Powerful Sample Prep and LC Column Solutions for Forensic Toxicology Applications

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Friendly Reminders Please use text chat functionality to submit your questions today. Providing Live Technical Support during today s event Poll Questions Audience participation Upon conclusion, follow up information will be available: http://www.waters.com/oct16 Recorded version of today s presentation PDF Copy of today s slides Application specific discount offers CORTECS, Oasis, Ostro, BEH Columns Product specific information and reference materials 2013 Waters Corporation 2

About Today s Presenter Jonathan Danaceau, Ph.D., Senior Applications Chemist, Waters Corporation Dr. Jonathan Danaceau is a Senior Applications Chemist within Waters Consumable Business Unit. Jon received his B.S. in Biology from Allegheny College (Meadville, PA) and his Ph.D. in Neuroscience from the University of Utah (Salt Lake City, UT). Jon has an extensive background in bioanalysis including experience in the pharmaceutical industry, forensic toxicology, and anti-doping analysis. Jon joined Waters Chemistry Applied Technology group in 2011 where he has been focusing on sample preparation and chromatography solutions for various applications, including forensic toxicology and clinical research. 2013 Waters Corporation 3

Sample Preparation and LC Column Solutions for Forensic Toxicology Applications Jonathan Danaceau, Ph.D. Senior Applications Chemist Waters Corporation 2013 Waters Corporation 4

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Opioids and metabolites in urine and oral fluid Synthetic cannabinoids in urine and whole blood THC and metabolites in whole blood Expanded Toxicology Panel from Urine Summary 2013 Waters Corporation 5

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Opioids and metabolites in urine, whole blood and oral fluid Synthetic cannabinoids in urine and whole blood Synthetic cathinones Bath Salts in urine THC and metabolites in whole blood Summary 2013 Waters Corporation 6

Goal of Sample Preparation Provides the target analyte(s) in solution Provides the analyte(s) at a concentration appropriate for detection or measurement Concentrating the analyte helps increase sensitivity and achieve lower limits of detection Removes interfering matrix elements (such as phospholipids, salts, proteins, nucleic acids, sugars, etc.) that alter the MS response or co-elute with the target analyte Matrix effects result in ion suppression (loss of signal) or ion enhancement (gain in signal) Matrix effects have a negative impact on the accuracy, precision, and robustness of the method; add to method variability 2013 Waters Corporation 7

Importance of Clean Samples Having cleaner samples means: Better chromatography Lower limits of detection Decreases assay variability; more robust assay o Reduced matrix effects o Fewer reanalyses o Less chance of false positives/negatives Longer column lifetime Less instrument downtime Minimize costs in manpower and equipment maintenance Sample Prep makes your analytical lab more productive! 2013 Waters Corporation 8

Sample Preparation Options Technique Advantages Disadvantages Appropriate Matrices Nonselective Dilution Simple Cheap Easy to automate No cleanup No enrichment Non-selective Urine Protein Precipitation Simple Quick Minimal method development Minimal selectivity; does not remove most matrix interferences No enrichment Substantial solvent evaporation may be needed Whole blood, plasma, serum Lipid/protein removal plates Simple, universal method Quick Minimal method development Minimal selectivity No enrichment Substantial solvent evaporation may be needed Whole blood, plasma, serum Liquid-Liquid Extraction Offers better clean up than protein precipitation Can be optimized for different compound classes Less selective than SPE; does not remove endogenous phospholipids Cumbersome; requires user intervention Difficult to automate Not ideal for highly polar drugs and metabolites Solvent evaporation needed Urine, plasma, serum, oral fluid Highly selective 2013 Waters Corporation 12

Sample Preparation Options Technique Advantages Disadvantages Appropriate Matrices Nonselective Dilution Simple Cheap Easy to automate No cleanup No enrichment Non-selective Urine Protein Precipitation Simple Quick Minimal method development Minimal selectivity; does not remove most matrix interferences No enrichment Substantial solvent evaporation may be needed Whole blood, plasma, serum Lipid/protein removal plates Simple, universal method Quick Minimal method development Minimal selectivity No enrichment Substantial solvent evaporation may be needed Whole blood, plasma, serum Liquid-Liquid Extraction Solid-Phase Extraction (SPE) Offers better clean up than protein precipitation Can be optimized for different compound classes Best cleanup option Fast; easy to automate Achieves the highest recovery and reproducibility Can be manipulated for optimum recovery and cleanup Variety of device formats and sorbent chemistries Less selective than SPE; does not remove endogenous phospholipids Cumbersome; requires user intervention Difficult to automate Not ideal for highly polar drugs and metabolites Solvent evaporation needed May require method development to optimize the protocol Perceived to be difficult and costly Urine, plasma, serum, oral fluid Urine, whole blood, plasma, serum, oral fluid Highly selective 2013 Waters Corporation 13

Sample Preparation Options Technique Advantages Disadvantages Appropriate Matrices Nonselective Dilution Simple Cheap Easy to automate No cleanup No enrichment Non-selective Urine Protein Precipitation Simple Quick Minimal method development Minimal selectivity; does not remove most matrix interferences No enrichment Substantial solvent evaporation may be needed Whole blood, plasma, serum Lipid/protein removal plates Simple, universal method Quick Minimal method development Minimal selectivity No enrichment Substantial solvent evaporation may be needed Whole blood, plasma, serum Liquid-Liquid Extraction Solid-Phase Extraction (SPE) Offers better clean up than protein precipitation Can be optimized for different compound classes Best cleanup option Fast; easy to automate Achieves the highest recovery and reproducibility Can be manipulated for optimum recovery and cleanup Variety of device formats and sorbent chemistries Less selective than SPE; does not remove endogenous phospholipids Cumbersome; requires user intervention Difficult to automate Not ideal for highly polar drugs and metabolites Solvent evaporation needed May require method development to optimize the protocol Perceived to be difficult and costly Urine, plasma, serum, oral fluid Urine, whole blood, plasma, serum, oral fluid Highly selective 2013 Waters Corporation 14

Solid-Phase Extraction (SPE) SPE is gaining acceptance in clinical and forensic labs Cleans up, concentrates, and produces a final sample that is in an appropriate solvent for further analysis Uses polymeric or silica-based chromatographic particles packed into a variety of formats 96-well plates, cartridges, etc. SPE is considered to be a very versatile sample preparation technique for various analytes in complex matrices Blood, serum, plasma, oral fluid, tears, nasal fluid, CSF, urine, feces, meconium, postmortem samples, and many more! It s the best technique for minimizing matrix interferences including proteins, phospholipids, salts, and other endogenous compounds 2013 Waters Corporation 15

