moeller proteomics course
DESCRIPTION
Quantitative Proteomics presentation from our quantitative proteomics short courses at the UC Davis Genome Center Proteomics Core FacilityTRANSCRIPT
1
Practical Aspects of Quantitation with Triple-Quadrupole Mass
SpectrometersBen Moeller, PhD Candidate
University of California – DavisK.L. Maddy Equine Analytical Chemistry Laboratory
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Triple Quadrupole use in Quantitation
Absolute Quantitation of Analyte in Matrix
Testosterone 500 pg/ml
RT: 0.00 - 2.51 SM: 11G
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RT: 1.59AA: 1222176
NL: 1.20E5m/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
C7_003 #379-403 RT: 1.55-1.64 AV: 5 NL: 1.31E5F: + 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]
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TestosteroneY = 0.0222582+0.00197076*X R^2 = 0.9982 W: 1/X
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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 useEx) 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.
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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
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
processing and variations in instrument performance
6
Ideal MS Quantitative Method
Isotope Dilution Mass Spectrometry (IDMS) – use of a isotopically labeled internal standard.
Sample
Internal standard (IS)
1. Take aliquot
2. Add IS
3. Process Sample
4. Analyze by LC-MS/MS
5. Integrate and calculate areas of IS and analyte peaks – Quantitate using analyte/IS area ratio
IS
Analyte
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 purchaseProteomics
Purchase heavy peptides from vendors Express protein in culture with heavy media
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Analyte to SIS Area Count Ratio
Calibrator 6 500 pg/ml nominal
Analyte/SIS ratio = 1.07 533 pg/ml based on
calibration curve
C6_001 #384-414 RT: 1.58-1.70 AV: 7 NL: 8.38E4F: + 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]
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C6_001 #384-404 RT: 1.58-1.65 AV: 4 NL: 6.06E4F: + c ESI sid=5.00 SRM ms2 292.260 [97.071-97.081]
97.072 97.074 97.076 97.078 97.080m/z
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RT: 1.00 - 2.47 SM: 7G
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RT: 1.63AA: 606660
RT: 1.62AA: 603473
NL: 8.82E4m/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
NL: 8.45E4m/z= 96.50-97.50 F: + c ESI sid=5.00 SRM ms2 292.260 [97.071-97.081] MS ICIS C6_001
TestosteroneY = 0.0194483+0.00197195*X R^2 = 0.9982 W: 1/X
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TestosteroneY = 0.0194483+0.00197195*X R^2 = 0.9982 W: 1/X
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Testosterone289.2 -> 97.1
SISD3-Testosterone292.2 -> 97.1
Extracted Ion ChromatogramMoeller et al (2009)
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Types of MS scan in Quantitation
Four MS scan types used in quantitative analysisFull scan MSSelect ion monitoring (SIM)Product ion MS/MSSelect reaction monitoring (SRM)
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Quantitation using MS
Types of MS commonly used in quantitationSingle QuadIon Traps (2D and 3D)TOF, QqTOFOrbitrap type MSMagnetic SectorsTriple Quadrupole – the “gold standard”
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Advantages Targeted Analyte
Monitoring High Duty Cycle “Simultaneous”
Monitoring of Multiple Transitions
Disadvantage No “advanced”
structural information
Fixed m/zFragmentFixed m/z
Q1 Q2 Q3
Why use Select Reaction Monitoring (SRM)?
Also known as multiple reaction monitoring (MRM)
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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/zFragmentFixed m/z
Q1 Q2 Q3Quadrupole 3 (Q3) selects specific
fragmentation ions (product ions) which are counted in the detector
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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).
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Determining SRM transitions
Infusion of pure substance – Preferably commercially obtained with certificates of analysis (traceability).
In Silico - predicted product ions from software (Proteomics).
