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

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 Hormone Hormones LC-MS/MS Plasma Serum Steroid Steroid Hormones Steroid metabolite Steroids Urine

This product sheet compares automated sample preparation using the Biotage®Extrahera™ to an equivalent manual method utilizing a vacuum manifold. A selection of beta blocker drugs were extracted from pooled stripped plasma using a supported liquid extraction procedure.

96-well plate Alprenolol Beta blockers Clinical English Extrahera Forensic LC-MS/MS Metoprolol Pharma Pindolol Plasma Product Notes Propranolol β-blockers

Automated sample preparation using the Biotage®Extrahera™ was compared to an equivalent manual method utilizing a vacuum manifold. Analytes were extracted from pooled stripped plasma using a supported liquid extraction procedure.

25-OH Vitamin D2 25-OH Vitamin D3 25-hydroxy-vitamin D 96-well plate Clinical English Extrahera LC-MS/MS Plasma Product Notes Vitamin D

This document compares automated sample preparation using the Biotage® Extrahera™ to an equivalent manual method utilizing a vacuum manifold. A selection of non-steroidal anti-inflammatory drugs (NSAIDS) were extracted from pooled stripped plasma using a supported liquid extraction procedure.

96-well plate Clinical English Extrahera Forensic Indomethacin Ketoprofen LC-MS/MS NSAIDs Pharma Plasma Product Notes Sulindac

This poster compares the use of anion and cation exchange SPE approaches for the extraction of these important clinical biomarkers. MSACL Europe 2014

96-well plate Clinical English LC-MS/MS Plasma Posters Reverse Tri-iodothyronine (rT3) Thyroid Hormone Thyroxine (T4) Tri-iodothyronine (T3) rT3

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 evaluates the performance of a novel 96-well filter plate (ISOLUTE PLD+ Protein and Phospholipid Removal Plate) for the simultaneous removal of proteins and phospholipids from serum samples prior to LC-MS/MS analysis. ASMS2014

96-well plate Acidic Amitriptyline Analgesics Atenolol Basic Bretylium Clinical English Fluoxetine Forensic Ketoprofen LC-MS/MS Metoprolol Mianserin NSAIDs Naltrexone Pharma Plasma Posters Procainamide Quinidine Ranitidine Salbutamol Serum Sulindac Tricyclic Antidepressant β-blockers

This poster presents the ISOLUTE PLD+ Protein and Phospholipid removal plate, highlighting its ease of use and excellent performance with respect to sample clean up, analyte recovery and elimination of back pressure build up in the UPLC system. IMSC 2014

96-well plate Acetaminophen Acidic Amitriptyline Atenolol Basic Bretylium Brompheniramine Clinical English Fluoxetine Forensic Ketoprofen LC-MS/MS Metoprolol Mianserin Naltrexone Neutral Pharma Pharmaceuticals Plasma Posters Procainamide Quinidine Ranitidine Salbutamol Sulindac p-toluamide

Miniaturisation within various fields of analytical chemistry is not a new phenomenon. Early phase small animal drug trials have largely been the driver due to limited sample sizes available. However, this trend is gaining popularity in forensic/clinical toxicology with respect to alleviating patient discomfort, particularly in paediatrics. Increased LC-MS/MS sensitivity has reignited this focus area. This poster will evaluate a novel low-volume 96-well solid phase extraction (SPE) format and potential application to forensic and clinical toxicology. ASMS, 2020.

6-MAM Alprazolam Amphetamine Buprenorphine Clinical Clonazepam Cocaine Codeine Diazepam Drugs of Abuse EDDP Estazolam Fentanyl Flunitrazepam Flurazepam Forensic Hydrocodone Hydromorphone Ketamine Lorazepam Lysergic acid diethylamide (LSD) MDA MDEA MDMA Mephedrone Methadone Methamphetamine Midazolam Mikro plate Morphine Nitrazepam Norbuprenorphine Nordiazepam Norfentanyl Norketamine Oxazepam Oxycodone Phencyclidine (PCP) Plasma Posters Temazepam Triazolam Zolpidem Zopiclone

Matrix components, particularly salts, proteins and phospholipids, can lead to ion suppression resulting in inaccurate quantitation and reduced detection limits. Resin-based mixed-mode cation exchange SPE sorbents are widely used for the extraction of basic compounds from biological fluids. The dual retention mechanism allows a two stage interference wash protocol, which results in extremely clean extracts. This poster will investigate the performance of EVOLUTE™ CX for the extraction of a wide range of basic drugs from plasma, showing high analyte recovery and advanced extract cleanliness. The analyte suite was selected to cover a variety of basic analytes with wide ranging polarities (LogP values). Extract cleanliness experiments were performed showing overall ion suppression, then individual matrix components examined in terms of protein and phospholipid removal.