Oasis Family of Sorbents: Reversed-Phase and Mixed-ModeMode Sorbent ALWAYS Charged (-) Sorbent ALWAYS Charged (+) Selective for Basic Compounds Selective for Acidic Compounds Selective for Strong Basic Compounds For wide range of acidic, basic, and neutral compounds Selective for Strong Acidic Compounds Sorbent charged (-) at high ph; unionized at low ph Sorbent charged (+) at Low ph; unionized at high ph 2013 Waters Corporation 16

Waters SPE Device Formats Formats 96-well plates (with 5, 10, 30, 60 mg of sorbent) Syringe barrel cartridges Glass cartridges Online columns µelution plates How to process samples? Gravity Pressure Vacuum Automation 2013 Waters Corporation 18

Ostro 96-Well Sample Preparation Plate Cleanup of phospholipids and proteins in plasma and serum (also blood) Fast, easy in-well protein precipitation; precipitated proteins and phospholipids are left behind in the wells Significant time savings; protocol eliminates extract transfer and evaporation steps (also in plate format) Generic protocol; no method development Extracts can often be directly injected and analyzed Suitable for a wide variety of acidic, basic, and neutral compounds Pass-through method 2013 Waters Corporation 19

Phospholipids Remaining in the Extract: Ostro vs. LLE and PPT 100 % LLE with 5% NH 4 OH in MTBE MRM of m/z 184-184 2.29 2.21 2.10 184.4 > 184.4 (Lipid 184) 2.00e8 2.60 2.78 2.72 2.88 1.90 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 100 LLE with MTBE 2.27 184.4 > 184.4 (Lipid 184) 2.00e8 2.56 2.62 2.682.80 % 1.90 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 100 Ostro 184.4 > 184.4 (Lipid 184) 2.00e8 % 0 1.90 1.77 1.96 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 100 % PPT 1.381.42 1.51 1.32 1.63 1.75 1.96 2.21 184.4 > 184.4 (Lipid 184) 2.00e8 2.84 0 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 Time Ostro is superior to LLE and PPT for phospholipid removal 2013 Waters Corporation 20

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Opioids and Metabolites in Urine Summary 2013 Waters Corporation 21

Opioids and Metabolites in Urine Assay Use Quantification of opioids and metabolites in urine Analytes 26 natural opiate drugs, semi-synthetic opioids, and synthetic narcotic analgesic compounds Goals Increase sample cleanliness, sensitivity and accuracy compared to sample dilution Accurate quantification of a comprehensive panel of opioid drugs and metabolites Direct analysis of glucuronide metabolites without hydrolysis 2013 Waters Corporation 22

Comprehensive opioid panel Compound 1 Morphine-3β-D-glucuronide 2 Oxymorphone-3β-D-glucuronide 3 Hydromorphone-3β-D- glucuronide 4 Morphine-6β-D-glucuronide 5 Morphine 6 Oxymorphone 7 Hydromorphone 8 Codeine-6β-D-glucuronide 9 Dihydrocodeine 10 Codeine 11 Oxycodone 12 6-Acetylmorphone (6-AM) 13 O-desmethyl Tramadol 14 Hydrocodone 15 Norbuprenorphine-glucuronide 16 Norfentanyl 17 Tramadol 18 Normeperedine 19 Meperidine 20 Buprenorphine-glucuronide 21 Norbuprenorphine 22 Fentanyl 23 Buprenorphine 24 EDDP+ 25 Propoxyphene 26 Methadone Natural opiates and metabolites Semi-synthetic opioids Synthetic narcotic analgesics All bases Oasis MCX 2013 Waters Corporation 23

Extraction Methodologies for Urine Oasis MCX µelution Plate Protocol (Mixed-mode strong cation exchange SPE) Sample Pretreatment 100 µl urine + 100 µl 4% H 3 PO 4 + 100 µl IS Condition Plate 200 µl MeOH then 200 µl Water Load 300 µl pretreated sample Wash 200 µl Water, then 200 µl MeOH Elute 2 x 50 µl (60:40 ACN:MeOH + 5% NH 4 OH) Sample Dilution Protocol 100 µl urine Add 100 µl IS (dissolved in water) Vortex Inject 10 µl A short evaporation step (<5 min) implemented to evaporate and reconstitute in the mobile phase Prevents solvent effects for early eluting compounds Evaporate under N 2 @ 37 o C Reconstitute in 50 µl of starting mobile phase (2% ACN/0.1% FA) Inject 10 µl 2013 Waters Corporation 24

UPLC and MS conditions UPLC Conditions MS Conditions Column ACQUITY BEH C 18 1.7 µm; 2.1 x 100 LC ACQUITY UPLC Flow Rate 0.4 ml/min MPA Water with 0.1% FA MPB ACN with 0.1% FA Column Temp 30 C Inj. Vol 10 µl XEVO TQD ESI+ Capillary: 1.0 kv Source Temp: 150 C Desolvation Temp: 350 C Cone Gas Flow: 50 L/Hr Desolvation Gas Flow: 900 L/Hr MRM Transitions optimized individually Time Flow %A %B Curve 0 0.4 98 2 6 6 0.4 47.2 52.8 6 6.5 0.4 98 2 11 8 0.4 98 2 11 2013 Waters Corporation 25

Opioid Chromatography % Separation of isobaric compounds (highlighted compounds) 1, 3 2 4, 5 6 7 9 8 10 12 11 13 14 15,16 17 18,19 20 ACQUITY BEH C 18 1.7 µm, 2.1 x 100mm 0 1.00 1.50 2.00 2.50 3.00 3.50 4.00 4.50 5.00 21 Compound 1 Morphine-3β-D-glucuronide 2 Oxymorphone-3β-D-glucuronide 3 Hydromorphone-3β-D- glucuronide 4 Morphine-6β-D-glucuronide 5 Morphine 6 Oxymorphone 7 Hydromorphone 8 Codeine-6β-D-glucuronide 9 Dihydrocodeine 10 Codeine 11 Oxycodone 2013 Waters Corporation 26 22 23 24 25 26 Time 12 6-Acetylmorphone (6-AM) 13 O-desmethyl Tramadol 14 Hydrocodone 15 Norbuprenorphine-glucuronide 16 Norfentanyl 17 Tramadol 18 Normeperedine 19 Meperidine 20 Buprenorphine-glucuronide 21 Norbuprenorphine 22 Fentanyl 23 Buprenorphine 24 EDDP+ 25 Propoxyphene 26 Methadone

% Recovery on Oasis MCX SPE 6 Lots of Urine 120% 100% 80% %R Recovery 60% 40% 20% 0% 2013 Waters Corporation 27

