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"If the value for the theoretical plates for an HPLC column decreases below 2000, should the column be discarded? And which parameters should be considered when deciding when to discard a column?"
"Unfortunately the answer to your question is - it depends.
Deciding when to get rid of an HPLC column is difficult and may depend on what
the column is being used for and whether it is being used for a single type of
method or lots of different methods and samples. The theoretical plate count
(N) is a good indication of how well a column is working but it is relative,
this means that you need to monitor the value over the course of the column
lifetime and correlate the value with the decrease in separating power.
chromatography for a method looks good and the peaks are still separated
adequately for quantitative analysis then I would not discard a column because
the value of N went below 2000. However, from experience for a particular
column you may know that the method does not work as well once N is below a
certain value and thus this could be your indicator that the column should be
discarded. It is definitely easier to use this approach if the column has only
been used for one type of analysis. Once a column is used for lots of different
samples then it is very difficult to predict when it will fail.
There are a few things to be aware of if you are using plate
number. The value is specific to a particular analyte and thus it will be
different for each peak in your chromatogram, this can make it tricky to say a
column has a particular number. Also, it is only useful if you are using
isocratic elution methods, it is not meaningful for gradient methods.
Typical indicators that a column has reached the end
of its life are:
Resolution – if the peaks that you are interested in are not
separated adequately then the column may need to be discarded, you would expect
this to build up over time. A sudden loss of resolution may have a different
source. A typical value of resolution when the separation is satisfactory is Rs > 2 although a value of around 1.5 is usually considered baseline separated. Therefore a value < 1.5 indicates that the separation is not adequate.
Peak shape – peak tailing (especially on large peaks) is
usually associated with the age of the column and at some stage will be too
great for reasonable quantitative analysis. A typical value of an acceptable tailing
factor is T < 2. Therefore when the tailing factor is above this value the column may be no longer performing at an adequate level. Peak shoulders may also appear (on all peaks) which indicate
that there is a build-up on the column inlet that means the column is no longer
fit for use.
Theoretical plate count, N – in the region of about 2000 is
usually accepted as a limit but be careful not to throw away a column that it
Pressure – The back pressure of the column will usually
build up over time and may determine the lifetime of the column."
When setting up a stability programme for a pharmaceutical substance or product, analytical methods are selected to allow appropriate testing at the required time-points. A number of different types of methods may be selected but the quantitative measure of the active pharmaceutical ingredient (API) and its degradation products is probably the most important.
Since these methods are being used to interpret the effects of the stability study then it is self evident that they need to be stability indicating but how can you be sure that this is the case? The challenge is to demonstrate that each method is, in the words of ICH Q2(R1), "suitable for its intended use".
To demonstrate suitability, and therefore show that your method is stability indicating, you will need to be able to quantify the API and its degradation products so that you can monitor the expected decrease in the amount of the API and the corresponding increase in the amount of degradation products. Therefore, samples of the degradation products will be necessary to show the method is capable. These are usually sourced by applying stress to the samples in a forced degradation study. Although the principle is straightforward, this type of investigation can be very problematic and requires a thorough understanding of the chemical properties of the API and predicted degradation products.
When carrying out an audit which involves stability testing, I will often ask the question "Is this analytical method stability indicating?" What I expect to see is a section in the validation report which details the evidence that demonstrates that the method is stability indicating.
Since the analysis of an API and its degradation products most often requires the use of HPLC, Mourne Training Services has created a training course which focuses on how to develop stability indicating HPLC methods. Strategies for generation of suitable degradation products via forced degradation is combined with a thorough step by step guide to HPLC method development.