Validation of Quantitative Methods

Opioids (Hydrocodone, Hydromorphone, Oxycodone, Analyte Oxymorphone, Codeine, Morphine, 6-Acetylmorphine) Units of Measure ng/mL

Analyst Performing Validation Studies Melissa Lloyd

Responsible Supervisor Dayong Lee

Start Date May 5, 2016

Completion Date November 18, 2016

Primary Matrix Blood

Secondary Matrix Urine 20 ng/mL (Hydrocodone, Hydromorphone, Oxycodone, Low Calibrator Concentration Oxymorphone, Codeine, Morphine); 5 ng/mL (6-Acetylmorphine) 800 ng/mL (Hydrocodone, Hydromorphone, Highest Calibrator Concentration Oxycodone, Oxymorphone, Codeine, Morphine); 200 ng/mL (6-Acetylmorphine) Equipment/Instrument GCMS-2

Instrument Serial Number CN10937127

Method Hydoxy.M and Morph.M

Validation Report Date January 4, 2018

Validation Approval

Analyst: Date

Responsible Supervisor: Date

Quality Director: Date METHOD VALIDATION PROTOCOL AND RESULTS Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Hydoxy.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS

Data analysis was processed using 1/x weighting even though 1/x2 gave Unweighted: = 208.83 the lowest %RE because all other 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 145.13 validation parameters met 1/x2 Weighting: = 144.08 acceptance criteria when 1/x was used.

95% CI of slope includes 1 95% CI of Slope = 0.9982 - 1.0064 1 Linearity 95% CI of intercept includes 0 95% CI of Intercept = -0.9196 - 0.3887

95% CI of slope includes 1 95% CI of Slope = 0.9915 - 1.0143 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -6.9182 - 4.4580

Max %RE low calibrator = -2.80 1 Validation Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) Max %RE other calibrators = 3.98

Not performed. Validation of 6 calibrators 1 Casework Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) will be used for casework.

Comparison of Validation Calibration to 95% CI of slope includes 1 Not performed. Validation of 6 calibrators 1 Casework Calibration 95% CI of intercept includes 0 will be used for casework.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three levels did not consistently LOD = 20 meet ion ratio criteria (red values Signal to Noise ≥ 3 S:N = >245.228; S:N not documented for 2 Limit of Detection (LOD) above indicate failed ion ratios). Acceptable detection and identification criteria 20 ng/mL because S:N of 18 ng/mL was > Therefore, the LOD and LOQ will be 3. 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments.

Bias: ≤ 25% Bias = 4.39% Replicate in red failed to meet ion 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-Run Imprecision = 2.76% ratio acceptance criteria. Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 6.24%

Bias Bias: ≤ 20% Max Bias = -5.51% Red values indicate ion ratios not meeting acceptance criteria. Low 4 Within-Run Imprecision Within-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.00% recovery on 8/29/16 caused multiple ion ratios to fail; those data points were excluded from the calculation. Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 4.70%

All ion ratios for hydrocodone in the Negative (in red) do not meet 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 21.70% acceptance criteria. Therefore, the method is considered to have no significant carryover. 6 Matrix Interference Response of blank matrix ≤ 10% of the average response of LOQ No matrix interference was identified.

6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is no interference from labeled IS.

There are 0 compounds tested in this study Concentration of analytes of interest within ± 20% of average for bias 6 Exogenous Substances Interferences that cause significiant interferences with the and imprecision analyte of interest.

Red values indicate ion ratios that did not meet acceptance criteria. Given the quantified concentrations 7 Dilution Integrity Concentration within ± 20% of target Max % of target = 13.82 are acceptable, up to 10x dilution can be used for the lower curve and 4x for the upper curve if ion ratios are acceptable.

Concentration within 20% of the average value obtained in the Bias 8 In-Process Stability Stable for 3 hours and Imprecision studies

Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, Concentration within 20% of the average value obtained in the Bias Unpunctured Vials:Stable for 24 hours 8 Autosampler Stability respectively. Red values indicate ion and Imprecision studies Punctured Vials:Stable for 24 hours ratios that did not meet acceptance criteria. The results will not be reported if ion ratios are not acceptable.

Study not performed; storage stability is documented in Disposition 8 Storage Stability Concentration within 20% of average time 0 of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Blt Extraction efficiency and matrix effects were not performed for If method achieves desired limit of quantification and/or detection for hydrocodone, hydromorphone, 9 Extraction Efficiency & Matrix Effect the analyte, its extraction efficiency and matrix effect are considered Study not performed oxycodone, or oxymorphone acceptable. because derivatization needs to occur prior to extraction for these four analytes.

10 Blind Sample Analysis Percent difference of the expected vs. determined values ≤ 20% 100% of samples ≤ 20% of target value

* Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 208.83 1/x Weighting: 145.13 1/x2 Weighting: 144.08

Comments: Data analysis was processed using 1/x weighting even though 1/x2 gave the lowest %RE because all other validation parameters met acceptance criteria when 1/x was used. Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 20 0.122238 1 2.444769 20 0.12223846 400 0.014942 17.83944 -10.8028 10.80279 40 0.252139 1 10.08555 40 0.25213883 1600 0.063574 38.40348 -3.99129 3.991294 100 0.642177 1 64.21771 100 0.64217711 10000 0.412391 100.149 0.148981 0.148981 300 1.861663 1 558.4988 300 1.86166264 90000 3.465788 293.2012 -2.26628 2.266278 500 2.964891 1 1482.445 500 2.9648908 250000 8.790577 467.8491 -6.43018 6.430183 800 4.83622 1 3868.976 800 4.83621977 640000 23.38902 764.0922 -4.48848 4.488479 20 0.136542 1 2.730836 20 0.1365418 400 0.018644 20.10375 0.518749 0.518749 40 0.288275 1 11.53099 40 0.28827467 1600 0.083102 44.12401 10.31003 10.31003 100 0.679789 1 67.97895 100 0.67978948 10000 0.462114 106.1033 6.103255 6.103255 300 2.059975 1 617.9925 300 2.05997509 90000 4.243497 324.5953 8.198423 8.198423 500 3.392311 1 1696.155 500 3.3923109 250000 11.50777 535.5124 7.102472 7.102472 800 5.294404 1 4235.523 800 5.29440415 640000 28.03072 836.6256 4.578202 4.578202 20 0.138976 1 2.779528 20 0.13897642 400 0.019314 20.48916 2.445823 2.445823 40 0.287135 1 11.48541 40 0.28713518 1600 0.082447 43.94362 9.859058 9.859058 100 0.72712 1 72.71199 100 0.72711994 10000 0.528703 113.596 13.59596 13.59596 300 2.096524 1 628.9571 300 2.09652369 90000 4.395412 330.3811 10.12705 10.12705 500 3.513023 1 1756.512 500 3.51302334 250000 12.34133 554.6219 10.92438 10.92438 800 5.530289 1 4424.231 800 5.53028856 640000 30.58409 873.9676 9.245949 9.245949 20 0.137445 1 2.748902 20 0.13744511 400 0.018891 20.24675 1.233748 1.233748 40 0.288453 1 11.53814 40 0.28845341 1600 0.083205 44.15231 10.38077 10.38077 100 0.705419 1 70.54186 100 0.70541862 10000 0.497615 110.1605 10.16051 10.16051 300 2.195368 1 658.6103 300 2.1953677 90000 4.819639 346.0288 15.34292 15.34292 500 3.457183 1 1728.592 500 3.45718311 250000 11.95212 545.782 9.156407 9.156407 800 5.759065 1 4607.252 800 5.7590649 640000 33.16683 910.1843 13.77304 13.77304 20 0.125838 1 2.516767 20 0.12583835 400 0.015835 18.40933 -7.95337 7.95337 40 0.257592 1 10.30368 40 0.25759208 1600 0.066354 39.26677 -1.83308 1.833084 100 0.638742 1 63.87422 100 0.63874221 10000 0.407992 99.60522 -0.39478 0.394784 300 1.976372 1 592.9115 300 1.97637151 90000 3.906044 311.3603 3.786766 3.786766 500 3.422353 1 1711.176 500 3.42235258 250000 11.7125 540.2681 8.053629 8.053629 800 5.347044 1 4277.635 800 5.3470437 640000 28.59088 844.9588 5.619848 5.619848 Sum 8800 59.13456 5 33252.96 8800 59.1345641 4960000 223.6713 208.8262 Slope 0.00631687 Intercept 0.00954903 R2 0.99314045 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.122238 0.05 0.122238 1 0.00611192 20 0.000747 17.93382 -10.3309 10.33091 40 0.252139 0.025 0.252139 1 0.00630347 40 0.001589 37.28275 -6.79313 6.793133 100 0.642177 0.01 0.642177 1 0.00642177 100 0.004124 95.37975 -4.62025 4.620245 300 1.861663 0.003333 1.861663 1 0.00620554 300 0.011553 277.0246 -7.65845 7.658451 500 2.964891 0.002 2.964891 1 0.00592978 500 0.017581 441.3528 -11.7294 11.72945 800 4.83622 0.00125 4.83622 1 0.00604527 800 0.029236 720.0911 -9.98861 9.988615 20 0.136542 0.05 0.136542 1 0.00682709 20 0.000932 20.06433 0.321651 0.321651 40 0.288275 0.025 0.288275 1 0.00720687 40 0.002078 42.66525 6.663137 6.663137 100 0.679789 0.01 0.679789 1 0.00679789 100 0.004621 100.9822 0.982195 0.982195 300 2.059975 0.003333 2.059975 1 0.00686658 300 0.014145 306.5637 2.187899 2.187899 500 3.392311 0.002 3.392311 1 0.00678462 500 0.023016 505.0179 1.003574 1.003574 800 5.294404 0.00125 5.294404 1 0.00661801 800 0.035038 788.3386 -1.45768 1.457676 20 0.138976 0.05 0.138976 1 0.00694882 20 0.000966 20.42697 2.134856 2.134856 40 0.287135 0.025 0.287135 1 0.00717838 40 0.002061 42.49553 6.238813 6.238813 100 0.72712 0.01 0.72712 1 0.0072712 100 0.005287 108.0322 8.032165 8.032165 300 2.096524 0.003333 2.096524 1 0.00698841 300 0.014651 312.0077 4.002562 4.002562 500 3.513023 0.002 3.513023 1 0.00702605 500 0.024683 522.9982 4.599647 4.599647 800 5.530289 0.00125 5.530289 1 0.00691286 800 0.03823 823.4741 2.934258 2.934258 20 0.137445 0.05 0.137445 1 0.00687226 20 0.000945 20.19888 0.994402 0.994402 40 0.288453 0.025 0.288453 1 0.00721134 40 0.00208 42.69188 6.729698 6.729698 100 0.705419 0.01 0.705419 1 0.00705419 100 0.004976 104.7997 4.799709 4.799709 300 2.195368 0.003333 2.195368 1 0.00731789 300 0.016065 326.7307 8.910235 8.910235 500 3.457183 0.002 3.457183 1 0.00691437 500 0.023904 514.6807 2.936144 2.936144 800 5.759065 0.00125 5.759065 1 0.00719883 800 0.041459 857.5508 7.193847 7.193847 20 0.125838 0.05 0.125838 1 0.00629192 20 0.000792 18.47003 -7.64986 7.649857 40 0.257592 0.025 0.257592 1 0.0064398 40 0.001659 38.09502 -4.76245 4.76245 100 0.638742 0.01 0.638742 1 0.00638742 100 0.00408 94.86812 -5.13188 5.13188 300 1.976372 0.003333 1.976372 1 0.00658791 300 0.01302 294.1108 -1.96308 1.963077 500 3.422353 0.002 3.422353 1 0.00684471 500 0.023425 509.4926 1.898528 1.898528 800 5.347044 0.00125 5.347044 1 0.0066838 800 0.035739 796.1794 -0.47758 0.47758 Sum 8800 59.13456 0.457917 59.13456 30 0.20224897 8800 0.398682 145.1266 Slope 0.00671357 Intercept 0.00183853 R2 0.99788719 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.122238 0.0025 0.00611192 0.05 0.000306 1 3.74E-05 18.30164 -8.49178 8.491783 40 0.252139 0.000625 0.00630347 0.025 0.000158 1 3.97E-05 37.50099 -6.24752 6.247519 100 0.642177 0.0001 0.00642177 0.01 6.42E-05 1 4.12E-05 95.14887 -4.85113 4.851125 300 1.861663 1.11E-05 0.00620554 0.003333 2.07E-05 1 3.85E-05 275.3895 -8.20349 8.203487 500 2.964891 0.000004 0.00592978 0.002 1.19E-05 1 3.52E-05 438.4473 -12.3105 12.31054 800 4.83622 1.56E-06 0.00604527 0.00125 7.56E-06 1 3.65E-05 715.0308 -10.6211 10.62115 20 0.136542 0.0025 0.00682709 0.05 0.000341 1 4.66E-05 20.41568 2.078424 2.078424 40 0.288275 0.000625 0.00720687 0.025 0.00018 1 5.19E-05 42.84189 7.104726 7.104726 100 0.679789 0.0001 0.00679789 0.01 6.8E-05 1 4.62E-05 100.708 0.708005 0.708005 300 2.059975 1.11E-05 0.00686658 0.003333 2.29E-05 1 4.71E-05 304.7002 1.566745 1.566745 500 3.392311 0.000004 0.00678462 0.002 1.36E-05 1 4.6E-05 501.6202 0.324047 0.324047 800 5.294404 1.56E-06 0.00661801 0.00125 8.27E-06 1 4.38E-05 782.7507 -2.15616 2.156161 20 0.138976 0.0025 0.00694882 0.05 0.000347 1 4.83E-05 20.77552 3.877612 3.877612 40 0.287135 0.000625 0.00717838 0.025 0.000179 1 5.15E-05 42.67347 6.683682 6.683682 100 0.72712 0.0001 0.0072712 0.01 7.27E-05 1 5.29E-05 107.7035 7.703474 7.703474 300 2.096524 1.11E-05 0.00698841 0.003333 2.33E-05 1 4.88E-05 310.1021 3.36738 3.36738 500 3.513023 0.000004 0.00702605 0.002 1.41E-05 1 4.94E-05 519.4616 3.892321 3.892321 800 5.530289 1.56E-06 0.00691286 0.00125 8.64E-06 1 4.78E-05 817.6146 2.201821 2.201821 20 0.137445 0.0025 0.00687226 0.05 0.000344 1 4.72E-05 20.54919 2.745974 2.745974 40 0.288453 0.000625 0.00721134 0.025 0.00018 1 5.2E-05 42.86831 7.170772 7.170772 100 0.705419 0.0001 0.00705419 0.01 7.05E-05 1 4.98E-05 104.496 4.496008 4.496008 300 2.195368 1.11E-05 0.00731789 0.003333 2.44E-05 1 5.36E-05 324.7113 8.237113 8.237113 500 3.457183 0.000004 0.00691437 0.002 1.38E-05 1 4.78E-05 511.2084 2.241677 2.241677 800 5.759065 1.56E-06 0.00719883 0.00125 9E-06 1 5.18E-05 851.4278 6.42848 6.42848 20 0.125838 0.0025 0.00629192 0.05 0.000315 1 3.96E-05 18.83371 -5.83146 5.83146 40 0.257592 0.000625 0.0064398 0.025 0.000161 1 4.15E-05 38.30699 -4.23253 4.232535 100 0.638742 0.0001 0.00638742 0.01 6.39E-05 1 4.08E-05 94.6412 -5.3588 5.358804 300 1.976372 1.11E-05 0.00658791 0.003333 2.2E-05 1 4.34E-05 292.3436 -2.55214 2.552141 500 3.422353 0.000004 0.00684471 0.002 1.37E-05 1 4.68E-05 506.0604 1.212083 1.212083 800 5.347044 1.56E-06 0.0066838 0.00125 8.35E-06 1 4.47E-05 790.5309 -1.18364 1.183641 8800 59.13456 0.016208 0.20224897 0.457917 0.003072 30 0.001368 144.0807 Slope 0.00676587 Intercept -0.00158814 R2 0.99719109 Validation Study 1 LINEARITY Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0023 Intercept -0.2654

20 ng/mL 20160505_MRL 20.00 19.49 Std err in slope, Sb 0.0020 Std err in intercept 0.3219 40 ng/mL 20160505_MRL 40.00 40.26 Degrees freedom 34 Degrees freedom 34 100 ng/mL 20160505_MRL 100.00 102.62 Confidence level 95% Confidence level 95% 300 ng/mL 20160505_MRL 300.00 297.60 Student t 2.0322 Student t 2.0322 20 ng/mL 20160512_MRL 20.00 19.44 Confidence interval 0.0041 Confidence interval 0.6542 40 ng/mL 20160512_MRL 40.00 41.59 Slope 1.0023 ± 0.0041 Intercept -0.2654 ± 0.6542 100 ng/mL 20160512_MRL 100.00 98.74 Range 0.9982 - 1.0064 Lower -0.9196 - 0.3887 300 ng/mL 20160512_MRL 300.00 300.22 PASS PASS 20 ng/mL 20160829_MRL 20.00 20.01 40 ng/mL 20160829_MRL 40.00 40.66 100 ng/mL 20160829_MRL 100.00 97.68 300 ng/mL 20160829_MRL 300.00 301.62 20 ng/mL 20161005_MRL 20.00 20.16 350.00 40 ng/mL 20161005_MRL 40.00 40.20 y = 1.0023x - 0.2654 100 ng/mL 20161005_MRL 100.00 98.13 300.00 R² = 0.9999 300 ng/mL 20161005_MRL 300.00 301.48 20 ng/mL 20161011_MRL 20.00 20.50 250.00 40 ng/mL 20161011_MRL 40.00 38.84 100 ng/mL 20161011_MRL 100.00 100.20 200.00 300 ng/mL 20161011_MRL 300.00 300.44 20 ng/mL 20161013_MRL 20.00 20.61 150.00 40 ng/mL 20161013_MRL 40.00 39.43 100 ng/mL 20161013_MRL 100.00 97.51 100.00 300 ng/mL 20161013_MRL 300.00 302.43 20 ng/mL 20161021_MRL 20.00 20.39 50.00 40 ng/mL 20161021_MRL 40.00 39.89 100 ng/mL 20161021_MRL 100.00 97.54 0.00 300 ng/mL 20161021_MRL 300.00 302.16 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 20 ng/mL 20161026_MRL 20.00 20.65 40 ng/mL 20161026_MRL 40.00 38.50 100 ng/mL 20161026_MRL 100.00 100.25 300 ng/mL 20161026_MRL 300.00 300.58 20 ng/mL 20161027_MRL 20.00 19.51 40 ng/mL 20161027_MRL 40.00 40.77 100 ng/mL 20161027_MRL 100.00 100.91 300 ng/mL 20161027_MRL 300.00 298.80

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 UPPER LINEARITY Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0029 Intercept -1.2301

100 ng/mL 20160505_MRL 100.00 99.68 Std err in slope, Sb 0.0056 Std err in intercept 2.7989 300 ng/mL 20160505_MRL 300.00 304.80 Degrees freedom 34 Degrees freedom 34 500 ng/mL 20160505_MRL 500.00 490.37 Confidence level 95% Confidence level 95% 800 ng/mL 20160505_MRL 800.00 805.14 Student t 2.0322 Student t 2.0322 100 ng/mL 20160512_MRL 100.00 98.24 Confidence interval 0.0114 Confidence interval 5.6881 300 ng/mL 20160512_MRL 300.00 305.42 Slope 1.0029 ± 0.0114 Intercept -1.2301 ± 5.6881 500 ng/mL 20160512_MRL 500.00 505.41 Range 0.9915 - 1.0143 Lower -6.9182 - 4.4580 800 ng/mL 20160512_MRL 800.00 790.92 PASS PASS 100 ng/mL 20160829_MRL 100.00 99.31 300 ng/mL 20160829_MRL 300.00 308.29 500 ng/mL 20160829_MRL 500.00 484.91 800 ng/mL 20160829_MRL 800.00 807.47 900.00 y = 1.0029x - 1.2301 100 ng/mL 20161005_MRL 100.00 99.77 800.00 300 ng/mL 20161005_MRL 300.00 297.48 R² = 0.9989 500 ng/mL 20161005_MRL 500.00 509.59 700.00 800 ng/mL 20161005_MRL 800.00 793.14 600.00 100 ng/mL 20161011_MRL 100.00 100.17 300 ng/mL 20161011_MRL 300.00 301.70 500.00 500 ng/mL 20161011_MRL 500.00 493.19 400.00 800 ng/mL 20161011_MRL 800.00 804.91 100 ng/mL 20161013_MRL 100.00 98.88 300.00 300 ng/mL 20161013_MRL 300.00 302.44 200.00 500 ng/mL 20161013_MRL 500.00 506.95 100.00 800 ng/mL 20161013_MRL 800.00 791.77 100 ng/mL 20161021_MRL 100.00 99.82 0.00 300 ng/mL 20161021_MRL 300.00 304.06 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 500 ng/mL 20161021_MRL 500.00 490.76 800 ng/mL 20161021_MRL 800.00 805.34 100 ng/mL 20161026_MRL 100.00 102.56 300 ng/mL 20161026_MRL 300.00 298.43 500 ng/mL 20161026_MRL 500.00 474.40 800 ng/mL 20161026_MRL 800.00 824.59 100 ng/mL 20161027_MRL 100.00 102.83 300 ng/mL 20161027_MRL 300.00 287.82 500 ng/mL 20161027_MRL 500.00 500.78 800 ng/mL 20161027_MRL 800.00 808.56

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 20 19.49 -2.55 Level 2 40 40.26 0.65 Level 3 100 102.62 2.62 Level 4 300 297.60 -0.80 5/5/16 Upper-Level 3 100 99.68 -0.32 Upper-Level 4 300 304.80 1.60 Level 5 500 490.37 -1.93 Level 6 800 805.14 0.64 Level 1 20 19.44 -2.80 Level 2 40 41.59 3.98 Level 3 100 98.74 -1.26 Level 4 300 300.22 0.07 5/12/16 Upper-Level 3 100 98.24 -1.76 Upper-Level 4 300 305.42 1.81 Level 5 500 505.41 1.08 Level 6 800 790.92 -1.14 Level 1 20 20.01 0.05 Level 2 40 40.66 1.65 Level 3 100 97.68 -2.32 Level 4 300 301.62 0.54 8/29/16 Upper-Level 3 100 99.31 -0.69 Upper-Level 4 300 308.29 2.76 Level 5 500 484.91 -3.02 Level 6 800 807.47 0.93 Level 1 20 20.16 0.80 Level 2 40 40.20 0.50 Level 3 100 98.13 -1.87 Level 4 300 301.48 0.49 10/5/16 Upper-Level 3 100 99.77 -0.23 Upper-Level 4 300 297.48 -0.84 Level 5 500 509.59 1.92 Level 6 800 793.14 -0.86 Level 1 20 20.50 2.50 Level 2 40 38.84 -2.90 Level 3 100 100.20 0.20 Level 4 300 300.44 0.15 10/11/16 Upper-Level 3 100 100.17 0.17 Upper-Level 4 300 301.70 0.57 Level 5 500 493.19 -1.36 Level 6 800 804.91 0.61

Max %RE LOQ = -2.80 Max %RE other calibrators = 3.98

Comments:

Acceptance Criteria: %RE Calibrators ± 20% of target (25% at lowest calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GC-MS 2 Matrix: Blood

6 Run One LOWER Curve UPPER Curve Slope 0.00625 Slope 0.00595 4 Intercept 0.000275 Intercept 0.0496 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Two Slope 0.00685 Slope 0.00666 4 Intercept 0.00337 Intercept 0.0253 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Three Slope 0.00731 Slope 0.00714 4 Intercept 0.00895 Intercept 0.00335 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 8 Run Four 6 Slope 0.00658 Slope 0.00679 Intercept 0.00683 Intercept 0.0366 4 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Five Slope 0.00681 Slope 0.00677 4 Intercept 0.0017 Intercept 0.00935 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GC-MS 2 Matrix: Blood

Cnom y (Response Ratio) 7 Combined Regression 20 0.122238463 40 0.25213883 6 100 0.642177107 300 1.861662635 500 2.964890797 5 800 4.836219772 20 0.1365418 40 0.288274668 4 100 0.67978948 300 2.059975088 500 3.392310897 3 800 5.294404146 Response Ratio Response 20 0.138976416 40 0.287135178 2 100 0.727119938 300 2.096523689 500 3.513023337 1 800 5.53028856 20 0.137445112 40 0.288453414 0 100 0.705418624 0.000 100.000 200.000 300.000 400.000 500.000 600.000 700.000 800.000 900.000 300 2.195367697 Concentration 500 3.457183112 800 5.759064902 Slope 0.0067 20 0.125838346 Intercept 0.0018 40 0.257592081 R2 0.9979 100 0.638742214 300 1.976371508 500 3.422352584 800 5.347043702 Validation Study 2 LOD Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 18.00 15.00 10.00 Source 1-1 385.1 171.6 276.5 Source 1-2 782.6 232.9 371.9 Source 2-1 755.9 477.1 264.1 5/5/16 Source 2-2 120.9 304.1 413.1 Source 3-1 357.7 371.1 476.9 Source 3-2 193.1 536.9 197.5 Source 1-1 157.4 471.7 545.7 Source 1-2 362.3 495.5 504.8 Source 2-1 133.8 664.1 346.7 5/12/16 Source 2-2 354.4 422.5 423.9 Source 3-1 410.2 570.8 324.2 Source 3-2 223.2 237.5 477.5 Source 1-1 35.7 41.7 25.5 Source 1-2 30.1 20.8 77.7 Source 2-1 21.6 24.1 N/A *Poor extraction recovery *8/29/16 Source 2-2 36.7 27.4 27.6 Source 3-1 26.2 24.8 24.3 Source 3-2 27.2 22.1 22.1

Mean Signal to Noise: 245.228 284.261 282.353

Established LOD: 20.00 ng/mL S:N of LOD: >245.228; S:N not documented for 20 ng/mL because S:N of 18 ng/mL was > 3. The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three Comments: levels did not consistently meet ion ratio criteria (red values above indicate failed ion ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments.

Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 20.00 8/29/16 1-1 23.28 1-2 23.32 1-3 23.22 Within Run Mean 23.27 SD 0.05 %CV 0.22% % Bias 16.37% 10/5/16 2-1 20.01 2-2 19.78 2-3 20.08 Within Run Mean 19.96 SD 0.16 %CV 0.79% % Bias -0.22% 10/11/16 3-1 20.09 3-2 19.79 3-3 20.57 Within Run Mean 20.15 SD 0.39 %CV 1.95% % Bias 0.75% 10/13/16 4-1 19.56 4-2 20.62 4-3 20.39 Within Run Mean 20.19 SD 0.56 %CV 2.76% % Bias 0.95% 10/26/16 5-1 20.83 5-2 20.77 5-3 20.85 Within Run Mean 20.82 SD 0.04 %CV 0.20% % Bias 4.08%

Mean 20.88 Imprecision Within-Run 2.76% (%CV) Between-Run 6.24% % Bias 4.39%

Comments: Replicate in red failed to meet ion ratio acceptance criteria.

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 25.00 250.00 250.00 400.00 8/29/16 1-1 28.63* 240.90* 246.06* 415.25* 1-2 28.40* 259.63* 265.26* 418.40* 1-3 28.44* 275.30* 281.31* 382.89* Within Run Mean SD %CV % Bias 10/5/16 2-1 23.97 230.09 228.21 382.90 2-2 24.69 236.71 234.81 376.15 2-3 24.13 231.11 229.06 372.25 Within Run Mean 24.26 232.64 230.69 377.10 SD 0.38 3.56 3.59 5.39 %CV 1.56% 1.53% 1.56% 1.43% % Bias -2.95% -6.95% -7.72% -5.73% 10/11/16 3-1 26.10 247.72 249.20 374.69 3-2 25.90 230.73 232.07 367.71 3-3 24.18 220.39 221.34 370.61 Within Run Mean 25.39 232.95 234.20 371.00 SD 1.06 13.80 14.05 3.51 %CV 4.16% 5.92% 6.00% 0.95% % Bias 1.57% -6.82% -6.32% -7.25% 10/13/16 4-1 27.44 246.96 247.32 372.88 4-2 24.80 231.75 232.21 381.19 4-3 25.16 230.51 230.98 374.36 Within Run Mean 25.80 236.41 236.84 376.14 SD 1.43 9.16 9.10 4.43 %CV 5.55% 3.87% 3.84% 1.18% % Bias 3.20% -5.44% -5.27% -5.96% 10/26/16 5-1 25.49 247.05 246.10 369.40 5-2 25.68 241.54 240.73 370.87 5-3 25.14 249.65 248.64 376.24 Within Run Mean 25.44 246.08 245.16 372.17 SD 0.27 4.14 4.04 3.60 %CV 1.08% 1.68% 1.65% 0.97% % Bias 1.75% -1.57% -1.94% -6.96% 11/18/16 5-1 24.45 252.37 241.58 395.60 5-2 23.79 250.20 239.56 392.64 5-3 24.48 261.61 250.17 392.19 Within Run Mean 24.24 254.73 243.77 393.48 SD 0.39 6.06 5.63 1.85 %CV 1.61% 2.38% 2.31% 0.47% % Bias -3.04% 1.89% -2.49% -1.63%

Mean 25.03 240.56 238.13 377.98 SD 0.9774 11.31 9.04 9.00 Imprecision Max Within-Run 5.55% 5.92% 6.00% 1.43% (%CV) Between-Run 3.91% 4.70% 3.79% 2.38% % Bias 0.11% -3.78% -4.75% -5.51%

Comments: Red values indicate ion ratios not meeting acceptance criteria. Low recovery on 8/29/16 caused multiple ion ratios to fail; those data points were excluded from the calculation.

Bias: ≤ 20% Acceptance Criteria: Within-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 20.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/5/16 10/11/16 High Calibrator 800.000 805.14 790.92 807.47 793.14 804.91 Negative 0.27 -0.02 4.33 1.14 2.58 % of Low Calibrator 1.4% -0.1% 21.7% 5.7% 12.9% Maximum Carryover: 21.7%

Comments: All ion ratios for hydrocodone in the Negative (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 27631 Run 2 91540 Run 3 101186 Run 4 108564 Run 5 49958 Average 75776

Identification Peak Response Comment Blank Matrix (NO IS) 1 419 Pass Blank Matrix (NO IS) 2 883 Pass Blank Matrix (NO IS) 3 764 Pass Blank Matrix (NO IS) 4 68 Pass Blank Matrix (NO IS) 5 234 Pass Blank Matrix (NO IS) 6 943 Pass Blank Matrix (NO IS) 7 949 Pass Blank Matrix (NO IS) 8 518 Pass Blank Matrix (NO IS) 9 388 Pass Blank Matrix (NO IS) 10 912 Pass Blank Matrix (with IS) 1 2476 Pass Blank Matrix (with IS) 2 3654 Pass Blank Matrix (with IS) 3 4038 Pass Blank Matrix (with IS) 4 504 Pass Blank Matrix (with IS) 5 473 Pass Blank Matrix (with IS) 6 2103 Pass Blank Matrix (with IS) 7 1574 Pass Blank Matrix (with IS) 8 227 Pass Blank Matrix (with IS) 9 158 Pass Blank Matrix (with IS) 10 2777 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is no interference from labeled IS. Comments:

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 25.03 ng/mL Control Acceptance (%): 20 Run Date: 10/21/16

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration 7-Aminoclonazepam Nordiazepam Oxazepam Diazepam 1 Benzodiazepines Mix Lorazepam 1,000 ng/mL 26.35 Pass Temazepam Clonazepam Alpha-hydroxyalprazolam Alprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 24.10 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 24.43 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 25.83 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 26.94 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 24.09 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 23.99 Pass

Conclusions: There are 0 compounds tested in this study that cause significiant interferences with the analyte of interest.

Comments:

Acceptance Criteria: Concentration of analytes of interest within ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated %Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 250.00 500.00 125.00 500.00 50.00 500.00 250.00 500.00 125.00 500.00 Run 1 1-1 238.41 476.82 -4.64 118.18 472.72 -5.46 48.90 489.00 -2.20 240.04 480.08 -3.98 118.45 473.80 -5.24 10/11/16 1-2 220.04 440.08 -11.98 119.70 478.80 -4.24 49.33 493.30 -1.34 221.28 442.56 -11.49 119.73 478.92 -4.22 Run 2 2-1 268.74 537.48 7.50 138.96 555.84 11.17 55.36 553.60 10.72 268.97 537.94 7.59 140.01 560.04 12.01 10/13/16 2-2 264.81 529.62 5.92 134.76 539.04 7.81 56.91 569.10 13.82 265.06 530.12 6.02 135.83 543.32 8.66 Run 3 3-1 249.29 498.58 -0.28 121.40 485.60 -2.88 51.13 511.30 2.26 248.28 496.56 -0.69 123.17 492.68 -1.46 10/26/16 3-2 246.54 493.08 -1.38 122.05 488.20 -2.36 51.44 514.40 2.88 245.60 491.20 -1.76 123.78 495.12 -0.98

Mean: 495.94 503.37 521.78 496.41 507.31 SD: 35.67 34.97 32.56 34.80 35.69 Between-Run Imprecision (%CV): 7.19 6.95 6.24 7.01 7.04 %Bias: -0.81 0.67 4.36 -0.72 1.46

Comments: Red values indicate ion ratios that did not meet acceptance criteria. Given the quantified concentrations are acceptable, up to 10x dilution can be used for the lower curve and 4x for the upper curve if ion ratios are acceptable.