Improved Matrix Factors and %CV with Oasis MCX SPE vs. dilution - 6 Lots of Urine 1.40 1.20 Mixed-mode SPE Dilution * * * Matrix Factors 1.00 0.80 0.60 0.40 0.20 * * * * * * * * * 0.00 * Statistically significant difference in Matrix Factors (12/26 compounds) - reduced %CV in 23/26 compounds 2013 Waters Corporation 28

QC Results Diluted Urine QC Concentration(ng/mL) 7.5 75 250 400 %CV Bias %CV Bias %CV Bias %CV Bias Morphine-3-gluc 10.3% -5.7% 6.1% -2.3% 2.3% -4.2% 6.2% -5.0% Oxymorphone-3-gluc 18.1% -8.7% 6.8% -2.8% 4.0% -8.1% 7.0% -8.5% Hydromorphone-3-gluc 14.5% 3.3% 4.5% 4.1% 6.9% -5.4% 5.8% -9.3% Morphine-6-gluc 23.1% 4.7% 17.5% -1.4% 9.3% -0.4% 3.5% -10.4% Morphine 26.9% -29.7% 7.9% 1.3% 9.4% 7.0% 16.6% 2.7% Oxymorphone 23.3% 19.7% 9.7% 9.9% 5.8% 0.7% 5.4% -10.0% Hydromorphone 14.1% 8.3% 5.0% 5.7% 5.1% 0.7% 3.4% -4.7% Codeine-6-β-d-gluc 11.5% -14.0% 7.0% -4.5% 8.0% -9.3% 4.4% -10.4% Dihydrocodeine 9.4% 10.0% 8.0% 14.8% 5.6% -2.1% 5.3% -3.2% Codeine 10.5% 5.3% 4.7% 2.0% 8.0% -5.5% 3.9% -8.5% Oxycodone 20.4% 0.3% 6.8% 5.6% 3.4% -2.8% 3.4% -4.9% 6-Acetylmorphone 7.7% -13.3% 9.5% -8.9% 2.8% -13.8% 5.2% -7.1% O-desmethyl Tramadol 3.6% -0.7% 4.9% 5.9% 3.3% -3.9% 2.5% -7.8% Hydrocodone 8.2% -10.0% 3.6% -4.2% 6.4% -9.1% 5.8% -14.7% Norbuprenorphine-gluc 5.3% -3.3% 2.7% 2.8% 5.0% -6.2% 3.0% -12.4% Norfentanyl 11.2% -18.7% 3.7% 6.9% 3.7% -3.6% 0.6% -7.9% Tramadol 1.5% -13.0% 3.6% -6.9% 1.3% -12.8% 0.8% -16.1% Normeperedine 4.6% -0.7% 5.1% 5.7% 3.1% -6.2% 0.7% -10.8% Meperidine 1.7% -2.3% 7.0% 3.2% 2.1% -5.5% 2.7% -8.2% Buprenorphine-gluc 4.5% -36.0% 3.6% -12.3% 4.9% -15.6% 2.1% -18.2% Norbuprenorphine 9.2% -4.7% 2.8% 6.2% 5.6% -3.0% 1.7% -9.0% Fentanyl 3.3% -6.7% 2.9% 3.9% 3.9% -4.1% 1.4% -8.2% Buprenorphine 6.4% -9.3% 3.8% 0.6% 3.7% -7.6% 2.3% -10.9% EDDP+ 1.7% -0.7% 3.3% 4.4% 1.0% -4.3% 2.1% -8.7% Propoxyphene 8.2% -6.7% 2.2% 1.2% 2.8% -8.1% 4.5% -12.5% Methadone 6.0% -7.0% 2.5% 0.7% 3.4% -6.9% 4.4% -12.6% Mean %CV @ 7.5 ng/ml = 10.2% 11 compounds >10% 2013 Waters Corporation 29

QC Results Oasis MCX SPE QC Concentration(ng/mL) 7.5 75 250 400 %CV Bias %CV Bias %CV Bias %CV Bias Morphine-3-gluc 8.3% -5.3% 5.2% -0.7% 2.2% 0.0% 3.6% -3.4% Oxymorphone-3-gluc 9.7% -1.0% 3.0% 2.5% 4.9% -4.0% 3.7% -7.0% Hydromorphone-3-gluc 7.8% 6.3% 5.8% 1.9% 2.9% 0.9% 3.7% -0.5% Morphine-6-gluc 8.7% 10.7% 6.7% -0.1% 5.1% -3.7% 4.0% -5.8% Morphine 10.1% 8.7% 7.7% 0.8% 5.1% -13.2% 4.3% -2.2% Oxymorphone 5.1% 4.7% 4.2% -2.3% 4.7% -2.6% 4.5% -3.6% Hydromorphone 1.6% 5.7% 3.0% 0.9% 3.7% -0.9% 1.2% -2.8% Codeine-6-β-d-gluc 4.0% 3.7% 3.8% -1.9% 5.0% 2.9% 2.6% 5.4% Dihydrocodeine 0.8% 2.0% 1.1% 1.1% 0.6% -2.5% 2.8% -5.5% Codeine 4.7% 2.3% 0.6% 1.1% 1.9% -1.9% 0.9% -3.7% Oxycodone 5.2% 1.0% 2.3% 0.7% 3.4% -2.2% 2.8% -5.5% 6-Acetylmorphone 5.3% 2.7% 4.3% 1.6% 2.3% -1.7% 0.7% -2.1% O-desmethyl Tramadol 1.9% 4.3% 1.3% 0.0% 0.7% -1.2% 0.7% -3.8% Hydrocodone 1.9% 1.3% 1.3% -0.7% 1.6% -2.3% 0.9% -4.7% Norbuprenorphine-gluc 3.6% 4.0% 3.1% 1.8% 3.9% 2.0% 1.3% 0.5% Norfentanyl 0.0% 1.3% 2.3% 3.3% 1.2% 0.4% 2.2% 1.7% Tramadol 0.0% 1.3% 0.3% 2.4% 0.8% -3.8% 0.5% -7.7% Normeperedine 2.0% -0.3% 1.6% 0.4% 1.2% -4.5% 1.4% -7.2% Meperidine 0.7% -1.0% 0.5% -2.5% 2.4% -3.1% 1.7% -3.0% Buprenorphine-gluc 2.7% 7.7% 1.8% 3.7% 1.6% 6.8% 1.3% 10.3% Norbuprenorphine 1.2% 3.0% 3.8% 3.6% 1.5% -1.6% 1.0% -5.7% Fentanyl 0.0% 1.3% 1.1% 2.4% 1.0% -2.7% 1.0% -3.2% Buprenorphine 2.3% 0.7% 1.9% 2.9% 1.9% -1.1% 1.3% -0.7% EDDP+ 1.3% 2.0% 1.1% 0.0% 0.9% -2.7% 1.1% -3.1% Propoxyphene 0.8% 0.7% 0.5% 4.5% 0.9% -2.6% 1.9% -5.3% Methadone 0.7% 1.0% 1.5% 4.3% 1.0% -1.4% 1.2% -3.4% Mean %CV @ 7.5 ng/ml= 3.5% 1 point >10% Reduced Total Analytical Error vs. Dilute and Shoot 2013 Waters Corporation 30

