Pain management therapy warrants constant monitoring of therapeutic levels of prescribed drug levels in patient urine samples. The number of samples being submitted for analysis has increased dramatically in the last 10 years with improvements in high throughput automated screening capabilities. Patient samples analysis is complicated by the need for an effective sample preparation methodology that can extract target analytes from complex matrices with good efficiency. Further complicating the process is the need to enzymatically hydrolyse the glucoronidated metabolites prior to extraction from the urine matrix. A fully automated sample preparation process using a TECAN Freedom EVO® 100 was designed to incorporate both the enzymatic hydrolysis and subsequent sample preparation assay as one continuous workflow. Supported Liquid Extraction (ISOLUTE SLE+) which offers an efficient alternative to traditional liquid-liquid extraction (LLE) and solid phase extraction (SPE) techniques was used to extract a suite of pain management drugs from spiked urine samples. A recovery and quantitation assay was run on the TECAN Freedom EVO® 100 using mock patient samples to demonstrate utility of automation process. MSACL, Pain Management, Biotage, SPE, SLE, LLE, Supported Liquid Extraction, Drugs, MSACL, San Diego, 2013

6-acetylmorphine (MAM) 96-well plate Benzodiazepines Clinical Clonazepam Codeine Diazepam Dihydrocodeine (DHC) Drugs of Abuse Flunitrazepam Hydrocodone Hydrolysed urine Hydromorphone LC-MS/MS Methadone Methamphetamine Morphine Nitrazepam Opiates Oxycodone Oxymorphone Posters Urine

The ability to extract a broad range of different drugs from a biological matrix allows for the expedited analysis of a patient sample using LC-MS/MS. Typically small molecules are extracted from matrices like urine based on their polarities. A fast and reliable sample preparation method that could be implemented to extract drugs of different polarities from urine could be used as a screening tool to quickly identify the presence of illicit drugs in patient samples using LC-MS-MS. This poster demonstrates the utility of supported liquid extraction for the extraction of over 30 different acidic, basic and neutral drugs in urine prior to LC-MS/MS. MSACL 2016

11-hydroxy- Δ9-tetrahydrocannabinol (THC-OH) 11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 6-acetyl codeine 96-well plate Acidic Alprazolam Amphetamine Barbiturates Basic Benzodiazepines Benzoylecgonine Buprenorphine Butalbital Clinical Clonazepam Codeine Dextromethorphan Diazepam Drugs of Abuse EDDP English Fentanyl Flunitrazepam Forensic Hydrocodone Hydrolysed urine Hydromorphone LC-MS/MS MDEA Morphine Naltrexone Neutral Nitrazepam Norbuprenorphine Norcodeine Norfentanyl Opiates Oxazepam Oxycodone Oxymorphone Pain Management Pentazocine Pentobarbital Phenobarbital Posters Propoxyphene Secobarbital THC THC metabolites THC-COOH Temazepam Δ9-tetrahydrocannabinol (THC)

This poster describes a supported liquid extraction method for extraction of a class of novel psychoactive substances (NBOMes) with analysis using laser diode thermal desorption (LDTD) MS/MS. Samples are oral fluid collected using Salivettes. TAMS 2014

25B-NBOMe 25C-NBOMe 25D-NBOMe 25E-NBOMe 25H-NBOMe 25I-NBOMe 25N-NBOMe 25T2-NBOMe 96-well plate Drugs of Abuse English Forensic LDTD-MS/MS Legal highs NBOMes Oral fluid Phenethylamines Posters

This application note describes the extraction of acidic, basic, and neutral drugs from urine for screening purposes using ISOLUTE® SLE+ supported liquid extraction plates prior to LC-MS/MS analysis.

11-hydroxy- Δ9-tetrahydrocannabinol (THC-OH) 11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 6-acetyl codeine 96-well plate Acidic Alprazolam Amphetamine Application Notes Barbiturates Basic Benzodiazepines Benzoylecgonine Buprenorphine Butalbital Clinical Clonazepam Codeine Dextromethorphan Diazepam Drugs of Abuse EDDP English Fentanyl Flunitrazepam Forensic Hydrocodone Hydrolysed urine Hydromorphone LC-MS/MS MDEA Naltrexone Neutral Nitrazepam Norbuprenorphine Norcodeine Norfentanyl Normeperidine Opiates Oxazepam Oxycodone Oxymorphone Pain Management Pentazocine Pentobarbital Phenobarbital Propoxyphene Secobarbital THC metabolites Temazepam Urine Δ9-tetrahydrocannabinol (THC)