Acceptance Criteria: Concentration within ± 20% of target Validation Study 8 IN-PROCESS STABILITY Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20%

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 23.92 Pass 24.13 20.02 30.03 LQC-2 24.34 Pass MQC-1 252.32 Pass 237.57 192.45 288.67 MQC-2 222.81 Pass 10/11/16 *MQC-1 253.83 Pass *Upper curve 238.64 190.51 285.76 *MQC-2 223.44 Pass *Upper curve HQC-1 359.95 Pass 358.87 302.38 453.57 HQC-2 357.78 Pass

Overall Acceptance: Stable for 3 hours

Comments:

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20%

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 20.00 19.35 N/A 16.00 24.00 Pass 40.00 38.95 N/A 32.00 48.00 Pass 100.00 103.97 N/A 80.00 120.00 Pass 300.00 321.46 N/A 240.00 360.00 Pass 100.00 105.54 N/A 80.00 120.00 Pass *Upper curve 300.00 309.21 N/A 240.00 360.00 Pass *Upper curve 500.00 490.75 N/A 400.00 600.00 Pass Injected on 10/26/16; 800.00 869.69 N/A 640.00 960.00 Pass Reinjected on 26.98 10/27/16 25.03 26.25 20.02 30.03 Pass 25.51 259.97 240.56 255.25 192.45 288.67 Pass 250.53 251.31 *Upper curve 238.13 246.96 190.51 285.76 Pass 242.61 377.96 377.98 380.41 302.38 453.57 Pass 382.86

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 25.60 Pass 25.53 20.02 30.03 LQC-2 Punctured 25.45 Pass MQC-1 Punctured 254.69 Pass Injected on 251.68 192.45 288.67 10/27/16; MQC-2 Punctured 248.66 Pass Reinjected on *MQC-1 Punctured 246.48 Pass *Upper Curve 10/28/16 243.93 190.51 285.76 *MQC-2 Punctured 241.37 Pass *Upper Curve HQC-1 Punctured 381.73 Pass 378.32 302.38 453.57 HQC-2 Punctured 374.90 Pass LQC-1 Unpunctured 24.97 Pass 24.98 20.02 30.03 LQC-2 Unpunctured 24.98 Pass MQC-1 Unpunctured 251.24 Pass Extracted on 246.22 192.45 288.67 10/27/16; MQC-2 Unpunctured 241.19 Pass Injected on *MQC-1 Unpunctured 243.36 Pass *Upper Curve 10/28/16 238.59 190.51 285.76 *MQC-2 Unpunctured 233.81 Pass *Upper Curve HQC-1 Unpunctured 380.14 Pass 375.99 302.38 453.57 HQC-2 Unpunctured 371.84 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively. Red values indicate ion ratios that did not meet acceptance criteria. The results will not be reported if ion ratios are not acceptable.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 Room Temperature LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Room Temperature LQC-1 w/ Light Protection LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Refrigerated (2 to 8oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Frozen (-5 to -20oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Peak Extraction Average Type of Sample Sample ID Response of Efficiency Matrix Effect Response Drug (%Recovery)

Group 1-Internal Standard and drug analyte fortified before extraction

Group 2-Internal Standard and analyte fortified after extraction

Group 3-Internal Standard and analyte fortified to elution solvent/no extraction

Extraction efficiency and matrix effects were not performed for hydrocodone, hydromorphone, Comments: oxycodone, or oxymorphone because derivatization needs to occur prior to extraction for these four analytes.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction efficiency Criteria: and matrix effect are considered acceptable. Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 50.00 59.95 40-60 19.9 Pass Blind Sample 3 100.00 100.56 80-120 0.6 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 120.00 120.29 96-144 0.2 Pass Blind Sample 7 400.00 439.41 320-480 9.9 Pass Blind Sample 8 250.00 290.38 200-300 16.2 Pass Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 500.00 521.77 400-600 4.4 Pass 11/9/16 Blind Sample 11 600.00 621.80 480-720 3.6 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 800.00 825.53 640-960 3.2 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 160.00 162.31 98-192 1.4 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 400.00 435.43 320-480 8.9 Pass

Comments:

Acceptance Percent difference of the expected vs. determined values ≤ 20% Criteria: SUMMARY OF VALIDATION PERFORMANCE Analyte: Hydrocodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason August 2, 2016 There were multiple interferences with ions causing quantitative values, ion ratios, and overall chromatography to not be acceptable. Autosampler stability was attempted on this day, but the extraction vials October 13, 2016 dried out prior to injection so the experiment was repeated on October 27, 2016.

Deviations from SOP:

Other Observations: Per reccomendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of hydrocodone confirmation analysis. METHOD VALIDATION PROTOCOL AND RESULTS Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Hydoxy.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS

Data analysis was processed using 1/x weighting for the lower Unweighted: = 329.33 curve and unweighted for the 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 150.5 upper curve even though 1/x2 1/x2 Weighting: = 123.39 gave the lowest %RE because all other validation parameters met acceptance criteria.

95% CI of slope includes 1 95% CI of Slope = 0.9916 - 1.0009 1 Linearity 95% CI of intercept includes 0 95% CI of Intercept = -0.3119 - 1.1664

95% CI of slope includes 1 95% CI of Slope = 0.9867 - 1.0134 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -6.6488 - 6.6343

Max %RE low calibrator = -5.30 1 Validation Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) Max %RE other calibrators = -16.58

Not performed. Validation of 6 calibrators 1 Casework Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) will be used for casework.

Comparison of Validation Calibration 95% CI of slope includes 1 Not performed. Validation of 6 calibrators 1 to Casework Calibration 95% CI of intercept includes 0 will be used for casework.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three levels did not LOD = 20 consistently meet ion ratio criteria Signal to Noise ≥ 3 S:N = >140.667; S:N not documented for (red values indicate failed ion 2 Limit of Detection (LOD) Acceptable detection and identification criteria 20 ng/mL because S:N of 18 ng/mL was < ratios). Therefore, the LOD and 3. LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for the LOD experiments.

Bias: ≤ 25% Bias = 0.77% 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-Run Imprecision= 2.31% Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 1.96% Bias Bias: ≤ 20% Max Bias = -4.04% 4 Within-Run Imprecision Within-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.31% Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 5.06%

All ion ratios for Hydromorphone in the Negative (in red) do not meet acceptance criteria. 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 15.1% Therefore, the method is considered to have no significant carryover.

6 Matrix Interference Response of blank matrix ≤ 10% of the average response of LOQ No matrix interference was identified.

6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is no interference from labeled IS.

There are 0 compounds tested in this study Concentration of analytes of interest within ± 20% of average for bias and 6 Exogenous Substances Interferences that cause significant interference with the imprecision analyte of interest. Up to 10x dilution can be used for 7 Dilution Integrity Concentration within 20% of target Max % of target = 14.92 the lower curve and 4x for the upper curve.

Concentration within 20% of the average value obtained in the Bias and 8 In-Process Stability Stable for 3 hours Imprecision studies

Punctured vials in Experiment 1 were stable for 24 hours. Concentration within 20% of the average value obtained in the Bias and Unpunctured Vials:Stable for 24 hours 8 Autosampler Stability Punctured and unpunctured vials Imprecision studies Punctured Vials:Stable for 24 hours in Experiment 2 were stable for 21 and 24 hours, respectively.

Study not performed; storage stability is documented in 8 Storage Stability Concentration within 20% of average time 0 Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Extraction efficiency and matrix effects were not performed for If method achieves desired limit of quantification and/or detection for the hydrocodone, hydromorphone, analyte, its extraction efficiency and matrix effect are considered 9 Extraction Efficiency & Matrix Effect Study not performed oxycodone, and oxymorphone acceptable. because derivatization needs to occur prior to extraction for these four analytes.

Blind Sample 2 did not meet acceptance criteria. The quality controls ran with the blind samples quantitated on target for Percent difference of the expected vs. determined values ≤ 20% 10 Blind Sample Analysis 95% of samples ≤ 20% of target value hydromorphone (< ± 3%). Blind Sample 2 quantitated high for all drugs. Therefore, the high quantitation can be attributed to over-spiking the blind sample.

* Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 329.33 1/x Weighting: 150.50 1/x2 Weighting: 123.39

Comments: Data analysis was processed using 1/x weighting for the lower curve and unweighted for the upper curve even though 1/x2 gave the lowest %RE because all other validation parameters met acceptance criteria.

Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 20 0.117023 1 2.340467 20 0.11702335 400 0.013694 10.59903 -47.0049 47.00487 40 0.250661 1 10.02645 40 0.25066128 1600 0.062831 33.46179 -16.3455 16.34553 100 0.632814 1 63.28145 100 0.6328145 10000 0.400454 98.84053 -1.15947 1.159471 300 1.795105 1 538.5315 300 1.79510485 90000 3.222401 297.6851 -0.77165 0.771646 500 2.813898 1 1406.949 500 2.81389793 250000 7.918022 471.9801 -5.60398 5.603982 800 4.461032 1 3568.826 800 4.46103188 640000 19.90081 753.7716 -5.77855 5.778547 20 0.12708 1 2.541601 20 0.12708007 400 0.016149 12.31953 -38.4024 38.40237 40 0.273151 1 10.92605 40 0.27315124 1600 0.074612 37.30937 -6.72657 6.726574 100 0.644615 1 64.46153 100 0.64461531 10000 0.415529 100.8594 0.859411 0.859411 300 1.916885 1 575.0656 300 1.91688517 90000 3.674449 318.5192 6.173076 6.173076 500 3.067019 1 1533.51 500 3.06701905 250000 9.406606 515.284 3.056806 3.056806 800 4.554611 1 3643.689 800 4.55461094 640000 20.74448 769.7811 -3.77736 3.77736 20 0.132683 1 2.653655 20 0.13268277 400 0.017605 13.27804 -33.6098 33.60982 40 0.267795 1 10.71182 40 0.26779538 1600 0.071714 36.39309 -9.01727 9.017274 100 0.656048 1 65.60477 100 0.65604771 10000 0.430399 102.8153 2.815265 2.815265 300 1.912219 1 573.6656 300 1.91221876 90000 3.656581 317.7209 5.906966 5.906966 500 3.082931 1 1541.465 500 3.08293096 250000 9.504463 518.0062 3.601248 3.601248 800 4.731126 1 3784.901 800 4.73112606 640000 22.38355 799.9793 -0.00259 0.002586 20 0.128757 1 2.575142 20 0.1287571 400 0.016578 12.60643 -36.9678 36.96783 40 0.271073 1 10.84292 40 0.27107293 1600 0.073481 36.95381 -7.61546 7.615465 100 0.646938 1 64.69378 100 0.64693778 10000 0.418528 101.2567 1.25674 1.25674 300 1.958545 1 587.5636 300 1.95854544 90000 3.8359 325.6465 8.548821 8.548821 500 3.025103 1 1512.552 500 3.02510327 250000 9.15125 508.1131 1.622617 1.622617 800 4.831844 1 3865.475 800 4.83184432 640000 23.34672 817.2102 2.151273 2.151273 20 0.117519 1 2.350382 20 0.11751908 400 0.013811 10.68383 -46.5808 46.58083 40 0.245724 1 9.828947 40 0.24572367 1600 0.06038 32.61707 -18.4573 18.45734 100 0.61461 1 61.46096 100 0.61460963 10000 0.377745 95.72604 -4.27396 4.273959 300 1.876043 1 562.8128 300 1.87604253 90000 3.519536 311.5319 3.843957 3.843957 500 3.17381 1 1586.905 500 3.17381044 250000 10.07307 533.5539 6.710779 6.710779 800 4.763378 1 3810.702 800 4.76337772 640000 22.68977 805.4969 0.687116 0.687116 Sum 8800 53.09004 5 29476.91 8800 53.0900411 4960000 175.4911 329.3295 Slope 0.00584522 Intercept 0.05506970 R2 0.99670860 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.117023 0.05 0.117023 1 0.00585117 20 0.000685 16.8241 -15.8795 15.87951 40 0.250661 0.025 0.250661 1 0.00626653 40 0.001571 39.18348 -2.04129 2.041291 100 0.632814 0.01 0.632814 1 0.00632814 100 0.004005 103.1228 3.122754 3.122754 300 1.795105 0.003333 1.795105 1 0.00598368 300 0.010741 297.5892 -0.80358 0.803584 500 2.813898 0.002 2.813898 1 0.0056278 500 0.015836 468.0468 -6.39065 6.390649 800 4.461032 0.00125 4.461032 1 0.00557629 800 0.024876 743.634 -7.04575 7.045753 20 0.12708 0.05 0.12708 1 0.006354 20 0.000807 18.50672 -7.46641 7.466408 40 0.273151 0.025 0.273151 1 0.00682878 40 0.001865 42.94635 7.365877 7.365877 100 0.644615 0.01 0.644615 1 0.00644615 100 0.004155 105.0972 5.097185 5.097185 300 1.916885 0.003333 1.916885 1 0.00638962 300 0.012248 317.9647 5.988233 5.988233 500 3.067019 0.002 3.067019 1 0.00613404 500 0.018813 510.3973 2.079451 2.079451 800 4.554611 0.00125 4.554611 1 0.00569326 800 0.025931 759.291 -5.08863 5.088627 20 0.132683 0.05 0.132683 1 0.00663414 20 0.00088 19.44412 -2.77938 2.779381 40 0.267795 0.025 0.267795 1 0.00669488 40 0.001793 42.05024 5.125612 5.125612 100 0.656048 0.01 0.656048 1 0.00656048 100 0.004304 107.01 7.009976 7.009976 300 1.912219 0.003333 1.912219 1 0.00637406 300 0.012189 317.1839 5.727983 5.727983 500 3.082931 0.002 3.082931 1 0.00616586 500 0.019009 513.0595 2.611906 2.611906 800 4.731126 0.00125 4.731126 1 0.00591391 800 0.027979 788.8243 -1.39696 1.396964 20 0.128757 0.05 0.128757 1 0.00643785 20 0.000829 18.78731 -6.06346 6.06346 40 0.271073 0.025 0.271073 1 0.00677682 40 0.001837 42.59862 6.496557 6.496557 100 0.646938 0.01 0.646938 1 0.00646938 100 0.004185 105.4858 5.485766 5.485766 300 1.958545 0.003333 1.958545 1 0.00652848 300 0.012786 324.935 8.31167 8.31167 500 3.025103 0.002 3.025103 1 0.00605021 500 0.018302 503.3842 0.676838 0.676838 800 4.831844 0.00125 4.831844 1 0.00603981 800 0.029183 805.6758 0.709473 0.709473 20 0.117519 0.05 0.117519 1 0.00587595 20 0.000691 16.90704 -15.4648 15.4648 40 0.245724 0.025 0.245724 1 0.00614309 40 0.00151 38.35736 -4.10661 4.106606 100 0.61461 0.01 0.61461 1 0.0061461 100 0.003777 100.0768 0.076839 0.076839 300 1.876043 0.003333 1.876043 1 0.00625348 300 0.011732 311.1312 3.710396 3.710396 500 3.17381 0.002 3.17381 1 0.00634762 500 0.020146 528.2649 5.652973 5.652973 800 4.763378 0.00125 4.763378 1 0.00595422 800 0.028362 794.2204 -0.72245 0.722448 Sum 8800 53.09004 0.457917 53.09004 30 0.18684581 8800 0.321029 150.499 Slope 0.00597682 Intercept 0.01646882 R2 0.99868766 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.117023 0.0025 0.00585117 0.05 0.000293 1 3.42E-05 18.22179 -8.89103 8.891026 40 0.250661 0.000625 0.00626653 0.025 0.000157 1 3.93E-05 39.93933 -0.15167 0.151672 100 0.632814 0.0001 0.00632814 0.01 6.33E-05 1 4E-05 102.0432 2.043158 2.043158 300 1.795105 1.11E-05 0.00598368 0.003333 1.99E-05 1 3.58E-05 290.9273 -3.02424 3.024237 500 2.813898 0.000004 0.0056278 0.002 1.13E-05 1 3.17E-05 456.4916 -8.70167 8.701673 800 4.461032 1.56E-06 0.00557629 0.00125 6.97E-06 1 3.11E-05 724.1678 -9.47902 9.479021 20 0.12708 0.0025 0.006354 0.05 0.000318 1 4.04E-05 19.85611 -0.71943 0.719428 40 0.273151 0.000625 0.00682878 0.025 0.000171 1 4.66E-05 43.59418 8.985454 8.985454 100 0.644615 0.0001 0.00644615 0.01 6.45E-05 1 4.16E-05 103.9609 3.960911 3.960911 300 1.916885 1.11E-05 0.00638962 0.003333 2.13E-05 1 4.08E-05 310.7178 3.572614 3.572614 500 3.067019 0.000004 0.00613404 0.002 1.23E-05 1 3.76E-05 497.6264 -0.47472 0.474716 800 4.554611 1.56E-06 0.00569326 0.00125 7.12E-06 1 3.24E-05 739.3754 -7.57808 7.578076 20 0.132683 0.0025 0.00663414 0.05 0.000332 1 4.4E-05 20.76661 3.833054 3.833054 40 0.267795 0.000625 0.00669488 0.025 0.000167 1 4.48E-05 42.7238 6.809498 6.809498 100 0.656048 0.0001 0.00656048 0.01 6.56E-05 1 4.3E-05 105.8188 5.818794 5.818794 300 1.912219 1.11E-05 0.00637406 0.003333 2.12E-05 1 4.06E-05 309.9595 3.319834 3.319834 500 3.082931 0.000004 0.00616586 0.002 1.23E-05 1 3.8E-05 500.2123 0.042454 0.042454 800 4.731126 1.56E-06 0.00591391 0.00125 7.39E-06 1 3.5E-05 768.0609 -3.99239 3.992386 20 0.128757 0.0025 0.00643785 0.05 0.000322 1 4.14E-05 20.12865 0.643247 0.643247 40 0.271073 0.000625 0.00677682 0.025 0.000169 1 4.59E-05 43.25644 8.141089 8.141089 100 0.646938 0.0001 0.00646938 0.01 6.47E-05 1 4.19E-05 104.3383 4.338337 4.338337 300 1.958545 1.11E-05 0.00652848 0.003333 2.18E-05 1 4.26E-05 317.4881 5.829354 5.829354 500 3.025103 0.000004 0.00605021 0.002 1.21E-05 1 3.66E-05 490.8147 -1.83707 1.837065 800 4.831844 1.56E-06 0.00603981 0.00125 7.55E-06 1 3.65E-05 784.4287 -1.94642 1.946417 20 0.117519 0.0025 0.00587595 0.05 0.000294 1 3.45E-05 18.30236 -8.48822 8.488221 40 0.245724 0.000625 0.00614309 0.025 0.000154 1 3.77E-05 39.13692 -2.1577 2.157699 100 0.61461 0.0001 0.0061461 0.01 6.15E-05 1 3.78E-05 99.08468 -0.91532 0.915321 300 1.876043 1.11E-05 0.00625348 0.003333 2.08E-05 1 3.91E-05 304.0805 1.360164 1.360164 500 3.17381 0.000004 0.00634762 0.002 1.27E-05 1 4.03E-05 514.9811 2.996224 2.996224 800 4.763378 1.56E-06 0.00595422 0.00125 7.44E-06 1 3.55E-05 773.3021 -3.33723 3.337232 8800 53.09004 0.016208 0.18684581 0.457917 0.002897 30 0.001167 123.3884 Slope 0.00615346 Intercept 0.00489632 R2 0.99774145 Validation Study 1 LINEARITY Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 0.9962 Intercept 0.4272

20 ng/mL 20160505_MRL 20.00 18.94 Std err in slope, Sb 0.0023 Std err in intercept 0.3637 40 ng/mL 20160505_MRL 40.00 41.00 Degrees freedom 34 Degrees freedom 34 100 ng/mL 20160505_MRL 100.00 104.09 Confidence level 95% Confidence level 95% 300 ng/mL 20160505_MRL 300.00 295.95 Student t 2.0322 Student t 2.0322 20 ng/mL 20160512_MRL 20.00 19.09 Confidence interval 0.0046 Confidence interval 0.7391 40 ng/mL 20160512_MRL 40.00 41.93 Slope 0.9962 ± 0.0046 Intercept 0.4272 ± 0.7391 100 ng/mL 20160512_MRL 100.00 100.01 Range 0.9916 - 1.0009 Lower -0.3119 - 1.1664 300 ng/mL 20160512_MRL 300.00 298.94 PASS PASS 20 ng/mL 20160829_MRL 20.00 19.49 40 ng/mL 20160829_MRL 40.00 41.32 100 ng/mL 20160829_MRL 100.00 99.23 300 ng/mL 20160829_MRL 300.00 299.95 20 ng/mL 20161005_MRL 20.00 19.93 40 ng/mL 20161005_MRL 40.00 40.38 100 ng/mL 20161005_MRL 100.00 99.22 300 ng/mL 20161005_MRL 300.00 300.45 350.00 20 ng/mL 20161011_MRL 20.00 20.26 300.00 y = 0.9962x + 0.4272 40 ng/mL 20161011_MRL 40.00 39.07 R² = 0.9998 100 ng/mL 20161011_MRL 100.00 101.17 250.00 300 ng/mL 20161011_MRL 300.00 299.49 20 ng/mL 20161013_MRL 20.00 20.36 200.00 40 ng/mL 20161013_MRL 40.00 39.65 150.00 100 ng/mL 20161013_MRL 100.00 98.58 300 ng/mL 20161013_MRL 300.00 301.39 100.00 20 ng/mL 20161021_MRL 20.00 19.88 50.00 40 ng/mL 20161021_MRL 40.00 40.06 100 ng/mL 20161021_MRL 100.00 100.56 0.00 300 ng/mL 20161021_MRL 300.00 299.48 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 20 ng/mL 20161026_MRL 20.00 20.38 40 ng/mL 20161026_MRL 40.00 38.79 100 ng/mL 20161026_MRL 100.00 101.23 300 ng/mL 20161026_MRL 300.00 299.58 20 ng/mL 20161027_MRL 20.00 19.07 40 ng/mL 20161027_MRL 40.00 40.84 100 ng/mL 20161027_MRL 100.00 103.74 300 ng/mL 20161027_MRL 300.00 296.33

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 UPPER LINEARITY Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0000 Intercept -0.0073

100 ng/mL 20160505_MRL 100.00 95.12 Std err in slope, Sb 0.0066 Std err in intercept 3.2681 300 ng/mL 20160505_MRL 300.00 308.97 Degrees freedom 34 Degrees freedom 34 500 ng/mL 20160505_MRL 500.00 496.42 Confidence level 95% Confidence level 95% 800 ng/mL 20160505_MRL 800.00 799.48 Student t 2.0322 Student t 2.0322 100 ng/mL 20160512_MRL 100.00 83.42 Confidence interval 0.0134 Confidence interval 6.6416 300 ng/mL 20160512_MRL 300.00 312.01 Slope 1 ± 0.013 Intercept -0.007 ± 6.642 500 ng/mL 20160512_MRL 500.00 518.64 Range 0.9867 - 1.0134 Lower -6.6488 - 6.6343 800 ng/mL 20160512_MRL 800.00 785.91 PASS PASS 100 ng/mL 20160829_MRL 100.00 92.93 300 ng/mL 20160829_MRL 300.00 313.25 500 ng/mL 20160829_MRL 500.00 494.40 800 ng/mL 20160829_MRL 800.00 799.41 100 ng/mL 20161005_MRL 100.00 89.81 300 ng/mL 20161005_MRL 300.00 302.03 500 ng/mL 20161005_MRL 500.00 520.36 800 ng/mL 20161005_MRL 800.00 787.78 900.00 100 ng/mL 20161011_MRL 100.00 92.72 800.00 y = 1x - 0.0073 300 ng/mL 20161011_MRL 300.00 309.20 R² = 0.9985 700.00 500 ng/mL 20161011_MRL 500.00 501.64 800 ng/mL 20161011_MRL 800.00 796.43 600.00 100 ng/mL 20161013_MRL 100.00 87.65 500.00 300 ng/mL 20161013_MRL 300.00 307.45 400.00 500 ng/mL 20161013_MRL 500.00 516.29 300.00 800 ng/mL 20161013_MRL 800.00 788.53 100 ng/mL 20161021_MRL 100.00 94.21 200.00 300 ng/mL 20161021_MRL 300.00 309.14 100.00 500 ng/mL 20161021_MRL 500.00 498.25 0.00 800 ng/mL 20161021_MRL 800.00 798.38 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 100 ng/mL 20161026_MRL 100.00 99.40 300 ng/mL 20161026_MRL 300.00 309.74 500 ng/mL 20161026_MRL 500.00 485.15 800 ng/mL 20161026_MRL 800.00 805.69 100 ng/mL 20161027_MRL 100.00 98.46 300 ng/mL 20161027_MRL 300.00 293.51 500 ng/mL 20161027_MRL 500.00 514.38 800 ng/mL 20161027_MRL 800.00 793.63

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 20 18.94 -5.30 Level 2 40 41.00 2.50 Level 3 100 104.09 4.09 Level 4 300 295.95 -1.35 5/5/16 Upper-Level 3 100 95.12 -4.88 Upper-Level 4 300 308.97 2.99 Level 5 500 496.42 -0.72 Level 6 800 799.48 -0.06 Level 1 20 19.09 -4.55 Level 2 40 41.93 4.83 Level 3 100 100.01 0.01 Level 4 300 298.94 -0.35 5/9/16 Upper-Level 3 100 83.42 -16.58 Upper-Level 4 300 312.01 4.00 Level 5 500 518.64 3.73 Level 6 800 785.91 -1.76 Level 1 20 19.49 -2.55 Level 2 40 41.32 3.30 Level 3 100 99.23 -0.77 Level 4 300 299.95 -0.02 8/29/16 Upper-Level 3 100 92.93 -7.07 Upper-Level 4 300 313.25 4.42 Level 5 500 494.40 -1.12 Level 6 800 799.41 -0.07 Level 1 20 19.93 -0.35 Level 2 40 40.38 0.95 Level 3 100 99.22 -0.78 Level 4 300 300.45 0.15 10/5/16 Upper-Level 3 100 89.81 -10.19 Upper-Level 4 300 302.03 0.68 Level 5 500 520.36 4.07 Level 6 800 787.78 -1.53 Level 1 20 20.26 1.30 Level 2 40 39.07 -2.33 Level 3 100 101.17 1.17 Level 4 300 299.49 -0.17 10/11/16 Upper-Level 3 100 92.72 -7.28 Upper-Level 4 300 309.20 3.07 Level 5 500 501.64 0.33 Level 6 800 796.43 -0.45

Max %RE LOQ = -5.30 Max %RE other calibrators = -16.58

Comments:

Acceptance Criteria: %RE Calibrators ± 20% of target (25% at lowest calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

6 Run One LOWER Curve UPPER Curve Slope 0.00606 Slope 0.00544 4 Intercept 0.00224 Intercept 0.116 R2 0.999 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Two Slope 0.0064 Slope 0.00557 4 Intercept 0.00494 Intercept 0.18 R2 1.000 R2 0.996 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Three Slope 0.0065 Slope 0.00592 4 Intercept 0.171 Intercept 0.0965 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Four Slope 0.00627 Slope 0.00594 4 Intercept 0.00743 Intercept 0.0807 R2 1.000 R2 0.997 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Five Slope 0.00658 Slope 0.00603 4 Intercept 0.0198 Intercept 0.087 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Cnom y (Response Ratio) 6 Combined Regression 20 0.11702335 40 0.250661275 100 0.632814498 5 300 1.795104853 500 2.813897931 800 4.46103188 4 20 0.127080065 40 0.273151239 100 0.644615308 300 1.916885168 3 500 3.067019054 800 4.554610943 Response Ratio Response 20 0.132682765 2 40 0.267795378 100 0.65604771 300 1.912218763 500 3.082930964 1 800 4.731126055 20 0.128757098 40 0.271072932 0 100 0.646937783 0.000 100.000 200.000 300.000 400.000 500.000 600.000 700.000 800.000 900.000 300 1.958545435 Concentration 500 3.025103267 800 4.831844319 Slope 0.0060 20 0.117519079 Intercept 0.0165 40 0.245723674 R2 0.9987 100 0.614609625 300 1.876042525 500 3.173810444 800 4.763377724 Validation Study 2 LOD Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 18.00 15.00 10.00 Source 1-1 117.6 144.5 95.5 Source 1-2 161.1 155.5 107.9 Source 2-1 118.1 151.7 92.4 5/5/16 Source 2-2 746.5 99.2 94.5 Source 3-1 115.6 120.7 144.1 Source 3-2 101.3 138.8 125.6 Source 1-1 107.2 84.2 135.0 Source 1-2 138.3 110.6 134.7 Source 2-1 85.0 122.8 95.0 5/12/16 Source 2-2 166.4 114.4 125.2 Source 3-1 92.9 98.2 97.8 Source 3-2 138.0 94.3 106.4 Source 1-1 64.6 65.4 80.5 Source 1-2 90.7 122.0 139.8 Source 2-1 86.1 44.7 N/A *Poor extraction recovery 8/29/16 Source 2-2 63.6 118.2 50.2 Source 3-1 42.8 29.1 68.7 Source 3-2 96.2 71.8 59.3

Mean Signal to Noise: 140.667 104.783 103.094

Established LOD: 20.00 ng/mL S:N of LOD: >140.667; S:N not documented for 20 ng/mL because S:N of 18 ng/mL was < 3.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three levels Comments: did not consistently meet ion ratio criteria (red values indicate failed ion ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for the LOD experiments.

Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 20.00 8/29/16 1-1 20.23 1-2 20.22 1-3 19.75 Within Run Mean 20.07 SD 0.27 %CV 1.37% % Bias 0.33% 10/5/16 2-1 19.89 2-2 19.73 2-3 19.85 Within Run Mean 19.82 SD 0.08 %CV 0.42% % Bias -0.88% 10/11/16 3-1 19.76 3-2 19.79 3-3 20.21 Within Run Mean 19.92 SD 0.25 %CV 1.26% % Bias -0.40% 10/13/16 4-1 19.79 4-2 20.72 4-3 20.36 Within Run Mean 20.29 SD 0.47 %CV 2.31% % Bias 1.45% 10/26/16 5-1 20.46 5-2 20.67 5-3 20.88 Within Run Mean 20.67 SD 0.21 %CV 1.02% % Bias 3.35%

Mean 20.15 Imprecision Max Within-Run 2.31% (%CV) Between-Run 1.96% % Bias 0.77%

Comments:

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 25.00 250.00 250.00 400.00 8/29/16 1-1 24.03 221.71 227.37 387.22 1-2 23.56 230.93 237.50 387.67 1-3 22.61 230.02 236.50 356.32 Within Run Mean 23.40 227.55 233.79 377.07 SD 0.72 5.08 5.58 17.97 %CV 3.09% 2.23% 2.39% 4.77% % Bias -6.40% -8.98% -6.48% -5.73% 10/5/16 2-1 24.05 231.29 229.09 392.47 2-2 25.27 238.49 236.68 385.67 2-3 24.54 233.94 231.89 382.99 Within Run Mean 24.62 234.57 232.55 387.04 SD 0.61 3.64 3.84 4.89 %CV 2.49% 1.55% 1.65% 1.26% % Bias -1.52% -6.17% -6.98% -3.24% 10/11/16 3-1 26.55 247.16 252.08 382.14 3-2 26.73 228.88 232.13 370.44 3-3 24.59 220.51 222.99 377.08 Within Run Mean 25.96 232.18 235.73 376.55 SD 1.19 13.63 14.88 5.87 %CV 4.57% 5.87% 6.31% 1.56% % Bias 3.83% -7.13% -5.71% -5.86% 10/13/16 4-1 27.96 251.06 252.83 380.50 4-2 25.55 234.23 234.94 392.66 4-3 25.79 234.05 234.51 388.68 Within Run Mean 26.43 239.78 240.76 387.28 SD 1.33 9.77 10.46 6.20 %CV 5.02% 4.07% 4.34% 1.60% % Bias 5.73% -4.09% -3.70% -3.18% 10/26/16 5-1 25.90 252.02 259.30 386.05 5-2 26.15 244.68 251.52 383.46 5-3 25.30 254.82 262.28 388.48 Within Run Mean 25.78 250.51 257.70 386.00 SD 0.44 5.24 5.56 2.51 %CV 1.69% 2.09% 2.16% 0.65% % Bias 3.13% 0.20% 3.08% -3.50% 11/18/16 5-1 25.47 250.95 246.15 401.45 5-2 24.38 251.07 246.26 398.81 5-3 25.13 262.31 257.08 399.21 Within Run Mean 24.99 254.78 249.83 399.82 SD 0.56 6.52 6.28 1.42 %CV 2.23% 2.56% 2.51% 0.36% % Bias -0.03% 1.91% -0.07% -0.04%

Mean 25.20 239.90 241.73 385.63 SD 1.27 12.13 11.90 10.69 Imprecision Max Within-Run 5.02% 5.87% 6.31% 4.77% (%CV) Between-Run 5.03% 5.06% 4.92% 2.77% % Bias 0.79% -4.04% -3.31% -3.59%

Comments:

Bias: ≤ 20% Acceptance Criteria: Within-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 20.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/5/16 10/11/16 High Calibrator 800.00 799.48 785.91 799.41 787.78 796.43 Negative -0.17 -0.56 1.24 1.19 3.01 % of Low Calibrator -0.9% -2.8% 6.2% 6.0% 15.1% Maximum Carryover: 15.1%

Comments: All ion ratios for hydromorphone in the Negative (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 21366 Run 2 86166 Run 3 86540 Run 4 101483 Run 5 50202 Average 69151

Identification Peak Response Comment Blank Matrix (NO IS) 1 16 Pass Blank Matrix (NO IS) 2 53 Pass Blank Matrix (NO IS) 3 16 Pass Blank Matrix (NO IS) 4 15 Pass Blank Matrix (NO IS) 5 22 Pass Blank Matrix (NO IS) 6 42 Pass Blank Matrix (NO IS) 7 17 Pass Blank Matrix (NO IS) 8 27 Pass Blank Matrix (NO IS) 9 24 Pass Blank Matrix (NO IS) 10 20 Pass Blank Matrix (with IS) 1 862 Pass Blank Matrix (with IS) 2 1377 Pass Blank Matrix (with IS) 3 1685 Pass Blank Matrix (with IS) 4 35 Pass Blank Matrix (with IS) 5 35 Pass Blank Matrix (with IS) 6 937 Pass Blank Matrix (with IS) 7 526 Pass Blank Matrix (with IS) 8 25 Pass Blank Matrix (with IS) 9 61 Pass Blank Matrix (with IS) 10 1600 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is no interference from labeled IS. Comments:

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 25.20 ng/mL Control Acceptance (%): 20 Run Date: 10/21/2016

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration 7-Aminoclonazepam Nordiazepam Oxazepam Diazepam 1 Benzodiazepines Mix Lorazepam 1,000 ng/mL 26.72 Pass Temazepam Clonazepam Alpha-hydroxyalprazolam Alprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 23.93 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 24.36 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 26.19 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 26.25 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 24.32 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 23.63 Pass

Conclusions: There are 0 compounds tested in this study that cause significant interference with the analyte of interest.

Comments:

Acceptance Criteria: Concentration of analytes of interest within ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated %Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 250.00 500.00 125.00 500.00 50.00 500.00 250.00 500.00 125.00 500.00 Run 1 1-1 236.37 472.74 -5.45 119.17 476.68 -4.66 50.18 501.80 0.36 240.30 480.60 -3.88 112.27 449.08 -10.18 10/11/16 1-2 220.35 440.70 -11.86 120.68 482.72 -3.46 50.49 504.90 0.98 222.81 445.62 -10.88 114.01 456.04 -8.79 Run 2 2-1 271.14 542.28 8.46 142.32 569.28 13.86 56.61 566.10 13.22 274.97 549.94 9.99 135.36 541.44 8.29 10/13/16 2-2 269.33 538.66 7.73 139.25 557.00 11.40 57.46 574.60 14.92 273.32 546.64 9.33 131.56 526.24 5.25 Run 3 3-1 252.62 505.24 1.05 126.02 504.08 0.82 54.18 541.80 8.36 259.93 519.86 3.97 125.69 502.76 0.55 10/26/16 3-2 250.48 500.96 0.19 125.77 503.08 0.62 52.70 527.00 5.40 257.67 515.34 3.07 125.42 501.68 0.34

Mean: 500.10 515.47 536.03 509.67 496.21 SD: 38.91 38.68 30.49 40.18 37.03 Between-Run Imprecision (%CV): 7.78 7.50 5.69 7.88 7.46 %Bias: 0.02 3.09 7.21 1.93 -0.76

Comments: Up to 10x dilution can be used for the lower curve and 4x for the upper curve.