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Opioids and Metabolites in Oral Fluid Summary 2013 Waters Corporation 31

Opioids and Metabolites in Oral Fluid Oral fluid advantages Increasingly popular techniques Non-invasive Easily observed collection Reflective of recent use/impairment Can be correlated with plasma concentrations o Bases may be present at higher conc. than plasma Oral fluid considerations Limited sample volume Contamination from oral administration Collection challenges (devices/inconsistency) Protein content of oral fluid (0.3%) Salts, matrix components from stabilization buffers 2013 Waters Corporation 32

Extraction Methodology for Oral Fluid Oasis MCX µelution 96-Well Plate (Mixed-mode strong cation exchange SPE) Sample Pretreatment 1 ml oral fluid + 3 ml stabilizing buffer* Remove a 400 µl aliquot (100 µl oral fluid) Add 200 µl of 4% H 3 PO 4 + 20 µl IS (500 ng/ml) Condition Plate 200 µl MeOH then 200 µl Water Load 100 µl pretreated sample Wash 200 µl of 2% formic acid; then 200 µl MeOH; then 200 µl IPA Elute 2 x 50 µl (60:40 ACN:IPA + 5% NH 4 OH) Selective cleanup for basic compounds µelution format Designed for small volumes Samples and elution volumes Evaporate under N 2 @ 37 o C Reconstitute in 50 µl of starting mobile phase (2% ACN/0.1% FA) Inject 10 µl * Oral fluid collected using QuantiSal from Immunalysis. 2013 Waters Corporation 33

%Recovery Using Oasis MCX µelution Plate 120% Recovery of Opioids from Oral Fluid 100% 80% 60% 40% 20% 0% Average Recovery = 91% 2013 Waters Corporation 34

Linearity and calibration curve results Compound R 2 Max. %CV Max. %Bias LOQ (ng/ml) Morphine 0.989 16.4% 9.7% 5 Oxymorphone 0.997 9.4% 1.9% 5 Hydromorphone 0.997 11.8% 1.6% 5 Dihydrocodeine 0.996 6.5% 5.9% 5 Codeine 0.994 6.7% 5.9% 5 Oxycodone 0.996 5.7% 6.8% 5 6-Acetylmorphone (6-AM) 0.996 7.6% 4.2% 5 O-desmethyl Tramadol 0.999 4.3% 1.7% 5 Hydrocodone 0.998 5.0% 2.3% 5 Norfentanyl 0.998 4.2% 3.2% 1.25 Tramadol 0.999 3.3% 3.6% 5 Normeperedine 0.999 3.9% 1.8% 5 Meperidine 0.999 2.6% 2.0% 5 Norbuprenorphine 0.996 5.5% 6.5% 5 Fentanyl 0.999 2.9% 1.5% 1.25 Buprenorphine 0.999 3.3% 2.0% 5 EDDP+ 0.999 2.5% 1.2% 5 Propoxyphene 0.999 3.6% 2.3% 5 Methadone 0.999 2.9% 2.2% 5 5-500 ng/ml Calib. Curve (1.25-125 for Fentanyl and norfentanyl All R 2 Values 0.99 2013 Waters Corporation 35

QC Results - Oral fluid QC Concentration (ng/ml) (N=4) 7.5 25 150 300 Compound %CV Bias %CV Bias %CV Bias %CV Bias Morphine 15.3% 19.7% 2.7% 18.2% 12.2% 11.6% 6.9% 5.9% Oxymorphone 9.2% 2.7% 6.4% 3.3% 2.6% 4.1% 2.7% 5.1% Hydromorphone 7.7% 1.1% 3.6% 5.1% 3.2% 5.4% 3.8% 6.4% Dihydrocodeine 2.3% 6.7% 3.6% 11.4% 2.7% 4.4% 2.2% 1.5% Codeine 8.7% 7.2% 3.7% 11.7% 3.8% 4.3% 3.9% 1.4% Oxycodone 7.0% 5.3% 5.6% 10.6% 5.1% 7.5% 2.7% 2.2% 6-Acetylmorphone (6-AM) 5.3% 5.4% 3.6% 8.5% 3.6% 3.3% 7.1% 4.5% O-desmethyl Tramadol 5.6% 6.1% 2.5% 7.7% 2.1% 5.8% 1.7% 5.4% Hydrocodone 5.6% 6.4% 3.4% 4.6% 2.7% 4.7% 3.0% 6.6% Norfentanyl 7.0% 0.8% 3.9% 8.3% 2.9% 2.6% 3.3% 4.9% Tramadol 4.8% 6.4% 3.1% 8.8% 2.6% 6.7% 2.2% 4.8% Normeperedine 4.8% -0.7% 3.3% 3.5% 2.2% 3.1% 2.8% 2.4% Meperidine 5.5% 5.2% 4.1% 4.9% 2.6% 6.6% 2.5% 6.2% Norbuprenorphine 5.9% 5.4% 3.6% 8.3% 2.3% 4.8% 1.5% 2.9% Fentanyl 4.6% 4.8% 2.5% 7.4% 2.7% 6.8% 1.5% 6.4% Buprenorphine 4.5% 6.5% 2.8% 8.1% 3.0% 7.9% 1.5% 7.5% EDDP+ 4.7% 4.8% 2.4% 5.8% 2.7% 6.8% 2.5% 7.3% Propoxyphene 3.8% 6.8% 3.0% 8.6% 2.4% 7.0% 2.2% 7.0% Methadone 5.3% 6.1% 3.2% 8.0% 3.0% 6.8% 2.4% 6.5% 5-500 ng/ml Calib. Curve Mean % Bias = 5.3% Mean %CV = 4.5% 2013 Waters Corporation 36

Sample Preparations Strategies for Opioids - Conclusions Urine and Oral Fluid Highly selective clean up Mixed mode SPE (MCX) Improved accuracy and precision vs. dilution µelution format ideal for limited sample volume (OF) Excellent linearity, analytical accuracy, and precision. 2013 Waters Corporation 37

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Synthetic Cannabinoids in Urine Summary 2013 Waters Corporation 38

