Drug Bioanalysis 1 Flashcards

1
Q

A) What is drug bioanalysis?

B) What is qualitative bioanalysis?

C) What is quantitative bioanalysis?

A

A) Qualitative and Quantitative methods for analysis of drugs in biological samples

  • Experimental testing (typically in the developmental phase) to establish a drugs compounds bioacitvity/ potency using in vitro and/ or in vivo

B)

  • Typically employed for drug detection in biofluids - usually fairly simple/inexpensive

C)

  • normally used for measuring a LOW drug concentration in biofluids - in comparison can be relatively time consuming/expensive
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2
Q

What are the three major applications for drug bioanalysis?

A
  • THERAPEUTIC DRUG MONITORING (TDM)
  • DRUG RESEARCH AND DEVELOPMENT
  • FORENSIC ANALYSIS AND TOXICOLOGY
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3
Q

For Therapeutic Drug Monitoring (TDM);

A) What does it involve/do?

B) Can it be used for all drugs?

C) What is its main purpose? (real life use)

D) What are some other non-general uses?

E) When is the biosample collected?

F) What is the assay accuracy level required and is it expensive to do

A

A)

  • Involves interval measurement of a patient drug plasma concentration to maintain a relatively constant and safe drug level within the patient (check to see if drug level in patient is asafe and effective)

B)

  • No, TDM is only required for specific and limited range of drugs
  • Included in this range of drugs, are drugs with a small therapeutic window of concentration i.e. possess narrow therapeutic range TR

C)

  • Normally used for assessment of long-term drug therapy in patients with chronic diseases/conditions

D)

  • Monitoring of patient drug compliance
  • Monitoring of patients with hepatic/renal issues
  • Identifying the effects of drug interactions
  • Dose tailoring for a specific patient

E)

  • Timing of patient biosample collection is important and usually obtained when ‘steady state’ drug level is achieved

F)

  • Drug bioanalyis must be specific, relatively cheap and fairly rapid
  • May sacrifice asssay accuracy
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4
Q

For Drug Research and Development (R and D);

A) What is it used for?

B) Give some examples of real life use

C) Is the timing of the sample important?

D) What is the assay accuracy level required and is it expensive to do?

A

A)

  • Drug bioanalysis for pharmaceutical studies relating to pharmacokinetics, drug bioavailability and bioequivalence

B)

  • Important aspect of drug candidates during early discovery/development stages and clinical trials
  • New generic drugs require comparative bioequivalence studies against existing drug products

C)

  • As for TDM the timing of sample collection is crucial

D)

  • High level of assay accuracy usually required but often assay cost is not very important
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5
Q

For Forensic analysis and toxicology;

A) What does it involve/do?

B) What is it used for? (real life purposes)

C) How is it different from TDM and drug ‘R and D’

D) What does routine drug screening/testing involve?

A

A)

  • Involves drug screening/testing procedures for the detection of legal, illegal and banned drug substances

B)

  • Criminal cases e.g. medical negligence, murder
  • Workplace/occupational issues e.g. safety, pre-employment
  • Sporting events e.g. Olympic games
  • Monitoring of narcotic use e.g. in addicts and school children
  • Routinely employed for use in clinical and criminal cases to establish drug poisoning (for ‘Tox. Screening’)

C)

  • Typically involves the use of a broader range of biosample types than TDM and drug ‘R & D’

D)

  • Most routine drug screening/testing involves the use of noninvasive sampling e.g. testing of urine sample
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6
Q

A) What are some of the common biosample types that are used for testing?

B) What are the two most common biosmample types for bioanalysia from part a

C) What are biosample choices influenced by

D) What does the Window of Detection (WOD) mean? What happens if there is a long window of detection and influecnes the WOD?

A

A)

  • Blood, urine, hair, saliva, sweat, tissue (biopsy), faeces (stool), bile extract, gastric contents

B)

  • Blood and urine due to ease of handling, reliability and level of sample preparation

C)

  • Influenced by analysis of prupose, sample collection issues and length of the ‘WOD’ for the drug/sample type

D)

  • WOD = time frame within which a drug can be detected since its last use/administration
  • Long WOD = drugs persists for longer in sample/in vivo and it s detectable over a longer period after its intake
  • WOD depends on drug level/ length of use and biosample type
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7
Q

For blood samples;

A) What type of blood is used?

B) What does the bioanalysis designed to assess?

C) What is the main advantage of using blood samples?

D) What are some problems/issues

E) Is pre-treatment required? If so, why?

