This application note describes the extraction of 96 licit and illicit drugs of abuse from urine prior to UPLC-MS/MS analysis using EVOLUTE® HYDRO CX 96-well plates. EVOLUTE® HYDRO CX plates offer an efficient way to perform hydrolysis in the well of the extraction plate. This method provides high analyte recovery, reduced extraction time due to the elimination of a sample transfer step as well as the elimination of the column conditioning and equilibration steps, and a reduced risk for sample carryover or cross-contamination due to the elimination of the sample transfer step.

6-acetyl codeine 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam 96-well plate Alprazolam Amitriptyline Amphetamine Amphetamines Analgesics Application Notes Aripiprazole Atomoxetine Barbiturates Benzodiazepines Benzoylecgonine Buprenorphine Bupropion Buspirone Butalbital Carbamate hypnotic Carbamate muscle relaxant Carbamates Carbazepine Carisoprodol Chlordiazepoxide Chlorpheniramine Chlorpromazine Clinical Clomipramine Clonazepam Clonidine Clozapine Cocaethylene Cocaine Cocaines Codeine Cyclobenzaprine Desalkylflurazepam Designer stimulants Dextromethorphan Diazepam Dihydrocodeine (DHC) Drugs of Abuse Duloxetine EDDP English Ethyl Sulfate (EtS) Extrahera Fentanyl Fluoxetine Forensic Gabapentin Haloperidol Hydrocodone Hydrolysed urine Hydromorphone Hydroxyalprazolam Hydroxybupropion Hydroxymidazolam Hydroxytriazolam Imipramine Ketamine LC-MS/MS Lamotrigine Levetiracetam Lidocaine Lorazepam MDA MDEA MDMA Meperidine Meprobamate Methadone Methamphetamine Methaqualone Methcathinone Midazolam Morphine N-des-tapentadol N-desmethylclomipramine N-desmethyltapentadol Naloxone Norbuprenorphine Nordiazepam Norfentanyl Norhydrocodone Norketamine Normeperidine Noroxymorphone Norpropoxyphene Nortryptiline Opiates Opioid Opioid agonist Oxazepam Oxcarbazepine Oxycodone Oxymorphone Pain Management Pentobarbital Pharmaceuticals Phencyclidine (PCP) Phenobarbital Pregabalin Quetiapine Ritalinic acid Secobarbital Stimulants THC-COOH Tapentadol Temazepam Topiramate Tramadol Triazolam Trimipramine Urine Venlafaxine Zolpidem Zolpidem-phenyl-4-COOH mCPP z-drugs

Analysis of drug panels in urine samples can be challenging, and the trend towards larger panels including multiple drug classes compounds the issues faced during method development. This white paper examines a number of aspects of sample preparation, and their impact on the success of subsequent LC-MS/MS analysis of broad urine panels. Section 1 examines the applicability of various sample preparation techniques: supported liquid extraction, reverse phase SPE and mixed-mode SPE, to the various classes of drugs extracted. In addition, hydrolysis approaches: enzyme type and protocol used (time, temperature), are compared. Mixed-mode reverse phase/cation exchange SPE is widely used for extraction of basic drug classes from urine, but the inclusion of drugs and metabolites that exhibit ‘non-typical’ functionality within urine panels can be problematic. Section 2 examines the impact of various parameters (interference wash strength, elution solvent composition) on analyte retention, elution and extract cleanliness with particular focus on zwitterionic (gabapentin, pregabalin) and non-ionic (carisoprodol, meprobamate) drugs.