Synthetic Cannabinoids Designer drugs that mimic the psychoactive effects of natural cannabinoids Often referred to or marketed as Spice compounds Labeled as not for human consumption and marketed as a legal alternative to natural cannabis Popularity has risen substantially in the last several years A growing challenge for law enforcement agencies and forensic laboratories Recent legislation has banned some of these compounds, but minor modifications are made to existing structures to circumvent existing laws Quantitative analysis in urine and whole blood 2013 Waters Corporation 39

Synthetic Cannabinoids in Urine Oasis HLB µelution Need to extract neutrals, bases, and acidic metabolites Clean up urine matrix Concentrate samples o No need for evaporation High recovery and sensitivity High throughput CORTECS Solid Core Columns 1.6 µm solid-core particles Highest efficiency column available 2013 Waters Corporation 40

Synthetic Cannabinoids and Metabolites No. Compound 1 AM2233 2 RCS-4, M10 3 RCS-4, M11 4 AM 1248 5 JWH-073 4-butanoic acid met. 6 JWH-073 4-hydroxybutyl met. 7 JWH-018 5-pentanoic acid met. 8 JWH-073 (+/-) 3-hydroxybutyl met. 9 JWH-018 5-hydroxypentyl met. 10 JWH-018 (+/-) 4-hydroxypentyl met. 11 JWH-015 12 RCS-4 14 JWH-022 13 JWH-073 15 XLR-11 16 JWH-203 17 JWH-018 18 RCS-8 19 UR-144 20 JWH-210 21 AB 001 22 AKB 48 Acids Bases Neutrals 2013 Waters Corporation 41

Extraction Methodology with the Oasis HLB µelution Plate Condition Plate 200 µl MeOH then 200 µl Water Sample Pretreatment Mix 1 ml urine + 0.5 ml of 0.8 potassium phosphate (ph 7.0) Add 10 µl of β-glucuronidase and incubate at 40 C for 1 hr Add 1.5 ml of 4% H 3 PO 4 Load 600 µl pretreated sample (200 µl urine) Wash 200 µl water, then 200 µl 50:50 H 2 O:MeOH Elute 2 x 50 µl 60:40 ACN:IPA Dilute with 75 µl of water Inject 5 µl 2013 Waters Corporation 42

UPLC and MS conditions UPLC Conditions MS Conditions Column CORTECS C 18 1.6 µm; 2.1 x 100 XEVO TQD ESI+ LC ACQUITY UPLC I-Class Capillary: 1.0 kv Flow Rate 0.6 ml/min Source Temp: 150 C MPA Water with 0.1% FA Desolvation Temp: 600 C MPB ACN with 0.1% FA Cone Gas Flow: 0 L/Hr Column Temp 30 C Desolvation Gas Flow: 900 L/Hr Inj. Vol 5 µl MRM Transitions optimized individually Time Flow %A %B Curve 0 0.6 70 30 6 2.0 0.6 50 50 6 3.0 0.6 50 50 6 7.0 0.6 10 90 6 7.2 0.6 70 30 6 8.5 0.6 70 30 6 2013 Waters Corporation 43

Chromatogram for 22 Synthetic Cannabinoids and Metabolites 100 % 100 1 2 3 4 0 0.50 1.00 1.50 2.00 2.50 3.00 3.50 % 5 6 8 7 9, 10 11, 17 18 13, 12 14 19 21 15 16 20 Baseline separation of isobaric metabolites 22 Time 1) AM 2223 2) RCS4, M10 3) RCS-4, M11 4) AM 1248 5) JWH-073 4-COOH met. 6) JWH-073 4-OH met. 7) JWH-018 5-COOH met. 8) JWH-073 (+/-) 3-OH met. 9) JWH-018 5-OH met. 10) JWH-018 (+/-) 4-OH met. 11) JWH-015 12) RCS-4 13) JWH-073 14) JWH-022 15) XLR-11 16) JWH-203 17) JWH-018 18) RCS-8 19) UR-144 20) JWH-210 21) AB 001 22) AKB 48 0 4.00 4.50 5.00 5.50 6.00 6.50 7.00 Time Column: CORTECS UPLC C 18, 2.1 x 100 mm; 1.6 µm 2013 Waters Corporation 44

Improved Resolution of CORTECS 6 100 8 9, 10 5) JWH-073 4-COOH met. 6) JWH-073 4-OH met. 7) JWH-018 5-COOH met. 8) JWH-073 (+/-) 3-OH met. 9) JWH-018 5-OH met. 10) JWH-018 (+/-) 4-OH met. % 5 7 ACQUITY UPLC BEH C 18 2.1 x 100 mm 0 2.40 2.60 2.80 3.00 3.20 3.40 Time 100 6 8 9, 10 % 5 7 CORTECS UPLC C 18 2.1 x 100 mm 0 Time 2.20 2.40 2.60 2.80 3.00 3.20 2013 Waters Corporation 45

Recovery and Matrix Effects from Urine: Oasis HLB µelution Plates 120.0% 100.0% 80.0% Recovery Matrix Effects 60.0% 40.0% 20.0% 0.0% -20.0% -40.0% -60.0% -80.0% Average recovery was 74%. Matrix effects ranged from -49% (ion suppression) to 32% (enhancement), although most were less than 20%. Even in instances in which recovery was comparatively low, there was more than adequate sensitivity for the purposes of this assay. 2013 Waters Corporation 46

