Bioanalytical Sciences Sample Collection
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Questions and Answers

What matrix should be analyzed to determine drugs consumed in the last 2 days?

Blood, saliva, urine, sweat

What is the recommended matrix for analyzing drugs consumed in the last 6 months?

Hair or nails

Which type of compounds should be targeted when analyzing for drugs in urine?

  • No specific compounds
  • Only metabolites
  • Parent drugs and some metabolites (correct)
  • Only parent drugs

    • Hair analysis primarily reveals parent drugs.

    <p>False</p> Signup and view all the answers

    Which of these biological matrices can be used in bioanalytical sciences? (Select all that apply)

    <p>Blood-based matrices</p> Signup and view all the answers

    What is bioanalysis?

    <p>A sub-discipline of analytical chemistry covering the quantitative measurement of xenobiotics and biotics in biological systems.</p> Signup and view all the answers

    Whole blood contains red blood cells, white blood cells, and platelets.

    <p>True</p> Signup and view all the answers

    What factors can influence the choice between plasma and serum?

    <p>The compounds of interest, the anticoagulant used, and the specific requirements of the analytical method.</p> Signup and view all the answers

    What is one characteristic of urine as a sample for analysis?

    <p>Easy to collect and non-invasive</p> Signup and view all the answers

    In bioanalysis, blood can be separated into ______ and serum.

    <p>plasma</p> Signup and view all the answers

    What is an example of a Phase II metabolite?

    <p>Conjugation with glucuronide</p> Signup and view all the answers

    What can cause degradation of analytes in urine samples?

    <p>Micro-organisms can contaminate urine at collection, causing degradation.</p> Signup and view all the answers

    Which of the following is a topic covered in the bioanalytical sciences group work?

    <p>Post-mortem analysis of fentanyl analogues in vitreous humour</p> Signup and view all the answers

    Vitreous humour is a recommended matrix for the analysis of substances like fentanyl.

    <p>True</p> Signup and view all the answers

    What is the maximum number of students allowed in each group for the bioanalytical sciences topic?

    <p>5</p> Signup and view all the answers

    Chiral analysis of ______ and derivatives in saliva is one of the groups' topics.

    <p>ketamine</p> Signup and view all the answers

    Match the following group topics with their descriptions:

    <p>Post-mortem analysis of fentanyl analogues = Analyzing substances in vitreous humour Analysis of FA and FMA isomers = Examining dried blood spots Post-mortem analysis of amphetamine-derivatives = Evaluating metabolites in nails Chiral analysis of ketamine = Studying saliva samples</p> Signup and view all the answers

    Which of the following substances requires the use of anticoagulants during plasma collection?

    <p>Plasma</p> Signup and view all the answers

    Cerebrospinal fluid is considered a biological matrix for analysis.

    <p>True</p> Signup and view all the answers

    What biological matrix would you use to analyze the composition of sweat?

    <p>Sweat</p> Signup and view all the answers

    Blood can be separated into ______ and serum.

    <p>plasma</p> Signup and view all the answers

    Match the following biological matrices with their respective types:

    <p>Blood = Fluid matrix Hair = Tissue matrix Teeth = Solid matrix Urine = Liquid matrix</p> Signup and view all the answers

    Which matrix would you analyze to detect drugs taken in the last 6 months?

    <p>Hair</p> Signup and view all the answers

    Parent drugs are often analyzed from hair or nails.

    <p>True</p> Signup and view all the answers

    Name one method used in sample preparation for bioanalytical studies.

    <p>Liquid-liquid extraction</p> Signup and view all the answers

    For therapeutic drug monitoring, analytes are typically separated from samples using ______.

    <p>solid-phase extraction</p> Signup and view all the answers

    Match the following matrices with their respective detection timeframes:

    <p>Blood = Last 2 days Urine = Last 2 days Hair = Last 6 months Nails = Last 6 months</p> Signup and view all the answers

    Which of the following methods is commonly used for sample handling?

    <p>Protein precipitation</p> Signup and view all the answers

    Sweat analysis can detect drugs taken over the last month.

    <p>False</p> Signup and view all the answers

    What is one target compound for urine analysis in a toxicology screening?

    <p>Metabolites</p> Signup and view all the answers

    The procedure for separating blood components involves separating into plasma and ______.

    <p>serum</p> Signup and view all the answers

    Which of these is a risk associated with storage of samples?

    <p>Degradation of analytes</p> Signup and view all the answers

    Which of the following is a common reaction associated with Phase I metabolites?

    <p>Reduction</p> Signup and view all the answers

    Phase II metabolites are typically formed through oxidation processes.

    <p>False</p> Signup and view all the answers

    What is a characteristic of Phase II metabolites in drug metabolism?

    <p>Conjugation with another substance.</p> Signup and view all the answers

    The substance ______ is a Phase I metabolite associated with heroin.

    <p>6-monoacetylmorphine</p> Signup and view all the answers

    Match the drug with its primary metabolite:

    <p>Heroin = 6-monoacetylmorphine Morphine = Morphine-3-glucuronide Caffeine = Paraxanthine Alcohol = Acetaldehyde</p> Signup and view all the answers

    Which of the following metabolites can be detected in urine up to 3 days after heroin use?

    <p>6-monoacetylmorphine</p> Signup and view all the answers

    Hair analysis provides information primarily about parent drugs rather than metabolites.

    <p>True</p> Signup and view all the answers

    In the context of bioanalysis, what does the acronym ADME stand for?

    <p>Absorption, Distribution, Metabolism, Excretion</p> Signup and view all the answers

    What is a common issue linked to protein presence in urine?

    <p>Proteinuria</p> Signup and view all the answers

    Results obtained from serum can be directly compared to those from plasma.

    <p>False</p> Signup and view all the answers

    What type of fluid is considered sterile but can become contaminated at the moment it exits the body?

    <p>urine</p> Signup and view all the answers

    The detection window for oral fluid is similar to that of ______.

    <p>urine</p> Signup and view all the answers

    Match the following biological matrices with their characteristics.

    <p>Urine = Easily collected, non-invasive CSF = Very invasive procedure Hair = Long-term detection Feces = Information on the microbiome</p> Signup and view all the answers

    Which of the following is NOT a characteristic of urine as a sample for analysis?

    <p>Contains high protein concentration</p> Signup and view all the answers

    Nails and hair can provide short-term data for analysis.

    <p>False</p> Signup and view all the answers

    What is a common taste description associated with urine in cases of diabetes mellitus?

    <p>sweet</p> Signup and view all the answers

    Urine has a pH range between ______ and ______.

    <p>3.5, 8</p> Signup and view all the answers

    Which of the following matrices requires the smallest volume for analysis?

    <p>Nails</p> Signup and view all the answers

    Sweat is considered a non-invasive biological matrix for testing.

    <p>True</p> Signup and view all the answers

    What is a common visual sign in urine that may indicate kidney diseases?

    <p>red or foamy urine</p> Signup and view all the answers

    CSF provides information with the closest connection to the ______.

    <p>brain</p> Signup and view all the answers

    What should be considered when analyzing xenobiotics?

    <p>Both parent compound and metabolites</p> Signup and view all the answers

    Both skin and hair can be used to detect substances postmortem.

    <p>True</p> Signup and view all the answers

    Which of the following substances is known for its carcinogenicity?

    <p>Benzene</p> Signup and view all the answers

    Green chemistry aims to reduce the use of hazardous reagents.

    <p>True</p> Signup and view all the answers

    What method is used to assess the greenness of analytical procedures?

