2_BAS_2024-2025_SamplePreparation.pdf

Bioanalytical Sciences Sample collection, handling & preparation Dr. Isabelle Kohler | 6th September 2024 1 Bioanalytical Sciences | [email protected]  Bioanalytical Sciences Sample collection, handling & preparation Reminder t...

Bioanalytical Sciences Sample collection, handling & preparation Dr. Isabelle Kohler | 6th September 2024 1 Bioanalytical Sciences | [email protected]  Bioanalytical Sciences Sample collection, handling & preparation Reminder to myself Dr. Isabelle Kohler | 6th September 2024 2 Bioanalytical Sciences | [email protected]  Group # Topic name Announcements 1 2 Post-mortem analysis of fentanyl analogues in vitreous humour Analysis of FA and FMA isomers in dried blood spots Groups are formed! 3 Post-mortem analysis of amphetamine-derivatives and metabolites in nails 4 Post-mortem analysis of nitazene and other new synthetic opioids in nails (new!) One new topic added 5 Chiral analysis of ketamine and derivatives in saliva 4-5 students per group You can start working! 6 Quantitative analysis of DMT in rat brain samples 7 Analysis of sulfate- and glucuronide-conjugated androgen steroids in urine for doping purpose Tutorials available 8 Quantitative analysis of benzofurans 4-APB, 5-APB, 6-APB and their derivatives in plasma How to prepare and present a poster 9 Workplace drug testing for cocaine and heroine consumption via oral fluid analysis How to make an awesome video 10 Development of a method for the TDM of antiretroviral HIV drugs in DBS How to prepare the methodology presentation 11 Monitoring and detecting the consumption of THC and synthetic cannabinoids in hair 12 Quantitative analysis of designer benzodiazepines in serum in the context of drug-facilitated sexual assault This afternoon: Self-study 13 Quantitative analysis of psilocybin, psilocin, 4-HIAA, and psilocin glucuronide in dried blood spots 14 Monitoring of cannabis, cigarette and alcohol (ab)use in breastfeeding mothers with breastmilk analysis 3 Bioanalytical Sciences | [email protected]  Bioanalytical workflow Forensic Toxicology Therapeutic Drug Monitoring Study design & Sample handling Analytes Analytes Data analysis & sampling & preparation separation detection interpretation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery 4 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 5 Bioanalytical Sciences | [email protected]  Bioanalytical workflow | Lectures II & III Forensic Toxicology Therapeutic Drug Monitoring Study design & Sample handling sampling & preparation Doping Control Clinical Toxicology & Chemistry Biomarker Discovery “Bioanalysis is a sub-discipline of analytical chemistry covering the quantitative measurement of xenobiotics (drugs and their metabolites, and biological molecules in unnatural locations or concentrations) and biotics (macromolecules, proteins, DNA, (Dead or alive) large molecule drugs, metabolites) in biological systems.” 6 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO Biological matrices Which biological matrices can we use in bioanalytical sciences? 7  Q&A INTERMEZZO Biological matrices …. 8  Q&A INTERMEZZO Biological matrices Blood-based Urine Cerebrospinal Tissues Hair Vaginal swab & Oral fluid matrices fluid sperm Nails Feces Sweat Teeth/bones Vitreous & aqueous Cerumen Cells (2D, 3D models) 9 humour  Blood, plasma and serum Plasma Serum collection collection Whole blood 10 Bioanalytical Sciences | [email protected]  Blood collection | Plasma or serum? Addition of anticoagulants Plasma (heparine, citrate, EDTA) collection (activated by heparin) 11 Bioanalytical Sciences | [email protected]  Blood collection | Plasma or serum? Addition of anticoagulants (heparine, citrate, EDTA) Choice? 12 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO Selection of collection tube How do I know whether I should use whole blood, plasma, or serum for my analysis? If I use plasma, which anticoagulant should I use? 13  Q&A INTERMEZZO Selection of collection tube Whole blood Contains red blood cells (RBC, 40-45% of the volume), white blood cells and platelets Some compounds are strongly influenced by the RBC content; for these compounds it is important to measure whole blood Also preferred in forensic labs to help in the interpretation of drugs concentrations More complicated to guarantee sample integrity during storage and handling 14 RBCs can rupture/lyse over time (hemolysis), which affects the results  Q&A INTERMEZZO Selection of collection tube Plasma and serum Easier to handle and store than whole blood, but concentrations may be less representative Are not interchangeable: some compounds (especially metabolites influenced by the coagulation process) may lead totally different concentrations (up to 100 times!). Little information about the possible differences in metabolites concentration is available in the literature The anticoagulant used can influence the results as well, so always check which one has been used. 15 Choice between plasma & serum: should be investigated for each method/application (but often depends on what the clinicians/toxicologists/biobanks give you..)  