Validation of a HPLC Method for Quantification of Thiamine and Its Phosphate Esters in Rat Brain Tissue

Abstract

The present data show a fast and efficient biological sample processing method for the extraction of thiamine (vitamin B1) and its mono-(TMP) and di-(TDP) phosphate esters from hippocampus, thalamus and prefrontal cortex (PFC) and blood sample of the rodents. In addition, using the hippocampus and standards of these three compounds we validated an isocratic fluorescence HPLC procedure for a simultaneous detection of them in a single chromatogram within a total run time of about 12 min. Reproducibility for TDP, TMP and B1 was 2.66%, 4.50% and 7.43% (intraday) and 37.54%, 25.39% and 25.87% (interday), respectively. Recovery assays were between 96.0% and 101.7%. The calibration curves were linear and the concentrations of the three compounds, all in nanomolar range, were determined in the brain areas and in the blood samples. When compared to the current methods in the literature, this new method provides information on essential variables, such as linearity range and limit of detection, reproducibility and stability of thiamine, TMP and TDP in rat brain samples. The present data on sample processing and B1 and its phosphate ester level determinations are the first to be validated using hippocampus samples of rats.

Available calcium levels in central nervous system for Arzenazo III method

“…The processing of the samples involved the use of the denaturing agent TCA at 10% and, it is known that the use of this chemical reagent releases and solubilizes calcium ions associated with proteins, allowing their quantification in aqueous solution. The proposal was to use samples processed according to NUNES et al, (2017) (26), lyophilized overnight and resuspended in 100 mM HCl, to measure calcium.…”

The Potential of a Site-Specific Delivery of Thiamine Hydrochloride as a Novel Insect Repellent Exerting Long-Term Protection on Human Skin: In-vitro, Ex-vivo Study and Clinical Assessment

“…The calibration curve was obtained by plotting the area under the curve in function of the concentration of the injected solution. Six solutions were tested with theoretical concentrations of 0.5,1, 2.5,5,10 and 20 mg/ml.. 38 After conducting the permeation study, the effective diffusion area of the skin was separated, washed several times with normal saline to remove the excess formula, and then cut into small pieces in a glass vial, vortexed with 5 ml methanol, soaked for 24 h to ensure maximum extraction of the retained TH in the skin. The resulting mixture was subjected to centrifugation at 10,000 rpm for 15 min, then the supernatant was filtered using a membrane filter (0.45 mm), and TH concentration was analyzed by HPLC to determine the amount of TH deposited within the skin.…”