Beyond Westgard Rules: Quality Control for Mass Spectrometry Dr Russell Grant VP R&D Laboratory Corporation of America® Holdings.
Beyond Westgard Rules: Quality Control for Mass Spectrometry
Dr Russell GrantVP R&D
Laboratory Corporation of America® Holdings.
Assay Validation Criteria and Acceptance
Validation Process Materials Requirements and Acceptance Criteria
Accuracy (Intra and Inter-assay)
Min. 2 levels of spiked QC’s including LLOQ and ULOQ; Min 20 measurements each in a single run and across 25 days. Bias % <15% or 20% @LLOQ
Precision (Intra and Inter-assay)
Min. 2 levels of spiked QC’s including LLOQ and ULOQ; Min 20 measurements each in a single run and across 25 days. CV % 15%/20% @LLOQ
Standard Curve Fit 6-10 levels run in each assay, evaluated over 5 assays (days), R>0.995, Slope CV<5%
Drug Selectivity Up to 350 potentially interfering endogenous and exogenous analytes spiked supraphysiological concentrations, Response < 20% LLOQ when spiked at Maximum Normal x10 ideal, minimally <20% LLOQ @ upper normal range.
Materials Selectivity Blank matrix test 6 or more lots Response<20% LLOQ in 5 of 6.
Matrix Interference Effect of lipid, Icteria and Hemolysis. Bias % <15%/20% @LLOQTest anticoagulants Bias % <15%/20% @LLOQAlternate sample types Deming regression 0.9-1.1, R >0.9 for same reference interval. Matrix effects (sample mixing) Bias<15%
Assay Validation Criteria and AcceptanceValidation Process Materials Requirements and Acceptance Criteria
Carry-over After ULOQ – 3 runs including <20% LLOQ Stress test (10-100x ULOQ), how many for <20% LLOQ
Dilutional Linearity 5 Level sample mixing - Accuracy % 85% to 115%1:1 – 1:50000 dilution - Accuracy % 85% to 115%Serial 100-10% dilution - Accuracy % 85% to 115%, (80/120%@LLOQ)
Sample Stability All conditions tested from arm to final resultMean Bias < 15%/20%@ LLOQ, %CV < 15%/20%@ LLOQ, Deming regression 0.9-1.1, R >0.9
Reference Interval 120 in each reference interval 20 (95% CI) for range transfer 1/20 outside existing range
Batch Size Maximum 184 samples prior to recalibrate. Bias % <15%/20% @LLOQ, % CV % <15%/20% @LLOQ, Carry-over <20% LLOQ Repeat samples n = 2 (first and 2nd plate) Deming regression 0.9-1.1, R >0.9
Inter-assay Comparison Min 20 – External Disparate assays – For information onlyInternal assays same cals – Deming regression 0.9-1.1, R >0.9Same assay different system – Deming regression 0.95-1.05, R >0.95Same assay Manual/Automation – Deming regression 0.95-1.05, R >0.95
Transition ratio monitoring Bias from extracted calibrators in each batch <20% up to 3x LLOQ, < 15% after that for ratio>0.5
Verification Process Materials Requirements and Acceptance Criteria
Materials Selectivity Blank matrix <20% LLOQ in 5 runs.
