MLS418: CLINICAL CHEMISTRY LECTURE
TOXICOLOGY: METHODS OF DETECTION IN SERUM AND URINE
Bernardo Ordaneza, Jr., RMT March 18, 2021
OUTLINE → after the addition of the Drug-Enzyme complex,
I. Introduction III. Chromatographic the DE complex will be free (will not attach to the
II. Immunochemical Methods anti-drug antibody)
Methods A. Thin Layer → addition of the substrate will result in its
A. Enzyme-Mediated Chromatography attachment to the DE complex, producing a
Immunologic Technique B. Hih-Performance Liquid product, usually a color reaction
B. Fluorescence Chromatography → the product will detach itself from the enzyme. The
Polarization C.Capillary Electrophoresis enzyme will have free active sites ready for other
Immunoassay D.GC-MS substrates (substrates are in excess in assays)
C.Drug-Binding to E. LC-MS → Recall:
Antibodies F. GC/IR [E] + [S] ⇒ [ES] ⇒ [E] + [S]
Note: Use Henry’s as a reference → color reaction corresponds to the concentration or
amount of free drug
I. INTRODUCTION ● If the patient is NOT a drug-user
● study of substances introduced exogenously into the body → The anti-drug antibody is added
● this also involves the study of the biological effects and → no amphetamine molecules will react with the
methods for detection of exogenous chemical compounds antibody
that profoundly influence bodily functions* → addition of the DE complex will allow reaction with
→ could be deleterious or therapeutic the anti-drug antibody
● Four areas: → active sites are blocked by the anti-drug antibody,
→ drugs of abuse preventing the attachment of substrate to the DE
→ therapeutic drugs complex
→ environmental carcinogens ▪ therefore no product is formed, no color
▪ mutagenic - induces DNA mutation reaction
→ toxins and acute poisons
● Basic Techniques:
→ Immunochemical Methods (Immunoassays)
→ Chromatographic Techniques
II. IMMUNOCHEMICAL METHODS
● used for screening tests
● Types:
→ Enzyme-Mediated (or Multiplied) Immunologic
Technique)
→ Fluorescence Polarization Immunoassay
● Note: Much of the drug testing performed today is using
homogenous immunoassays
→ all performed in a solution; no need for phase separation
or washing
→ allowed rapid testing
A. ENZYME-MEDIATED IMMUNOLOGIC TECHNIQUE
● a ‘marker’ drug (drug-enzyme (D-E) complex) is used
→ drug covalently attached to an enzyme (e.g., G6PD)
● exogenous drug (serum/urine) competes with the D-E
complex for the anti-drug antibody
→ more exogenous drugs mean more free D-E, thus an ● Note: The Syva Corporation pioneered EMIT
increase in enzymatic activity ● Mouse monoclonal antibodies ⇒ anti-drug antibody to
● the anti-drug antibody (present in the testing kit) attaches to D-amphetamine and D-methamphetamine
a specific molecule of the drug ● Amphetamines labelled with bacterial G6PDH as
→ eg. a test kit for amphetamine drug has an antidrug substrates
antibody specifically attaches to amphetamine molecule ● Other ingredients: Tris buffer, BSA, Preservatives,
stabilizers
B. FLUORESCENCE POLARIZATION IMMUNOASSAY
● a ‘marker’ drug (drug-probe complex) is used
● drug covalently attached to a fluorescent probe molecule
(fluorophore)
● uses polarized light to detect if the probe is…
Simplified Mechanism → immobilized by the anti-drug antibody (emitted light with
● The anti-drug antibody is added to a cuvette the same polarization as exciting light); or
● The patient sample (urine/serum) is added → exogenous drug competes with the D-P complex, and
● If the patient is a drug-user for amphetamine D-P “tumbles” in the solution (decreased polarization)
→ free amphetamine molecule present in the patient ● Polarizing filters → block other waves that are not
sample will bind to the anti-drug antibody perpendicular to the polarizing filter to allow only the
polarized light to pass through
