Beyond Westgard Rules: Quality Control for Mass Spectrometry Dr Russell Grant VP R&D Laboratory Corporation of America Holdings.
Assay Validation Criteria and Acceptance Validation Process Accuracy (Intra and Interassay) Precision (Intra and Interassay) Standard Curve Fit Drug Selectivity Materials Selectivity Matrix Interference Materials Requirements and Acceptance Criteria 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 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 6-10 levels run in each assay, evaluated over 5 assays (days), R>0.995, Slope CV<5% 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. Blank matrix test 6 or more lots Response<20% LLOQ in 5 of 6. Effect of lipid, Icteria and Hemolysis. Bias % <15%/20% @LLOQ Test anticoagulants Bias % <15%/20% @LLOQ Alternate sample types Deming regression 0.9-1.1, R >0.9 for same reference interval. Matrix effects (sample mixing) Bias<15%
Validation Process Assay Validation Criteria and Acceptance 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 Inter-assay Comparison Reference Interval All conditions tested from arm to final result Mean Bias < 15%/20%@ LLOQ, %CV < 15%/20%@ LLOQ, Deming regression 0.9-1.1, R >0.9 Min 20 External Disparate assays For information only Internal assays same cals Deming regression 0.9-1.1, R >0.9 Same assay different system Deming regression 0.95-1.05, R >0.95 Same assay Manual/Automation Deming regression 0.95-1.05, R >0.95 120 in each reference interval 20 (95% CI) for range transfer 1/20 outside existing range Transition ratio monitoring Bias from extracted calibrators in each batch <20% up to 3x LLOQ, < 15% after that for ratio>0.5 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 2 nd plate) Deming regression 0.9-1.1, R >0.9
Assay Translation (R&D Ops) (Assay Viability QC Ranges Generation n=25 over 5 runs) Verification Process Materials Selectivity Standard Linearity Carry-over Precision (Intra and Interassay) Materials Requirements and Acceptance Criteria Blank matrix <20% LLOQ in 5 runs. All Cals used, Accuracy % 85% to 115%, 80 120% @LLOQ and R>0.995, Slope CV<5% After ULOQ 3 runs including <20% LLOQ Stress test (10-100x ULOQ), how many for <20% LLOQ 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) (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 Power Failure Leak detection Pressure beyond pumping Air bubble Column Degradation Complete Sample volume injected Injector blockage Contamination Internal QC Action Shutdown (without UPS) Shutdown* Shutdown Shutdown or No monitoring No monitoring No monitoring No monitoring* No monitoring MS System Power Failure Gas failure Vacuum failure Temperature effects Contamination Resolution change Sensitivity drift Selectivity Internal QC Action Shutdown (without UPS) Shutdown Shutdown Shutdown in extreme conditions No monitoring No monitoring No monitoring No monitoring
System Suitability Test (SST) The Doctor Urine Cortisol SST 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 LLOQ Acquired correctly? Retention time 5% variance Sensitivity S:N >20:1 Peak shape As 0.8-1.2 Multi-channel acceptance column-column/channelchannel/system-system Urine Cortisol LLOQ SST is key part of troubleshooting experiments Ignore the first (or second) injection