11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 6-acetylmorphine (MAM) 7-Aminoclonazepam 96-well plate Alprazolam Amitriptyline Amphetamine Amphetamines Anaesthetic Anticonvulsant Application Notes Barbiturates Benzodiazepines Benzoylecgonine Buprenorphine Butalbital Cannabinoid Carbamate hypnotic Carbamate muscle relaxant Carbamates Carisoprodol Chlordiazepoxide Clinical Clonazepam Cocaine Cocaines Codeine Diazepam Dihydrocodeine (DHC) EDDP English Fentanyl Forensic Gabapentin Hallucinogen Hydrocodone Hydrolysed urine Hydromorphone Ketamine LC-MS/MS Lorazepam MDMA Meperidine Meprobamate Methadone Methamphetamine Morphine N-des-tapentadol Naloxone Norbuprenorphine Nordiazepam Norfentanyl Norhydrocodone Norketamine Normeperidine Noroxymorphone Norpropoxyphene Nortryptiline O-desmethyltramadol Opiates Opioid Opioid agonist Oxazepam Oxycodone Oxymorphone Pentobarbital Phencyclidine Phencyclidine (PCP) Phenobarbital Pregabalin Product Notes Ritalinic acid Secobarbital Stimulants Sympathomimetic amine THC metabolites THC-COOH Tapentadol Temazepam Tramadol Tricyclic Antidepressant White Papers Zolpidem Zolpidem-phenyl-4-COOH z-drugs α-OH alprazolam

This poster discusses the potential for a single extraction protocol for various drugs of abuse classes in whole blood prior to UPLC-MS/MS analysis. SOFT 2017, Boca Raton also presented at SFTA, Marseille, France, 2018

Amphetamines Benzodiazepines Cannabinoid Cathinones Clinical Cocaines Column Drugs of Abuse English Extrahera Forensic LC-MS/MS Opiates Posters THC metabolites Whole blood z-drugs

This poster demonstrates the extraction of a range of drugs of abuse from oral fluid, collected with common collection devices, prior to UPLC-MS/MS analysis. The target analyte list includes benzodiazepines, z drugs, amphetamines, cathinones, opiates, cocaine, buprenorphine, PCP, THC-COOH, fentanyl and ketamine. SOFT 2014

11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 2-OH-Et-Flurazepam 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam Alprazolam Amphetamine Amphetamines Benzodiazepines Benzoylecgonine Bromazepam Buprenorphine Cathinones Clonazepam Cocaine Codeine Column Diazepam Dihydrocodeine (DHC) EDDP English Estazolam Fentanyl Flunitrazepam Flurazepam Forensic Hydrocodone Hydromorphone Ketamine LC-MS/MS Lorazepam MDA MDEA MDMA Mephedrone Methadone Methamphetamine Midazolam Morphine Nitrazepam Norbuprenorphine Nordiazepam Norfentanyl Norketamine Opiates Opiates Oral fluid Oxazepam Oxycodone Oxymorphone Phencyclidine (PCP) Posters Saliva Temazepam Triazolam Zaleplone Zolpidem Zopiclone z-drugs α-OH Triazolam α-OH alprazolam

This poster compares the performance of manual processing to a novel automated sample preparation system prior to GC/MS or LC-MS/MS analysis in forensic toxicology applications. Emphasis is placed on the potential for 96-well cross contamination and strategies for its elimination. TIAFT 2016 SOFT 2016

2-Hydroxyethylflurazepam 2-OH-Et-Flurazepam 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam 96-well plate Alprazolam Amphetamine Amphetamines Benzodiazepines Bromazepam Buprenorphine Clinical Clonazepam Codeine Column Diazepam Dihydrocodeine (DHC) Drugs of Abuse EDDP English Estazolam Extrahera Fentanyl Flunitrazepam Flurazepam Forensic GC-MS Hydrocodone Hydromorphone Ketamine LC-MS/MS Lorazepam MDA MDEA MDMA Mephedrone Methadone Methamphetamine Midazolam Morphine Nitrazepam Norbuprenorphine Nordiazepam Norfentanyl Norketamine Opiates Opiates Oxazepam Oxycodone Oxymorphone Phencyclidine (PCP) Posters Temazepam Triazolam Urine Whole blood Zaleplone Zolpidem Zopiclone z-drugs α-OH Triazolam α-OH alprazolam

This poster describes an improved workflow for the analysis of drugs of abuse from hair. TIAFT 2018, Ghent, Belgium