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

Most drugs, both licit and illicit, are excreted in urine as glucuronide conjugates. Hydrolysis using beta-glucuronidase converts the glucuronidated metabolites back to their “free” or non-conjugated form, increasing sensitivity for LC-MS/MS analysis. Hydrolysis using red abalone, abalone, and recombinant beta-glucuronidase enzymes was evaluated using an in-well hydrolysis plate to determine which provided the most efficient hydrolysis of glucuronide metabolites without affecting overall recovery of nonconjugated compounds. A glucuronide control containing 8 glucuronide compounds was used to determine the extent of hydrolysis that occurred. A non-conjugated control containing 96 licit and illicit drugs of abuse was evaluated to determine if signal suppression occurred as a result of enzyme hydrolysis. ASMS 2017

96-well plate Clinical Codeine Drugs of Abuse English Forensic Hydrolysed urine Hydromorphone LC-MS/MS Lorazepam Morphine Norbuprenorphine Oxazepam Oxymorphone Pain Management Posters Urine

The method described in this application note achieves high recoveries of THC and an extended suite of common metabolites in oral fluid from Quantisal (Immunalysis) oral fluid collection devices. This application note describes effective and efficient ISOLUTE SLE+ protocols optimized for sample loading volumes of either 300 μL or 800 μL. The simple sample preparation procedure delivers clean extracts and analyte recoveries greater than 64% with RSDs of <10% for all analytes.

11-hydroxy- Δ9-tetrahydrocannabinol (THC-OH) 11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) 11-nor-9-carboxy- Δ9-tetrahydrocannabivarin (THC-V-COOH) 11-nor-9-carboxy- Δ9tetrahydrocannabinol glucuronide (THC-COOH-g 11-nor-9-carboxy-Δ9-THC glucuronide Application Notes Cannabidiol Cannabigerol Column Drugs of Abuse English Forensic LC-MS/MS Oral fluid Saliva THC THC metabolites THC-COOH Δ9-tetrahydrocannabinol (THC) Δ9-tetrahydrocannabivarin (THCV)

When analyzing human urine for drugs of abuse, one of the most common tests is for the cocaine metabolite Benzoylecgonine (BZE). A contract laboratory has automated this labor intensive procedure using the RapidTrace.

Application Notes Benzoylecgonine Column Drugs of Abuse Forensic GC-MS RapidTrace Urine

This poster presents a method for detection of approximately 100 drugs and metabolites in urine using mixed-mode polymeric cation exchange solid phase extraction (SPE). Four different glucuronidase enzymes and several incubation times and temperatures were evaluated to determine optimum hydrolysis conditions for seven commonly detected glucuronide metabolites. Samples were subjected to offline hydrolysis and SPE (EVOLUTE® EXPRESS CX, Biotage, Charlotte, NC) and results compared to an SPE plate that incorporates in-well hydrolysis (EVOLUTE HYDRO CX, Biotage). MSACL 2018, Palm Springs, CA

96-well plate Clinical Drugs of Abuse English Forensic Hydrolysed urine LC-MS/MS Pain Management Posters Urine

This poster compares two different procedures for quantification of drugs of abuse from postmortem brain tissue.

7-Aminoflunitrazepam Amphetamine Benzoylecgonine Brain Cocaine Codeine Column Diazepam Drugs of Abuse English Forensic GC-MS LC-MS/MS Methadone Morphine Posters

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 that a large urine panel, comprised of 43 DOAs, from multiple drug classes, can be simultaneously screened by mixed-mode cation exchange SPE (using EVOLUTE EXPRESS CX 96 well plates) despite their disparate intermolecular traits, by thoughtfully selecting appropriate organic wash and elution conditions that simultaneously enable sample isolation and detection along with minimizing sample matrix effects. The extraction method is automated using the Biotage® Extrahera™ Automated sample Preparation Platform. MSACL 2017, Palm Springs SOFT 2017, Boca Raton

96-well plate Benzodiazepines Carbamates Carisoprodol Clinical Drugs of Abuse English Extrahera Forensic Gabapentin Hydrolysed urine LC-MS/MS Meprobamate Opiates Pain Management Posters Pregabalin Stimulants Urine