    <p>Eco-Scale</p> Signup and view all the answers

    Dichloromethane and chloroform can cause __________ of the central nervous system.

    <p>depression</p> Signup and view all the answers

    Match the following principles of green chemistry to their descriptions:

    <p>Prevent waste = Design synthetic methods to maximize incorporation of all materials Maximize efficiency = Use catalytic reagents, as selective as possible Use renewable feedstocks = Employ raw materials that are renewable Design safer chemicals = Design chemicals to minimize toxicity</p> Signup and view all the answers

    Which of the following is a typical result of using high solvent consumption in analysis?

    <p>Higher costs</p> Signup and view all the answers

    The Twelve Principles of Green Analytical Chemistry were established in 2013.

    <p>True</p> Signup and view all the answers

    Name one health hazard associated with methanol ingestion.

    <p>Blindness</p> Signup and view all the answers

    Which of the following methods is used for sample preparation in bioanalytical sciences?

    <p>Liquid-liquid extraction</p> Signup and view all the answers

    Solid-phase extraction is considered an automated method of sample preparation.

    <p>True</p> Signup and view all the answers

    Name one benefit of using green chemistry techniques in sample preparation.

    <p>Reducing the use of hazardous reagents</p> Signup and view all the answers

    The process of __________ involves collecting samples like blood and preparing them for analysis.

    <p>sample handling</p> Signup and view all the answers

    Match the following extraction techniques with their descriptions:

    <p>Microextractions = Techniques primarily used for small-volume samples Protein precipitation = Method used to separate proteins from solutions Solid-phase extraction = Uses solid adsorbents to isolate analytes Liquid-liquid extraction = Separates compounds based on solubility in different liquids</p> Signup and view all the answers

    Which of the following methods is commonly associated with reducing the use of hazardous reagents in sample preparation?

    <p>Microextractions</p> Signup and view all the answers

    Supercritical fluid extraction is a method that uses toxic solvents.

    <p>False</p> Signup and view all the answers

    Name a non-toxic extraction agent that can be used in green chemistry.

    <p>Ionic liquids</p> Signup and view all the answers

    The principle of ______ involves the reduction of the volume of solvents used in sample procedures.

    <p>miniaturization</p> Signup and view all the answers

    Match the following microextraction techniques with their characteristics:

    <p>SPME = Uses solid phase for extraction Liquid-based microextractions = Utilizes solvents to extract compounds Solid-based microextractions = Involves the use of solid sorbents Headspace analysis = Analyzes vapors above a sample</p> Signup and view all the answers

    Which extraction technique does NOT typically use solvents?

    <p>Solid phase microextraction</p> Signup and view all the answers

    Miniaturization of procedures leads to an increase in sample size requirements.

    <p>False</p> Signup and view all the answers

    What is the primary objective of green chemistry in the context of sample preparation?

    <p>To minimize the use of hazardous chemicals and reduce environmental impact.</p> Signup and view all the answers

    Which type of microextraction involves a packed sorbent?

    <p>Microextraction by packed sorbent</p> Signup and view all the answers

    Dispersive liquid-liquid microextraction (DLLME) incorporates a single drop for extraction.

    <p>False</p> Signup and view all the answers

    What is the primary application of SDME (Single drop microextraction)?

    <p>Sample extraction in analytical chemistry</p> Signup and view all the answers

    ___ is a method of sample handling that involves separating different phases in a sample.

    <p>Electroextraction</p> Signup and view all the answers

    Match the following extraction methods with their descriptions:

    <p>MEPS = Microextraction by packed sorbent DLLME = Dispersive liquid-liquid microextraction SDME = Single drop microextraction Electroextraction = Utilizes electrical fields for extraction</p> Signup and view all the answers

    At what pH value does MDMA need to be adjusted to ensure it is 99% ionized?

    <p>pH 7.8</p> Signup and view all the answers

    If the pH of a sample is 9.3, the percentage of amphetamine that is under its neutral form is 0%.

    <p>False</p> Signup and view all the answers

    What is the pKa of the amine group of MDMA?

    <p>9.9</p> Signup and view all the answers

    MDMA is considered to be _____% ionized at a pH lower than 7.9.

    <p>99</p> Signup and view all the answers

    Match the following compounds with their respective pKa values:

    <p>Amphetamine = pKa not provided in content MDMA = 9.9 Urine = Varies between 3.5 - 8</p> Signup and view all the answers

    What is the significance of pKa in sample preparation?

    <p>It defines the acidity or basicity of a compound.</p> Signup and view all the answers

    Salicylic acid has a pKa of 2.79.

    <p>True</p> Signup and view all the answers

    Name one method used in sample preparation for forensic toxicology.

    <p>Liquid-liquid extraction</p> Signup and view all the answers

    In forensic toxicology, __________ is a process used for removing proteins from a sample.

    <p>protein precipitation</p> Signup and view all the answers

    Match the following drugs with their associated activities:

    <p>Fentanyl = Pain management MDMA = Recreational drug Morphine = Opioid analgesic Paclitaxel = Cancer treatment</p> Signup and view all the answers

    What is the dissociation constant (pKa) value for salicylic acid?

    <p>2.79</p> Signup and view all the answers

    The concentration of the protonated and deprotonated forms of an analyte are equal when pH is above the pKa.

    <p>False</p> Signup and view all the answers

    Name one method used in sample preparation for bioanalytical studies.

    <p>Liquid-liquid extraction</p> Signup and view all the answers

    At pH 2.79, the concentration of salicylic acid is ______% protonated and ______% deprotonated.

    <p>50, 50</p> Signup and view all the answers

    Match the following sample preparation methods with their descriptions:

    <p>Protein precipitation = Removal of proteins from sample Liquid-liquid extraction = Separation based on solubility in two immiscible liquids Solid-phase extraction = Separation using a solid sorbent material Doping control = Ensuring athletes do not use prohibited substances</p> Signup and view all the answers

    Which of the following is NOT a type of sample handling method commonly used in bioanalytical sciences?

    <p>Therapeutic Drug Monitoring</p> Signup and view all the answers

    Salicylic acid is in its deprotonated form at a pH that is higher than its pKa.

    <p>True</p> Signup and view all the answers

    Identify one area in which bioanalytical sciences are applied.

    <p>Forensic Toxicology</p> Signup and view all the answers

    At which pH level is 99% of an acid group ionized?

    <p>pH = pKa + 2</p> Signup and view all the answers

    At a pH of 4.79, 90% of the acid group is neutral.

    <p>False</p> Signup and view all the answers

    To achieve 99% ionization of an acid, what should be the pH in relation to pKa?

    <p>pKa + 2</p> Signup and view all the answers

    99% of the basic group is neutral when pH = pKa + _____

    <p>2</p> Signup and view all the answers

    Match the pH levels with the percentage of ionization for an acid:

    <p>pH 0.79 = 99% AH, 1% A- pH 2.79 = 50% AH, 50% A- pH 3.79 = 10% AH, 90% A- pH 5.79 = 0.1% AH, 99.9% A-</p> Signup and view all the answers

    At what pH is 10% of the acid group ionized?

    <p>pH 4.79</p> Signup and view all the answers

    At pH 3.79, 90% of the acid group is in its ionized form.

    <p>True</p> Signup and view all the answers

    What percentage of AH is present at pH 1.79?

    <p>90%</p> Signup and view all the answers

    Explain the significance of pKa in relation to sample preparation for drug analysis.

    <p>pKa is significant as it indicates the pH at which a drug will exist in equal concentrations of its ionized and non-ionized forms, influencing its solubility and extraction efficiency.</p> Signup and view all the answers

    What are the main techniques used for sample preparation in forensic toxicology?