HPLC Pressure Trace The Heartbeat Goal Pre-batch and per-injection check of appropriate LC performance Design: SOP Example Annotate an acceptable pressure trace Inject/Focus Detect Gradient Wash Recondition Pressure profile Start and overall maximum pressure Changes in pressure profile Air bubbles in A or B Dead volume differences Bubble in A Column dimensions change Full injection Injector/filter/column/lines blockage Post column blockage (bypass/source probe) Bubble in B
Internal QA Pre-Batch LC Solvent Contamination XIC of +MRM (6 pairs): 184.2/107.1 Da from Sample 2 (Double Blank) of Validation Batch 2.wiff (Turbo Spray) Max. 550 cps. XIC of +MRM (6 pairs): 184.2/107.1 Da from Sample 4 (Standard 1) of Validation Batch 2.wiff (Turbo Spray) Max. 645 cps. XIC of +MRM (6 pairs): 184.2/107.1 Da from Sample 1 (Blank) of SST 082908.wiff (Turbo Spray) Max. 1.5e4 cps. 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. 5500 5000 4500 0.18 0.25 0.29 6450 6000 5500 5000 1.5e4 1.4e4 1.3e4 1.2e4 9 0.26 0.30 9347 9000 8500 8000 7500 C 0.26 E 4000 3500 4500 4000 1.1e4 1.0e4 900 7000 6500 6000 5500 3000 2500 2000 0.50 3500 3000 2500 2000 800 700 600 500 0.61 5000 4500 4000 3500 3000 1500 1000 0.40 0.46 0.48 0.51 0.76 1500 1000 400 300 200 0.40 0.42 0.54 0.58 0.44 0.49 2500 2000 1500 500 500 100 1000 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 Blank, Clean Epinephrine LLOQ Contaminant Resolved Air Bubble/Column or MP Degradation/Injector blockage 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. XIC 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. 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. 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. 1.5e4 3.8e6 1.05e6 1.10e6 1.4e4 1.3e4 1.2e4 3.6e6 3.4e6 3.2e6 3.0e6 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 1.05e6 1.00e6 9.50e5 9.00e5 8.50e5 1.1e4 2.8e6 7.50e5 8.00e5 1.0e4 900 800 700 600 500 400 300 200 100 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5 0.51 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 0.66 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 0.55 5 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 0.75 0 5 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 0.75 0 5 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 0.75 5 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 0.75 Air Bubble SST SST Day 1 SST Day 4 SST Day 4 post LC flush/new MPB
Internal QA Pre and During Batch MS System Source contamination Pre-run XIC of -MRM (8 pairs): 197.0/137.0 Da from Sample 1 (sst-3) of sst_072111_1.wiff (Turbo Spray) XIC of -MRM (8 pairs): 20/137.0 Da from Sample 1 (sst-1) of sst_072011a1_1.wiff (Turbo Spray) Max. 450 cps. Max. 610 cps. 1.7e5 1.6e5 1.8e6 1.7e6 1.5e5 1.6e6 1.4e5 1.5e6 1.3e5 1.4e6 1.2e5 1.3e6 1.2e6 1.1e5 1.1e6 1.0e5 9.0e4 8.0e4 7.0e4 1.0e6 9.0e5 8.0e5 7.0e5 6.0e4 6.0e5 5.0e4 5.0e5 4.0e4 4.0e5 3.0e4 3.0e5 2.0e4 2.0e5 1.0e5 1.0e4 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 0.24 0.30 0.37 0.40 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 0.65 HVA, VMA, 5-HIAA 1 st Sample, source fouling HVA, VMA, 5-HIAA Appropriate SST LC-MS/MS Column Degradation RT3 SST at LLOQ XIC of -MRM (8 pairs): 649.9/126.8 Da from Sample 3 (SST) of rt3 MD 070711.wiff (Turbo Spray) 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. 5 1.14e4 0.42 1.8e4 1.10e4 1.05e4 1.7e4 1.00e4 1.6e4 9500 1.5e4 9000 1.4e4 8500 T3 rt3 0.38 Max. 1.1e4 cps. 1.3e4 1.2e4 1.1e4 1.0e4 900 800 700 600 3 8000 7500 7000 6500 6000 5500 5000 4500 4000 3500 500 400 300 200 0.73 0.21 100 5 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 SST at LLOQ - Acceptable 3000 2500 2000 1500 1000 500 0 0.23 5 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 Reduction in sensitivity and resolution of T3 from reverse T3 0.52
