Assay quantifaction of two active peaks..please help

[seamoro]

I am working on an HPLC assay at the moment with 2 active peaks (Penicllin 90% and 4-Hydroxypenicillin 10%). we want to quantify the 2 peaks. but the problem is my company wants to only use one standard (Penicillin 90%) to quantify to cut cost. So what can i do?...We have both standards here so could i work out an response factor ? how do i do that?. I could weight out 15mg of both standards and analyze them and see there UV area response.?

[Consumer Products Guy]

I'd bet that trying to cut costs in that type of manner would not be looked on favorably by FDA. Yes, USP controls standards, and sells them for relatively high price, kind of self-serving or "conflict of interests", like it's their ball, and "if you don't like it, don't play".

FDA might say: you don't have a standard to line up retention times for the 4-Hydroxypenicillin, so how do you prove that's the correct material???

[grzesiek]

but who really wants to cut costs?
I'm sure it's not QA and I'm sure they don't even know about your supervisor's or whoeveritwas's idea
cutting costs is not the best way to assure quality in my opinion

you calculate Response Factor by dividing calibration lines slopes of these compounds, so you've got to make two response functions (cal lines) first - and this is a part of validation after all

but... your SST solution would probably need both compounds to show resolution, tailing, retention times or whatever... so I think you will have to use the second standard even if your company doesen't want to

[Ron]

If you have protocols and standard methods of analysis I would not want to be involved in anything like this. There is no way that this would be acceptable in a regulated laboratory environment. This is the kind of thing that leads to major fines and penalties, and that does nothing to cut costs.

[seamoro]

Guys.......is there not another way of doing?....you are giving me no answers here!

[Consumer Products Guy]

There are pharmaceutical regulations; in your case, this is NOT the place to save costs....

You're an ethical guy, and your company should also have ethics...do it right, these are internal drugs, not some topical OTC item....

[seamoro]

yeah your right...**** the company, **** the system. Im Keanu Reeves in the Matrix .....Spoon Boy: "Do not try and bend the spoon. That's impossible. Instead, only try to realize the truth." Neo: "What truth?" Spoon Boy: "There is no spoon." Neo: "There is no spoon?" Spoon Boy: "Then you will see that it is not the spoon that bends, it is only yourself."

[let's keep it "family friendly" as we do have some younger chromatographers in here now and then]

[DR]

If you must save $, I think the only way to do it is to make a retention marker solution and freeze lots of aliquots. You can't use this as a standard, but you can use it to re-establish your retention times. As long as both components absorb well enough at a single wavelength that is not in a steep section of either component's UV spectrum, a simple absorbance per conc. unit ratio can be applied to yield a fixed relative response factor to be used when running a single component standard.

I wouldn't do it this way, unless it was for product development, assay, informal stability, dissolution... type testing only. nothing having to do with product to be released for consumption, not for exhibit or clinical lots etc. etc.

[grzesiek]

as I said before - you surely need SST solution, you can calculate response factors in the way I described but surely you are going to face the question - why don't you quantify both substances using respective standards - what will be your answer? to cut the costs? well this would be unacceptable answer to me

make the metod faster, use methanol, less organic. etc, but your way of cutting costs is not going to work in my opinion