Background: Uremia is the outcome of the remaining of nitrogenous waste products that are normally removed by the kidneys. Para-cresol (4-methylphenol) can be regarded as a proteinbound uremic toxin. The p-cresol determination in sera is necessary since it is a marker of cardiovascular risk and overall mortality in hemodialysis patients. Among the reported methods, chromatographic ones especially HPLC techniques due to the high sensitivity, selectivity and reproducibility have been extensively exploited in analysis of p-cresol in complex mixtures. However, an appropriate sample preparation prior to analysis is necessary for obtaining accurate and precise results.
Methods: In this study, the appropriate precipitating agent for p-cresol determination in plasma samples was investigated. Then, in situ surfactant-based solid phase microextraction followed by HPLCFL detection was developed and validated for the quantification of p-cresol in plasma samples.
Results: According to the results, HCl/heat precipitation method was used for p-cresol microextraction from from plasma samples. In situ surfactant-based solid phase microextraction using cetyltrimethylammonium bromide as extraction medium was proposed for pretreatment of plasma samples prior to analysis. The separation was achieved by isocratic elution with sodium acetate buffer (pH 3.8) and acetonitrile (20:80, v/v). Linearity was found to be acceptable over the concentration ranges of 0.5 to 8 μg mL-1 with the limit of detection and quantification of 0.324 and 0.422 μg mL-1, respectively. The variations for intra-day and inter-day precisions were both less than 8.2% and the extraction recoveries were more than 97%.
Conclusion: A validated ISS-SPME followed by HPLC-FL detection reported to determine the total p-cresol concentration of human plasma samples. The traditional liquid-liquid extraction techniques are normally time consuming and require the use of large amounts of toxic organic solvents. In addition, the evaporation of extraction solvent and dissolving the analyte in the mobile phase is commonly used before HPLC analysis. Such a requirement makes the sample preparation process even more tedious and time consuming. ISS-SPME that is the developed ISS-SPE in micro scale, is a simple, rapid and effective sample preparation technique that is appropriate for HPLC-FL analysis.
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