Internal QA Pre-Batch Checking 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 15744 1/4/2008 2015 2467 258 12828 374 16164 1/6/2008 292 13820 424 17413 1/8/2008 305 12584 442 15856 1/9/2008 284 13391 412 16873 1/11/2008 2410 2943 307 12664 445 15957 1/13/2008 296 13804 430 17393 1/15/2008 289 12706 419 16010 1/16/2008 245 13454 355 16952 1/18/2008 1913 2312 234 10090 339 12713 1/20/2008 198 9034 287 11383 1/22/2008 1208 1315 130 7023 189 8849 1/23/2008 98 5012 142 6315
Batch Data Review Formula #1 = Tr analyte / Tr internal standard, as a percentage
IS Contamination Internal QA LC-MS/MS Batch/Assay Review XIC of +MRM (4 pairs): 271.2/213.1 Da from Sample 1 (Double Blank) of 186065.wiff (Heated Nebulizer) Max. 132 cps. XIC of +MRM (4 pairs): 271.2/213.1 Da from Sample 13 (Blank w/is) of 186065.wiff (Heated Nebulizer) Max. 342 cps. XIC of +MRM (4 pairs): 271.2/213.1 Da from Sample 3 (Std 1 20 ng/dl) of 186065.wiff (Heated Nebulizer) Max. 278 cps. 1300 1250 1200 0.21 0.24 0.29 0.31 3400 3200 0.19 2780 2600 0.19 1150 0.15 3000 2400 1100 1050 0.22 0.18 0.23 0.19 0.13 9 0.27 2800 2200 1000 950 900 4 4 0.14 2600 2400 0 2000 850 800 750 700 650 600 550 500 450 3 2 2200 2000 1800 1600 1400 1200 1800 1600 1400 1200 1000 400 350 300 250 200 1000 800 600 3 4 5 6 8 0.10 0.12 0.13 0.23 0.25 0.28 0.29 0.37 800 0 0.25 0.39 0.29 0.31 600 7 2 5 8 0.27 0.33 0.12 4 400 150 400 100 50 200 200 0 5 0.10 0.15 0.20 0.25 0.30 0.35 DHEA Double Blank 0 5 0.10 0.15 0.20 0.25 0.30 0.35 DHEA Blank with IS 0 5 0.10 0.15 0.20 0.25 0.30 0.35 DHEA LLOQ 20ng/dL Sample recovery/injected IS Peak Area vs Index (004102 DHEA)- "Mean" Regression ("No" weighting): mean = 8.11e+004 x (std. dev. = 1.44e+004) 192005.rdb (004706 Androstenedione): "Linear" Regression ("1 / x" weighting): y = 0419 x + 502 (r = 0.9995) 1.00e5 9.50e4 2.2 2.1 9.00e4 2.0 8.50e4 1.9 8.00e4 7.50e4 7.00e4 6.50e4 6.00e4 5.50e4 5.00e4 4.50e4 4.00e4 3.50e4 3.00e4 2.50e4 Analyte Area / IS Area 1.8 1.7 1.6 1.5 1.4 1.3 1.2 1.1 1.0 0.9 0.8 0.7 0.6 0.5 Androstenedione Cal 5 Expected = 250 Measured = 299.322 Accuracy (%) = 119.729 2.00e4 0.4 1.50e4 0.3 1.00e4 0.2 5000 0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 Index 0.1 0 50 100 150 200 250 300 350 400 450 500 Analyte Conc. / IS Conc.
IS Peak Area vs Index (Phenylalanine)- "Mean" Regression ("No" weighting): mean = 8.33e+005 x (std. dev. = 2.9e+004) Internal QA LC-MS/MS Batch/Assay Review MS System Sensitivity Drift - Internal Standard Response Plot IS Peak Area vs Index (Phenylalanine)- "Mean" Regression ("No" weighting): mean = 1.98e+006 x (std. dev. = 6.24e+005) 1.00e6 9.50e5 9.00e5 8.50e5 8.00e5 7.50e5 7.00e5 6.50e5 6.00e5 5.50e5 5.00e5 4.50e5 4.00e5 3.50e5 3.00e5 2.50e5 2.00e5 1.50e5 1.00e5 5.00e4 0 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 Index Phenylalanine D5 IS Plot, IS increase at 97 th aqueous calibrator injection (intra-assay inaccuracy batch, n=20 at 8 levels) 2.9e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5 0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 Index Phenylalanine D5 IS Plot, real samples MS System Source contamination within Run XIC 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. 