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13. 4. 2012.

Testing methods in doping

Methods that are used for substance detection in biological agences are:
             I.      Chromatography methods
          II.      Mass spectrometry
       III.      Immunochemical methods
       IV.      Other methods

I. Chromatography methods

Gas chromatography(GC) and liquid chromatography(LC), and also high pressure liquid chromatography(HPLC), are mostly contained in medicaments testing in biology material and that will probably occur in the future, too. Their highest value lies in the fact that they posses unique combination of separation, identification and quantity analysis – metabolites and medicines determination at relatively low concentrations.
  1. Gas chromatography – GC
Operating principe – substances entered into gas chromatograph evaporate at high temperature and carried by inert gass- carrier over the huge surface of non-evaporatable, fluid(stationary) phase in which they are disposed. Effluent of substance that is detected after that process passes through detector.
  1. Liquid chromatography – LC
Operating principe – substances entered into liquid chromatograph under high pressure are separating on column by mechanism of adsorption, distribution, combined effect of these two mechanisms, ion exchange or mechanism of ione couples. After that effluents detection is done. Analysis is done on room or light increased temperature(till 50 degrees). These technique is today more and more in use, due to, separated from gas chromatography, no problems here are evaporation and termostability of the substances and other.
  1. Thin layer chromatography – TLC
Thin layer chromatography – TLC is today widely used for quantitative analysis(separation and identification), also for quantitative analysis. Determination of substances amount in some sample is measured directly by spectroscopic method – densitometry. Measuring of light absorption of specified wave length, measuring of denied or released light intensity, gives specified densitogram. Efficiency of thin layer chromatography responds to gas and liquid chromatography, during which increased sensitivity can be achieved by fluorescent densitometria. Advantage of TLC is possibility to question more examples at the same time.

II. Mass spectrometry(MS)

Mass spectrometry is in farmacokinetic researches one of the most powerful analytic techniques and it can be used alone or in combination with some other technique. Gas chromatography or highly efficient fluid chromatography represent a good combination with mass spectrometry. In this case mass spectrometer is very sensitive and selective detector. Also, it is possible to apply dual mass spectrometry, where two mass spectrometers are connected(MS-MS). Mass spectrometry represents important complementary method to other methods, including NMR(nuclear magnet resonance).
Operating principemass spectral analysis is based on creating and separating of ione molecules or ione molecule fragments of tested substance(medicament) in vacuum under heavy pressure. Creation of positive and negative ions is done through the use of ionization energy of proper power, and separation of iones through application of magnetic and electrofields.

III. Immunochemical methods

These methods are based on princips of specific antibodies principe according to antigene molecules. At this type of test it is needed to have antibodies capable to connect substance that is questioned. Immunochemical methods are shared according to the parameter that is measured:
1)      RadioImmunoAssays – RAI methods, where antigen is marked by responsive radioisotope(125J or 3H), a number of radioactive decays;
2)      EnzymeImmunoAssays – EIA methods, where antigen and antibody are marked with enzyme which appliance to the substrate cannot be measured, by determing UV absorption or in visible area;
Appliance of enzyme immunodetermination(EIA) identifies or determines antigens or antibodies in biological fluids.
This method can be used as qualitative, semiquantitative or quantitative.
3)      FluoroImmunoAssays – FIA methods, or luminoimmunochemical methods are the methods where intensity of light created by fluorescence, or lamination is measured.
Antigene is here marked by luminescent group.

IV. Other methods

1)      Isotope methods
Isotopes are the pairs or atom groups with same atom number, but different atom masses. The ones that broadcast radiation are radioactive, opposite of the stabile ones.
2)      Spectrometric and fluorometric methods
Spectrometric methods( UV spectromethria – 180-400nm and colorimethria – visible area, 400-700nm) measure absorption of light with specified wave length features for determined connection of connections between specified atoms in some molecul.
Fluoromethric method measures light intensity that broadcasts some part of organic molecule after illumination with light of specified wave length.
These are the oldest analytic methods that are used in pharmacology researches.

“Doping in sport”, Marina Djordjevic Nikic

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