I injected the sample obtained after cell lysis but I think the intracellular uptake is in nanogram and the LOQ I obtained after running the linearity is something around 5 µg/mL.
So, how to enhance the peak do I need to spike the specified quantity of the known same drug? or something else please suggest.
The procedure is as follow
For intracellular uptake of lipid vesicular entrapped drug formulations, HaCaT cells were seeded (2x 105 cells) on a 6-well flat-bottom culture plate. The confluence of the cell after incubation was washed with buffer and treated with lipid-based vesicular entrapped drug at non-toxic concentration, blank transfersomes, and cell alone was considered as the negative control. The plate was incubated for 24 h in a 5% CO2 incubator. After incubation, the cells from each well were trypsinized (200µL) and transferred to a sterile centrifuge tube (1.5 mL). The cells were lysed using 1% v/v triton x (100 µL) and centrifuged at 15000 rpm for 30 min at 4 °C. The supernatant was filtered through a 0.22-micron nylon filter and the volume was made up to 800 µL using methanol then sonicated for 30min. A 20µL was subjected to ultra-performance liquid chromatography (UHPLC) techniques to quantify the permeated curcuminoids content within the cells.
Thanks in advance
It seems fluorescence (immunofluorescence, label, dye, etc) seems to be more common. I found the paper below that may be helpful"
Article HPLC-MS/MS Shows That the Cellular Uptake of All-Trans-Retin...
Dear Sudarshan Singh , an LOQ of 5 µg/ml can be high or low depending on the absorptivity of the analyte or "substance of interest".
I dare to say that such an LOQ is due to the analyte showing a low UV-Vis absorptivity (due to low number/intensity of chromophores) and not to the use of low precision instrumentation/method.
As such, have you tried the suggestion of Shin Murakami or a "post column" derivatization procedure (sometimes they make miracles!).
Kind regards, Luis.
- Badges
- Science topic
- Similar topics
- Mathematical Sciences
- Graphs