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Moeller proteomics course

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

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Moeller proteomics course

  1. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 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. 35. Acknowledgements University of California, Davis  Scott Stanley, PhD  Heather Knych, DVM PhD  EACL Staff 35
  36. 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.

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