F) What type of studies is it used for

A

A)

  • Pre-treated blood products derived from patient/subject blood samples (plasma or serum)

> Plasma: Pale yellow liquid component of unclotted blood (after centrifugation) which contains clotting factors e.g. fibrinogen

> Serum: Blood plasma minus clotting factors

B)

  • Drug level in blood typically represents that present in tissues
  • Bioanalysis is designed to assess total drug in the blood but drugs can be bound to plasma proteins e.g. albumin

C)

  • Drug level in blood typically represents that present in tissues
  • Most accurate sample for establishing in vivo drug concentration

D)

  • Major issue is the short window of drug detection
  • Biosamples will contain a complex mixture of endgoneous substances
  • Samplng procedure is invasive
  • Analysis is the most laborious, time-consuming and expensive of the assay procdures

E)

  • Pre-treatment is required to extract and isolate drugs (workup procedure) as biosamples contain endogenous substances

F)

  • Used almost exclusively for TDM and most ‘R & D’ studies
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8
Q

For urine samples;

A) Is the drug concentration higher in blood or kidneys

B) Is the drug level in urine represantative of in vivo concentrations?

C) Does it have a smaller or higher window of detection compared to blood?

D) What are some advantages of urine samples?

E) What are some disadvantages of urine samples?

F) Does it require pre-treatment like blood?

G) What is it used for?

A

A)

  • Drug concentration in urine sample is usually higher than for blood products due to kidneys that serve to concentrate xenobiotics

B)

  • Drug level in urine is NOT representative of in vivo concentrations

C)

  • Much larger window of detection compared to blood

D)

  • Sampling procedure = non-invasive
  • Large quantity of sample can be collected
  • Samples are cleaner than blood samples
  • Assays are less intensive/time consuming and represent the cheapest form of drug bioanalysis

E)

  • Prone to physiological variables and sample tampering

F)

  • Yes, but not as much as blood –> samples are cleaner

G)

  • Most commonly used for ‘routine’ forensic drug detection/screening
  • Limited use in TDM due to above issue of in vivo representation and also drug output is dependent on patient fluid intake
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9
Q

For Hair samples;

A) What are some advantages?

B) What are some disadvantages?

C) What is it used for?

A

A)

  • Largest limit of detection of all biosamples (months to years)
  • Long term drug detection is possible and samples are also unaffected by prolonged periods of drug abstinence
  • Sample collection is non-invasive and only small amount required

B)

  • Laborious/time-consuming ‘workup’ - may need sample decontamination of cosmetics resulting in fairly expensive bioassays

C)

  • Normally reserved for forensic/historical purposes
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10
Q

For Saliva Samples;

A) What are some advantages?

B) What are some disadvantages?

C) What is it used for?

A

A)

  • Unbound ‘free’ drug can be measured and also permits the detection of more recent drug use/administration
  • Non-invasive sample collection (but can be problematic)
  • Less expensive than blood and hair

B)

  • Short window of detection
  • Requires time-consuming workup (more expensive than urine)

C)

  • Normally employed for narcotic/illegal drug testing purposes
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11
Q

For sweat samples;

A) What are some advantages?

B) What are some disadvantages?

C) Does it have long or short window of detection?

A

A)

  • Typically used for ‘long term’ sample collection periods

B)

  • Considered intrusive due to the use of sweat patches for sample collection but induced sweating and skin wipes are less intrusive
  • Sweat patches are prone to tampering
  • Uncommon sample type –> mostly used when other sample types are not suitable (e.g. pediatric patients)

C)

  • Short window of drug detection
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12
Q

Disucss when other sample types (tissue, faeces, bile extract, gastric contents) are used

A
  • Uncommon/rare usage
  • Mostly employed for case dependent forensic purposes
  • Biosample collection and processing is considered significantly less agreeable than for biofluids
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13
Q

What are the two types of bioanalytical methods? Discuss the factors that dictate the choice of method

A
  1. CHEMICAL METHODS
  2. IMMUNOASSAYS

Choice of method is dictate by wide range of factors:

  • DRUG PHYSIO-CHEMICAL PROPERTIES: Profoundly influences assay/detection technique and sample preparation procedures. Most influential include drug stability and solubility.
  • Drug concentration: Depends on various factors such as drug stability, metabolism, plasma protein binding and presence of other drugs. Varies significantly for clinically available drugs from reasonably high level ( ug/ml) to very low level (pg/ml).
  • Drug and its related substances: Bioanalysis may require the detection/measurement of drug and/or its metabolites
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14
Q

For chemical bioanalysis;

A) Describe it uses

B) What are the methods used

C) Why do these chemical methods require a workup (clean up) procedure before biosample analysis can be started

D) What are the common workup procedures?