Accuracy, Precision, and Sensitivity QC concentration (ng/ml) 2.5 7.5 75 R 2 % Acc %CV %Acc %CV %Acc %CV Mean AM2233 0.996 95.25 7.59 109.78 8.45 102.48 6.52 102.50 RCS4, M10 0.998 99.00 3.65 103.13 1.97 96.63 4.33 99.58 RCS4, M11 0.999 102.30 2.40 103.53 0.68 96.43 4.11 100.75 AM 1248 0.987 111.43 3.79 110.70 1.73 98.98 2.97 107.03 JWH-073 4-COOH 0.997 104.68 3.29 108.43 1.13 94.58 3.87 102.56 JWH-073 4-OH Butyl 0.999 105.40 2.53 110.30 0.99 93.78 2.59 103.16 JWH-018, 5-COOH 0.998 102.10 4.94 104.53 1.62 97.53 4.61 101.38 JWH-073, 3-OH Butyl 0.999 103.63 3.89 108.00 0.35 98.95 2.47 103.53 JWH-018, 5-OH Met 0.999 103.40 4.65 107.40 1.88 100.58 3.50 103.79 JWH-018, 4-OH Met 0.999 103.63 2.11 108.60 1.15 100.70 2.75 104.31 JWH-015 0.994 96.65 3.39 99.53 1.81 93.23 3.60 96.47 RCS-4 0.992 98.05 2.27 97.88 2.24 91.85 3.02 95.93 JWH-022 0.993 100.80 3.69 93.50 5.63 93.28 5.68 95.86 JWH-073 0.982 95.48 7.19 88.30 4.51 103.23 6.01 95.67 XLR-11 0.987 105.20 8.37 103.55 1.96 90.85 2.87 99.87 JWH-203 0.990 97.35 5.39 85.65 2.85 93.65 3.00 92.22 JWH-018 0.996 98.48 2.11 86.60 9.38 95.95 6.25 93.68 RCS-8 0.992 98.58 4.09 93.48 10.85 96.23 6.38 96.09 UR-144 0.989 114.30 9.22 94.35 4.15 94.65 2.31 101.10 JWH-210 0.991 89.95 10.86 90.78 14.52 99.80 8.28 93.51 AB 001 0.988 100.28 4.02 86.38 9.66 97.45 5.96 94.70 AKB 48 0.985 104.28 3.58 87.55 5.79 94.35 5.07 95.39 Calibration range 1-100 ng/ml All accuracies within 15% of expected values. Most % CVs less than 10% and none greater than 15%. Limits of detection were as low as 0.1 ng/ml ; none greater than 2 ng/ml. 2013 Waters Corporation 47

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Synthetic Cannabinoids in Whole Blood Summary 2013 Waters Corporation 48

Whole Blood Extraction Methodology with Ostro Plate*

% Recovery and matrix effects of Synthetic Cannabinoids from Whole Blood Using Ostro Plates 130.0% 110.0% 90.0% 70.0% 50.0% 30.0% Recovery Matrix Effect 10.0% -10.0% -30.0% An average recovery of 92% for all compounds. Matrix effects were minimal. 2013 Waters Corporation 50

Linearity and Analytical Sensitivity QC concentrations(ng/ml) 7.5 75 300 Mean % Acc. R 2 %Acc. %CV %Acc. %CV %Acc. %CV AM2233 0.997 100.5 2.0% 103.6 3.3% 100.5 2.0% 101.5 RCS4, M10 0.986 97.5 3.9% 106.1 5.7% 101.7 8.4% 101.7 RCS4, M11 0.991 91.3 16.3% 108.8 5.1% 96.8 12.0% 98.9 AM 1248 0.993 83.1 10.0% 106.1 5.7% 105.4 6.4% 98.2 JWH-073 4-COOH 0.991 96.1 9.8% 99.3 7.4% 106.2 9.1% 100.5 JWH-073 4-OH Butyl 0.996 88.7 21.3% 98.1 3.5% 102.2 3.9% 96.3 JWH-018, 5-COOH 0.992 90.7 15.2% 97.8 3.8% 103.7 10.6% 97.4 JWH-073, 3-OH Butyl 0.993 79.0 8.6% 92.9 8.3% 96.6 2.9% 89.5 JWH-018, 5-OH Met 0.995 82.8 10.3% 100.0 10.4% 100.1 3.4% 94.3 JWH-018, 4-OH Met 0.992 82.3 17.9% 103.1 6.3% 96.0 1.9% 93.8 JWH-015 0.993 87.1 4.3% 101.8 3.9% 101.3 2.1% 96.8 RCS-4 0.993 92.5 8.1% 99.6 5.0% 97.3 3.6% 96.4 JWH-022 0.993 85.3 4.9% 100.3 4.8% 97.8 4.2% 94.5 JWH-073 0.994 89.6 6.5% 99.4 6.6% 97.6 4.9% 95.5 XLR-11 0.993 101.4 10.4% 99.6 2.8% 99.7 5.0% 100.2 JWH-203 0.990 82.1 12.2% 96.1 12.2% 94.6 9.3% 91.0 JWH-018 0.994 88.4 2.9% 97.2 3.9% 98.8 3.6% 94.8 RCS-8 0.992 94.3 2.6% 101.9 4.6% 99.4 4.7% 98.5 UR-144 0.994 85.1 5.4% 97.0 6.7% 99.2 3.7% 93.8 JWH-210 0.994 92.7 6.4% 96.3 4.5% 95.6 5.3% 94.8 AB 001 0.992 84.4 8.1% 101.0 4.7% 100.2 10.6% 95.2 AKB 48 0.992 92.8 9.9% 98.5 4.8% 97.7 8.4% 96.4 Mean % Acc. 89.4 100.2 99.5 R 2 values of >0.99 for 21 of the 22 compounds Most % CVs less than 10% and none greater than 13%. Accurate at all QC levels 2013 Waters Corporation 51

Synthetic Cannabinoids - Conclusions Urine Single method for extracting neutral, acidic, and basic compounds o Possibility of using the same technique for related compounds Removes salts, enzymes, and buffers Rapid and simple sample preparation o 96-well plates utilized Achieved excellent recovery and sensitivity No evaporation and reconstitution steps necessary o µelution format Whole Blood Clean up highly complex matrix (Ostro) Removal of proteins, cellular debris, and phospholipids Rapid, universal method with minimal method development Excellent recovery with minimal matrix effects Excellent accuracy and precision 2013 Waters Corporation 52

Expanded Toxicology Panel Expanded Toxicology Panel Replace immunoassay screening o Single analysis vs. multiple methods o Improved specificity and sensitivity HPLC Column Comparison Attributes of CORTECS 2.7 µm and XBridge BEH Phenyl XP 2.5 µm columns Simplified Sample Preparation Strategy Simplified procedure using MCX µelution plates 2013 Waters Corporation 53

HPLC Column Comparisons Compound Key Expanded Tox Panel 1) Amphetamine 2) MDA 3) Methamphetamine 4) MDMA 5) Phentermine 6) MDEA 7) BZE 8) PCP 9) Nitrazepam 10) Oxazepam 11) Alprazolam 12) Lorazepam 13) Clonazepam 14) Flunitrazepam 15) Temazepam 16) Diazepam amines benzodiazepines 17) Morphine 18) Oxymorphone 19) Hydromorphone 20) Dihydrocodeine 21) Codeine 22) Oxycodone 23) 6-AM 24) O-desmethyl Tramadol 25) Hydrocodone 26) Norfentanyl 27) Tramadol 28) Normeperedine 29) Meperedine 30) Norbuprenorphine 31) Fentanyl 32) Buprenorphine 33) EDDP 34) Propoxyphene 35) Methadone opioids 2013 Waters Corporation 54