→ has a net effect of decreasing the intensity of light
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Simplified Mechanism Takeaway
● Patient sample: POSITIVE ● Once the Antibody concentration is saturated, the
→ Amphetamine molecules bind to the Fab portion D-Ab complex will no longer increase despite the
of the immobilized anti-drug antibodies in the test presence of free drug molecules
kit Langmuir Expression
→ Drug-probe complex is used, which will randomly
move around the solution (since no attachment
occurred with the anti-drug antibodies)
→ excitation (polarized) light is applied
▪ take note that the polarized light is traveling in ● Ab0 = total concentration of Ab
the same direction → variable
→ the fluorophore gets excited and emits light that is ● k = equilibrium constant (D-Ab complex)
no longer polarized ● n = number of Ab binding sites/molecule of Ab
▪ therefore when the emitted light from the → constant
fluorophore passes through another polarizing ● D = drug concentration
filter, some light may not pass through ➢ The response of the system is directly proportional to the
therefore there is a net decrease in light drug concentration up to a certain extent
intensity detected → Recall Concept of Michaelis Menten Equation
▪ a decrease in polarization is proportional to the
amount of drug present in the sample ⇒
POSITIVE
● Patient sample: NEGATIVE
→ immobilized antibodies present in the test kit will
bind to the Drug-Probe complex
▪ no free DP complex will be found in the solution
→ Remember that the antibodies are immobilized, III. CHROMATOGRAPHIC METHODS
therefore the orientation (?) of light will not be ● separation and qualitative detection of drugs of abuse and
changed once the fluorophore emits light toxins, and to a lesser extent, determination of therapeutic
→ upon reaching the second polarization filter, the drug levels
polarized light will be the same as the initial ● used for confirmatory tests
polarized light → confirmation of a positive immunoassay test result
▪ no decrease in polarization ⇒ NEGATIVE ● Major Types:
→ Thin Layer Chromatography
→ High-Performance Liquid Chromatography
→ Gas Chromatography-Mass Spectrometry
▪ the gold standard for the detection, quantitation of
volatile drugs
● Newer Analytic Techniques:
→ Capillary Electrophoresis
→ Liquid Chromatography-Mass Spectrometry
→ Tandem Mass Spectrometry
A. THIN LAYER CHROMATOGRAPHY
● qualitative detection of drugs
● Principle: Separation of mixtures into pure molecules of the
same compound
● compounds separated based on their relative affinities for:
→ a polar solid stationary phase (usually hydrated silicate)
→ a nonpolar liquid mobile phase (such as 10% CH 3 OH
in chloroform)
● different compounds adsorb to the stationary phase at
different positions as the mobile phase migrate up
C. DRUG-BINDING TO ANTIBODIES
● the concentration of free marker* drug is a measure of
D-Ab
● the concentration of D-Ab is related to D
→ the more D is present, the more D-Ab is formed
● however… because Ab concentration is constant…
● at high concentrations of D, all Ab will be saturated, and no
further D-Ab could form
● There is a non-linear relationship between the → speed of migration of compounds depends on the affinity
concentration of the drug and the response of FPIA and to the silica gel strip (polar) or the eluting substance
EMIT (nonpolar)
→ EMIT ⇒ intensity of the color reaction ▪ the more polar the substance is, the higher the affinity
→ FPIA ⇒ decrease in polarization to the silica gel strip therefore the distance travelled is
less than the non polar substances
− polar ⇒ migrates slowest
− non polar ⇒ migrates fastest
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● For a given solvent system, this ratio is constant for the ● Identification of Specific Drugs