Standard Linearity All Cals used, Accuracy % 85% to 115%, 80 – 120% @LLOQ and R>0.995, Slope CV<5%
Carry-over After ULOQ – 3 runs including <20% LLOQ Stress test (10-100x ULOQ), how many for <20% LLOQ
Precision (Intra and Inter-assay)
Total 25 QC’s over 5 runs %CV <15% or 20% @LLOQ
Inter-assay Comparison Minimum n=20 per batch – Deming regression 0.90-1.00, R >0.90
Do not use best technicians for this step Add multiple columns/materials lots /systems, time to emulate reality
Assay Translation (R&D – Ops)(Assay Viability – QC Ranges Generation n=25 over 5 runs)
Assay Translation (R&D – Ops)(Check Manufacturing Process)
Plasma Metanephrine
Slope = 1.148
R = 1
Plasma Normetanephrine
Slope = 1.128
R = 1
Internal QC for LC-MS/MS
LC System Internal QC Action
Power FailureLeak detection
Pressure beyond pumpingAir bubble
Column DegradationComplete Sample volume injected
Injector blockageContamination
Shutdown (without UPS)Shutdown*Shutdown
Shutdown or No monitoringNo monitoringNo monitoringNo monitoring*No monitoring
MS System Internal QC Action
Power FailureGas failure
Vacuum failureTemperature effects
ContaminationResolution change
Sensitivity driftSelectivity
Shutdown (without UPS)ShutdownShutdown
Shutdown – in extreme conditionsNo monitoringNo monitoringNo monitoringNo monitoring
System Suitability Test (SST) – “The Doctor”
Goal – System check prior to analysis to confirm appropriateness of test system for analysis/ or DRIFT
Design:
Neat solution stored under stable conditions –preferably at the assay LLOQAcquired correctly? Retention time – 5% varianceSensitivity – S:N >20:1Peak shape – As 0.8-1.2Multi-channel acceptance – column-column/channel-channel/system-system
SST is key part of troubleshooting experiments
Ignore the first (or second) injection
Urine Cortisol SST
Urine Cortisol LLOQ
HPLC Pressure Trace – “The Heartbeat”
Goal – Pre-batch and per-injection check of appropriate LC performance
Design:
SOP Example – Annotate an acceptable pressure trace
Pressure profile – Start and overall maximum pressure
Changes in pressure profile –
Air bubbles in A or B
Dead volume differences
Column dimensions change
Full injection
Injector/filter/column/lines blockage
Post column blockage (bypass/source probe)
Bubble in A
Bubble in B
Inject/FocusGradient
DetectWash
Recondition
Internal QA – Pre-Batch
LC Solvent Contamination
XIC of +MRM (4 pairs): 166.1/134.1 Da from Sample 1 (SST) of SST 072111 sys 4.wiff (Turbo Spray) Max. 1.5e4 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
Inte
nsity
, cps
Air Bubble/Column or MP Degradation/Injector blockageXIC of +MRM (4 pairs): 166.1/134.1 Da from Sample 3 (SST) of SST 071811 sys 4.wiff (Turbo Spray) Max. 7.0e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
3.6e6
3.8e6
Inte
nsity
, cps
0.51
XIC of +MRM (4 pairs): 166.1/134.1 Da from Sample 2 (SST) of SST 072111 sys 4.wiff (Turbo Spray) Max. 1.7e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75Time, min
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
1.05e6
Inte
nsity
, cps
0.66
XIC of +MRM (4 pairs): 166.1/134.1 Da from Sample 7 (SST) of SST 072111 sys 4.wiff (Turbo Spray) Max. 1.7e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70 0.75Time, min
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
1.05e6
1.10e6
Inte
nsity
, cps
0.55
Air Bubble SST
SST Day 4 SST Day 4 post LC flush/new
MPB
SST Day 1
XIC of +MRM (6 pairs): 184.2/107.1 Da from Sample 2 (Double Blank) of Validation Batch 2.wiff (Turbo Spray) Max. 5500.0 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
Inte
nsity
, cps
0.25
0.18
0.29
0.50
0.40 0.480.46
0.510.76
Blank, Clean
XIC of +MRM (9 pairs): 184.2/107.1 Da from Sample 3 (Standard 1) of 186026.wiff (Turbo Spray), Smoothed, Smoothed, Smoothed Max. 9346.9 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6500
7000
7500
8000
8500
9000
9347
Inte
nsity
, cps
0.26
ResolvedContaminant