A

A)

  • ‘standard’ and most routinely employed approach for the vast majority of drug bioanalysis
  • Common method for research studies and forensic science but its use in TDM is decreasing
  • Assays are usually specific to a drug or drug group

B)

  • Spectrophotometry: Occasionally used for rapid drug detection but lacks specificity/sensitivity. Most favourable in terms of costs and technical skill requirement.
  • Fluroimetry: - Relatively uncommon technique despite good specificity/sensitivity. Only applicable to limited number of drugs
  • HPLC: Premier method for bioanalysis (esp. LC-MS) due to its separative/isolative capability, high specificity/sensitivity and very accurate for effective drug detection/quantification. Hampered to a certain degree by equipment costs and technical skill requirements.

C)

  • Workup is necessary due to the ‘mixed/composite’ nature of many biosample types that may contain a range of ‘contaminant’ and potentially interfering endogenous substances
  • Intergral aspect of sample preparation due to its ability to extract/isolate drug analyte and improve assay sensitivity
  • May increase assay length and cause ‘in sample’ drug loss can be accounted for with internal standard (IS)

D)

  • PROTEIN PRECIPITATION
  • LIQUID-LIQUID EXTRACTION (LLE)
  • SOLID-PHASE EXTRACTION (SPE)
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15
Q

Describe how protein precipitation works (chemical bioanalysis)

A
  • Simplest of the bioanalytical workup procedures
  • Involves the addition of various chemical reagents to induce precipitation of proteins
  • Reagents include acetonitrile (CH3CN), trichloroacetic acid (CCl3CO2H) and inorganic salts (e.g. ZnSO4, (NH4)2SO4)
  • After addition of the precipitating reagent, the biosample is subjected to centrifugation to give a supernatant for bioanalysis
  • Proces also involves dilution and therefore generally used for easily detected drugs at high concentration
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16
Q
A
  • Removes sample contaminants by selective solvent extraction coupled with pH adjustment using acids/bases
  • Drug extraction involves converting/maintaining drug in unionised form with acid or base followed by solubilisation in organic solvent
  • Solvent evaporation can also be undertaken to increase assay sensitivity then drug ‘back-extraction’ into aqueous media
  • Each workup step involves minor drug loss which can be tracked/evaluated via the addition of a defined small amount of an internal standard (IS) prior to workup
17
Q

Describe how solid-phase extraction works (SPE) (chemical bioanalysis)

A
  • Very popular approach due to its ease of use/rapidity with similar principle to LLE but much simpler and uses less solvents
  • Employs a small cartridge system containing suitable adsorbent material (mainly silica-based) to remove assay interferants
  • Interferants may either pass through the adsorbent (and the drug is retained on the adsorbent) or retained (and drug passes through)
  • SPE cartridges can be fitted directly onto HPLC system
18
Q

For Immunoassays;

A) What is it

B) What is it used for

C) what is used to detect/measure

D) Describe how drug IA works and what they are used for

A

A)

  • Biochemical test used to detect/measure the level of a substance via a binding reaction of an antibody to its antigen to give an antibody-antigen complex

B)

  • Widely utilised in clinical chemistry for diagnostic and detection purposes e.g. HIV, insulin, pregnancy testing

C)

  • detect/measure an antigen (i.e. drug or endogenous substance) or antibody against a pathogen
  • For most antigen assessment a monoclonal antibody is utilised which recognizes a single site on the antigen
  • Antigen detection necessitates the use of a labelled replica or conjugated version of the antigen

D)

  • For a drug IA the drug molecule serves as an ‘antigen’ that can bind to a specifically designed drug antibody
  • Drug IAs are extensively used for ‘rapid’ drug detection purposes and semi-quantitative drug analysis
19
Q

Immunoassays can involve either a COMPETITIVE or NON-COMPETITIVE binding process

Describe how competitive binding IA works

A
  • Analyte antigen (Ag) within the biosample competes with the added labelled antigen for binding to the antibodies (Ab)
  • Amount of ‘labelled Ag-Ab’ complex is then measured and is INVERSELY proportional to the amount of the analyte antigen that was originally present within the biosample
  • FOR A DRUG IA: drug molecules in the biosample act as the 1st Antigen which compete for antibody access with the labelled drug molecules that serves as the 2nd Antigen
  • After a washing step the amount of labelled drug bound to the antibodies is determined and will be INVERSELY PROPORTIONAL TO DRUG AMOUNT IN THE SAMPLE
20
Q

