Drug Bioanalysis 1 Flashcards
A) What is drug bioanalysis?
B) What is qualitative bioanalysis?
C) What is quantitative bioanalysis?
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
What are the three major applications for drug bioanalysis?
- THERAPEUTIC DRUG MONITORING (TDM)
- DRUG RESEARCH AND DEVELOPMENT
- FORENSIC ANALYSIS AND TOXICOLOGY
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)
- 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
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)
- 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
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)
- 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
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)
- 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
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)
- 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
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)
- 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
For Hair samples;
A) What are some advantages?
B) What are some disadvantages?
C) What is it used for?
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
For Saliva Samples;
A) What are some advantages?
B) What are some disadvantages?
C) What is it used for?
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
For sweat samples;
A) What are some advantages?
B) What are some disadvantages?
C) Does it have long or short window of detection?
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
Disucss when other sample types (tissue, faeces, bile extract, gastric contents) are used
- Uncommon/rare usage
- Mostly employed for case dependent forensic purposes
- Biosample collection and processing is considered significantly less agreeable than for biofluids
What are the two types of bioanalytical methods? Discuss the factors that dictate the choice of method
- CHEMICAL METHODS
- 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
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)
- ‘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)
Describe how protein precipitation works (chemical bioanalysis)
- 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
- 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
Describe how solid-phase extraction works (SPE) (chemical bioanalysis)
- 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
Immunoassays can involve either a COMPETITIVE or NON-COMPETITIVE binding process
Describe how competitive binding IA works
- 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
Immunoassays can involve either a COMPETITIVE or NON-COMPETITIVE binding process
Describe how non-competitive binding IA works
- 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
