bamhi ggg met bira f  (Addgene inc)

Journal: Analytical Chemistry

Article Title: An Integrated Platform for High-Throughput Extraction and Mass Spectrometry-Based Quantification of Cholesterol and Sphingosine

doi: 10.1021/acs.analchem.4c06628

Figure Lengend Snippet: RF-MS method development for cholesterol quantification. (A) A titration of native cholesterol was prepared at concentrations ranging from 19.5 to 10,000 ng/mL and injected into RapidFire MS (red trace) in the absence or presence of a constant amount of 13 C-cholesterol at 500 ng/mL (blue trace). The top concentrations of native cholesterol in the titration are indicated above the trace. (B) MS integrated area for serial dilutions (156 ng/mL to 5 μg/mL) of native cholesterol from (A) without (solid circle) or with (open circle) 13 C-cholesterol included at a fixed concentration. (C) Ratios of integrated areas from native and 13 C-cholesterol from (B) were plotted against the concentration of native cholesterol. (D) Compatibility of 384-well plates from different vendors was tested in the semiautomated cholesterol extraction protocol using MTBE containing 1 μg/mL 13 C-cholesterol in absence of cells. 13 C-cholesterol added directly to the destination plate without going through the extraction process served as control to compare levels of 13 C-cholesterol after RF-MS quantification ( n = 12).

Article Snippet: The plate was returned to the Bravo station, and 20 μL of the upper MTBE layer was transferred to a destination plate (ThermoFisher 264573) without disturbing the lower aqueous phase.

Techniques: Titration, Injection, Concentration Assay, Extraction, Control