Optimization of SRM Transitions 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
Progesterone #1-38 RT: 0.00-0.32 AV: 38 NL: 3.11E6F: + c APCI Q1MS [170.000-400.000]
180 200 220 240 260 280 300 320 340 360 380 400m/z
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315.22
356.25
211.13
225.12
181.11214.11 313.21
297.20193.11311.21271.19202.13 338.33 353.28255.20 279.18245.17 326.22 381.97370.62 394.25
Precursor Ion OptimizationProgesterone –APCI
SRM Optimization
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Common SRM Settings
Number of SRM Transitions• 3 minimum per analyte, 5 recommended.• 20% deviation in relative intensities allowed
SS_QC3_003 #384-403 RT: 1.59-1.65 AV: 4 NL: 2.59E5F: + 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]
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77.06
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RT: 0.00 - 2.50 SM: 5G
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NL: 7.02E5TIC 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
Precursor ProductCollision Energy
Relative Abundanc
e289.2 77.05 52 18289.2 79.1 39 29289.2 81.09 35 17289.2 97.05 21 100289.2 109.08 23 91
Testosterone SRM
Moeller et al (2009)
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Selectivity of SRM
RT: 0.00 - 2.50 SM: 5G
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100 NL: 4.81E4m/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
NL: 5.03E4m/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
NL: 2.09E4m/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
NL: 1.07E5m/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
NL: 3.52E5m/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
C7_003 #269-291 RT: 1.13-1.19 AV: 4 NL: 1.10E5F: + 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]
77.05m/z
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121.07m/z
269.18m/z
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• 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.
EIC used for quantitation
Extracted Ion Chromatograms (EIC)
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Common SRM Settings
Scan Time/Dwell Time• 0.01 – 0.2 seconds• Need > 9 scans per peak
RT: 1.40 - 1.90 SM: 5G
1.4 1.5 1.6 1.7 1.8Time (min)
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NL: 2.82E5m/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 SS_QC3_003
RT: 1.40 - 1.90 SM: 5G
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NL: 7.02E5TIC 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• 15 scans per peak
• 50 msec dwell time• 21 SRM transitions
monitored for 6 analytes
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SRM Method Setup
• Segments for Multiple Analytes
• Scan Width• 1 dalton
• Peak Width• 0.7 daltons• Enhanced
Resolution QqQ 0.1 – 0.2 daltons35 analytes using
Thermo TSQ Vantage using Xcalibur software
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Method Development and Validation
Limit of Detection (LOD)
Limit of Quantitation (LOQ)
Linearity of CalibrationCalibration RangePrecisionAccuracySelectivityRobustness and
Reproducibility
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Limit of Detection
RT: 0.00 - 2.51 SM: 7G
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RT: 2.28MA: 17172
1.84 1.95
1.101.28
1.63
0.281.390.19 0.480.43
0.090.65
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NL: 2.39E3m/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
LOD of Stanozolol - 25 pg/ml with S/B = 3:1
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 Quantitative Analysis by Mass Spectrometry.
Signal
Background
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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 Boyd R, Basic C, Bethem R (2008) Trace
Quantitative Analysis by Mass Spectrometry.
