratio
 C8 – 10,000 pg/ml
12
8
6
4
2
0
0
SIS used: D3-Testosterone
2000
4000
6000
pg/ml
8000
10000
31

32. Assay Precision
Inter assay (n=36)
Testosterone Stanozolol Nandrolone
Trenbolone
Boldenone
QC level 1
Average (pg/mL)
609.7
601.0
662.1
594.5
588.0
(600 pg/mL)
%CV
6.8
6.3
13.4
14.2
9.5
QC level 2
Average (pg/mL)
1176.2
1196.8
1170.2
1160.0
1179.3
(1200 pg/mL)
%CV
6.8
5.1
12.8
8.0
6.0
QC level 3
Average (pg/mL)
4184.7
4234.6
4417.7
3958.7
4185.1
(4000 pg/mL)
%CV
8.3
8.5
10.5
10.9
7.8
Trenbolone
Boldenone
Intra assay (n=12)
Testosterone Stanozolol Nandrolone
QC level 1
Average (pg/mL)
621.1
611.5
662.1
529.2
583.4
(600 pg/mL)
%CV
4.6
6.8
11.8
12.5
7.1
QC level 2
Average (pg/mL)
1251.0
1225.5
1086.6
1106.5
1219.9
(1200 pg/mL)
%CV
3.2
4.6
10.7
6.5
3.2
QC level 3
Average (pg/mL)
4472.1
4369.8
4250.1
3576.3
4332.8
(4000 pg/mL)
%CV
9.7
12.9
7.6
8.6
8.9
32

33. Assay Accuracy
Inter assay (n=36)
Testosterone Stanozolol Nandrolone
Trenbolone
Boldenone
QC level 1
Average (pg/mL)
609.7
601.0
622.7
594.5
588.0
(600 pg/mL)
%of nominal
101.6 %
100.2 %
103.8 %
99.1 %
98.0 %
QC level 2
Average (pg/mL)
1176.2
1196.8
1170.2
1160.0
1179.3
(1200 pg/mL)
%of nominal
98.0 %
99.7 %
97.5 %
96.7 %
98.3 %
QC level 3
Average (pg/mL)
4184.7
4234.6
4417.7
3958.7
4185.1
(4000 pg/mL)
%of nominal
104.6 %
105.9 %
110.4 %
99.0 %
104.6 %
Intra assay (n=12)
Testosterone Stanozolol Nandrolone Trenbolone
Boldenone
QC level 1
Average (pg/mL)
621.1
611.5
662.1
529.2
583.4
(600 pg/mL)
%of nominal
103.5 %
101.9 %
110.3%
88.2 %
97.2 %
QC level 2
Average (pg/mL)
1251.0
1225.5
1086.6
1106.5
1219.9
(1200 pg/mL)
%of nominal
104.3 %
102.1 %
90.5%
92.2 %
101.7 %
QC level 3
Average (pg/mL)
4472.1
4369.8
4250.1
3576.3
4332.8
(4000 pg/mL)
%of nominal
111.8 %
109.2 %
106.2%
89.4 %
108.3 %
33

34. Summary
 Validation is key
 Reproducibility
 Defined quantitation limits (LLOQ/ULOQ)
 Selectivity – Qualitative ID (3 or more SRM)
 Accuracy and Precision
 Need Quality Control samples
 Inter- and Intra-Day
Robustness
34

35. Acknowledgements
University of California, Davis
 Scott Stanley, PhD
 Heather Knych, DVM PhD
 EACL Staff
35

36. Questions?
References
1)
2)
3)
4)
5)
Lee JM et al. (2006) Fit-for-Purpose Method Development and
Validation for Successful Biomarker Measurement. Pharmaceutical
Research. 23(2) 312-328.
US Food and Drug Administration (2001) Guidance for Industry:
Bioanalytical Method Validation.
http://www.fda.gov/cder/guidance/index.htm
Boyd R, Basic C, Bethem R (2008) Trace Quantitative Analysis by Mass
Spectrometry. West Sussex: John Wiley & Sons.
Krull I, Kissinger PT, Swartz M (2008) Analytical Method Validation in
Proteomics and Peptidomics Studies. LCGC 26 (11)
Moeller BC, Stanley SD (2009) Quantitative Analysis of Testosterone,
Nandrolone, Boldenone and Stanozolol using Liquid Chromatography –
Tandem Mass Spectrometry by Highly Selective Reaction Monitoring.
36
Manuscript in preparation.