Help on: TOC vs HPLC for Cleaning Validation

MTS HELPDESK

Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

Question:
"I want to know whether TOC analysis is better than HPLC for cleaning validation?"

Answer:
"The use of Total Organic Carbon (TOC) as an analytical test for cleaning validation has increased rapidly in recent years. This is due to a number of factors including:

• It is very easy to use;
• The actual testing can be carried out very quickly;
• TOC testing requires very little method development and is easy to validate;
• One method can be used for all cleaning samples; and
• It is a cost effective analytical technique.

TOC measures all the carbon in the sample and is thus a non-specific technique; this can be viewed as both an advantage and disadvantage. On one hand it means that all carbon which can be oxidised under TOC conditions can be detected and thus the result provides a measure of all contaminants. On the other hand it does not determine the amount due to the active substance. The result may be considered as ‘worst case’, assuming that the entire residue is due to the active substance.

To use TOC for cleaning analysis, you need to establish that the contaminating material is organic and contains carbon that can be oxidised under the TOC test conditions; some organic compounds cannot be reliably detected using TOC. Use of TOC requires that the contaminant is at least slightly soluble in water.

As to which is better, TOC or HPLC, it will depend on the contaminating substances you are testing for. The advantages of TOC make it preferable but there are times when it will not be suitable, in which case HPLC may provide a better option. The effort involved in validation of cleaning means that it is not usually beneficial to switch established processes from HPLC to TOC. However for new processes TOC may provide a good solution.”

MTS Recommends... LC Trends at Pittcon

‘New Chromatography Columns and Accessories at Pittcon 2010: Part I’ by Ronald E. Majors in the March 2010 issue of LCGC North America.

Every year after Pittcon, Ron Majors writes a great summary of the trends and new introductions at the conference relating to chromatography. This provides an easy way to keep up to date with the latest developments in LC so that you can evaluate their usefulness in your laboratory.

This year Ron observes ‘that there was a further increase in columns and accessories designed to work with ultrahigh-pressure liquid chromatography (UHPLC) products’. New column introductions included more sub 2 micron columns for UHPLC but also a number of manufacturers have introduced superficially porous particles (SPP) which have a solid core of non-porous silica coated with a thin shell of silica and can achieve improved efficiency comparable to UHPLC using standard HPLC systems. There were over 57 new reversed phase bonded phases added in Pittcon 2010, dominated, as in previous years, by silica based columns with C18 bonded phases.

All these identified trends in the very latest LC developments are already incorporated into training courses available from MTS so you can be sure of up to date and relevant training when you register for one of our courses.

Help on: Developing a HPLC Method for Ertapenem

MTS HELPDESK

Do you have any problems relating to analytical chemistry for pharmaceuticals or training? Send your questions to the MTS helpdesk using our contact form.

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."

MTS Recommends... A History of LC-MS

‘The fascinating history of the development of LC-MS; a personal perspective’, by Frank Pullen, in the March 2010 issue of Chromatography Today.