3_BAS_2024-2025_SamplePreparation_PartII.pdf

Bioanalytical Sciences Sample collection, handling & preparation Dr. Isabelle Kohler | 10th September 2024 1 Bioanalytical Sciences | [email protected]  Bioanalytical workflow | Lectures II + III Lecture I...

Bioanalytical Sciences Sample collection, handling & preparation Dr. Isabelle Kohler | 10th September 2024 1 Bioanalytical Sciences | [email protected]  Bioanalytical workflow | Lectures II + III Lecture I Forensic Toxicology Therapeutic Drug Monitoring Study design & Sample handling Analytes Analytes Data analysis & sampling & preparation separation detection interpretation Doping Control Clinical Toxicology & Chemistry Lecture II Lecture IV Lecture VI Biomarker Discovery Lecture III Lecture V Lecture VII Lecture VIII Lecture IX Lecture X 2 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sampling Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Liquid-liquid extraction Solid-phase extraction 3 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sampling Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Risks Storage of samples 4 Bioanalytical Sciences | [email protected]  Working with biological samples | Safety HCV HIV Acute: «flu-like» symptoms Acute: «flu-like» symptoms Hepatitis C HIV Chronic HCV infection, cirrhosis (0.2-0.4%) Chronic: Immunodeficience (ca. 1%) Antiviral therapy very expensive Antriretroviral therapy, no cure Hepatitis B HBV (4-5%) Acute: «flu-like» symptoms. Chronic liver infection, cirrhosis, liver cancer No cure 5 Bioanalytical Sciences | [email protected]  HBV, HCV and HIV | Risks and how to lower them Blood samples obtained from biobanks? 10’ 6 Bioanalytical Sciences | [email protected]  HBV, HCV and HIV | Vaccination Hepatitis C HIV (ca. 1%) (0.2-0.4%) Vaccine available? HIV  NO HCV  NO HBV  YES Check your vaccination record Hepatitis B Discuss with your supervisor (4-5%) (early enough! > 1 month before starting) The World Health Organization (WHO) recommends… “Healthcare workers and others who may be exposed to blood and blood products through their work should be vaccinated” 7 Bioanalytical Sciences | [email protected]  Biosamples| Storage and degradation Interferences Analyte decomposition Biosamples (all) Micro-organisms Rule of thumb: -80˚C Storage at -80˚C as fast as possible after collection (snap freeze the sample in liquid nitrogen) Temperature -20˚C: some compounds are stable, some not (lipids) Prepared samples: OK at -20˚C for few days Adjuvants Method development includes stability tests 8 Bioanalytical Sciences | [email protected]  Biosamples| Storage and degradation Interferences Analyte decomposition Biosamples (all) Micro-organisms If stability studies show that analytes are not stable: addition of anti-oxidants Vitamin A Temperature Vitamin E Butylated hydroxytoluene (BHT) Adjuvants 9 Bioanalytical Sciences | [email protected]  Biosamples| Storage and degradation Interferences Analyte decomposition Biosamples (all) Micro-organisms As a colleague of mine used to say… “Would you even consider eating meatballs that have been stored for 5 years in your freezer? No? So why Temperature would you even consider analyzing samples which have been stored for so long in a freezer?” Adjuvants 10 Bioanalytical Sciences | [email protected]  Storage and degradation | an example Geometric mean ratio (y-axis) = ratio of the plasma metabolite level to the level seen at 0 min at room temperature (25˚C) or at 1 h at cold temperature (4˚C). Nishiumi et al., J Biosci Bioeng 125 (2018) 613 11 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO How to handle urine samples? I am interested in analysing estradiol glucuronide in the urine sample of one of my colleagues. My colleague gives me a fresh urine sample (just collected), that I put on the lab bench (temperature 25˚C). Right at that time, I get an important call and totally forget about the urine sample. I am back in the lab the day after (+ 24h), and wonder whether it still makes sense to analyse this sample. 12 Does is make sense to analyse this sample? Can I trust the data I will get???  Q&A INTERMEZZO How to handle urine samples? Room temperature Glucuronidases from the microorganisms growing in the urine sample will rupture the bond estradiol – glucuronide; releasing the free from. 13 Urine is sterile but only in the bladder!  Q&A INTERMEZZO How to handle urine samples? Store (and handle) your samples correctly Always take the time to think and define what is needed for your sample, depending on the application In case of doubts: -80˚C as fast as possible after sample collection 14  Q&A INTERMEZZO How to handle and store samples? 15  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Liquid-liquid extraction Solid-phase extraction 16 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Govert will appear on the slides containing information Solid-phase you will further discuss during extraction his lecture 17 Bioanalytical Sciences | [email protected]  Sample preparation | Why? Plasma Serum Urine Saliva Volume 1-10 1-10 5-100 0.1-3 [ml] pH 7.4 7.4 3-8 6.7 Water 91.5 91.5 98.0 98.0 [%] Proteins 7.5 7.7 0.6 0.3 [g/100ml] 18 Bioanalytical Sciences | [email protected]  Sample preparation | Plasmatic proteins 19 Bioanalytical Sciences | [email protected]  Sample preparation | Why? Elimination of biological elements not compatible with the analytical system Pressure Release the free fraction if bound to Free from Bound form plasmatic proteins Elimination of possible interferents Sample concentration (increase sensitivity) 20 Bioanalytical Sciences | [email protected]  Sample preparation | How? Sample Selective procedures Non-selective procedures “sample preparation” “sample pre-treatment” LLE SPE Dilution PP 21 Bioanalytical Sciences | [email protected]  Protein precipitation (PP) | Procedure 1. PP agent (1000 µL) 2. plasma (500 µL) 5. Collection of the supernatant Non-selective technique of choice 3. agitation 6. 