Sample Preparation for Forensic Toxicology and Clinical Research

Sample Preparation for Forensic Toxicology and Clinical Research Part 1: Sample Preparation Options For Forensic Use

Agenda E-seminar Series Introduction Challenges for Today s Forensic Toxicology Laboratories Agilent Sample Preparation Technologies An Overview Conclusions and Options 2

Agilent Sample Preparation e-seminar Series Part 1: An Introduction to Agilent s sample preparation products Part 2: A practical approach to selecting the right sample preparation product for your needs Part 3: Tips and tools for solving common analytical challenges using sample preparation For Forensic Use 3

CHALLENGES FOR TODAY S FORENSIC TOXICOLOGY LABS Evolving analytical approaches Market changes Doing more with less For Forensic Use 4

The Ideal Analyzer Analyzer Sample Report 5

The Reality: Complex Workflows, With Complicated Challenges Technician and technologist capabilities Complex samples that can vary dramatically from donor to donor, and even from the same donor from day to day Reporting requirements Validation burden Cost and time pressures Consolidation and increased competition For Forensic Use 6

Application Challenge: Transitioning Existing Methods from GC-MS to LC-MS LC-MS Potential Advantages Promise of reduced sample prep transition from a complex sample prep protocol to using little to no sample prep Is this because there is no derivatization step in LC-MS? Increased selectivity tandem mass spectrometry or highresolution mass spectrometry Improved sensitivity lower limits of detection, increased injection volumes Speed of analysis 40-80 or more compounds in one run How Do I Select the Right Sample Prep Product to Exploit My New Instrument s Capabilities? 7

Application Challenge: Broader Range of Sample Types and Lower Detection and Quantitation Limits Urine, plasma, serum, and whole blood are common samples Use of hair, oral fluids, meconium, and other tissues is rising Lower limits of quantitation ranging from ng/ml to pg/ml and fg/ml How Do I Select the Right Sample Prep Method to Match My Sample Type? For Forensic Use 8

Application Challenge: Emerging Drugs of Abuse and New Compounds to Analyze Spice drugs and bath salts Multi-component screening and confirmation in a single analysis Increase in prescription drug abuse New or continually evolving designer drugs showing up in different geographies How Do I Develop New Methods to Stay On Pace With the Illicit Drug Chemists? For Forensic Use 9

Application Challenge: Updating or Creating New Methods on Existing Instrumentation Implementing new testing and assays for existing GC or LC instruments Updating current methods to improve results, reduce costs, increase throughput, optimize effectiveness, etc. Starting point current sample prep methods or find new ones? Method acceptance requirements what needs to be done to get the method ready for use Where Can I Go to Find Out About My Sample Preparation Options? 10

AGILENT SAMPLE PREPARATION OPTIONS An overview of available technologies 11

Objectives of Sample Preparation Removal of interferences which would affect detection of analyte Removal of interferences that would affect instrument or column lifetime Concentration of an analyte to a detectable concentration Solvent Switching into an analytically more compatible solvent 12

Striking the Right Balance in Sample Preparation Just Right Ideal Quality of Results Realistic Effort & Investment 13

Agilent Sample Preparation Techniques For Today s Discussion 1. Dilute and Shoot 2. Captiva Filtration Basic particulate removal from ALL kinds of samples Useful when additional step of lipid content removal is needed 3. Chem Elut Solid supported liquid extraction (SLE) Increased productivity using liquid/liquid extraction principle and the concept of automation Ideal for aqueous sample or samples 4. Bond Elut Solid phase extraction (SPE) Ultra-clean sample preparation for analysis when high selectivity and sensitivity are required 14

Sample Prep Options: An Overview Direct injection Dilute & Shoot Filtration Liquid/liquid extraction (LLE, SLE) QuEChERS Solid phase extraction (SPE) MIPS and Immunoaffinity Columns Less Selective More Selective For Forensic Use 15

Dilute and Shoot Simple sample dilution Advantages Fast and easy High throughput Limitations Interferences are not removed Concentration is reduced Instrument and column contamination Matrix interferences ion suppression or poor peak shapes 16

Agilent Captiva Filtration A Straightforward Companion to Dilute-and-Shoot Basic sample prep method for biological samples Can be the first choice of sample prep or secondary step Mechanical filtration for particulate interference removal - Captiva Filter Vial Syringeless filters - Captiva Syringe Filters - Agilent Captiva cartridge and 96-well plate formats - Agilent Captiva ND Non-drip cartridge and 96-well plates Mechanical filtration + extraction by sorbent for lipid removal - Agilent Captiva ND Lipids cartridge and 96-well plates For Forensic Use 17