XIC of +MRM (6 pairs): 184.2/107.1 Da from Sample 1 (Blank) of SST 082908.wiff (Turbo Spray) Max. 1.5e4 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
Inte
nsity
, cps
0.26
0.30
0.09
0.61
0.420.40 0.580.540.44 0.49
C E
Epinephrine LLOQ
XIC of +MRM (6 pairs): 184.2/107.1 Da from Sample 4 (Standard 1) of Validation Batch 2.wiff (Turbo Spray) Max. 6450.0 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8Time, min
0
500
1000
1500
2000
2500
3000
3500
4000
4500
5000
5500
6000
6450
Inte
nsity
, cps
Internal QA – Pre and During Batch
MS System Source contamination Pre-runXIC of -MRM (8 pairs): 200.0/137.0 Da from Sample 1 (sst-1) of sst_072011A1_1.wiff (Turbo Spray) Max. 4500.0 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e51.7e5
Inte
nsity
, cps
XIC of -MRM (8 pairs): 197.0/137.0 Da from Sample 1 (sst-3) of sst_072111_1.wiff (Turbo Spray) Max. 6100.0 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
1.8e6
Inte
nsity
, cps
0.24 0.370.30 0.40
HVA, VMA, 5-HIAA1st Sample, source fouling
HVA, VMA, 5-HIAAAppropriate SST
LC-MS/MS Column Degradation RT3 SST at LLOQXIC of -MRM (8 pairs): 649.9/126.8 Da from Sample 3 (SST) of rT3 MD 070711.wiff (Turbo Spray) Max. 1.1e4 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
5000.00
5500.00
6000.00
6500.00
7000.00
7500.00
8000.00
8500.00
9000.00
9500.00
1.00e4
1.05e4
1.10e41.14e4 0.05
0.38
0.520.23
XIC of -MRM (8 pairs): 649.9/126.8 Da from Sample 9 (SST) of rT3 MD 070711.wiff (Turbo Spray) Max. 1.8e4 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.70Time, min
0.0
1000.0
2000.0
3000.0
4000.0
5000.0
6000.0
7000.0
8000.0
9000.0
1.0e4
1.1e4
1.2e4
1.3e4
1.4e4
1.5e4
1.6e4
1.7e4
1.8e40.42
0.03
0.73
0.21
SST at LLOQ - Acceptable Reduction in sensitivity and resolution of T3 from reverse T3
T3 rT3
Internal QA – Pre-BatchChecking Instrument Sensitivity with SST at LLOQ
Date Unextract A D2
Unextract A D3 Std A D2 IS A D2 Std A D3 IS A D3
1/2/2008 300 12495 435 157441/4/2008 2015 2467 258 12828 374 161641/6/2008 292 13820 424 174131/8/2008 305 12584 442 158561/9/2008 284 13391 412 16873
1/11/2008 2410 2943 307 12664 445 159571/13/2008 296 13804 430 173931/15/2008 289 12706 419 160101/16/2008 245 13454 355 169521/18/2008 1913 2312 234 10090 339 127131/20/2008 198 9034 287 113831/22/2008 1208 1315 130 7023 189 88491/23/2008 98 5012 142 6315
Batch Data Review
Formula #1 = Tr analyte / Tr internal standard, as a percentage
Internal QA – LC-MS/MS Batch/Assay Review
IS ContaminationXIC of +MRM (4 pairs): 271.2/213.1 Da from Sample 1 (Double Blank) of 186065.wiff (Heated Nebulizer) Max. 1320.0 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35Time, min
0
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
Inte
nsity
, cps
0.290.24
0.310.21
0.150.22
0.230.180.19
0.130.09 0.27
0.140.04
0.04
0.030.02
DHEA Double Blank DHEA LLOQ 20ng/dL
XIC of +MRM (4 pairs): 271.2/213.1 Da from Sample 3 (Std 1 20 ng/dL) of 186065.wiff (Heated Nebulizer) Max. 2780.0 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2780
Inte
nsity
, cps
0.19
0.00 0.390.25 0.310.290.070.330.08 0.270.050.02 0.12
0.04
DHEA Blank with IS
XIC of +MRM (4 pairs): 271.2/213.1 Da from Sample 13 (Blank w/IS) of 186065.wiff (Heated Nebulizer) Max. 3420.0 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35Time, min
0
200
400
600
800
1000
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
Inte
nsity
, cps
0.19
0.00
0.290.23 0.250.28
0.370.03
0.100.08 0.130.120.04 0.060.05
Sample recovery/injectedIS Peak Area vs Index (004102 DHEA)- "Mean" Regression ("No" weighting): mean = 8.11e+004 x (std. dev. = 1.44e+004)
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95Index
0.00
5000.00
1.00e4
1.50e4
2.00e4
2.50e4
3.00e4
3.50e4
4.00e4
4.50e4
5.00e4
5.50e4
6.00e4
6.50e4
7.00e4
7.50e4
8.00e4
8.50e4
9.00e4
9.50e4
1.00e5
192005.rdb (004706 Androstenedione): "Linear" Regression ("1 / x" weighting): y = 0.00419 x + 0.0502 (r = 0.9995)
0 50 100 150 200 250 300 350 400 450 500Analyte Conc. / IS Conc.
