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High concentrations of a drug in a sample means milligrams of drug in a liter or kilogram of sample matrix. The needle (1 mg) in a haystack (1 kg) means looking for and accurately measuring 1 part in 1,000,000. Low drug concentration make this ratio 10 to 1000 times larger. Separating the drug from most of the background material can be very useful in the determination of drug concentrations. The inclusion of similar compounds such as precursors or metabolites makes the separation even more of a challenge.
Some of these techniques include:
A significant separation can be achieved at times by simple centrifugation. Centrifugation of whole blood after clot formation produces serum with no red blood cells and fewer plasma proteins. Centrifugation of whole blood pretreated with anticoagulants such as heparin, EDTA or citrate produces plasma. Protein can be removed from plasma by centrifugation after the addition of trichloroacetic acid (TCA) or acetonitrile.
Extraction can be used to remove unwanted interfering compounds or to concentrate the compound(s) of interest. The use of various organic solvents of differing polarity and/or aqueous buffers can provide excellent resolution based on the solubility of the free compounds of interest and/or their salt forms. Extraction may be used to remove lipophilic interfering compounds or to remove the desirable compounds into a cleaner environment.
One method of separating free drug from plasma samples is ultrafiltration. The sample can be forced (by centrifugation) through a membrane filter. The filtrate is protein free solution of the compound.
Chromatography can be used in a clean-up mode or in a sensitive analytical mode. In the clean-up mode the compound of interest may be adsorbed tightly onto a column while the interfering compounds are washed through it and subsequently flushed from the column with a 'stronger' eluting solvent. In the analytical mode the challenge is to choose the right column (stationary phase) and mobile phase so that the compound is eluted within a reasonable time and well resolved from other components in the sample.
The very tight, strong binding between a compound and an antibody can be very useful as a method drug analysis. Using controlled, limited amounts of antibody and labelled drug it is possible analyse the drug sample.
Copyright 2001-3 David W. A. Bourne (david@boomer.org)