By Bharat Kumar Agarwal on Thursday, May 6, 2004 - 04:55 am:

Repeatability: Repeatability should be assessed using:
a) a minimum of 9 determinations covering the specified range for the procedure (e.g.
3 concentrations/3 replicates each)
or
b) a minimum of 6 determinations at 100% of the test concentration.

Please explain me above statement of ICH

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By Bharat Kumar Agarwal on Thursday, May 6, 2004 - 05:34 am:

How we measure the column effecincy or number of theoritical plate ? If we made 1000 run during validation of a method then is it necessary to calculate the column effecincy or number of theoritical plate for every run ?

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By Bharat kumar agarwal on Thursday, May 6, 2004 - 05:21 am:

Hi everybody,

If two drug have not the same Maximum UV absorpation suppose one shows the 322 and another shows 254 nm, in that conditions can we develope a simultaneous HPLC method.What is the basic criteria for develope a simultaeous method developement?

Please suggest me ?

Thanks in advance

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By tom jupille on Thursday, May 6, 2004 - 08:50 am:

Re: ICH. the statement means just what it says. You have a choice:
a) do three runs at each of three concentrations, or
b) do 6 runs at the "target" concentration of your analyte.

Re: wavelength. No simple answer here, depends on what interferences are present and how well your two compounds are separated. For method development, use a photodiode array detector; you can then reprocess the data at different wavelengths to get an idea of how to proceed. Safest would be to switch wavelengths between the two peaks (but they must be reasonably well resolved for this to work). An intermediate wavelength (e.g., 280 nm) may also work, but may have robustness problems.

Re: plate number. That is a *very* basic question. We have a free on-line course called "Getting Started in HPLC" that will answer it (and many more!). You can access it here:
http://www.lcresources.com/resources/reslinksform.html

If you measure retention times and resolutions in each run, then plate number need not be specified separately. On the other hand, most data systems will do the calculation for you automatically, so why not?

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By Uwe Neue on Saturday, May 8, 2004 - 09:23 pm:

Here is a bit of basic thought:
In order to determine the quality of your separation, the general measure is the resolution between peaks. Resolution has several components: the retention time difference between the peaks, and the peak width(s). In order to get at retention time difference, you need to know the retention times. Retention times are one set of the important parameters from the standpoint of troubleshooting: did one peak move, did both move, etc.? The other set of important parameters are the peak widths. Did one peak become wider, or both? Is the peak width where it is expected to be?
Retention time differences are primarily caused by changes in the chemistry of the separation - differences in the mobile phase or aging of the stationary phase or similar things.
Peak width differences are often caused by changes in the physics of the separation - deterioration of plate count, differences in the sample make-up, differences in the plumbing of the system etc.
While these are not 100% solid rules, they are good starting points for troubleshooting.
Since you are getting all these other pieces of information for free when you calculate resolution, it is worth keeping an eye on the more basic parameters.