This application note describes the extraction of nine phthalate metabolites from human urine using ISOLUTE® ENV+ solid phase extraction columns.

Application Notes Biomarker Clinical Column English LC-MS/MS Mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) Mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) Mono(2-ethylhexyl) phthalate (MEHP) Monobenzyl phthalate (MBzP) Monobutyl phthalate (MBP) Monoethyl phthalate (MEP) Monohexyl phthalate (MHxP) Monoisononyl phthalate (MiNP) Monomethyl phthalate (MMP) Phthalate Metabolites Phthalates Urine

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

This application note describes the extraction of ethyl glucuronide and ethyl sulfate from human urine using ISOLUTE® NH2 solid phase extraction columns. The method has been applied real patient samples that had been previously analyzed with a validated referee method. The results of the orthogonal measurements agreed, to provide similar diagnostic values.

Alcohol metabolite Application Notes Clinical Column Ethyl Sulfate (EtS) Ethyl glucuronide (EtG) Forensic LC-MS/MS Urine

This report details a “load, wash, elute” weak cation exchange solid phase extraction procedure amenable to both plasma and urine samples. The extracts are subsequently injected into an LC-MS/MS system. The preliminary sample preparation method was developed at the Biotage US Applications lab (Charlotte, NC).The method was then transferred to Ionics (Bolton, ON, Canada) to facilitate the nmole/L measurements of the selected biomarkers by laminar flow tandem mass spectrometry. The SPE-LC-ESIMS/ MS method parameters were first optimized using pooled mixed gender plasma. A set of patient samples (n=32) was later supplied by the Mayo Clinic (Rochester, MN) that had previously been analyzed by a validated referee method. The population represented measured values across a range of clinical relevance. AACC 2014

96-well plate Catecholamines Clinical English LC-MS/MS Metanephrine Normetanephrine Plasma Posters Urine

This poster describes the use of ISOLUTE ENV+ columns to extract important phthalate metabolites from human urine. MSACL Europe 2014

Clinical Column LC-MS/MS Mono(2-ethyl-5-carboxypentyl) phthalate (MECPP) Mono(2-ethyl-5-hydroxyhexyl) phthalate (MEHHP) Mono(2-ethylhexyl) phthalate (MEHP) Monobenzyl phthalate (MBzP) Monobutyl phthalate (MBP) Monoethyl phthalate (MEP) Monohexyl phthalate (MHxP) Monoisononyl phthalate (MiNP) Monomethyl phthalate (MMP) Phthalate Metabolites Phthalates Posters Urine

The objective was to develop a GC-MS assay for the determination of free benzodiazepines using Supported Liquid Extraction (SLE). The SLE extraction mechanism is very efficient, delivering higher analyte recoveries and cleaner extracts than equivalent LLE methods. ISOLUTE, SLE, SLE+, Supported Liquid Extraction, Benzodiazepines, Forensic, Drugs, DOA, Drugs of Abuse, SOFT,

4-Hydroxynordiazepam 7-Aminoclonazepam 7-Aminoflunitrazepam 7-Aminonitrazepam Benzodiazepines Bromazepam Column Demethylflunitrazepam Flurazepam Forensic GC-MS Hydroxyalprazolam Hydroxymidazolam Hydroxytriazolam Midazolam Nitrazepam Posters Urine

The use of LC/MS analysis in the clinical lab has increased exponentially over the last 10 years. Modern mass spectrometers are extremely sensitive allowing low level detection of many target analytes. However, this sensitivity can come at a price, in that levels of contamination not previously detected with less sensitive instruments can now have larger impact on analysis. The complexity of common matrices such as plasma/serum and urine while presenting different challenges can have a marked influence on method performance. As a result sample preparation is an extremely important part of the process in order to provide robust methods. This seminar focuses on some of the method development challenges our lab faced when looking at two clinical assays: Endogenous steroid hormone extraction from serum, and catecholamine extraction from plasma and urine. Particular emphasis is placed on the various sample preparation options we looked at for the extraction of these analytes. During optimization of the extraction process we investigated analyte recovery, co-extractable matrix components, HPLC column degradation, calibration curve performance and limits of quantitation. MSACL US 2018 Breakfast Seminar

