Endogenous metabolites, liquid

With BBSs, the internal standard is present in the extraction solvent and can only equilibrate with the endogenous metabolites that are extracted from the DBS. Therefore, isotope dilution principles do not apply to the extraction step since they are not in the blood (blood spot) to begin with. However, subsequent steps such as derivatization, sample transfer, and so on do indeed follow the principles of isotope dilution. To note the differences between analyzing liquid versus BBSs using stable isotope standards and the fact that quantification is less accurate using BBSs, the term pseudoisotope dilution is often used. 

Imperato, A., and Di Chiara, G. Transtriatal dialysis coupled to reverse-phase high performance liquid chromatography with electrochemical detection A new method for the study of the in vivo release of endogenous dopamine and metabolites. J Neurosci A.966-911, 1984. 

Xu X, Roman JM, Issaq HJ, Keefer LK, Veenstra TD, Ziegler RG (2007) Quantitative measurement of endogenous estrogens and estrogen metabolites in human serum by liquid chromatography-tandem mass spectrometry. Anal Chem 79 7813-7821... 

Xu X, Keefer LK, Ziegler RG, Veenstra TD (2007) A liquid chromatography-mass spectrometry method for the quantitative analysis of urinary endogenous estrogen metabolites. Nat Protoc 2 1350-1355... 

High performance liquid chromatography (HPLC) can rapidly separate drugs and metabolites from endogenous compounds in biological fluids. Fractions are readily collected and thus HPLC has been used in cannabinoid work to purify biological fluid extracts prior to analysis by techniques offering either more sensitive or specific detection than has been available for LC. 

Levels of drug and/or its metabolite have to be determined in plasma samples collected during the course of a toxicity study in order to evaluate the toxicokinetics of that compound. The UV absorption properties of the compound are used to determine the compound of interest. Since many of the endogenous compounds also show absorption in that wavelength range, a tailored sample work-up has to provide additional selectivity for the compound of interest. In addition, the plasma proteins have to be removed by the work-up in order to avoid blocking of the HPLC columns. Three principles are generally applied to remove the proteins from the sample Protein precipitation, liquid/liquid extraction and liquid/solid extraction. 

After generation, the metabolites must be extracted and isolated. A variety of sample pretreatment methods can be applied, such as protein precipitation, liquid extraction, liquid-liquid extraction (LLE), solid-phase extraction (SPE), and microdialysis. Cleanup prior to LC-MS analysis is essential to reduce endogenous interferences and small peaks at every m/z in the mass spectra. The cleanup step also facilitates the data processing and peak detection in the chromatogram. 

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