    <p>The main techniques for sample preparation include protein precipitation, liquid-liquid extraction, and solid-phase extraction.</p> Signup and view all the answers

    Why is pH control essential during the sample handling phase in bioanalysis?

    <p>pH control is essential because it affects the stability of the analytes and the efficiency of extraction methods used in sample preparation.</p> Signup and view all the answers

    Discuss the role of sampling in therapeutic drug monitoring.

    <p>Sampling is crucial in therapeutic drug monitoring as it determines the drug concentration in biological matrices, guiding dosing decisions.</p> Signup and view all the answers

    What is the impact of sample storage conditions on the integrity of biological samples?

    <p>Improper storage conditions can lead to degradation of analytes, affecting the reliability of analytical results.</p> Signup and view all the answers

    To obtain 99% ionization of MDMA in urine, what pH should the urine sample be adjusted to?

    <p>The urine sample should be adjusted to a pH lower than 7.9.</p> Signup and view all the answers

    If the pKa of amphetamine is 9.5, to what pH range should urine be adjusted for 99% ionization?

    <p>Urine should be adjusted to a pH lower than 7.5.</p> Signup and view all the answers

    At a pH of 9.3, what percentage of amphetamine is in its neutral form?

    <p>At pH 9.3, amphetamine would be approximately 90% in its neutral form.</p> Signup and view all the answers

    Describe the significance of adjusting urine pH before performing solid-phase extraction (SPE).

    <p>Adjusting urine pH ensures that the target compound is primarily in its ionized form, improving extraction efficiency.</p> Signup and view all the answers

    For effective extraction of amphetamine, what is the general pH range of urine samples typically found in healthy individuals?

    <p>The typical pH range of urine in healthy individuals is between 4 and 6.5.</p> Signup and view all the answers

    Explain the significance of pKa in the context of sample preparation.

    <p>The pKa indicates the pH at which a compound exists in equal amounts of protonated and deprotonated forms, affecting its solubility and stability during analysis.</p> Signup and view all the answers

    What role does pH play in the dissociation of salicylic acid?

    <p>At the pH equal to its pKa (2.79), salicylic acid exists in a 50% protonated and 50% deprotonated state, influencing its detection.</p> Signup and view all the answers

    Describe two common methods used for sample preparation in bioanalytical sciences.

    <p>Common methods include protein precipitation and solid-phase extraction, which are used to isolate analytes from biological matrices.</p> Signup and view all the answers

    How does liquid-liquid extraction function in the context of bioanalysis?

    <p>Liquid-liquid extraction separates analytes based on their solubility in two different immiscible liquids, enhancing purity for analysis.</p> Signup and view all the answers

    Identify one critical factor that must be considered during sample handling to ensure analyte integrity.

    <p>Temperature control is crucial, as improper temperatures can lead to degradation or alteration of the analyte.</p> Signup and view all the answers

    What is the primary objective of therapeutic drug monitoring in bioanalysis?

    <p>The primary objective is to ensure optimal drug dosing by adjusting levels based on the measured concentrations in biological samples.</p> Signup and view all the answers

    Discuss why solid-phase extraction is preferred over other extraction methods in some cases.

    <p>Solid-phase extraction offers greater selectivity and efficiency, allowing for the purification of analytes with minimal sample volume.</p> Signup and view all the answers

    In what way does the choice of biological matrix influence bioanalytical results?

    <p>The biological matrix affects analyte stability, recovery rates, and potential interferences during analysis.</p> Signup and view all the answers

    At what pH value do you need to adjust your sample to ensure that 99% of amphetamine is ionized?

    <p>pH 9.3 - 2 = pH 7.3.</p> Signup and view all the answers

    If the pH of a sample is 9.3, what percentage of amphetamine would be in its neutral form?

    <p>Approximately 99%.</p> Signup and view all the answers

    Define the relationship between pH and pKa for an acid to be 99% ionized.

    <p>The acid must be at pH = pKa + 2.</p> Signup and view all the answers

    What is the expected percentage of amphetamine in its neutral form when the pH is adjusted to pKa + 2?

    <p>1%.</p> Signup and view all the answers

    At pH 4.79, what are the predominant forms of an acid if 99% is ionized?

    <p>1% of the acid (AH) and 99% of its conjugate base (A-).</p> Signup and view all the answers

    Explain the significance of pH being equal to pKa ± 2 for an acid's ionization.

    <p>It indicates the range in which an acid is predominantly ionized or neutral.</p> Signup and view all the answers

    When an acid is 99% ionized, what does this indicate about the environmental pH relative to its pKa?

    <p>It suggests that the pH is two units above the pKa.</p> Signup and view all the answers

    What will be the state of an acid at pH equal to pKa - 2?

    <p>99% of the acid group will be neutral (AH).</p> Signup and view all the answers

    What is the primary advantage of using solid-phase extraction (SPE) in sample preparation?

    <p>SPE allows for improved selectivity and sensitivity by selectively retaining target analytes while eliminating interferents.</p> Signup and view all the answers

    How does sample concentration enhance the sensitivity of bioanalytical procedures?

    <p>Sample concentration increases the amount of the target analyte present, making it more detectable by analytical methods.</p> Signup and view all the answers

    Name one method commonly employed for the elimination of interferents in bioanalytical sample preparation.

    <p>Protein precipitation (PP) is a common method used to remove proteins and other compounds that can interfere with analysis.</p> Signup and view all the answers

    What role do extraction supports play in sample preparation for bioanalytical analysis?

    <p>Extraction supports provide a substrate for analyte binding, facilitating the selective extraction of target compounds from samples.</p> Signup and view all the answers

    In protein precipitation, what is the relationship between the volume of precipitant and sample under typical procedures?

    <p>The typical ratio involves using 1000 µL of PP agent to 500 µL of plasma, ensuring effective precipitation.</p> Signup and view all the answers

    What is the primary advantage of using solid-phase extraction (SPE) over traditional liquid-liquid extraction?

    <p>SPE offers increased sensitivity and reproducibility by concentrating analytes and reducing solvent use.</p> Signup and view all the answers

    Describe one common technique for sample concentration in bioanalytical methods.

    <p>One common technique is evaporative concentration, which reduces the sample volume by removing solvents through gentle heating or vacuum.</p> Signup and view all the answers

    What is one method used for the elimination of interferents in bioanalytical sample preparation?

    <p>A common method is solid-phase extraction (SPE), which selectively retains the target analytes while allowing interferents to be washed away.</p> Signup and view all the answers

    What role do extraction supports play in solid-phase extraction processes?

    <p>Extraction supports provide a surface for the interaction and retention of target analytes, enhancing separation from the matrix.</p> Signup and view all the answers

    Explain the mechanism of protein precipitation in sample preparation.

    <p>Protein precipitation involves adding a precipitating agent that disrupts protein solubility, causing them to aggregate and separate from the solution.</p> Signup and view all the answers

    Why is solid-phase extraction often preferred in therapeutic drug monitoring?

    <p>SPE allows for selective extraction and provides cleaner samples, which is crucial for accurate quantification of drugs.</p> Signup and view all the answers

    Identify a factor that can affect the efficiency of solid-phase extraction.

    <p>The choice of solvent and its polarity can significantly impact the ability of the SPE to retain or elute the analytes.</p> Signup and view all the answers

    What is the purpose of using a washing step in solid-phase extraction?

    <p>The washing step removes unwanted matrix components while retaining the target analytes on the extraction medium.</p> Signup and view all the answers

    What principle does Solid-Phase Extraction (SPE) rely on for separating analytes from a sample?