96-well plate Catecholamines Clinical English LC-MS/MS Plasma Serum Steroid Hormones Steroid metabolite Urine

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

This application note describes the fully-automated extraction of opiates from acid-hydrolyzed urine and non-hydrolyzed urine prior to GC/MS analysis. The methods were automated using Biotage® Extrahera™ configured for use with ISOLUTE® SLE+ supported liquid extraction columns. Using the Biotage® Extrahera™, 24 hydrolyzed samples were extracted in under 31 minutes and 24 non-hydrolyzed samples were extracted in under 36 minutes. The limits of quantitation meet or exceed the sensitivity requirements set by SAMHSA and EWDTS for workplace testing applications.

6-acetylmorphine (MAM) 96-well plate Application Notes Codeine Dihydrocodeine (DHC) English Extrahera Forensic GC-MS Hydrocodone Hydrolysed urine Hydromorphone Morphine Opiates Opiates Oxycodone Oxymorphone Urine

This application note describes the extraction of a range of SPICE drugs and metabolites in urine which are typically screened in forensic toxicology panels using ISOLUTE® SLE+ in a 96-well plate format. Both manual (Biotage Pressure+ 96) and automated (TECAN Freedom EVO® 100) processing conditions are described.

96-well plate Application Notes English Forensic JWH 018-N-(4-hydroxypentyl) JWH-018 JWH-018 JWH-018-(5-hydroxypentyl) JWH-018-N-5-(pentanoic acid) JWH-073 JWH-073-N-(3-hydroxybutyl) JWH-200 JWH-250 JWH-250-N-(5-hydroxypentyl) LC-MS/MS Synthetic Cannabinoids (SPICE) TECAN UR-144 UR-144-(5-hydroxypentyl) UR-144-(pentanoic acid) UR144-(5-chloropentyl) Urine XLR-11

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

An extraction protocol using a 10 mg mixed-mode cation exchange sorbent (EVOLUTE® EXPRESS CX) was developed and various sample sizes were assessed to determine the optimal sample volume for a 98-compound DOA panel. A method involving microelution off of the sorbent requiring no evaporation or reconstitution steps were also developed. ASMS 2019

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

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

This poster demonstrates two fast and robust methods for the extraction of buprenorphine and norbuprenorphine from urine, oral fluid and whole blood. SOFT 2015

96-well plate Buprenorphine Clinical Column English Forensic LC-MS/MS Norbuprenorphine Oral fluid Pharmaceuticals Posters Saliva Urine Whole blood

Buprenorphine and Norbuprenorphine are typically problematic for analysis due to analyte stability issues during sample preparation. This poster will demonstrate two fast and robust methods for the extraction of buprenorphine and norbuprenorphine in urine (using EVOLUTE EXPRESS CX), oral fluid (using EVOLUTE EXPRESS CX) and whole blood (using ISOLUTE SLE+). MSACL 2016

96-well plate Buprenorphine Clinical Column English Forensic LC-MS/MS Norbuprenorphine Oral fluid Pain Management Posters Saliva Synthetic urine Urine Whole blood

Endogenous phospholipids present in biological fluids are a major problem in LCMS/ MS analysis as they are often very difficult to remove during sample preparation. When phospholipids are not removed, they retain very strongly on reversed phase analytical columns. If high organic (end of run) washes are not incorporated into the LC methods these matrix components may elute in subsequent analyses causing regions of suppression/enhancement leading to inaccurate quantitation. This poster evaluates the use of polymer-based solid phase extraction (SPE) sorbents, incorporating hydrophobic and various mixedmode retention mechanisms to address the problems associated with phospholipid removal. Phospholipid, EVOLUTE, STRATA X, OASIS, WATERS, AX, WAX, CX, WCX, ABN, ASMS 2011

Application Notes Aqueous Biological fluids Column LC-MS/MS Plasma RapidTrace Serum Urine Whole blood

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