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Tracing horse doping with GC/MS.

Testing race horses for illicit drugs and their metabolites is as old as the practice of doping' itself. Nowadays, there are reliable methods to detect tampering with the animals' performance and for identifying illegal substances.

The Challenge: Sample Size

The challenge to the analyst is twofold. There is: a) the lengthly list of structurally very different substances to look for; and b) the requirement to detect them and their metabolites reliably from only a few milliliters of blood. To complicate matters, the kinetics of psychoactive drugs and steroids produce only very low concentrations in the blood stream.

Here's the problem: considering what is known about the relationship of drug concentration and pharmacological effect, are the analytical methods sensitive enough to detect all doping substances? Some substances listed as target drugs simply can't be detected at all in a routine analysis.

The two interrelated key limitations of analytical success, then, are:

* detection limits of the method and instrumentation

* concentration of the unknown substance in the animal's blood.

The Key Issues

Translated into the realities of detecting horse drugging, successful analysis depends on two major issues - proper sample handling and the reliability and accuracy of the analytical instrumentation. Drugs and their metabolites are usually identified with GC/MS. Sample treatment must promote optimal separation of the target drug from the matrix - allowing for extraction, derivatization, sample volume, and drug recovery. Last but not least: short analysis times are essential as well.

Considering Serum Concentration

Taking all these requirements into account, substances are classified as basic, acidic, or neutral, and the first step is a solid-phase extraction. Additional purification of the extract may cause heavy loss of the target substance, and can be undetaken only when the concentration is known to be high. A high level of impurities, on the other hand, can overlap a low concentration of target substance to make it impossible to detect by mass spectrometry.

Salicylic Acid - a Case in Point

A weak analgesic, salicylic acid, serves to illustrate the challenge in routine doping analysis with GC/MS. Of the three isomers of hydroxybenzoic acid, only 2-hydroxybenzoic acid (salicylic acid) is an analgesic. The other two, 3-hydroxybenzoic acid and 4-hydroxybenzoic acid, may nevertheless be contained in certain medications and feed.

In preparing the sample for GC/MS (methylation), all three isomers of hydrobenzoic acid are treated equally, that is, the injection sample contains all three isomers of methoxy benzoic methyl esters. A reliable identification of derivatized salicylic acid requires identification of all isomers. Since 4-methoxybenzoic acid has an unmistakably longer GC retention time than the other two isomers, the analytical focus is narrowed to separation of the derivatized salicylic acid and 3-methoxybenzoic acid methyl ester.

The mass spectrum (Figure 1) shows dimethylated salicylic acid (molecular weight 166) of a doping sample; the ion 105 is characteristic. The 3-methoxybenzoic acid methyl ester contained in the same sample does not show ion 105, but instead identifies that fragment as 107 (see Figure 2) - the two isomers are clearly differentiated. A comparison of the retention times Figure 3) provides further differentiation.

To sum up, clear chromatographic separation of the isomers is essential for reliable identification with MS. The long list of illegal substances encountered in horse drugging provides many similar analytical challenges, and the quality of the analytical instrumentation used must match the difficult task.

Are There Alternatives?

The frontiers of detecting drugging in harness racing are defined by the low serum concentrations dealt with, especially for pharmaceuticals with very high receptor affinity. Even the introduction of techniques like chemical ionization (CI) won't solve the problem. Although CI may increase sensitivity, the technique is less suitable for routine analysis. And even CI is no match for some of substances with extremely high receptor affinities. For these drugs, the switch to analyzing urine samples would not necessarily help. The very low concentrations involved will continue to challenge the detection limits of even the most sensitive instrumentation.
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Title Annotation:gas chromatography/mass spectrometry
Author:Schulz, Rudiger
Publication:Canadian Chemical News
Date:Oct 1, 1990
Words:653
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