Toxicological samples, matrices

Several methods are available for the analysis of tetrachloroethylene in biological media. The method of choice depends on the nature of the sample matrix required precision, accuracy, and detection limit cost of analysis and turnaround time of the method. Since tetrachloroethylene is metabolized in the human body to trichloroacetic acid (TCA), TCA may be quantified in blood and urine as an indirect measure of tetrachloroethylene exposure (Monster et al. 1983). It should be pointed out that the determination of TCA may not provide unambiguous proof of tetrachloroethylene exposure since it is also a metabolite of trichloroethylene. Trichloroethanol has also been thought to be a metabolite of tetrachloroethylene, identified following occupational exposure (Bimer et al. 1996 Ikeda et al. 1972 Monster et al. 1983). However, rather than being a metabolite of tetrachloroethylene, it is more likely that trichloroethanol is formed from trichloroethylene, which is often found as a contaminant of tetrachloroethylene (Skender et al. 1991). Methods for the determination of trichloroethylene and trichloroethanol are summarized in the Toxicological Profile for Trichloroethylene (ATSDR 1993). 

Toxicology samples that we might need to consider may include blood or urine, both of which contain the analytes of interest in a complex matrix that might interfere in an HPLC analysis. 

Quality control Run quality control samples (commercial kit supplied or spiked sample matrix) as part of precision testing during validation. This data can be used to confirm and/or establish QC ranges that will be used to monitor the assay when used on toxicology studies Mean 2SD/3SD or 20% nominal (expected) values... 

GC/MS has been employed by Demeter et al. (1978) to quantitatively detect low-ppb levels of a- and P-endosulfan in human serum, urine, and liver. This technique could not separate a- and P-isomers, and limited sensitivity confined its use to toxicological analysis following exposures to high levels of endosulfan. More recently, Le Bel and Williams (1986) and Williams et al. (1988) employed GC/MS to confirm qualitatively the presence of a-endosulfan in adipose tissue previously analyzed quantitatively by GC/ECD. These studies indicate that GC/MS is not as sensitive as GC/ECD. Mariani et al. (1995) have used GC in conjunction with negative ion chemical ionization mass spectrometry to determine alpha- and beta-endosulfan in plasma and brain samples with limits of detection reported to be 5 ppb in each matrix. Details of commonly used analytical methods for several types of biological media are presented in Table 6-1. 

The analysis of codeine, morphine, 6-monoacetylmorphine (6-MAM, a metabohte of heroin), and cocaine is important for many toxicology labs to determine illicit drug use. When analyzing opiates in urine samples, frequently the matrix chosen for drug screening, the conjugated metabolites must be hydrolyzed however, this process can break down 6-MAM (Christophersen et al., 1987). These compounds can be derivatized to increase sensitivity, and both BCD and NPD are used for these assays. Derivatizations used include reaction with N-methyl-N-trimethylsilyltrifluoroacetamide followed by GC-FID (Lin et al., 1994) or with N,0-bis(trimethylsilyl)trifluoroacetamide (Christophersen et al., 1987 Lee and Lee, 1991), PFPA (Christophersen et al., 1987), or heptafluorobutyric anhydride (HFBA) followed by GC-ECD. All these methods show good sensitivity and selectivity. 

Plasma is the main biological sample used in clinical and toxicological analysis, as concentrations found in this matrix are correlated to the pharmacological effect, as well as to the side and toxic effects. However, oral fluid has also been employed in some specific applications because of the advantages associated to this alternative specimen easy, painless, and noninvasive collection, which does not require qualified personnel, it represents the free analyte fraction, and it has a window of detection similar to that in plasma. Within the possible applications of oral fluid analysis, two are of special relevance ... 

The specificity of an assay evaluates the potential interferences from matrix components and from the different animal species to be used in pharmacology and toxicology experiments. Samples from each species to be studied are analyzed neat (with no added compound) and fortified with known amounts of the candidate, and the results are calculated by means of a standard curve prepared in assay diluent. The response obtained from the neat samples indicate the level of interference from each matrix, and the calculated amounts in the fortified samples show the difference in absolute recovery from the matrix compared with the candidate in buffer. 

Sequential extraction procedures include successive sample treatment with a series of extractants selected on the basis of their ability to dissolve analytes bound to different components of the matrix. The use of sequential extraction procedures simulating natural phenomena, (e.g., acid rain), can deliver detailed information about the origin, mode of occurrence, physiological availability, and mobilization of trace elements. It is also possible to estimate toxicological risk ensuing from the presence of different forms of metals and various phases containing those metals. A... 

The use of matrix-based calibration standards seems also important in toxicological analysis, in order to account for matrix effects during sample workup and measurement (e.g. by chromatographic methods). Consensus should be reached on how many concentration levels and how many replicates per level should be analysed. From our own experience six levels with six replicates each seems reasonable. Weighted calibration models will gen-... 

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