1.

IMMUNOCHEMICAL METHODS

a. Enzyme-Mediated (or Multiplied) Immunologic Technique (EMIT)

- drug–enzyme complex is used as the marker

-used competitive binding process

-concentrations of drug proportional to free enzyme activity

-EMIT have a wide application in therapeutic and illicit drug monitoring In this type of assay, a sample of
interest with the analyte (drug) is added to a fixed quantity of enzyme-bound drug, and the anti-drug antibody.
After the addition of substrate, absorbance measurements are taken at time intervals to determine the speed
of the enzyme reaction.

The more free drug in the sample, the faster the enzyme reaction because only unbound enzyme-drug
complexes are capable of binding the substrate.

In other words, antigen being measured competes for the antibody binding sites with the antigen that has
been labeled with an enzyme the antibody reagent blocks any enzymatic activity when it is bound with the
reagent enzyme antigen complex (thus preventing the formation of the product with a substrate) then the
free antigen-enzyme complexes compete with the antigen (in the sample) forming a color change which are
proportional to the concentration of the antigen present in the tested sample.

******The increase in absorbance is proportional to the concentration of lithium in the sample. The
intensity of the dye is measured by reflectance spectrophotometry at the end of incubation.

2. FLOURESCENCE POLARIZATION IMMUNOASSAY (FPIA)

-homogenous, competitive immunoassay

Rather than being linked to an enzyme, as in Figure 23-1, A, the drug is covalently attached to a fluorescent probe molecule.
2. CHROMATOGRAPHIC METHODS

- have been applied mainly to the qualita- tive detection of drugs of abuse and toxins, and less to the determination of levels of therapeutic drugs.

- The three major methods are

a. thin-layer chromatography (TLC),

b. high-performance liquid chromatography (HPLC), and

c. gas chromatography–mass spectroscopy (GC-MS).

NOTE: GC-MS is considered the “gold standard” for detection and quantitation of volatile drugs and poisons, newer analytic techniques such as capillary
electrophoresis (CE) and liquid chromatography–mass spectroscopy (LC-MS) are available.

A. THIN-LAYER CHROMATOGRAPHY

- Many compounds can be separated from one another with this method, based on their relative affinities for a polar solid stationary phase (usually

a hydrated silicate) and a mobile liquid phase that is nonpolar (such as 10% methanol in chloroform).

- Depending on these affinities, different com- pounds adsorb to the hydrated silicate at different positions as the nonpolar solvent migrates up the stationary hydrated
silicate.

- For a given solvent system, the ratio of the distance traversed by the compound to the distance traversed by the solvent front is a constant for the compound and can
be used to identify the compound in a mixture. This ratio is called the rf. This technique is central to iden- tifying different drugs of abuse, many of which can be
separated from one another using TLC. The method has been packaged in the form of Toxi- Lab (Irvine, Calif.) kits in which the user is supplied with discrete strips of
silicate, extraction solvents, and color-developing solutions.