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. 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. 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. 0.32 1.13e4 1.10e4 0.24 1.05e4 0.47 1.00e4 9500 9000 8500 8000 7500 7000 6500 0.39 6000 2 0.42 0.54 5500 5000 4500 4000 3500 3000 2500 2000 1500 1000 500 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 4.4e6 4.2e6 4.0e6 3.8e6 3.6e6 3.4e6 3.2e6 3.0e6 2.8e6 2.6e6 2.4e6 2.2e6 2.0e6 1.8e6 1.6e6 1.4e6 1.2e6 1.0e6 8.0e5 6.0e5 4.0e5 2.0e5 0.33 0.24 0.46 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 2.2e6 2.1e6 2.0e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 1.0e5 0.47 0.32 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 4.8e4 4.6e4 4.4e4 4.2e4 4.0e4 3.8e4 3.6e4 3.4e4 3.2e4 3.0e4 2.8e4 2.6e4 2.4e4 2.2e4 2.0e4 1.8e4 1.6e4 1.4e4 1.2e4 1.0e4 800 600 400 200 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 LLOQ for allo-isoleucine (A,100nm/L) MSUD Patient Normal Patient following MSUD Expanded normal view
Internal QA LC-MS/MS Batch/Assay Review Matrix Effects in Individual samples Signal:Noise Sample Analyte Conc RT Analyte Peak Calc Ion Ratio IS Response Area conc Ratio Flag? Std D D3 200 2.84 13902 12597 1.10 201.1 1.4 NO Std C D3 100 2.82 5210 10349 0.50 99.8 1.3 NO Std B D3 30 2.88 2128 11640 0.18 33.3 1.4 NO Std A D3 1 2.87 249 10274 2 1.1 1.4 NO Ctrl HI D3 2.88 808 13878 6 10.7 1.4 NO Ctrl LO D3 2.88 3190 12186 0.26 48.4 1.4 NO M64598 D3 2.89 2809 8234 0.34 63.0 1.3 NO M67899 D3 2.86 1408 13192 0.11 19.7 1.5 NO M70133 D3 2.84 1504 13469 0.11 20.6 1.5 NO M70134 D3 2.82 1443 10960 0.13 24.3 1.2 NO M70135 D3 2.90 1703 12058 0.14 26.1 1.3 NO M71222 D3 2.80 1774 13004 0.14 25.2 1.4 NO M72090 D3 2.84 1932 11747 0.16 30.4 1.4 NO T80031 D3 2.83 2034 2312 0.88 162.6 1.4 NO T80100 D3 2.83 2178 13075 0.17 30.8 1.3 NO T81070 D3 2.89 4508 12678 0.36 65.7 1.2 NO T90909 D3 2.83 3488 11378 0.31 56.7 1.4 NO T91002 D3 2.80 2912 12790 0.23 42.1 1.4 NO T91114 D3 2.84 2600 12610 0.21 38.1 1.5 NO
Internal QA LC-MS/MS Batch/Assay Review Selectivity Variance - Matrix Effects 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. 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. 4.3e5 4.2e5 0.15 1.7e5 1.7e5 0.15 4.0e5 3.8e5 3.6e5 1.6e5 1.5e5 3.4e5 3.2e5 3.0e5 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 Partial Suppression from co-elutor 1.4e5 6.0e4 1.2e5 5.0e4 1.0e5 4.0e4 8.0e4 6.0e4 3.0e4 4.0e4 2.0e4 2.0e4 1.0e4 2 4 6 8 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 2 4 6 8 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 D3-Metanephrine IS at 4.3 e5 cps D3-Metanephrine IS at 1.7 e5 cps 40% of earlier sample Selectivity Variance - Internal Standard Transition ratio plots