Acceptance Criteria: Concentration within 20% of target Validation Study 8 IN-PROCESS STABILITY Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20%

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 24.05 Pass 24.22 20.16 30.24 LQC-2 24.39 Pass MQC-1 251.35 Pass 237.11 191.92 287.87 MQC-2 222.86 Pass 10/11/16 *MQC-1 256.65 Pass *Upper curve 241.11 193.38 290.07 *MQC-2 225.56 Pass *Upper curve HQC-1 362.90 Pass 362.25 308.50 462.75 HQC-2 361.60 Pass

Overall Acceptance: Stable for 3 hours

Comments:

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20%

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 20.00 18.66 N/A 16.00 24.00 Pass 40.00 38.23 N/A 32.00 48.00 Pass 100.00 102.94 N/A 80.00 120.00 Pass 300.00 313.49 N/A 240.00 360.00 Pass 100.00 97.65 N/A 80.00 120.00 Pass *Upper Curve 300.00 310.88 N/A 240.00 360.00 Pass *Upper Curve 500.00 495.10 N/A 400.00 600.00 Pass Injected on 10/26/16; 800.00 844.51 N/A 640.00 960.00 Pass Reinjected on 24.11 10/27/16 25.20 24.31 20.16 30.24 Pass 24.51 256.11 239.90 252.00 191.92 287.87 Pass 247.88 252.78 *Upper Curve 241.73 248.61 193.38 290.07 Pass 244.44 384.62 385.63 387.44 308.50 462.75 Pass 390.26

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 24.70 Pass 24.72 20.16 30.24 LQC-2 Punctured 24.73 Pass MQC-1 Punctured 250.52 Pass Injected on 248.50 191.92 287.87 10/27/16; MQC-2 Punctured 246.48 Pass Reinjected on *MQC-1 Punctured 247.11 Pass *Upper Curve 10/28/16 245.07 193.38 290.07 *MQC-2 Punctured 243.02 Pass *Upper Curve HQC-1 Punctured 387.31 Pass 384.38 308.50 462.75 HQC-2 Punctured 381.44 Pass LQC-1 Unpunctured 24.77 Pass 24.40 20.16 30.24 LQC-2 Unpunctured 24.02 Pass MQC-1 Unpunctured 244.61 Pass Extracted on 241.33 191.92 287.87 10/27/16; MQC-2 Unpunctured 238.05 Pass Injected on *MQC-1 Unpunctured 241.12 Pass *Upper Curve 10/28/16 237.80 193.38 290.07 *MQC-2 Unpunctured 234.48 Pass *Upper Curve HQC-1 Unpunctured 384.75 Pass 378.45 308.50 462.75 HQC-2 Unpunctured 372.15 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 Room Temperature LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Room Temperature LQC-1 w/ Light Protection LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Refrigerated (2 to 8oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Frozen (-5 to -20oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Peak Extraction Average Type of Sample Sample ID Response of Efficiency Matrix Effect Response Drug (%Recovery)

Group 1-Internal Standard and drug analyte fortified before extraction

Group 2-Internal Standard and analyte fortified after extraction

Group 3-Internal Standard and analyte fortified to elution solvent/no extraction

Extraction efficiency and matrix effects were not performed for hydrocodone, hydromorphone, Comments: oxycodone, and oxymorphone because derivatization needs to occur prior to extraction for these four analytes.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction efficiency Criteria: and matrix effect are considered acceptable. Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 50.0 60.25 40-60 20.5 Fail Blind Sample 3 100.0 102.33 80-120 2.3 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 120.0 122.53 96-144 2.1 Pass Blind Sample 7 400.0 445.95 320-480 11.5 Pass Blind Sample 8 250.0 299.90 200-300 20.0 Pass Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 500.0 536.25 400-600 7.3 Pass 11/9/16 Blind Sample 11 600.0 625.94 480-720 4.3 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 800.0 814.83 640-960 1.9 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 160.0 161.88 98-192 1.2 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 400.0 438.89 320-480 9.7 Pass

Blind Sample 2 did not meet acceptance criteria. The quality controls ran with the blind samples quantitated on target for Comments: hydromorphone (< ± 3%). Blind Sample 2 quantitated high for all drugs. Therefore, the high quantitation can be attributed to over-spiking the blind sample.

Acceptance Criteria: Percent difference of the expected vs. determined values ≤ 20% SUMMARY OF VALIDATION PERFORMANCE Analyte: Hydromorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason There were multiple interferences with ions causing quantitative values, ion August 2, 2016 ratios, and overall chromatography to not be acceptable.

Autosampler stability was attempted on this day, but the extraction vials October 13, 2016 dried out prior to injection so the experiment was repeated on October 27, 2016.

Deviations from SOP:

Other Observations: Per reccomendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of hydromorphone confirmation analysis. METHOD VALIDATION PROTOCOL AND RESULTS Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Hydoxy.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS Data analysis was processed using 1/x weighting for the lower curve and Unweighted: = 361.5 unweighted for the upper curve even though 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 163.38 1/x2 gave the lowest %RE because all other 1/x2 Weighting: = 138.38 validation parameters met acceptance criteria.

95% CI of the slope does not include 1. However, linearity is acceptable based on 95% CI of slope includes 1 95% CI of Slope = 0.9908 - 0.9992 evaluation of the standard residual plot, 1 Linearity 95% CI of intercept includes 0 95% CI of Intercept = -0.0989 - 1.2450 correlation coefficients, standard error, and % difference of individual calibrator values from the target concentration.

95% CI of slope includes 1 95% CI of Slope = 0.9862 - 1.0138 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -6.8650 - 6.8607

Max %RE low calibrator = -4.60 1 Validation Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) Max %RE other calibrators = -18.11

Not performed. Validation of 6 calibrators will 1 Casework Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) be used for casework.

Comparison of Validation Calibration to 95% CI of slope includes 1 Not performed. Validation of 6 calibrators will 1 Casework Calibration 95% CI of intercept includes 0 be used for casework.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three LOD = 20 levels did not consistently meet ion ratio Signal to Noise ≥ 3 2 Limit of Detection (LOD) S:N = >669.711; S:N not documented for 20 criteria (red values above indicate failed ion Acceptable detection and identification criteria ng/mL because S:N of 18 ng/mL was > 3. ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments.

Replicates in red failed to meet ion ratio Bias: ≤ 25% Bias = -2.08% acceptance crieteria. Given that the 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-Run Imprecision= 2.83% quantified concentrations are acceptable, 20 Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 3.22% ng/mL will be used as the LOQ.

Bias Bias: ≤ 20% Max Bias = 6.04% Red values indicate ion ratios not meeting 4 Within-Run Imprecision Within-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.11% acceptance criteria. Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 5.98%

All ion ratios for oxycodone in the Negative (in red) do not meet acceptance criteria. 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 8.10% Therefore, the method is considered to have no significant carryover.

6 Matrix Interference Response of blank matrix ≤ 10% of the average response of LOQ No matrix interference was identified. The Blank Matrix (with IS) 10 (in red) was > 10% of the average response of the LOQ. However, ion ratios did not meet acceptance 6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is no interference from labeled IS. criteria. Therefore, the method is considered to have no significant carryover.

The red value indicates ion ratios that failed There are 0 compounds tested in this study to meet acceptance criteria. Concentration of analytes of interest within ± 20% of average for bias 6 Exogenous Substances Interferences that cause significant interference with the Benzodiazepines may have contributed to and imprecision analyte of interest. the failed ion ratio. If ion ratios are not acceptable, results will not be reported.

Up to 10x dilution can be used for the lower 7 Dilution Integrity Concentration within ± 20% of target Max % of target = 16.30 curve and 4x for the upper curve.

Red values indicate ion ratios that did not meet acceptance criteria. Given the Concentration within 20% of the average value obtained in the Bias and quantified concentrations are acceptable, up 8 In-Process Stability Stable for 3 hours Imprecision studies to a 3 hour delay can be used. If ion ratios are not acceptable, results will not be reported.

Punctured vials in Experiment 1 were stable Concentration within 20% of the average value obtained in the Bias and Unpunctured Vials:Stable for 24 hours for 24 hours. Punctured and unpunctured 8 Autosampler Stability Imprecision studies Punctured Vials:Stable for 24 hours vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Study not performed; storage stability is documented in Disposition of Toxic Drugs 8 Storage Stability Concentration within 20% of average time 0 and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Extraction efficiency and matrix effects were not performed for hydrocodone, If method achieves desired limit of quantification and/or detection for the hydromorphone, oxycodone, or 9 Extraction Efficiency & Matrix Effect analyte, its extraction efficiency and matrix effect are considered Study not performed oxymorphone because derivatization needs acceptable to occur prior to extraction for these four analytes.

All Blind Samples had to be reinjected for oxycodone because the drug was not within its SIM window in the original injection. All 10 Blind Sample Analysis Percent difference of the expected vs. determined values ≤ 20% 100% of samples ≤ 20% of target value reinjection values fell within ± 20% of the target and were therefore deemed acceptable. * Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 361.50 1/x Weighting: 163.38 1/x2 Weighting: 138.38

Comments: Data analysis was processed using 1/x weighting for the lower curve and unweighted for the upper curve even though 1/x2 gave the lowest %RE because all other validation parameters met acceptance criteria.

Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 20 0.147743 1 2.954864 20 0.14774322 400 0.021828 11.17784 -44.1108 44.11079 40 0.299004 1 11.96017 40 0.29900414 1600 0.089403 35.31462 -11.7135 11.71346 100 0.754255 1 75.42549 100 0.75425491 10000 0.5689 107.9592 7.95919 7.95919 300 2.119052 1 635.7155 300 2.11905171 90000 4.49038 325.7404 8.580146 8.580146 500 3.26425 1 1632.125 500 3.26424998 250000 10.65533 508.4802 1.69605 1.69605 800 5.116501 1 4093.201 800 5.11650084 640000 26.17858 804.0448 0.505596 0.505596 20 0.145806 1 2.916114 20 0.14580569 400 0.021259 10.86867 -45.6567 45.65666 40 0.308618 1 12.34471 40 0.30861763 1600 0.095245 36.84864 -7.87839 7.87839 100 0.718842 1 71.88418 100 0.71884179 10000 0.516734 102.3083 2.308301 2.308301 300 2.133646 1 640.0937 300 2.13364566 90000 4.552444 328.0692 9.3564 9.3564 500 3.376152 1 1688.076 500 3.37615248 250000 11.39841 526.3366 5.267317 5.267317 800 5.001375 1 4001.1 800 5.00137497 640000 25.01375 785.6741 -1.79074 1.79074 20 0.153453 1 3.069064 20 0.15345322 400 0.023548 12.08899 -39.5551 39.55505 40 0.297748 1 11.9099 40 0.29774753 1600 0.088654 35.1141 -12.2148 12.21476 100 0.703397 1 70.33969 100 0.70339691 10000 0.494767 99.84375 -0.15625 0.156245 300 2.019421 1 605.8263 300 2.01942109 90000 4.078062 309.8423 3.280779 3.280779 500 3.184667 1 1592.334 500 3.18466743 250000 10.14211 495.7812 -0.84375 0.843755 800 4.922971 1 3938.377 800 4.92297072 640000 24.23564 773.1631 -3.35462 3.354615 20 0.145945 1 2.918898 20 0.14594492 400 0.0213 10.89089 -45.5456 45.54557 40 0.300562 1 12.02248 40 0.30056211 1600 0.090338 35.56322 -11.0919 11.09195 100 0.696137 1 69.61367 100 0.69613673 10000 0.484606 98.68525 -1.31475 1.314755 300 2.064139 1 619.2417 300 2.064139 90000 4.26067 316.978 5.659331 5.659331 500 3.189768 1 1594.884 500 3.18976755 250000 10.17462 496.5951 -0.68099 0.680989 800 5.113119 1 4090.495 800 5.11311858 640000 26.14398 803.5051 0.438132 0.438132 20 0.133311 1 2.66622 20 0.13331101 400 0.017772 8.874887 -55.6256 55.62557 40 0.274636 1 10.98542 40 0.27463557 1600 0.075425 31.42611 -21.4347 21.43472 100 0.660704 1 66.07036 100 0.66070362 10000 0.436529 93.03117 -6.96883 6.968833 300 1.97684 1 593.0521 300 1.97684042 90000 3.907898 303.0477 1.015906 1.015906 500 3.299639 1 1649.819 500 3.29963892 250000 10.88762 514.1273 2.825456 2.825456 800 4.957164 1 3965.731 800 4.95716398 640000 24.57347 778.6193 -2.67259 2.672586 Sum 8800 57.47886 5 31767.15 8800 57.4788623 4960000 203.7393 361.502 Slope 0.00626682 Intercept 0.07769365 R2 0.99792371 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.147743 0.05 0.147743 1 0.00738716 20 0.001091 18.13461 -9.32696 9.326961 40 0.299004 0.025 0.299004 1 0.0074751 40 0.002235 41.67627 4.190677 4.190677 100 0.754255 0.01 0.754255 1 0.00754255 100 0.005689 112.5297 12.52974 12.52974 300 2.119052 0.003333 2.119052 1 0.00706351 300 0.014968 324.9414 8.313805 8.313805 500 3.26425 0.002 3.26425 1 0.0065285 500 0.021311 503.1756 0.635127 0.635127 800 5.116501 0.00125 5.116501 1 0.00639563 800 0.032723 791.4528 -1.0684 1.068403 20 0.145806 0.05 0.145806 1 0.00729028 20 0.001063 17.83306 -10.8347 10.83471 40 0.308618 0.025 0.308618 1 0.00771544 40 0.002381 43.17248 7.931191 7.931191 100 0.718842 0.01 0.718842 1 0.00718842 100 0.005167 107.0182 7.018174 7.018174 300 2.133646 0.003333 2.133646 1 0.00711215 300 0.015175 327.2128 9.07092 9.07092 500 3.376152 0.002 3.376152 1 0.0067523 500 0.022797 520.5917 4.118341 4.118341 800 5.001375 0.00125 5.001375 1 0.00625172 800 0.031267 773.535 -3.30812 3.308121 20 0.153453 0.05 0.153453 1 0.00767266 20 0.001177 19.02329 -4.88355 4.883545 40 0.297748 0.025 0.297748 1 0.00744369 40 0.002216 41.4807 3.701743 3.701743 100 0.703397 0.01 0.703397 1 0.00703397 100 0.004948 104.6144 4.614393 4.614393 300 2.019421 0.003333 2.019421 1 0.0067314 300 0.013594 309.4353 3.145098 3.145098 500 3.184667 0.002 3.184667 1 0.00636933 500 0.020284 490.7897 -1.84206 1.842057 800 4.922971 0.00125 4.922971 1 0.00615371 800 0.030295 761.3325 -4.83344 4.833438 20 0.145945 0.05 0.145945 1 0.00729725 20 0.001065 17.85473 -10.7264 10.72636 40 0.300562 0.025 0.300562 1 0.00751405 40 0.002258 41.91875 4.796869 4.796869 100 0.696137 0.01 0.696137 1 0.00696137 100 0.004846 103.4844 3.484447 3.484447 300 2.064139 0.003333 2.064139 1 0.00688046 300 0.014202 316.395 5.465005 5.465005 500 3.189768 0.002 3.189768 1 0.00637954 500 0.020349 491.5835 -1.6833 1.683305 800 5.113119 0.00125 5.113119 1 0.0063914 800 0.03268 790.9264 -1.1342 1.134204 20 0.133311 0.05 0.133311 1 0.00666555 20 0.000889 15.88843 -20.5578 20.55783 40 0.274636 0.025 0.274636 1 0.00686589 40 0.001886 37.88364 -5.2909 5.290896 100 0.660704 0.01 0.660704 1 0.00660704 100 0.004365 97.96977 -2.03023 2.030225 300 1.97684 0.003333 1.97684 1 0.00658947 300 0.013026 302.8082 0.936067 0.936067 500 3.299639 0.002 3.299639 1 0.00659928 500 0.021775 508.6834 1.736686 1.736686 800 4.957164 0.00125 4.957164 1 0.00619645 800 0.030717 766.6542 -4.16822 4.168225 Sum 8800 57.47886 0.457917 57.47886 30 0.20705527 8800 0.37644 163.3766 Slope 0.00642524 Intercept 0.03122394 R2 0.99883368 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.147743 0.0025 0.00738716 0.05 0.000369 1 5.46E-05 19.7263 -1.3685 1.368504 40 0.299004 0.000625 0.0074751 0.025 0.000187 1 5.59E-05 42.47709 6.192729 6.192729 100 0.754255 0.0001 0.00754255 0.01 7.54E-05 1 5.69E-05 110.9503 10.95027 10.95027 300 2.119052 1.11E-05 0.00706351 0.003333 2.35E-05 1 4.99E-05 316.2261 5.408693 5.408693 500 3.26425 0.000004 0.0065285 0.002 1.31E-05 1 4.26E-05 488.4726 -2.30548 2.305479 800 5.116501 1.56E-06 0.00639563 0.00125 7.99E-06 1 4.09E-05 767.0652 -4.11685 4.116848 20 0.145806 0.0025 0.00729028 0.05 0.000365 1 5.31E-05 19.43488 -2.8256 2.825602 40 0.308618 0.000625 0.00771544 0.025 0.000193 1 5.95E-05 43.92303 9.807582 9.807582 100 0.718842 0.0001 0.00718842 0.01 7.19E-05 1 5.17E-05 105.6239 5.623865 5.623865 300 2.133646 1.11E-05 0.00711215 0.003333 2.37E-05 1 5.06E-05 318.4211 6.140373 6.140373 500 3.376152 0.000004 0.0067523 0.002 1.35E-05 1 4.56E-05 505.3036 1.060718 1.060718 800 5.001375 1.56E-06 0.00625172 0.00125 7.81E-06 1 3.91E-05 749.7494 -6.28132 6.281324 20 0.153453 0.0025 0.00767266 0.05 0.000384 1 5.89E-05 20.58513 2.925638 2.925638 40 0.297748 0.000625 0.00744369 0.025 0.000186 1 5.54E-05 42.28809 5.72022 5.72022 100 0.703397 0.0001 0.00703397 0.01 7.03E-05 1 4.95E-05 103.3008 3.300838 3.300838 300 2.019421 1.11E-05 0.0067314 0.003333 2.24E-05 1 4.53E-05 301.2409 0.413626 0.413626 500 3.184667 0.000004 0.00636933 0.002 1.27E-05 1 4.06E-05 476.5028 -4.69944 4.699443 800 4.922971 1.56E-06 0.00615371 0.00125 7.69E-06 1 3.79E-05 737.9568 -7.7554 7.755399 20 0.145945 0.0025 0.00729725 0.05 0.000365 1 5.32E-05 19.45582 -2.72089 2.720892 40 0.300562 0.000625 0.00751405 0.025 0.000188 1 5.65E-05 42.71142 6.778556 6.778556 100 0.696137 0.0001 0.00696137 0.01 6.96E-05 1 4.85E-05 102.2089 2.208852 2.208852 300 2.064139 1.11E-05 0.00688046 0.003333 2.29E-05 1 4.73E-05 307.9668 2.655597 2.655597 500 3.189768 0.000004 0.00637954 0.002 1.28E-05 1 4.07E-05 477.2699 -4.54602 4.546024 800 5.113119 1.56E-06 0.0063914 0.00125 7.99E-06 1 4.08E-05 766.5565 -4.18044 4.180438 20 0.133311 0.0025 0.00666555 0.05 0.000333 1 4.44E-05 17.55558 -12.2221 12.22208 40 0.274636 0.000625 0.00686589 0.025 0.000172 1 4.71E-05 38.81187 -2.97032 2.970315 100 0.660704 0.0001 0.00660704 0.01 6.61E-05 1 4.37E-05 96.87944 -3.12056 3.120558 300 1.97684 1.11E-05 0.00658947 0.003333 2.2E-05 1 4.34E-05 294.8364 -1.72119 1.721193 500 3.299639 0.000004 0.00659928 0.002 1.32E-05 1 4.36E-05 493.7954 -1.24093 1.240926 800 4.957164 1.56E-06 0.00619645 0.00125 7.75E-06 1 3.84E-05 743.0997 -7.11253 7.112533 8800 57.47886 0.016208 0.20705527 0.457917 0.003313 30 0.001436 138.3751 Slope 0.00664860 Intercept 0.01659094 R2 0.99741157 Validation Study 1 LINEARITY Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 0.9950 Intercept 0.5731

20 ng/mL 20160505_MRL 20.00 19.18 Std err in slope, Sb 0.0021 Std err in intercept 0.3307 40 ng/mL 20160505_MRL 40.00 40.42 Degrees freedom 34 Degrees freedom 34 100 ng/mL 20160505_MRL 100.00 104.36 Confidence level 95% Confidence level 95% 300 ng/mL 20160505_MRL 300.00 296.03 Student t 2.0322 Student t 2.0322 20 ng/mL 20160512_MRL 20.00 19.08 Confidence interval 0.0042 Confidence interval 0.6720 40 ng/mL 20160512_MRL 40.00 42.02 Slope 0.995 ± 0.0042 Intercept 0.5731 ± 0.672 100 ng/mL 20160512_MRL 100.00 99.80 Range 0.9908 - 0.9992 Lower -0.0989 - 1.2450 300 ng/mL 20160512_MRL 300.00 299.08 FAIL PASS 20 ng/mL 20160829_MRL 20.00 19.22 40 ng/mL 20160829_MRL 40.00 41.80 100 ng/mL 20160829_MRL 100.00 99.57 300 ng/mL 20160829_MRL 300.00 299.39 20 ng/mL 20161005_MRL 20.00 19.54 40 ng/mL 20161005_MRL 40.00 41.06 100 ng/mL 20161005_MRL 100.00 99.72 300 ng/mL 20161005_MRL 300.00 299.66 350.00 y = 0.995x + 0.5731 20 ng/mL 20161011_MRL 20.00 19.99 R² = 0.9999 40 ng/mL 20161011_MRL 40.00 39.44 300.00 100 ng/mL 20161011_MRL 100.00 101.81 250.00 300 ng/mL 20161011_MRL 300.00 298.74 20 ng/mL 20161013_MRL 20.00 20.05 200.00 40 ng/mL 20161013_MRL 40.00 39.89 150.00 100 ng/mL 20161013_MRL 100.00 99.99 300 ng/mL 20161013_MRL 300.00 300.06 100.00 20 ng/mL 20161021_MRL 20.00 19.90 50.00 40 ng/mL 20161021_MRL 40.00 40.17 100 ng/mL 20161021_MRL 100.00 100.12 0.00 300 ng/mL 20161021_MRL 300.00 299.79 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 20 ng/mL 20161026_MRL 20.00 20.17 40 ng/mL 20161026_MRL 40.00 39.12 100 ng/mL 20161026_MRL 100.00 101.64 300 ng/mL 20161026_MRL 300.00 299.06 20 ng/mL 20161027_MRL 20.00 19.18 40 ng/mL 20161027_MRL 40.00 40.86 100 ng/mL 20161027_MRL 100.00 102.89 300 ng/mL 20161027_MRL 300.00 297.04

Comments: 95% CI of the slope does not include 1. However, linearity is acceptable based on evaluation of the standard residual plot, correlation coefficients, standard error, and % difference of individual calibrator values from the target concentration. 95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 1 - Standard Residuals

Regression Statistics Multiple R 0.999926354 R Square 0.999852714 Adjusted R S0.999848382 Standard Er 1.376743999 Observation 36

ANOVA df SS MS F Significance F Regression 1 437481.9981 437481.9981 230809.5652 9.81245E-67 Residual 34 64.44441729 1.895424038 Total 35 437546.4425

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0% Intercept 0.575480878 0.330720626 1.740081605 0.090890495 -0.096624299 1.247586055 -0.096624299 1.247586055 X Variable 1 0.994988572 0.002071053 480.426441 9.81245E-67 0.990779686 0.999197458 0.990779686 0.999197458

RESIDUAL OUTPUT

Observation Predicted Y Residuals Standard Residuals 1 20.47525232 -1.29525232 -0.954543556 2 40.37502376 0.044976239 0.033145495 3 100.0743381 4.285661914 3.158342899 Standard Residuals Plot 4 299.0720525 -3.032052501 -2.234488318 4 5 20.47525232 -1.38525232 -1.020869568 6 40.37502376 1.644976239 1.212274586 3 7 100.0743381 -0.274338086 -0.202175011 8 299.0720525 0.017947499 0.013226512 2 9 20.47525232 -1.25525232 -0.925065329 10 40.37502376 1.434976239 1.057513893 1 11 100.0743381 -0.504338086 -0.371674818 0

12 299.0720525 0.327947499 0.241682773 Residuals 0 50 100 150 200 250 300 350 13 20.47525232 -0.93525232 -0.689239511 -1 14 40.37502376 0.694976239 0.512166688 15 100.0743381 -0.354338086 -0.261131465 -2 16 299.0720525 0.587947499 0.43329125 -3 17 20.47525232 -0.47525232 -0.350239897 X Variable 1 18 40.37502376 -0.935023761 -0.689071073 19 100.0743381 1.745661914 1.286475467 20 299.0720525 -0.332052501 -0.244707977 21 20.47525232 -0.42525232 -0.313392113 22 40.37502376 -0.485023761 -0.357441017 23 100.0743381 -0.084338086 -0.062153431 24 299.0720525 0.987947499 0.728073523 25 20.47525232 -0.57525232 -0.423935465 26 40.37502376 -0.205023761 -0.151093426 27 100.0743381 0.055661914 0.041020364 28 299.0720525 0.727947499 0.536465046 29 20.47525232 -0.30525232 -0.224957431 30 40.37502376 -1.255023761 -0.924896892 31 100.0743381 1.565661914 1.153823444 32 299.0720525 -0.012052501 -0.008882159 33 20.47525232 -1.29525232 -0.954543556 34 40.37502376 0.494976239 0.364775552 35 100.0743381 2.825661914 2.082387603 36 299.0720525 -2.022052501 -1.490163079 Validation Study 1 UPPER LINEARITY Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0000 Intercept -0.0021

100 ng/mL 20160505_MRL 100.00 91.85 Std err in slope, Sb 0.0068 Std err in intercept 3.3770 300 ng/mL 20160505_MRL 300.00 312.65 Degrees freedom 34 Degrees freedom 34 500 ng/mL 20160505_MRL 500.00 497.91 Confidence level 95% Confidence level 95% 800 ng/mL 20160505_MRL 800.00 797.57 Student t 2.0322 Student t 2.0322 100 ng/mL 20160512_MRL 100.00 81.89 Confidence interval 0.0138 Confidence interval 6.8628 300 ng/mL 20160512_MRL 300.00 314.30 Slope 1 ± 0.0138 Intercept -0.0021 ± 6.8628 500 ng/mL 20160512_MRL 500.00 518.41 Range 0.9862 - 1.0138 Lower -6.8650 - 6.8607 800 ng/mL 20160512_MRL 800.00 785.39 PASS PASS 100 ng/mL 20160829_MRL 100.00 94.04 300 ng/mL 20160829_MRL 300.00 312.59 500 ng/mL 20160829_MRL 500.00 492.89 800 ng/mL 20160829_MRL 800.00 800.45 100 ng/mL 20161005_MRL 100.00 89.47 300 ng/mL 20161005_MRL 300.00 303.51 500 ng/mL 20161005_MRL 500.00 518.70 800 ng/mL 20161005_MRL 800.00 788.30 900.00 100 ng/mL 20161011_MRL 100.00 90.99 800.00 y = 1x - 0.0021 300 ng/mL 20161011_MRL 300.00 311.01 R² = 0.9984 700.00 500 ng/mL 20161011_MRL 500.00 502.65 800 ng/mL 20161011_MRL 800.00 795.33 600.00 100 ng/mL 20161013_MRL 100.00 86.88 500.00 300 ng/mL 20161013_MRL 300.00 307.71 400.00 500 ng/mL 20161013_MRL 500.00 517.84 300.00 800 ng/mL 20161013_MRL 800.00 787.62 200.00 100 ng/mL 20161021_MRL 100.00 91.25 300 ng/mL 20161021_MRL 300.00 307.00 100.00 500 ng/mL 20161021_MRL 500.00 508.72 0.00 800 ng/mL 20161021_MRL 800.00 793.01 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 100 ng/mL 20161026_MRL 100.00 100.88 300 ng/mL 20161026_MRL 300.00 304.91 500 ng/mL 20161026_MRL 500.00 489.73 800 ng/mL 20161026_MRL 800.00 804.46 100 ng/mL 20161027_MRL 100.00 96.47 300 ng/mL 20161027_MRL 300.00 296.68 500 ng/mL 20161027_MRL 500.00 513.75 800 ng/mL 20161027_MRL 800.00 793.08

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 20 19.18 -4.10 Level 2 40 40.42 1.05 Level 3 100 104.36 4.36 Level 4 300 296.03 -1.32 5/5/16 Upper-Level 3 100 91.85 -8.15 Upper-Level 4 300 312.65 4.22 Level 5 500 497.91 -0.42 Level 6 800 797.57 -0.30 Level 1 20 19.08 -4.60 Level 2 40 42.02 5.05 Level 3 100 99.80 -0.20 Level 4 300 299.09 -0.30 5/12/16 Upper-Level 3 100 81.89 -18.11 Upper-Level 4 300 314.30 4.77 Level 5 500 518.41 3.68 Level 6 800 785.39 -1.83 Level 1 20 19.22 -3.90 Level 2 40 41.80 4.50 Level 3 100 99.57 -0.43 Level 4 300 299.39 -0.20 8/29/16 Upper-Level 3 100 94.04 -5.96 Upper-Level 4 300 312.59 4.20 Level 5 500 492.89 -1.42 Level 6 800 800.45 0.06 Level 1 20 19.54 -2.30 Level 2 40 41.06 2.65 Level 3 100 99.72 -0.28 Level 4 300 299.66 -0.11 10/5/16 Upper-Level 3 100 89.47 -10.53 Upper-Level 4 300 303.51 1.17 Level 5 500 518.70 3.74 Level 6 800 788.30 -1.46 Level 1 20 19.99 -0.05 Level 2 40 39.44 -1.40 Level 3 100 101.81 1.81 Level 4 300 298.74 -0.42 10/11/16 Upper-Level 3 100 90.99 -9.01 Upper-Level 4 300 311.01 3.67 Level 5 500 502.65 0.53 Level 6 800 795.33 -0.58

Max %RE LOQ = -4.60 Max %RE other calibrators = -18.11

Comments:

Acceptance Criteria: %RE Calibrators ± 20% of target (25% at low calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

6 Run One LOWER Curve UPPER Curve Slope 0.00712 Slope 0.00618 4 Intercept 0.0112 Intercept 0.1860 R2 0.999 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Two Slope 0.0071 Slope 0.00609 4 Intercept 0.00103 Intercept 0.2200 R2 0.999 R2 0.993 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Three Slope 0.00684 Slope 0.00625 4 Intercept 0.0145 Intercept 0.1070 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Four Slope 0.00658 Slope 0.00615 4 Intercept 0.00433 Intercept 0.1110 R2 1.000 R2 0.998 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Five Slope 0.00712 Slope 0.00638 4 Intercept 0.00501 Intercept 0.1400 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Cnom y (Response Ratio) 6 Combined Regression 20 0.147743218 40 0.299004139 100 0.754254914 5 300 2.119051709 500 3.264249976 800 5.116500844 4 20 0.145805685 40 0.308617625 100 0.718841789 300 2.133645655 3 500 3.376152483 800 5.001374969 Response Ratio Response 20 0.153453224 2 40 0.29774753 100 0.703396909 300 2.019421092 500 3.184667429 1 800 4.922970723 20 0.14594492 40 0.300562111 0 100 0.696136732 0.000 100.000 200.000 300.000 400.000 500.000 600.000 700.000 800.000 900.000 300 2.064138998 Concentration 500 3.189767553 800 5.113118579 Slope 0.0064 20 0.133311006 Intercept 0.0312 40 0.274635573 R2 0.9988 100 0.660703617 300 1.97684042 500 3.299638916 800 4.957163982 Validation Study 2 LOD Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 18.00 15.00 10.00 Source 1-1 1027.3 179.9 175.9 Source 1-2 833.9 219.3 845.4 Source 2-1 1400.0 565.7 1914.7 5/5/16 Source 2-2 1035.5 583.8 198.8 Source 3-1 652.3 701.8 315.2 Source 3-2 432.1 867.0 732.6 Source 1-1 1302.5 806.5 425.2 Source 1-2 768.2 628.1 264.7 Source 2-1 1125.3 991.7 500.9 5/12/16 Source 2-2 799.8 883.4 191.0 Source 3-1 1142.5 557.1 606.1 Source 3-2 1159.3 636.7 602.6 Source 1-1 69.2 41.8 51.9 Source 1-2 90.9 54.2 48.4 Source 2-1 52.2 25.9 N/A *Poor extraction recovery *8/29/16 Source 2-2 64.1 47.9 58.3 Source 3-1 35.2 40.8 79.3 Source 3-2 64.5 81.7 23.8

Mean Signal to Noise: 669.711 439.628 413.812

Established LOD: 20.00 ng/mL S:N of LOD: >669.711; S:N not documented for 20 ng/mL because S:N of 18 ng/mL was > 3.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three Comments: levels did not consistently meet ion ratio criteria (red values above indicate failed ion ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments. Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 20.00 8/29/16 1-1 18.80 1-2 18.59 1-3 18.85 Within Run Mean 18.75 SD 0.14 %CV 0.74% % Bias -6.27% 10/5/16 2-1 19.51 2-2 19.18 2-3 19.52 Within Run Mean 19.40 SD 0.19 %CV 1.00% % Bias -2.98% 10/11/16 3-1 19.36 3-2 19.09 3-3 19.80 Within Run Mean 19.42 SD 0.36 %CV 1.85% % Bias -2.92% 10/13/16 4-1 19.54 4-2 20.66 4-3 20.28 Within Run Mean 20.16 SD 0.57 %CV 2.83% % Bias 0.80% 10/26/16 5-1 19.93 5-2 20.16 5-3 20.48 Within Run Mean 20.19 SD 0.28 %CV 1.37% % Bias 0.95%

Mean 19.58 Imprecision Max Within-Run 2.83% (%CV) Between-Run 3.22% % Bias -2.08%

Replicates in red failed to meet ion ratio acceptance crieteria. Given that the quantified Comments: concentrations are acceptable, 20 ng/mL will be used as the LOQ.

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 25.00 250.00 250.00 400.00 8/29/16 1-1 24.89 227.84 234.34 413.82 1-2 24.60 236.57 243.88 403.09 1-3 23.49 237.64 245.05 371.98 Within Run Mean 24.33 234.02 241.09 396.30 SD 0.74 5.38 5.87 21.73 %CV 3.04% 2.30% 2.44% 5.48% % Bias -2.69% -6.39% -3.56% -0.93% 10/5/16 2-1 25.70 247.87 248.07 429.88 2-2 26.46 256.58 257.39 421.66 2-3 25.59 253.17 253.75 418.11 Within Run Mean 25.92 252.54 253.07 423.22 SD 0.47 4.39 4.70 6.04 %CV 1.83% 1.74% 1.86% 1.43% % Bias 3.67% 1.02% 1.23% 5.80% 10/11/16 3-1 27.82 267.64 276.27 421.26 3-2 27.65 252.95 259.85 412.03 3-3 25.50 241.46 247.01 416.36 Within Run Mean 26.99 254.02 261.04 416.55 SD 1.29 13.12 14.67 4.62 %CV 4.79% 5.17% 5.62% 1.11% % Bias 7.96% 1.61% 4.42% 4.14% 10/13/16 4-1 29.92 272.97 277.77 425.07 4-2 26.65 255.81 258.86 431.76 4-3 27.41 255.53 258.58 426.88 Within Run Mean 27.99 261.44 265.07 427.90 SD 1.71 9.99 11.00 3.46 %CV 6.11% 3.82% 4.15% 0.81% % Bias 11.97% 4.57% 6.03% 6.98% 10/26/16 5-1 27.77 263.48 268.50 404.73 5-2 27.42 257.99 261.70 422.45 5-3 27.08 270.64 275.16 428.90 Within Run Mean 27.42 264.04 268.45 418.69 SD 0.35 6.34 6.73 12.52 %CV 1.26% 2.40% 2.51% 2.99% % Bias 9.69% 5.61% 7.38% 4.67% 11/18/16 5-1 26.84 276.18 274.95 447.87 5-2 25.97 274.28 272.98 448.57 5-3 26.42 284.77 283.85 445.92 Within Run Mean 26.41 278.41 277.26 447.45 SD 0.44 5.59 5.79 1.37 %CV 1.65% 2.01% 2.09% 0.31% % Bias 5.64% 11.36% 10.90% 11.86%

Mean 26.51 257.41 261.00 421.69 SD 1.47 15.40 14.00 18.08 Imprecision Max Within-Run 6.11% 5.17% 5.62% 5.48% (%CV) Between-Run 5.53% 5.98% 5.36% 4.29% % Bias 6.04% 2.96% 4.40% 5.42%

Comments: Red values indicate ion ratios not meeting acceptance criteria.