Q&A INTERMEZZO Selection of collection tube Plasma and serum: one example The difference can be pronounced for some metabolites (way more than proteins) 16 Nishiumi et al., J Biosci Bioeng 125 (2018) 613  Q&A INTERMEZZO Selection of collection tube Anticoagulants for plasma The anticoagulant used can influence the results as well, so always check which one has been used. Some logical selection: if you are interested in the TCA cycle, don’t use citrate as anticoagulant. Again, it often depends on what the clinicians/toxicologists/biobanks give you.. 17  Q&A INTERMEZZO Selection of collection tube 18  Q&A INTERMEZZO Selection of collection tube Use an analytical method only for the matrix it has been validated for Do not compare results obtained between plasma and serum, or only if studies have demonstrated that they gave comparable results 19  Urine Easily collected, non-invasive Large volumes collected (> 100 mL for human beings; rodent models are a bit more complicated) Low protein concentration (! Proteinuria [presence of protein in urine] is a common issue linked to many pathologies) Within-day variability Large pH range (between 3.5 – 8) dependent on the diet and many factors, large variability in salts concentrations, dilution of analytes Urine is sterile but contaminated with micro-organisms at the moment it exits the urethra ( analytes degradation) Possible adulteration (doping, workplace drug testing) 20 Bioanalytical Sciences | [email protected]  Urine | The urine wheel Thomas Willis (English physician), in 1674: “…wonderfully sweet as if it were imbued with honey or sugar.” Common diseases found Diabetes mellitus (sweet taste) Jaundice (brownish urine tint) Kidney diseases (red or foamy urine) UT tumors (blood in urine) 21 Bioanalytical Sciences | [email protected]  Other and “alternative” matrices Body fluid with the closest Long-term detection connection to the (living) brain Stability of the matrix CSF Very invasive procedure Nails & hair Postmortem analysis Information on the microbiome Postmortem analysis Gut-brain axis Teeth/bones Less postmortem redistribution Feces VH & HA Easy to collect, non-invasive Pre-clinical studies Tests at home or on the road In-vitro experiments Oral fluid Tissues Sweat Detection window similar to Cells (2D, 3D models) Cerumen urine 22 Bioanalytical Sciences | [email protected]  Other and “alternative” matrices Long-term detection Stability of the matrix Small volume required (µL range) Nails & hair Postmortem analysis Easily transported (spot dried on paper) Pay attention during the Video Easy to collect, non-invasive presentations to learn more Tests at home or on the road about these matrices Oral fluid Sweat Detection window similar to Cerumen urine 23 Bioanalytical Sciences | [email protected]  Other and “alternative” matrices | An example 24 Bioanalytical Sciences | [email protected]  Which one to choose | Detection window When analyzing xenobiotics, think about which analytes to measure: Parent compound Product(s) of the metabolism (metabolites) Parent compound and metabolite(s) Think about the application of the developed method Detection window 25 Bioanalytical Sciences | [email protected]  Which one to choose | Metabolites? What’s that? Phase I and Phase II metabolites ADME 26 Bioanalytical Sciences | [email protected]  Which one to choose | Metabolites? What’s that? Phase I and Phase II metabolites Phase I metabolites Phase II metabolites Oxidation Conjugation with glucuronide Reduction Conjugation with sulfate Dealkylation Conjugation with glutathione Hydroxylation … …. Phase I & Phase II metabolites are often as important as the parent drug; sometimes even more! 27 Bioanalytical Sciences | [email protected]  Which one to choose | An example: 2C-B 4-bromo-2,5- dimethoxyphenethylamine Phase I metabolites Phase II metabolites 28 Bioanalytical Sciences | [email protected]  Which one to choose | An example: heroin & morphine Detection Parent drug Drug metabolites window (metabolites) Heroin versus morphine consumption: Heroin Both are consumed as illicit drugs! How do we differentiate between them? Half-life: few minutes 6-monoacetylmorphine Detected up to 3 days in only! Detected up to 8h in urine urine 29 Bioanalytical Sciences | [email protected]  Q&A INTERMEZZO Which matrix for which application? I am interested in the drug(s) that a drug user has consumed in the last 2 days. Which matrix should I analyse? And which target compounds? I am interested in the drug(s) that a drug user has consumed in the last 6 months. Which matrix should I analyse? And which target compounds? 30  Q&A INTERMEZZO Which matrix for which application? I am interested in the drug(s) that a drug user has consumed in the last 2 days. Which matrix should I analyse? And which target compounds? Blood, saliva, urine, sweat, … Especially parent drug but also some metabolites (check literature!) 31  Q&A INTERMEZZO Which matrix for which application? I am interested in the drug(s) that a drug user has consumed in the last 6 months. Which matrix should I analyse? And which target compounds? Hair or nails Mostly metabolites but parent drugs can also be incorporated 32  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 Risks extraction Storage of samples Solid-phase extraction 33 Bioanalytical Sciences | [email protected]  Bioanalytical workflow | Next week Tuesday 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 is pre-recorded Lecture X 34 Bioanalytical Sciences | [email protected]