RT: 0.00 - 2.51 SM: 5G
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RT: 2.27MA: 51558
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1.651.211.100.30
0.21 0.47 1.02 1.360.870.69 0.76
NL: 9.49E3m/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
LOQ of Stanozolol-150 pg/ml
B=665 counts
S = 9,500 counts
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Generation of Calibration Curve
Optimize for an expected concentration range (if known)
Develop method around that range
RosiglitazoneY = 0.0313243+0.00277886*X R^2 = 0.9949 W: Equal
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Detector saturation
ULOQ
Y = mx + b
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Generation of Calibration Curveand Quality Control Samples
Sample
C11 ng/mlLOQ
C25 ng/ml
C310 ng/ml
C425 ng/ml
C550 ng/ml
C675 ng/ml
C7100 ng/mlULOQ
Internal Standard
QC13 ng/ml
QC215 ng/ml
QC360 ng/ml
n=6 n=6 n=6
Quality Control Samples
Calibrators
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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
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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 intra-assay (same run) precision and accuracy
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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)
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TestosteroneY = 0.010179+0.00178017*X R^2 = 0.9991 W: 1/X
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Testosterone (n=6 per level) Average
StandardDeviation %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
RT: 0.00 - 2.50 SM: 5G
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RT: 1.62AA: 1018291
RT: 2.04AA: 26482
NL: 1.64E5m/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
RT: 0.00 - 2.52 SM: 5G
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RT: 1.62MA: 37906
2.03
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0.98 1.10
1.190.92 1.29
0.77 2.220.551.45 2.35 2.47
0.22 0.630.470.41
0.10
NL: 7.71E3m/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
Accuracy and Precision
210 injections over 19 hours
LOQ – 25 pg/ml
QC2 – 750 pg/ml
Moeller et al (2009)
28
Quantitation Software
Peak IntegrationDetermine settings in validation
and use throughout study Integration must be consistent for
Calibrators, QC’s, and samplesAvoid manual integration
Set up Calibrator and QC levelsFail runs that fall outside expected
concentration and % CVFail runs with calibration curves R2
<0.98
Thermo Quan Browser
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
30
RT: 0.00 - 2.51 SM: 7G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time (min)
0
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Re
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e A
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RT: 2.28MA: 17172
1.84 1.95
1.101.28
1.63
0.281.390.19 0.480.43
0.090.65
0.84
NL: 2.39E3m/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
Validated Quantitative Multiple Analyte Method
Analyte LOD (pg/mL) LOQ (pg/mL)Stanozolol 25 150Testosterone 20 150Boldenone 50 250Nandrolone 150 250Trenbolone 150 250
Stanozolol25 pg/ml
Stanozolol150 pg/ml
NC Serum
RT: 0.00 - 2.51 SM: 5G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time (min)
0
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25
30
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Re
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RT: 2.27MA: 51558
1.47
0.58
1.95
0.37 1.78
1.651.211.100.30
0.21 0.47 1.02 1.360.870.69 0.76
NL: 9.49E3m/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
RT: 0.00 - 2.51 SM: 5G
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4Time (min)
0
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Re
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e A
bu
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an
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2.04
2.21
2.27
2.121.87
1.82
2.381.11
1.561.52
1.691.360.61 0.91
0.220.19
0.560.340.850.670.11
NL: 2.39E3m/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
LOD LOQ
31
Calibration Curves
Calibrator Concentrations C1 - 150 pg/ml C2 - 250 pg/ml C3 - 500 pg/ml C4 - 750 pg/ml C5 – 1,000 pg/ml C6 – 2,500 pg/ml C7 – 5,000 pg/ml C8 – 10,000 pg/ml
StanozololY = 0.113603+0.00136788*X R^2 = 0.9976 W: Equal
0 2000 4000 6000 8000 10000pg/ml
0
2
4
6
8
10
12
14
Are
a R
atio
TestosteroneY = -0.0183096+0.00114189*X R^2 = 0.9987 W: Equal
0 2000 4000 6000 8000 10000pg/ml
0
2
4
6
8
10
12
Are
a R
atio
SIS used: D3-Testosterone
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
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) %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
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 %
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
35
Acknowledgements
University of California, Davis Scott Stanley, PhD Heather Knych, DVM PhD EACL Staff
36
Questions?
References1) Lee JM et al. (2006) Fit-for-Purpose Method Development and
Validation for Successful Biomarker Measurement. Pharmaceutical Research. 23(2) 312-328.
2) US Food and Drug Administration (2001) Guidance for Industry: Bioanalytical Method Validation. http://www.fda.gov/cder/guidance/index.htm
3) Boyd R, Basic C, Bethem R (2008) Trace Quantitative Analysis by Mass Spectrometry. West Sussex: John Wiley & Sons.
4) Krull I, Kissinger PT, Swartz M (2008) Analytical Method Validation in Proteomics and Peptidomics Studies. LCGC 26 (11)
5) Moeller BC, Stanley SD (2009) Quantitative Analysis of Testosterone, Nandrolone, Boldenone and Stanozolol using Liquid Chromatography –Tandem Mass Spectrometry by Highly Selective Reaction Monitoring. Manuscript in preparation.