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
1.4
1.5
1.6
1.7
1.8
1.9
2.0
2.1
2.2
Anal
yte
Area
/ IS
Are
a
Androstenedione Cal 5 Expected = 250
Measured = 299.322 Accuracy (%) = 119.729
IS Peak Area vs Index (Phenylalanine)- "Mean" Regression ("No" weighting): mean = 8.33e+005 x (std. dev. = 2.9e+004)
50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800Index
0.00
5.00e4
1.00e5
1.50e5
2.00e5
2.50e5
3.00e5
3.50e5
4.00e5
4.50e5
5.00e5
5.50e5
6.00e5
6.50e5
7.00e5
7.50e5
8.00e5
8.50e5
9.00e5
9.50e5
1.00e6
Phenylalanine D5 IS Plot, IS increase at 97th aqueous calibrator injection (intra-assay inaccuracy batch, n=20 at 8 levels)
MS System Sensitivity Drift - Internal Standard Response PlotIS Peak Area vs Index (Phenylalanine)- "Mean" Regression ("No" weighting): mean = 1.98e+006 x (std. dev. = 6.24e+005)
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480Index
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
2.9e6
Phenylalanine D5 IS Plot, real samples
Internal QA – LC-MS/MS Batch/Assay Review
XIC of +MRM (10 pairs): 132.1/86.1 Da from Sample 29 (Sample 7_ ) of Validation Batch 3RP2.wiff (Turbo Spray) Max. 4.4e6 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1Time, min
0.0
2.0e5
4.0e5
6.0e5
8.0e5
1.0e6
1.2e6
1.4e6
1.6e6
1.8e6
2.0e6
2.2e6
2.4e6
2.6e6
2.8e6
3.0e6
3.2e6
3.4e6
3.6e6
3.8e6
4.0e6
4.2e6
4.4e6
Inte
nsity
, cps
0.33
0.24
0.46
MSUD Patient
XIC of +MRM (10 pairs): 132.1/86.1 Da from Sample 30 (Sample 8_ ) of Validation Batch 3RP2.wiff (Turbo Spray) Max. 2.2e6 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
1.8e6
1.9e6
2.0e6
2.1e62.2e6
Inte
nsity
, cps
0.47
0.32
Normal Patient following MSUD
XIC of +MRM (10 pairs): 132.1/86.1 Da from Sample 30 (Sample 8_ ) of Validation Batch 3RP2.wiff (Turbo Spray), Smoothed, Smoothe... Max. 2.0e6 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1Time, min
0.0
2000.0
4000.0
6000.0
8000.0
1.0e4
1.2e4
1.4e4
1.6e4
1.8e4
2.0e4
2.2e4
2.4e4
2.6e4
2.8e4
3.0e4
3.2e4
3.4e4
3.6e4
3.8e4
4.0e4
4.2e4
4.4e4
4.6e4
4.8e4
Inte
nsity
, cps
Expanded normal view
MS System Source contamination within RunXIC of +MRM (10 pairs): 132.1/86.1 Da from Sample 3 (Standard 1_ ) of Validation Batch 3RP2.wiff (Turbo Spray), Smoothed, Smoothe... Max. 1.1e4 cps.
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1Time, min
0.00
500.00
1000.00
1500.00
2000.00
2500.00
3000.00
3500.00
4000.00
4500.00
5000.00
5500.00
6000.00
6500.00
7000.00
7500.00
8000.00
8500.00
9000.00
9500.00
1.00e4
1.05e4
1.10e41.13e4
Inte
nsity
, cps
0.32
0.240.47
0.390.540.02 0.42
LLOQ for allo-isoleucine (A,100nm/L)
Sample Analyte Conc RT Analyte Peak Area IS Response Calc
concIon
RatioRatio Flag?
Std D D3 200 2.84 13902 12597 1.10 201.1 1.4 NOStd C D3 100 2.82 5210 10349 0.50 99.8 1.3 NOStd B D3 30 2.88 2128 11640 0.18 33.3 1.4 NOStd A D3 1 2.87 249 10274 0.02 1.1 1.4 NOCtrl HI D3 2.88 808 13878 0.06 10.7 1.4 NOCtrl LO D3 2.88 3190 12186 0.26 48.4 1.4 NOM64598 D3 2.89 2809 8234 0.34 63.0 1.3 NOM67899 D3 2.86 1408 13192 0.11 19.7 1.5 NOM70133 D3 2.84 1504 13469 0.11 20.6 1.5 NOM70134 D3 2.82 1443 10960 0.13 24.3 1.2 NOM70135 D3 2.90 1703 12058 0.14 26.1 1.3 NOM71222 D3 2.80 1774 13004 0.14 25.2 1.4 NOM72090 D3 2.84 1932 11747 0.16 30.4 1.4 NOT80031 D3 2.83 2034 2312 0.88 162.6 1.4 NOT80100 D3 2.83 2178 13075 0.17 30.8 1.3 NOT81070 D3 2.89 4508 12678 0.36 65.7 1.2 NOT90909 D3 2.83 3488 11378 0.31 56.7 1.4 NOT91002 D3 2.80 2912 12790 0.23 42.1 1.4 NOT91114 D3 2.84 2600 12610 0.21 38.1 1.5 NO
Internal QA – LC-MS/MS Batch/Assay Review Matrix Effects in Individual samples – Signal:Noise
Selectivity Variance - Matrix EffectsInternal QA – LC-MS/MS Batch/Assay Review
XIC of +MRM (4 pairs): 183.1/151.1 Da from Sample 16 (Calibrator 1_10pg/mL_6__) of PMETS Validation Batch 2_04282011.wiff (Turb... Max. 4.3e5 cps.