    <p>SPE relies on the principle of adsorption and desorption of analytes on a solid sorbent.</p> Signup and view all the answers

    What is the role of pH in sample preparation for bioanalytical analyses?

    <p>pH affects the ionization of analytes, which influences their retention and separation during extraction.</p> Signup and view all the answers

    Name two common extraction supports used in Solid-Phase Extraction.

    <p>Common extraction supports include silica gel and ion-exchange resins.</p> Signup and view all the answers

    What modifications occur in protein structures due to the presence of acids during sample preparation?

    <p>Acids can modify protein structures through electrostatic interactions, altering their solubility and retention.</p> Signup and view all the answers

    Describe the significance of using mixed-mode extraction supports in bioanalytical methods.

    <p>Mixed-mode supports provide better selectivity by utilizing multiple interaction mechanisms for analyte retention.</p> Signup and view all the answers

    What problem can arise from the presence of salts during the extraction process?

    <p>Salts can lead to increased surface tension, which may hinder the retention and elution of analytes.</p> Signup and view all the answers

    Explain the importance of utilizing appropriate solvents in the precipitation of proteins.

    <p>Appropriate solvents, like organic solvents, help to selectively precipitate proteins, removing them for better analyte analysis.</p> Signup and view all the answers

    In Solid-Phase Extraction, what is the effect of flow rate on ion-exchange interactions?

    <p>A low flow rate is required for ion-exchange interactions to ensure sufficient time for analyte binding.</p> Signup and view all the answers

    What are the two main solvent mixtures used to activate SPE cartridges?

    <p>Methanol (MeOH) and water.</p> Signup and view all the answers

    Why is it important to pre-treat a sample before loading it onto an SPE cartridge?

    <p>Pre-treatment helps remove protein clogs and ensures the sample pH is suitable for analyte retention.</p> Signup and view all the answers

    What is the difference in charging between mixed-mode strong cation exchange (MCX) and mixed-mode weak cation exchange (WCX)?

    <p>MCX is always charged, while WCX is charged depending on the pH.</p> Signup and view all the answers

    What flow rate should be maintained during the loading step of SPE, and why?

    <p>The flow rate should be slow to accommodate electrostatic interactions.</p> Signup and view all the answers

    What pH condition is indicated by the formula pH = pKa - 2 in relation to cation exchange?

    <p>This formula suggests a condition where cations are retained due to acidic pH levels.</p> Signup and view all the answers

    How does the presence of a protein-rich matrix impact the solid phase extraction process?

    <p>It can clog the sorbent, necessitating the precipitation of proteins before extraction.</p> Signup and view all the answers

    In the elution phase of SPE, what is a common strategy used to ensure proper analyte recovery?

    <p>Elution is often performed with a stronger solvent to displace the analytes from the sorbent.</p> Signup and view all the answers

    What role does reading the manufacturer's manual (RTFM) play when using SPE cartridges?

    <p>It provides essential instructions for activating and using the cartridges correctly.</p> Signup and view all the answers

    What does the phrase 'Like dissolves like' imply in the context of solid-phase extraction?

    <p>It implies that polar compounds will have a greater affinity for polar stationary phases than apolar elution phases.</p> Signup and view all the answers

    How does adjusting the pH of the sample affect solid-phase extraction?

    <p>Adjusting the pH can influence the charge of the analyte, affecting its interactions with the sorbent during the loading step.</p> Signup and view all the answers

    When performing solid-phase extraction, what factors should you consider when choosing the washing and elution solvents?

    <p>Consider the polarity and charge of both the stationary phase and the analyte of interest.</p> Signup and view all the answers

    What role does logP (or logD) play in the selection of compounds during the solid-phase extraction process?

    <p>LogP indicates the polarity of the compounds, guiding the choice of stationary and elution phases for optimal interaction.</p> Signup and view all the answers

    What preprocessing steps should be taken with plasma before loading it onto the sorbent in solid-phase extraction?

    <p>The plasma sample may need to be conditioned or adjusted for pH before it is loaded onto the sorbent.</p> Signup and view all the answers

    Describe the importance of selecting the correct type of sorbent in solid-phase extraction.

    <p>The sorbent's polarity and charge are crucial as they determine how effectively the analytes will bind and be separated from matrix components.</p> Signup and view all the answers

    In solid-phase extraction, why is it important to understand the polarity properties of the compounds involved?

    <p>Understanding the polarity helps in optimizing the interaction between analytes and the stationary phase for improved extraction efficiency.</p> Signup and view all the answers

    What effect does the composition of the washing phase have on the solid-phase extraction process?

    <p>The washing phase can selectively remove impurities while retaining the target analytes, which is critical for clean extraction.</p> Signup and view all the answers

    What is the significance of protein precipitation in the extraction of MA and MDMA from plasma?

    <p>It prevents clogging of the sorbent during extraction.</p> Signup and view all the answers

    Why is it important to control the pH of the sample during the Solid Phase Extraction (SPE) process?

    <p>The pH affects the retention of analytes of interest, ensuring optimal extraction efficiency.</p> Signup and view all the answers

    What are the potential consequences of not filtering plasma samples prior to extraction?

    <p>Inadequate filtration can lead to sorbent clogging and inaccurate analytical results.</p> Signup and view all the answers

    Which organic solvent is typically recommended for protein precipitation when extracting substances from plasma?

    <p>Acetonitrile is commonly used for this purpose.</p> Signup and view all the answers

    What is the role of perchloric acid in sample preparation for extracting MA and MDMA

    <p>Perchloric acid can be used to facilitate the protein precipitation process.</p> Signup and view all the answers

    How does the extraction technique applied influence the choice of acids used in sample preparation?

    <p>The specific requirements of the SPE procedure dictate the selection of appropriate acids for optimal extraction.</p> Signup and view all the answers

    What is the preferred condition for loading samples in terms of pH for the extraction of MA and MDMA?

    <p>Samples should be loaded at a pH lower than approximately 7.9.</p> Signup and view all the answers

    Why are strong but slow electrostatic interactions important in the context of bioanalytical sample processing?

    <p>These interactions help stabilize the analytes during the extraction process, impacting the efficiency of their recovery.</p> Signup and view all the answers

    What is a significant composition difference between plasma and serum?

    <p>Plasma contains a significant number of platelets, while serum does not.</p> Signup and view all the answers

    How do collection tubes impact the quality of metabolomic studies?

    <p>Collection tubes can release plasticizers that interfere with the ionization process in mass spectrometry.</p> Signup and view all the answers

    Why is it necessary to stick to one manufacturer for collection tubes in a study?

    <p>Additives from different manufacturers may contain interfering compounds that affect analytical quality.</p> Signup and view all the answers

    What is the role of anticoagulants in blood collection for analysis?

    <p>Anticoagulants prevent coagulation, allowing for the collection of plasma without forming clots.</p> Signup and view all the answers

    What should be assessed before starting metabolomics studies concerning blood sample collection?

    <p>The types of collection tubes and their potential impact on the metabolome should be evaluated.</p> Signup and view all the answers

    What problem can arise from using plastic collection tubes during analysis?

    <p>Plastic collection tubes can introduce plasticizers that lead to sample contamination.</p> Signup and view all the answers

    What factor should be considered regarding the labeling of collection tubes?

    <p>Labels must resist very low temperatures during long-term storage to avoid loss of information.</p> Signup and view all the answers

    What is the consequence of not handling samples properly during collection and storage?

    <p>Improper handling can lead to degradation of analytes, impacting the validity of analytical results.</p> Signup and view all the answers

    What is the ideal clotting time for serum collection?