Method Parameters UPLC MS MPA MPB Col Temp I-Class, FL with Column Manager (CMA) Xevo TQD 0.1% Formic Acid 0.1% Formic Acid in ACN 30 C Inj. Vol 10 µl Columns CORTECS C 18, 2.7 µm; 3.0 x 50 mm XBridge BEH Phenyl XP, 2.5 µm; 3.0 x 50 mm Time (min) Flow (ml/min) % MPA % MPB 0.0 0.6 95 5 4.0 0.6 40 60 4.1 0.6 95 5 5.0 0.6 95 5 2013 Waters Corporation 55

Opioid Chromatography 100 % 0 100 % 0 23 20 21 17 18 19 22 24 25 26 27 29 28 30 CORTECS C 18 2.7 µm 3.0 x 50 mm 0.50 1.00 1.50 2.00 2.50 3.00 3.50 1718 19 20,21 24 22 23 25 26 27 29 28 1.00 1.50 2.00 2.50 3.00 3.50 30 31 32 33 34 35 Time XBridge BEH Phenyl XP 2.7 µm, 3.0 x 50 mm 31 32 33 35 34 Time 17) Morphine 18) Oxymorphone 19) Hydromorphone 20) Dihydrocodeine 21) Codeine 22) Oxycodone 23) 6-AM 24) O-desmethyl Tramadol 25) Hydrocodone 26) Norfentanyl 27) Tramadol 28) Normeperedine 29) Meperedine 30) Norbuprenorphine 31) Fentanyl 32) Buprenorphine 33) EDDP 34) Propoxyphene 35) Methadone 2013 Waters Corporation 56

Enhanced Retention of Opiates 17) Morphine 100 CORTECS C 18 2.7 µm 3.0 x 50 mm 25 19) Hydromorphone 21) Codeine 25) Hydrocodone % 17 19 21 0 Time 0.20 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 100 % XBridge BEH Phenyl XP 2.7 µm, 3.0 x 50 mm 17 19 21 25 0 Time 0.40 0.60 0.80 1.00 1.20 1.40 1.60 1.80 2013 Waters Corporation 57

Amine Chromatography 100 % 1 2 3 4 5 6 7 8 CORTECS C 18 2.7 µm 3.0 x 50 mm 0 Time 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 1) Amphetamine 2) MDA 3) Methamphetamine 4) MDMA 5) Phentermine 6) MDEA 7) BZE 8) PCP 100 8 XBridge BEH Phenyl XP 2.7 µm, 3.0 x 50 mm % 1 3 4 2 5 6 7 0 Time 1.00 1.20 1.40 1.60 1.80 2.00 2.20 2.40 2.60 2.80 3.00 2013 Waters Corporation 58

Benzodiazepine Chromatography 100 % CORTECS C 18 2.7 µm 3.0 x 50 mm 9 10 11 13 12 14 15 16 9) Nitrazepam 10) Oxazepam 11) Alprazolam 12) Lorazepam 0 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 Time 13) Clonazepam 14) Flunitrazepam 15) Temazepam 16) Diazepam 100 XBridge BEH Phenyl XP 2.7 µm, 3.0 x 50 mm 15 % 10 13 9 1211 14 16 0 Time 2.40 2.60 2.80 3.00 3.20 3.40 3.60 3.80 4.00 2013 Waters Corporation 59

Performance Statistics and Conclusions Column Particle Size (µm) Pressure (Max) Mean Peak Width (sec) CORTECS C 18 2.7 2206 2.52 XBridge BEH Phenyl XP 2.5 3274 2.94 CORTECS High efficiency, narrow peak widths, and low pressure XBridge BEH Phenyl XP Enhanced retention of opiates and other polars 2013 Waters Corporation 60

Expanded Toxicology Panel Expanded Toxicology Panel Replace immunoassay screening o Single analysis vs. multiple methods o Improved specificity and sensitivity HPLC Column Comparison Attributes of CORTECS 2.7 µm and BEH Phenyl XP 2.5 µm columns Simplified Sample Preparation Strategy Universal simplified procedure using MCX µelution plates 2013 Waters Corporation 61

Simplified MCX procedure Compound Key Expanded Tox Panel 1 Amphetamine 2 MDA 3 MDMA 4 MDEA 5 BZE 6 PCP 7 7-Aminoclonazepam 8 alpha-oh Alprazolam 9 Oxazepam 10 Lorazepam 11 Clonazepam 12 Alprazolam 13 Temazepam 14 Diazepam 15 Morphine-3-gluc 16 Morphine-6-gluc 17 Morphine 18 Oxymorphone 19 Hydromorphone 20 Naloxone 21 Dihydrocodeine 22 Codeine 23 Noroxycodone 24 O-desmethyl Tramadol 25 6-MAM 26 Oxycodone 27 Hydrocodone 28 Norfentanyl 29 Tramadol 30 Tapentadol 31 Norbuprenorphine 32 Fentanyl 33 Buprenorphine 34 EDDP 35 Methadone 2013 Waters Corporation 62

Instrument Parameters UPLC MS MPA MPB Column Col Temp I-Class, FL with Column Manager (CMA) Xevo TQD 0.1% Formic Acid 0.1% Formic Acid in ACN BEH Phenyl; 1.7 µm, 2.1 x 100mm 40 C Inj. Vol 15 µl Time (min) Flow (ml/min) % MPA % MPB 0.0 0.6 95 5 5.0 0.6 37.5 62.5 5.1 0.6 95 5 6.0 0.6 95 5 2013 Waters Corporation 63

Chromatography 100 Amines, BZE, and PCP 100 Benzodiazepines 14 6 13 3 % 1 2 4 5 % 7 9 10 11 12 8 0 Time 0 0.50 1.00 1.50 2.00 2.50 3.00 2.00 2.50 3.00 3.50 4.00 Time 100 24 29 30 Opioids and Others % 0 15 16 17 18 22 20 21 23 25 26 19 27 28 33 Time 0.25 0.50 0.75 1.00 1.25 1.50 1.75 2.00 2.25 2.50 2.75 3.00 3.25 3.50 3.75 31 32 34 35 2013 Waters Corporation 64

Extraction Methodology for Urine Oasis MCX µelution Plate Protocol (Mixed-mode strong cation exchange SPE) In Well Hydrolysis 50 µl urine + 50 µl 0.1 M NH 4 OAc + 10 µl β-glucuronidase Add 200 µl 4% H 3 PO 4 Load Sample Wash 200 µl 0.02 N HCl in 20% MeOH Elute 2 x 50 µl (60:40 ACN:MeOH + 5% NH 4 OH) Pre-conditioning steps Eliminated Wash Steps Consolidated Evaporate Reconsititute with 50 ul 2% ACN/1% FA Inject 15 µl 2013 Waters Corporation 65