0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
3.0e5
3.2e5
3.4e5
3.6e5
3.8e5
4.0e5
4.2e54.3e5
Inte
nsity
, cps
0.15
D3-Metanephrine IS at 4.3 e5 cps
XIC of +MRM (4 pairs): 183.1/151.1 Da from Sample 20 (LC_4__) of PMETS Validation Batch 2_04282011.wiff (Turbo Spray), Smoothe... Max. 1.7e5 cps.
0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e51.7e5
Inte
nsity
, cps
0.15
D3-Metanephrine IS at 1.7 e5 cps 40% of earlier sample
Partial Suppression from co-elutor
Selectivity Variance - Internal Standard Transition ratio plots
Internal QA – LC-MS/MS Batch/Assay Review Transition Ratio Monitoring –Exploring Outliers
-30.00
-20.00
-10.00
0.00
10.00
20.00
30.00
0 5 10 15 20 25 30 35 40
Calculated Concentration (ng/dL)
Tran
sist
ion
Rat
io B
ias
(%)
LLOQ sample Calculated Conc 1.9ng/dL
XIC of +MRM (4 pairs): 289.2/97.1 Da from Sample 27 (S1_5__2.5ng/dL_) of Validation Batch 1.wiff (Heated Nebulizer), Smoothed, S... Max. 1472.5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
0.19
0.01
0.12
0.350.11
XIC of +MRM (4 pairs): 289.2/97.1 Da from Sample 113 (Sample 96__) of SLE 081010.wiff (Heated Nebulizer), Smoothed, Smoothed Max. 2168.8 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35Time, min
0
100
200
300
400
500
600
700
800
900
1000
1100
1200
1300
1400
1500
1600
1700
1800
1900
2000
21002169 0.13
0.020.32
0.35
Concentration 5.3ng/dL
Sample Analyte Conc RT Analyte Peak Area IS Response Calc
concIon
RatioRatio Flag?
Std D D3 200 2.81 12870 12370 1.04 198.9 1.5 NOStd C D3 100 2.82 5990 12335 0.49 100.1 1.5 NOStd B D3 30 2.88 2288 13237 0.17 32.9 1.5 NOStd A D3 1 2.88 252 13704 0.02 0.9 1.4 NOCtrl HI D3 2.90 780 13109 0.06 11.6 1.4 NOCtrl LO D3 2.86 3501 13932 0.25 48.7 1.5 NOW54634 D3 2.83 1650 13081 0.13 24.5 1.5 NOW58132 D3 2.86 1249 12703 0.10 19.1 1.5 NOW66023 D3 2.89 1641 13211 0.12 24.1 1.5 NOW62743 D3 2.81 2423 12636 0.19 37.2 1.5 NOW66817 D3 2.86 2208 13543 0.16 31.6 1.5 NOH66438 D3 2.83 2359 12163 0.19 37.6 1.4 NOH62633 D3 2.81 2057 13559 0.15 29.4 1.5 NOH78948 D3 2.82 1593 12762 0.12 24.2 1.4 NOH78388 D3 2.82 2836 12824 0.22 42.9 1.4 NOH74054 D3 2.87 1257 12469 0.10 19.6 1.5 NOH87727 D3 2.67 1362 12944 0.11 20.4 1.9 YESH89927 D3 2.85 1851 12528 0.15 28.7 1.4 NOH88721 D3 2.89 2731 13284 0.21 39.9 1.4 NO
Internal QA – LC-MS/MS Batch/Assay Review Transition Ratio Monitoring –Exploring Outliers
External QC 3-Plex Linked Analytes Plasma MetanephrinesThe importance of Automation
IS Peak Area vs Index (Normetanephrine)- "Mean" Regression ("No" weighting): mean = 1.15e+005 x (std. dev. = 5.54e+004)
10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190Index
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
9.0e4
1.0e5
1.1e5
1.2e5
1.3e5
1.4e5
1.5e5
1.6e5
1.7e5
1.8e5
1.9e5
2.0e5
2.1e5
2.2e5
