HPLC method for quantification of creatinine and ketamine in C. elegans: A novel approach for studying excretory function and drug metabolism
Start Date
2023 3:00 PM
Location
Alter Hall 211
Abstract
Caenorhabditis elegans (C. elegans) represent an excellent model organism of human pathology, physiology, and pharmacology. Much of the C. elegans genome and proteome are conserved in higher organisms, including humans. Creatinine is a waste product generated by muscles as they break down creatine, which is a molecule involved in energy production. In humans, creatinine output is used clinically to measure glomerular filtration to determine kidney function. In C. elegans, creatinine output could be used as a model of overall excretory function in these animals to inform pharmacokinetic studies done in this model. Ketamine is a dissociative anesthetic that has recently been approved for treatment resistant depression. It is important to fully characterize the pharmacokinetics of this drug throughout development and identify factors that may impact its metabolism. We therefore aim to use C. elegans as a model organism to study the pharmacokinetics of ketamine by measuring creatinine output and the breakdown of ketamine. The first step towards this goal is to develop robust analytical methods for both ketamine and creatinine in these animals. We report on a simple dilute-and-shoot high performance liquid chromatographic (HPLC) method for the quantification of creatinine output and ketamine degradation in C. elegans, without the need for precolumn derivatization or use of mobile phase buffers. Chromatographic separation is obtained using a C18 column and isocratic mobile phase of 80:20 (H2O, acetonitrile and 0.1% formic acid), at a detection wavelength of 233 nm for creatinine and 210 nm for ketamine with a runtime of 4 minutes. A calibration curve of ketamine was generated with an R2 of 0.97 in triplicate with a linear range of 5����g/mL to 3000����g/mL. A calibration curve of creatinine was generated with an R2 of 0.95 in triplicate with a linear range of 2.5 ����g/mL to 3000 ����g/mL. This creatinine method showed minimal matrix effects when used to detect creatinine in C. elegans media after 12 hours of worm incubation. Accumulation of creatinine in the worm solution can be seen increasing until a plateau at 12 hours when worms in the sample tube died. In the future, this method will be used to study the clearance pharmacokinetics of ketamine by measuring ketamine degradation while simultaneously quantifying overall excretion rates of creatinine output.
HPLC method for quantification of creatinine and ketamine in C. elegans: A novel approach for studying excretory function and drug metabolism
Alter Hall 211
Caenorhabditis elegans (C. elegans) represent an excellent model organism of human pathology, physiology, and pharmacology. Much of the C. elegans genome and proteome are conserved in higher organisms, including humans. Creatinine is a waste product generated by muscles as they break down creatine, which is a molecule involved in energy production. In humans, creatinine output is used clinically to measure glomerular filtration to determine kidney function. In C. elegans, creatinine output could be used as a model of overall excretory function in these animals to inform pharmacokinetic studies done in this model. Ketamine is a dissociative anesthetic that has recently been approved for treatment resistant depression. It is important to fully characterize the pharmacokinetics of this drug throughout development and identify factors that may impact its metabolism. We therefore aim to use C. elegans as a model organism to study the pharmacokinetics of ketamine by measuring creatinine output and the breakdown of ketamine. The first step towards this goal is to develop robust analytical methods for both ketamine and creatinine in these animals. We report on a simple dilute-and-shoot high performance liquid chromatographic (HPLC) method for the quantification of creatinine output and ketamine degradation in C. elegans, without the need for precolumn derivatization or use of mobile phase buffers. Chromatographic separation is obtained using a C18 column and isocratic mobile phase of 80:20 (H2O, acetonitrile and 0.1% formic acid), at a detection wavelength of 233 nm for creatinine and 210 nm for ketamine with a runtime of 4 minutes. A calibration curve of ketamine was generated with an R2 of 0.97 in triplicate with a linear range of 5����g/mL to 3000����g/mL. A calibration curve of creatinine was generated with an R2 of 0.95 in triplicate with a linear range of 2.5 ����g/mL to 3000 ����g/mL. This creatinine method showed minimal matrix effects when used to detect creatinine in C. elegans media after 12 hours of worm incubation. Accumulation of creatinine in the worm solution can be seen increasing until a plateau at 12 hours when worms in the sample tube died. In the future, this method will be used to study the clearance pharmacokinetics of ketamine by measuring ketamine degradation while simultaneously quantifying overall excretion rates of creatinine output.