2-OH-Et-Flurazepam 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam 96-well plate Alprazolam Amphetamine Amphetamines Benzodiazepines Benzoylecgonine Bromazepam Buprenorphine Clonazepam Cocaine Codeine Column Diazepam Dihydrocodeine (DHC) EDDP English Estazolam Extrahera Fentanyl Flunitrazepam Flurazepam Forensic Hair Hydrocodone Hydromorphone Ketamine LC-MS/MS Lorazepam Lysera Lysergic acid diethylamide (LSD) MDA MDEA MDMA Methadone Methamphetamine Midazolam Morphine Nitrazepam Norbuprenorphine Nordiazepam Norfentanyl Norketamine Opiates Opioid Oxazepam Oxycodone Oxymorphone PCP Posters Temazepam Triazolam Zaleplone Zolpidem Zopiclone z-drugs α-OH Triazolam α-OH alprazolam

Most drugs are excreted in urine as glucuronide conjugates. Hydrolysis using a beta-glucuronidase enzyme to convert the metabolites to their “free” form for analysis increases sensitivity. Red abalone (Kura Biotech), abalone (Campbell Scientific), and recombinant (IMCSzyme) beta-glucuronidase enzymes were evaluated to determine which provided the most complete hydrolysis of glucuronide metabolites without effecting the overall recovery of non-conjugated compounds. EVOLUTE EXPRESS CX 96-well plates were used to extract hydrolysed urine samples, and the impact of th enzymes was compared. MSACL 2017, Palm Springs SOFT 2017, Boca Raton

6-acetylmorphine (MAM) 7-Aminoclonazepam 96-well plate Alprazolam Amitriptyline Amphetamine Amphetamines Analgesics Barbiturates Benzodiazepines Benzoylecgonine Butalbarbital Carbamates Carisoprodol Chlordiazepoxide Clinical Clonazepam Cocaine Cocaines Codeine Diazepam Dihydrocodeine (DHC) Drugs of Abuse EDDP English Fentanyl Forensic Gabapentin Hydrocodone Hydrolysed urine Hydromorphone Ketamine LC-MS/MS Lorazepam MDMA Meperidine Meprobamate Methadone Methamphetamine Morphine N-des-tapentadol Naloxone Norbuprenorphine Nordiazepam Norfentanyl Norhydrocodone Norketamine Normeperidine Nortryptiline Opiates Over the counter pharmaceuticals Oxazepam Oxycodone Oxymorphone Pain Management Pharmaceuticals Phencyclidine (PCP) Phenobarbital Posters Pregabalin Ritalinic acid Secobarbital Stimulants THC metabolites THC-COOH Tapentadol Zolpidem Zolpidem-phenyl-4-COOH z-drugs α-OH alprazolam

Product Note for EVOLUTE® HYDRO Solid Phase Extraction Plates with In-Well Hydrolysis Capability

96-well plate Alcohol metabolite Amphetamines Anaesthetic Analgesics Barbiturates Benzodiazepines Carbamate hypnotic Carbamate muscle relaxant Carbamates Clinical Designer stimulants Drugs of Abuse English Forensic GC-MS Hydrolysed urine LC-MS/MS NSAIDs Opiates Opioid Opioid agonist Pain Management Pharmaceuticals Product Notes Stimulants Urine z-drugs

This application note describes a Supported Liquid Extraction (SLE) protocol for the extraction of various drugs of abuse from hydrolyzed urine prior to LC-MS/MS analysis. drugs of abuse, sle, multiclass

2-Hydroxyethylflurazepam 7-Aminoclonazepam 96-well plate Alprazolam Amphetamine Amphetamines Anhydroecgonine methyl ester Application Notes Benzodiazepines Benzoylecgonine Bromazepam Buprenorphine Clonazepam Cocaethylene Cocaine Cocaines Codeine Column Diazepam Ecgonine methyl ester Estazolam Fentanyl Flunitrazepam Hydrolysed urine Hydroxyalprazolam Hydroxytriazolam Ketamine Methamphetamine Midazolam Morphine Nitrazepam Norbuprenorphine Norcocaine Nordiazepam Norfentanyl Norketamine Opiates Opioid Oxazepam Temazepam Triazolam Zaleplone Zolpidem Zopiclone z-drugs

This application note describes a Supported Liquid Extraction (SLE) protocol for the extraction of various drugs of abuse from non- hydrolyzed urine prior to LC-MS/MS analysis. drugs of abuse, sle, multiclass