In postmortem cases, where drugs or pesticides have been used for their poisonous properties, traditional matrices such as urine and whole blood may be inappropriate for qualitative and quantitative analysis. As the site of metabolism for most drugs and toxins, the liver may provide more insight to cause of death than other bodily fluids. This poster describes the use of ISOLUTE SLE+ supported liquid extraction columns to extract a range of drug and pesticide classes form homogenised liver using a simple, streamlined workflow. SOFT 2016

Alprazolam Amphetamine Atrazine Bead Ruptor Bendiocarb Biphenthrin Butabarbital Chlorothalonil Clinical Clonazepam Cocaine Column Diazepam Drugs of Abuse English Estazolam Forensic GC-MS Ketamine Liver MDMA Malathion Methadone Methamphetamine Midazolam Nitrazepam Pesticides Phenobarbital Posters Propanil Secobarbital THC Triazolam

This poster demonstrates the extraction of a range of drugs of abuse from oral fluid collection devices using supported liquid extraction suitable for UPLC-MS/MS analysis. Unlike some sample preparation techniques, SLE allows for the simultaneous extraction of cross-functional analytes in a single extraction protocol without forfeiting extract cleanliness. The target analyte list includes benzodiazepines, z drugs, amphetamines, cathinones, opiates, cocaine, buprenorphine, PCP, THC-COOH, fentanyl and ketamine. TIAFT 2015

2-OH-Et-Flurazepam 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam Alprazolam Amphetamine Amphetamines Benzodiazepines Benzoylecgonine Bromazepam Buprenorphine Cathinones Clonazepam Cocaine Cocaines Codeine Column Diazepam Dihydrocodeine (DHC) Drugs of Abuse EDDP English Estazolam Fentanyl Flunitrazepam Flurazepam Forensic Hydrocodone Hydromorphone LC-MS/MS Lorazepam MDA MDMA Mephedrone Methadone Midazolam Morphine Norbuprenorphine Nordiazepam Norfentanyl Norketamine Opiates Oral fluid Oxazepam Oxycodone Oxymorphone Pain Management Phencyclidine (PCP) Posters Temazepam Triazolam Zaleplone Zolpidem Zopiclone α-OH Triazolam α-OH alprazolam

This poster demonstrates a fast, reliable protocol to extract multiple drug of abuse panels from whole blood using a common supported liquid extraction methodology. This benefits laboratory workflow where multiple assays are run each day, saving both worker hours and consumable costs. SOFT 2015 ASMS 2016 TIAFT 2016

2-Hydroxyethylflurazepam 2C-B 5-APB 6-APB 6-acetylmorphine (MAM) 7-Aminoclonazepam 7-Aminoflunitrazepam Alprazolam Amobarbital Amphetamine Anhydroecgonine Methyl Ester AEME BZP Benzoylecgonine Bromazepam Butabarbital Butalbarbital Butylone Clinical Clonazepam Cocaethylene Cocaine Cocaines Codeine Column Diazepam Dihydrocodeine (DHC) Drugs of Abuse Ecgonine Methyl Ester EME English Estazolam Ethylone Flunitrazepam Flurazepam Forensic GC-MS Hexobarbital Hydrocodone Hydromorphone Lorazepam MDA MDEA MDMA MDPV Mephedrone Methamphetamine Methcathinone Methedrone Methylone Midazolam Morphine Naphyrone Nitrazepam Nordiazepam Oxazepam Oxycodone Oxymorphone Pentobarbital Phenobarbital Posters Secobarbital TFMPP Temazepam Triazolam Whole blood mCPP pMMA α-OH Triazolam α-OH alprazolam

This poster evaluates the extraction of a range of drugs of abuse from hydrolyzed and nonhydrolyzed urine using a novel flow through scavenging product, ISOLUTE® HYDRO DME+. Matrix component removal in terms of creatinine and urea, salt residue, pigmentation associated with urobillin content and protein removal will be demonstrated. SOFT 2018

96-well plate Barbiturates Benzodiazepines Clinical Cocaines Column Drugs of Abuse English Forensic LC-MS/MS Opiates Opioid Posters

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

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 poster describes a simple supported liquid extraction (SLE) method for extraction of THC and metabolites from whole blood and urine, suitable for GC-MS analysis. SOFT 2015

11-hydroxy- Δ9-tetrahydrocannabinol (THC-OH) 11-nor- Δ9-carboxy tetrahydrocannabinol (THC-COOH) Column Drugs of Abuse English Forensic GC-MS Posters THC THC-COOH Urine Whole blood Δ9-tetrahydrocannabinol (THC)