compound and can be used to identify the compound in the ➢ standard color reaction
mixture. ▪ dipped successively in different solvents → color
Distance f rom the origin to the substance reaction
Distance f rom the origin to the solvent f ront
= rf value
➢ ultraviolet (UV) light
▪ excites fluorescence in some compounds
● Compare with Reference Patterns based on:
➢ rf
➢ Characteristic color change
➢ Presence/absence of fluorescence
Reliability of the Method
● sensitivity of the method is limited by the ability of the
Toxi-Lab® Drug ID System naked eye to detect color changes and/or the presence of
● Extraction procedure fluorescence
1. Acidic drugs are separated from basic drugs → level of detection is on the order of 1 μg/mL of
compound present on the strip
● extraction procedures are occasionally inefficient
● extraction and evaporation procedures are time-consuming
(~30 mins)
● cocaine has a number of polar metabolites that barely
migrate from the origin
→ converted completely to polar metabolites before
excretion
● some difficulty in distinguishing among various opiates may
occur
→ rf values are can be close to one another
[Left] Acidic Drug i.e. phenobarbital
▪ although experienced personnel can make the
[Right] Basic Drug i.e. MDMA or Ecstacy
disctinction
▪ Almost all drugs of abuse are basic drugs ● nontoxic drugs may give cx color changes and rf values
− amine-derivatives that are similar to those for drugs of abuse
▪ Acidic drugs are almost exclusively barbiturates → e.g., antihistamines appear on the A strip very similar to
▪ The common sample used by chromatography: urine amphetamines
− non-invasive; can be collected in large quantities
B. HIGH-PERFORMANCE LIQUID
2. To isolate the basic drugs, urine is treated with base;
CHROMATOGRAPHY (HPLC)
▪ to isolate acidic drug, urine is treated with acid
● quantitative detection and sharper separation of therapeutic
drugs and drugs of abuse
→ sensitivity: nanomolar to micromolar range
● stationary phase : uniform, ultrafine particles (make sure
that the absorptive surface area is greatly increased)
▪ amine will behave as a base or acid depending on packed into a column
what substance it will react to → polar (silicic acid)
▪ R-NH3+ is not soluble in nonpolar eluent → nonpolar (C-18 columns) reverse phase chromatography
▪ R-NH2 is soluble in nonpolar solvent ● resistance to flow in the column is high
▪ for the basic drug to be extracted using a nonpolar → high pressures* needed to deliver a constant flow of
solvent, the amine to be used must be in the form of mobile phase
NH2 not NH3 → Liquid chromatography used to utilize gravity
3. a nonpolar organic phase is used to extract the ● internal standard
compound → a compound similar in structure to the drug of interest
→ added to the specimen to be analyzed in a known
● Evaporation procedure
concentration
1. a small paper disk is added to the organic extraction
▪ to know how much of the internal standard is
mixture
recovered from the column in the eluate
2. a solvent is then evaporated so that all basic drugs
adsorb onto the paper disk Uses
● Separation procedure ● therapeutic drug monitoring (TDM)
1. perform thin layer chromatography ● detection of cocaine and heroin in urine
▪ paper disk is placed at the “origin” of the silicate strip ● separation and quantitation of tricyclic antidepressants* and
▪ strip is placed in the migrating nonpolar solvent their metabolites
− Strips are labelled as A and B → amitriptyline → tricyclic antidepressant
− A ⇒ basic drugs → protriptyline → internal standard
− B ⇒ acidic drugs (barbiturates)
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C. CAPILLARY ELECTROPHORESIS
● a variant of TLC that has the advantages of HPLC
→ may not be true (?)