Captiva Filtration Product Recommendation Filtration Needs/Situation Agilent Captiva Product High Throughput Vacuum processing In-well protein precipitation Particulate filtration only Automation Friendly Lipid removal Larger sample size Single cartridge X X X X Single ND cartridge X X can be done X X Single ND Lipids cartridge X X X X X 96-well plate X X X X 96-well ND plate X X X can be done X 96-well ND Lipids plate X X X X X Syringe filter can be done X can be done X For Forensic Use 18

Agilent Chem Elut Solid Supported Liquid Extraction (SLE) Extraction mechanism: same as traditional liquid/liquid extraction (LLE) Simple, time-saving process Apply aqueous sample to the solid bed Extract with water-immiscible solvent (MTBE, dichloromethane, ethyl acetate) Analyze extract or evaporate and reconstitute as needed Convert LLE methods to Chem Elut SLE to save time and money, and increase throughput 19

Chem Elut SLE - Benefits No emulsions easier extractions No special glassware lower cost per sample Less time, minimal method development faster implementation Reduced technique dependence better ruggedness Increased reproducibility better results Automatable enables batch processing 20

Disc Technology: SPEC Disc-Based SPE Pre-Filter SPEC Disc SPEC Solid structure with through pores Higher surface area than packed beds leads to: Higher capacity per bed mass (so smaller bed masses) Smaller elution volumes Suitable for low sample volumes Monolithic-disc, low bed mass Capacity ~10% of bed mass Void volume ~1 3 µl/mg of bed mass 21

Bond Elut Solid Phase Extraction Types of SPE - Reversed phase SPE - Cation exchange SPE - Anion exchange SPE - Mixed mode SPE - Specialty SPE - SPEC Disc-based SPE Capabilities - Very selective - Highly clean samples - Wide range of applicability - Automation friendly Condition Load Wash Elute 22

Select a Format that Meets Your Needs Tubes 1 ml to 60 ml Straight Barrel (50 mg 10 g) Bond Elut Jr (500 mg 1 g) LRC (Large Reservoir Capacity) (100-500 mg) Mega Bond Elut 12-150 ml (2 g - 70 g) Multi-Array Well Plates 1 ml, 2 ml 96 Well 1.8 ml VersaPlate OMIX 96 SPEC 1 ml, 2 ml Automation Hamilton TomTEC Gilson ASPEC Gerstel Spark Holland Prospekt 23

Bond Elut Packed Bed Phases Non-polar Plexa ENV, LMS, PPL, FOCUS NEXUS C18, C8, C2, C1 C18 variations in carbon load and end-capping EnvirElut CH cyclohexyl CN-E endcapped cyano PH phenyl Anion Exchange SAX quaternary amine PSA primary and secondary amine NH2 aminopropyl DEA diethylaminopropyl Polar PSA - primary and secondary amine NH2 - aminopropyl CN-U unendcapped cyano DEA - diethylaminopropyl Diol Si - silica Mixed mode IEX/NP Plexa PCX Plexa PAX Certify SCX/C8 Certify II SAX/C8 Cation Exchange SCX benzenesulfonic acid PRS propylsulfonic acid CBA carboxylic acid Reversible Covalent PBA phenylboronic acid Specialty Phases AccuCAT Atrazine Etc.. Inorganic, Non-Silica, and Dual-Phase Alumina aluminum oxide Florisil magnesium-silica Carbon Carbon/NH2 24

Bond Elut Plexa Polymeric SPE Family Plexa Polymeric SPE Products Plexa reversed phase polymeric SPE Plexa PCX cation exchange mixed mode SPE Plexa PAX anion exchange mixed mode SPE Plexa Polymeric SPE Advantages Rugged methods provide cleaner extracts Simple method development Consistent, fast flow for high throughput 25

SUMMARY AND NEXT STEPS Summary of available products Learn more Contact us 26

Agilent Sample Preparation Products Bond Elut Solid Phase Extraction Bond Elut SPE Bond Elut Plexa Polymeric SPE SPEC Bond Elut QuEChERS Captiva Filtration Chem Elut SLE Chem Elut SLE Tox Elut SLE Chem Elut Plus SLE Combilute SLE Hydromatrix Captiva Syringe Filters, cartridges, and plates Captiva Non-Drip and ND Lipids cartridges and plates For Forensic Use 27