Immunoassays can involve either a COMPETITIVE or NON-COMPETITIVE binding process

Describe how non-competitive binding IA works

A
  • Analyte antigen (Ag) present within the biosample binds to the antibodies (Ab) to generate antigen-antibody complex
  • Labelled antibody is subsequently added and binds to the ‘bound’ antigen of the antigen-antibody complex
  • Labelled antibody does NOT compete with the antigen
  • Amount of ‘labelled Ab-Ag-Ab’ complex is then measured and is DIRECTLY proportional to the amount of analyte antigen that was originally located in the biosample
  • Also known as a ‘Sandwich assay’ because the analyte antigen is sandwiched between the antibody and labelled Ab
  • Represent a very sensitive and specific IA
  • NOT generally used for drug bioanalysis but common for detection of certain biological/pathological markers
21
Q
  • ‘primary’ antibody components are ‘surface bound’ to the assay vessel, multi-well assay plate (used for assessment of multiple samples) or polymer beads
  • IAs are relatively sensitive BUT may occasionally give ‘False Positive’ results due to ‘cross-reactivity’ (lack of specificity) arising from binding to ‘non-analyte’ molecules

What two classes can IAs be divided into? Describe these classes

A

HETEROGENOUS IA

  • Require an extra separation step to remove any unbound ‘free’ antigen. Represent the standard form of IA (especially for drug bioanalysis) which include radioimmunoassays (RIAs) and ELISA.

HOMOGENOUS IA

  • Do NOT require a separation step to remove unbound ‘free’ antigen. Typically faster and easier to perform. Several types of IA available (e.g. EMIT, SLFIA) but limited use for most drug bioanalysis but utilised for certain illicit drug and biomarker testing.
22
Q

For radioimmunoassay RIA;

A) How does it work?

B) Is it commonly used?

A

A)

  • Uses radioactive/radiolabelled version of the analyte antigen
  • Usually 3H (T), 14C or 125I radiolabelled forms are employed
  • Involves pre-mixing of known quantity of radioactive antigen with the antibody then addition of the biosample with the unlabelled/non-radioactive ‘cold’ analyte antigen
  • Both antigens compete for antibody and at higher levels of ‘cold’ antigen more of it binds and displaces the radiovariant
  • Separation of ‘bound’ Ab complex (with labelled/unlabelled antigen) is required from the unbound ‘free’ antigens
  • Separate via filtration/centrifugation or precipitation using an absorption/binding process (e.g. coated charcoal, 2nd Ab)
  • Amount of radioactive antigen (both the ‘free’ and ‘bound’ forms) is then measured using standard techniques for radiolabelled substances e.g. LSC

B)

  • relatively uncommon due to high costs, use of radioactive materials and need for speciliased treatment
  • Extemely senstive/specific bioanalytical tool and represents the oldest form of IA
  • Above is major method for plasma level evaluation of many hormones, digoxin and certain drugs of abuse
23
Q

For Enzyme-linked immunosorbent assay (elisa) ;

A) How does it work?

B) What are the different types?

A

A)

  • Widely applied and popular form of IA - used for drugs, disease/pathogenic biomarkers and food allergens
  • Most common IA in drug analysis - especially for illicit drugs
  • Very sensitive IA but limited use in TDM and ‘R & D’ due to inadequate accuracy for quantitative analysis
  • Adopts similar approach to RIA but applies either an antigen-enzyme or antibody-enzyme conjugate
  • Enzyme converts a substrate to a coloured product that can be detected/measured via spectrophotometry or fluorimetry
  • ELISA represents a form of colorimetric assay

B)

  • In competitive and direct/indirect ELISA: amount of coloured product is inversely proportional to analyte in the biosample
  • In non-competitive or ‘Sandwich’ ELISA: amount of coloured product is directly proportional to analyte in the biosample
24
Q

What is Enzyme Multiplied Immunoassay Technique (EMIT)

A
  • Used mostly for drug and protein biomarker detection
  • Like ELISA uses an analyte-enzyme conjugate and colorimetry
  • Biosample, antibody and enzyme substrate are mixed then an analyte-enzyme bound conjugate is added
  • If analyte absent in the biosample: Antibodies will bind to analyteenzyme conjugate and cause enzyme deactivation (no colour rxn.)
  • If analyte present: Antibodies bind to analyte and the ‘free’ enzyme conjugate can converts substrate to detectable coloured product​
25
Q

What is Substrate Labelled Fluorescence Immunoassay (SLFIA)?

A
  • Similar applications to EMIT but uses fluorimetry and an analyte-substrate conjugate which is mixed with biosample and antibod
  • Enzyme is added and if analyte is absent in sample the antibodies bind to the conjugate and substrate is unavailable to the enzyme
  • If analyte is present the antibodies bind to it and ‘free’ substrate conjugate is converted by the enzyme to a fluorescent product