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