Химия и химические науки /2.Теоретическая химия

Panasenko O. I., Samura T. O., Postol N. A., Filatova O. O.,

Gotsulya A. S., Safonov A. A., Buryak V. P., Shcherbyna R. O., Salionov V. O.

Zaporizhzhia State Medical University

MODERN ANALYTICAL TOXICOLOGY

The last 25 years have seen many advances in methods for detecting identifying and measuring drugs and other poisons in biological fluids with consequent improvement in the scope and reliability of analytical results. The value of certain emergency assays and their contribution to therapeutic intervention has been clarified. Some such assays are performed for clinical purposes, but have overt medico legal implications and require a high degree of analytical reliability. Examples include “brain death” and child abuse screening and instances of suspected iatrogenic poisoning. In addition, demand for the measurement of plasma drug and sometimes metabolite concentrations to aid treatment [therapeutic drug monitoring (TDM)], for drug of abuse and laxative/diuretic screening, and for laboratory analyses to monitor occupational exposure to certain chemicals, has increased. Nowadays, a range of powerful analytical methods, typically chromatographic methods, ligand immunoassays and other techniques are available to the analytical toxicologist. However, it remains impossible to look for all poisons in all sample at the sensitivity required. It is vital therefore that the reason for any analysis is kept clearly in view. Although the underlying principles remain the same in the different branches of analytical toxicology, the nature and the amount of specimen available can vary widely, as may the time-scale over which the result is required and purpose for which the result is to be used. All these factors may, in burn, influence the choice of method for a particular analysis. Gas-solid chromatography (GSC) and, more commonly, gas-liquid chromatography (GLC) have made a notable contribution to both the qualitative and quantitative analysis of drugs and other organic.

Poisons, especially since the introduction of sensitive detectors such as the flame ionization detector (FID) and the electron-capture detector (ECD). These detectors ate often complementary because the ECD shows a selective response to certain compounds or derivatives compounds, in practice those containing either a halogen or some other electronegative or “electron-capturing” species such as a nitro moiety, while the FID responds to most organic compounds. More recently, the introduction of nitrogen-selective detectors (NSD), also known as alkali flame-ionization (AFID) and nitrogen-phosphorus (NPD) detectors, which show on enhanced and selective response to compounds containing C-N bonds or phosphorus, further extended the scope of GLC. However, the use of gas chromatography in combination with mass spectrometry (GS-MS) sometimes referred to as an example of a “hyphenated technique”, provides high sensitivity together with unparalleled selectivity and can identify unequivocally many compounds using only nanogram quantities of material, and has largely supplanted the use of the NPD, certainly as far as qualitative work in concerned.

High performance (originally high pressure) liquid chromatography (HPLC) has achieved wide application in analytical toxicology since the early 1970s. Gases and very volatile solvents excepted, most analytic are amenable to analysis by HPLC or a variant of the basic procedure, in contrast to GC which is restricted to the analysis of compounds which are both stable and volatile at temperatures up to approximately 350 °C. However, the use of HPLC in the qualitative analyses of drugs is restricted to a certain extent by the lack of a sensitive universal detector analogous to the FID in GC, although a range of sensitive and reliable detectors [notably UV absorption, fluorimetric, electrochemical (ED) and MS] of varying sensitivities and selectivity are now available. In addition, fractions of column corresponding to the chromatographic peaks of interest may be collected and analyzed off-line, for example, by immunoassay. Immunoassay have also found wide application, whether radioimmunoassay (RIA) or more recent variants, for example, enzyme multiplied immunoassay technique (EMIT) and cloned enzyme donor immunoassay (CEDIA), and are often highly sensitive. Enzyme-based assays, such as that for paracetamol (acetaminophen), have also been described. However, all of these assays have the disadvantage that antibodies, enzyme, or specified binding proteins have to be prepared for each analyses or group of analyses before an analysis is possible. On the other band, these and similar assays may often be used directly in small volumes of aqueous media (homogenous assay), in contrast to chromatographic methods which often require some form of purification procedure, for example, solvent extraction, prior to the analysis. Although, immunoassays can be very sensitive, some may be poorly selective, that is the antibody may recognize several structurally similar molecules. Sometimes, thus cross-reactivity can be exploded as in screening for classes of abused drugs.