Bias: ≤ 20% Acceptance Criteria: Within-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 20.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/5/16 10/11/16 High Calibrator 800.000 797.57 785.39 800.45 788.30 795.330 Negative -0.77 -0.66 0.04 0.23 1.62 % of Low Calibrator -3.9% -3.3% 0.2% 1.2% 8.1% Maximum Carryover: 8.1%

Comments: All ion ratios for oxycodone in the Negative (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 7404 Run 2 37233 Run 3 36004 Run 4 33130 Run 5 22345 Average 27223

Identification Peak Response Comment Blank Matrix (NO IS) 1 16 Pass Blank Matrix (NO IS) 2 18 Pass Blank Matrix (NO IS) 3 38 Pass Blank Matrix (NO IS) 4 7 Pass Blank Matrix (NO IS) 5 11 Pass Blank Matrix (NO IS) 6 18 Pass Blank Matrix (NO IS) 7 8 Pass Blank Matrix (NO IS) 8 5 Pass Blank Matrix (NO IS) 9 11 Pass Blank Matrix (NO IS) 10 19 Pass Blank Matrix (with IS) 1 1238 Pass Blank Matrix (with IS) 2 2091 Pass Blank Matrix (with IS) 3 763 Pass Blank Matrix (with IS) 4 1555 Pass Blank Matrix (with IS) 5 1334 Pass Blank Matrix (with IS) 6 1193 Pass Blank Matrix (with IS) 7 949 Pass Blank Matrix (with IS) 8 777 Pass Blank Matrix (with IS) 9 618 Pass Blank Matrix (with IS) 10 5326 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is no interference from labeled IS. Comments: The Blank Matrix (with IS) 10 (in red) was > 10% of the average response of the LOQ. However, ion ratios did not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 26.51 ng/mL Control Acceptance (%): 20 Run Date: 10/21/2016

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration 7-Aminoclonazepam Nordiazepam Oxazepam Diazepam 1 Benzodiazepines Mix Lorazepam 1,000 ng/mL 27.01 Pass Temazepam Clonazepam Alpha-hydroxyalprazolam Alprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 24.83 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 25.28 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 26.78 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 27.65 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 25.32 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 24.54 Pass

Conclusions: There are 0 compounds tested in this study that cause significant interference with the analyte of interest.

Comments: The red value indicates ion ratios that failed to meet acceptance criteria. Benzodiazepines may have contributed to the failed ion ratio. If ion ratios are not acceptable, results will not be reported.

Acceptance Criteria: Concentration of analytes of interest within ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated %Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 250.00 500.00 125.00 500.00 50.00 500.00 250.00 500.00 125.00 500.00 Run 1 1-1 259.04 518.08 3.62 130.69 522.76 4.55 54.34 543.40 8.68 266.66 533.32 6.66 123.26 493.04 -1.39 10/11/16 1-2 240.99 481.98 -3.60 133.17 532.68 6.54 54.23 542.30 8.46 246.50 493.00 -1.40 126.03 504.12 0.82 Run 2 2-1 269.04 538.08 7.62 141.10 564.40 12.88 56.13 561.30 12.26 273.40 546.80 9.36 132.28 529.12 5.82 10/13/16 2-2 267.06 534.12 6.82 137.27 549.08 9.82 57.16 571.60 14.32 271.41 542.82 8.56 128.05 512.20 2.44 Run 3 3-1 272.75 545.50 9.10 138.93 555.72 11.14 58.15 581.50 16.30 277.32 554.64 10.93 139.42 557.68 11.54 10/26/16 3-2 268.05 536.10 7.22 140.56 562.24 12.45 57.96 579.60 15.92 272.45 544.90 8.98 141.09 564.36 12.87

Mean: 525.64 547.81 563.28 535.91 526.75 SD: 23.21 16.76 17.36 22.12 29.11 Between-Run Imprecision (%CV): 4.42 3.06 3.08 4.13 5.53 %Bias: 5.13 9.56 12.66 7.18 5.35

Comments: Up to 10x dilution can be used for the lower curve and 4x for the upper curve.

Acceptance Criteria: Concentration within ± 20% of target Validation Study 8 IN-PROCESS STABILITY Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20%

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 25.16 Pass 25.33 21.21 31.81 LQC-2 25.50 Pass MQC-1 273.24 Pass 258.68 205.93 308.89 MQC-2 244.12 Pass 10/11/16 *MQC-1 282.53 Pass *Upper Curve 266.26 208.80 313.20 *MQC-2 249.99 Pass *Upper Curve HQC-1 404.09 Pass 403.45 337.35 506.02 HQC-2 402.81 Pass

Overall Acceptance: Stable for 3 hours

Comments: Red values indicate ion ratios that did not meet acceptance criteria. Given the quantified concentrations are acceptable, up to a 3 hour delay can be used. If ion ratios are not acceptable, results will not be reported.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 20.00 18.89 N/A 16.00 24.00 Pass 40.00 40.15 N/A 32.00 48.00 Pass 100.00 103.91 N/A 80.00 120.00 Pass 300.00 308.16 N/A 240.00 360.00 Pass 100.00 97.47 N/A 80.00 120.00 Pass *Upper Curve 300.00 308.20 N/A 240.00 360.00 Pass *Upper Curve 500.00 487.76 N/A 400.00 600.00 Pass Injected on 10/26/16; 800.00 814.74 N/A 640.00 960.00 Pass Reinjected on 26.34 10/27/16 26.51 26.4550 21.21 31.81 Pass 26.57 278.81 257.41 271.2900 205.93 308.89 Pass 263.77 277.49 *Upper Curve 261.00 269.9700 208.80 313.20 Pass 262.45 427.12 421.69 420.4400 337.35 506.02 Pass 413.76

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 27.17 Pass 27.20 21.21 31.81 LQC-2 Punctured 27.22 Pass MQC-1 Punctured 267.61 Pass Injected on 262.13 205.93 308.89 10/27/16; MQC-2 Punctured 256.64 Pass Reinjected on *MQC-1 Punctured 266.36 Pass *Upper Curve 10/28/16 260.75 208.80 313.20 *MQC-2 Punctured 255.13 Pass *Upper Curve HQC-1 Punctured 409.56 Pass 410.06 337.35 506.02 HQC-2 Punctured 410.56 Pass LQC-1 Unpunctured 26.08 Pass 26.00 21.21 31.81 LQC-2 Unpunctured 25.92 Pass MQC-1 Unpunctured 265.07 Pass Extracted on 261.83 205.93 308.89 10/27/16; MQC-2 Unpunctured 258.59 Pass Injected on *MQC-1 Unpunctured 263.83 Pass *Upper Curve 10/28/16 260.45 208.80 313.20 *MQC-2 Unpunctured 257.06 Pass *Upper Curve HQC-1 Unpunctured 410.87 Pass 409.64 337.35 506.02 HQC-2 Unpunctured 408.40 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 Room Temperature LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Room Temperature LQC-1 w/ Light Protection LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Refrigerated (2 to 8oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Frozen (-5 to -20oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Peak Extraction Average Type of Sample Sample ID Response of Efficiency Matrix Effect Response Drug (%Recovery)

Group 1-Internal Standard and drug analyte fortified before extraction

Group 2-Internal Standard and analyte fortified after extraction

Group 3-Internal Standard and analyte fortified to elution solvent/no extraction

Extraction efficiency and matrix effects were not performed for hydrocodone, hydromorphone, Comments: oxycodone, or oxymorphone because derivatization needs to occur prior to extraction for these four analytes.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction Criteria: efficiency and matrix effect are considered acceptable Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 50.00 59.57 40-60 19.1 Pass Blind Sample 3 100.00 100.54 80-120 0.5 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 120.00 120.46 96-144 0.4 Pass Blind Sample 7 400.00 444.53 320-480 11.1 Pass Blind Sample 8 250.00 295.39 200-300 18.2 Pass Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 500.00 526.26 400-600 5.3 Pass 11/9/16 Blind Sample 11 600.00 620.75 480-720 3.5 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 800.00 805.48 640-960 0.7 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 160.00 163.85 98-192 2.4 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 400.00 440.34 320-480 10.1 Pass

Comments: All Blind Samples had to be reinjected for oxycodone because the drug was not within its SIM window in the original injection. All reinjection values fell within ± 20% of the target and were therefore deemed acceptable.

Acceptance Percent difference of the expected vs. determined values ≤ 20% Criteria: SUMMARY OF VALIDATION PERFORMANCE Analyte: Oxycodone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason There were multiple interferences with ions causing quantitative values, ion August 2, 2016 ratios, and overall chromatography to not be acceptable. Autosampler stability was attempted on this day, but the extraction vials dried out prior to injection so the experiment was repeated on October 27, October 13, 2016 2016.

Deviations from SOP:

Other Observations: Throughout validation, oxycodone ions 459.3 and 385.2 were seen to be concentration dependent. Therefore, the acceptance criteria for these ions will be ± 2 standard deviations from the average of the calibrators used in the calibration curve. Please see supplemental Opioids SIM Quantification QC Worksheets for more details. Per reccomendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of oxycodone confirmation analysis. METHOD VALIDATION PROTOCOL AND RESULTS Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Hydoxy.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS

Data analysis was processed using 1/x weighting for the lower curve and Unweighted: = 519.7 unweighted for the upper curve even 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 218.53 though 1/x2 gave the lowest %RE 1/x2 Weighting: = 170.18 because all other validation parameters met acceptance criteria.

95% CI of the slope does not include 1. However, linearity is acceptable based on evaluation of the standard 95% CI of slope includes 1 95% CI of Slope = 0.9888 - 0.9986 1 Linearity residual plot, correlation coefficient, 95% CI of intercept includes 0 95% CI of Intercept = -0.0680 - 1.4971 standard error, and % difference of individual calibrator values from the target concentration.

100 ng/mL Calibrator in batch 95% CI of slope includes 1 95% CI of Slope = 0.9790 - 1.0210 20150512_MRL did not back calculate 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -10.4714 - 10.4371 to within ± 20% of the target. This replicate was deemed to be an outlier.

100 ng/mL Calibrator in batch Max %RE low calibrator = -6.65 20150512_MRL did not back calculate 1 Validation Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) Max %RE other calibrators = -17.10 to within ± 20% of the target. This replicate was deemed to be an outlier.

Not performed. Validation of 6 calibrators will be 1 Casework Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) used for casework.

Comparison of Validation Calibration to 95% CI of slope includes 1 Not performed. Validation of 6 calibrators will be 1 Casework Calibration 95% CI of intercept includes 0 used for casework.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three levels did not consistently meet LOD = 20 ion ratio criteria (red values above Signal to Noise ≥ 3 2 Limit of Detection (LOD) S:N = >561.228; S:N not documented for 20 indicate failed ion ratios). Therefore, Acceptable detection and identification criteria ng/mL because the S:N of 18 ng/mL was > 3. the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using the three different sources as required for LOD experiment.

Bias: ≤ 25% Bias = -0.34% Replicate in red failed to meet ion ratio 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-Run Imprecision= 2.67% acceptance criteria. Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 1.83%

Bias Bias: ≤ 20% Max Bias = -5.30% Red values indicate ion ratios not 4 Within-Run Imprecision Witnin-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.71% meeting acceptance criteria. Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 5.79%

All ion ratios for oxymorphone in the Negative (in red) do not meet 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 11.8% acceptance criteria. Therefore, the method is considered to have no significant carryover.

6 Matrix Interference Response of blank matrix≤ 10% of the average response of LOQ No matrix interference was identified.

6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is no interference from labeled IS.

There are 0 compounds tested in this study that Concentration of analytes of interest within ± 20% of average for bias and 6 Exogenous Substances Interferences cause significant interference with the analyte of imprecision interest.

Up to 10x dilution can be used for the 7 Dilution Integrity Concentration within ± 20% of target Max % of target = 15.90 lower curve and 4x for the upper curve.

Concentration within 20% of the average value obtained in the Bias and 8 In-Process Stability Stable for 3 hours Imprecision studies

Punctured vials in Experiment 1 were stable for 24 hours. Punctured and Concentration within 20% of the average value obtained in the Bias and Unpunctured Vials:Stable for 24 hours 8 Autosampler Stability unpunctured vials in Experiment 2 Imprecision studies Punctured Vials:Stable for 24 hours were stable for 21 and 24 hours, respectively.

Study not performed; storage stability is documented in Disposition of Toxic 8 Storage Stability Concentration within 20% of average time 0 Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Extraction efficiency and matrix effects were not performed for hydrocodone, If method achieves desired limit of quantification and/or detection for the hydromorphone, oxycodone, or 9 Extraction Efficiency & Matrix Effect Study not performed analyte, its extraction efficiency and matrix effect are considered acceptable. oxymorphone because derivatization needs to occur prior to extraction for these four analytes.

Blind Samples 2 and 8 did not meet acceptance criteria. The quality controls run with the blind samples quantitated on target for oxymorphone 10 Blind Sample Analysis Percent difference of the expected vs. determined values≤ 20% 90% of samples ≤ 20% of target value (< ± 5%). Blind Samples 2 and 8 quantitated high for all drugs. Therefore, the high quantitation can be attributed to over-spiking these blind samples.

* Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 519.70 1/x Weighting: 218.53 1/x2 Weighting: 170.18

Comments: Data analysis was processed using 1/x weighting for the lower curve and unweighted for the upper curve even though 1/x2 gave the lowest %RE because all other validation parameters met acceptance criteria.

Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 20 0.13145 1 2.628992 20 0.13144959 400 0.017279 6.706825 -66.4659 66.46588 40 0.277052 1 11.08206 40 0.27705156 1600 0.076758 33.15909 -17.1023 17.10228 100 0.674663 1 67.46634 100 0.67466345 10000 0.455171 105.3953 5.395297 5.395297 300 1.898565 1 569.5696 300 1.89856521 90000 3.60455 327.7479 9.249288 9.249288 500 2.896619 1 1448.31 500 2.89661926 250000 8.390403 509.0695 1.813902 1.813902 800 4.497953 1 3598.363 800 4.49795328 640000 20.23158 799.9922 -0.00098 0.000981 20 0.132387 1 2.647733 20 0.13238664 400 0.017526 6.877063 -65.6147 65.61469 40 0.283972 1 11.35889 40 0.2839722 1600 0.08064 34.4164 -13.959 13.95901 100 0.663117 1 66.31173 100 0.66311728 10000 0.439725 103.2976 3.297646 3.297646 300 1.915988 1 574.7964 300 1.91598815 90000 3.671011 330.9132 10.30439 10.30439 500 3.046168 1 1523.084 500 3.04616842 250000 9.279142 536.2389 7.247776 7.247776 800 4.395083 1 3516.066 800 4.39508284 640000 19.31675 781.3031 -2.33711 2.337107 20 0.132326 1 2.646522 20 0.13232609 400 0.01751 6.866062 -65.6697 65.66969 40 0.271541 1 10.86162 40 0.27154057 1600 0.073734 32.15788 -19.6053 19.60531 100 0.647247 1 64.72469 100 0.64724686 10000 0.418928 100.4144 0.414383 0.414383 300 1.813003 1 543.901 300 1.81300338 90000 3.286981 312.2034 4.067802 4.067802 500 2.890314 1 1445.157 500 2.89031433 250000 8.353917 507.9241 1.584812 1.584812 800 4.314879 1 3451.904 800 4.31487947 640000 18.61818 766.7322 -4.15848 4.158477 20 0.131906 1 2.638122 20 0.13190608 400 0.017399 6.789758 -66.0512 66.05121 40 0.279854 1 11.19416 40 0.27985391 1600 0.078318 33.66821 -15.8295 15.82949 100 0.648983 1 64.89834 100 0.64898341 10000 0.421179 100.7299 0.729873 0.729873 300 1.92028 1 576.0839 300 1.92027983 90000 3.687475 331.6929 10.56429 10.56429 500 2.915292 1 1457.646 500 2.91529174 250000 8.498926 512.4618 2.492367 2.492367 800 4.48464 1 3587.712 800 4.48464013 640000 20.112 797.5735 -0.30331 0.303315 20 0.120328 1 2.406555 20 0.12032775 400 0.014479 4.686261 -76.5687 76.56869 40 0.247754 1 9.910143 40 0.24775357 1600 0.061382 27.83637 -30.4091 30.40908 100 0.604288 1 60.42876 100 0.60428756 10000 0.365163 92.60975 -7.39025 7.390254 300 1.797811 1 539.3434 300 1.79781131 90000 3.232125 309.4434 3.147794 3.147794 500 2.940921 1 1470.46 500 2.94092097 250000 8.649016 517.118 3.423607 3.423607 800 4.299704 1 3439.763 800 4.29970387 640000 18.48745 763.9752 -4.50311 4.503106 Sum 8800 51.27409 5 28133.36 8800 51.2740887 4960000 159.9747 519.7018 Slope 0.00550433 Intercept 0.09453302 R2 0.99623564 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.13145 0.05 0.13145 1 0.00657248 20 0.000864 17.09303 -14.5348 14.53485 40 0.277052 0.025 0.277052 1 0.00692629 40 0.001919 42.58677 6.466921 6.466921 100 0.674663 0.01 0.674663 1 0.00674663 100 0.004552 112.2054 12.20542 12.20542 300 1.898565 0.003333 1.898565 1 0.00632855 300 0.012015 326.5008 8.833607 8.833607 500 2.896619 0.002 2.896619 1 0.00579324 500 0.016781 501.2521 0.250418 0.250418 800 4.497953 0.00125 4.497953 1 0.00562244 800 0.025289 781.6328 -2.29589 2.295894 20 0.132387 0.05 0.132387 1 0.00661933 20 0.000876 17.2571 -13.7145 13.7145 40 0.283972 0.025 0.283972 1 0.00709931 40 0.002016 43.79852 9.496293 9.496293 100 0.663117 0.01 0.663117 1 0.00663117 100 0.004397 110.1838 10.18378 10.18378 300 1.915988 0.003333 1.915988 1 0.00638663 300 0.012237 329.5514 9.850479 9.850479 500 3.046168 0.002 3.046168 1 0.00609234 500 0.018558 527.4369 5.48739 5.48739 800 4.395083 0.00125 4.395083 1 0.00549385 800 0.024146 763.6211 -4.54737 4.547368 20 0.132326 0.05 0.132326 1 0.0066163 20 0.000876 17.2465 -13.7675 13.76751 40 0.271541 0.025 0.271541 1 0.00678851 40 0.001843 41.62184 4.054594 4.054594 100 0.647247 0.01 0.647247 1 0.00647247 100 0.004189 107.405 7.404999 7.404999 300 1.813003 0.003333 1.813003 1 0.00604334 300 0.010957 311.5196 3.839876 3.839876 500 2.890314 0.002 2.890314 1 0.00578063 500 0.016708 500.1481 0.029629 0.029629 800 4.314879 0.00125 4.314879 1 0.0053936 800 0.023273 749.5781 -6.30274 6.302739 20 0.131906 0.05 0.131906 1 0.0065953 20 0.00087 17.17296 -14.1352 14.13521 40 0.279854 0.025 0.279854 1 0.00699635 40 0.001958 43.07744 7.693595 7.693595 100 0.648983 0.01 0.648983 1 0.00648983 100 0.004212 107.7091 7.709056 7.709056 300 1.92028 0.003333 1.92028 1 0.00640093 300 0.012292 330.3029 10.10096 10.10096 500 2.915292 0.002 2.915292 1 0.00583058 500 0.016998 504.5215 0.904298 0.904298 800 4.48464 0.00125 4.48464 1 0.0056058 800 0.02514 779.3018 -2.58727 2.587272 20 0.120328 0.05 0.120328 1 0.00601639 20 0.000724 15.14568 -24.2716 24.27158 40 0.247754 0.025 0.247754 1 0.00619384 40 0.001535 37.45692 -6.35769 6.357689 100 0.604288 0.01 0.604288 1 0.00604288 100 0.003652 99.88317 -0.11683 0.116829 300 1.797811 0.003333 1.797811 1 0.0059927 300 0.010774 308.8596 2.953206 2.953206 500 2.940921 0.002 2.940921 1 0.00588184 500 0.017298 509.009 1.801793 1.801793 800 4.299704 0.00125 4.299704 1 0.00537463 800 0.023109 746.921 -6.63488 6.634879 Sum 8800 51.27409 0.457917 51.27409 30 0.1868282 8800 0.300056 218.5326 Slope 0.00571128 Intercept 0.03382645 R2 0.99799732 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.13145 0.0025 0.00657248 0.05 0.000329 1 4.32E-05 19.38868 -3.05659 3.056592 40 0.277052 0.000625 0.00692629 0.025 0.000173 1 4.8E-05 43.67376 9.184394 9.184394 100 0.674663 0.0001 0.00674663 0.01 6.75E-05 1 4.55E-05 109.9918 9.991781 9.991781 300 1.898565 1.11E-05 0.00632855 0.003333 2.11E-05 1 4.01E-05 314.1274 4.709132 4.709132 500 2.896619 0.000004 0.00579324 0.002 1.16E-05 1 3.36E-05 480.5937 -3.88126 3.881265 800 4.497953 1.56E-06 0.00562244 0.00125 7.03E-06 1 3.16E-05 747.6815 -6.53981 6.539808 20 0.132387 0.0025 0.00661933 0.05 0.000331 1 4.38E-05 19.54497 -2.27514 2.275139 40 0.283972 0.000625 0.00709931 0.025 0.000177 1 5.04E-05 44.82806 12.07014 12.07014 100 0.663117 0.0001 0.00663117 0.01 6.63E-05 1 4.4E-05 108.066 8.065987 8.065987 300 1.915988 1.11E-05 0.00638663 0.003333 2.13E-05 1 4.08E-05 317.0334 5.677794 5.677794 500 3.046168 0.000004 0.00609234 0.002 1.22E-05 1 3.71E-05 505.5371 1.107422 1.107422 800 4.395083 1.56E-06 0.00549385 0.00125 6.87E-06 1 3.02E-05 730.5237 -8.68454 8.684539 20 0.132326 0.0025 0.0066163 0.05 0.000331 1 4.38E-05 19.53487 -2.32563 2.325634 40 0.271541 0.000625 0.00678851 0.025 0.00017 1 4.61E-05 42.75457 6.886435 6.886435 100 0.647247 0.0001 0.00647247 0.01 6.47E-05 1 4.19E-05 105.4189 5.418945 5.418945 300 1.813003 1.11E-05 0.00604334 0.003333 2.01E-05 1 3.65E-05 299.8565 -0.04785 0.047845 500 2.890314 0.000004 0.00578063 0.002 1.16E-05 1 3.34E-05 479.5421 -4.09159 4.091585 800 4.314879 1.56E-06 0.0053936 0.00125 6.74E-06 1 2.91E-05 717.1465 -10.3567 10.35669 20 0.131906 0.0025 0.0065953 0.05 0.00033 1 4.35E-05 19.46482 -2.6759 2.675899 40 0.279854 0.000625 0.00699635 0.025 0.000175 1 4.89E-05 44.14116 10.35291 10.35291 100 0.648983 0.0001 0.00648983 0.01 6.49E-05 1 4.21E-05 105.7086 5.708587 5.708587 300 1.92028 1.11E-05 0.00640093 0.003333 2.13E-05 1 4.1E-05 317.7492 5.916398 5.916398 500 2.915292 0.000004 0.00583058 0.002 1.17E-05 1 3.4E-05 483.7081 -3.25838 3.258385 800 4.48464 1.56E-06 0.0056058 0.00125 7.01E-06 1 3.14E-05 745.461 -6.81737 6.817372 20 0.120328 0.0025 0.00601639 0.05 0.000301 1 3.62E-05 17.53366 -12.3317 12.3317 40 0.247754 0.000625 0.00619384 0.025 0.000155 1 3.84E-05 38.78712 -3.0322 3.0322 100 0.604288 0.0001 0.00604288 0.01 6.04E-05 1 3.65E-05 98.25373 -1.74627 1.746272 300 1.797811 1.11E-05 0.0059927 0.003333 2E-05 1 3.59E-05 297.3226 -0.89248 0.892478 500 2.940921 0.000004 0.00588184 0.002 1.18E-05 1 3.46E-05 487.9828 -2.40344 2.40344 800 4.299704 1.56E-06 0.00537463 0.00125 6.72E-06 1 2.89E-05 714.6154 -10.6731 10.67308 8800 51.27409 0.016208 0.1868282 0.457917 0.002992 30 0.00117 170.1799 Slope 0.00599553 Intercept 0.01520412 R2 0.99613000 Validation Study 1 LINEARITY Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 0.9937 Intercept 0.7145

20 ng/mL 20160505_MRL 20.00 18.72 Std err in slope, Sb 0.0024 Std err in intercept 0.3850 40 ng/mL 20160505_MRL 40.00 41.55 Degrees freedom 34 Degrees freedom 34 100 ng/mL 20160505_MRL 100.00 103.90 Confidence level 95% Confidence level 95% 300 ng/mL 20160505_MRL 300.00 295.82 Student t 2.0322 Student t 2.0322 20 ng/mL 20160512_MRL 20.00 18.67 Confidence interval 0.0049 Confidence interval 0.7825 40 ng/mL 20160512_MRL 40.00 42.36 Slope 0.9937 ± 0.0049 Intercept 0.7145 ± 0.7825 100 ng/mL 20160512_MRL 100.00 101.60 Range 0.9888 - 0.9986 Lower -0.0680 - 1.4971 300 ng/mL 20160512_MRL 300.00 297.36 FAIL PASS 20 ng/mL 20160829_MRL 20.00 18.99 40 ng/mL 20160829_MRL 40.00 42.18 100 ng/mL 20160829_MRL 100.00 99.97 300 ng/mL 20160829_MRL 300.00 298.85 20 ng/mL 20161005_MRL 20.00 19.56 40 ng/mL 20161005_MRL 40.00 40.85 100 ng/mL 20161005_MRL 100.00 100.28 300 ng/mL 20161005_MRL 300.00 299.29 350.00 20 ng/mL 20161011_MRL 20.00 19.98 300.00 y = 0.9937x + 0.7145 40 ng/mL 20161011_MRL 40.00 39.31 R² = 0.9998 100 ng/mL 20161011_MRL 100.00 102.31 250.00 300 ng/mL 20161011_MRL 300.00 298.37 20 ng/mL 20161013_MRL 20.00 19.89 200.00 40 ng/mL 20161013_MRL 40.00 40.15 150.00 100 ng/mL 20161013_MRL 100.00 100.22 300 ng/mL 20161013_MRL 300.00 299.72 100.00 20 ng/mL 20161021_MRL 20.00 20.12 50.00 40 ng/mL 20161021_MRL 40.00 39.99 100 ng/mL 20161021_MRL 100.00 99.13 0.00 300 ng/mL 20161021_MRL 300.00 300.74 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 20 ng/mL 20161026_MRL 20.00 20.51 40 ng/mL 20161026_MRL 40.00 38.53 100 ng/mL 20161026_MRL 100.00 101.17 300 ng/mL 20161026_MRL 300.00 299.77 20 ng/mL 20161027_MRL 20.00 18.77 40 ng/mL 20161027_MRL 40.00 41.62 100 ng/mL 20161027_MRL 100.00 103.29 300 ng/mL 20161027_MRL 300.00 296.30

Comments: 95% CI of the slope does not include 1. However, linearity is acceptable based on evaluation of the standard residual plot, correlation coefficient, standard error, and % difference of individual calibrator values from the target concentration. 95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 1 - Standard Residuals

Regression Statistics Multiple R 0.999899926 R Square 0.999799862 Adjusted R S0.999793976 Standard Er 1.602901833 Observation 36

ANOVA df SS MS F Significance F Regression 1 436391.6733 436391.6733 169848.8475 1.80156E-64 Residual 34 87.3560057 2.569294285 Total 35 436479.0293

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0% Intercept 0.714546277 0.385048127 1.855732382 0.072177324 -0.067965664 1.497058219 -0.067965664 1.497058219 X Variable 1 0.993747907 0.002411265 412.1272225 1.80156E-64 0.988847627 0.998648187 0.988847627 0.998648187

RESIDUAL OUTPUT

Observation Predicted Y Residuals Standard Residuals 1 20.58950441 -1.869504413 -1.1833525 2 40.46446255 1.085537452 0.687119779 3 100.089337 3.810663046 2.412060444 4 298.8389183 -3.018918307 -1.910904571 5 20.58950441 -1.919504413 -1.215001329 X Variable 1 Residual Plot 6 40.46446255 1.895537452 1.199830804 3 7 100.089337 1.510663046 0.956214321 2.5 8 298.8389183 -1.478918307 -0.936120645 2 9 20.58950441 -1.599504413 -1.012448825 1.5 10 40.46446255 1.715537452 1.085895021 1 11 100.089337 -0.119336954 -0.075537496 0.5 12 298.8389183 0.011081693 0.007014452 0

13 20.58950441 -1.029504413 -0.651652177 Residuals -0.5 0 50 100 150 200 250 300 350 14 40.46446255 0.385537452 0.244036176 -1 15 100.089337 0.190663046 0.120685242 -1.5 16 298.8389183 0.451081693 0.285524145 -2 17 20.58950441 -0.609504413 -0.385802016 -2.5 X Variable 1 18 40.46446255 -1.154462548 -0.73074775 19 100.089337 2.220663046 1.405627689 20 298.8389183 -0.468918307 -0.296814304 21 20.58950441 -0.699504413 -0.442769907 22 40.46446255 -0.314462548 -0.199047427 23 100.089337 0.130663046 0.082706647 24 298.8389183 0.881081693 0.557704072 25 20.58950441 -0.469504413 -0.297185295 26 40.46446255 -0.474462548 -0.300323679 27 100.089337 -0.959336954 -0.60723782 28 298.8389183 1.901081693 1.203340179 29 20.58950441 -0.079504413 -0.050324431 30 40.46446255 -1.934462548 -1.224469478 31 100.089337 1.080663046 0.684034394 32 298.8389183 0.931081693 0.589352901 33 20.58950441 -1.819504413 -1.151703672 34 40.46446255 1.155537452 0.731428139 35 100.089337 3.200663046 2.025944733 36 298.8389183 -2.538918307 -1.607075815 Validation Study 1 UPPER LINEARITY Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0000 Intercept -0.0172

100 ng/mL 20160505_MRL 100.00 89.13 Std err in slope, Sb 0.0103 Std err in intercept 5.1442 300 ng/mL 20160505_MRL 300.00 315.33 Degrees freedom 34 Degrees freedom 34 500 ng/mL 20160505_MRL 500.00 499.78 Confidence level 95% Confidence level 95% 800 ng/mL 20160505_MRL 800.00 795.74 Student t 2.0322 Student t 2.0322 100 ng/mL 20160512_MRL 100.00 78.40 Confidence interval 0.0210 Confidence interval 10.4542 300 ng/mL 20160512_MRL 300.00 314.15 Slope 1 ± 0.021 Intercept -0.0172 ± 10.4542 500 ng/mL 20160512_MRL 500.00 526.81 Range 0.9790 - 1.0210 Lower -10.4714 - 10.4371 800 ng/mL 20160512_MRL 800.00 780.63 PASS PASS 100 ng/mL 20160829_MRL 100.00 84.72 300 ng/mL 20160829_MRL 300.00 319.60 500 ng/mL 20160829_MRL 500.00 502.96 800 ng/mL 20160829_MRL 800.00 792.70 100 ng/mL 20161005_MRL 100.00 82.90 300 ng/mL 20161005_MRL 300.00 308.55 500 ng/mL 20161005_MRL 500.00 525.62 800 ng/mL 20161005_MRL 800.00 782.91 900.00 100 ng/mL 20161011_MRL 100.00 85.33 800.00 y = 1x - 0.0172 300 ng/mL 20161011_MRL 300.00 315.17 R² = 0.9964 700.00 500 ng/mL 20161011_MRL 500.00 508.92 800 ng/mL 20161011_MRL 800.00 790.56 600.00 100 ng/mL 20161013_MRL 100.00 80.53 500.00 300 ng/mL 20161013_MRL 300.00 312.78 400.00 500 ng/mL 20161013_MRL 500.00 523.63 300.00 800 ng/mL 20161013_MRL 800.00 782.7 200.00 100 ng/mL 20161021_MRL 100.00 80.78 300 ng/mL 20161021_MRL 300.00 319.54 100.00 500 ng/mL 20161021_MRL 500.00 512.25 0.00 800 ng/mL 20161021_MRL 800.00 787.41 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 100 ng/mL 20161026_MRL 100.00 88.24 300 ng/mL 20161026_MRL 300.00 312.15 500 ng/mL 20161026_MRL 500.00 507.17 800 ng/mL 20161026_MRL 800.00 792.42 100 ng/mL 20161027_MRL 100.00 86.73 300 ng/mL 20161027_MRL 300.00 302.49 500 ng/mL 20161027_MRL 500.00 526.8 800 ng/mL 20161027_MRL 800.00 783.97

Comments: 100 ng/mL Calibrator in batch 20150512_MRL did not back calculate to within ± 20% of the target. This replicate was deemed to be an outlier.

