Drugs of Abuse – Analytical methods Flashcards
1
Q
What are Sample Adulteration methods?
A
- Biological: Dilute sample by drinking large amounts H2O prior to test
- Chemical: Acid, bleach, oxidants
- Addition of drugs: eg spiking with methadone to demonstrate compliance
- Urine substitution: Synthetic urine, Someone else’s, Pet’s, Other liquid
2
Q
How do we check for Sample Adulteration?
A
Measure urine creatinine/electrolytes
- Creatinine >1.8 mmol/L
- Creatinine <2.0 mmol/L ‘dilute’
- Osmolality >50 mosm/kg,
pH
- Normal urine pH ~4.5-9.0
Specific gravity
- Normal 1.0010-1.0200
Appearance
- Temperature on collection
- Green -? methadone spike
- Odour -? bleach
Measure metabolites
- e.g methadone detected but no EDDP – adulterated
- Measure cotinine – metabolite of nicotine. If they are known smokers and have no cotinine in the sample, raises suspicion that there was a substitution with another individuals sample
3
Q
What are the purposes of Thin Layer Chromatography as a Drug Screening Tool?
A
- Formerly used as initial screening test prior to confirmatory technique
- Can detect parent drugs and their metabolites in most cases
- Specialist manual assay requires considerable experience for interpretation of results
- Off the shelf TLC method ‘Toxi-Lab’ widely cited in older literature but now withdrawn
4
Q
What are features of Thin Layer Chromatography?
A
Precision / Accuracy
- Qualitative assay
- Semi-quantitative assays possible with use of standards and densitometry
Sensitivity
- Relatively poor
Specificity
- Poor; overlapping spots e.g methadone/EDDP
- User variability in interpretation
Sample types
- Primarily urine
- Little/no sample preparation required
Speed
- Long assays – at least 3-4 hr/plate, up to 20 samples/plate
- Difficult to automate
Cost
- TLC plates & solvents relatively cheap
- Standards more expensive
Instrumentation
- Requires fume cupboard for toxic solvents / developing
Staffing / Ease of use
- Experienced staff required for interpretation
- Health and safety aspects of solvents and staining
POCT
- Not suitable
5
Q
What are the purposes of Immunoassays?
A
- Usually used as initial screening test prior to confirmatory method
- Typically, homogenous competitive assays as drug molecules too small for 2 site sandwich assays
- Screening test as it has poor specificity. Results of the immunoassays require confirmation
6
Q
What causes reduced specificity in immunoassays?
A
- Cross reaction is common and unable to separate drugs. Cross-reactivity of immunoassays to a broad class of drugs can hamper their utility.
- Immunoassays are calibrated against a single drug - other drugs in the same class may have greater or lesser degrees of cross-reactivity.
- Opiates are the top and benzodiazepines at the bottoms and can be difficult to differentiate
7
Q
What are the types of immunoassays?
A
- CEDIA: Cloned Enzyme Donor Immunoassay
- EMIT: Enzyme Multiplied Immunoassay Technique
- FPIA: Fluorescence Polarisation Immunoassay
8
Q
What are the features of immunoassays?
A
Precision / Accuracy
- Good precision <10% on most automated analysers
- Lack of specificity impacts on accuracy due to cross reactivity to structurally related drugs
Sensitivity
- Depends on antibody
Specificity
- Limited – drug group specific rather than single drug specific, can also get cross reactivity between drug groups
Sample Types
- Primarily urine
- No sample preparation required.
- Low sample volume
Speed
- Quick <1 hr
- Amenable to large batches
Cost
- Immunoassay kits relatively expensive due to cost of Abs
Instrumentation
- Existing automated immunoassay analysers
Staffing / Ease of use
- Can be added to existing IA repertoire with little or no change in staffing/training
- Minimal additional H&S concerns other than handling liquid reagents
POCT
- Most POCT devices IA based – see later
9
Q
What is the purpose of Gas Chromatography in Drug Screening?
A
- Gold standard for DOA analysis prior to LC-MS
- Reproducible retention times and Excellent chromatographic resolving power
- Remains a common choice for confirmatory toxicology analysis
- Many detector options but typically FID or MS
10
Q
What are the diadvanatgase of using urine DOA screening?
A
- Extensive sample preparation – need for derivatisation (to render analytes less polar and therefore more volatile) and extraction
- User expertise in method development, troubleshooting, interpretation
- Long run times
11
Q
What are the features of Gas Chromatography for Drug Screening?
A
Precision / Accuracy
- Good
Sensitivity
- Flame ionisation / MS detectors very high sensitivity
Specificity
- Detectors used in GC very high specificity
Sample types
- Most sample types amenable to GC i.e. urine, blood, serum, plasma, hair
- Forensics - Fluids, Tissue extracts
Speed
- Long sample preparation due to hydrolysis/derivitisation/extraction
- Chromatography run times at least 10 min/sample
Cost
- Expensive - Hardware, Gases, Columns, Software (libraries)
Instrumentation
- GC, columns, detector, gases, sample prep/derivitisation materials
Staffing / Ease of use
- Extensive experience required for method development, troubleshooting and interpretation
- H&S aspects of gases and solvents
POCT
- Not suitable
12
Q
What are the advanatges of using LC-MS over GC-MS?
A
Has become the standard clinical laboratory tool for confirmatory DOA screening
Advantages over GC:
- Allows simultaneous detection of multiple compounds
- Can analyse polar, non-volatile, heat labile compounds
- No need to derivatise
- Quicker run times
Disadvantages over GC:
- Still requires extensive sample preparation i.e hydrolysis and extraction
- Instrumentation is more expensive
13
Q
What are features of LC-MS?
A
Precision / Accuracy
- Very good typically <10% CV
Sensitivity
- Good
- Most urine DOA guideline cut-off concentrations well above functional sensitivity of MS
Specificity
- Excellent if using MRM, optimisation of chromatography required to ensure maximum resolution and separation of multiple drug peaks
Sample types
- Urine and oral fluid
- May require extensive sample prep (hydrolysis & extraction)
- Does not require derivatisation
- Potential for matrix effects (ion suppression)
Speed
- 5-6 min sample injection time, 100 samples ~10 hr (overnight run)
Cost
- Very expensive hardware, columns, analytical grade purity solvents
- Internal standards expensive
Instrumentation
- Specialist LC-MS equipment required, fume cupboard for solvent waste
Staffing / Ease of use
- Considerable expertise required for method development, troubleshooting and interpretation
- H&S aspects of solvents/gases
POCT
- Not suitable
14
Q
How is Ful screening for Unknown Drugs carried out?
A
Systematic Toxicological Analysis by LC-MS/MS
- Full scan acquisition
- MS1 scans over user defined mass range
- No collision gas, MS2 operated in RF mode only
- All parent ions in selected mass range travel through to detector
- Very short inter scan delay allows rapid switching between cone voltages
- Ramping up cone voltage causes parent molecule fragmentation = Cone Voltage Fragmentation
- Patterns from multiple cone voltage spectra matched to specific library of ~1000 different compounds
- Similar process may be performed by GC-MS – matching characteristic ion spectra to published libraries.
15
Q
What are the benefits and limitations of using Toxicological Analysis by LC-MS/MS?
A
Advantages
- Can be used on existing triple quad mass spec and GC-MS systems
Disadvantages
- Limited libraries – newer drugs might not be in them.
- Many drugs/compounds not detected due to incompatibility with: Chromatography system, Ionisation source, Instrument polarity, Relatively subjective