Capillary GC, often with MS detection, is widely used both in systematic toxicological analysis (STA) and in the specific analyses, although packed column GC may still find a place for certain applications. ILPLC, often nowadays in conjunction with MS, is used to analyze specific compounds or groups of compounds, although STA procedures based on diode-array detectors (DAD) and wavelength rationing techniques are also used. The problem in STA (poisons screening drug screening, unknown screening) is simply to detect reliably as wide a range of compounds as possible in as little sample (plasma/serum/whole blood, urine, vitreous humor, stomach contents or vomit, or tissues) as
possible at high sensitivity, but with no false positives. Ideally some sample should be left to permit confirmation of the results using another technique and also quantitation of any poison(s) present to aid clinical interpretation of the results.
When screening for unknown substances in is important to adopt a systematic approach in order to eliminate possible contenders and to “home in” of the compounds present. STA can be divided into three key stages.

The aim of the sample preparation step in to retain all the toxicologically important substances whilst removing potentially interfering sample matrix components. Thus, as wide a range as possible of analytes of interest, including lipophilic and moderately polar, acidic, basic and neutral species, should be isolated. To increase the yield of analyte, the sample may be treated with β-glucuronidase/arylsulfatase to hydrolyze conjugated metabolites.

The aim of the differentiation/detection step is identify the relevant compounds in the minimum amount of time. This requires a combination of relatively nonspecific (“universal”) assays with highly specific methods. Immunoassays, particularly is the antibody has wide cross-reactivity, are useful for identifying classes of drugs. TLC has the advantage that all the nonvolatile materials in the extract remain on the plate, whereas with GC and HPLC there always the possibility that compounds have not been eluted from the column. Obviously, one analytical technique cannot separate and identify all the possible compounds of interest; for example, only a finite number of compounds can be resolved on a single TLC plate. The ability of a given analytical method to identify a compound from a given set of test compounds is known as the identification power. One approach in quantifying identification power is the use of discriminating power (DP):

DP = 1 - (1)

where m is the number of pairs of compounds which are not resolved and N is the number of compounds examined. The concept of discriminating power was introduced by with the aim of quantifying the ability of paper chromatography, TLC and GLC to give unequivocal identification of unknowns. When
this approach was applied to an investigation of the separation of 34 neutral compounds in 15 TLC systems, it was shown that one system had the greatest DP (0,75). However, by combining the results from two of the systems, the DP could be increased to 0,88. As the identification power increases so the DP increases towards 1,0. A second approach to define the identification power is the mean list length (MLL). A list length is defined as the number of feasible candidates for a particular analytical parameter, for example, the retention index in a GC system. The average of all list lengths gives the MLLs can be calculated for a combination of systems MLLs are > 1,0, but will approach 1,0 as identification power increases. In both cases (DP and MLL), examination of a low number of test compounds will give an overestimation of the identification power of the method [1]. The greater the number and range of techniques that are available to the analyst, the greater the probability that unknown substance will be identified correctly. Investigation of the responses of various analytic to different detector, for example FID/ECD, can provide valuable information about the nature of a compound. HPLC-DAD not only provides spectral information, but also can confirm peak purity via multiple scanning of an eluting peak. Hyphenated techniques such as GC-MS can provide robust analytic identification, particularly when combined with computerized libraries of electron ionization (EI) fragmentation data that can be searched rapidly to confirm compound identity. In addition, chemical ionization (CI) MS can be used to obtain the M_r of a substance. Analyses may be chemically modified to improve their chromatographic properties or “detectability”, but derivatization can also give useful qualitative information. One old, but classic, example is the so-called “acetone-shift” (reaction of acetone with a primary amine to give the corresponding Schiff`s base). Amphetamine, for example, reacts with acetone to form N-(1-methyl-2-phenylethyl)propanimine.