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 20 18.72 -6.40 Level 2 40 41.55 3.87 Level 3 100 103.90 3.90 Level 4 300 295.82 -1.39 5/5/16 Upper-Level 3 100 89.13 -10.87 Upper-Level 4 300 315.33 5.11 Level 5 500 499.78 -0.04 Level 6 800 795.74 -0.53 Level 1 20 18.67 -6.65 Level 2 40 42.36 5.90 Level 3 100 101.60 1.60 Level 4 300 297.36 -0.88 5/12/16 Upper-Level 3 100 78.40 -21.60 Upper-Level 4 300 314.15 4.72 Level 5 500 526.81 5.36 Level 6 800 780.63 -2.42 Level 1 20 18.99 -5.05 Level 2 40 42.18 5.45 Level 3 100 99.97 -0.03 Level 4 300 298.85 -0.38 8/29/16 Upper-Level 3 100 84.72 -15.28 Upper-Level 4 300 319.60 6.53 Level 5 500 502.96 0.59 Level 6 800 792.70 -0.91 Level 1 20 19.56 -2.20 Level 2 40 40.85 2.13 Level 3 100 100.28 0.28 Level 4 300 299.29 -0.24 10/5/16 Upper-Level 3 100 82.90 -17.10 Upper-Level 4 300 308.55 2.85 Level 5 500 525.62 5.12 Level 6 800 782.91 -2.14 Level 1 20 19.98 -0.10 Level 2 40 39.31 -1.72 Level 3 100 102.31 2.31 Level 4 300 298.37 -0.54 10/11/16 Upper-Level 3 100 85.33 -14.67 Upper-Level 4 300 315.17 5.06 Level 5 500 508.92 1.78 Level 6 800 790.56 -1.18

Max %RE LOQ = -6.65 Max %RE other calibrators = -17.10

Comments: 100 ng/mL Calibrator in batch 20150512_MRL did not back calculated to within ± 20% of the target. This replicate was deemed to be an outlier. Acceptance Criteria: %RE Calibrators ± 20% of target (25% at lowest calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

6 Run One LOWER Curve UPPER Curve Slope 0.00638 Slope 0.00541 4 Intercept 0.121 Intercept 0.192 R2 0.999 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Two Slope 0.0064 Slope 0.00531 4 Intercept 0.129 Intercept 0.246 R2 0.999 R2 0.993 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Three Slope 0.0064 Slope 0.00542 4 Intercept 0.104 Intercept 0.191 R2 0.999 R2 0.997 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Four Slope 0.00599 Slope 0.00528 4 Intercept 0.00228 Intercept 0.165 R2 1.000 R2 0.995 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Five Slope 0.00653 Slope 0.00542 4 Intercept 0.118 Intercept 0.176 R2 1.000 R2 0.998 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Cnom y (Response Ratio) 5 Combined Regression 20 0.131449593

5 40 0.277051561 100 0.674663445

4 300 1.898565208 500 2.896619257 800 4.49795328 4 20 0.132386639 40 0.2839722 3 100 0.663117284 300 1.915988153 3 500 3.046168421 800 4.395082836 Response Ratio Response 2 20 0.132326089 40 0.271540567 2 100 0.647246855 300 1.813003378 1 500 2.890314329 800 4.314879468 1 20 0.131906084 40 0.279853914 0 100 0.648983412 0.000 100.000 200.000 300.000 400.000 500.000 600.000 700.000 800.000 900.000 300 1.920279827 Concentration 500 2.915291736 800 4.484640133 Slope 0.0057 20 0.120327745 Intercept 0.0338 40 0.247753566 R2 0.9980 100 0.604287562 300 1.797811307 500 2.940920974 800 4.299703874 Validation Study 2 LOD Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 18.00 15.00 10.00 Source 1-1 387.4 427.7 135.2 Source 1-2 489.8 620.2 373.9 Source 2-1 361.1 693.1 313.8 5/5/16 Source 2-2 484.1 587.2 429.7 Source 3-1 533.8 484.8 282.3 Source 3-2 344.5 345.2 377.3 Source 1-1 1383.2 821.6 527.4 Source 1-2 1232.1 567.8 203.1 Source 2-1 887.2 890.1 353.9 5/12/16 Source 2-2 1635.9 636.8 513.2 Source 3-1 1009.7 383.1 232.6 Source 3-2 747.6 288.8 234.0 Source 1-1 104.2 106.9 101.1 Source 1-2 101.3 59.3 89.4 Source 2-1 157.6 56.1 N/A *Poor extraction recovery *8/29/16 Source 2-2 105.7 108.5 75.7 Source 3-1 52.7 45.1 159.9 Source 3-2 84.2 140.7 41.5

Mean Signal to Noise: 561.228 403.500 261.412

Established LOD: 20.00 ng/mL S:N of LOD: >561.228; S:N not documented for 20 ng/mL because the S:N of 18 ng/mL was > 3.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three Comments: levels did not consistently meet ion ratio criteria (red values above indicate failed ion ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using the three different sources as required for LOD experiment. Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 20.00 8/29/16 1-1 19.99 1-2 19.68 1-3 19.69 Within Run Mean 19.79 SD 0.18 %CV 0.89% % Bias -1.07% 10/5/16 2-1 19.69 2-2 19.54 2-3 19.56 Within Run Mean 19.60 SD 0.08 %CV 0.42% % Bias -2.02% 10/11/16 3-1 19.69 3-2 19.74 3-3 20.22 Within Run Mean 19.88 SD 0.29 %CV 1.47% % Bias -0.58% 10/13/16 4-1 19.48 4-2 20.54 4-3 20.14 Within Run Mean 20.05 SD 0.54 %CV 2.67% % Bias 0.27% 10/26/16 5-1 20.10 5-2 20.45 5-3 20.48 Within Run Mean 20.34 SD 0.21 %CV 1.04% % Bias 1.72%

Mean 19.93 Imprecision Max Within-Run 2.67% (%CV) Between-Run 1.83% % Bias -0.34%

Comments: Replicate in red failed to meet ion ratio acceptance criteria.

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 25.00 250.00 250.00 400.00 8/29/16 1-1 22.85 212.98 218.19 383.59 1-2 23.38 219.09 225.41 380.10 1-3 21.97 220.26 226.78 349.17 Within Run Mean 22.73 217.44 223.46 370.95 SD 0.71 3.91 4.62 18.95 %CV 3.13% 1.80% 2.07% 5.11% % Bias -9.07% -13.02% -10.62% -7.26% 10/5/16 2-1 23.55 230.35 230.38 400.17 2-2 24.63 238.28 239.37 394.77 2-3 24.10 232.92 233.30 394.21 Within Run Mean 24.09 233.85 234.35 396.38 SD 0.54 4.05 4.59 3.29 %CV 2.24% 1.73% 1.96% 0.83% % Bias -3.63% -6.46% -6.26% -0.90% 10/11/16 3-1 26.35 247.97 256.08 394.05 3-2 26.74 230.22 235.28 382.35 3-3 24.32 221.10 224.58 387.21 Within Run Mean 25.80 233.10 238.65 387.87 SD 1.30 13.66 16.02 5.88 %CV 5.04% 5.86% 6.71% 1.52% % Bias 3.21% -6.76% -4.54% -3.03% 10/13/16 4-1 27.46 249.38 254.20 384.76 4-2 25.01 233.22 235.47 397.45 4-3 25.39 231.52 233.46 393.36 Within Run Mean 25.95 238.04 241.04 391.86 SD 1.32 9.86 11.44 6.48 %CV 5.08% 4.14% 4.75% 1.65% % Bias 3.81% -4.78% -3.58% -2.04% 10/26/16 5-1 25.11 241.24 245.67 373.01 5-2 25.78 238.56 243.28 396.91 5-3 25.12 247.22 253.27 402.80 Within Run Mean 25.34 242.34 247.41 390.91 SD 0.38 4.43 5.22 15.78 %CV 1.52% 1.83% 2.11% 4.04% % Bias 1.35% -3.06% -1.04% -2.27% 11/18/16 5-1 24.74 250.39 250.26 418.97 5-2 23.89 251.12 251.10 414.18 5-3 24.52 265.83 266.99 410.41 Within Run Mean 24.38 255.78 256.12 414.52 SD 0.44 8.71 9.43 4.29 %CV 1.81% 3.41% 3.68% 1.03% % Bias -2.47% 2.31% 2.45% 3.63%

Mean 24.72 236.76 240.17 392.08 SD 1.37 13.70 13.20 16.07 Imprecision Max Within-Run 5.08% 5.86% 6.71% 5.11% (%CV) Between-Run 5.53% 5.79% 5.49% 4.10% % Bias -1.13% -5.30% -3.93% -1.98%

Comments: Red values indicate ion ratios not meeting acceptance criteria.

Bias: ≤ 20% Acceptance Criteria: Witnin-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 20.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/5/16 10/11/16 High Calibrator 800.000 795.74 780.63 792.70 782.91 790.560 Blank -1.53 -1.50 0.16 0.17 2.35 % of Low Calibrator -7.7% -7.5% 0.8% 0.9% 11.8% Maximum Carryover: 11.8%

Comments: All ion ratios for oxymorphone in the Negative (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 7723 Run 2 46012 Run 3 46687 Run 4 45700 Run 5 25461 Average 34317

Identification Peak Response Comment Blank Matrix (NO IS) 1 18 Pass Blank Matrix (NO IS) 2 17 Pass Blank Matrix (NO IS) 3 7 Pass Blank Matrix (NO IS) 4 12 Pass Blank Matrix (NO IS) 5 5 Pass Blank Matrix (NO IS) 6 15 Pass Blank Matrix (NO IS) 7 19 Pass Blank Matrix (NO IS) 8 17 Pass Blank Matrix (NO IS) 9 5 Pass Blank Matrix (NO IS) 10 10 Pass Blank Matrix (with IS) 1 340 Pass Blank Matrix (with IS) 2 1120 Pass Blank Matrix (with IS) 3 559 Pass Blank Matrix (with IS) 4 1148 Pass Blank Matrix (with IS) 5 996 Pass Blank Matrix (with IS) 6 922 Pass Blank Matrix (with IS) 7 543 Pass Blank Matrix (with IS) 8 501 Pass Blank Matrix (with IS) 9 373 Pass Blank Matrix (with IS) 10 2113 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is no interference from labeled IS. Comments:

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 24.72 ng/mL Control Acceptance (%): 20 Run Date: 10/21/16

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration 7-Aminoclonazepam Nordiazepam Oxazepam Diazepam 1 Benzodiazepines Mix Lorazepam 1,000 ng/mL 26.32 Pass Temazepam Clonazepam Alpha-hydroxyalprazolam Alprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 24.07 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 24.52 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 26.43 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 26.93 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 24.33 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 23.74 Pass

Conclusions: There are 0 compounds tested in this study that cause significant interference with the analyte of interest.

Comments:

Acceptance Criteria: Concentration of analytes of interest within ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated %Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 250.00 500.00 125.00 500.00 50.00 500.00 250.00 500.00 125.00 500.00 Run 1 1-1 239.41 478.82 -4.24 121.65 486.60 -2.68 50.98 509.80 1.96 246.05 492.10 -1.58 108.00 432.00 -13.60 10/11/16 1-2 223.06 446.12 -10.78 122.90 491.60 -1.68 51.48 514.80 2.96 226.88 453.76 -9.25 109.46 437.84 -12.43 Run 2 2-1 266.23 532.46 6.49 141.49 565.96 13.19 56.56 565.60 13.12 273.83 547.66 9.53 128.66 514.64 2.93 10/13/16 2-2 264.39 528.78 5.76 136.70 546.80 9.36 57.95 579.50 15.90 271.72 543.44 8.69 123.05 492.20 -1.56 Run 3 3-1 258.22 516.44 3.29 127.54 510.16 2.03 54.76 547.60 9.52 265.22 530.44 6.09 117.93 471.72 -5.66 10/26/16 3-2 247.79 495.58 -0.88 130.00 520.00 4.00 54.34 543.40 8.68 253.67 507.34 1.47 120.78 483.12 -3.38

Mean: 499.70 520.19 543.45 512.46 471.92 SD: 33.24 31.14 27.42 35.80 31.99 Between-Run Imprecision (%CV): 6.65 5.99 5.05 6.99 6.78 %Bias: -0.06 4.04 8.69 2.49 -5.62

Comments: Up to 10x dilution can be used for the lower curve and 4x for the upper curve.

Acceptance Criteria: Concentration within ± 20% of target Validation Study 8 IN-PROCESS STABILITY Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20%

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 23.72 Pass 23.88 19.77 29.66 LQC-2 24.03 Pass MQC-1 253.15 Pass 239.48 189.41 284.11 MQC-2 225.81 Pass 10/11/16 *MQC-1 262.15 Pass *Upper curve 246.13 192.14 288.20 *MQC-2 230.11 Pass *Upper curve HQC-1 374.41 Pass 373.34 313.67 470.50 HQC-2 372.26 Pass

Overall Acceptance: Stable for 3 hours

Comments:

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20%

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 20.00 18.76 N/A 16.00 24.00 Pass 40.00 39.32 N/A 32.00 48.00 Pass 100.00 102.33 N/A 80.00 120.00 Pass 300.00 304.11 N/A 240.00 360.00 Pass 100.00 85.60 N/A 80.00 120.00 Pass *Upper curve 300.00 311.25 N/A 240.00 360.00 Pass *Upper curve Injected on 500.00 494.51 N/A 400.00 600.00 Pass 10/26/2016; 800.00 812.97 N/A 640.00 960.00 Pass Reinjected on 23.70 10/27/2016 24.72 23.83 19.77 29.66 Pass 23.96 248.82 236.76 244.16 189.41 284.11 Pass 239.49 249.25 *Upper curve 240.17 244.03 192.14 288.20 Pass 238.80 380.70 392.08 383.09 313.67 470.50 Pass 385.48

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 23.95 Pass 24.14 19.77 29.66 LQC-2 Punctured 24.33 Pass MQC-1 Punctured 240.58 Pass Injected on 236.64 189.41 284.11 10/27/16; MQC-2 Punctured 232.69 Pass Reinjected on *MQC-1 Punctured 240.22 Pass *Upper Curve 10/28/16 235.92 192.14 288.20 *MQC-2 Punctured 231.62 Pass *Upper Curve HQC-1 Punctured 380.28 Pass 377.14 313.67 470.50 HQC-2 Punctured 374.00 Pass LQC-1 Unpunctured 24.12 Pass 23.88 19.77 29.66 LQC-2 Unpunctured 23.63 Pass MQC-1 Unpunctured 237.35 Pass Extracted on 235.80 189.41 284.11 10/27/16; MQC-2 Unpunctured 234.24 Pass Injected on *MQC-1 Unpunctured 236.93 Pass *Upper Curve 10/28/16 235.18 192.14 288.20 *MQC-2 Unpunctured 233.42 Pass *Upper Curve HQC-1 Unpunctured 375.68 Pass 372.90 313.67 470.50 HQC-2 Unpunctured 370.12 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 Room Temperature LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Room Temperature LQC-1 w/ Light Protection LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Refrigerated (2 to 8oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Frozen (-5 to -20oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Peak Extraction Average Type of Sample Sample ID Response Efficiency Matrix Effect Response of Drug (%Recovery)

Group 1-Internal Standard and drug analyte fortified before extraction

Group 2-Internal Standard and analyte fortified after extraction

Group 3-Internal Standard and analyte fortified to elution solvent/no extraction

Extraction efficiency and matrix effects were not performed for hydrocodone, hydromorphone, Comments: oxycodone, or oxymorphone because derivatization needs to occur prior to extraction for these four analytes.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction Criteria: efficiency and matrix effect are considered acceptable. Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 50.0 60.57 40-60 21.1 Fail Blind Sample 3 100.0 106.05 80-120 6.1 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 120.0 126.84 96-144 5.7 Pass Blind Sample 7 400.0 459.25 320-480 14.8 Pass Blind Sample 8 250.0 310.74 200-300 24.3 Fail Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 500.0 551.70 400-600 10.3 Pass 11/9/16 Blind Sample 11 600.0 632.22 480-720 5.4 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 800.0 812.46 640-960 1.6 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 160.0 165.33 98-192 3.3 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 400.0 457.04 320-480 14.3 Pass

Comments: Blind Samples 2 and 8 did not meet acceptance criteria. The quality controls run with the blind samples quantitated on target for oxymorphone (< ± 5%). Blind Samples 2 and 8 quantitated high for all drugs. Therefore, the high quantitation can be attributed to over-spiking these blind samples.

Acceptance Percent difference of the expected vs. determined values ≤ 20% Criteria: SUMMARY OF VALIDATION PERFORMANCE Analyte: Oxymorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason There were multiple interferences with ions causing quantitative values, ion August 2, 2016 ratios, and overall chromatography to not be acceptable. Autosampler stability was attempted on this day, but the extraction vials dried out prior to injection so the experiment was repeated on October 27, October 13, 2016 2016.

Deviations from SOP:

Other Observations: Per recommendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of oxymorphone confirmation analysis. METHOD VALIDATION PROTOCOL AND RESULTS Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Morph.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS

Data analysis was processed using 1/x Unweighted: = 144.03 weighting even though 1/x2 gave the 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 106.72 lowest %RE because all other 1/x2 Weighting: = 96.32 validation parameters met acceptance criteria when 1/x was used.

95% CI of slope includes 1 95% CI of Slope = 0.9926 - 1.0018 1 Linearity 95% CI of intercept includes 0 95% CI of Intercept = -0.4191 - 1.0456

95% CI of slope includes 1 95% CI of Slope = 0.9900 - 1.0143 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -6.9453 - 5.1454

Max %RE low calibrator = -3.80 1 Validation Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) Max %RE other calibrators = 4.90

Not performed. Validation of 6 calibrators will 1 Casework Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) be used for casework.

Comparison of Validation Calibration to 95% CI of slope includes 1 Not performed. Validation of 6 calibrators will 1 Casework Calibration 95% CI of intercept includes 0 be used for casework.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N >3). However, all three levels did not consistently meet ion ratio LOD = 20 Signal to Noise ≥ 3 criteria (red values above indicate failed 2 Limit of Detection (LOD) S:N = >154.017; S:N not documented for 20 Acceptable detection and identification criteria ion ratios). Therefore, the LOD and ng/mL because S:N of 18 ng/mL was > 3. LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments.

Bias: ≤ 25% Bias = -1.63% Replicates in red failed to meet ion ratio 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-run Imprecision= 5.14% acceptance criteria. Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 3.51%

Bias Bias: ≤ 20% Max Bias = -8.53%

Within-Run Imprecision Within-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.22% 4

Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 5.04%

All ion ratios for codeine in the Negative (in red) do not meet acceptance 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 5.00% criteria. Therefore, the method is considered to have no significant carryover. 6 Matrix Interference Response of blank matrix ≤ 10% of the average response of LOQ No matrix interference was identified.

6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is no interference from labeled IS.

There are 0 compounds tested in this study Concentrations of analytes of interest within ± 20% of average for bias 6 Exogenous Substances Interferences that cause significant interference with the and imprecision analyte of interest. Up to 10x dilution can be used for the 7 Dilution Integrity Concentration within ± 20% of target Max % of target = -13.71 lower curve and 4x for the upper curve.

Concentration within 20% of the average value obtained in the Bias 8 In-Process Stability Stable for 3 hours and Imprecision studies

Punctured vials in Experiment 1 were stable for 24 hours. Punctured and Concentration within 20% of the average value obtained in the Bias Unpunctured Vials:Stable for 24 hours 8 Autosampler Stability unpunctured vials in Experiment 2 were and Imprecision studies Punctured Vials:Stable for 24 hours stable for 21 and 24 hours, respectively.

Study not performed; storage stability is documented in Disposition of Toxic 8 Storage Stability Concentration within 20% of average time 0 Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Extraction Efficiency = (mean peak area of samples fortified before If method achieves desired limit of quantification and/or detection for extraction/mean peak area of samples Extraction Efficiency = 73 - 92% 9 Extraction Efficiency & Matrix Effect the analyte, its extraction efficiency and matrix effect are considered fortified after extraction) x 100 Matrix Effect = 16 - 37% acceptable. Matrix Effect = (mean peak area of samples fortified after extraction/mean peak area of neat samples-1) x 100

Blind Sample 2 did not meet acceptance criteria. The quality controls run with the blind samples quantified on target for codeine (< ± 8%). Blind 10 Blind Sample Analysis Percent difference of the expected vs. determined values≤ 20% 95% of samples ≤ 20% of target value Sample 2 quantified high for all drugs. Therefore, the high quantification can be attributed to over-fortifying the blind sample.

* Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 144.03 1/x Weighting: 106.72 1/x2 Weighting: 96.32

Comments: Data analysis was processed using 1/x weighting even though 1/x2 gave the lowest %RE because all other validation parameters met acceptance criteria when 1/x was used. Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 20 0.131233 1 2.624654 20 0.13123268 400 0.017222 16.44735 -17.7633 17.76327 40 0.26307 1 10.5228 40 0.26306995 1600 0.069206 37.01893 -7.45267 7.452666 100 0.67193 1 67.19303 100 0.67193025 10000 0.45149 100.8166 0.816563 0.816563 300 1.876499 1 562.9497 300 1.87649907 90000 3.521249 288.7747 -3.74175 3.741755 500 2.961384 1 1480.692 500 2.9613839 250000 8.769795 458.0577 -8.38846 8.388461 800 4.819513 1 3855.611 800 4.81951317 640000 23.22771 747.9959 -6.50051 6.500506 20 0.14316 1 2.863191 20 0.14315957 400 0.020495 18.30839 -8.45805 8.458049 40 0.296163 1 11.84653 40 0.29616333 1600 0.087713 42.18275 5.456873 5.456873 100 0.67807 1 67.80705 100 0.67807046 10000 0.45978 101.7747 1.774666 1.774666 300 2.039331 1 611.7993 300 2.03933093 90000 4.158871 314.1826 4.727549 4.727549 500 3.340377 1 1670.188 500 3.34037677 250000 11.15812 517.1949 3.438975 3.438975 800 5.162808 1 4130.246 800 5.1628078 640000 26.65458 801.5629 0.195358 0.195358 20 0.137188 1 2.743764 20 0.13718818 400 0.018821 17.37663 -13.1169 13.11686 40 0.277721 1 11.10885 40 0.27772116 1600 0.077129 39.30507 -1.73731 1.737315 100 0.670161 1 67.01611 100 0.67016107 10000 0.449116 100.5405 0.540503 0.540503 300 1.986351 1 595.9053 300 1.98635102 90000 3.94559 305.9158 1.971928 1.971928 500 3.243326 1 1621.663 500 3.24332575 250000 10.51916 502.0513 0.410251 0.410251 800 5.085668 1 4068.535 800 5.0856682 640000 25.86402 789.5262 -1.30923 1.309228 20 0.139598 1 2.791956 20 0.13959778 400 0.019488 17.75262 -11.2369 11.23691 40 0.289703 1 11.58811 40 0.28970279 1600 0.083928 41.17466 2.936653 2.936653 100 0.67344 1 67.34397 100 0.67343968 10000 0.453521 101.0521 1.05209 1.05209 300 2.01504 1 604.5121 300 2.01504032 90000 4.060387 310.3924 3.464132 3.464132 500 3.178814 1 1589.407 500 3.17881394 250000 10.10486 491.985 -1.603 1.603004 800 5.383427 1 4306.742 800 5.38342693 640000 28.98129 835.9878 4.498471 4.498471 20 0.132523 1 2.650463 20 0.13252314 400 0.017562 16.64871 -16.7565 16.75647 40 0.273901 1 10.95604 40 0.27390094 1600 0.075022 38.70898 -3.22756 3.227562 100 0.651372 1 65.13716 100 0.65137162 10000 0.424285 97.60864 -2.39136 2.391359 300 1.974598 1 592.3794 300 1.97459813 90000 3.899038 304.0819 1.36063 1.36063 500 3.401334 1 1700.667 500 3.40133356 250000 11.56907 526.7064 5.341287 5.341287 800 5.273711 1 4218.969 800 5.27371138 640000 27.81203 818.868 2.3585 2.3585 Sum 8800 57.17141 5 32014.46 8800 57.1714134 4960000 206.9705 144.0278 Slope 0.00640871 Intercept 0.02582646 R2 0.99670744 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.131233 0.05 0.131233 1 0.00656163 20 0.000861 18.11831 -9.40846 9.408463 40 0.26307 0.025 0.26307 1 0.00657675 40 0.00173 38.56575 -3.58563 3.585628 100 0.67193 0.01 0.67193 1 0.0067193 100 0.004515 101.9784 1.97837 1.97837 300 1.876499 0.003333 1.876499 1 0.006255 300 0.011737 288.8022 -3.73258 3.732585 500 2.961384 0.002 2.961384 1 0.00592277 500 0.01754 457.0636 -8.58728 8.587278 800 4.819513 0.00125 4.819513 1 0.00602439 800 0.029035 745.2521 -6.84348 6.843483 20 0.14316 0.05 0.14316 1 0.00715798 20 0.001025 19.96812 -0.1594 0.159398 40 0.296163 0.025 0.296163 1 0.00740408 40 0.002193 43.6984 9.246004 9.246004 100 0.67807 0.01 0.67807 1 0.0067807 100 0.004598 102.9307 2.930691 2.930691 300 2.039331 0.003333 2.039331 1 0.00679777 300 0.013863 314.0568 4.685609 4.685609 500 3.340377 0.002 3.340377 1 0.00668075 500 0.022316 515.8439 3.168782 3.168782 800 5.162808 0.00125 5.162808 1 0.00645351 800 0.033318 798.4958 -0.18803 0.188027 20 0.137188 0.05 0.137188 1 0.00685941 20 0.000941 19.04198 -4.79009 4.790095 40 0.277721 0.025 0.277721 1 0.00694303 40 0.001928 40.83809 2.095232 2.095232 100 0.670161 0.01 0.670161 1 0.00670161 100 0.004491 101.704 1.703976 1.703976 300 1.986351 0.003333 1.986351 1 0.00662117 300 0.013152 305.8399 1.946617 1.946617 500 3.243326 0.002 3.243326 1 0.00648665 500 0.021038 500.7917 0.158336 0.158336 800 5.085668 0.00125 5.085668 1 0.00635709 800 0.03233 786.5317 -1.68353 1.683533 20 0.139598 0.05 0.139598 1 0.00697989 20 0.000974 19.4157 -2.92149 2.921491 40 0.289703 0.025 0.289703 1 0.00724257 40 0.002098 42.6964 6.740993 6.740993 100 0.67344 0.01 0.67344 1 0.0067344 100 0.004535 102.2125 2.212475 2.212475 300 2.01504 0.003333 2.01504 1 0.0067168 300 0.013535 310.2894 3.429816 3.429816 500 3.178814 0.002 3.178814 1 0.00635763 500 0.02021 490.7862 -1.84277 1.84277 800 5.383427 0.00125 5.383427 1 0.00672928 800 0.036227 832.7129 4.089117 4.089117 20 0.132523 0.05 0.132523 1 0.00662616 20 0.000878 18.31845 -8.40774 8.407736 40 0.273901 0.025 0.273901 1 0.00684752 40 0.001876 40.24559 0.613979 0.613979 100 0.651372 0.01 0.651372 1 0.00651372 100 0.004243 98.78981 -1.21019 1.210192 300 1.974598 0.003333 1.974598 1 0.00658199 300 0.012997 304.017 1.339008 1.339008 500 3.401334 0.002 3.401334 1 0.00680267 500 0.023138 525.2981 5.059614 5.059614 800 5.273711 0.00125 5.273711 1 0.00659214 800 0.034765 815.6965 1.962061 1.962061 Sum 8800 57.17141 0.457917 57.17141 30 0.20002836 8800 0.372087 106.7214 Slope 0.00644762 Intercept 0.01441277 R2 0.99897355 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.131233 0.0025 0.00656163 0.05 0.000328082 1 4.3055E-05 18.7473 -6.26351 6.263511 40 0.26307 0.000625 0.00657675 0.025 0.000164419 1 4.32536E-05 38.92338 -2.69155 2.691548 100 0.67193 0.0001 0.0067193 0.01 6.7193E-05 1 4.5149E-05 101.4945 1.494452 1.494452 300 1.876499 1.11111E-05 0.006255 0.003333 2.085E-05 1 3.9125E-05 285.839 -4.72034 4.720338 500 2.961384 0.000004 0.00592277 0.002 1.18455E-05 1 3.50792E-05 451.8673 -9.62653 9.62653 800 4.819513 1.5625E-06 0.00602439 0.00125 7.53049E-06 1 3.62933E-05 736.2313 -7.97108 7.971085 20 0.14316 0.0025 0.00715798 0.05 0.000357899 1 5.12367E-05 20.57256 2.86281 2.86281 40 0.296163 0.000625 0.00740408 0.025 0.000185102 1 5.48204E-05 43.98792 9.969795 9.969795 100 0.67807 0.0001 0.0067807 0.01 6.7807E-05 1 4.5978E-05 102.4341 2.434135 2.434135 300 2.039331 1.11111E-05 0.00679777 0.003333 2.26592E-05 1 4.62097E-05 310.7584 3.586137 3.586137 500 3.340377 0.000004 0.00668075 0.002 1.33615E-05 1 4.46325E-05 509.8676 1.973515 1.973515 800 5.162808 1.5625E-06 0.00645351 0.00125 8.06689E-06 1 4.16478E-05 788.7684 -1.40395 1.403954 20 0.137188 0.0025 0.00685941 0.05 0.00034297 1 4.70515E-05 19.65871 -1.70643 1.706433 40 0.277721 0.000625 0.00694303 0.025 0.000173576 1 4.82057E-05 41.16557 2.913921 2.913921 100 0.670161 0.0001 0.00670161 0.01 6.70161E-05 1 4.49116E-05 101.2237 1.2237 1.2237 300 1.986351 1.11111E-05 0.00662117 0.003333 2.20706E-05 1 4.38399E-05 302.6505 0.883495 0.883495 500 3.243326 0.000004 0.00648665 0.002 1.29733E-05 1 4.20766E-05 495.0151 -0.99698 0.99698 800 5.085668 1.5625E-06 0.00635709 0.00125 7.94636E-06 1 4.04125E-05 776.9631 -2.87961 2.879613 20 0.139598 0.0025 0.00697989 0.05 0.000348994 1 4.87189E-05 20.02747 0.137372 0.137372 40 0.289703 0.000625 0.00724257 0.025 0.000181064 1 5.24548E-05 42.99921 7.498029 7.498029 100 0.67344 0.0001 0.0067344 0.01 6.7344E-05 1 4.53521E-05 101.7255 1.725451 1.725451 300 2.01504 1.11111E-05 0.0067168 0.003333 2.23893E-05 1 4.51154E-05 307.041 2.34701 2.34701 500 3.178814 0.000004 0.00635763 0.002 1.27153E-05 1 4.04194E-05 485.1424 -2.97153 2.971528 800 5.383427 1.5625E-06 0.00672928 0.00125 8.4116E-06 1 4.52833E-05 822.5314 2.816429 2.816429 20 0.132523 0.0025 0.00662616 0.05 0.000331308 1 4.3906E-05 18.94479 -5.27606 5.276065 40 0.273901 0.000625 0.00684752 0.025 0.000171188 1 4.68886E-05 40.58093 1.452326 1.452326 100 0.651372 0.0001 0.00651372 0.01 6.51372E-05 1 4.24285E-05 98.34821 -1.65179 1.651795 300 1.974598 1.11111E-05 0.00658199 0.003333 2.194E-05 1 4.33226E-05 300.8518 0.283949 0.283949 500 3.401334 0.000004 0.00680267 0.002 1.36053E-05 1 4.62763E-05 519.1963 3.839253 3.839253 800 5.273711 1.5625E-06 0.00659214 0.00125 8.24017E-06 1 4.34563E-05 805.7408 0.717601 0.717601 8800 57.17141 0.016208368 0.20002836 0.457917 0.003133705 30 0.0013366 96.31876 Slope 0.00653433 Intercept 0.00873157 R2 0.99850332 Validation Study 1 LINEARITY Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 0.9972 Intercept 0.3132 20 ng/mL 20160505_MRL 20.00 19.24 Std err in slope, Sb 0.0023 Std err in intercept 0.3604 40 ng/mL 20160505_MRL 40.00 40.12 Degrees freedom 34 Degrees freedom 34 100 ng/mL 20160505_MRL 100.00 104.89 Confidence level 95% Confidence level 95% 300 ng/mL 20160505_MRL 300.00 295.73 Student t 2.0322 Student t 2.0322 20 ng/mL 20160512_MRL 20.00 19.29 Confidence interval 0.0046 Confidence interval 0.7324 40 ng/mL 20160512_MRL 40.00 41.96 Slope 0.9972 ± 0.0046 Intercept 0.3132 ± 0.7324 100 ng/mL 20160512_MRL 100.00 98.53 Range 0.9926 - 1.0018 Lower -0.4191 - 1.0456 300 ng/mL 20160512_MRL 300.00 300.19 PASS PASS 20 ng/mL 20160829_MRL 20.00 19.37 40 ng/mL 20160829_MRL 40.00 41.73 100 ng/mL 20160829_MRL 100.00 98.71 300 ng/mL 20160829_MRL 300.00 300.16 20 ng/mL 20161005_MRL 20.00 19.63 350.00 40 ng/mL 20161005_MRL 40.00 41.18 100 ng/mL 20161005_MRL 100.00 98.72 y = 0.9972x + 0.3132 300.00 300 ng/mL 20161005_MRL 300.00 300.45 R² = 0.9998 20 ng/mL 20161011_MRL 20.00 19.94 250.00 40 ng/mL 20161011_MRL 40.00 39.70 100 ng/mL 20161011_MRL 100.00 101.36 200.00 300 ng/mL 20161011_MRL 300.00 298.99 20 ng/mL 20161013_MRL 20.00 20.31 150.00 40 ng/mL 20161013_MRL 40.00 39.87 100 ng/mL 20161013_MRL 100.00 98.22 100.00 300 ng/mL 20161013_MRL 300.00 301.59 20 ng/mL 20161021_MRL 20.00 19.83 50.00 40 ng/mL 20161021_MRL 40.00 40.46 100 ng/mL 20161021_MRL 100.00 99.67 0.00 300 ng/mL 20161021_MRL 300.00 300.02 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 20 ng/mL 20161026_MRL 20.00 20.08 40 ng/mL 20161026_MRL 40.00 39.64 100 ng/mL 20161026_MRL 100.00 100.57 300 ng/mL 20161026_MRL 300.00 299.69 20 ng/mL 20161027_MRL 20.00 19.00 40 ng/mL 20161027_MRL 40.00 41.58 100 ng/mL 20161027_MRL 100.00 101.80 300 ng/mL 20161027_MRL 300.00 297.60

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 LINEARITY Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0021 Intercept -0.9000

100 ng/mL 20160505_MRL 100.00 99.74 Std err in slope, Sb 0.0060 Std err in intercept 2.9747 300 ng/mL 20160505_MRL 300.00 304.83 Degrees freedom 34 Degrees freedom 34 500 ng/mL 20160505_MRL 500.00 489.53 Confidence level 95% Confidence level 95% 800 ng/mL 20160505_MRL 800.00 805.88 Student t 2.0322 Student t 2.0322 100 ng/mL 20160512_MRL 100.00 97.66 Confidence interval 0.0122 Confidence interval 6.0454 300 ng/mL 20160512_MRL 300.00 307.14 Slope 1.0021 ± 0.0122 Intercept -0.9 ± 6.0454 500 ng/mL 20160512_MRL 500.00 507.36 Range 0.9900 - 1.0143 Lower -6.9453 - 5.1454 800 ng/mL 20160512_MRL 800.00 787.81 PASS PASS 100 ng/mL 20160829_MRL 100.00 100.71 300 ng/mL 20160829_MRL 300.00 304.02 500 ng/mL 20160829_MRL 500.00 480.53 800 ng/mL 20160829_MRL 800.00 814.73 100 ng/mL 20161005_MRL 100.00 99.57 300 ng/mL 20161005_MRL 300.00 297.75 500 ng/mL 20161005_MRL 500.00 511.21 800 ng/mL 20161005_MRL 800.00 791.46 900.00 100 ng/mL 20161011_MRL 100.00 99.63 800.00 y = 1.0021x - 0.9 300 ng/mL 20161011_MRL 300.00 302.96 R² = 0.9988 700.00 500 ng/mL 20161011_MRL 500.00 495.98 800 ng/mL 20161011_MRL 800.00 801.40 600.00 100 ng/mL 20161013_MRL 100.00 98.54 500.00 300 ng/mL 20161013_MRL 300.00 302.44 400.00 500 ng/mL 20161013_MRL 500.00 510.02 300.00 800 ng/mL 20161013_MRL 800.00 788.97 200.00 100 ng/mL 20161021_MRL 100.00 100.42 300 ng/mL 20161021_MRL 300.00 302.32 100.00 500 ng/mL 20161021_MRL 500.00 488.64 0.00 800 ng/mL 20161021_MRL 800.00 808.61 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 100 ng/mL 20161026_MRL 100.00 101.51 300 ng/mL 20161026_MRL 300.00 302.51 500 ng/mL 20161026_MRL 500.00 475.38 800 ng/mL 20161026_MRL 800.00 820.59 100 ng/mL 20161027_MRL 100.00 102.51 300 ng/mL 20161027_MRL 300.00 288.31 500 ng/mL 20161027_MRL 500.00 503.09 800 ng/mL 20161027_MRL 800.00 806.07

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 20 19.24 -3.80 Level 2 40 40.12 0.30 Level 3 100 104.89 4.89 Level 4 300 295.73 -1.42 5/5/16 Upper-Level 3 100 99.74 -0.26 Upper-Level 4 300 304.83 1.61 Level 5 500 489.53 -2.09 Level 6 800 805.88 0.73 Level 1 20 19.29 -3.55 Level 2 40 41.96 4.90 Level 3 100 98.53 -1.47 Level 4 300 300.19 0.06 5/12/16 Upper-Level 3 100 97.66 -2.34 Upper-Level 4 300 307.14 2.38 Level 5 500 507.36 1.47 Level 6 800 787.81 -1.52 Level 1 20 19.37 -3.15 Level 2 40 41.73 4.32 Level 3 100 98.71 -1.29 Level 4 300 300.16 0.05 8/29/16 Upper-Level 3 100 100.71 0.71 Upper-Level 4 300 304.02 1.34 Level 5 500 480.53 -3.89 Level 6 800 814.73 1.84 Level 1 20 19.63 -1.85 Level 2 40 41.18 2.95 Level 3 100 98.72 -1.28 10/5/16 Level 4 300 300.45 0.15 Upper-Level 3 100 99.57 -0.43 Upper-Level 4 300 297.75 -0.75 Level 5 500 511.21 2.24 Level 6 800 791.46 -1.07 Level 1 20 19.94 -0.30 Level 2 40 39.70 -0.75 Level 3 100 101.36 1.36 Level 4 300 298.99 -0.34 10/11/16 Upper-Level 3 100 99.63 -0.37 Upper-Level 4 300 302.96 0.99 Level 5 500 495.98 -0.80 Level 6 800 801.40 0.17

Max %RE LOQ = -3.80 Max %RE other calibrators = 4.90

Comments:

Acceptance Criteria: %RE Calibrators ± 20% of target (25% at lowest calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

6 Run One LOWER Curve UPPER Curve Slope 0.00631 Slope 0.00587 4 Intercept 0.00978 Intercept 0.0861 R2 0.999 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Two Slope 0.00675 Slope 0.0065 4 Intercept 0.1290 Intercept 0.0434 R2 0.999 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Three Slope 0.00668 Slope 0.0066 4 Intercept 0.1080 Intercept 0.00887 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Four Slope 0.00656 Slope 0.00668 4 Intercept 0.00375 Intercept 0.0141 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 6 Run Five Slope 0.00683 Slope 0.00664 4 Intercept 0.00475 Intercept 0.0260 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Cnom y (Response Ratio) 6 Combined Regression 20 0.131232682 40 0.263069954 100 0.671930252 5 300 1.876499068 500 2.961383899 800 4.819513167 4 20 0.143159568 40 0.296163333 100 0.678070457 300 2.039330927 3 500 3.340376772 800 5.1628078 Response Ratio Response 20 0.137188176 2 40 0.277721158 100 0.670161067 300 1.986351016 500 3.243325745 1 800 5.085668203 20 0.139597784 40 0.289702794 0 100 0.673439675 0.000 100.000 200.000 300.000 400.000 500.000 600.000 700.000 800.000 900.000 300 2.015040315 Concentration 500 3.178813936 800 5.383426925 Slope 0.0064 20 0.132523143 Intercept 0.0144 40 0.273900936 R2 0.9990 100 0.651371624 300 1.974598127 500 3.401333563 800 5.273711378 Validation Study 2 LOD Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 18.00 15.00 10.00 Source 1-1 223.2 137.0 211.1 Source 1-2 464.0 181.1 424.6 Source 2-1 146.3 206.2 118.4 5/5/16 Source 2-2 201.9 172.2 161.7 Source 3-1 210.3 178.3 159.1 Source 3-2 136.3 192.9 123.9 Source 1-1 253.9 217.6 254.3 Source 1-2 167.0 148.4 120.1 Source 2-1 148.8 148.2 192.8 5/12/16 Source 2-2 106.3 219.4 163.2 Source 3-1 152.2 926.0 484.6 Source 3-2 115.8 138.7 225.7 Source 1-1 42.8 66.8 40.0 Source 1-2 40.7 134.9 113.8 Source 2-1 106.0 51.9 29.1 *8/29/16 Source 2-2 88.7 136.8 30.7 Source 3-1 72.3 64.3 49.3 Source 3-2 95.8 35.7 46.9

Mean Signal to Noise: 154.017 186.467 163.850

Established LOD: 20.00 ng/mL S:N of LOD: >154.017; S:N not documented for 20 ng/mL because S:N of 18 ng/mL was > 3.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N >3). However, all three Comments: levels did not consistently meet ion ratio criteria (red values above indicate failed ion ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments. Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 20.00 8/29/16 1-1 19.60 1-2 19.39 1-3 17.82 Within Run Mean 18.94 SD 0.97 %CV 5.14% % Bias -5.32% 10/5/16 2-1 19.63 2-2 19.20 2-3 19.65 Within Run Mean 19.49 SD 0.25 %CV 1.30% % Bias -2.53% 10/11/16 3-1 19.45 3-2 19.32 3-3 20.06 Within Run Mean 19.61 SD 0.40 %CV 2.01% % Bias -1.95% 10/13/16 4-1 19.25 4-2 20.23 4-3 20.29 Within Run Mean 19.92 SD 0.58 %CV 2.93% % Bias -0.38% 10/26/16 5-1 20.21 5-2 20.33 5-3 20.69 Within Run Mean 20.41 SD 0.25 %CV 1.22% % Bias 2.05%

Mean 19.67 Imprecision Within-Run 5.14% (%CV) Between-Run 3.51% % Bias -1.63%

Comments: Replicates in red failed to meet ion ratio acceptance criteria.