Manual Preparation First Run with SPE Automated First Run With SPEIS Peak Area vs Index (Normetanephrine)- "Mean" Regression ("No" weighting): mean = 6.16e+005 x (std. dev. = 1.62e+005)
5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95Index
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e5
Recovery Variance ADDS to Imprecision which makes Westgard Rules Impossible to applyUse Automation OR Train Technicians VERY well
External QC - Calibrator Stability
Cocaine ProfileULOQ (1000 ng/mL)
0.05 0.15 0.25 0.35 0.45 Time, min0.0
1.0e6
2.0e6
3.0e6
4.0e6
5.0e6
Intensity, cps
B
EC
Cocaine Profile LOQ (1 ng/mL)
0.05 0.15 0.25 0.35 Time, min0.0
2000
4000
6000
8000
1.0e4
Intensity, cps
0.45
B
E
C
XIC of +MRM (12 pairs): 304.2/82.0 Da from Sample 33 (Plasma Low QC_1) of Cocaine Validation Batch 2.wiff (Turbo Spray), Smoothe... Max. 2.2e4 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e48.2e4
0.15
XIC of +MRM (12 pairs): 200.1/182.1 Da from Sample 34 (Urine QC_1) of Cocaine Validation Batch 2.wiff (Turbo Spray), Smoothed, Sm... Max. 7.7e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e57.7e5 0.47
XIC of +MRM (12 pairs): 304.2/82.0 Da from Sample 35 (Wh Blood QC_1) of Cocaine Validation Batch 2.wiff (Turbo Spray), Smoothed,... Max. 4.3e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e55.7e5
0.16
XIC of +MRM (12 pairs): 200.1/182.1 Da from Sample 36 (Organic QC_1) of Cocaine Validation Batch 2.wiff (Turbo Spray), Smoothed, ... Max. 8.6e5 cps.
0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
7.0e5
7.5e5
8.0e5
8.5e50.47
Cocaine Profile Plasma QC1 (5 ng/mL)
Cocaine Profile Urine QC
(100-110 ng/mL)
Cocaine Profile Organic QC
(105-110 ng/mL)
Cocaine Profile Blood QC
(90-105 ng/mL)
Cocaine Stability
0.000
1.000
2.000
3.000
4.000
5.000
6.000
0 5 10 15
Days
Con
cent
ratio
n (n
g/m
L)
Plasma QC
Plasma QC stability at 2-8C inc 1% NaFNeed Alternate Matrix to assess Calibrator
Stability
Column Verification
CAP Checklist, Gas Chromatography and High Performance Liquid Chromatography Section
CHM.16850 Column Verification Phase IINew columns are verified for performance before use.
Evidence of Compliance:✓ Written procedure for column verification AND✓ Records of column verification
“The 3 R’s”: Verifiable Column Performance Characteristics
Retention – Are all analytes properly adsorbed to the new stationary phase
Resolution – Are all analytes exhibiting appropriate desorption from the new stationary phase
Response – Are any components of the new column affecting MS ionization efficiency
Verification Procedure:
1. System Suitability Test Solutions (At concentrations slightly higher than the assay’s extracted LLOQ)
2. Replicate Injections (More than 3)
3. DO NOT USE THE FIRST INJECTION (or the second, and possibly the third, dependent on the particular column passivation required). Set the appropriate limits.