    <p>The ideal clotting time should be between 30 and 60 minutes.</p> Signup and view all the answers

    Why is temperature control crucial after clot formation in serum collection?

    <p>Temperature control is crucial because lower temperatures reduce cellular metabolism, preserving the integrity of the sample.</p> Signup and view all the answers

    How do clot-activating agents affect serum collection time?

    <p>Clot-activating agents allow for a shorter clotting time of less than 30 minutes.</p> Signup and view all the answers

    What is a significant benefit of using plasma over serum in collection methods?

    <p>Plasma samples can be placed on ice quickly after collection, eliminating the need for clotting time at room temperature.</p> Signup and view all the answers

    What impact do anticoagulants have on plasma component collection?

    <p>Anticoagulants like heparin, EDTA, and citrate significantly influence the quality of plasma data.</p> Signup and view all the answers

    How can prolonged clotting time be detrimental to serum analysis?

    <p>Prolonged clotting time can lead to lysis of cells, releasing cellular components into the serum.</p> Signup and view all the answers

    What are two common anticoagulants used in clinical plasma collection?

    <p>Common anticoagulants include heparin and ethylenediaminetetraacetic acid (EDTA).</p> Signup and view all the answers

    Why should clotting time and temperature be consistent across samples in a study?

    <p>Consistency in clotting time and temperature ensures comparability and accuracy of results in metabolomics studies.</p> Signup and view all the answers

    What key process occurs during serum collection that influences metabolite presence?

    <p>The blood coagulation cascade is activated, transforming fibrinogen into fibrin, forming a blood clot.</p> Signup and view all the answers

    How do the collection tubes used impact the quality of serum and plasma samples?

    <p>Collection tubes with anticoagulants prevent clotting and allow for plasma collection, while non-anticoagulant tubes yield serum after clot formation.</p> Signup and view all the answers

    Why is the use of anticoagulants essential in plasma collection?

    <p>Anticoagulants prevent clotting, which allows for the analysis of all soluble components in the plasma.</p> Signup and view all the answers

    What sample handling consideration is critical to maintain the integrity of blood samples during analysis?

    <p>Proper temperature control is essential to prevent degradation of metabolites and ensure accurate measurement.</p> Signup and view all the answers

    What is a major challenge associated with analyzing whole blood in metabolomics?

    <p>Whole blood is complex due to the presence of cellular components that can confound metabolite profiles.</p> Signup and view all the answers

    How is the supernatant in serum collection obtained?

    <p>After blood clot formation, centrifugation separates the supernatant, which is the serum, from the cellular components.</p> Signup and view all the answers

    What are the implications of using different anticoagulants in plasma collection?

    <p>Different anticoagulants can affect the stability and concentration of metabolites measured in plasma samples.</p> Signup and view all the answers

    What happens to fibrinogen during the serum collection process?

    <p>Fibrinogen is transformed into fibrin, which contributes to the clotting process and the formation of a blood clot.</p> Signup and view all the answers

    Study Notes

    Sample Collection, Handling & Preparation

    • Emphasis on the importance of proper sample collection, handling, and preparation in bioanalytical sciences for accurate results. These processes are critical as even minor deviations can lead to significant variations in data, potentially skewing the results of experiments and clinical diagnostics. Rigor in these practices ensures the integrity and reliability of the findings, which are paramount in research and applied biomedical science.
    • Various biological matrices used, including blood, urine, tissues, hair, nails, oral fluid, and more. Each type of matrix offers unique advantages and challenges, necessitating specific collection protocols and storage conditions to prevent degradation or contamination. Understanding the characteristics of each matrix is essential for selecting the appropriate analytical techniques.

    Biological Matrices

    • Blood-based Matrices: This category encompasses three vital components: whole blood, which contains red blood cells, white blood cells, and platelets; plasma, the liquid component in which blood cells and other substances are suspended; and serum, the liquid that remains after coagulation, which contains antibodies and proteins relevant for various tests. These matrices are commonly used for a range of diagnostic and therapeutic purposes, allowing for thorough analysis of biochemical markers.
    • Urine: The collection of urine for analysis is favored because it is a non-invasive process, allowing for easy and repeated sampling. Its ability to yield a large volume makes it particularly useful in many screening tests. However, pitfalls exist, such as the potential for contamination from skin or external sources. Additionally, variability can arise due to factors like hydration status and dietary influences, which can affect test outcomes.
    • Alternative Matrices: These alternatives to blood and urine provide valuable insights in specific situations. Cerebrospinal fluid (CSF) is crucial for analyzing conditions affecting the central nervous system, while feces can be assessed for gastrointestinal disorders. Hair and nails serve as interesting matrices for monitoring chronic exposure to drugs or toxins, as they provide a historical record of substance intake over extended periods. Oral fluid, often collected via saliva, is increasingly utilized for drug testing and hormone analysis due to its convenience.

    Blood Collection

    • Whole Blood: This type of blood is a complete fluid that contains a mixture of cell types including red blood cells (erythrocytes), which are crucial for oxygen transport; white blood cells (leukocytes), which play key roles in the immune response; and platelets (thrombocytes), which are essential for blood clotting. In forensic analysis, whole blood is particularly valuable because it allows for accurate interpretation of drug concentrations, providing insights into substance usage at the time of sample collection.
    • Plasma vs. Serum: Plasma is the liquid component of blood that is obtained when blood is collected with anticoagulants such as heparin, citrate, or EDTA, which prevent clotting. This allows for the evaluation of a wide range of biochemical analytes. In contrast, serum is the liquid that remains after blood has clotted and is devoid of fibrinogen. It is important to note that the choice of using plasma or serum can significantly influence the concentrations of various analytes, thus affecting diagnostic outcomes.

    Analytes and Metabolites

    • Xenobiotics: Encompasses drugs, their metabolites, and biological molecules in unnatural concentrations.
    • Focus on both parent compounds and metabolites, crucial for understanding drug metabolism and activity.
    • Phase I Metabolites: Involves oxidation and reduction processes.
    • Phase II Metabolites: Involves conjugation processes with substances such as glucuronide and sulfate.

    Detection Windows

    • Different biological matrices provide varying detection windows for substances, influencing method application based on needed time frame.
    • Short-term interrogation may involve blood or saliva, whereas longer-term assessment may use hair or nails.

    Methodology and Applications

    • Bioanalytical workflow includes study design, sample handling, analyte separation, detection, data analysis, and interpretation across various toxicological contexts.
    • Application of methodologies must consider the selected matrix and previous validation studies.

    Forensic and Therapeutic Context

    • Forensic Toxicology: This specialized field of science focuses on the post-mortem analysis of biological specimens, such as blood, urine, and tissues. It plays a critical role in detecting not only common medications but also synthetic drugs and their analogues, which may have contributed to a person's death. By identifying these substances, forensic toxicologists can provide vital information in legal cases. Their work assists law enforcement, legal professionals, and medical examiners in understanding the circumstances surrounding fatalities.
    • Therapeutic Drug Monitoring: This process is essential in the field of medicine as it involves the systematic measurement of specific drug concentrations within a patient's bloodstream. By regularly assessing these levels, healthcare providers can ensure that patients receive the optimal therapeutic effect from their medication. This monitoring helps to tailor treatments to individual patient needs, avoiding subtherapeutic dosing or potential toxicity, thereby fostering better health outcomes and minimizing adverse effects.

    Practical Considerations

    • Collection tubes and anticoagulants significantly influence analyte integrity and concentration.
    • Results obtained from plasma and serum should only be compared if validated due to possible discrepancies in metabolites.