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 25.00 250.00 250.00 400.00 8/29/16 1-1 22.28 210.49 212.33 362.60 1-2 21.81 225.01 227.68 370.57 1-3 21.64 222.01 223.76 329.77 Within Run Mean 21.91 219.17 221.26 354.31 SD 0.33 7.67 7.98 21.63 %CV 1.51% 3.50% 3.60% 6.10% % Bias -12.36% -12.33% -11.50% -11.42% 10/5/16 2-1 22.88 224.69 223.20 377.20 2-2 23.65 228.59 226.77 369.85 2-3 22.97 227.28 225.41 368.03 Within Run Mean 23.17 226.85 225.13 371.69 SD 0.42 1.98 1.80 4.85 %CV 1.82% 0.87% 0.80% 1.31% % Bias -7.33% -9.26% -9.95% -7.08% 10/11/16 3-1 24.65 242.70 244.99 371.48 3-2 24.49 228.02 229.88 357.78 3-3 23.03 216.76 218.29 361.88 Within Run Mean 24.06 229.16 231.05 363.71 SD 0.89 13.01 13.39 7.03 %CV 3.71% 5.68% 5.79% 1.93% % Bias -3.77% -8.34% -7.58% -9.07% 10/13/16 4-1 26.12 243.36 244.06 365.96 4-2 23.45 228.70 229.31 379.13 4-3 23.54 228.31 229.00 374.87 Within Run Mean 24.37 233.46 234.12 373.32 SD 1.52 8.58 8.61 6.72 %CV 6.22% 3.67% 3.68% 1.80% % Bias -2.52% -6.62% -6.35% -6.67% 10/26/16 5-1 24.45 236.09 238.31 361.63 5-2 24.21 230.58 232.75 367.03 5-3 24.19 242.36 244.64 370.29 Within Run Mean 24.28 236.34 238.57 366.32 SD 0.14 5.89 5.95 4.37 %CV 0.60% 2.49% 2.49% 1.19% % Bias -2.87% -5.46% -4.57% -8.42%

Mean 23.56 229.00 230.03 365.87 SD 1.19 9.28 9.59 11.63 Imprecision Max Within-Run 6.22% 5.68% 5.79% 6.10% (%CV) Between-Run 5.04% 4.05% 4.17% 3.18% % Bias -5.77% -8.40% -7.99% -8.53%

Comments:

Bias: ≤ 20% Acceptance Criteria: Within-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 20.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/5/16 10/11/16 High Calibrator 800.00 805.88 787.81 814.73 791.46 801.40 Blank -1.19 -1.49 -0.40 -0.18 0.99 % of Low Calibrator -6.0% -7.5% -2.0% -0.9% 5.0% Maximum Carryover: 5.0%

Comments: All ion ratios for codeine in the Negative (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 38199 Run 2 116665 Run 3 127068 Run 4 151331 Run 5 47474 Average 96148

Identification Peak Response Comment Blank Matrix (NO IS) 1 78 Pass Blank Matrix (NO IS) 2 194 Pass Blank Matrix (NO IS) 3 310 Pass Blank Matrix (NO IS) 4 93 Pass Blank Matrix (NO IS) 5 48 Pass Blank Matrix (NO IS) 6 304 Pass Blank Matrix (NO IS) 7 78 Pass Blank Matrix (NO IS) 8 166 Pass Blank Matrix (NO IS) 9 0 Pass Blank Matrix (NO IS) 10 177 Pass Blank Matrix (with IS) 1 3012 Pass Blank Matrix (with IS) 2 4116 Pass Blank Matrix (with IS) 3 1840 Pass Blank Matrix (with IS) 4 2529 Pass Blank Matrix (with IS) 5 1734 Pass Blank Matrix (with IS) 6 2361 Pass Blank Matrix (with IS) 7 1327 Pass Blank Matrix (with IS) 8 835 Pass Blank Matrix (with IS) 9 833 Pass Blank Matrix (with IS) 10 542 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is no interference from labeled IS. Comments:

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 23.56 ng/mL Control Acceptance (%): 20 Run Date: 10/21/2016

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration 7-Aminoclonazepam Nordiazepam Oxazepam Diazepam 1 Benzodiazepines Mix Lorazepam 1,000 ng/mL 23.55 Pass Temazepam Clonazepam Alpha-hydroxyalprazolam Alprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 22.39 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 22.80 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 23.95 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 24.99 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 22.47 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 22.79 Pass

Conclusions: There are 0 compounds tested in this study that cause significant interference with the analyte of interest.

Comments:

Acceptance Criteria: Concentrations of analytes of interest within ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated % Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 250.00 500.00 125.00 500.00 50.00 500.00 250.00 500.00 125.00 500.00 Run 1 1-1 227.97 455.94 -8.81 116.09 464.36 -7.13 46.89 468.90 -6.22 229.57 459.14 -8.17 114.81 459.24 -8.15 10/11/16 1-2 215.73 431.46 -13.71 118.04 472.16 -5.57 48.29 482.90 -3.42 217.21 434.42 -13.12 116.76 467.04 -6.59 Run 2 2-1 264.15 528.30 5.66 136.94 547.76 9.55 53.91 539.10 7.82 264.91 529.82 5.96 137.39 549.56 9.91 10/13/16 2-2 263.11 526.22 5.24 133.73 534.92 6.98 54.83 548.30 9.66 263.90 527.80 5.56 134.16 536.64 7.33 Run 3 3-1 241.47 482.94 -3.41 120.51 482.04 -3.59 50.77 507.70 1.54 243.74 487.48 -2.50 121.63 486.52 -2.70 10/26/16 3-2 239.11 478.22 -4.36 122.44 489.76 -2.05 50.50 505.00 1.00 241.36 482.72 -3.46 123.59 494.36 -1.13

Mean: 483.85 498.50 508.65 486.90 498.89 SD: 38.28 34.52 30.84 37.57 36.75 Between-Run Imprecision (%CV): 7.91 6.93 6.06 7.72 7.37 %Bias: -3.23 -0.30 1.73 -2.62 -0.22

Comments: Up to 10x dilution can be used for the lower curve and 4x for the upper curve.

Acceptance Criteria: Concentration within ± 20% of target Validation Study 8 IN-PROCESS STABILITY Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20%

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 22.37 Pass 22.62 18.85 28.27 LQC-1 22.86 Pass MQC-1 242.41 Pass 228.52 183.20 274.80 MQC-1 214.63 Pass 10/11/16 *MQC-1 244.43 Pass *Upper curve 230.15 184.02 276.03 *MQC-1 215.86 Pass *Upper curve HQC-1 347.44 Pass 347.26 292.70 439.05 HQC-2 347.07 Pass

Overall Acceptance: Stable for 3 hours

Comments:

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 20.00 18.68 N/A 16.00 24.00 Pass 40.00 39.92 N/A 32.00 48.00 Pass 100.00 106.79 N/A 80.00 120.00 Pass 300.00 323.48 N/A 240.00 360.00 Pass 100.00 107.25 N/A 80.00 120.00 Pass *Upper curve 300.00 312.90 N/A 240.00 360.00 Pass *Upper curve Injected on 500.00 498.50 N/A 400.00 600.00 Pass 10/26/16; 800.00 876.74 N/A 640.00 960.00 Pass Reinjected on 22.59 10/27/16 23.56 22.66 18.85 28.27 Pass 22.73 255.27 229.00 252.70 183.20 274.80 Pass 250.12 248.14 *Upper curve 230.03 245.71 184.02 276.03 Pass 243.28 374.40 365.87 379.87 292.70 439.05 Pass 385.33

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 23.25 Pass 23.65 18.85 28.27 LQC-2 Punctured 24.05 Pass MQC-1 Punctured 248.78 Pass Injected on 245.76 183.20 274.80 10/27/16; MQC-2 Punctured 242.73 Pass Reinjected on *MQC-1 Punctured 242.02 Pass *Upper Curve 10/28/16 239.13 184.02 276.03 *MQC-2 Punctured 236.24 Pass *Upper Curve HQC-1 Punctured 371.70 Pass 370.97 292.70 439.05 HQC-2 Punctured 370.24 Pass LQC-1 Unpunctured 22.96 Pass 22.77 18.85 28.27 LQC-2 Unpunctured 22.57 Pass MQC-1 Unpunctured 241.77 Pass Extracted on 236.39 183.20 274.80 10/27/16; MQC-2 Unpunctured 231.01 Pass Injected on *MQC-1 Unpunctured 235.37 Pass *Upper Curve 10/28/16 230.26 184.02 276.03 *MQC-2 Unpunctured 225.14 Pass *Upper Curve HQC-1 Unpunctured 370.95 Pass 366.38 292.70 439.05 HQC-2 Unpunctured 361.81 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 Room Temperature LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Room Temperature LQC-1 w/ Light Protection LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Refrigerated (2 to 8oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Frozen (-5 to -20oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date: 11/7/16 Peak Extraction Average Type of Sample Sample ID Response of Efficiency Matrix Effect Response Drug (%Recovery) 25 ng/mL-1 37741 25 ng/mL-2 43334 25 ng/mL-3 41907 39943 76 37 25 ng/mL-4 37091 25 ng/mL-5 39643 Group 1-Internal 250 ng/mL-1 361247 Standard and 250 ng/mL-2 397976 drug analyte 250 ng/mL-3 400678 393537 73 16 fortified before 250 ng/mL-4 419275 extraction 250 ng/mL-5 388509 400 ng/mL-1 760964 400 ng/mL-2 791042 400 ng/mL-3 826058 802123 92 25 400 ng/mL-4 841234 400 ng/mL-5 791316 25 ng/mL-1 44376 25 ng/mL-2 52359 25 ng/mL-3 53514 52221 25 ng/mL-4 55578 25 ng/mL-5 55276 250 ng/mL-1 552475 Group 2-Internal 250 ng/mL-2 544341 Standard and 250 ng/mL-3 552479 538166 analyte fortified 250 ng/mL-4 519406 after extraction 250 ng/mL-5 522128 400 ng/mL-1 884545 400 ng/mL-2 862953 400 ng/mL-3 784289 871800 400 ng/mL-4 897363 400 ng/mL-5 929850 25 ng/mL-1 38812 25 ng/mL-2 42893 25 ng/mL-3 39250 38147 25 ng/mL-4 37679 25 ng/mL-5 32101 Group 3-Internal 250 ng/mL-1 461734 Standard and 250 ng/mL-2 447032 analyte fortified 250 ng/mL-3 442191 462081 to elution 250 ng/mL-4 442682 solvent/no 250 ng/mL-5 516767 extraction 400 ng/mL-1 705172 400 ng/mL-2 661218 400 ng/mL-3 665260 698658 400 ng/mL-4 705114 400 ng/mL-5 756528

Extraction Efficiency = (mean peak area of samples fortified before extraction/mean peak area of Comments: samples fortified after extraction) x 100. Matrix Effect = (mean peak area of samples fortified after extraction/mean peak area of neat samples-1) x 100.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction efficiency and matrix effect are Criteria: considered acceptable. Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 50.0 60.22 40-60 20.4 Fail Blind Sample 3 100.0 102.42 80-120 2.4 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 120.0 122.46 96-144 2.0 Pass Blind Sample 7 400.0 443.64 320-480 10.9 Pass Blind Sample 8 250.0 294.76 200-300 17.9 Pass Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 500.0 525.97 400-600 5.2 Pass 11/9/16 Blind Sample 11 600.0 630.50 480-720 5.1 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 800.0 827.09 640-960 3.4 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 160.0 165.13 98-192 3.2 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 400.0 440.12 320-480 10.0 Pass

Blind Sample 2 did not meet acceptance criteria. The quality controls run with the blind samples quantified on target for Comments: codeine (< ± 8%). Blind Sample 2 quantified high for all drugs. Therefore, the high quantification can be attributed to over- fortifying the blind sample.

Acceptance Percent difference of the expected vs. determined values ≤ 20% Criteria: SUMMARY OF VALIDATION PERFORMANCE Analyte: Codeine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason There were multiple interferences with ions causing quantitative values, ion August 2, 2016 ratios, and overall chromatography to not be acceptable. Autosampler stability was attempted on this day, but the extraction vials October 13, 2016 dried out prior to injection so the experiment was repeated on October 27, 2016.

Deviations from SOP:

Other Observations: Throughout validation codeine ions 343.1 and 372.2 were seen to be concentration dependent. Therefore, the acceptance criteria for these ions will be ± 2 standard deviations from the average of the calibrators used in the calibration curve. Please see supplemental Opioids SIM Quantification QC Worksheets for more details. Per reccomendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of codeine confirmation analysis. METHOD VALIDATION PROTOCOL AND RESULTS Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Morph.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS

Data analysis was processed using 1/x Unweighted: = 140.46 weighting even though 1/x2 gave the 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 122.4 lowest %RE because all other validation 1/x2 Weighting: = 122.21 parameters met acceptance criteria when 1/x was used.

95% CI of the slope does not include 1. However, linearity is acceptable based on evaluation of the standard residual 95% CI of slope includes 1 95% CI of Slope = 1.0014 - 1.0135 1 Linearity plot, correlation coefficient, standard 95% CI of intercept includes 0 95% CI of Intercept = -1.8271 - 0.1097 error, and % difference of individual calibrator values from the target concentration.

95% CI of slope includes 1 95% CI of Slope = 0.9962 - 1.0270 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -12.6026 - 2.7394

Max %RE low calibrator = 3.90 1 Validation Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) Max %RE other calibrators = -5.15

Not performed. Validation of 6 calibrators 1 Casework Calibration %RE Calibrators ± 20% of target (25% at lowest calibrator) will be used for casework.

Comparison of Validation Calibration to 95% CI of slope includes 1 Not performed. Validation of 6 calibrators 1 Casework Calibration 95% CI of intercept includes 0 will be used for casework.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three LOD = 20 levels did not consistently meet ion ratio Signal to Noise ≥ 3 S:N = >109.789; S:N not documented for 20 criteria (red values above indicate failed 2 Limit of Detection (LOD) Acceptable detection and identification criteria ng/mL because the S:N of 18 ng/mL was ion ratios). Therefore, the LOD and >3. LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments.

Bias: ≤ 25% Bias = 2.48% 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-Run Imprecision= 3.47% Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 2.94% Bias Bias: ≤ 20% Max Bias = -10.59% 4 Within-Run Imprecision Within-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.98% Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 6.02%

All ion ratios for morphine in the Negative (in red) do not meet 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 24.90% acceptance criteria. Therefore, the method is considered to have no significant carryover.

6 Matrix Interference Response of blank matrix ≤ 10% of the average response of LOQ No matrix interference was identified.

6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is no interference from labeled IS. There are 0 compounds tested in this study Concentration of analytes of interest withing ± 20% of average for 6 Exogenous Substances Interferences that cause significant interference with the bias and imprecision analyte of interest.

Up to 10x dilution can be used for the 7 Dilution Integrity Concentration within ± 20% of target Max % of target = 19.62 lower curve and 4x for the upper curve.

Concentration within 20% of the average value obtained in the Bias 8 In-Process Stability Stable for 3 hours and Imprecision studies

Punctured vials in Experiment 1 were Concentration within 20% of the average value obtained in the Bias Unpunctured Vials:Stable for 24 hours stable for 24 hours. Punctured and 8 Autosampler Stability and Imprecision studies Punctured Vials:Stable for 24 hours unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Study not performed; storage stability is documented in Disposition of Toxic 8 Storage Stability Concentration within 20% of average time 0 Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Extraction Efficiency = (mean peak area of samples fortified before If method achieves desired limit of quantification and/or detection extraction/mean peak area of samples Extraction Efficiency = 55 - 63% 9 Extraction Efficiency & Matrix Effect for the analyte, its extraction efficiency and matrix effect are fortified after extraction) x 100 Matrix Effect = 13 - 29% considered acceptable. Matrix Effect = (mean peak area of samples fortified after extraction/mean peak area of neat samples-1) x 100.

10 Blind Sample Analysis Percent difference of the expected vs. determined values ≤ 20% 100% of samples ≤ 20% of target value

* Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 140.46 1/x Weighting: 122.40 1/x2 Weighting: 122.21

Comments: Data analysis was processed using 1/x weighting even though 1/x2 gave the lowest %RE because all other validation parameters met acceptance criteria when 1/x was used. Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 20 0.12676 1 2.535208 20 0.12676041 400 0.016068 16.96127 -15.1936 15.19364 40 0.276171 1 11.04683 40 0.27617083 1600 0.07627 36.57286 -8.56784 8.56784 100 0.694823 1 69.48233 100 0.69482328 10000 0.482779 91.52513 -8.47487 8.474871 300 2.07859 1 623.5769 300 2.0785895 90000 4.320534 273.1581 -8.9473 8.947299 500 3.365478 1 1682.739 500 3.36547784 250000 11.32644 442.0749 -11.585 11.58502 800 5.757745 1 4606.196 800 5.75774511 640000 33.15163 756.0836 -5.48955 5.489552 20 0.140279 1 2.805574 20 0.14027868 400 0.019678 18.73568 -6.32161 6.321609 40 0.303842 1 12.15368 40 0.30384204 1600 0.09232 40.20498 0.512457 0.512457 100 0.738005 1 73.80053 100 0.73800531 10000 0.544652 97.1932 -2.8068 2.806802 300 2.289864 1 686.9591 300 2.28986361 90000 5.243475 300.8899 0.296638 0.296638 500 3.852462 1 1926.231 500 3.85246212 250000 14.84146 505.9964 1.199278 1.199278 800 6.037348 1 4829.879 800 6.03734839 640000 36.44958 792.7843 -0.90197 0.901965 20 0.146569 1 2.931376 20 0.14656879 400 0.021482 19.56132 -2.19341 2.193414 40 0.308423 1 12.33692 40 0.3084229 1600 0.095125 40.80627 2.015666 2.015666 100 0.741549 1 74.15492 100 0.7415492 10000 0.549895 97.65837 -2.34163 2.341633 300 2.311584 1 693.4751 300 2.31158358 90000 5.343419 303.7409 1.246958 1.246958 500 3.946911 1 1973.456 500 3.94691142 250000 15.57811 518.3938 3.678759 3.678759 800 6.262054 1 5009.643 800 6.26205403 640000 39.21332 822.2791 2.784889 2.784889 20 0.14497 1 2.899402 20 0.1449701 400 0.021016 19.35147 -3.24263 3.242632 40 0.312112 1 12.48449 40 0.31211231 1600 0.097414 41.29054 3.226344 3.226344 100 0.748931 1 74.89305 100 0.74893053 10000 0.560897 98.62724 -1.37276 1.37276 300 2.376487 1 712.9461 300 2.37648692 90000 5.64769 312.2601 4.086693 4.086693 500 3.793588 1 1896.794 500 3.79358835 250000 14.39131 498.2686 -0.34627 0.346275 800 6.636274 1 5309.019 800 6.63627428 640000 44.04014 871.3992 8.924901 8.924901 20 0.13203 1 2.640596 20 0.13202979 400 0.017432 17.65293 -11.7354 11.73535 40 0.282386 1 11.29545 40 0.28238636 1600 0.079742 37.38871 -6.52822 6.528216 100 0.703593 1 70.35929 100 0.70359289 10000 0.495043 92.67623 -7.32377 7.323772 300 2.239758 1 671.9274 300 2.23975802 90000 5.016516 294.3131 -1.89565 1.895646 500 3.945501 1 1972.75 500 3.94550074 250000 15.56698 518.2086 3.641725 3.641725 800 6.310382 1 5048.306 800 6.31038208 640000 39.82092 828.6226 3.57783 3.57783 Sum 8800 67.00447 5 38079.72 8800 67.0044694 4960000 293.1213 140.4604 Slope 0.00761847 Intercept -0.00245861 R2 0.99981253 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.12676 0.05 0.12676 1 0.00633802 20 0.000803 18.8371 -5.81451 5.81451 40 0.276171 0.025 0.276171 1 0.00690427 40 0.001907 38.30459 -4.23852 4.238518 100 0.694823 0.01 0.694823 1 0.00694823 100 0.004828 92.85309 -7.14691 7.146907 300 2.07859 0.003333 2.07859 1 0.00692863 300 0.014402 273.1515 -8.9495 8.949497 500 3.365478 0.002 3.365478 1 0.00673096 500 0.022653 440.8272 -11.8346 11.83456 800 5.757745 0.00125 5.757745 1 0.00719718 800 0.04144 752.5287 -5.93392 5.933916 20 0.140279 0.05 0.140279 1 0.00701393 20 0.000984 20.59847 2.992335 2.992335 40 0.303842 0.025 0.303842 1 0.00759605 40 0.002308 41.91002 4.775061 4.775061 100 0.738005 0.01 0.738005 1 0.00738005 100 0.005447 98.47952 -1.52048 1.520485 300 2.289864 0.003333 2.289864 1 0.00763288 300 0.017478 300.6796 0.226519 0.226519 500 3.852462 0.002 3.852462 1 0.00770492 500 0.029683 504.279 0.855801 0.855801 800 6.037348 0.00125 6.037348 1 0.00754669 800 0.045562 788.9597 -1.38004 1.380038 20 0.146569 0.05 0.146569 1 0.00732844 20 0.001074 21.41804 7.090197 7.090197 40 0.308423 0.025 0.308423 1 0.00771057 40 0.002378 42.50689 6.267226 6.267226 100 0.741549 0.01 0.741549 1 0.00741549 100 0.005499 98.94127 -1.05873 1.058733 300 2.311584 0.003333 2.311584 1 0.00770528 300 0.017811 303.5096 1.169857 1.169857 500 3.946911 0.002 3.946911 1 0.00789382 500 0.031156 516.5853 3.317063 3.317063 800 6.262054 0.00125 6.262054 1 0.00782757 800 0.049017 818.2378 2.279727 2.279727 20 0.14497 0.05 0.14497 1 0.00724851 20 0.001051 21.20974 6.048688 6.048688 40 0.312112 0.025 0.312112 1 0.00780281 40 0.002435 42.9876 7.469008 7.469008 100 0.748931 0.01 0.748931 1 0.00748931 100 0.005609 99.90302 -0.09698 0.096979 300 2.376487 0.003333 2.376487 1 0.00792162 300 0.018826 311.9662 3.988727 3.988727 500 3.793588 0.002 3.793588 1 0.00758718 500 0.028783 496.608 -0.6784 0.678396 800 6.636274 0.00125 6.636274 1 0.00829534 800 0.05505 866.997 8.374626 8.374626 20 0.13203 0.05 0.13203 1 0.00660149 20 0.000872 19.52367 -2.38163 2.381632 40 0.282386 0.025 0.282386 1 0.00705966 40 0.001994 39.11445 -2.21388 2.21388 100 0.703593 0.01 0.703593 1 0.00703593 100 0.00495 93.99573 -6.00427 6.004266 300 2.239758 0.003333 2.239758 1 0.00746586 300 0.016722 294.151 -1.94966 1.949657 500 3.945501 0.002 3.945501 1 0.007891 500 0.031134 516.4015 3.280302 3.280302 800 6.310382 0.00125 6.310382 1 0.00788798 800 0.049776 824.5347 3.066842 3.066842 Sum 8800 67.00447 0.457917 67.00447 30 0.22208967 8800 0.51163 122.404 Slope 0.00767487 Intercept -0.01781180 R2 0.99834528 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 20 0.12676 0.0025 0.00633802 0.05 0.000317 1 4.02E-05 18.43215 -7.83925 7.839254 40 0.276171 0.000625 0.00690427 0.025 0.000173 1 4.77E-05 38.06854 -4.82866 4.828661 100 0.694823 0.0001 0.00694823 0.01 6.95E-05 1 4.83E-05 93.09027 -6.90973 6.909726 300 2.07859 1.11E-05 0.00692863 0.003333 2.31E-05 1 4.8E-05 274.9529 -8.34904 8.349041 500 3.365478 0.000004 0.00673096 0.002 1.35E-05 1 4.53E-05 444.0832 -11.1834 11.18336 800 5.757745 1.56E-06 0.00719718 0.00125 9E-06 1 5.18E-05 758.4889 -5.18889 5.188888 20 0.140279 0.0025 0.00701393 0.05 0.000351 1 4.92E-05 20.2088 1.043996 1.043996 40 0.303842 0.000625 0.00759605 0.025 0.00019 1 5.77E-05 41.70525 4.263116 4.263116 100 0.738005 0.0001 0.00738005 0.01 7.38E-05 1 5.45E-05 98.76551 -1.23449 1.234492 300 2.289864 1.11E-05 0.00763288 0.003333 2.54E-05 1 5.83E-05 302.7197 0.906583 0.906583 500 3.852462 0.000004 0.00770492 0.002 1.54E-05 1 5.94E-05 508.0855 1.617106 1.617106 800 6.037348 1.56E-06 0.00754669 0.00125 9.43E-06 1 5.7E-05 795.236 -0.5955 0.595502 20 0.146569 0.0025 0.00732844 0.05 0.000366 1 5.37E-05 21.03548 5.177409 5.177409 40 0.308423 0.000625 0.00771057 0.025 0.000193 1 5.95E-05 42.30729 5.768225 5.768225 100 0.741549 0.0001 0.00741549 0.01 7.42E-05 1 5.5E-05 99.23127 -0.76873 0.768734 300 2.311584 1.11E-05 0.00770528 0.003333 2.57E-05 1 5.94E-05 305.5743 1.858104 1.858104 500 3.946911 0.000004 0.00789382 0.002 1.58E-05 1 6.23E-05 520.4986 4.099721 4.099721 800 6.262054 1.56E-06 0.00782757 0.00125 9.78E-06 1 6.13E-05 824.7681 3.096013 3.096013 20 0.14497 0.0025 0.00724851 0.05 0.000362 1 5.25E-05 20.82537 4.126865 4.126865 40 0.312112 0.000625 0.00780281 0.025 0.000195 1 6.09E-05 42.79217 6.980434 6.980434 100 0.748931 0.0001 0.00748931 0.01 7.49E-05 1 5.61E-05 100.2014 0.201364 0.201364 300 2.376487 1.11E-05 0.00792162 0.003333 2.64E-05 1 6.28E-05 314.1043 4.701429 4.701429 500 3.793588 0.000004 0.00758718 0.002 1.52E-05 1 5.76E-05 500.348 0.069599 0.069599 800 6.636274 1.56E-06 0.00829534 0.00125 1.04E-05 1 6.88E-05 873.9503 9.243789 9.243789 20 0.13203 0.0025 0.00660149 0.05 0.00033 1 4.36E-05 19.12468 -4.37659 4.376594 40 0.282386 0.000625 0.00705966 0.025 0.000176 1 4.98E-05 38.88542 -2.78646 2.786459 100 0.703593 0.0001 0.00703593 0.01 7.04E-05 1 4.95E-05 94.24283 -5.75717 5.757172 300 2.239758 1.11E-05 0.00746586 0.003333 2.49E-05 1 5.57E-05 296.1346 -1.28847 1.288473 500 3.945501 0.000004 0.007891 0.002 1.58E-05 1 6.23E-05 520.3132 4.062641 4.062641 800 6.310382 1.56E-06 0.00788798 0.00125 9.86E-06 1 6.22E-05 831.1197 3.889958 3.889958 8800 67.00447 0.016208 0.22208967 0.457917 0.003266 30 0.00165 122.2127 Slope 0.00760886 Intercept -0.01348714 R2 0.99785018 Validation Study 1 LINEARITY Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0074 Intercept -0.8587

20 ng/mL 20160505_MRL 20.00 19.48 Std err in slope, Sb 0.0030 Std err in intercept 0.4775 40 ng/mL 20160505_MRL 40.00 40.86 Degrees freedom 36 Degrees freedom 36 100 ng/mL 20160505_MRL 100.00 100.79 Confidence level 95% Confidence level 95% 300 ng/mL 20160505_MRL 300.00 298.85 Student t 2.0281 Student t 2.0281 20 ng/mL 20160512_MRL 20.00 19.74 Confidence interval 0.0061 Confidence interval 0.9684 40 ng/mL 20160512_MRL 40.00 41.15 Slope 1.0074 ± 0.0061 Intercept -0.8587 ± 0.9684 100 ng/mL 20160512_MRL 100.00 97.98 Range 1.0014 - 1.0135 Lower -1.8271 - 0.1097 300 ng/mL 20160512_MRL 300.00 301.10 FAIL PASS 20 ng/mL 20160829_MRL 20.00 19.97 40 ng/mL 20160829_MRL 40.00 41.12 100 ng/mL 20160829_MRL 100.00 96.51 300 ng/mL 20160829_MRL 300.00 302.38 20 ng/mL 20161005_MRL 20.00 20.26 40 ng/mL 20161005_MRL 40.00 40.40 100 ng/mL 20161005_MRL 100.00 96.80 300 ng/mL 20161005_MRL 300.00 302.51 350.00 20 ng/mL 20161011_MRL 20.00 20.78 y = 1.0074x - 0.8587 300.00 40 ng/mL 20161011_MRL 40.00 38.75 R² = 0.9997 100 ng/mL 20161011_MRL 100.00 98.53 250.00 300 ng/mL 20161011_MRL 300.00 301.92 20 ng/mL 20161013_MRL 20.00 20.83 200.00 40 ng/mL 20161013_MRL 40.00 39.86 150.00 100 ng/mL 20161013_MRL 100.00 94.56 300 ng/mL 20161013_MRL 300.00 304.72 100.00 20 ng/mL 20161021_MRL 20.00 20.50 50.00 40 ng/mL 20161021_MRL 40.00 39.88 100 ng/mL 20161021_MRL 100.00 96.82 0.00 300 ng/mL 20161021_MRL 300.00 302.78 0.00 50.00 100.00 150.00 200.00 250.00 300.00 350.00 20 ng/mL 20161026_MRL 20.00 20.84 40 ng/mL 20161026_MRL 40.00 39.43 100 ng/mL 20161026_MRL 100.00 95.95 300 ng/mL 20161026_MRL 300.00 303.77 20 ng/mL 20161027_MRL 20.00 19.47 40 ng/mL 20161027_MRL 40.00 41.37 100 ng/mL 20161027_MRL 100.00 99.20 300 ng/mL 20161027_MRL 300.00 299.94

Comments: 95% CI of the slope does not include 1. However, linearity is acceptable based on evaluation of the standard residual plot, correlation coefficient, standard error, and % difference of individual calibrator values from the target concentration.