4. Have confidence in a working system prior to column verification run
Critical Data Features Direct Observations
Sample NameAnalyte Peak Name
Analyte Peak Area (counts) ResponseAnalyte Retention Time (min) Retention
Analyte Exp. RT (min)Analyte Peak Width (min)
Analyte Peak Asym. Peak ShapeIS Peak Name
IS Peak Area (counts) ResponseIS Retention Time (min) Retention
IS Exp. RT (min)IS Peak Asym. Peak Shape
Calculated Observations Criteria
Response Predefined from Working System/Validation
RetentionPredefined Tr of Analyte and
Internal Standard in Validation
ResolutionSeparation of Critical
Pairs/Isobars (i.e. Tr1+0.5Pw1>Tr2-0.5Pw2)
XIC of +MRM (111 pairs): 314.100/210.100 amu Expected RT: 2.9 ID: Alprazolam-d5.IS from Sample 5 (SST 1) of SST 05312013.wiff (T... Max. 1.8e4 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
2.0e4
4.0e4
6.0e4
8.0e4
1.0e5
1.2e5
1.4e5
1.6e5
1.8e5
2.0e5
2.2e5
2.4e5
2.6e5
2.8e5
Inte
nsity
, cps
2.97
Multi-analyte panel, API 3200 (Scheduled MRM) Col Verification, SST Injection #3
Column Verification
Test Name:Example Panel
Analyte Analyte Retention Time
IS Retention Time
Retention Time Alignment
Critical Pair Separation Analyte Asymmetry Sensitivity
6-MAM Pass Pass Fail NA Fail Fail7-Aminoclonazepam Pass Pass Pass NA Pass PassAlprazolam Pass Pass Pass NA Pass PassAmphetamine Pass Fail Pass NA Fail PassBenzoylecgonine Pass Pass Pass NA Fail PassBuprenorphine Pass Pass Pass NA Pass PassCarisoprodol Pass Pass Pass NA Pass PassClonazepam Fail Fail Pass NA Pass PassCodeine Pass Pass Fail Pass Pass PassDiazepam Fail Fail Pass NA Pass PassEDDP Pass Pass Pass NA Pass PassFentanyl Pass Pass Pass NA Pass PassGabapentin Pass Pass Fail NA Fail PassHydrocodone Pass Pass Pass Pass Pass PassHydromorphone Pass Pass Fail Fail Fail FailHydroxyalprazolam Pass Pass Pass NA Pass PassLorazepam Pass Pass Pass NA Pass PassMDMA Pass Pass Fail NA Fail PassMeperidine Pass Pass Pass NA Fail PassMeprobamate Pass Pass Pass NA Pass PassMethadone Pass Pass Pass NA Fail PassMethamphetamine Pass Pass Fail NA Fail PassMorphine Pass Pass Pass Fail Fail PassNaltrexone Pass Pass Pass NA Pass PassNorbuprenorphine Pass Pass Pass NA Pass PassNordiazepam Pass Pass Pass NA Pass PassNorfentanyl Pass Pass Pass NA Pass PassNorhydrocodone Fail Fail Fail NA Pass PassNoroxycodone Pass Pass Fail NA Pass PassOxycodone Pass Pass Pass NA Fail PassOxymorphone Pass Pass Pass NA Fail PassPCP Pass Pass Pass NA Pass PassPregabalin Fail Fail Pass NA Fail FailTapentadol Pass Pass Pass NA Pass PassTemazepam Pass Pass Pass NA Pass PassTramadol Pass Pass Pass NA Pass PassZolpidem Fail Fail Pass NA Pass PassNumber of Failures 5 6 8 2 13 3
Column Serial Number 111-111-1111-FVM Date Installed 2/7/1964
Column Manufacturer Flintstones Vit-Min
Initials JPGR
Stationary Phase PolyPro-Dibenzophenol Taffy
Dimensions (L x W, Dp) 11 x 1.753 mm, 6.2 um
Verification Data
XIC of +MRM (111 pairs): 314.100/210.100 amu Expected RT: 2.9 ID: Alprazolam-d5.IS from Sample 10 (SST 1) of SST 05312013.wiff ... Max. 7435.6 cps.