How do Sample Prep Options Compare? Agilent Bond Elut Solid Phase Extraction (SPE) - Very clean, allows for trace analysis, built-in concentration - Potential for greater selectivity (and hence cleanliness) Investment Agilent Chem Elut Solid Supported Liquid Extraction (SLE) - Relatively clean and inexpensive Complexity Agilent Captiva Syringe Filters, Tubes, Plates, and Vials Filtration - Mechanical removal of particulates or functional filtration to remove particulates and lipids Cleanliness Dilute and shoot For Forensic Use 28

Learn More E-Seminar Series Part 2: Selecting the Right Sample Preparation Product Agilent Sample Preparation Portfolio Guide Publication Number 5991-2954EN E-Seminar Series Part 3: Applying Sample Preparation to Solve Common Analytical Challenges Agilent Sample Preparation and Analysis for Drug Screening and Confirmation Compendium: www. agilent.com/chem/drugscreeningand Confirmation For Forensic Use 29

Contact Us To learn more about the Agilent Sample Preparation portfolio, visit agilent.com/chem/sampleprep To find your local Agilent Representative or Agilent Authorized Distributor, visit agilent.com/chem/contactus U.S. and Canada: 1-800-227-9770 agilent_inquiries@agilent.com Europe: info_agilent@agilent.com Asia Pacific: adinquiry_aplsca@agilent.com India: lsca-india_marketing@agilent.com 30

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Sample Preparation for Forensic Toxicology and Clinical Research Part 2: Selecting the Right Sample Preparation Product For Forensic Use

Agenda E-seminar Series Introduction Agilent Sample Preparation Product Review Selecting the Right Sample Prep A Practical Approach Conclusions and Options 2

AGILENT SAMPLE PREPARATION OPTIONS An overview of available technologies 4

Striking the Right Balance in Sample Preparation Just Right Ideal Quality of Results Realistic Effort & Investment 6

SELECTING THE RIGHT PRODUCT Considerations and options 8

Sample Preparation Considerations We often talk about a triangle but the questions about sample prep and SPE are more complex than this simple model Sorbent Analyte Matrix Other Sample Prep Considerations Analytical goals Published methods Instrument availability Skill and expertise Regulations Sample Size Detection limits Cost per sample Lab setup and supplies investment Automation needs 9

Practical Approaches to Selecting a Sample Preparation Method Interferences to Remove Application Type Matrix or Sample Format for Automation C18 Current Product Alternative Journal Article, Application Note, or Book Interactive Selection Guides 10

Sample Preparation Selection: Interference Removal Needs Sample Prep Technique Interference Removed More Specific Instrument Separation and Detection Specificity Less Specific Less Specific Sample Preparation Specificity More Specific Dilute & Shoot Filtration Liquid/Liquid Extractions Supported Liquid Extractions (SLE) Dried Matrix Spotting Precipitation filtration QuEChERS Precipitation- Lipid Removal Hybrid' Filtration Lipids No No No Some No No Yes Yes Yes Solid Phase Extraction Oligomeric Surfactants No No No No No No No Yes Yes Particulates No Yes No Some No Yes Yes Yes Yes Pigments No No No Some No No Yes No Yes Polar Organic Acids No No Yes Yes No No Yes No Proteins No No Yes Yes Yes Yes Yes Yes Yes Salts No No Yes Yes No No No No Yes Filtration is suggested with any LC or GC method of sample preparation For Forensic Use 11

Sample Preparation Selection: Application Type or Market Area For Forensic Use 12

Sample Preparation Selection: Format for Automation or Sample Size 13

Sample Preparation Selection: Current Product Alternative Step 1: Locate your current product Step 2: Identify the comparable Agilent product 14

Sample Preparation Selection: Journal Article, Application Note, or Book For Forensic Use 15

Agilent Product Citation Reference Library Citations for Agilent products demonstrate utility and performance in a wide range of applications, in peerreviewed journals For Forensic Use 16

Agilent LC Column and Sample Preparation Navigator Tool www.agilent.com/chem/navigator For Forensic Use 17

Agilent Syringe Filter Selection Tool www.agilent.com/chem/selectfilters 18

SUMMARY AND NEXT STEPS Selection process overview Learn more Contact us 19

Additional Resources and Options Selecting the right sample preparation method lays the foundation for your analytical success. You can count on Agilent to support your quest to find the products that are just right for your needs: Agilent e-library Agilent technical support and your local Agilent representative Agilent Sample Preparation 2013-2014 Product catalog Agilent Sample Preparation Handbook 21

Learn More E-Seminar Series Part 1: Sample Preparation Options Agilent Sample Preparation Portfolio Guide Publication Number 5991-2954EN E-Seminar Series Part 3: Applying Sample Preparation to Solve Common Analytical Challenges Agilent Sample Preparation and Analysis for Drug Screening and Confirmation Compendium: www. agilent.com/chem/drugscreeningand Confirmation For Forensic Use 22

