[jasmine0507]

Hi all,

I am basically a biologist, but I have some courses on analytical techniques. So here, I am dealing with
LC/MS of a compound. I read an article where To quantify the levels of DSB (3-O-(3
,3
-dimethylsuccinyl)-betulinic acid)
present in HIV-1 particles, suspensions of virions were extracted with 2ml of a 3:1 (v/v) solution of chloroform and methanol. Prior to extraction, a fixed quantity (50 pmol) of a betulinic acid derivative (LH91; compound 4 in Ref 9) was added as an internal control for extraction efficiency. LH91 has an additional CH2 in the side chain of DSB, thus allowing for simultaneous
detection of both molecules by mass spectrometry.

Questions :
1. they have done sucrose density gradient centrifugation to obtain viral pellet, what is the use of chloroform/methanol extraction after that ?
2. what is the internal control LH91 for ?

Pls help me.
Thank you.
I have to make a presntation on this, day after.

Thanks again.

[JGK]

Questions :
1. they have done sucrose density gradient centrifugation to obtain viral pellet, what is the use of chloroform/methanol extraction after that ?

The viral pellet will contain a lot of "junk" material as well as the compound of interest DSB. the further extraction with chloroform/methanol will remove the DSB and leave a lot of the "junk" behind. It's an additional purification step. LC-MS units are not cheap and last longer if you don't flood them with "dirty" samples

2. what is the internal control LH91 for ?

Bioanalytical extractions are notoriously fickle things. For example if you had 2 samples containing the same concentration of DSB, one sample may extract with 100% efficiency the next may be at 30%. So If you are quantitating based on peak area v concentration you would get highly variable results for samples of the same concentration. However, if you add a fixed amount of LH91 to all of the samples and standards and calibrate using peak area ratio (area DSB/area LH91) vs concentration, any varation due to extraction efficience is eliminated as both materials are extracted with virtually the same efficiency. i.e. For the peak area ratio is the same regardless of extraction efficiency.