Plasma Posters

Sample preparation is generally regarded as a pain point for many labs as it can be laborious, time consuming and relatively expensive. In a high throughput setting performing offline chemistry also leads to increases in sample turnaround time and reporting. This poster evaluates simplified sample preparation workflows using optimized sample hold-up technology allowing chemistry in-situ. Reduced labour, extraction complexity, processing time, solvent usage and resultant instrument contamination and downtime can offset upfront costs associated with sample preparation. ASMS 2019

96-well plate Clinical Drugs of Abuse English Forensic Hydrolysed urine LC-MS/MS Over the counter pharmaceuticals Pharmaceuticals Plasma Posters Urine

This application note describes the use of the ISOLUTE® PLD+ Protein and Phospholipid Removal Plate for clean-up of a range of acidic, basic and neutral drugs from plasma. ISOLUTE® PLD+ Protein and Phospholipid Removal Plates are suitable for clean-up of a range of analytes with widely differing functionality and polarity characteristics from plasma, giving high recoveries, good reproducibility and excellent extract cleanliness.

96-well plate Acetaminophen Acidic Amitriptyline Application Notes Atenolol Basic Bretylium Brompheniramine Clinical English Fluoxetine Forensic Ketoprofen LC-MS/MS Metoprolol Mianserin NSAIDs Naltrexone Neutral Pharma Plasma Procainamide Quinidine Ranitidine Salbutamol Sulindac Tricyclic Antidepressant p-toluamide β-blockers

This application note describes a solid phase extraction protocol for the extraction of three catecholamine metabolites (vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid) from plasma prior to LC-MS/MS detection. The method described in this application achieves high reproducible recoveries and excellent linearity (>0.99) in the range 10-200 ng/mL.

5-hydroxyindoleacetic acid (5-HIAA) 96-well plate Acidic catecholamine metabolites Application Notes Clinical English Homovanillic acid (HVA) LC-MS/MS Plasma Vanillylmandelic acid (VMA)

This application note describes a supported liquid extraction protocol for the extraction of three acidic catecholamine metabolites (vanillylmandelic acid, homovanillic acid and 5-hydroxyindoleacetic acid) from plasma prior to LC-MS/MS detection. The method described in this application note achieves high reproducible recoveries (>80%, RSD < 5%) for these analytes in plasma with linearity > 0.995 in the range 2–200 ng/mL.

5-hydroxyindoleacetic acid (5-HIAA) 96-well plate Acidic catecholamine metabolites Application Notes Clinical English Homovanillic acid (HVA) LC-MS/MS Plasma Vanillylmandelic acid (VMA)

This Application Note describes the extraction of a variety of basic drugs from plasma using EVOLUTE® CX mixed-mode resin-based SPE. The analyte suite includes basic drugs with wide ranging pKa and logP values. Basic Drugs, Biotage, Ion Exchange, EVOLUTE, Pharmaceuticals

Amitriptyline Application Notes Atenolol Basic Clinical Fluoxetine LC-MS/MS Metoprolol Mianserin Naltrexone Plasma Procainamide Quinidine Ranitidine Salbutamol

Catecholamine metabolites vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5-HIAA) are biomarkers for neuroblastoma and catecholaminesecreting tumors. This poster describes optimization of the method development process to maximize analyte sensitivity in the extraction and quantitation of these metabolites from plasma. Parameters investigated include MRM transitions, chromatography and sample preparation protocols. MSACL EU 2017

5-hydroxyindoleacetic acid (5-HIAA) 96-well plate Acidic catecholamine metabolites Catecholamines Clinical English Homovanillic acid (HVA) LC-MS/MS Plasma Posters Vanillylmandelic acid (VMA)

Catecholamine metabolites vanillylmandelic acid (VMA), homovanillic acid (HVA) and 5-hydroxyindoleacetic acid (5 HIAA) are biomarkers for neuroblastoma and catecholamine-secreting tumors. This poster presents optimization of the method development process to maximize analyte sensitivity in the extraction and quantitation of these metabolites from plasma. Sample preparation protocols using solid phase extraction (EVOLUTE EXPRESS ABN and AX) and supported liquid extraction (ISOLUTE SLE+) are presented. MSACL NA 2018 Palm Springs, CA

5-hydroxyindoleacetic acid (5-HIAA) 96-well plate Acidic catecholamine metabolites Catecholamines Clinical English Forensic Homovanillic acid (HVA) LC-MS/MS Plasma Posters Vanillylmandelic acid (VMA)

The following methods have been developed and optimized for the extraction of chloramphenicol from a variety of sample matrixes including milk, plasma, honey, urine, and shrimp/prawns for subsequent LC-MS/MS analysis. The methods are highly reproducible and offer low limits of detection.

Antibiotics Application Notes Chloramphenicol Column Food Honey LC-MS/MS Milk Plasma Prawns Shellfish Shrimps Urine Veterinary drugs

This Application Note highlights the use of ISOLUTE® SLE+ supported-liquid extraction plates for the extraction of corticosteroids from human plasma, specifically Triamcinolone, Prednisolone, Hydrocortisone, Prednisone, Betamethasone, Cortisone, Dexamethasone, Flumethasone, Corticosterone, Beclomethasone, Triamcinolone, Fluocinolone, Budesonide 1, Budesonide 2 and 5-Pregnen-3β−ol-20-one.

5-pregnen-3β-ol-20-one Application Notes Beclomethasone Betamethasone Budesonide Structural Isomer 1 Budesonide Structural Isomer 2 Clinical Corticosteroids Corticosterone Cortisone Dexamethasone Flumethasone Fluocinolone Acetonide Hormone Hormones Hydrocortisone LC-MS/MS Plasma Prednisolone Prednisone Steroid Steroid Hormones Steroids Triamcinolone Triamcinolone Acetonide