Thursday, 18 March 2010

Help on: Developing a HPLC Method for Ertapenem

MTS HELPDESK

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Question:
"Can you suggest a HPLC method for Ertapenem Injection?”

Answer:
“A quick search on an internet search engine reveals a multitude of HPLC methods in the literature developed for the antibiotic Ertapenem in a variety of samples, but mostly biological matrices. This type of information can be very useful when developing a new HPLC method: it’s reassuring to know that HPLC can be used for analysis of your molecule and; the conditions used by other operators can point you in the direction of the most suitable conditions for your requirements. I recommend a 5 step strategy for HPLC method development, as taught on the MTS training course ‘HPLC Analytical Method Development for Pharmaceutical Analysis’. The 5 steps involved are:

Step 1: What are your goals?
Step 2: What do you already know?
Step 3: What sample(s) will you use to develop the method?
Step 4: What conditions will you use for the method?
Step 5: What method parameters will you use?


Step 1
In the case of Ertapenem injection the first step involves a consideration of the goals of the required method. This simply involves establishing the following for the required method: the analyte(s); the type of method (e.g. assay, impurities, etc.); the nature of the samples; and the purpose of the method (e.g. stability analysis, clinical study, etc.). You will also want to evaluate the level of effort you want to invest in the method and the resources that you have available, together with any specific requirements, such as a short analysis time.

Step 2
The second step involves assessing all the information you currently have on the analyte(s) of interest. For example, the structure of Ertapenem is shown below.


The size of the molecule and the nature of its functional groups means that it will probably be amenable to reversed phase HPLC and this will usually be our first preference. From the structure we can deduce that it is an ionisable compound since it contains amine groups and carboxylic acids groups and thus requires a pH controlled mobile phase for reproducible chromatography. The actual pKas of the molecule may be helpful to select a suitable buffer pH but this information is not always known. You can use predictive software to calculate pKa if it is available. The aromatic nature of the molecule together with the presence of double bonds indicates that the molecule will be suitable for detection by UV.

Other information that you may have access to relating to previous work, sample preparation, and impurities (to give just a few examples) should be evaluated fully. From a brief literature search I found a method for Ertapenem using a C18 column with a mobile phase of methanol and phosphate buffer at pH 6.5 using UV detection at 300nm. This could provide a convenient starting point for method development.

Step 3
The third step involves considering the nature of your samples which will be analysed by the new method and considering what samples you will use for the actual method development. This is likely to be more complex when you have multiple analytes, e.g. an impurities method. You may need to use specific samples containing analytes of interest or you may need to degrade your main analyte to obtain samples of degradation products.

Step 4
The fourth step involves a simple gradient run to test combinations of stationary phase and mobile phase for the analyte(s) of interest. In complex mixtures a number of different combinations may be tried to investigate which gives the best separation. Each set of conditions will be chosen to provide very different environments for the sample and thus provide a range of different selectivities. For a simple analysis involving a single analyte a good first choice is to run a gradient scouting run on a familiar C18 column, in the case of Ertapenem the conditions found in the literature would be a suitable choice using a gradient of 5 to 90% methanol. The information from this run indicates whether a gradient or isocratic run will be possible. If you have a single analyte and no interferences then it is likely that you will be able to run an isocratic method, the most suitable mobile phase proportions can be determined from the gradient scouting run.

Step 5
In the final step the most promising conditions from the scouting experiments are investigated fully and the best mobile phase conditions selected. Other system parameters such as temperature, injection volume, flow rate and column dimensions can be optimised.

This 5 step strategy allows the analyst to develop a suitable method for their purpose, maximising the potential for success by taking a flexible but structured approach to the task."

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