    Guidelines for Selection

    • Choosing the correct matrix depends on the analytes of interest and application requirements, especially in clinical or forensic settings.
    • Consideration of the metabolic phase is fundamental in selecting target compounds for analysis.

    Conclusion

    • Understanding bioanalytical techniques and appropriate sample handling is essential for accurate and reliable results in clinical and forensic applications.### Sample Collection and Preparation in Bioanalytical Sciences
    • Hair and nails can incorporate metabolites and parent drugs.
    • Sample collection is crucial for accurate forensic toxicology and therapeutic drug monitoring.

    Sample Handling Techniques

    • Techniques include:
      • Protein precipitation: This technique involves the removal of proteins from a solution by adding a precipitating agent, which causes the proteins to aggregate and separate from the liquid phase. It is commonly used in sample preparation to reduce interference from proteins in analytical procedures.
      • Liquid-liquid extraction: This method utilizes two immiscible liquids to separate compounds based on their solubility. It is effective for isolating specific analytes from complex mixtures, making it a crucial step in environmental and pharmaceutical analyses.
      • Solid-phase extraction: This technique involves passing a liquid sample through a solid adsorbent material that selectively retains target analytes. This process enhances the concentration of analytes and reduces matrix effects, which is particularly useful in preparing samples for chromatographic analysis.

    Risks in Sample Handling

    • Proper storage of samples is vital to avoid contamination and degradation.

    Bioanalytical Workflow

    • Key phases include:
      • Study design and sampling
      • Sample handling and preparation
      • Analyte separation and detection
      • Data analysis and interpretation

    Lecture Structure

    • Series of lectures are organized into specific themes, covering:

      • Forensic Toxicology involves the application of toxicology in legal contexts, particularly focusing on the detection and analysis of drugs, alcohol, and poisons in biological samples for legal investigations and court cases. It plays a crucial role in criminal justice, helping to establish cause of death, impairment in driving, and determination of toxic substances involved in various crimes.
      • Therapeutic Drug Monitoring is a clinical practice aimed at measuring specific drug levels in patients' blood to ensure efficacy while minimizing toxicity. It is essential in maintaining optimal therapeutic ranges, particularly for drugs with narrow therapeutic indices, and supports personalized medicine approaches.
      • Doping Control involves systematic testing and analysis to detect the presence of prohibited substances or methods in athletes. This process is vital to promoting fair competition in sports and maintaining athlete health, as it identifies those who may be using performance-enhancing drugs.
      • Clinical Toxicology & Chemistry focuses on the diagnosis and management of poisoning and overdose cases. It encompasses the study of chemical substances that can cause harmful effects in humans, utilizing various analytical techniques to identify toxic agents quickly and effectively.
      • Biomarker Discovery is a rapidly evolving field aimed at identifying biological markers that indicate specific disease states or conditions. This area of research is critical for advancing diagnostic methods, monitoring disease progression, and evaluating treatment responses through non-invasive testing techniques.

    Sample Collection and Preparation

    • Sample collection is crucial in bioanalytical sciences, focusing on various biological matrices.
    • Types of samples include blood, urine, saliva, hair, nails, tissues, and alternative matrices like sweat and feces.

    Biological Matrices

    • Biological matrices are categorized into:
      • Blood-based samples – These include whole blood, plasma, and serum, which are crucial for a multitude of diagnostic tests. Whole blood includes all components, while plasma is the liquid portion after cells are removed, containing proteins, electrolytes, and hormones. Serum, on the other hand, is plasma without clotting factors, often used in serology for antibody detection.
      • Urine – Urine samples are valuable for assessing metabolic function and screening for various diseases, due to the waste elimination process of the kidneys, revealing important information about a person's health status.
      • Cerebrospinal fluid (CSF) – This fluid surrounds the brain and spinal cord, and its analysis can assist in diagnosing neurological disorders, infections, and cancers affecting the central nervous system.
      • Tissues – Both two-dimensional (2D) and three-dimensional (3D) models of tissues are essential in studying cellular behaviors, interactions, and responses to treatments in a controlled environment.
      • Hair, nails, and other unconventional samples – These include biological materials like cerumen (earwax) and feces, which can provide insight into exposure to drugs, environmental toxins, and gastrointestinal health, broadening the scope of biomarker analysis and potential diagnostic applications.

    Blood Collection

    • Plasma collection involves the use of anticoagulants such as heparin, citric acid, and ethylenediaminetetraacetic acid (EDTA). These substances prevent clotting and are crucial for preserving the integrity of blood components for analysis.
    • The choice between whole blood, plasma, or serum is dictated by the specific analytical methods employed and the requirements of the tests to be performed. This decision is critical as each type of sample can yield different results due to the presence or absence of clotting factors and cellular components.
    • It is essential to avoid comparing results between plasma and serum unless there has been a thorough validation process. The biochemical composition of these fluids can differ significantly, which may lead to inaccurate conclusions if comparisons are made without appropriate standardization.

    Urine Characteristics

    • Urine is a non-invasive sample, easily collected and allows for large volumes.
    • Commonly exhibits variability in pH (3.5–8) and concentration of salts based on diet and health.
    • Immediate contamination issues occur at exit from the body, affecting analyte integrity.

    Other Biological Matrices

    • Cerebrospinal fluid provides insights into brain activity but requires invasive collection.
    • Nails and hair enable long-term drug metabolite detection, useful in postmortem analyses.
    • Oral fluids (from saliva or sweat) allow easy collection with similar detection windows as urine.

    Metabolites in Drug Analysis

    • Focus on both parent compounds and their metabolites during analysis.
    • Classifications of metabolites:
      • Phase I consists of oxidation, reduction, hydrolysis processes.
      • Phase II involves conjugation with substances like glucuronides and sulfates.
    • Metabolites can sometimes be more indicative of drug use than the parent compounds.

    Specific Drug Examples

    • Heroin is metabolized into 6-monoacetylmorphine, detectable in urine for up to 3 days.
    • 2C-B, a psychoactive substance, has distinct Phase I and Phase II metabolites important for analysis.

    Choosing an appropriate matrix for analys is is vital to obtain reliable and relevant results. Different types of biological matrices include whole blood, plasma, serum, urine, saliva, and tissue samples, each offering distinct advantages based on the substances being monitored. For example, plasma is often preferred for the analysis of proteins, while urine may be selected for metabolite profiling. The matrix chosen impacts the sensitivity, specificity, and overall accuracy of the biomarker analyses. Furthermore, considerations such as the stability of the analytes within the chosen matrix, the method of sample collection, and the potential for interference from other substances must also be taken into account to ensure optimal analytical outcomes.

    • For short-term detection (last 2 days), blood, saliva, urine, or sweat should be analyzed for the parent drug and specific metabolites.
    • For long-term detection (last 6 months), hair or nails are preferred, primarily focusing on metabolites.

    Sample Handling Techniques

    • Key techniques for sample handling include:
      • Protein precipitation: This method involves the addition of a precipitating agent to a protein solution, causing proteins to aggregate and separate from the liquid. It is commonly used to purify proteins or remove them from analytical samples, facilitating more accurate analysis of other components.
      • Liquid-liquid extraction: This technique separates compounds based on their different solubilities in two immiscible liquids, typically water and an organic solvent. It is particularly effective for isolating low molecular weight compounds from complex biological matrices.
      • Solid-phase extraction: In this process, a liquid sample is passed through a solid adsorbent material. Target analytes are retained on the solid phase while impurities are washed away, followed by elution of the analytes for further analysis. This method is crucial for concentrating analytes and improving the sensitivity of subsequent analytical techniques.
    • Storage conditions of samples are crucial to prevent degradation.

