Chromatography Forum

The sample prep involves weighing up 60 mg of tablet and qs to 25 mL with solvent, then take 10 mL of the mixture and dilute 1/2, then rotovap, then add acetic anhydride + pyridine to the residue @0.5 mL ea, reflux condenser for 1 hr, ostensibly this converts the active ingredient into a form that is analyzable, then qs that residual volume to 3 mL with ethyl acetate, vial it up and shoot on LC. The std is 20 mg of pure api 99.6% potency--- dissolved in 10mL of solvent.
What is the dilution factor? and what should value should be put into the empower run setup to make it generate a final value in ug per mg as % w/w of the active ingredient in the tested substanceinthe final 'amount densitiy" of the results table?
For some reason I can't follow the units here? Connectivity betw std and sx conc'n is ambiguous?
Any insight or experience on this ?

Hi Meerkat,

The value that should be entered into Alter Sample -- Amount for the standard aught to be 2.0 mg x 0.996 = 19.92 mg. Seems to me that you're running a one-point "standard curve", yes?

After re-processing, SampleWeight is a divisor, so it seems to me that the SampleWeight value aught to represent your API Label Claim value in mg--you note the tablet mass is 60 mg, you want to enter the mass within that 60 mg of tablet that is the API. Just occurred to me that I may be in error here...either it will be the Label Claim for the API or the mass of the sample(s)...the "60 mg" or so that which was actually weighed out...the latter seems to me much more likely now that I think of it a bit more.

After re-processing, Dilution is a multiplier, so the Dilution value aught to represent all manipulations you've placed your sample through. Here, though, quite a bit has been done...you take 60 mg of tablet, dilute it into 25 mL, then take 10 mL of the initial dilution and place it in 20 mL...and then rotovap this second dilution to dryness, correct?

That is just a way of saying "start with 60 mg of tablet, take out 24 mg for later work-up". So far, a "net dilution" factor of 2.5.

You do the acetylation on ca. 24 mg of tablet and work it up into 3 mL of ethyl acetate...okay, then you've "removed" 24 mg of the original 60 mg of tablet, and worked that 24 mg into 3 mL...you need to return to a unit of mass at this point, so 2.5 x 3 = 7.5, this is the value for dilution factor, to return to the mass of API in the 60 mg sample.

Please, see what you think.

Hey Meerkat,

Just wondered if these Dilution and SampleWeight values worked out for you. Is everything copasetic in the world of Empower and acetylation of drug products?

Both are not as exciting as Zen and the Art of Motorcycle Maintenance or Hitchhiker's Guide to the Galaxy.