Figure. Reaction of amphetamine with acetone

The third step in STA is to compare the observed data with validated database information. Clearly, databases used in compound identification need to be regularly updated, and must include information on not only parent compounds, but also contaminants. It is important that the analytical techniques used in establishing such databases are reproducible, but within and between laboratories.
Enzymatic methods for blood ethanol using alcohol dehydrogenase with spectrophotometric measurement of a coenzyme are available in kit form such as that available for the Abbott TD_x/AD_x. GC analysis of ethanol either by diluted with deionized water [3], or by static headspace sampling, is also widely used, particularly in forensic work. GC is advantageous because methanol, 2-propanol and acetone may be separated and measured simultaneously. Methanol poisoning from ingestion of synthetic alcoholic drinks is one of the few cause of acute poisoning “epidemics” and measurement of blood methanol us important in confirming the diagnosis and in monitoring treatment.

More that 20 additional volatile compounds may be encountered in acute poisoning cases arising, for example, from deliberate inhalation of vapor in order to become intoxicated [“glue sniffing”, solvent abuse, inhalant abuse, volatile substance abuse (VSA)]. Some of these volatile compounds have metabolites that may be measured in urine in order to assess exposure, notably hippuric and methylhippuric (toluric) acids (from toluene and the xylenes, respectively) and trichloracetic acid (from trichloroethylene). However, most volatile substances are excreted unchanged in exhaled air, and thus whole blood is the best sample in which to detect and identify these compounds [2]. In order to help diagnose chronic poisoning, where elevations of only a few mgL^-1 (parts per billion, ppb, i.e. parts per thousand million) of blood or serum can be important, good accuracy and reproducibility are essential [4].

Sample contamination during collection (e.g. from sample tubes, or even from syringe needles in the case of chromium) and manganese and within the laboratory itself can be serious sources of error. This applies particularly to common elements such as lead and aluminium. Modern methods for measuring toxic metals in biological materials very enormously in terms of complexity, cost accuracy and sensitivity. Some techniques (isotope dilution MS, neutron activation analysis) are in reality reference methods. Atomic absorption spectrometry with either flame or electrothermal atomization using a graphite furnace has seen employed widely, but us furnace has been employed widely, but us being superseded by inductively coupled plasma-mass spectrometry (ICP-MS). In the case of serum iron, however, reliable kits based on the formation of a colored complex remain widely used in clinical chemistry. ISP-MS is a multi-element technique that can detect and measure elements with detection limits of mg·L^-1 to ng·L^-1. Different isotopes of an element can also by measured. For some elements, the relative abundance of the isotopes depends upon the source of the metal. Therefore, by measuring the isotope ratios of an element such as lead in a sample from a chronically poisoned patient with those found in material present in the patients immediate environment it may be possible to localize the source of exposure. Ethnic cosmetics such as surma may contain from 0 to 80% elemental lead as either the oxide or sulfide and such products are important causes of lead poisoning. So-called “traditional” medicines may also contain toxic doses of salts of lead or other toxic metals [4].

SUMMARY. Analytical toxicology is concerned with the detection, identification and measurement of drugs and other foreign compounds (xenobiotics) and their metabolites in biological and related specimens. The analytical toxicologist can play a useful role in the diagnosis, management and, in some cases, the prevention of poisoning, but to do so a basic knowledge of clinical and forensic toxicology is essential. Moreover the analyst must be able to communicate effectively with clinicians, pathologist, police and, possibly, others.

REFERENCE

1. Boone C. M. Evaluation of capillary electrophoretic techniques towards systematic toxicological analysis / Y. P. Franke, R. A. Zeeuw, K. Ensing // J. Chromatogr. – 1999. – Vol. 838. – P. 259 – 272.

2. Braithwaite R. A. Clinical and sub-clinical lead poisoning: A laboratory perspective / R. A. Braithwaite, S. S. Brown // Human Toxicol. – 1988. – Vol. 7. – P. 503 – 513.

3. Curry A. S. Determination of ethanol on blood by gas chromatography / A. S. Curry, G. W. Walker, G. S. Simpson // Analyst. – 1966. – Vol. 91. – P. 742.

4. Delves H. T. Measurement of total lead concentrations and lead isotope ratios in whole blood by use of inductively coupled plasma source mass spectrometry / H. T. Delves, M. J. Campbell // J. Anal. At. Spectrom. – 1988. – Vol. 3. – P. 343 – 348.