HPLC UV Assay

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. 

A typical method using liquid/liquid extraction was described by Shum et al. (1994) for the determination of an ACAT inhibitor in rat plasma. Internal standard, 

1 mL water and 1 mL of n-pentane were added to 0.25 mL volume of plasma. After shaking for 15 min and centrifugation for 15 min at approx. 1200 g, the upper organic phase was transferred and evaporated. The residue was reconstituted in 200 xL of mobile phase, and 100 xL was injected onto the HPLC column for analysis. The HPLC system consisted of a Bio Sil ODS-5S column (150 x 4 mm 5 xm particle size) and a UV detector operating at 250 nm. The mobile phase consisted of 55 % acetonitrile, 27 % methanol, and 18% water. The flow rate was 1.9 mL/min. The column temperature was ambient temperature. 

An appropriate tool to enhance the selectivity and the feasibility of the work-up procedure using liq-uid/liquid extraction is the subsequent back extraction into aqueous phase. For example, Los et al. (1996) described a toxicokinetic assay for diltiazem and its two metabolites. To 0.2 mL rat plasma, internal standard solution and a pH 7.3 phosphate buffer and methyl-t-butyl ether were added. The analytes were 

Kawauchi T, Matsumoto H, Yano S (2001) Determination of a new thymidine phosphorylase inhibitor, TPI, in dog and rat plasma by reversed-phase ion-pair high-performance liquid chromatography. J Chromatogr Biomed Appl 751(2) 325-330 

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