Can metabolite standards be produced from human hepatic cells for use in forensic toxicology?Previously researchers have used rat plasma and urine for the detection of drug metabolites. In order to detect the metabolites, high performance liquid chromatography High-performance liquid chromatography (HPLC) is a form of column chromatography used frequently in biochemistry and analytical chemistry. It is also sometimes referred to as high-pressure liquid chromatography. (HPLC HPLC high-performance liquid chromatography. HPLC high performance liquid chromatography. HPLC High-performance liquid chromatography Lab instrumentation A highly sensitive analytic method in which analytes are placed ) was used. The purpose of this project was to find a more direct source for detecting drug metabolites by using human hepatic cells. The researcher hypothesized that drug metabolites could be produced from human hepatic cells since the liver should provide the purest form for metabolites. The human hepatic cells (Hepa C1C7) were grown in DMEM media supplemented with 10% fetal bovine serum Fetal bovine serum ( or foetal bovine serum) is serum taken from the fetuses of cows. Fetal Bovine Serum (or FBS) is the most widely used serum in the culturing of cells. In some papers the expression foetal calf serum is used. , 200 U/ml penicillin G sodium, and 200 mg/ml of streptomycin sulfates. The cells were maintained under standard incubator conditions (95% air, 5% carbon dioxide, 37 degrees Celsius). The cells were incubated for 24 hours Adv. 1. for 24 hours - without stopping; "she worked around the clock" around the clock, round the clock , and then treated with 40 [micro]ls fluoxetine fluoxetine /flu·ox·e·tine/ (floo-ok´se-ten) a selective serotonin reuptake inhibitor used as the hydrochloride salt in the treatment of depression, obsessive-compulsive disorder, bulimia nervosa, and premenstrual dysphoric disorder. or 40 [micro]ls of theophylline theophylline /the·oph·yl·line/ (the-of´i-lin) a xanthine derivative found in tea leaves and prepared synthetically; its salts and derivatives act as smooth muscle relaxants, central nervous system and cardiac muscle stimulants, and along with 1 micromolar of dexamethazone or ICZ to control a P450 enzyme system. After being exposed for 24 hours, the media was pulled off and frozen. The frozen media was analyzed by liquid-liquid extraction and gas chromatography-mass spectrometry. Fluoxetine's main metabolite Norfluoxetine was not produced in any of the media that was exposed to it. Theophylline's metabolite caffeine was produced in all of the media that was exposed to it. The theophylline along with ICZ and Dexamethazone produced less caffeine than the control theophylline. Also Dexamethazone alone produced more caffeine than ICZ alone and the control theophylline. Megan A. Hamilton Timberland High School |
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