Detection of Drugs of Abuse

Drug of abuse could be done on prescription drugs and even the counter drugs can be
abused
- Cough syrups can be used as a drug of abuse
- not only limited to delicit or designer drugs

Drug abuse
- synonymous to drug overdose
- habitual use of drugs exceeding the therapeutic purpose, their physical and mood are
altered.
- alcohol is part of drugs of abuse = most prevalent (alcohol intoxication)
- stoppage causes withdrawal causing mood sways
- drugs are taken to stimulate them
- ex. drivers take certain drugs causing them to have dilated pupils and red eyes
= taking stimulants
- drug assessment is treated with medical interests
- drugs that can have negative effect
- important to identify the responsible agent to the addiction
- identify drugs in non-overdose situations = for rationale treatment of addiction
- employ use of body fluids and even hair
- qualitative screening detects the presence and absence of the drugs
Disadvantages
- problem, same biochemical composition of other substances would be detected
by the assay itself
- it is important to look for the clinical course as well as the history
accompanied with a confirmatory test
- positive result in single urine sample, do not discard, store until 7 days then
recommend for confirmatory test
- only detects recent drug use
- urine sample is the specimen
- abstinence are being done by drug abusers = not identifiable for short
term drugs
- positive drug screen cannot discriminate between single casual use and chronic
abuse
- confirmatory test provides clear difference between recent and long-term
use
- positive drug screen does not determine the time frame or dose of the drug
taken
 - we cannot determine when did they intake the drugs

The laboratory must ensure that data are legally admissible and defendable
- needs proper documentation and collection of samples is within the premises of the
laboratory
- needs required form that are filled up
> use analytic methods which are validated as accurate and precise
- we need to eliminate the false positives and negatives
> documentation of specimen security
- there is a designated comfort room, the opening has filter so that the patients cannot
get water
- there should be no sinks and other cleaning materials that are present
- water and bleach are commonly adulterized
- measure the temperature, pH, volume is 60 mL
- single cubital so that single patient at a time
> Protocols and procedures must be established
> measurement of temperature, pH, SG and creatinine
- to determine if urine is really the sample given
- to determine adulterated specimen
> specimen collection is monitored, cahin of custody is establsihed
- 3 forms are filled
- 1 copy is submitted to confirmatory if positive

Drug Testing Process

1. Sample Collection
- female patients = we do not allow patients with menstruation
- sample is submitted to the reception and access the submilibility
- properly labeled, name, date and time of colection, age and gender
2. Sample accession
3. Drug screening
- single urne sample may be utilized for different drugs
4. Drug confirmation
- report as true positive if it is positive in confirmatory
- if negative result, check the cut-off value for the relevance of the substance
5. Sample Reporting
Specimen
1. Urine
- most preferred specimen analysis
2. Blood
- if impossible to collect urine sample
- ex. if patient has menstruation or rescheduled for next screening
- patient that are aggressive = for medicolegal purposes (preso)
- anticoagulant is sodium flouride = gray top
- 2 samples = 1 for screening and the other one is for confirmatory and reference
3. Hair
- utilized for medico-legala nd forensic
4. Oral fluids
5. Sweats

Drugs of Abuse
1. Amphetamines
2. Barbiturates
3. Benzodiazepines
4. Cannabinids
5. Cocaine
6. Methadone
7. Opiates
8. PCP

Prevalence rate
Alcohol = highest prevalence 58%
- collegiate level to midlife age
MJ or Marijuana = 16%
Cocaine = 2-3%

Testing of Drugs of abuse

Detected using 2-tiered approach
> screening tests are also refered to as spot tests
- required prior to work = required for employee which is newly hired
> simple, rapid, inexpensive and capable of being automated
- most common is immunochromatographic test kit
> have good analytical sensitivity with marginal specificity
- eliminate falase positives, rule out false-positive result (determine relevance of analyte
to a ceertain degree)
> calibrated at established cut-off concentrations
- if the dosage is lower or undergone abstinence, drugs cannot be measured
Confirrmatory
- do only for positive results
> have high sensitivity and specificity
> require method different from screening
- gold standard is GC-MS
- has lower cut-off values = presence of parent substance or active substance, it can still
be detected