30 Internal QA LC-MS/MS Batch/Assay Review Transition Ratio Monitoring Exploring Outliers 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. 1600 1500 0.19 1400 1300 1200 20 1100 1000 900 800 700 600 1 LLOQ sample Calculated Conc 1.9ng/dL 500 400 300 0.11 0.12 0.35 Transistion Ratio Bias (%) 10 0-10 200 100 0 5 0.10 0.15 0.20 0.25 0.30 0.35 0 5 10 15 20 25 30 35 40 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. 2169 2100 0.13-20 2000 1900 1800 1700 1600 1500 1400-30 1300 1200 1100 1000 900 800 700 600 500 400 300 2 0.32 0.35 Calculated Concentration (ng/dl) Concentration 5.3ng/dL 200 100 0 5 0.10 0.15 0.20 0.25 0.30 0.35
Internal QA LC-MS/MS Batch/Assay Review Transition Ratio Monitoring Exploring Outliers Sample Analyte Conc RT Analyte Peak Calc Ion Ratio IS Response Area conc Ratio Flag? Std D D3 200 2.81 12870 12370 1.04 198.9 1.5 NO Std C D3 100 2.82 5990 12335 0.49 100.1 1.5 NO Std B D3 30 2.88 2288 13237 0.17 32.9 1.5 NO Std A D3 1 2.88 252 13704 2 0.9 1.4 NO Ctrl HI D3 2.90 780 13109 6 11.6 1.4 NO Ctrl LO D3 2.86 3501 13932 0.25 48.7 1.5 NO W54634 D3 2.83 1650 13081 0.13 24.5 1.5 NO W58132 D3 2.86 1249 12703 0.10 19.1 1.5 NO W66023 D3 2.89 1641 13211 0.12 24.1 1.5 NO W62743 D3 2.81 2423 12636 0.19 37.2 1.5 NO W66817 D3 2.86 2208 13543 0.16 31.6 1.5 NO H66438 D3 2.83 2359 12163 0.19 37.6 1.4 NO H62633 D3 2.81 2057 13559 0.15 29.4 1.5 NO H78948 D3 2.82 1593 12762 0.12 24.2 1.4 NO H78388 D3 2.82 2836 12824 0.22 42.9 1.4 NO H74054 D3 2.87 1257 12469 0.10 19.6 1.5 NO H87727 D3 2.67 1362 12944 0.11 20.4 1.9 YES H89927 D3 2.85 1851 12528 0.15 28.7 1.4 NO H88721 D3 2.89 2731 13284 0.21 39.9 1.4 NO
External QC 3-Plex Linked Analytes Plasma Metanephrines The importance of Automation Manual Preparation First Run with SPE Automated First Run With SPE IS Peak Area vs Index (Normetanephrine)- "Mean" Regression ("No" weighting): mean = 1.15e+005 x (std. dev. = 5.54e+004) IS Peak Area vs Index (Normetanephrine)- "Mean" Regression ("No" weighting): mean = 6.16e+005 x (std. dev. = 1.62e+005) 2.2e5 2.1e5 2.0e5 1.9e5 1.8e5 1.7e5 1.6e5 1.5e5 1.4e5 1.3e5 1.2e5 1.1e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 8.5e5 8.0e5 7.5e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 1.5e5 1.0e5 5.0e4 10 20 30 40 50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 Index 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 Index Recovery Variance ADDS to Imprecision which makes Westgard Rules Impossible to apply Use Automation OR Train Technicians VERY well
External QC - Calibrator Stability Cocaine Stability 1.0e4 8000 6000 4000 C E B 5.0e6 4.0e6 3.0e6 2.0e6 C E B Concentration (ng/ml) 6.000 5.000 4.000 3.000 2.000 1.000 Plasma QC 2000 1.0e6 00 0 5 10 15 5 0.15 0.25 0.35 0.45 Cocaine Profile LOQ (1 ng/ml) 5 0.15 0.25 0.35 0.45 Cocaine Profile ULOQ (1000 ng/ml) Days Plasma QC stability at 2-8C inc 1% NaF Need Alternate Matrix to assess Calibrator Stability 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. 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. 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. 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. 8.2e4 8.0e4 7.7e5 7.5e5 0.47 8.5e5 0.47 5.7e5 5.5e5 7.5e4 7.0e4 7.0e5 6.5e5 8.0e5 7.5e5 5.0e5 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 7.0e5 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.5e5 4.0e5 3.5e5 3.0e5 0.16 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 0.15 3.5e5 3.0e5 2.5e5 2.0e5 4.0e5 3.5e5 3.0e5 2.5e5 2.0e5 2.5e5 2.0e5 1.5e5 1.5e4 1.5e5 1.5e5 1.0e5 1.0e4 500 1.0e5 5.0e4 1.0e5 5.0e4 5.0e4 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 5 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55 0.60 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)