Recovery Simplified Protocol 120% 100% 80% 60% 40% 20% 0% Recovery Mean Recovery = 100% Compound LOD (ng/ml) Morphine-3-gluc 10 Morphine-6-gluc 10 Morphine 10 Oxymorphone 10 Hydromorphone 10 Amphetamine 1 Naloxone 10 Dihydrocodeine 10 Codeine 10 MDA 1 Noroxycodone 10 O-desmethyl Tramadol 1 6-MAM 10 Oxycodone 10 MDMA 1 Hydrocodone 10 MDEA 1 7-Aminoclonazepam 1 Norfentanyl 1 BZE 1 Tramadol 1 Tapentadol 1 Norbuprenorphine 10 PCP 1 Fentanyl 0.1 Buprenorphine 1 alpha-oh Alprazolam 10 EDDP 1 Oxazepam 1 Lorazepam 1 Clonazepam 1 Alprazolam 1 Methadone 1 Temazepam 1 Diazepam 1 2013 Waters Corporation 66

Overview Goal of Sample Preparation Sample Preparation Options Application Examples THC and Metabolites in Blood Summary 2013 Waters Corporation 67

THC and Metabolites in Whole Blood Assay Use Highly sensitive assay for THC and metabolites in whole blood Analytes THC, COOH-THC, OH-THC Goals Clean up whole blood matrix Maximize recovery and minimize matrix effects Need excellent linearity and sensitivity Selective extraction of acidic compounds (Oasis MAX) Short analytical run times Elimination of derivatization step prior to analysis Improved sample throughput vs. GC/MS 2013 Waters Corporation 68

Sample Preparation PPT Followed by Mixed Mode SPE Sample pretreatment (PPT and pretreatment) Oasis MAX Cartridge Protocol (Mixed-mode strong anion exchange SPE) Sample 0.2 ml whole blood Precipitate 0.4 ml ACN (added dropwise) Centrifuge 400 x g for 10 Transfer Supernatent Transfer supernatent (0.4 ml) To 0.6 ml 1% NH 4 OH Condition Cartridge 1 ml MeOH then 1 ml 1% NH 4 OH Load 1 ml prepared sample Wash 0.5 ml 50% ACN Elute 1.5 ml of 49:49:2 hexane:ethyl acetate:acetic acid Evaporate under N 2 at 40 C Reconstitute in 0.133 ml 70% aqueous MeOH Inject 15 µl 2013 Waters Corporation 69

THC Chromatography 0.5 ng/ml in Whole Blood THC-COOH qualifier ion THC-COOH quantifier ion THC-OH qualifier ion LC System: ACQUITY UPLC Column: ACQUITY BEH C 18 1.7 µm, 2.1 x 100 mm Flow: 400 µl/min MPA: 0.1% formic acid MPB: ACN Gradient: 60% B to 90% B over 4 minutes Mass spectrometer: XEVO TQ-S Ionization: ESI + Acquisition: MRM THC-OH quantifier ion Calibration Range: 0.5-50 ng/ml THC qualifier ion THC quantifier ion 2013 Waters Corporation 70

Mean recovery and matrix effects 6 lots of whole blood 100.0 Recovery and Matrix Effects 80.0 60.0 40.0 20.0 Recovery Matrix Effects 0.0-20.0 THC-OH THC-COOH THC Mean % recovery and matrix effects for cannabinoids spiked in whole blood at low (0.5 ng/ml), medium (5.0 ng/ml), and high (25 ng/ml) concentrations. The error bars are standard deviations. 2013 Waters Corporation 71

Inter-Day Accuracy and Precision 3.33 ng/ml Inter-day Accuracy % Target 16.67 ng/ml 33.33 ng/ml 3.33 ng/ml Inter-day Precision %RSD 16.67 ng/ml 33.33 ng/ml THC-OH 104.1 100.9 97.5 5.0 4.5 6.3 THC-COOH 102.7 99.2 96.5 6.7 4.4 3.8 THC 106.5 102.5 97.7 5.8 4.7 4.3 Inter-day accuracy and precision assessed by analyzing three quality control (QC) concentrations over 5 different days. The mean achieved values for the QC replicates over the 5-day period at the three concentration levels were within 10% of target, and the %RSD was <10%. 2013 Waters Corporation 72

Conclusions The challenges posed by the matrix (whole blood) and analytical requirements were best met with a two step process PPT followed by MAX Excellent sensitivity, linearity, accuracy and precision Minimal matrix effects Rapid analytical run time 2013 Waters Corporation 73

Overview Goal of Sample Preparation Sample Preparation Options Application Examples Summary 2013 Waters Corporation 74

Summary of Applications and Sample Preparation Strategies Application Matrix Solution Benefits Opiates Urine 720004650EN Oral Fluid 720004838EN Urine Oral Fluid Oasis MCX µelution ACQUITY BEH C 18 Oasis MCX µelution ACQUITY BEH C 18 Good recovery for all compounds Improved linearity, accuracy and precision vs Dilution Reduced matrix effects Good recovery for all compounds Linear, accurate and precise data Limited sample volume Synthetic Cannabinoids Urine 720004780EN Whole Blood 720004708EN Expanded Tox Panel THC and Metabolites 720004700EN Urine Oasis HLB µelution Sorbent appropriate for multiple chemotypes Linear, accurate and precise CORTECS C 18 Effective cleanup Concentration without evaporation Whole Blood Urine Whole Blood Ostro CORTECS C 18 Oasis MCX µelution ACQUITY BEH Phenyl PPT and Oasis MAX ACQUITY BEH C 18 Protein and phospholipid removal Linear, accurate and precise Minimal matrix effects Fast and Easy Minimal method development Excellent recovery for all compound classes Simplified SPE Method Rapid Sample Prep and analysis Sensitive, linear, accurate and precise Good recovery and minimal matrix effects 2013 Waters Corporation 75

Acknowledgements Sherri Naughton Nebila Idris Erin Chambers Michelle Wood Robert Lee 2013 Waters Corporation 76

Thank You for Attending! Post-Event Landing Page - www.waters.com/oct16 40% Promotional Offer On CORTECS Columns and Oasis uelution Plates 30% Offer on Ostro Plates 20% offer on BEH Columns (ACQUITY and Xbridge) Full Webinar Recording of Today s Session w/pdf Slide Deck Compilation of TODAY S KEY Literature, Brochures etc For Questions and to Submit your Ideas for our Next Topic Please email - mychemrep@waters.com Visit Us at SOFT: Booth # 101/200, October 21st 23rd 2013 Waters Corporation 77

Thank You 2013 Waters Corporation 78