    Upcoming Lectures and Topics

    • A series of lectures scheduled, focusing on:
      • Study design and sampling techniques
      • Handling and preparation of samples
      • Analyte separation and detection methods
    • Topics related to forensic toxicology, clinical chemistry, and biomarker discovery will be covered.

    Green Chemistry Principles

    • Defined by Anastas and Warner (1999) for the chemical and pharmaceutical industry.
    • Namiesnik (2013) outlined the Twelve Principles of Green Analytical Chemistry in analytical chemistry.

    Hazards of Common Solvents

    • Dichloromethane/chloroform can depress the central nervous system (CNS).
    • Hexane is associated with neuropathy.
    • Common alcohols can irritate membranes and tissues.
    • Benzene is a known carcinogen.
    • Methanol ingestion can lead to blindness or ototoxicity.

    Greenness Evaluation

    • Eco-Scale is a tool for assessing the environmental friendliness of analytical procedures.

    Implementing Green Chemistry

    • Strategies include:
      • Substituting hazardous reagents with non-toxic alternatives.
      • Employing solventless techniques.
      • Miniaturizing procedures to reduce sample and solvent volumes.

    Microextractions Techniques

    • Microextraction methods represent critical techniques in analytical chemistry, particularly for isolating trace levels of analytes from complex matrices. Solid-phase microextraction (SPME) is a sampling technique where a fiber coated with an extracting phase absorbs target compounds from a sample. This method is advantageous due to its simplicity, cost-effectiveness, and ability to concentrate analytes. Dispersive liquid-liquid microextraction (DLLME), on the other hand, utilizes a combination of an appropriate organic solvent and an aqueous sample, enabling efficient extraction of analytes while requiring minimal sample volume and offering rapid processing times.
    • SPME specifically incorporates custom-made adapters that facilitate in vivo sampling, enabling researchers to obtain blood or tissue samples in small animals like mice or rats with minimal discomfort and risk. This technique reduces the necessity for traditional invasive procedures, improving animal welfare while still allowing for accurate analysis of biological samples.
    • .

    Sample Preparation and Handling

    • Important in various fields: forensic toxicology, therapeutic drug monitoring, doping control, clinical toxicology, and biomarker discovery.
    • Techniques used include:
      • Protein precipitation: A technique used in biochemistry to separate proteins from a solution by adding a precipitating agent, causing the proteins to aggregate and form a solid phase. This method is crucial for purifying proteins for analysis or further biochemical applications.
      • Liquid-liquid extraction: A separation method that involves the partitioning of compounds between two immiscible liquids, often an aqueous phase and an organic solvent. This technique is widely used to isolate target compounds from complex mixtures based on their solubility differences.
      • Solid-phase extraction: A sample preparation process where compounds are retained on a solid adsorbent while unwanted matrix components are washed away. This method enhances the purity and concentration of analytes prior to chromatographic analysis, making it essential for environmental and pharmaceutical applications.

    Sample Preparation Challenges

    • Factors affecting sample preparation include selectivity, cost, time, and difficulty.
    • Green chemistry aims to enhance efficiency while reducing harmful chemical use.
    • The field of microextractions is continuously evolving, promoting efficient and eco-friendly methods for sample collection and analysis.

    Sample Collection and Preparation

    • Essential for various applications: Forensic Toxicology, Therapeutic Drug Monitoring, Doping Control, Clinical Toxicology & Chemistry, and Biomarker Discovery.
    • Techniques include:
      • Protein Precipitation: Separates proteins from samples.
      • Liquid-Liquid Extraction: Utilizes two immiscible liquids for separation.
      • Solid-Phase Extraction: Uses a solid-phase medium to filter analytes from liquids.

    Importance of pKa and pH

    • The dissociation constant (pKa) indicates the strength of an acid; for Salicylic acid, pKa = 2.79.
    • At pH equal to pKa, protonated and deprotonated forms are equal (50% each).
    • Ionization Percentages:
      • At pH = pKa ± 2, an acid is either ≥99% ionized or neutral depending on acidity.
      • Rule of thumb: Acid is ≥99% ionized at pKa + 2; ≥99% neutral at pKa - 2.

    Percent Ionization Examples

    • For Acids:
      • pH 0.79: 99% AH, 1% A-
      • pH 1.79: 90% AH, 10% A-
      • pH 2.79: 50% AH, 50% A-
      • pH 3.79: 10% AH, 90% A-
      • pH 4.79: 1% AH, 99% A-
    • For Bases:
      • 99% ionization occurs at pH = pKa - 2.
      • 99% neutral occurs at pH = pKa + 2.

    Application to Specific Compounds

    • Amphetamine Extraction:

      • Required to adjust urine pH so that 99% is ionized.
      • For extraction, adjust urine sample to pH ≤ pKa - 2.
      • If starting pH is 9.3, a calculation is needed for the percentage of neutral form.

    • MDMA Extraction:

      • pKa of MDMA’s amine group is 9.9.
      • To ensure 99% ionization, adjust urine pH to below 7.9 (9.9 - 2).
      • Urine pH usually ranges from 3.5 to 8, ideally between 4 and 6.5 in healthy individuals.

    General Observations

    • Sample pH significantly impacts the extraction efficiency in bioanalytical processes.
    • Buffering samples helps maintain consistent pH levels for reliable results.

    Sample Collection and Preparation

    • Essential for various applications: Forensic Toxicology, Therapeutic Drug Monitoring, Doping Control, Clinical Toxicology & Chemistry, and Biomarker Discovery.
    • Techniques include:
      • Protein Precipitation: Separates proteins from samples.
      • Liquid-Liquid Extraction: Utilizes two immiscible liquids for separation.
      • Solid-Phase Extraction: Uses a solid-phase medium to filter analytes from liquids.

    Importance of pKa and pH

    • The dissociation constant (pKa) indicates the strength of an acid; for Salicylic acid, pKa = 2.79.
    • At pH equal to pKa, protonated and deprotonated forms are equal (50% each).
    • Ionization Percentages:
      • At pH = pKa ± 2, an acid is either ≥99% ionized or neutral depending on acidity.
      • Rule of thumb: Acid is ≥99% ionized at pKa + 2; ≥99% neutral at pKa - 2.

    Percent Ionization Examples

    • For Acids:
      • pH 0.79: 99% AH, 1% A-
      • pH 1.79: 90% AH, 10% A-
      • pH 2.79: 50% AH, 50% A-
      • pH 3.79: 10% AH, 90% A-
      • pH 4.79: 1% AH, 99% A-
    • For Bases:
      • 99% ionization occurs at pH = pKa - 2.
      • 99% neutral occurs at pH = pKa + 2.

    Application to Specific Compounds

    • Amphetamine Extraction:

      • Required to adjust urine pH so that 99% is ionized.
      • For extraction, adjust urine sample to pH ≤ pKa - 2.
      • If starting pH is 9.3, a calculation is needed for the percentage of neutral form.

    • MDMA Extraction:

      • pKa of MDMA’s amine group is 9.9.
      • To ensure 99% ionization, adjust urine pH to below 7.9 (9.9 - 2).
      • Urine pH usually ranges from 3.5 to 8, ideally between 4 and 6.5 in healthy individuals.

    General Observations

    • Sample pH significantly impacts the extraction efficiency in bioanalytical processes.
    • Buffering samples helps maintain consistent pH levels for reliable results.