2-Hydroxyethylflurazepam 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam Alprazolam Amphetamine Amphetamines Application Notes Benzodiazepines Benzoylecgonine Bromazepam Buprenorphine Clonazepam Cocaine Cocaines Codeine Diazepam Dihydrocodeine (DHC) EDDP English Estazolam Fentanyl Flunitrazepam Flurazepam Forensic Hydrocodone Hydromorphone Hydroxyalprazolam Hydroxytriazolam Ketamine LC-MS/MS MDA MDEA MDMA Methadone Methamphetamine Midazolam Morphine Nitrazepam Norbuprenorphine Nordiazepam Norfentanyl Norketamine Opiates Opioid Oxazepam Oxymorphone RapidTrace TECAN Temazepam Triazolam Urine Zaleplone Zolpidem Zopiclone z-drugs

This application note describes the extraction, using ISOLUTE SLE+ supported liquid extraction columns, of 49 drugs of abuse from whole blood, prior to UPLC-MS/MS analysis. High, reproducible drug recoveries are achieved, with sub ng/mL LOQs for most analytes. The method is easily automated using Biotage® Extrahera.

11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 2-Hydroxyethylflurazepam 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam Alprazolam Amphetamine Benzodiazepines Benzoylecgonine Bromazepam Buprenorphine Clonazepam Cocaine Cocaines Codeine Column Diazepam Dihydrocodeine (DHC) Drugs of Abuse EDDP English Estazolam Extrahera Fentanyl Flunitrazepam Flurazepam Forensic Hydrocodone Hydromorphone Ketamine LC-MS/MS Lorazepam Lysergic acid diethylamide (LSD) MDA MDEA MDMA Mephedrone Methadone Methamphetamine Midazolam Morphine Nitrazepam Norbuprenorphine Nordiazepam Norfentanyl Norketamine Opiates Oxazepam Oxycodone Oxymorphone Pain Management Phencyclidine (PCP) THC metabolites THC-COOH Temazepam Triazolam Whole blood Zaleplone Zolpidem Zopiclone z-drugs α-OH Triazolam α-OH alprazolam

This poster discusses the effect of various sample preparation protocols of the recovery of a range of different Drugs of abuse classes, extracted from oral fluid collected using the Quantisal collection device. MSACL NA 2018, Palm Springs CA ASMS 2018, San Diego, CA

Anaesthetic Anticonvulsant Benzodiazepines Cannabinoid Carbamates Clinical Column English Forensic LC-MS/MS Opioid Oral fluid Posters Saliva Stimulants Tricyclic Antidepressant z-drugs

This poster evaluates 3 different sample preparation approaches (ISOLUTE SLE+, EVOLUTE EXPRESS ABN, EVOLUTE EXPRESS CX) for extraction of large multi-drug urine panels. Each approach is assessed in terms of suitability for extraction of analytes with different different properties (pka, LogP etc). ASMS 2017

11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 6-acetylmorphine (MAM) 7-Aminoclonazepam 96-well plate Alprazolam Amitriptyline Amphetamine Amphetamines Analgesics Barbiturates Benzodiazepines Benzoylecgonine Buprenorphine Butalbital Cannabinoid Carbamate hypnotic Carbamate muscle relaxant Carisoprodol Chlordiazepoxide Clinical Clonazepam Cocaine Cocaines Codeine Diazepam Dihydrocodeine (DHC) Drugs of Abuse EDDP English Fentanyl Forensic Gabapentin Hydrocodone Hydrolysed urine Hydromorphone Ketamine LC-MS/MS Lorazepam MDMA Meperidine Meprobamate Methadone Methamphetamine Morphine N-des-tapentadol Naloxone Norbuprenorphine Nordiazepam Norfentanyl Norhydrocodone Norketamine Normeperidine Nortryptiline Opiates Opioid Oxazepam Oxycodone Oxymorphone Pain Management Phencyclidine Phenobarbital Posters Pregabalin Ritalinic acid Secobarbital Tapentadol Temazepam Tramadol Urine Zolpidem Zolpidem-phenyl-4-COOH z-drugs α-OH alprazolam