Column Verification CAP Checklist, Gas Chromatography and High Performance Liquid Chromatography Section CHM.16850 Column Verification Phase II New 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 Sample Name Analyte Peak Name Analyte Peak Area (counts) Analyte Retention Time (min) Analyte Exp. RT (min) Analyte Peak Width (min) Analyte Peak Asym. IS Peak Name IS Peak Area (counts) IS Retention Time (min) IS Exp. RT (min) IS Peak Asym. Direct Observations Response Retention Peak Shape Response Retention Peak Shape Calculated Observations Response Retention Resolution Criteria Predefined from Working System/Validation Predefined T r of Analyte and Internal Standard in Validation Separation of Critical Pairs/Isobars (i.e. T r1 +0.5P w1 >T r2-0.5p w2 )
Verification Data Column Verification 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... 2.8e5 2.6e5 2.4e5 2.2e5 2.0e5 1.8e5 1.6e5 1.4e5 1.2e5 1.0e5 8.0e4 6.0e4 4.0e4 2.0e4 Multi-analyte panel, API 3200 (Scheduled MRM) Col Verification, SST Injection #3 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 3.4 2.97 Test Name: Analyte Example Panel Analyte Retention Time IS Retention Time Retention Time Alignment Critical Pair Separation Analyte Asymmetry Sensitivity 6-MAM Pass Pass Fail NA Fail Fail 7-Aminoclonazepam Pass Pass Pass NA Pass Pass Alprazolam Pass Pass Pass NA Pass Pass Amphetamine Pass Fail Pass NA Fail Pass Benzoylecgonine Pass Pass Pass NA Fail Pass Buprenorphine Pass Pass Pass NA Pass Pass Carisoprodol Pass Pass Pass NA Pass Pass Clonazepam Fail Fail Pass NA Pass Pass Codeine Pass Pass Fail Pass Pass Pass Diazepam Fail Fail Pass NA Pass Pass EDDP Pass Pass Pass NA Pass Pass Fentanyl Pass Pass Pass NA Pass Pass Gabapentin Pass Pass Fail NA Fail Pass Hydrocodone Pass Pass Pass Pass Pass Pass Hydromorphone Pass Pass Fail Fail Fail Fail Hydroxyalprazolam Pass Pass Pass NA Pass Pass Lorazepam Pass Pass Pass NA Pass Pass MDMA Pass Pass Fail NA Fail Pass Meperidine Pass Pass Pass NA Fail Pass Meprobamate Pass Pass Pass NA Pass Pass Methadone Pass Pass Pass NA Fail Pass Methamphetamine Pass Pass Fail NA Fail Pass Morphine Pass Pass Pass Fail Fail Pass Naltrexone Pass Pass Pass NA Pass Pass Norbuprenorphine Pass Pass Pass NA Pass Pass Nordiazepam Pass Pass Pass NA Pass Pass Norfentanyl Pass Pass Pass NA Pass Pass Norhydrocodone Fail Fail Fail NA Pass Pass Noroxycodone Pass Pass Fail NA Pass Pass Oxycodone Pass Pass Pass NA Fail Pass Oxymorphone Pass Pass Pass NA Fail Pass PCP Pass Pass Pass NA Pass Pass Pregabalin Fail Fail Pass NA Fail Fail Tapentadol Pass Pass Pass NA Pass Pass Temazepam Pass Pass Pass NA Pass Pass Tramadol Pass Pass Pass NA Pass Pass Zolpidem Fail Fail Pass NA Pass Pass Number 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, D p) 11 x 1.753 mm, 6.2 um
