Could anybody help me on the question that how one can start analytical method development for a unknown drug substance ? i mean tha how to proceed sequencely and how to apply the things one by one ?

Find out its chemical structure and properties, like UV absorbance, solubility, etc. Then find application for something similar. If no UV or vis absorbance, try RI detector or ELSD. Then explore HPLC conditions using that detector, start with relatively simple conditions such as RP-18 with H2O/ACN. Make placebo product, check linearity, recovery, etc. Don't overlook GC, is your material volatile or can be derivatized? After method development, validate as per ICH/cGMP.

If you want a short-form "cookbook", go to the LC Resources web site (the link is in the upper left corner here), and click through to Software . . . DryLab . . . Develop Robust Methods using DryLab. (Alternatively, the direct link is http://www.lcresources.com/dlmd.htm). Although this is based on the use of modeling software to cut down the experimental effort involved, it *can* be implemented without the software and it does provide a systematic approach to reversed-phase method development.

If you want a more extensive discussion, check out the book "Practical HPLC Method Development" by Snyder, Glajch, and Kirkland. Published by Wiley, available from Amazon (but I think we actually have a better price than Amazon!). http://www.lcresources.com/books.htm.

-- Tom Jupille / LC Resources Inc.

Anon,

If you know the chemical structure, I'd be happy to help you try to predict the retention of your separation with ChromSword Auto for HPLC separation prediction and fully automatic HPLC method development. Please feel free to contact me at 703-689-4981.

Thanks,

Matt Kramer
Hitachi Instruments, Inc.

Hi. Looking for existing methods on ink and credit card analysis. Thanks, Stephanie de Wet

According to some references (e.g. 'Effect of buffer on silica-based column stability in reversed-phase hplc' by H.A. Claessens et al., J. Chrom. A, 728, 259ff) is the silica in RP columns already being dissolved at neutral pH values by the use of a phosphate buffer, even just at slightly increased temperatures like 40 C. Since there is no real choice of the buffer system, if you want to use a low detection wavelength with a buffer at pH 6-7, I would like to know whether anybody has a solution? I am having right now problems with a YMC Pro 18 (3 micro particles) at pH 6.4 with a phosphate buffer.

Thanks for any comment,
Dirk Redlich

Indeed, one does have column lifetime issues at elevated temperature with a phosphate buffer. However, at room temperature, phosphate buffers are just fine and column lifetime is at least in the order of 3 months. We have used columns at pH 7 without difficulties for a long time. One needs to realize that the reduction in column lifetime is in the order of a factor of 3 for every 10 degrees C. In our experience, we found column lifetimes of about 3 weeks with pH 7 phosphate buffers at 50 degrees C for a standard high-purity silica-based column. That makes it about 1.5 years at room temperature. Nothing to worry about...

Dear Uwe:

Thanks for your answer. Unfortunately, the YMC column last under the described conditions for just about 100 injections (with kind of 'uncomplicated' drug substance samples). This is indded a problem if you have to transfer this method to a stability testing lab or an operations site. By the way: the column is not altered by the analyte. I am now wondering whether our problem is according to the buffer (column temp. 35 C) or a problem of the manufacturer?

This does not appear to be a buffer/temperature problem. I have no concern about phosphate at 35 degrees, especially not for 100 injections.
What do you observe? What is the signal of the column death? Double peaks, tailing peaks, retention changes? We may need to chase some problem other than the column.

Hello Dirk,
when you say you have an uncomplicated drug, and you have to use pH6,4 and low wavelength, this sounds like a tricky method. Using phosphate at elevated temperatures some users have never had a problem, some had big problems like you. With pH6,4 you are in a very critical pH area, and therefore you run at 35°C to keep retention times stable. And your wavelength is 210nm or less? Any chance to use a higher wavelength? When you use low wavelength, is this just to have a higher detection sensitivity? Than you should try to optimize your HPLC system (if possible), also with a small flow cell volume, so you can use smaller ID columns. And finally you can use for example an organic buffer like MES or MOPSO. UV cut off is about 230nm, but in combination with organic modifiers like methanol UV cut off switch towards 210nm (with about 50% MeOH). With organic buffers sometimes you can increase column lifetime. Good luck!
Gerhard

Hi Gerhard !!!

Organic Buffer: MES ??, MOPSO ?? can you explain the meaning of this initials.

Thanks

KAF

Hello KAF, MES means 2-Morpholinoethanesulphonic acid CAS: 4432-31-9, MOPSO is 3-Morpholino-2-hydroxypropanesuphonic acid CAS: 68399-77-9.
Hope this helps you.

Gregor

Dear all:

Thank you very much for the support! Let me answer Uwe's question first: we have a critical peak triple, eluting after aproximately 22 min. With a new column s a baseline separation achieved. Unfortunately do we loose resolution after about 100 injections and we are no longer able to quantify those compounds properly.

Gerhard, thanks also for your remark. But since we are profiling impurities, we need to stick to 210 nm. Optimization of the system wouldn't be a good choice, since we will not see our impurities at higher wavelength at all. Organic buffers like MOPSO and MOPS resulted in baseline shifts, which were not acceptable according to our 'Best Practice'.


Alvast bedankt,
Dirk.

A couple of brute force approaches are:
a. Use a pre-column and discard as required.
b. If the problem really is silica dissolution, the use of a saturator column before the injector might help.

Does anything bring back performance?
Column rinses, back-flushing

How dirty is your sample?

ANYONE HAS DETERMINED CLARITHROMYCIN BY HPLC???

Yes, but no need to shout...
Check USP if you're analysing bulk drug or dosage forms. I can't help you if you're doing bioanalysis, but I'm sure there are numerous papers out there dealing with that.

Does anybody know of any organic buffer solution that can be used to replace PIC A reagent which contains Tetrabutylammonium Phosphate.

start with tetrabutylammonium hydroxide and neutralize with your organic acid of choice.