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Sample Preparation for Forensic Toxicology and Clinical Research Part 3: Applying Sample Preparation to Solve Common Analytical Challenges For Forensic Use

Agenda E-seminar Series Introduction Quick Review of Agilent Sample Preparation Options Tips and Tools for Solving Analytical Challenges Conclusions and Summary 2

AGILENT SAMPLE PREPARATION OPTIONS An overview of available technologies 4

Striking the Right Balance in Sample Preparation Just Right Ideal Quality of Results Realistic Effort & Investment 5

COMMON CHALLENGES IN BIOLOGICAL SAMPLE ANALYSIS Biological Sample Analysis 9

Agilent Sample Preparation Solutions Matrix Challenges Lab Goals & Needs 10

Tandem Mass Spectrometry and The Case of the Disappearing Matrix Selected ion monitoring looking at 100 fg HCB in matrix MRM s better selectivity makes the matrix disappear 11

Disappearing Matrix What Can Dirty Samples Do? Interference type Salt/Polar ionics Proteins/ Peptides Lyso-phosphatidylcholines Lipids and other hydrophobics Typical Elution Conditions (C18 column) At or near void with < 20% organic 10 s of column volumes at 40% - 70% organic 10 s of column volumes at 70% - 90% organic 10 s to 100 s of column volumes at > 90% organic Short term effect (single injection) Significant ion-suppression Significant ionsuppression Significant ion-suppression Some ion suppression, however, usually retained on LC column) Long term effect (multiple injections) Unknown Unknown Decreased sensitivity, Increased variability Decreased sensitivity, Increased variability Likely long term causes Ion source contamination Ion source contamination Ion source contamination, Some column build-up Ion source contamination, Column build-up 12

Challenge: Instrument Contamination Example 1 Salt build-up in LC-MS ion source from unextracted salts 13

Challenge: Instrument Contamination Example 2 Orthogonal ESI Ion Source Condition after 3000x Urine Dilute/shoot Injections Nebulizer/Sprayer Spray Shield/MS Inlet/Capillary For Forensic Use 14

Challenge: Instrument Contamination Example 3 GC System Component Contamination with Biological Samples GC Inlet Liner GC Inlet Seal 15

Solution: Select an Agilent Sample Preparation Product Based on Interference Removal Particulates Proteins Lipids Surfactants Salts Captiva Filtration Captiva ND Captiva ND Lipids Chem Elut SLE Bond Elut SPE For Forensic Use 16

Challenge: Lipids Cause Interference and System Cleanliness Issues R R - R m/z = 184 (+2H) m/z = 184 (+2H) phosphatidylcholine lysophosphatidylcholine Two major lipid interferences that exist during bioanalysis by LC/MS/MS. By soft ionization conditions, these two phospholipids can generate the common fragment, 184 m/z. Removal of ion suppressing lipids can be verified by monitoring 184 184 m/z transition. For Forensic Use 17

Solution: Targeted Lipid Removal Green = ppt only Red = lipid-stripped ppt with Captiva ND Lipids MS Transition 184 184 For Forensic Use 18

Solution: Chem Elut SLE Extraction of Beta-Blockers in Plasma Plasma spiked with beta blockers was diluted by 2% ammonia 1:1. Load 0.3 ml of spiked & diluted plasma to VersaPlate. Apply slight vacuum to initiate flow. When sample is soaked below the top frit stop vacuum. Wait for 5 min for aqueous sample adsorption. Elute with 2 X 0.9 ml of EtOAc. Apply vacuum to have 1 2 drops per sec. After elution increase vacuum for 30 sec. Evaporate and reconstitute in 0.15 ml of 30% MeOH for LC/MS/MS analysis. For Forensic Use 19

Beta-Blockers by SLE Chromatography Acebutolol (ISTD) Timolol Nadolol Metoprolol Propranolol Pindolol For Forensic Use 20

Solution: Remove Proteins & Lipids Using SPE Exploit the chemistry and physical properties of Agilent Bond Elut Plexa SPE sorbent Polar and non-polar drugs bind in the SDVB hydrophobic end of the pore structure Albumin and proteins of similar size will be excluded Lipids adsorb and do not elute under normal elution conditions For Forensic Use 21