● a capillary tube lined with silicate is used as the solid
support in an electrophoresis apparatus
→ driving force for separation is the voltage (on the order of
25 kV) rather than the pressure (in HPLC)
● highly versatile
→ can be used to separate serum proteins and small
molecules**
Advantages
● analyte selectivity is based on different physicochemical
principles of separation without the need to change
instrumental hardware
→ capillary zone electrophoresis
→ micellar electrokinetic capillary chromatography (MECK)
▪ make detergent as a buffer system
→ capillary isotachophoresis
→ capillary isoelectric focusing
→ capillary electrochromatography
→ capillary gel electrophoresis
Uses
● not (yet) used as widespread as immunoassay techniques
in clinical toxicology
Mechanism ● commonly used in analytic forensic toxicologic studies and
● HPLC Pump will pump the solvent and sample to the molecular dx studies
injector
● The substances will go along the HPLC column,
differentiated by their retention time (may be affected by
temperature)
→ high affinity to stationary phase ⇒ high retention time
● Eluate is subjected to different types of detectors
● Data will be acquired
D. GAS CHROMATOGRAPHY-
MASS SPECTROMETRY (GC-MS)
● •“gold standard” for drug testing
→ the best confirmatory testing procedure
→ highly sensitive and reliable
→ best for identifying volatile compounds
● two techniques
→ gas-liquid chromatography ⇒ for separation
→ mass spectrometry ⇒ for detection and identification
Gas-Liquid Chromatography
● compounds are directly heated into the gas phase or are
derivatized to make them labile to facilitate heating them
into the gas phase
● stationary phase: liquid
→ usually hydrocarbon or silicone oil that coats a solid
support in the column
● mobile phase: gas (carrier gas)
→ typically an inert gas such as nitrogen, helium, or argon
or a low mass gas such as hydrogen
→ gas has little affinity to substance
● separation is based on the ability of each compound to
adsorb to the stationary phase
→ which partially depends on the relative solubilities of the
compound in the gas versus the liquid phase
● the eluate will be detected and quantitated by the mass
spectrometer
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Mass Spectrometry Interface Methods
● molecular ionization of the eluate ● electrospray ionization (ESI)
→ eluate is bombarded with electrons, turning them → for ionizable analytes of high MW and/or high polarity
(mostly) into cations with charge 1+ → more clinical and forensic application compared to APCI
→ at high temperatures, the electrons of the compounds ● atmospheric pressure chemical ionization (APCI)
could be excited, and the excitation is susceptible to the → for analytes of lower MW and less polarity
loss of electrons and become positively charged ● Difference between two methods: molecular weight and
● some molecules may undergo fragmentation polarity of substance to be nebulized
→ some bonds may break, forming unconnected chemical
species
● molecule ions are then passed through an electric field
→ generated by four rods that are subjected to rapidly
alternating currents (quadrupole detector)
● depending on the frequency of the AC, certain molecule
ions with specific m/z can pass through the field to the
detector
● separation of molecule ions on the basis of their m/z
(mass/charge ratio)
→ molecular weight/charge
● must be in a vacuum to prevent interferences in the air
Uses
● confirm positive test results from screening assays
→ drugs of abuses assays
→ poisoning detection in acute or chronic intoxication
→ therapeutic drug identification and quantitation (TDM)
→ pharmacokinetic and drug metabolism studies
F. GAS CHROMATOGRAPHY COUPLED
WITH IR SPECTROSCOPY (GC/IR)
● uses IR (or FTIR) spectroscopy
→ uses the light of high λ (low f) that excites vibrionic
states of molecules involved in bond stretching and bond
angle bending
→ uses for IR spectroscopy for detection (makes use of
light and its interaction with the substance)
● eluate is subjected to infrared light and each compound has
a characteristic IR absorption pattern (“fingerprint”)
→ even closely related compounds can be distinguished
readily from one another
● used for detection of amphetamines
Fragmentogram of Cocaine for a Confirmatory Test
E. LIQUID-CHROMATOGRAPHY-
MASS SPECTROMETRY (LC-MS)
● can be utilized for nonvolatile c ompounds
● has limitations compared to GC-MS, but has become a
complementary method to GC-MS
● interface between LC and MS…
→ must volatilize nonvolatile compounds separated in LC
→ must remove liquid solvent from LC
→ must correct flow rate incompatibility between LC and Cocaine
MS
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