TDM technologiesVINEETHA MENON

High Pressure Liquid Chromatography

PRINCIPLE:

• HPLC column is filled with solid particles like silica and the sample mixture is separated into compounds as it interacts with the column particles.

• separation is influenced by liquid solvents condition, chemical interaction between sample mixture and liquid solvent, and chemical interaction between sample mixture and solid particles packed inside the separation column.

• 2 variants of HPLC:– Normal phase HPLC– Reverse phase HPLC

Normal Phase HPLC• Stationary phase: polar silica particles• Mobile phase: non polar hexane• Polar compounds in the sample mixture will stick to the polar silica and the non

polar compounds will pass out more quickly through the column.

Reverse Phase HPLC• Stationary phase: non polar silica• Mobile phase: methanol• Non polar compounds in the sample mixture will stick to the non polar silica

particles and the polar compounds will pass out more quickly through the column.

Retention time• Time taken for a particular compound to travel through the column to the detector.• Its measured from the time at which the sample is injected to the point at which

display shows a maximum peak height for that compound.

Detection• Common method used is UV absorption.• Amount of light absorbed gives the amount of drug present in the sample mixture.

Interpretation of results• UV spectroscopy: quantification• Retention time: identification• Chromatogram: identification, quantification

Gas-liquid chromatography

PRINCIPLE:

• In GLC, the mobile phase is inert gas like helium and stationary phase is a microscopic layer of liquid on an inert support.

• The GLC column is placed within an oven. Very high temperatures is used to cause sample vaporization. Vaporization separates the various molecules in the samples into their different fractions.

• The gaseous compounds being analyzed interact with the stationary phase (adsorption) which causes each compound to elute at different time (retention time) and the substances are identified qualitatively.

• Commonly used detectors are flame ionization detector and thermal conductivity detector.

• thermal conductivity detector: identification• Retention time: identification• Chromatogram: identification, quantification

Liquid Chromatography Mass Spectrometry

PRINCIPLE:

• column is filled with solid particles like silica and the sample mixture is separated into compounds as it interacts with the column particles.

• separation is influenced by liquid solvents condition, chemical interaction between sample mixture and liquid solvent, and chemical interaction between sample mixture and solid particles packed inside the separation column.

• Elute is analyzed by mass spectrometry.

• Elute is loaded into MS instrument and undergoes vaporization .

• The components of the sample are ionized by a variety of methods like impacting with electron beam resulting in the formation of ions.

• Ions are then separated according to their mass- to- charge ratio in an analyzer by electromagnetic fields.

Radioimmunometric assays

PRINCIPLE:

• The specimen is mixed with a specific antibody to the drug being analyzed as well as a known quantity of the same drug labeled with radioactive iodine.

• The drug in the sample and the radioactive labeled drug compete for sites on the antibody.

• The sample is washed to remove unbound radioactive drug. A gamma counter is then used to measure the amount of radioactivity in the sample as counts per minute (CPM).

• This number is inversely proportional to the amount of drug in the sample.

Particle Enhanced Turbidimetric Inhibition Immunoassay

PRINCIPLE:

• PETINIA is a homogenous competitive immunoassay.

• The latex particle-bound drug binds to the drug-specific antibody, forming insoluble light-scattering aggregates.

• This causes an increase in the turbidity of the reaction mixture.

• The rate of particle aggregation (turbidity) is inversely proportional to the concentration of drug in the sample.

Enzyme Immunoassay

• EIA uses a non-radioactive enzyme label.

• This eliminates the need for special handling and reduces disposal costs.

• Sample with unknown amount of antigen is immobilized on a solid support usually a polystyrene microtiter plate.

• After immobilization of the antigen, a specific antibody is added forming a complex with the antigen.

• the antigen in a sample competes for limited antibody binding sites with antigen conjugated to a reporter enzyme. 

• In the final step a substance containing the enzymes substrate is added.

• When chromogenic substrate is added to the assay to develop color, samples with high antigen concentration generate a lower  signal than those containing low antigen concentration, yielding the inverse correlation between antigen concentration in the sample and color development in the assay.

• Subsequent reaction produces a detectable signal, usually a color change in the substrate.

Enzyme Multiplied Immunoassay Technique

• Sample mixture containing the drug is mixed with a solution containing a known concentration of antibody and the enzyme drug substrate.

• Antibodies that do not become bound to drug in the sample bind instead to this enzyme-drug "conjugate".

• The conjugate is designed in such a way that when antibodies bind to its drug portion, the enzyme is deactivated.

• The more drug there is in the sample, the more of the antibody is bound to it and less is available to deactivate the conjugate.

• If an enzyme substrate is present that is converted to a colored or fluorescent product, the presence of drug will inhibit the formation of the detectible product to a degree related to the concentration of the drug.

Fluorescence Polarization Immunoassay

• Sample is incubated with a known quantity of the fluorescent-labeled drug and an antibody specific for the drug.

• Fluorescein-labelled drug competes with unlabelled drug for antibody.

• Sample excited with plane polarized light (490 nm).

• Polarized light is emitted in certain angles depending on whether the fluorescent-labeled drug is bound to antibodies or not.

• Small, free drug-fluorescein, rotates faster leading to less emission.

• Larger, antibody-drug-fluorescein, rotates slower and produces more emission.

• More drug in the sample; less fluorescein labelled drug bound to antibody; lower emission of plane polarized light.

Chemiluminescence

• Chemiluminescence  is the emission of light, as the result of a chemical reaction. 

• The most common chemiluminescent assay methods are either

1. enzyme-amplified (or)

2. direct chemiluminescent measurements.

In direct chemiluminescence, such as the ADVIA Centaur system:

• The sample is incubated with the AE-labeled drug, and the drug specific antibody.

• Once the AE is oxidized by hydrogen peroxide and the sample becomes alkaline, a light emission occurs.

• The lower the drug concentration in the sample, the greater the light emission.

• An example of an enzyme-amplified chemiluminescence method is the IMMULITE system.

Affinity Chrome-Mediated Immunoassay

• Magnetic particles coated with the drug are added to bind free antibody-enzyme conjugate.

• The reaction mixture is then separated magnetically. • Following separation, the supernatant containing the drug-antibody-

enzyme complex is transferred by the instrument to another cuvette and mixed with a substrate.

• B-galactosidase catalyzes the hydrolysis of CPRG (chloropenol red B-galactopyranoside) to produce CPR (chloropenol red) that absorbs light at 577 nm.

• The change at 577 nm due to the formation of CRP is directly proportional to the amount of drug present in the sample, and is measured by a bichromatic (577, 700 nm) rate technique.

Cloned Enzyme Donor Immunoassay

• The polypeptide chain of the beta-galactosidase enzyme from Escherichia coli is presented as two inactive fragments, the large fragment, termed enzyme acceptor (EA), and a small fragment, enzyme donor (ED), which contains the 5% of enzyme missing from the EA portion.

• By conjugating drug to the ED fragment, the addition of antibodies that bind the drug can prevent the formation of an intact and therefore active enzyme.

• Any drug present in the sample competes for antibody–binding sites, such that an increase in drug concentrations yields less binding of antibody to the ED fragment and therefore results in more enzyme activity which can be monitored spectrophotometrically.

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