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 1 - Standard Residuals

Regression Statistics Multiple R 0.999850247 R Square 0.999700516 Adjusted R Sq0.999691707 Standard Erro1.987870917 Observations 36

ANOVA df SS MS F Significance F Regression 1 448488.0733 448488.0733 113494.4 1.70385E-61 Residual 34 134.3554467 3.951630784 Total 35 448622.4287

Coefficients Standard Error t Stat P-value Lower 95% Upper 95% Lower 95.0% Upper 95.0% Intercept -0.85379045 0.477525172 -1.787948575 0.0827 -1.824238359 0.116657 -1.82423836 0.116657458 X Variable 1 1.00742668 0.002990379 336.88934 1.7E-61 1.0013495 1.013504 1.0013495 1.013503861

RESIDUAL OUTPUT

Observation Predicted Y Residuals Standard Residuals 1 19.29474315 0.185256845 0.094554162 Standard Residuals Plot 2 39.44327676 1.426723241 0.728192362 2 3 99.88887757 0.901122426 0.459928351 4 301.3742136 -2.514213623 -1.283241981 1.5 5 19.29474315 0.455256845 0.232360803 6 39.44327676 1.716723241 0.876206903 1 7 99.88887757 -1.898877574 -0.969177558 0.5 8 301.3742136 -0.264213623 -0.134853304 9 19.29474315 0.675256845 0.344647696 0 10 39.44327676 1.686723241 0.860895054 0 50 100 150 200 250 300 350 11 99.88887757 -3.378877574 -1.72456211 -0.5 12 301.3742136 1.005786377 0.513348305 Residuals -1 13 19.29474315 0.975256845 0.497766186 14 39.44327676 0.956723241 0.488306727 -1.5 15 99.88887757 -3.078877574 -1.57144362 16 301.3742136 1.145786377 0.584803601 -2 17 19.29474315 1.495256845 0.763171569 18 39.44327676 -0.693276759 -0.353844969 -2.5 19 99.88887757 -1.358877574 -0.693564275 -3 20 301.3742136 0.555786377 0.28367057 X Variable 1 21 19.29474315 1.545256845 0.788691318 22 39.44327676 0.426723241 0.217797395 23 99.88887757 -5.318877574 -2.714728347 24 301.3742136 3.355786377 1.712776479 25 19.29474315 1.215256845 0.620260978 26 39.44327676 0.436723241 0.222901344 27 99.88887757 -3.058877574 -1.56123572 28 301.3742136 1.405786377 0.717506292 29 19.29474315 1.545256845 0.788691318 30 39.44327676 -0.013276759 -0.006776391 31 99.88887757 -3.938877574 -2.010383292 32 301.3742136 2.405786377 1.22790126 33 19.29474315 0.185256845 0.094554162 34 39.44327676 1.926723241 0.983389846 35 99.88887757 -0.688877574 -0.351599647 36 301.3742136 -1.424213623 -0.726911466 Validation Study 1 UPPER LINEARITY Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0116 Intercept -4.9316

100 ng/mL 20160505_MRL 100.00 102.12 Std err in slope, Sb 0.0076 Std err in intercept 3.7746 300 ng/mL 20160505_MRL 300.00 298.13 Degrees freedom 34 Degrees freedom 34 500 ng/mL 20160505_MRL 500.00 480.43 Confidence level 95% Confidence level 95% 800 ng/mL 20160505_MRL 800.00 819.30 Student t 2.0322 Student t 2.0322 100 ng/mL 20160512_MRL 100.00 98.95 Confidence interval 0.0154 Confidence interval 7.6710 300 ng/mL 20160512_MRL 300.00 302.04 Slope 1.0116 ± 0.0154 Intercept -4.9316 ± 7.671 500 ng/mL 20160512_MRL 500.00 506.53 Range 0.9962 - 1.0270 Lower -12.6026 - 2.7394 800 ng/mL 20160512_MRL 800.00 792.46 PASS PASS 100 ng/mL 20160829_MRL 100.00 101.76 300 ng/mL 20160829_MRL 300.00 301.87 500 ng/mL 20160829_MRL 500.00 474.25 800 ng/mL 20160829_MRL 800.00 822.10 100 ng/mL 20161005_MRL 100.00 100.81 300 ng/mL 20161005_MRL 300.00 294.91 500 ng/mL 20161005_MRL 500.00 504.66 800 ng/mL 20161005_MRL 800.00 799.60 900.00 100 ng/mL 20161011_MRL 100.00 100.88 800.00 y = 1.0116x - 4.9316 300 ng/mL 20161011_MRL 300.00 300.25 R² = 0.9981 700.00 500 ng/mL 20161011_MRL 500.00 488.91 800 ng/mL 20161011_MRL 800.00 809.94 600.00 100 ng/mL 20161013_MRL 100.00 100.56 500.00 300 ng/mL 20161013_MRL 300.00 295.81 400.00 500 ng/mL 20161013_MRL 500.00 505.05 300.00 800 ng/mL 20161013_MRL 800.00 798.56 200.00 100 ng/mL 20161021_MRL 100.00 101.74 300 ng/mL 20161021_MRL 300.00 297.14 100.00 500 ng/mL 20161021_MRL 500.00 487.58 0.00 800 ng/mL 20161021_MRL 800.00 813.52 0.00 100.00 200.00 300.00 400.00 500.00 600.00 700.00 800.00 900.00 100 ng/mL 20161026_MRL 100.00 103.64 300 ng/mL 20161026_MRL 300.00 296.14 500 ng/mL 20161026_MRL 500.00 468.24 800 ng/mL 20161026_MRL 800.00 831.97 100 ng/mL 20161027_MRL 100.00 102.83 300 ng/mL 20161027_MRL 300.00 285.58 500 ng/mL 20161027_MRL 500.00 506.98 800 ng/mL 20161027_MRL 800.00 804.59

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 20 19.48 -2.60 Level 2 40 40.86 2.15 Level 3 100 100.79 0.79 Level 4 300 298.85 -0.38 5/5/16 Upper-Level 3 100 102.12 2.12 Upper-Level 4 300 298.13 -0.62 Level 5 500 480.43 -3.91 Level 6 800 819.30 2.41 Level 1 20 19.74 -1.30 Level 2 40 41.15 2.88 Level 3 100 97.98 -2.02 Level 4 300 301.10 0.37 5/12/16 Upper-Level 3 100 98.95 -1.05 Upper-Level 4 300 302.04 0.68 Level 5 500 506.53 1.31 Level 6 800 792.46 -0.94 Level 1 20 19.97 -0.15 Level 2 40 41.12 2.80 Level 3 100 96.51 -3.49 Level 4 300 302.38 0.79 8/29/16 Upper-Level 3 100 101.76 1.76 Upper-Level 4 300 301.87 0.62 Level 5 500 474.25 -5.15 Level 6 800 822.10 2.76 Level 1 20 20.26 1.30 Level 2 40 40.40 1.00 Level 3 100 96.80 -3.20 Level 4 300 302.51 0.84 10/5/16 Upper-Level 3 100 100.81 0.81 Upper-Level 4 300 294.91 -1.70 Level 5 500 504.66 0.93 Level 6 800 799.60 -0.05 Level 1 20 20.78 3.90 Level 2 40 38.75 -3.13 Level 3 100 98.53 -1.47 Level 4 300 301.92 0.64 10/11/16 Upper-Level 3 100 100.88 0.88 Upper-Level 4 300 300.25 0.08 Level 5 500 488.91 -2.22 Level 6 800 809.94 1.24

Max %RE LOQ = 3.90 Max %RE other calibrators = -5.15

Comments:

Acceptance Criteria: %RE Calibrators ± 20% of target (25% at lowest calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

8 Run One LOWER Curve UPPER Curve 6 Slope 0.00699 Slope 0.00706 Intercept 0.00936 Intercept 0.0261 4 R2 1 R2 0.998 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 8 Run Two 6 Slope 0.00764 Slope 0.00764 Intercept 0.0106 Intercept 0.0181 4 2 2 R 1.000 R 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 8 Run Three 6 Slope 0.00791 Slope 0.00817 Intercept 0.0120 Intercept 0.0822 4 2 2 R 0.999 R 0.997 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 8 Run Four 6 Slope 0.00747 Slope 0.00802 Intercept 0.0193 Intercept 0.1010 4 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration 8 Run Five 6 Slope 0.00806 Slope 0.0083 Intercept 0.0401 Intercept 0.0752 4 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 200.000 400.000 600.000 800.000 1000.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Cnom y (Response Ratio) 7 Combined Regression 20 0.126760411 40 0.276170832 6 100 0.694823276 300 2.078589503 500 3.365477841 5 800 5.757745106 20 0.140278683 40 0.303842038 4 100 0.738005314 300 2.289863607 500 3.852462123 3 800 6.037348392 Response Ratio Response 20 0.146568792 40 0.308422903 2 100 0.741549196 300 2.311583578 500 3.946911417 1 800 6.262054028 20 0.144970104 40 0.312112311 0 100 0.74893053 0.000 100.000 200.000 300.000 400.000 500.000 600.000 700.000 800.000 900.000 300 2.376486921 Concentration 500 3.793588349 800 6.636274279 Slope 0.0077 20 0.132029787 Intercept -0.0178 40 0.282386362 R2 0.9983 100 0.703592894 300 2.239758015 500 3.945500736 800 6.31038208 Validation Study 2 LOD Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 18.00 15.00 10.00 Source 1-1 89.4 31.4 32.2 Source 1-2 122.8 64.5 33.3 Source 2-1 124.1 37.4 34.9 5/5/16 Source 2-2 157.0 71.5 32.8 Source 3-1 100.1 51.3 40.9 Source 3-2 112.7 44.8 31.9 Source 1-1 109.0 114.6 56.6 Source 1-2 181.3 138.6 36.9 Source 2-1 181.8 120.1 99.1 5/12/16 Source 2-2 214.4 164.7 54.5 Source 3-1 131.8 89.8 94.3 Source 3-2 273.9 94.7 78.0 Source 1-1 23.7 172.6 123.7 Source 1-2 24.4 258.6 20.4 Source 2-1 39.7 173.7 4.8 **8/29/2016 Source 2-2 27.1 27.4 94.4 Source 3-1 22.8 40.0 184.3 Source 3-2 40.2 14.8 16.8

Mean Signal to Noise: 109.789 95.028 59.433

Established LOD: 20.00 ng/mL S:N of LOD: >109.789; S:N not documented for 20 ng/mL because the S:N of 18 ng/mL was >3.

The signal to noise comparison for 10 ng/mL, 15 ng/mL, and 18 ng/mL was acceptable (S:N > 3). However, all three Comments: levels did not consistently meet ion ratio criteria (red values above indicate failed ion ratios). Therefore, the LOD and LOQ will be 20 ng/mL; the LOQ experiment for 20 ng/mL was performed using three different sources as required for LOD experiments. Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 20.00 8/29/16 1-1 20.49 1-2 20.34 1-3 19.22 Within Run Mean 20.02 SD 0.69 %CV 3.47% % Bias 0.08% 10/5/16 2-1 20.28 2-2 20.09 2-3 20.06 Within Run Mean 20.14 SD 0.12 %CV 0.59% % Bias 0.72% 10/11/16 3-1 20.47 3-2 20.51 3-3 21.01 Within Run Mean 20.66 SD 0.30 %CV 1.46% % Bias 3.32% 10/13/16 4-1 20.77 4-2 21.85 4-3 21.29 Within Run Mean 21.30 SD 0.54 %CV 2.54% % Bias 6.52% 10/26/16 5-1 20.03 5-2 20.46 5-3 20.58 Within Run Mean 20.36 SD 0.29 %CV 1.42% % Bias 1.78%

Mean 20.50 Imprecision Within-Run 3.47% (%CV) Between-Run 2.94% % Bias 2.48%

Comments:

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 25.00 250.00 250.00 400.00 8/29/16 1-1 23.07 202.82 204.71 347.62 1-2 22.08 219.22 221.63 361.29 1-3 21.85 215.59 217.30 314.92 Within Run Mean 22.33 212.54 214.55 341.28 SD 0.65 8.61 8.79 23.83 %CV 2.90% 4.05% 4.10% 6.98% % Bias -10.67% -14.98% -14.18% -14.68% 10/5/16 2-1 23.51 223.74 219.71 370.90 2-2 24.45 233.19 228.49 365.08 2-3 23.78 226.78 222.56 363.16 Within Run Mean 23.91 227.90 223.59 366.38 SD 0.48 4.82 4.48 4.03 %CV 2.02% 2.12% 2.00% 1.10% % Bias -4.35% -8.84% -10.57% -8.40% 10/11/16 3-1 25.81 238.92 237.23 357.60 3-2 26.34 219.40 218.28 348.96 3-3 23.85 212.49 211.39 353.94 Within Run Mean 25.33 223.60 222.30 353.50 SD 1.31 13.71 13.38 4.34 %CV 5.18% 6.13% 6.02% 1.23% % Bias 1.33% -10.56% -11.08% -11.63% 10/13/16 4-1 27.22 242.36 237.87 353.77 4-2 25.12 226.07 222.74 366.09 4-3 25.15 225.51 222.21 368.74 Within Run Mean 25.83 231.31 227.61 362.87 SD 1.20 9.57 8.89 7.99 %CV 4.66% 4.14% 3.91% 2.20% % Bias 3.32% -7.47% -8.96% -9.28% 10/26/16 5-1 24.75 241.80 238.74 361.51 5-2 24.85 233.32 230.88 363.75 5-3 24.67 244.03 240.80 367.23 Within Run Mean 24.76 239.72 236.81 364.16 SD 0.09 5.65 5.23 2.88 %CV 0.36% 2.36% 2.21% 0.79% % Bias -0.97% -4.11% -5.28% -8.96%

Mean 24.43 227.02 224.97 357.64 SD 1.47 12.02 10.56 13.74 Imprecision Max Within-Run 5.18% 6.13% 6.02% 6.98% (%CV) Between-Run 6.02% 5.29% 4.69% 3.84% % Bias -2.27% -9.19% -10.01% -10.59%

Comments:

Bias: ≤ 20% Acceptance Criteria: Within-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 20.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/5/16 10/11/16 High Calibrator 800.000 819.30 792.46 822.10 799.60 809.940 Blank 1.36 1.44 3.38 2.64 4.98 % of Low Calibrator 6.8% 7.2% 16.9% 13.2% 24.9% Maximum Carryover: 24.9%

Comments: All ion ratios for morphine in the Negative (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 21207 Run 2 86123 Run 3 90756 Run 4 121162 Run 5 28500 Average 69550

Identification Peak Response Comment Blank Matrix (NO IS) 1 0 Pass Blank Matrix (NO IS) 2 0 Pass Blank Matrix (NO IS) 3 0 Pass Blank Matrix (NO IS) 4 0 Pass Blank Matrix (NO IS) 5 0 Pass Blank Matrix (NO IS) 6 0 Pass Blank Matrix (NO IS) 7 0 Pass Blank Matrix (NO IS) 8 0 Pass Blank Matrix (NO IS) 9 0 Pass Blank Matrix (NO IS) 10 0 Pass Blank Matrix (with IS) 1 94 Pass Blank Matrix (with IS) 2 281 Pass Blank Matrix (with IS) 3 1654 Pass Blank Matrix (with IS) 4 307 Pass Blank Matrix (with IS) 5 50 Pass Blank Matrix (with IS) 6 2839 Pass Blank Matrix (with IS) 7 445 Pass Blank Matrix (with IS) 8 1390 Pass Blank Matrix (with IS) 9 0 Pass Blank Matrix (with IS) 10 13 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is no interference from labeled IS. Comments:

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 25 ng/mL Control Acceptance (%): 20 Run Date: 10/21/16

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration Desalkylflurazepam Flurazepam Nordiazepam Oxazepam 1 Benzodiazepines Mix Diazepam 1,000 ng/mL 25.16 Pass Lorazepam Temazepam Clonazepam Alpha-hydroxyalprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 23.23 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 23.61 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 25.33 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 25.57 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 24.11 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 22.9 Pass

Conclusions: There are 0 compounds tested in this study that cause significant interference with the analyte of interest.

Comments:

Acceptance Criteria: Concentration of analytes of interest withing ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated %Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 250.00 500.00 125.00 500.00 50.00 500.00 250.00 500.00 125.00 500.00 Run 1 1-1 231.75 463.50 -7.30 112.38 449.52 -10.10 48.29 482.90 -3.42 230.31 460.62 -7.88 114.29 457.16 -8.57 10/11/16 1-2 210.68 421.36 -15.73 115.01 460.04 -7.99 48.49 484.90 -3.02 209.85 419.70 -16.06 116.51 466.04 -6.79 Run 2 2-1 273.17 546.34 9.27 141.66 566.64 13.33 58.08 580.80 16.16 266.50 533.00 6.60 144.31 577.24 15.45 10/13/16 2-2 270.62 541.24 8.25 139.05 556.20 11.24 59.81 598.10 19.62 264.13 528.26 5.65 141.89 567.56 13.51 Run 3 3-1 249.17 498.34 -0.33 124.19 496.76 -0.65 51.41 514.10 2.82 245.56 491.12 -1.78 129.80 519.20 3.84 10/26/16 3-2 250.56 501.12 0.22 125.39 501.56 0.31 53.27 532.70 6.54 246.85 493.70 -1.26 130.91 523.64 4.73

Mean: 495.32 505.12 532.25 487.73 518.47 SD: 47.41 48.17 48.37 42.67 49.80 Between-Run Imprecision (%CV): 9.57 9.54 9.09 8.75 9.60 %Bias: -0.94 1.02 6.45 -2.45 3.69

Comments: Up to 10x dilution can be used for the lower curve and 4x for the upper curve.

Acceptance Criteria: Concentration within 20% of Target Validation Study 8 IN-PROCESS STABILITY Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 23.49 Pass 23.71 19.55 29.32 LQC-2 23.93 Pass MQC-1 244.04 Pass 229.29 181.61 272.42 MQC-2 214.53 Pass 10/11/16 *MQC-1 242.34 Pass *Upper curve 227.92 179.98 269.96 *MQC-1 213.50 Pass *Upper curve HQC-1 345.45 Pass 343.32 286.11 429.16 HQC-2 341.18 Pass

Overall Acceptance: Stable for 3 hours

Comments:

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 20 19.91 N/A 16.00 24.00 Pass 40 39.17 N/A 32.00 48.00 Pass 100 98.17 N/A 80.00 120.00 Pass 300 313.95 N/A 240.00 360.00 Pass 100 101.89 N/A 80.00 120.00 Pass *Upper curve 300 298.33 N/A 240.00 360.00 Pass *Upper curve 500 487.22 N/A 400.00 600.00 Pass Injected on 10/26/16; 800 897.26 N/A 640.00 960.00 Pass Reinjected on 24.20 10/27/16 24.43 24.18 19.55 29.32 Pass 24.16 248.97 227.02 246.02 181.61 272.42 Pass 243.07 239.18 *Upper curve 224.97 236.50 179.98 269.96 Pass 233.81 365.03 357.64 368.64 286.11 429.16 Pass 372.25

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 24.12 Pass 23.91 19.55 29.32 LQC-2 Punctured 23.69 Pass MQC-1 Punctured 241.33 Pass Injected on 241.68 181.61 272.42 10/27/16; MQC-2 Punctured 242.03 Pass Reinjected on *MQC-1 Punctured 232.23 Pass *Upper curve 10/28/16 232.55 179.98 269.96 *MQC-2 Punctured 232.86 Pass *Upper curve HQC-1 Punctured 369.99 Pass 369.24 286.11 429.16 HQC-2 Punctured 368.48 Pass LQC-1 Unpunctured 23.94 Pass 23.48 19.55 29.32 LQC-2 Unpunctured 23.01 Pass MQC-1 Unpunctured 235.47 Pass Extracted on 235.14 181.61 272.42 10/27/16; MQC-2 Unpunctured 234.80 Pass Injected on *MQC-1 Unpunctured 226.89 Pass *Upper curve 10/28/16 226.59 179.98 269.96 *MQC-2 Unpunctured 226.28 Pass *Upper curve HQC-1 Unpunctured 364.83 Pass 358.46 286.11 429.16 HQC-2 Unpunctured 352.08 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 Room Temperature LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Room Temperature LQC-1 w/ Light Protection LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Refrigerated (2 to 8oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3 Frozen (-5 to -20oC) LQC-1 LQC-2 LQC-3 HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemical in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Peak Extraction Average Type of Sample Sample ID Response Efficiency Matrix Effect Response of Drug (%Recovery) 25 ng/mL-1 28356 25 ng/mL-2 32453 25 ng/mL-3 32543 27775 55 29 25 ng/mL-4 18206 25 ng/mL-5 27318 250 ng/mL-1 302084 Group 1-Internal Standard and 250 ng/mL-2 296631 drug analyte 250 ng/mL-3 334940 310233 56 13 fortified before 250 ng/mL-4 366194 extraction 250 ng/mL-5 251315 400 ng/mL-1 503413 400 ng/mL-2 690299 400 ng/mL-3 483136 576996 63 23 400 ng/mL-4 616100 400 ng/mL-5 592031 25 ng/mL-1 43127 25 ng/mL-2 51119 25 ng/mL-3 52086 50797 25 ng/mL-4 54117 25 ng/mL-5 53535 250 ng/mL-1 577919 Group 2-Internal 250 ng/mL-2 557116 Standard and 250 ng/mL-3 560948 551623 analyte fortified 250 ng/mL-4 527338 after extraction 250 ng/mL-5 534796 400 ng/mL-1 907923 400 ng/mL-2 887116 400 ng/mL-3 839824 909966 400 ng/mL-4 944279 400 ng/mL-5 970687 25 ng/mL-1 40533 25 ng/mL-2 44391 25 ng/mL-3 39909 39269 25 ng/mL-4 37804 25 ng/mL-5 33707 Group 3-Internal 250 ng/mL-1 489194 Standard and 250 ng/mL-2 472453 analyte fortified 250 ng/mL-3 475408 487712 to elution 250 ng/mL-4 469520 solvent/no 250 ng/mL-5 531986 extraction 400 ng/mL-1 739846 400 ng/mL-2 707077 400 ng/mL-3 714732 740682 400 ng/mL-4 747609 400 ng/mL-5 794147

Extraction Efficiency = (mean peak area of samples fortified before extraction/mean peak area of samples fortified after extraction) x 100. Matrix Effect = (mean peak area of samples fortified after extraction/mean peak area of neat samples-1) x 100.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction efficiency Criteria: and matrix effect are considered acceptable. Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 50.0 58.58 40-60 17.2 Pass Blind Sample 3 100.0 101.65 80-120 1.7 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 120.0 122.10 96-144 1.8 Pass Blind Sample 7 400.0 435.27 320-480 8.8 Pass Blind Sample 8 250.0 298.88 200-300 19.6 Pass Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 500.0 534.34 400-600 6.9 Pass Blind Sample 11 600.0 613.96 480-720 2.3 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 800.0 826.12 640-960 3.3 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 160.0 159.12 98-192 -0.5 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 400.0 422.01 320-480 5.5 Pass

Comments:

Acceptance Percent difference of the expected vs. determined values ≤ 20% Criteria: SUMMARY OF VALIDATION PERFORMANCE Analyte: Morphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason There were multiple interferences with ions causing quantitative values, ion August 2, 2016 ratios, and overall chromatography to not be acceptable. Autosampler stability was attempted on this day, but the extraction vials October 13, 2016 dried out prior to injection so the experiment was repeated on October 27, 2016.

Deviations from SOP:

Other Observations: Per reccomendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of morphine confirmation analysis. METHOD VALIDATION PROTOCOL AND RESULTS Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Method: Morph.M Matrix: Blood Instrument: GCMS-2 SOP REFERENCE v3.1 SOP CRITERIA ALTERNATE CRITERIA* RESULTS VALIDATION COMMENTS Unweighted: = 112.75 Data analysis will be processed using 1 Weight Verification Weighting minimizes Σ|%RE| 1/x Weighting: = 104.42 1/x. 1/x2 Weighting: = 107.7

95% CI of slope includes 1 95% CI of Slope = 0.9952 - 1.0074 1 Linearity 95% CI of intercept includes 0 95% CI of Intercept = -0.2859 - 0.2030

95% CI of slope includes 1 95% CI of Slope = 0.9917 - 1.0178 1 Upper Linearity 95% CI of intercept includes 0 95% CI of Intercept = -2.1272 - 1.1142

Max %RE low calibrator = -7.60 1 Validation Calibration %RE Calibrators ± 20% of target (25% at the lowest calibrator) Max %RE other calibrators = 5.20

Not performed. Validation of 6 calibrators will be 1 Casework Calibration %RE Calibrators ± 20% of target (25% at the lowest calibrator) used for casework.

Comparison of Validation Calibration to 95% CI of slope includes 1 Not performed. Validation of 6 calibrators will be 1 Casework Calibration 95% CI of intercept includes 0 used for casework.

The signal to noise comparison for 2.50 ng/mL, 3.75 ng/mL, and 4.50 ng/mL was acceptable (S:N > 3). However, all three levels did not consistently meet ion ratio LOD = 5 Signal to Noise ≥ 3 criteria (red values above indicate failed 2 Limit of Detection (LOD) S:N = >132.239; S:N not documented for 5.0 Acceptable detection and identification criteria ion ratios). Therefore, the LOD and LOQ ng/mL because the S:N of 4.50 ng/mL was > 3. will be 5 ng/mL; the LOQ experiment for 5 ng/mL was performed using three different sources as required for LOD experiments.

Bias: ≤ 25% Bias = 0.67% 3 Limit of Quantification (LOQ) Within-Run Imprecision: CV ≤ 25% Within-Run Imprecision= 3.39% Between-Run Imprecision: CV ≤ 25% Between-Run Imprecision = 3.17% Bias Bias: ≤ 20% Max Bias = 18.10% Replicates in red did not calculate to within 20% of the target. This can be Within-Run Imprecision Within-Run Imprecision: CV ≤ 20% Max Within-Run Imprecision = 6.36% attributed to the degradation of 6- 4 acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood. Between-Run Imprecision Between-Run Imprecision: CV ≤ 20% Max Between-Run Imprecision = 6.14%

All ion ratios for 6-acetylmorphine (in red) do not meet acceptance criteria. 5 Carryover Quantitative result ≤ 25% of LOQ Maximum Carryover = 11.00% Therefore, the method is considered to have no significant carryover.

6 Matrix Interference Response of blank matrix≤ 10% of the average response of LOQ No matrix interference was identified. The Blank Matrix with IS 2 and 5 (in red) were >10% of the average response of the LOQ. However, all ion ratios did not meet acceptance criteria. Therefore, the method is considered to have no 6 Labeled IS Interference Response ≤ 10% of the average response of LOQ There is interference from labeled IS. significant interference contribution from labeled IS. 6-Acetylmorphine will be reported only qualitatively in blood.

PCP may have caused the calculated control (in red) to not fall within 20% of the target. However, this could be Concentrations of analytes of interest within ± 20% of average for bias There are compounds tested in this study that attributed to the degradation of 6- 6 Exogenous Substances Interferences and imprecision may interfere with the analyte of interest. acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood.

Replicates in red did not calculate to within 20% of the target. This can be attributed to the degradation of 6- 7 Dilution Integrity Concentration within ± 20% of target Max % of target = 34.00 acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood.

Concentration within 20% of the average value obtained in the Bias and 6-Acetylmorphine will be reported only 8 In-Process Stability Stable for 3 hours Imprecision studies qualitatively in blood.

Punctured vials in Experiment 1 were stable for 24 hours. Punctured and Concentration within 20% of the average value obtained in the Bias and Unpunctured Vials:Stable for 24 hours unpunctured vials in Experiment 2 were 8 Autosampler Stability Imprecision studies Punctured Vials:Stable for 24 hours stable for 21 and 24 hours, respectively. 6-Acetylmorphine will be reported only qualitatively in blood.

Study not performed; storage stability is documented in Disposition of Toxic 8 Storage Stability Concentration within 20% of average time 0 Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Extraction Efficiency = (mean peak area of samples fortified before If method achieves desired limit of quantification and/or detection for the extraction/mean peak area of samples Extraction Efficiency = 70 - 90% 9 Extraction Efficiency & Matrix Effect analyte, its extraction efficiency and matrix effect are considered fortified after extraction) x 100. Matrix Effect = 12 - 40% acceptable Matrix Effect = (mean peak area of samples fortified after extraction/mean peak area of neat samples-1) x 100.

The MQC did not calculate to within 20% of the target. This can be attributed to the degradation of 6-acetylmorphine, or to 10 Blind Sample Analysis Percent difference of the expected vs. determined values≤ 20% 100% of samples ≤ 20% of target value possible incorrect preparation of the calibrator prepared on 10/04/16. 6- Acetylmorphine will be reported only qualitatively in blood.

* Alternate criteria is any deviation from that described in the SOP as determined by the Supervisor Validation Study 1 STANDARD CURVE WEIGHT VERIFICATION Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

│%RE│ Unweighted: 112.75 1/x Weighting: 104.42 1/x2 Weighting: 107.70

Comments: Data analysis will be processed using 1/x.

Acceptance Criteria: Weighting minimizes Σ|%RE|

2 2 Cnom y w wxy wx wy wx wy Cfound %RE │%RE│ 5 0.099998 1 0.49999 5 0.09999791 25 0.01 4.06766 -18.6468 18.64681 10 0.227 1 2.270003 10 0.22700029 100 0.051529 9.837321 -1.62679 1.626788 25 0.558479 1 13.96197 25 0.55847897 625 0.311899 24.89625 -0.415 0.415001 75 1.59442 1 119.5815 75 1.59442031 5625 2.542176 71.9586 -4.0552 4.055197 125 2.574142 1 321.7678 125 2.57414228 15625 6.626208 116.4669 -6.82645 6.826452 200 4.278852 1 855.7704 200 4.27885182 40000 18.30857 193.9111 -3.04444 3.044436 5 0.120998 1 0.604992 5 0.12099832 25 0.014641 5.021699 0.433975 0.433975 10 0.25134 1 2.5134 10 0.25134001 100 0.063172 10.94306 9.430635 9.430635 25 0.582576 1 14.56439 25 0.58257562 625 0.339394 25.99095 3.963798 3.963798 75 1.750466 1 131.2849 75 1.75046568 5625 3.06413 79.04767 5.396899 5.396899 125 2.891181 1 361.3976 125 2.89118097 15625 8.358927 130.8699 4.695889 4.695889 200 4.479444 1 895.8888 200 4.47944376 40000 20.06542 203.0239 1.511966 1.511966 5 0.116916 1 0.584581 5 0.11691614 25 0.013669 4.836247 -3.27506 3.27506 10 0.233696 1 2.336957 10 0.23369574 100 0.054614 10.14149 1.414921 1.414921 25 0.569155 1 14.22888 25 0.56915532 625 0.323938 25.38127 1.525086 1.525086 75 1.688997 1 126.6748 75 1.68899697 5625 2.852711 76.25518 1.673571 1.673571 125 2.763706 1 345.4632 125 2.76370559 15625 7.638069 125.0787 0.062969 0.062969 200 4.366304 1 873.2609 200 4.36630438 40000 19.06461 197.8841 -1.05797 1.05797 5 0.117148 1 0.585741 5 0.11714826 25 0.013724 4.846792 -3.06416 3.064156 10 0.243551 1 2.435507 10 0.24355075 100 0.059317 10.5892 5.892008 5.892008 25 0.548609 1 13.71523 25 0.5486092 625 0.300972 24.44787 -2.20852 2.208519 75 1.716501 1 128.7376 75 1.71650136 5625 2.946377 77.50469 3.339587 3.339587 125 2.711012 1 338.8765 125 2.71101207 15625 7.349586 122.6849 -1.85211 1.852106 200 4.541829 1 908.3657 200 4.54182862 40000 20.62821 205.858 2.929024 2.929024 5 0.107732 1 0.538659 5 0.1077318 25 0.011606 4.419007 -11.6199 11.61987 10 0.223603 1 2.23603 10 0.22360299 100 0.049998 9.682983 -3.17017 3.170167 25 0.531929 1 13.29822 25 0.53192899 625 0.282948 23.6901 -5.23962 5.239617 75 1.627001 1 122.0251 75 1.62700112 5625 2.647133 73.43873 -2.08169 2.081688 125 2.819656 1 352.4571 125 2.81965647 15625 7.950463 127.6205 2.096428 2.096428 200 4.404189 1 880.8378 200 4.404189 40000 19.39688 199.6051 -0.19743 0.19743 Sum 2200 48.74043 5 6846.764 2200 48.7404307 310000 151.3409 112.748 Slope 0.02201210 Intercept 0.01046017 R2 0.99834541 2 2 Cnom y w (1/x) wxy wx wy wx wy Cfound %RE │%RE│ 5 0.099998 0.2 0.099998 1 0.01999958 5 0.002 4.22674 -15.4652 15.4652 10 0.227 0.1 0.227 1 0.02270003 10 0.005153 9.98315 -0.1685 0.168495 25 0.558479 0.04 0.558479 1 0.02233916 25 0.012476 25.00749 0.029974 0.029974 75 1.59442 0.013333 1.59442 1 0.02125894 75 0.033896 71.96176 -4.05099 4.050987 125 2.574142 0.008 2.574142 1 0.02059314 125 0.05301 116.3679 -6.9057 6.905703 200 4.278852 0.005 4.278852 1 0.02139426 200 0.091543 193.6342 -3.1829 3.182899 5 0.120998 0.2 0.120998 1 0.02419966 5 0.002928 5.178588 3.571758 3.571758 10 0.25134 0.1 0.25134 1 0.025134 10 0.006317 11.08635 10.86353 10.86353 25 0.582576 0.04 0.582576 1 0.02330302 25 0.013576 26.09968 4.398717 4.398717 75 1.750466 0.013333 1.750466 1 0.02333954 75 0.040855 79.03455 5.379401 5.379401 125 2.891181 0.008 2.891181 1 0.02312945 125 0.066871 130.7377 4.590175 4.590175 200 4.479444 0.005 4.479444 1 0.02239722 200 0.100327 202.7261 1.363038 1.363038 5 0.116916 0.2 0.116916 1 0.02338323 5 0.002734 4.993562 -0.12876 0.128759 10 0.233696 0.1 0.233696 1 0.02336957 10 0.005461 10.28662 2.866228 2.866228 25 0.569155 0.04 0.569155 1 0.02276621 25 0.012958 25.4914 1.965606 1.965606 75 1.688997 0.013333 1.688997 1 0.02251996 75 0.038036 76.24847 1.664624 1.664624 125 2.763706 0.008 2.763706 1 0.02210964 125 0.061105 124.9599 -0.0321 0.032105 200 4.366304 0.005 4.366304 1 0.02183152 200 0.095323 197.598 -1.201 1.200996 5 0.117148 0.2 0.117148 1 0.02342965 5 0.002745 5.004083 0.081662 0.081662 10 0.243551 0.1 0.243551 1 0.02435507 10 0.005932 10.7333 7.333033 7.333033 25 0.548609 0.04 0.548609 1 0.02194437 25 0.012039 24.56014 -1.75942 1.759424 75 1.716501 0.013333 1.716501 1 0.02288668 75 0.039285 77.49511 3.326813 3.326813 125 2.711012 0.008 2.711012 1 0.0216881 125 0.058797 122.5715 -1.94278 1.942781 200 4.541829 0.005 4.541829 1 0.02270914 200 0.103141 205.5537 2.776841 2.776841 5 0.107732 0.2 0.107732 1 0.02154636 5 0.002321 4.57728 -8.4544 8.454401 10 0.223603 0.1 0.223603 1 0.0223603 10 0.005 9.829167 -1.70833 1.70833 25 0.531929 0.04 0.531929 1 0.02127716 25 0.011318 23.80411 -4.78356 4.783561 75 1.627001 0.013333 1.627001 1 0.02169335 75 0.035295 73.43849 -2.08201 2.08201 125 2.819656 0.008 2.819656 1 0.02255725 125 0.063604 127.4959 1.996684 1.996684 200 4.404189 0.005 4.404189 1 0.02202094 200 0.096984 199.3151 -0.34243 0.342432 Sum 2200 48.74043 1.831667 48.74043 30 0.67423653 2200 1.081028 104.4162 Slope 0.02206277 Intercept 0.00674431 R2 0.99932011 2 2 2 Cnom y w (1/x ) wxy wx wy wx wy Cfound %RE │%RE│ 5 0.099998 0.04 0.01999958 0.2 0.004 1 0.0004 4.332999 -13.34 13.34003 10 0.227 0.01 0.02270003 0.1 0.00227 1 0.000515 10.03907 0.390735 0.390735 25 0.558479 0.0016 0.02233916 0.04 0.000894 1 0.000499 24.93204 -0.27184 0.271843 75 1.59442 0.000178 0.02125894 0.013333 0.000283 1 0.000452 71.47572 -4.69904 4.699035 125 2.574142 0.000064 0.02059314 0.008 0.000165 1 0.000424 115.4935 -7.60517 7.605172 200 4.278852 0.000025 0.02139426 0.005 0.000107 1 0.000458 192.0842 -3.95789 3.957887 5 0.120998 0.04 0.02419966 0.2 0.00484 1 0.000586 5.276523 5.530469 5.530469 10 0.25134 0.01 0.025134 0.1 0.002513 1 0.000632 11.13263 11.3263 11.3263 25 0.582576 0.0016 0.02330302 0.04 0.000932 1 0.000543 26.01467 4.058699 4.058699 75 1.750466 0.000178 0.02333954 0.013333 0.000311 1 0.000545 78.48667 4.64889 4.64889 125 2.891181 0.000064 0.02312945 0.008 0.000185 1 0.000535 129.7377 3.790183 3.790183 200 4.479444 0.000025 0.02239722 0.005 0.000112 1 0.000502 201.0966 0.548299 0.548299 5 0.116916 0.04 0.02338323 0.2 0.004677 1 0.000547 5.093116 1.862311 1.862311 10 0.233696 0.01 0.02336957 0.1 0.002337 1 0.000546 10.33989 3.398922 3.398922 25 0.569155 0.0016 0.02276621 0.04 0.000911 1 0.000518 25.41172 1.646863 1.646863 75 1.688997 0.000178 0.02251996 0.013333 0.0003 1 0.000507 75.72495 0.966597 0.966597 125 2.763706 0.000064 0.02210964 0.008 0.000177 1 0.000489 124.0104 -0.79168 0.791678 200 4.366304 0.000025 0.02183152 0.005 0.000109 1 0.000477 196.0134 -1.99331 1.993314 5 0.117148 0.04 0.02342965 0.2 0.004686 1 0.000549 5.103545 2.070891 2.070891 10 0.243551 0.01 0.02435507 0.1 0.002436 1 0.000593 10.78267 7.826667 7.826667 25 0.548609 0.0016 0.02194437 0.04 0.000878 1 0.000482 24.4886 -2.04559 2.045594 75 1.716501 0.000178 0.02288668 0.013333 0.000305 1 0.000524 76.96069 2.614251 2.614251 125 2.711012 0.000064 0.0216881 0.008 0.000174 1 0.00047 121.6429 -2.68565 2.685647 200 4.541829 0.000025 0.02270914 0.005 0.000114 1 0.000516 203.8995 1.94974 1.94974 5 0.107732 0.04 0.02154636 0.2 0.004309 1 0.000464 4.680474 -6.39053 6.39053 10 0.223603 0.01 0.0223603 0.1 0.002236 1 0.0005 9.886437 -1.13563 1.135635 25 0.531929 0.0016 0.02127716 0.04 0.000851 1 0.000453 23.73918 -5.04329 5.043287 75 1.627001 0.000178 0.02169335 0.013333 0.000289 1 0.000471 72.93954 -2.74728 2.747276 125 2.819656 0.000064 0.02255725 0.008 0.00018 1 0.000509 126.5242 1.21937 1.21937 200 4.404189 0.000025 0.02202094 0.005 0.00011 1 0.000485 197.7155 -1.14226 1.142258 2200 48.74043 0.259334 0.67423653 1.831667 0.04169 30 0.015188 107.6984 Slope 0.02225740 Intercept 0.00355663 R2 0.99803040 Validation Study 1 LINEARITY Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0013 Intercept -0.0415