    Sample Preparation Overview

    • Elimination of biological elements incompatible with analytical systems is crucial for accurate results.
    • Free fractions must be released from plasma proteins to improve analysis.
    • Interference removal increases sensitivity, often requiring sample concentration.

    Sample Preparation Methods

    • Procedures can be categorized into selective (sample preparation) and non-selective (sample pre-treatment) methods.
    • Common techniques include:
      • Liquid-Liquid Extraction (LLE)
      • Solid-Phase Extraction (SPE)
      • Dilution
      • Protein Precipitation (PP)

    Protein Precipitation (PP) Technique

    • Involves using a PP agent (e.g., methanol, ethanol) with a set volume of plasma.
    • The procedure includes agitation, centrifugation, and collecting the supernatant.
    • Recognized as a non-selective technique suitable for blood-based matrices, with caution regarding protein presence in urine and cerebrospinal fluid (CSF).

    Sample Handling and Applications

    • Essential in fields such as:
      • Forensic Toxicology
      • Therapeutic Drug Monitoring
      • Doping Control
      • Clinical Toxicology & Chemistry
      • Biomarker Discovery

    Sample Composition

    • Plasma: Volume 1-10 mL, pH 7.4, water content 91.5%, protein content 7.5 g/100 mL
    • Serum: Similar composition as plasma, slightly higher protein content.
    • Urine: Volume 5-100 mL, pH ranges from 3-8, water content 98%, lower protein content (0.6 g/100 mL).
    • Saliva: Volume 0.1-3 mL, pH 6.7, water content 98%, very low protein content (0.3 g/100 mL).

    Precipitating Agents for Protein Precipitation

    • Organic Solvents: Methanol, ethanol, acetonitrile create electrostatic interactions with proteins.
    • Acids: Perchloric, trichloroacetic, acetic, m-phosphoric acids modify protein structures via electrostatic interactions.
    • Salts: Ammonium sulfates and copper sulfates typically modify surface tension or promote complexation.

    Solid-Phase Extraction (SPE)

    • Involves three main steps: Conditioning, Loading, and Elution/Wash.
    • Extraction Supports include:
      • Adsorbants: Utilize weak interactions for retention (e.g., silica gel).
      • Ion Exchangers: Facilitate strong electrostatic interactions for cation or anion retention, requiring a slow flow rate.
      • Partitioning: Employs reversed or normal phase modes for apolar and polar compounds respectively.
      • Mixed-Mode: Combines two mechanisms for better selectivity.

    Importance of pH and pKa

    • pH levels significantly influence the efficiency of extraction methods in bioanalysis.
    • Understanding pKa values is essential for optimizing conditions for sample preparation.

    Specific Application of SPE

    • SPE can be applied for analyzing substances like MDMA and Methamphetamine in plasma using LC-MS, highlighting its importance in drug testing and toxicology.

    Solid-Phase Extraction (SPE) Overview

    • Rule: "Like dissolves like," meaning polar compounds preferentially interact with polar stationary phases.
    • Polarity adjustments in washing and elution phases influence compound retention and elution.
    • Understanding the polarity of compounds is crucial; logP (or logD for ionized forms) is a key parameter.

    SPE Procedure Guidelines

    • Conditioning of the sorbent typically uses methanol (MeOH).
    • Prior pH adjustment of samples may be necessary for optimal loading.
    • Washing and elution solvent composition is influenced by stationary phase properties (polarity and charge) and analyte characteristics (polarity and charge).

    Extraction of MA and MDMA from Plasma

    • Critical factors for extraction include knowing the pKa, charge of molecules at given pH, logP, and type of sorbent.
    • Two types of cation exchange modes:
      • MCX (Mixed-mode strong Cation eXchange) - always charged.
      • WCX (Mixed-mode Weak Cation eXchange) - charge depends on pH.

    Sorbent Activation

    • New SPE cartridges/plates must be activated to ensure proper interaction with analytes.
    • Typical activation involves using MeOH followed by water, as specified by the vendor.

    Sample Pre-Treatment

    • Pre-treat samples, especially protein-rich matrices, to prevent clogging of the sorbent.
    • Protein precipitation is necessary for plasma samples, achievable with various solvents and acids (e.g., MeOH, TCA, acetic acid).

    Important Parameters for Loading

    • Considerations include the protein content of the matrix and ensuring the pH is suitable for analyte retention.
    • Slow flow rates (maximum of one drop per second) may be necessary to allow for effective electrostatic interactions.
    • Aim to load samples at a pH less than approximately 7.9 for optimal retention.

    Acidic Conditioning Agents

    • Various acids can be used for protein precipitation during sample preparation, including:
      • Perchloric acid
      • Trichloroacetic acid
      • Acetic acid
      • m-Phosphoric acid
    • The specific choice of acid will depend on the chosen SPE procedure.

    Anticoagulants and Coagulation

    • Anticoagulants prevent the activation of the coagulation cascade, leading to no fibrin clot formation.
    • Plasma is obtained after centrifugation, where blood cells remain in the pellets while supernatant is collected.
    • Plasma composition closely resembles circulating blood, excluding anticoagulants.
    • Plasma contains significant amounts of platelets which may interfere with analyses, unlike serum.

    Sample Handling

    • Collection tubes impact the metabolome and must be evaluated prior to metabolomics studies.
    • Venipuncture collection tubes are typically made of plastic; glass tubes are less common.
    • Tubes can contain clot activators, anticoagulants, or polymer-based gels for easier separation post-centrifugation.
    • Release of plasticizers from tubes can negatively affect mass spectrometry (MS) analysis quality.
    • Repeated use of the same tube type and manufacturer within a study is crucial to maintain data integrity.
    • Proper labeling of tubes is essential, with labels needing to withstand low temperatures (-80°C) during storage.

    Clotting Time and Temperature

    • The time between blood collection and serum separation impacts metabolome and lipidome quality.
    • Ideal clotting time is between 30 and 60 minutes; shorter times may yield incomplete coagulation, while longer may cause cell lysis.
    • Coagulation should occur at room temperature, but samples should be cooled to 4°C post-clot formation to reduce cellular metabolism activity.
    • Consistent clotting time and temperature across all samples is vital, particularly in multi-center studies.
    • Plasma collection does not require room temperature clotting, allowing for immediate cooling post-collection.

    Clotting Agents and Separator Gels

    • Clot-activating agents can speed up clot formation but may increase costs and interfere with analyses.
    • Separator gels and clot-activators have been documented to complicate results in techniques like NMR and LC-MS.

    Anticoagulants in Plasma Collection

    • Selecting the right anticoagulant is crucial for quality data in plasma analysis.
    • Common anticoagulants include heparin, EDTA, citrate, and EDTA fluoride.

    Blood-based Matrices

    • Whole blood, plasma, and serum are key matrices in clinical metabolomics as they reflect physiological states influenced by various factors.
    • Blood is preferred due to ease of collection and long-term storage for future studies, but it requires careful handling.
    • Whole blood, while comprehensive, is complex due to the high cellular content and active metabolism post-collection.

    Serum and Plasma Collection

    • Serum is collected without anticoagulants, triggering coagulation and forming a clot that traps cells.
    • The supernatant serum retains metabolites, excluding fibrinogen and coagulation factors.
    • Plasma is collected in tubes containing anticoagulants, allowing for the separation of plasma without clotting.

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    Description

    Explore the key practices in sample collection, handling, and preparation crucial to bioanalytical sciences. This quiz covers diverse biological matrices such as blood, urine, and alternative samples, highlighting their advantages and challenges. Gain insights into factors affecting analyte concentrations and the importance of proper techniques for accurate results.

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