Clinical Applications and Testing

Methodologies: Spectrophotometry
& Immunoassay

TDM/TOX (BMS 555)

 Testing Methodologies used in
TDM
Spectrophotometry
UV Spectrophotometry
Atomic Absorption spectrophotometry (AAS)
Flame Emission Spectrophotometry (FES)
Chromatography
Thin-Layer Chromatography (TLC)
Gas Chromatography (GC)
High Performance Liquid Chromatography (HPLC)
Immunoassay
Enzyme Immunoassays (EIA)
Fluorescence Polarization Immunoassay (FPIA)
 Atomic Absorption
Spectrophotometry (AAS)

Hollow
Cathode Chopper Monochromator Detector
(Light source) Flame
Entrance Exit
Specific for
Slit Slit
element tested
Flame Emission Spectrophotometry

Element in the sample is excited by flame

measure radiant energy emitted as element returns to
ground state
Use to measure Lithium
 Immunoassay
Heterogeneous vs. Homogeneous assays
Enzyme Immunoassays
Enzyme-linked Immunoassay (ELISA)
Enzyme-multiplied Immunoassay
(EMIT)
Fluorescence Polarization Immunoassay
(FPIA)
ELISA
EMIT
Ab + Ag-enzyme
-Ag No enzyme
activity

+Ag Enzyme
activity

Screening for particular

drugs or drug families
Cocaine, methodone
Amphetamines,
barbiturates,
benzodiazapines,
cannabinoids, opiates
 FPIA

Fluorescence Brownian motion

FPIA
 Chromatography

Planar Column

Thin Layer
Paper Gas (GC) Liquid (LC)
(TLC)

Separation of sample components based on distribution

between two phases
Stationary Phase: may be solid, gel, or liquid
Mobile Phase: may be gas or liquid
Separation based on adsorption or partition
Adsorption: attraction for stationary phase
Partition: distribution between phases
Chromatographic Sample Preparation
Sample extraction
Eliminates matrix interferences
Concentrates drugs for better sensitivity
Addition of internal standard to compensate for drug loss
during procedure
2 main types
Liquid/liquid
Solid Phase extraction
Sample derivatization
Chemically modify the structure of an analyte to change
its chromatographic properties
Increase volatility (GC): acylation, sialylation,
esterification, alkylation
Enhances specificity and sensitivity
Chromatographs
 Resolution
Column Efficiency
ability of stationary
phase to elute narrow
peaks (peak width)
Selectivity
Ability of system to
differentiate analytes
(absolute retention
time)
Rs = 2 (tr1-tr2)
Wb2+Wb1
Thin-Layer Chromatography (TLC)

Least sensitive chromatographic technique still in common

laboratory usage
Sensitivity: 0.5g/ml
Specimens utilized: Urine, gastric, liver, physical evidence
 TLC Results
Visual identification of spots
Match unknown drug spot with
adjacent standard
Size, shape, and staining
characteristics
Retention Factor (RF)
= Distance migration analyte
Distance migration
solvent front
 Gas Chromatography (GC)

A gaseous mobile phase is used to pass a mixture of volatile

solutes through a column containing the stationary phase
More sensitive than TLC: dependent on detection system
Qualitative or Quantitative
Specimens analyzed: urine, gastric, blood, liver, physical
evidence
 Detectors Used for GC
Type Selectivity Detection
Limit
Thermal Conductance Universal <400 pg
(TCD) propane/ml He
Flame Ionization (FID) Hydrocarbon 10-100pg CHO

Thermionic Selective Nitrogen 0.4-10pg N 0.1-

(NPD: Nitrogen- Phosphorous 1pg P
phosphorus detector)
Mass Spectrometer Charged Ion 1ng: scan mode;
(MS) Fragments 10pg in single
ion mode (SIM)
 GC Separation
Based on relative differences of solutes in vapor pressure and
interactions with stationary phase (retention time, tr)
Adjusted Retention time (tr): = tr- to
Qualitative identification: Relative Retention time (RRT, Tr)

(Retention Time of Analyte)

(Retention Time of Internal Standard)
Quantitative identification: Response factor, Relative response
factor (RRF)
(Response Factor of Analyte)
(Response Factor of Internal Standard)
GC/Mass Spectrometry (GC/MS)
Mixture is separated into its analyte components by GC
identification based on Tr
Analytes are ionized in MS, forming stable charged
fragments (ions)
Fragment ions are separated and detected by MS
according to mass-to-charge ratio (m/z)
Molecular Ion: unfragmented ion of parent molecule
Base Peak: Highest abundant ion in the mass
spectrum
Mass Spectra: Cocaine

Criteria to be reported as present

Right retention time
Corresponding ions present
Ion ratios match standard
GC/MS: Qualitative vs. Quantitative
Analysis
Qualitative: Scan Mode
Full scan spectra: analyte mass spectra
Identification of analyte utilizing library search
Target compound analysis
Quantitative: Selected Ion Mode (SIM)
Selective
focuses on analysis of a limited number of ions
Increased sensitivity
Limited # ions = more signal from each selected
ion can be collected
Quantitation of ion abundance measured as peak
area
High Performance Liquid Chromatography

Separation is based on the distribution of solutes between a

liquid mobile phase and a column-packed stationary phase
More sensitive than TLC: Qualitative & Quantitative
Specimens utilized: Urine, gastric, blood, liver
Blood & urine utilized for screening
Detects antiarrythmics, antibiotics, antiepileptics,
analgesics, TCA
 Detectors: HPLC
Absorbance Spectrophotometry:
Detects the greatest variety of compounds
Least sensitive
Fluorescence
Highly sensitive
May require derivatization
Mass Spectrometry
Very sensitive (femtomole amounts)
Very specific
 Analyte Identification: HPLC
Capacity Factor (k): Volume of liquid required to elute an
analyte from column; also termed retention volume.
Characteristic of specific analyte with given set of
conditions (mobile phase, To, stationary phase)
Associated time = retention time

tr = retention time of analyte

(tr to)
k =
to to = retention time of unretained
compund
Quantitation of Chromatography
External Standard
standard curve used to
calculate the analyte
concentration in sample
Internal standard
known concentrations of
analyte spiked with constant
amount of internal standard
Plot [peak height/area]analyte
[peak height/area]standard
amount of analyte in unknown
samples is extrapolated from
the curve