1. Chromogenic reactions
- reagents added to sample producing colored reaction indicating presnece of analytes
2. Immunoassay-based procedures
- can be employed for both screening and confirmatory assays
- test kits = for screening
- other forms = for confirmatory testing
3. Chromatographic techniques
A. thin-layer chromatography
- do not use instruments or analyzers
- inexpensive method for screening
B. GC and Liquid Chromatography
- labor is intensive and complicated

Immunochemical methods
1. Homogeneous Immunoassays
A. EMIT
- enzyme activity is directly proportional to the concentration of the drug of abuse
- in the EMIt immunoassay, utilize drug bound to an enzyme, incubate the enzyme drug
complex and antibody
- if there is no presence of drug, the drug bound to the enzyme will attach to the
antibody forming antibody drug complex
- causing decrease enzymatice activity
- if there is free drug, it will compete to the drug complexed to the enzyme
- higher concentration of free drug binds more to antibody forming drug-anttibody
complex
- this will produce more enzymatic activity
B. FPIA
- instead of being bound to enzyme, there would be a fluorescent probe-drug complex
- drug attached to chloropore
- inversely proportional relationship of the drug of abuse and polarization of light
- absence of elicit drug or free drug or liberated drug, the drug bound to fluorescent
porbe is the ones attached to the antibody
- presence of free drug, it binds to the antibody, the fluorescence or emitted light is
decreased due to increased concentration of drug

Chromatographic technqiues
- 3 major methods
1. Thin-layer chromatography
2. HPLC
3. GC-MS
Other methods
- CE and LC-MS

1. Thin-Layer Chromatography
- has 2 phases, stationary and mobile phase
- the stationary is solid and polar
- hydrated silicate
- mobile phase is liquid phase that is nonpolar
- 10% methanol in chloroform
- substance of abuse depends on how they migrate towards the strip
- then identify the RF or retention factor
- substances put on the eluent then measure the substances after they are absrobed
- computation: distance leading edge of component moves/total distance solvent front
moves
- for screening, we need to identify what is the present substance on the patient sample
- the silica gel test strip dipped t 3 different solutions or stains
- after dipping, we culd employ 3 ways to detect the presence of substance
- control or test strip can e used to compare the results
- employ utilization of UV light since there are ones that produce fluorescence ex.
amphetamines and metamphetamines

2. High-Performace Liquid Chromatography

- allows presence and absence of durg of abuse as well as quantitation of the amount of
substance
- employed for therapeutic drug monitoring but valuable for tricyclic antidepressants and
their metabolites
- quantitiation of concentration is on a nanomolar to micromolar range
Principle:
- it has stationary phase which can either be polar that uses silicic acid and nonpolar
which is C-18 columns.
- migration of substance with the application of consisten flow of liquid
- like FES and AAS, needs internal monitoring or internal standard must have the same
structure and mechanism to detect the target analyte of interest
- most common standard is protriptyline = less inactive tricyclic compound
- the test is highly reproducible

3. Gas Chromatography-Mass spectroscopy

- considered as the reference method or glod standard because of its sensitivity and
reliability
- employs 2 techniques
- compounds are heated into gas phase or derivitize to mke them liable facilitating
migration otwards liquid mobile phase
- depends on how substances would be absorbed on the stationary phase, we will have
idea on what type of substances are present and have an idea of how much of the
substance that are present

Interference
> immunoassays for amphetamines and metamphetamines, they have different cross-
reactivites, sympathomimetic amine can be positive
> cross reactivity in opiates is low in immunoassays
Adulterants
- in collection area, no presence of water
> change pH = any acid or base, lemon juice, soap, house cleaning products
> change oxidant level = bleach, hydrogen peroxide, PCC
> nitrites can also cause alterations