A Reversed-Phase Derivatization HPLC Method for the Detection of Creatinine in C. Elegans
Start Date
April 2024
Location
2nd floor - Library
Abstract
Creatinine is a waste product generated by the breakdown of creatine phosphate in muscles, and its levels are a reliable indicator of kidney function and overall metabolic function. Caenorhabditis elegans was selected as a model organism to study metabolism, using creatinine as a biomarker. To quantify creatinine in supernatant samples from C. elegans, a reversed-phase high-performance liquid chromatography (HPLC) method is developed. This method utilizes a pre-column derivatization technique for creatinine, using ethyl chloroformate (ECF) as a derivatizing agent to synthesize N-ethyloxycarbonyl-creatinine, a reagent that is more hydrophobic than creatinine. This enhanced hydrophobicity leads to enhanced retention and quantification of creatinine on a C18 stationary phase. The Vanquish HPLC instrument consists of method parameters that are adjusted to detect the derivatized creatinine product at the proximal wavelength of 242nm. Considering the parameters, the samples yielded a consistent retention time of 3.1 minutes indicating the presence of creatinine. Peak quantification of derivatized samples is still in progress to detect more consistent results.
A Reversed-Phase Derivatization HPLC Method for the Detection of Creatinine in C. Elegans
2nd floor - Library
Creatinine is a waste product generated by the breakdown of creatine phosphate in muscles, and its levels are a reliable indicator of kidney function and overall metabolic function. Caenorhabditis elegans was selected as a model organism to study metabolism, using creatinine as a biomarker. To quantify creatinine in supernatant samples from C. elegans, a reversed-phase high-performance liquid chromatography (HPLC) method is developed. This method utilizes a pre-column derivatization technique for creatinine, using ethyl chloroformate (ECF) as a derivatizing agent to synthesize N-ethyloxycarbonyl-creatinine, a reagent that is more hydrophobic than creatinine. This enhanced hydrophobicity leads to enhanced retention and quantification of creatinine on a C18 stationary phase. The Vanquish HPLC instrument consists of method parameters that are adjusted to detect the derivatized creatinine product at the proximal wavelength of 242nm. Considering the parameters, the samples yielded a consistent retention time of 3.1 minutes indicating the presence of creatinine. Peak quantification of derivatized samples is still in progress to detect more consistent results.