2-20 µL for blood-based matrices 4. centrifugation injection ! Remember that urine (and CSF) can also contain proteins! 22 Bioanalytical Sciences | [email protected]  PP | Precipitating agents Organic solvents Creation of new Methanol electrostatic Ethanol interactions Acetonitrile between proteins Acids Perchloric acid Modification of the structure III via Trichloroacetic acid electrostatic Acetic acid interactions m-phosphoric acid Salts Modification Ammonium sulfates of surface tension or complexation tension Copper sulfates Zinc (hydroxide - sulfate) 23 Bioanalytical Sciences | [email protected]  Solid-phase extraction (SPE) | Principle Condition Load Wash & Elute Reservoir Sorbent 24 Bioanalytical Sciences | [email protected]  Solid-phase extraction (SPE) | Extraction supports 1. Adsorbant Retention on the surface with weak interactions Large specific area is required Silica gel, alumina, etc 2. Ion exchanger Strong electrostatic interaction Cation or anion exchanger Low flow rate is required! (« slow » interaction) Cation exchange = retains cations Anion exchange = retains anions 25 Bioanalytical Sciences | [email protected]  Solid-phase extraction (SPE) | Extraction supports 3. Partition Reversed phase mode for apolar compounds Normal phase mode for polar compounds Weak interactions 4. Mixed-mode Better selectivity with 2 mechanisms of interaction 26 Bioanalytical Sciences | [email protected]  SPE | Very popular in bioanalysis: mixed-mode phases 27 Bioanalytical Sciences | [email protected]  Sample preparation | Importance of the pH and pKa 28 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO SPE procedure | Extraction of MDMA and Methamphetamine (MA) in plasma I am in the lab and have received the mission to analyse MA and MDMA in plasma samples using LC-MS. I need to set up a sample preparation method to extract these compounds from plasma samples. What will my sample preparation procedure look like? 29  Extraction MA and MDMA from plasma Condition Load Wash & Elute 30 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma How do I proceed?? Which information do I need? pKa of the molecules? Charge of the molecule depending on the pH? LogP of the molecule? Type of sorbent? ( which type of interactions?) Conditioning of the sorbent? What do I need to do with the plasma before loading it onto the sorbent? Washing steps? Elution? 31 Bioanalytical Sciences | [email protected]  Extraction MA and MDMA from plasma How do I proceed?? Which information do I need? pKa of the molecules? Charge of the molecule depending on the pH? LogP of the molecule? Type of sorbent? ( which type of interactions?) Conditioning of the sorbent? What do I need to do with the plasma before loading it onto the sorbent? Washing steps? Elution? 32 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation Forensic Toxicology Therapeutic Drug Monitoring Sample handling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery Protein precipitation Liquid-liquid extraction Govert will save you if you haven’t understood the Solid-phase fundaments of LLE and SPE extraction 33 Bioanalytical Sciences | [email protected]  Bioanalytical Sciences Sample collection, handling & preparation: Microextractions Dr. Isabelle Kohler 34 Bioanalytical Sciences | [email protected]  Sample collection, handling and preparation 35 Bioanalytical Sciences | [email protected]  Learning objectives Take home message Be aware of the risks encountered when working with biosamples, and how to lower them Know which storage conditions are the most appropriate Understand the advantages of each type of matrix, and the information they can bring Understand the difference between plasma and serum Understand and be able to explain why we need to prepare the samples before analyzing them List the most widely-used sample preparation methods in bioanalysis Understand the principles behind PP and SPE Be able to explain the different steps occurring in SPE method and why we need each step Have understood how to optimize the SPE conditions (sorbent phase and solvent mixtures) depending on the pKa and LogP value of the analytes of interest Remember the rule of thumb “pH = pKa ± 2” Have understood what microextractions can bring in the field of green analytical chemistry 36 Bioanalytical Sciences | [email protected]  Bioanalytical workflow | Lecture IV Lecture I Forensic Toxicology Therapeutic Drug Monitoring Study design & Sample handling Analytes Analytes Data analysis & sampling & preparation separation detection interpretation Doping Control Clinical Toxicology & Chemistry Lecture II Lecture IV Lecture VI Biomarker Discovery Lecture III Lecture V Lecture VII Lecture VIII Lecture IX Lecture X 37 Bioanalytical Sciences | [email protected]