5 ng/mL 20160505_MRL 5.00 4.62 Std err in slope, Sb 0.0030 Std err in intercept 0.1203 10 ng/mL 20160505_MRL 10.00 10.52 Degrees freedom 34 Degrees freedom 34 25 ng/mL 20160505_MRL 25.00 25.89 Confidence level 95% Confidence level 95% 75 ng/mL 20160505_MRL 75.00 73.95 Student t 2.0322 Student t 2.0322 5 ng/mL 20160512_MRL 5.00 4.83 Confidence interval 0.0061 Confidence interval 0.2444 10 ng/mL 20160512_MRL 10.00 10.44 Slope 1.0013 ± 0.0061 Intercept -0.0415 ± 0.2444 25 ng/mL 20160512_MRL 25.00 24.71 Range 0.9952 - 1.0074 Lower -0.2859 - 0.2030 75 ng/mL 20160512_MRL 75.00 75.00 PASS PASS 5 ng/mL 20160829_MRL 5.00 4.93 10 ng/mL 20160829_MRL 10.00 10.45 25 ng/mL 20160829_MRL 25.00 23.98 75 ng/mL 20160829_MRL 75.00 75.63 5 ng/mL 20161005_MRL 5.00 4.92 10 ng/mL 20161005_MRL 10.00 10.29 25 ng/mL 20161005_MRL 25.00 24.55 75 ng/mL 20161005_MRL 75.00 75.22 80.00 5 ng/mL 20161011_MRL 5.00 4.98 70.00 y = 1.0013x - 0.0415 10 ng/mL 20161011_MRL 10.00 10.02 R² = 0.9997 25 ng/mL 20161011_MRL 25.00 25.02 60.00 75 ng/mL 20161011_MRL 75.00 74.96 50.00 5 ng/mL 20161013_MRL 5.00 5.08 40.00 10 ng/mL 20161013_MRL 10.00 10.12 25 ng/mL 20161013_MRL 25.00 23.99 30.00 75 ng/mL 20161013_MRL 75.00 75.79 20.00 5 ng/mL 20161021_MRL 5.00 5.03 10 ng/mL 20161021_MRL 10.00 10.19 10.00 25 ng/mL 20161021_MRL 25.00 24.11 0.00 75 ng/mL 20161021_MRL 75.00 75.65 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 5 ng/mL 20161026_MRL 5.00 4.95 10 ng/mL 20161026_MRL 10.00 10.11 25 ng/mL 20161026_MRL 25.00 24.92 75 ng/mL 20161026_MRL 75.00 75.00 5 ng/mL 20161027_MRL 5.00 5.21 10 ng/mL 20161027_MRL 10.00 9.33 25 ng/mL 20161027_MRL 25.00 25.69 75 ng/mL 20161027_MRL 75.00 74.76

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 UPPER LINEARITY Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Conc Calc. Conc Slope 1.0047 Intercept -0.5065

25 ng/mL 20160505_MRL 25.00 25.32 Std err in slope, Sb 0.0064 Std err in intercept 0.7975 75 ng/mL 20160505_MRL 75.00 74.84 Degrees freedom 34 Degrees freedom 34 125 ng/mL 20160505_MRL 125.00 121.67 Confidence level 95% Confidence level 95% 200 ng/mL 20160505_MRL 200.00 203.16 Student t 2.0322 Student t 2.0322 25 ng/mL 20160512_MRL 25.00 24.52 Confidence interval 0.0130 Confidence interval 1.6207 75 ng/mL 20160512_MRL 75.00 76.30 Slope 1.0047 ± 0.013 Intercept -0.5065 ± 1.6207 125 ng/mL 20160512_MRL 125.00 126.87 Range 0.9917 - 1.0178 Lower -2.1272 - 1.1142 200 ng/mL 20160512_MRL 200.00 197.29 PASS PASS 25 ng/mL 20160829_MRL 25.00 24.92 75 ng/mL 20160829_MRL 75.00 76.75 125 ng/mL 20160829_MRL 125.00 121.02 200 ng/mL 20160829_MRL 200.00 202.29 25 ng/mL 20161005_MRL 25.00 25.01 75 ng/mL 20161005_MRL 75.00 74.05 125 ng/mL 20161005_MRL 125.00 127.45 250.00 200 ng/mL 20161005_MRL 200.00 198.47 y = 1.0047x - 0.5065 200.00 25 ng/mL 20161011_MRL 25.00 25.07 R² = 0.9986 75 ng/mL 20161011_MRL 75.00 75.21 125 ng/mL 20161011_MRL 125.00 123.54 150.00 200 ng/mL 20161011_MRL 200.00 201.16 25 ng/mL 20161013_MRL 25.00 24.84 100.00 75 ng/mL 20161013_MRL 75.00 74.68 125 ng/mL 20161013_MRL 125.00 127.63 50.00 200 ng/mL 20161013_MRL 200.00 197.82 25 ng/mL 20161021_MRL 25.00 24.71 75 ng/mL 20161021_MRL 75.00 77.36 0.00 125 ng/mL 20161021_MRL 125.00 121.74 0.00 50.00 100.00 150.00 200.00 250.00 200 ng/mL 20161021_MRL 200.00 201.17 25 ng/mL 20161026_MRL 25.00 25.87 75 ng/mL 20161026_MRL 75.00 73.70 125 ng/mL 20161026_MRL 125.00 118.45 200 ng/mL 20161026_MRL 200.00 206.97 25 ng/mL 20161027_MRL 25.00 25.81 75 ng/mL 20161027_MRL 75.00 71.28 125 ng/mL 20161027_MRL 125.00 125.83 200 ng/mL 20161027_MRL 200.00 202.06

Comments:

95% CI of slope includes 1 Acceptance Criteria: 95% CI of intercept includes 0 Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: 6-Acetylmorphone Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Weighted Run Date Run Order Target % of Target Result Level 1 5 4.62 -7.60 Level 2 10 10.52 5.20 Level 3 25 25.89 3.56 Level 4 75 73.95 -1.40 5/5/16 Upper-Level 3 25 25.32 1.28 Upper-Level 4 75 74.84 -0.21 Level 5 125 121.67 -2.66 Level 6 200 203.16 1.58 Level 1 5 4.83 -3.40 Level 2 10 10.44 4.40 Level 3 25 24.71 -1.16 Level 4 75 75.00 0.00 5/12/16 Upper-Level 3 25 24.52 -1.92 Upper-Level 4 75 76.30 1.73 Level 5 125 126.87 1.50 Level 6 200 197.29 -1.36 Level 1 5 4.93 -1.40 Level 2 10 10.45 4.50 Level 3 25 23.98 -4.08 Level 4 75 75.63 0.84 8/29/16 Upper-Level 3 25 24.92 -0.32 Upper-Level 4 75 76.75 2.33 Level 5 125 121.02 -3.18 Level 6 200 202.39 1.19 Level 1 5 4.92 -1.60 Level 2 10 10.29 2.90 Level 3 25 24.55 -1.80 Level 4 75 75.22 0.29 10/5/16 Upper-Level 3 25 25.01 0.04 Upper-Level 4 75 74.05 -1.27 Level 5 125 127.45 1.96 Level 6 200 198.47 -0.77 Level 1 5 4.98 -0.40 Level 2 10 10.02 0.20 Level 3 25 25.02 0.08 Level 4 75 74.96 -0.05 10/11/16 Upper-Level 3 25 25.07 0.28 Upper-Level 4 75 75.21 0.28 Level 5 125 123.54 -1.17 Level 6 200 201.16 0.58

Max %RE LOQ = -7.60 Max %RE other calibrators = 5.20

Comments:

Acceptance Criteria: %RE Calibrators ± 20% of target (25% at the lowest calibrator) Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GC-MS 2 Matrix: Blood

6 Run One LOWER Curve UPPER Curve Slope 0.0216 Slope 0.0209 4 Intercept 0.000189 Intercept 0.0287 R2 0.999 R2 0.999 2 Response Ratio Response 0 0.000 50.000 100.000 150.000 200.000 250.000 Concentration 6 Run Two Slope 0.0232 Slope 0.0226 4 Intercept 0.00876 Intercept 0.0293 R2 1.000 R2 0.999 2 Response Ratio Response 0 0.000 50.000 100.000 150.000 200.000 250.000 Concentration 6 Run Three Slope 0.0227 Slope 0.0225 4 Intercept 0.00479 Intercept 0.0151 R2 0.999 R2 0.999 2 Response Ratio Response 0 0.000 50.000 100.000 150.000 200.000 250.000 Concentration 6 Run Four Slope 0.0216 Slope 0.0223 4 Intercept 0.00121 Intercept 0.0244 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 50.000 100.000 150.000 200.000 250.000 Concentration 6 Run Five Slope 0.0226 Slope 0.0223 4 Intercept 0.00316 Intercept 0.0180 R2 1.000 R2 1.000 2 Response Ratio Response 0 0.000 50.000 100.000 150.000 200.000 250.000 Concentration Validation Study 1 VALIDATION CURVE CALIBRATION Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GC-MS 2 Matrix: Blood

Cnom y (Response Ratio) 5 Combined Regression 5 0.099997911

5 10 0.227000294 25 0.558478971

4 75 1.594420307 125 2.574142282 200 4.278851816 4 5 0.120998319 10 0.251340007 3 25 0.582575617 75 1.750465684 3 125 2.891180974 200 4.479443761 Response Ratio Response 2 5 0.116916136 10 0.233695735 2 25 0.569155323 75 1.688996974 1 125 2.763705587 200 4.36630438 1 5 0.117148259 10 0.243550745 0 25 0.548609197 0.000 50.000 100.000 150.000 200.000 250.000 75 1.716501355 Concentration 125 2.711012065 200 4.54182862 Slope 0.0221 5 0.107731798 Intercept 0.0067 10 0.223602992 R2 0.9993 25 0.531928989 75 1.627001119 125 2.819656473 200 4.404188997 Validation Study 2 LOD Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 75% 50% 25% Concentration (ng/mL): 4.50 3.75 2.50 Source 1-1 153.1 182.0 287.4 Source 1-2 240.8 224.7 240.8 Source 2-1 94.6 150.0 322.3 5/5/16 Source 2-2 67.9 139.3 240.9 Source 3-1 101.1 146.2 79.3 Source 3-2 100.2 75.4 151.6 Source 1-1 94.0 106.9 70.3 Source 1-2 98.4 109.6 68.1 Source 2-1 86.0 74.2 53.9 5/12/16 Source 2-2 79.6 68.3 69.3 Source 3-1 81.7 93.7 76.6 Source 3-2 84.7 127.2 83.0 Source 1-1 166.3 167.5 225.6 Source 1-2 236.7 283.4 144.0 Source 2-1 210.9 164.2 32.8 8/29/16 Source 2-2 192.1 139.6 444.1 Source 3-1 139.5 226.9 163.8 Source 3-2 152.7 123.9 170.1

Mean Signal to Noise: 132.239 144.611 162.439

Established LOD: 5.00 ng/mL S:N of LOD: >132.239; S:N not documented for 5.0 ng/mL because the S:N of 4.50 ng/mL was > 3.

The signal to noise comparison for 2.50 ng/mL, 3.75 ng/mL, and 4.50 ng/mL was acceptable (S:N > 3). However, all three Comments: levels did not consistently meet ion ratio criteria (red values above indicate failed ion ratios). Therefore, the LOD and LOQ will be 5 ng/mL; the LOQ experiment for 5 ng/mL was performed using three different sources as required for LOD experiments. Signal to Noise ≥ 3 Acceptance Criteria: Acceptable detection and identification criteria Validation Study 3 LOQ Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LOQ Target Concentration (ng/mL): 5.00 8/29/16 1-1 5.13 1-2 5.16 1-3 4.86 Within Run Mean 5.05 SD 0.17 %CV 3.27% % Bias 1.00% 10/5/16 2-1 5.01 2-2 4.93 2-3 5.00 Within Run Mean 4.98 SD 0.04 %CV 0.88% % Bias -0.40% 10/11/16 3-1 5.13 3-2 4.88 3-3 5.21 Within Run Mean 5.07 SD 0.17 %CV 3.39% % Bias 1.47% 10/13/16 4-1 5.05 4-2 5.29 4-3 5.25 Within Run Mean 5.20 SD 0.13 %CV 2.47% % Bias 3.93% 10/26/16 5-1 4.77 5-2 5.00 5-3 4.83 Within Run Mean 4.87 SD 0.12 %CV 2.45% % Bias -2.67%

Mean 5.03 Imprecision Within-Run 3.39% (%CV) Between-Run 3.17% % Bias 0.67%

Comments:

Bias: ≤ 25% Acceptance Criteria: Within-Run Imprecision: CV ≤ 25% Between-Run Imprecision: CV ≤ 25% Validation Study 4 BIAS AND IMPRECISION Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date Run Order LQC MQC MQC-Upper Curve HQC Target Concentration (ng/mL): 6.25 62.50 62.50 100.00 8/29/16 1-1 6.89 62.66 63.97 108.79 1-2 6.59 67.68 68.54 110.32 1-3 6.56 66.01 67.27 98.65 Within Run Mean 6.68 65.45 66.59 105.92 SD 0.18 2.56 2.36 6.34 %CV 2.73% 3.91% 3.54% 5.99% % Bias 6.88% 4.72% 6.55% 5.92% 10/5/16 2-1 7.24 70.21 69.21 116.61 2-2 7.45 72.57 71.47 114.86 2-3 7.41 70.86 69.77 114.12 Within Run Mean 7.37 71.21 70.15 115.20 SD 0.11 1.22 1.18 1.28 %CV 1.51% 1.71% 1.68% 1.11% % Bias 17.87% 13.94% 12.24% 15.20% 10/11/16 3-1 7.70 76.15 76.21 114.51 3-2 7.72 70.82 70.92 112.58 3-3 7.07 67.12 67.17 113.02 Within Run Mean 7.50 71.36 71.43 113.37 SD 0.37 4.54 4.54 1.01 %CV 4.93% 6.36% 6.36% 0.89% % Bias 19.95% 14.18% 14.29% 13.37% 10/13/16 4-1 8.25 77.28 76.11 109.63 4-2 7.52 71.56 70.61 114.75 4-3 7.70 71.84 70.88 111.83 Within Run Mean 7.82 73.56 72.53 112.07 SD 0.38 3.22 3.10 2.57 %CV 4.86% 4.38% 4.27% 2.29% % Bias 25.17% 17.70% 16.05% 12.07% 10/26/16 5-1 7.50 76.33 74.96 113.44 5-2 7.56 74.54 73.26 116.20 5-3 7.56 77.58 76.16 117.68 Within Run Mean 7.54 76.15 74.79 115.77 SD 0.03 1.53 1.46 2.15 %CV 0.46% 2.01% 1.95% 1.86% % Bias 20.64% 21.84% 19.67% 15.77%

Mean 7.38 71.55 71.10 112.47 SD 0.45 4.39 3.68 4.59 Imprecision Max Within-Run 4.93% 6.36% 6.36% 5.99% (%CV) Between-Run 6.10% 6.14% 5.18% 4.08% % Bias 18.10% 14.48% 13.76% 12.47%

Comments: Replicates in red did not calculate to within 20% of the target. This can be attributed to the degradation of 6-acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood.

Bias: ≤ 20% Acceptance Criteria: Within-Run Imprecision: CV ≤ 20% Between-Run Imprecision: CV ≤ 20% Validation Study 5 CARRYOVER Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Low Calibrator Target: 5.00 ng/mL

Results Sample Order Target Concentration Day 1 Day 2 Day 3 Day 4 Day 5 (ng/mL) Date 5/5/16 5/12/16 8/29/16 10/11/16 10/13/16 High Calibrator 200.00 203.16 197.29 202.29 198.47 201.16 Blank 0.17 0.18 0.31 0.24 0.55 % of Low Calibrator 3.4% 3.6% 6.2% 4.8% 11.0% Maximum Carryover: 11.0%

Comments: All ion ratios for 6-acetylmorphine (in red) do not meet acceptance criteria. Therefore, the method is considered to have no significant carryover.

Acceptance Criteria: Quantitative result ≤ 25% of LOQ Validation Study 6 MATRIX & IS INTERFERENCE Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LOQ Response Run 1 18984 Run 2 24891 Run 3 30495 Run 4 29976 Run 5 9176 Average 22704

Identification Peak Response Comment Blank Matrix (NO IS) 1 35 Pass Blank Matrix (NO IS) 2 27 Pass Blank Matrix (NO IS) 3 19 Pass Blank Matrix (NO IS) 4 25 Pass Blank Matrix (NO IS) 5 9 Pass Blank Matrix (NO IS) 6 115 Pass Blank Matrix (NO IS) 7 115 Pass Blank Matrix (NO IS) 8 63 Pass Blank Matrix (NO IS) 9 20 Pass Blank Matrix (NO IS) 10 14 Pass Blank Matrix (with IS) 1 1434 Pass Blank Matrix (with IS) 2 5163 Interference Blank Matrix (with IS) 3 1678 Pass Blank Matrix (with IS) 4 1719 Pass Blank Matrix (with IS) 5 3545 Interference Blank Matrix (with IS) 6 1631 Pass Blank Matrix (with IS) 7 888 Pass Blank Matrix (with IS) 8 563 Pass Blank Matrix (with IS) 9 20 Pass Blank Matrix (with IS) 10 302 Pass

Matrix Interference: No matrix interference was identified. IS Interference: There is interference from labeled IS. Comments: The Blank Matrix with IS 2 and 5 (in red) were >10% of the average response of the LOQ. However, all ion ratios did not meet acceptance criteria. Therefore, the method is considered to have no significant interference contribution from labeled IS. 6-Acetylmorphine will be reported only qualitatively in blood.

Acceptance Criteria: Response of blank matrix ≤ 10% of the average response of LOQ Validation Study 6 EXOGENOUS SUBSTANCES INTERFERENCE Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Target Concentration: 7.38 ng/mL Control Acceptance (%): 20 Run Date: 10/21/16

Calculated Control Sample I.D. Identification Compound Concentration Comment Concentration 7-Aminoclonazepam Nordiazepam Oxazepam Diazepam 1 Benzo Mix Lorazepam 1,000 ng/mL 7.74 Pass Temazepam Clonazepam Alpha-hydroxyalprazolam Alprazolam Methamphetamine Amphetamine Ephedrine 2 Amphetamines Mix Pseudoephedrine 1,000 ng/mL 6.81 Pass MDEA MDA MDMA Cocaine Benzoylecgonine 3 Cocaine Mix 1,000 ng/mL 7.41 Pass Ecgonine Methyl Ester Cocaethylene Butalbital Secobarbital 4 Acid-Neutral Mix Phenobarbital 2,000 ng/mL 7.59 Pass Carisoprodol Meprobamate Methadone 4,000 ng/mL Doxylamine 2,000 ng/mL Fluoxetine 16,000 ng/mL Zolpidem 1,000 ng/mL Phenytoin 5,000 ng/mL Trazodone 4,000 ng/mL Diphenhydramine 1,000 ng/mL Tramadol 4,000 ng/mL Amitriptyline 2,000 ng/mL Nortriptyline 8,000 ng/mL Imipramine 2,000 ng/mL 5 Base Mix 8.07 Pass Cyclobenzaprine 2,000 ng/mL Venlafaxine 2,000 ng/mL Sertraline 2,000 ng/mL Meperidine 2,000 ng/mL Ketamine 1,000 ng/mL Zaleplon 2,000 ng/mL Propoxyphene 2,000 ng/mL Phenobarbital 1,000 ng/mL Benzylpiperdine 4,000 ng/mL Secobarbital 10,000 ng/mL Butalbital 10,000 ng/mL THC 6 Cannabinoids Mix Carboxy THC 1,000 ng/mL 7.16 Pass Hydoxy THC 7 PCP Mix Phencyclidine 100 ng/mL 5.71 Interference

Conclusions: There are compounds tested in this study that may interfere with the analyte of interest.

Comments: PCP may have caused the calculated control (in red) to not fall within 20% of the target. However, this could be attributed to the degradation of 6-acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood.

Acceptance Criteria: Concentrations of analytes of interest within ± 20% of average for bias and imprecision Validation Study 7 DILUTION INTEGRITY Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date 2x Calculated %Difference 4x Calculated %Difference 10x Calculated %Difference 2x-Upper Calculated %Difference 4x-Upper Calculated %Difference

Target Concentration (ng/mL): 62.50 125.00 31.25 125.00 12.50 125.00 62.50 125.00 31.25 125.00 Run 1 1-1 73.11 146.22 16.98 36.39 145.56 16.45 15.08 150.80 20.64 73.12 146.24 16.99 36.07 144.28 15.42 10/11/16 1-2 66.32 132.64 6.11 37.16 148.64 18.91 15.21 152.10 21.68 67.43 134.86 7.89 36.87 147.48 17.98 Run 2 2-1 66.86 133.72 6.98 34.52 138.08 10.46 13.78 137.80 10.24 66.09 132.18 5.74 34.98 139.92 11.94 10/13/16 2-2 66.72 133.44 6.75 33.44 133.76 7.01 14.08 140.80 12.64 65.95 131.90 5.52 33.94 135.76 8.61 Run 3 3-1 79.66 159.32 27.46 40.16 160.64 28.51 16.75 167.50 34.00 78.15 156.30 25.04 40.42 161.68 29.34 10/26/16 3-2 78.99 157.98 26.38 40.63 162.52 30.02 16.62 166.20 32.96 77.50 155.00 24.00 40.87 163.48 30.78

Mean: 143.89 148.20 152.53 142.75 148.77 SD: 12.50 11.64 12.40 11.29 11.42 Between-Run Imprecision (%CV): 8.69 7.86 8.13 7.91 7.68 %Bias: 15.11 18.56 22.03 14.20 19.01

Comments: Replicates in red did not calculate to within 20% of the target. This can be attributed to the degradation of 6-acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood.

Acceptance Criteria: Concentration within ± 20% of target Validation Study 8 IN-PROCESS STABILITY Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 3 hours Acceptable Limit (%): 20%

Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 6.83 Pass 6.99 5.91 8.86 LQC-2 7.15 Pass MQC-1 77.36 Pass 72.98 57.24 85.86 MQC-2 68.60 Pass 10/11/16 *MQC-1 77.46 Pass *Upper curve 73.04 56.88 85.32 *MQC-2 68.62 Pass *Upper curve HQC-1 109.94 Pass 109.79 89.97 134.96 HQC-2 109.63 Pass

Overall Acceptance: Stable for 3 hours

Comments: 6-Acetylmorphine will be reported only qualitatively in blood.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 AUTOSAMPLER STABILITY Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Length of Time Delay: 24 hours Acceptable Limit (%): 20

Experiment 1: Stability of Punctured Calibration Curve and Controls Acceptable Range Run Date Target Results Mean Pass/Unstable Low High 5.00 5.24 N/A 4.00 6.00 Pass 10.00 10.39 N/A 8.00 12.00 Pass 25.00 25.41 N/A 20.00 30.00 Pass 75.00 77.02 N/A 60.00 90.00 Pass 25.00 25.55 N/A 20.00 30.00 Pass *Upper curve 75.00 73.38 N/A 60.00 90.00 Pass *Upper curve Injected on 125.00 119.01 N/A 100.00 150.00 Pass 10/26/16; 200.00 210.27 N/A 160.00 240.00 Pass Reinjected on 8.21 10/27/16 7.38 8.17 5.91 8.86 Pass 8.12 78.57 71.55 77.37 57.24 85.86 Pass 76.16 74.82 *Upper curve 71.10 73.70 56.88 85.32 Pass 72.58 113.15 112.47 114.30 89.97 134.96 Pass 115.45

Experiment 2: Stability of Punctured and Unpunctured Controls Acceptable Range Run Date Sample I.D. Results Mean Pass/Unstable Low High LQC-1 Punctured 7.95 Pass 7.88 5.91 8.86 LQC-2 Punctured 7.80 Pass MQC-1 Punctured 77.77 Pass Injected on 76.84 57.24 85.86 10/27/16; MQC-2 Punctured 75.90 Pass Reinjected on *MQC-1 Punctured 74.07 Pass *Upper curve 10/28/16 73.21 56.88 85.32 *MQC-2 Punctured 72.34 Pass *Upper curve HQC-1 Punctured 114.83 Pass 113.79 89.97 134.96 HQC-2 Punctured 112.74 Pass LQC-1 Unpunctured 7.85 Pass 7.74 5.91 8.86 LQC-2 Unpunctured 7.63 Pass MQC-1 Unpunctured 76.23 Pass Extracted on 75.09 57.24 85.86 10/27/16; MQC-2 Unpunctured 73.95 Pass Injected on *MQC-1 Unpunctured 72.64 Pass *Upper curve 10/28/16 71.59 56.88 85.32 *MQC-2 Unpunctured 70.53 Pass *Upper curve HQC-1 Unpunctured 112.44 Pass 111.77 89.97 134.96 HQC-2 Unpunctured 111.10 Pass

Unpunctured Vials: Stable for 24 hours Punctured Vials: Stable for 24 hours

Comments: Punctured vials in Experiment 1 were stable for 24 hours. Punctured and unpunctured vials in Experiment 2 were stable for 21 and 24 hours, respectively. 6- Acetylmorphine will be reported only qualitatively in blood.

Acceptance Criteria: Concentration within 20% of the average value obtained in the Bias and Imprecision studies Validation Study 8 STORAGE STABILITY Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

LQC Day 0 HQC Day 0

Mean Acceptance %:

Storage Conditions Sample ID Day 1 Day 2 Day 7 Day 14 Day 30 LQC-1 LQC-2 LQC-3 Room Temperature HQC-1 HQC-2 HQC-3 LQC-1 LQC-2 Room Temperature w/ LQC-3 Light Protection HQC-1 HQC-2 HQC-3 LQC-1 LQC-2 LQC-3 Refrigerated (2 to 8oC) HQC-1 HQC-2 HQC-3 LQC-1 LQC-2 LQC-3 Frozen (-5 to -20oC) HQC-1 HQC-2 HQC-3

Summary: Days Room Temperature (RT): RT Light Protected: Refrigerated: Frozen:

Comments: Study not performed; storage stability is documented in Disposition of Toxic Drugs and Chemicals in Man, Tenth Edition by Randall C. Baselt.

Acceptance Criteria: Concentration within 20% of average time 0 Validation Study 9 EXTRACTION EFFICIENCY & MATRIX EFFECT Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Run Date: 11/7/16 Peak Extraction Average Type of Sample Sample ID Response of Efficiency Matrix Effect Response Drug (%Recovery) 25 ng/mL-1 9837 25 ng/mL-2 11819 25 ng/mL-3 11450 10393 70 40 25 ng/mL-4 9826 25 ng/mL-5 9032 Group 1-Internal 250 ng/mL-1 101059 Standard and 250 ng/mL-2 110496 drug analyte 250 ng/mL-3 112960 111862 71 12 fortified before 250 ng/mL-4 121854 extraction 250 ng/mL-5 112940 400 ng/mL-1 217259 400 ng/mL-2 227175 400 ng/mL-3 233616 229867 90 21 400 ng/mL-4 244216 400 ng/mL-5 227070 25 ng/mL-1 12207 25 ng/mL-2 14948 25 ng/mL-3 15369 14860 25 ng/mL-4 16117 25 ng/mL-5 15659 250 ng/mL-1 162819 Group 2-Internal 250 ng/mL-2 158552 Standard and 250 ng/mL-3 159950 157143 analyte fortified 250 ng/mL-4 151522 after extraction 250 ng/mL-5 152874 400 ng/mL-1 255443 400 ng/mL-2 250322 400 ng/mL-3 240208 255879 400 ng/mL-4 262545 400 ng/mL-5 270877 25 ng/mL-1 11163 25 ng/mL-2 11990 25 ng/mL-3 10755 10630 25 ng/mL-4 10102 25 ng/mL-5 9139 Group 3-Internal 250 ng/mL-1 142560 Standard and 250 ng/mL-2 135063 analyte fortified 250 ng/mL-3 136749 140669 to elution 250 ng/mL-4 137138 solvent/no 250 ng/mL-5 151833 extraction 400 ng/mL-1 210188 400 ng/mL-2 202934 400 ng/mL-3 208085 212079 400 ng/mL-4 213714 400 ng/mL-5 225473 Extraction Efficiency = (mean peak area of samples fortified before extraction/mean peak area of samples fortified after extraction) x 100. Matrix Comments: Effect = (mean peak area of samples fortified after extraction/mean peak area of neat samples-1) x 100.

Acceptance If method achieves desired limit of quantification and/or detection for the analyte, its extraction Criteria: efficiency and matrix effect are considered acceptable Validation Study 10 BLIND SAMPLE ANALYSIS Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

Expected Acceptable Run Date Sample I.D. Reported Results %Difference Pass/Fail Results Range

Blind Sample 1 ND ND <20 --- Pass Blind Sample 2 12.5 14.54 10-15 16.3 Pass Blind Sample 3 25.0 25.25 20-30 1.0 Pass Blind Sample 4 ND ND <20 --- Pass Blind Sample 5 ND ND <20 --- Pass Blind Sample 6 30.0 30.35 24-36 1.2 Pass Blind Sample 7 100.0 109.08 80-120 9.1 Pass Blind Sample 8 62.5 73.60 50-75 17.8 Pass Blind Sample 9 ND ND <20 --- Pass Blind Sample 10 125.0 131.42 100-150 5.1 Pass 11/9/16 Blind Sample 11 150.0 151.94 120-180 1.3 Pass Blind Sample 12 ND ND <20 --- Pass Blind Sample 13 ND ND <20 --- Pass Blind Sample 14 200.0 201.09 160-240 0.5 Pass Blind Sample 15 ND ND <20 --- Pass Blind Sample 16 ND ND <20 --- Pass Blind Sample 17 ND ND <20 --- Pass Blind Sample 18 40.0 39.75 32-48 -0.6 Pass Blind Sample 19 ND ND <20 --- Pass Blind Sample 20 100.0 106.72 80-120 6.7 Pass

Comments: The MQC did not calculate to within 20% of the target. This can be attributed to the degradation of 6-acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. 6-Acetylmorphine will be reported only qualitatively in blood.

Acceptance Percent difference of the expected vs. determined values ≤ 20% Criteria: SUMMARY OF VALIDATION PERFORMANCE Analyte: 6-Acetylmorphine Analyst: Melissa Lloyd Units: ng/mL Study Dates: 5/5/16 to 11/18/16 Instrument: GCMS-2 Matrix: Blood

The intent of this summary is to capture and document important information about the performance of this method outside the required measurements for validation. Please complete the below items as thoroughly as possible and add any special observations, which may not be listed.

Failed Runs (include dates/reasons):

Date Reason There were multiple interferences with ions causing quantitative values, ion August 2, 2016 ratios, and overall chromatography to be not acceptable. Autosampler stability was attempted on this day, but the extraction vials October 13, 2016 dried out prior to injection so the experiment was repeated on October 27, 2016.

Deviations from SOP:

Other Observations: Although 6-acetylmorphine failed to calculate within ± 20% of the target, the positive controls were positive and the negative controls were negative deeming the method appropriate for qualitative confirmation. 6- Acetylmorphine failing to calculate within ± 20% of the target can be attributed to the degradation of 6-acetylmorphine, or to possible incorrect preparation of the calibrator prepared on 10/04/16. Per reccomendation of the GC-MS trainer in June 2016 the purge flow to split vent changed from 50 mL/min to 150 mL/min and the EMV mode changed from "relative" to a gain factor of 5 to improve the robustness of the method. Two validation batches (20160505_MRL and 20160512_MRL) were performed under the old settings, while the subsequent validation batches were performed under the new settings. The changes did not make a difference in method performance.

Recommended Maximum Run Length (# unknown samples): 30

Conclusion: This method is fit for casework of 6-acetylmorphine qualitative confirmation analysis.