0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 2.6 2.8 3.0 3.2 3.4Time, min
0.0
5000.0
1.0e4
1.5e4
2.0e4
2.5e4
3.0e4
3.5e4
4.0e4
4.5e4
5.0e4
5.5e4
6.0e4
6.5e4
7.0e4
7.5e4
8.0e4
8.5e4
9.0e4
9.5e4
1.0e5
1.1e5
1.1e5
1.2e5
1.2e5
1.3e5
1.3e5
Inte
nsity
, cps
Multi-analyte panel, API 3200 (Scheduled MRM) Col Verification, SST Injection #3 Post-
Guard Column Replacement
Column Verification
Test Name: Example Panel
Analyte Analyte Retention Time
IS Retention Time
Retention Time Alignment
Critical Pair Separation
Analyte Asymmetry Sensitivity
6-MAM Pass Pass Pass NA Pass Pass7-Aminoclonazepam Pass Pass Pass NA Pass PassAlprazolam Pass Pass Pass NA Pass PassAmphetamine Pass Pass Pass NA Pass PassBenzoylecgonine Pass Pass Pass NA Pass PassBuprenorphine Pass Pass Pass NA Pass PassCarisoprodol Pass Pass Pass NA Pass PassClonazepam Pass Pass Pass NA Pass PassCodeine Pass Pass Pass Pass Pass PassDiazepam Pass Pass Pass NA Pass PassEDDP Pass Pass Pass NA Pass PassFentanyl Pass Pass Pass NA Pass PassGabapentin Pass Pass Pass NA Pass PassHydrocodone Pass Pass Pass Pass Pass PassHydromorphone Pass Pass Pass Pass Pass PassHydroxyalprazolam Pass Pass Pass NA Pass PassLorazepam Pass Pass Pass NA Pass PassMDMA Pass Pass Pass NA Pass PassMeperidine Pass Pass Pass NA Pass PassMeprobamate Pass Pass Pass NA Pass PassMethadone Pass Pass Pass NA Pass PassMethamphetamine Pass Pass Pass NA Pass PassMorphine Pass Pass Pass Pass Pass PassNaltrexone Pass Pass Pass NA Pass PassNorbuprenorphine Pass Pass Pass NA Pass PassNordiazepam Pass Pass Pass NA Pass PassNorfentanyl Pass Pass Pass NA Pass PassNorhydrocodone Pass Pass Pass NA Pass PassNoroxycodone Pass Pass Pass NA Pass PassOxycodone Pass Pass Pass NA Pass PassOxymorphone Pass Pass Pass NA Pass PassPCP Pass Pass Pass NA Pass PassPregabalin Pass Pass Pass NA Pass PassTapentadol Pass Pass Pass NA Pass PassTemazepam Pass Pass Pass NA Pass PassTramadol Pass Pass Pass NA Pass PassZolpidem Pass Pass Pass NA Pass PassNumber of Failures 0 0 0 0 0 0
Column Serial Number 111-111-1111-FVM Date Installed 2/7/1964
Column Manufacturer Flintstones Vit-MinInitials JPGR
Stationary Phase PolyPro-Dibenzophenol Taffy
Dimensions (L x W, Dp) 11 x 1.753 mm, 6.2 um
Verification Data, New Guard Column
XIC of +MRM (59 pairs): 110.100/93.100 Da ID: GABA D6 from Sample 18 (Pickering Neat) of RP1_09132013.wiff (Turbo Spray), Smoo... Max. 4.1e6 cps.
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
6.0e5
6.5e5
Inte
nsity
, cps
0.67*
Neat Pickering QC No IS added
Methionine D3Cystathionine D8
GABA D6
XIC of +MRM (59 pairs): 153.100/107.100 Da ID: Methionine D3 from Sample 35 (Pickering x10) of RP1_09132013.wiff (Turbo Spray), ... Max. 8.7e5 cps.
0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7Time, min
0.0
1.0e5
2.0e5
3.0e5
4.0e5
5.0e5
6.0e5
7.0e5
8.0e5
9.0e5
1.0e6
1.1e6
1.2e6
1.3e6
1.4e6
1.5e6
1.6e6
1.7e6
1.8e6
1.9e6
2.0e6
2.1e6
2.2e6
2.3e6
2.4e6
Inte
nsity
, cps
1.21
0.53
10 x Diluted Pickering QC + IS
*
*
*
When Accuracy based QC isn’t accurate
External Accuracy based mixture (>50 analytes) in Phenol/water
Supra-physiological ~250uM
Measure Neat (no IS),
Neat diluted (class A)
Determine how to deploy “accuracy” based materials that
do not represent the clinically observed concentrations of
mixtures
Inter-Laboratory Correlation LabCorp and St Pauls, BC
Concentrations < 45ng/dL
N= 52
Intercept: -0.096
CI Intercept: [-0.557,0.488]
Slope: 0.985
CI Slope: [0.954,1.014]
R-squared: 0.990513
Mean Difference: -2.49%
SD Difference: 5.304%
Acknowledgement: Dr Daniel Holmes,
Grace Van Der Gugten
If [examined value] exceeds [a threshold], then flag with [text string].
Automated Data review
S L I D E 28
S L I D E 28
Acknowledgements
Patricia Holland, Matt Crawford, Stacy Dee, Mary Morr, Dr Marcia Eisenberg, Dr Walt Chandler
Brian Rappold
Randy Julian
Dr Andy Hoofnagle