Solid Phase Extraction of Buprenorphine and nor- Buprenorphine in Whole Blood SPE using Agilent Bond Elut Plexa PCX polymeric cation exchange buprenorphine at 0.2 ng/ml Buprenorphine and nor-buprenorphine were efficiently extracted from whole blood nor-buprenorphine at 0.2 ng/ml Extraction method was simple and used only 0.5 ml of whole blood Mixed-mode cation exchange SPE ensures removal of matrix and high recoveries of targets Precise, reproducible extraction with low detection limits buprenorphine-d4 (ISTD) at 10 ng/ml nor-buprenorphine-d3 (ISTD) at 10 ng/ml For Forensic Use 22

Challenge: Degradation of Sub-2-µm LC Column Performance Over Time 23

Solution: Filtration Extends Column Lifetime For Forensic Use 24

Challenge: Unexplained Peaks in Sample ESI-Positive Mode 7000000 Filtered 30% MeOH by Filter W 0.2µm filter IS 6000000 5000000 Filtered 30% MeOH by Filter M PES 0.2µm filter IS 4000000 Filtered 30% MeOH by Filter P PES 0.2µm filter IS 3000000 2000000 Filtered 30% MeOH by Agilent PES 0.2µm filter IS 1000000 Unfiltered 30% MeOH IS 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min 25

Solution: Use Certified Clean Filtration Products ESI-Positive Mode 7000000 Filtered 30% MeOH by Filter W 0.2µm filter IS 6000000 5000000 Filtered 30% MeOH by Filter M PES 0.2µm filter IS 4000000 Filtered 30% MeOH by Filter P PES 0.2µm filter IS 3000000 2000000 Filtered 30% MeOH by Agilent PES 0.2µm filter IS 1000000 Unfiltered 30% MeOH IS 0 0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 min 26

Challenge: Sample Throughput and Productivity 100s or 1000s of samples a day to be processed Consistency and simplicity to achieve high-quality results with few repeats Limit the operator-to-operator and day-to-day variation (ruggedness) Support for automation for increased throughput and unattended operation 27

Solution: Replace Conventional Protein Precipitation with Captiva ND Lipids Centrifugation Protein Precipitation (PPT) Time (min) Captiva ND Lipids Time (min) Add 0.6 ml of MeOH and 0.2 ml of plasma sample to Captiva ND 96-well plate. Centrifuge at 10,000 RPM for 10 min. Transfer supernatant to 2 ml injection vials (if tubes were used) or a new empty 96-well plate for analysis (if plate format was used). Total time required for sample preparation Lipids WILL build up during your sequence. 5 Add 0.6 ml of MeOH and 0.2 ml of plasma sample to Captiva ND 96-well plate. 11 Mix each well with a pipette 5 times and apply vacuum for filtration. 10 Directly transfer injection plate for analysis. Total time required for sample preparation Proteins AND lipids will be removed during your sample preparation. 5 0 For Forensic Use 28

Results: Captiva ND Lipids for Beta-blockers in Plasma Acebutolol (ISTD) Timolol Nadolol Atenolol Good chromatography Propranolol Pindolol For Forensic Use 29

Challenge: Converting GC-MS Confirmatory Methods to LC-MSMS Confirmation Methods GC-MS methods have long been the gold standard for confirmations Conversion is not typically straightforward not as simple as taking the samples as prepared for GC-MS and putting them onto the LC- MSMS Considerations when using LC-MSMS: Ion ratios Matrix effects including ion suppression and ion enhancement New sample prep and analysis methods Method acceptance 30

Solution: Confirmatory Methods and Applications for Major Classes of Drugs of Abuse Application notes describing sample preparation methods using Bond Elut Plexa PCX Polymeric cation exchange SPE with mixed mode capabilities leads to clean extracts Simple methods for high throughput Effective, efficient cleanup with low matrix effects Straightforward method acceptance criteria For Forensic Use 31

SUMMARY AND OPTIONS Conclusions Learn More Contact Us 32

Conclusions Sample preparation can be a valuable tool to address common analytical challenges Looking at the specific challenge you re having can help match the right Agilent sample preparation product to your issue Agilent offers a range of sample prep products that can be used not only to prepare samples Improve results Increase analytical uptime Maximize productivity and reduce costs Achieve accurate results, right from the start 33

Agilent Offers Total Workflow Solutions, from Extraction To Final Report Sample Preparation Separation Detection, Analysis, and Reporting www.agilent.com/chem/forensics Page 34

Learn More E-Seminar Series Part 1: Sample Preparation Options E-Seminar Series Part 2: Selecting the Right Sample Preparation Product Agilent Sample Preparation Portfolio Guide Publication Number 5991-2954EN Agilent Sample Preparation and Analysis for Drug Screening and Confirmation Compendium: www. agilent.com/chem/drugscreeningand Confirmation For Forensic Use 35

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