Verification Data, New Guard Column Column Verification 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... 1.3e5 1.3e5 1.2e5 1.2e5 1.1e5 1.1e5 1.0e5 9.5e4 9.0e4 8.5e4 8.0e4 7.5e4 7.0e4 6.5e4 6.0e4 5.5e4 5.0e4 4.5e4 4.0e4 3.5e4 3.0e4 2.5e4 2.0e4 1.5e4 1.0e4 500 Multi-analyte panel, API 3200 (Scheduled MRM) Col Verification, SST Injection #3 Post- Guard Column Replacement 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 3.4 Test Name: Analyte Example Panel Analyte Retention Time IS Retention Time Retention Time Alignment Critical Pair Separation Analyte Asymmetry Sensitivity 6-MAM Pass Pass Pass NA Pass Pass 7-Aminoclonazepam Pass Pass Pass NA Pass Pass Alprazolam Pass Pass Pass NA Pass Pass Amphetamine Pass Pass Pass NA Pass Pass Benzoylecgonine Pass Pass Pass NA Pass Pass Buprenorphine Pass Pass Pass NA Pass Pass Carisoprodol Pass Pass Pass NA Pass Pass Clonazepam Pass Pass Pass NA Pass Pass Codeine Pass Pass Pass Pass Pass Pass Diazepam Pass Pass Pass NA Pass Pass EDDP Pass Pass Pass NA Pass Pass Fentanyl Pass Pass Pass NA Pass Pass Gabapentin Pass Pass Pass NA Pass Pass Hydrocodone Pass Pass Pass Pass Pass Pass Hydromorphone Pass Pass Pass Pass Pass Pass Hydroxyalprazolam Pass Pass Pass NA Pass Pass Lorazepam Pass Pass Pass NA Pass Pass MDMA Pass Pass Pass NA Pass Pass Meperidine Pass Pass Pass NA Pass Pass Meprobamate Pass Pass Pass NA Pass Pass Methadone Pass Pass Pass NA Pass Pass Methamphetamine Pass Pass Pass NA Pass Pass Morphine Pass Pass Pass Pass Pass Pass Naltrexone Pass Pass Pass NA Pass Pass Norbuprenorphine Pass Pass Pass NA Pass Pass Nordiazepam Pass Pass Pass NA Pass Pass Norfentanyl Pass Pass Pass NA Pass Pass Norhydrocodone Pass Pass Pass NA Pass Pass Noroxycodone Pass Pass Pass NA Pass Pass Oxycodone Pass Pass Pass NA Pass Pass Oxymorphone Pass Pass Pass NA Pass Pass PCP Pass Pass Pass NA Pass Pass Pregabalin Pass Pass Pass NA Pass Pass Tapentadol Pass Pass Pass NA Pass Pass Temazepam Pass Pass Pass NA Pass Pass Tramadol Pass Pass Pass NA Pass Pass Zolpidem Pass Pass Pass NA Pass Pass Number of Failures 0 0 0 0 0 0 Column Serial Number 111-111-1111-FVM Date Installed 2/7/1964 Column Manufacturer Stationary Phase Flintstones Vit-Min PolyPro-Dibenzophenol Taffy Initials JPGR Dimensions (L x W, D p) 11 x 1.753 mm, 6.2 um
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... When Accuracy based QC isn t accurate Neat Pickering QC No IS added Max. 4.1e6 cps. External Accuracy based mixture (>50 analytes) in Phenol/water 6.5e5 6.0e5 5.5e5 5.0e5 4.5e5 4.0e5 * 0.67 3.5e5 3.0e5 2.5e5 Supra-physiological ~250uM 2.0e5 1.5e5 1.0e5 5.0e4 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 Measure Neat (no IS), Neat diluted (class A) Methionine D3 Cystathionine 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),... 2.4e6 2.3e6 10 x Diluted Pickering QC + IS * Max. 8.7e5 cps. 2.2e6 Determine how to deploy accuracy based materials that do not represent the clinically observed concentrations of mixtures 2.1e6 2.0e6 1.9e6 1.8e6 1.7e6 1.6e6 1.5e6 1.4e6 1.3e6 1.2e6 1.1e6 1.0e6 9.0e5 8.0e5 7.0e5 6.0e5 5.0e5 4.0e5 3.0e5 2.0e5 0.53 * * 1.21 1.0e5 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
Inter-Laboratory Correlation LabCorp and St Pauls, BC Concentrations < 45ng/dL N= 52 Intercept: -96 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
Automated Data review If [examined value] exceeds [a threshold], then flag with [text string].
Acknowledgements Patricia Holland, Matt Crawford, Stacy Dee, Mary Morr, Dr Marcia Eisenberg, Dr Walt Chandler Brian Rappold Randy Julian Dr Andy Hoofnagle S L I D E 28