Drug metabolites sample preparation

Because the instability of the N-oxide metabolite, which was subjected to decomposition during sample preparation (solvent evaporation during offline SPE), online SPE LC/MS became the method of choice for the application. Hsieh et al. (2004) built a system with two TFC cartridges and one analytical column, and another system with two TFC cartridges and two analytical columns for GLP quantitative bioanalysis of drug candidates. A Turbo C18 (50 x 1.0 mm, 5 /.mi, Cohesive Technologies), an Xterra MS C18 (30 x 2.0 mm, 2.5 /mi), and a guard column were used. Protein precipitation preceded injection. The cycle times for the two systems were 0.8 and 0.4 min. 

These methods have been used since the 1970s they usually require little or no sample preparation and are rapid and easy to use. However, immunoassay has two limitations. First, it does not differentiate between active drugs and similar molecules such as metabolites or co-administered drugs.9,11 Thus, cross-reactivity is a common problem. Second, its use is limited to only those drugs for which antibodies are available. 

HPLC has high-throughput capability when it can simultaneously determine multiple drugs and their metabolites or when coupled with a unique monolithic column or sample preparation technique. Some examples are summarized below. 

Except for bulk drug substances, samples can rarely be analyzed directly following simple preparation steps. In many cases, an active ingredient is found in a formulated form or in a physiological fluid or tissue. Likewise, interferences associated with the matrix and the presence of possible degradation products, metabolites, and other closely related compounds mean the target analyte is often highly diluted. Also, an analyte may be difficult to detect. Effective sample preparation steps serve up to three broad purposes (1) eliminate and/or minimize possible interferences, (2) concentrate the sample, and (3) render the analyte of interest into a more easily detectable form. 

Small sample preparation. For synthetic PFCs, this means synthesizing either a large amount of very small samples ( libraries ) obtained by combinatorial chemistry, or regular-size PFC samples for the use as drug candidates (respectively for their synthesis), for preparing impurities, metabolites, and other compounds. For natural PFCs it involves product extraction, purification, and characterization. 

Figure 3.1 A hair sample from a suspected drug user is prepared for forensic analysis. As hair grows, it incorporates small amounts of chemicals that are produced when drugs are broken down in the body. To identify these drugs, the hair is first cut into pieces and soaked in a liquid solvent The solvent removes the traces of drug metabolites from the hair so that they can be identified by chromatography and mass spectrometry.

If the application is for a biological system, wherein the objective is to isolate or quantitate a specific drug and its metabolite which is known, then the chemist can proceed to work with the compounds directly. In most cases, however, the chemist must be prepared to resolve the unexpected compound, which is not all that difficult, depending on the equipment available. The possibility that a thermal environment, such as GC, can initiate a reaction involving a labile drug or metabolite can be distressing, to say the least. Sample preparation usually includes some isolation technique which restricts classes of compounds. The need for sample cleanup will depend on the nature of the sample (see Section 12.3). The point here is that there will be some sample preparation as the first step in the analysis which will provide an initial "cleanup" of the sample. 

Maquille et al. [121], due to the physicochemical properties (i.e., polarity and ionization state) of the investigated drugs (opiates, amphetamine, cocaine and metabolites), concluded that LLE should be selected. To automate the sample preparation procedure, this team proposed urine extraction by supported liquid-liquid extraction (SLE), a promising technique that appeared in 1997 [122], which can be easily automated in a 96-well plate format. It has been demonstrated that matrix effect is significantly minimized. 

The sample preparation plays a very important role for the analysis of a drug and its metabolites in biological tissue sections using mass spectrometric imaging. Several factors in IMS sample preparation must be considered, from sample collection to surface treatment prior to analysis in order to produce high quality, reliable, and reproducible results. 

Springfield, A. C., CartmeU, L. W., Aufderheide, A. C., Buikstra, J., and Ho, J., Cocaine and metabolites in the hair of ancient Peruvian coca leaf chewers. Forensic Sci. Int., 63, 269, 1993. Baumgartner, W. A. and HiU, V. A., Sample preparation techniques. Forensic Sci. Int., 63,121,1993. Nakahara, Y. and Kikura, R., Hair analysis for drugs of abuse. VII. The incorporation rates of cocaine, benzoylecgonine cmd ecgonine methyl ester into rat hair and hydrolysis of cocaine in rat hair. Arch. Toxicol, 68, 54,1994. 

The analytical process can be divided into four major steps sample preparation, separation, detection, and data treatment. Eor the analysis of drugs and metabolites in biological matrices, sample preparation remains the most challenging task since the compounds of interest are often present at trace level in a complex matrix containing a large number of biomolecules (e.g. proteins) and other substances, such as salts. 

The direct injection of biological samples onto the chromatographic column without any sample preparation is in most cases highly problematic and may lead to an irreversible contamination of the separation columns, which reduce selectivity and column performance. A powerful asset to circumvent all the named problems is the implementation of RAM for sample preparation. RAMs, a combination of SEC with adsorptive chromatography, have foimd widespread use in the analysis of drug metabolites and other low-MW compounds, but have... 

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