US and Euro Pharmacopeia

Harnessing the Power of Core-Shell Technology to Improve Older, Validated HPLC Methods within Acceptable European Pharmacopeia Guidelines.

Guest Author: Dr. Jeff Layne

Welcome back to the second article on harnessing the power of core-shell technology to improve the quality and productivity of existing compendial methods. Before reading further, make sure to go back and read the first one, “Using Core-Shell Technology to Improve HPLC Methods Within USP: Part 2”.

The key point I hope to illustrate is that coupling allowable adjustments in USP and EP methods (outlined in USP Chapter <621> and Ph. Eur. Chapter 2.2.64) with the performance benefits of ultra-high efficiency core-shell media, analysis times can be significantly decreased, while maintaining or even improving chromatography (e.g. sensitivity or resolution).

In the previous article, we took a USP assay method for Propranolol, which called for a 250 x 4.6 mm L7 column packed with 5 µm media, and simply changed to a 100 x 4.6 mm L7 column packed with 2.6 µm media. This is change is within the acceptable adjustment ratio of column length to particle size (-25% and +50% of the starting ratio value) as stated in Chapter <621>, which also reduced our total analysis time by a factor of four.  That is an amazing increase in productivity by just swapping out a column, and made possible by the efficiency advantage provided by moving from traditional fully porous media to core-shell media.

Now let’s look at another method improvement, this time with several modifications, all within the allowable adjustments.  The monograph for Metoprolol and related substances from the European Pharmacopeia (Ph. Eur. Monograph 1028) calls for a 150 x 3.9 mm C18 column packed with 5 µm media and run at a flow rate of 1 mL/min.  The full details for the official monograph are in Figure 1 below, and the allowable adjustments for Ph. Eur. methods are shown in Figure 2.

Figure 1.  Metoprolol tartrate and related substances Ph. Eur. Method

Metoprolol tartrate

Figure 2.  Allowable adjustments to Isocratic European Pharmacopeia methods

European Pharmacopeia

As you can see, the Ph. Eur. allows a wide variety of adjustments to be made to existing isocratic methods, if system suitability requirements are maintained.  Our first adjustment will be to change the column inner diameter from the 150 x 3.9 mm column specified in the monograph to a more conventional 150 x 4.6 mm format, a change that is well within the allowable adjustment of ± 25%.  The resulting chromatogram, obtained using a conventional fully porous 5 µm C18 column, is show in Figure 3.  Using this format, the elution time of the last peak is about 13 minutes and we are easily able to meet our minimum resolution (Rs) requirement of 6.0 with an actual resolution of about 10.

Figure 3.  Metoprolol and related substances using Luna 5 µm C18(2) 150 x 4.6mm.  Please note that the flow rate was held constant when moving from the stated 3.9 mm ID column in the monograph to a 4.6 mm ID column.

European Pharmacopeia

Now, let’s run that same method, but simply switch from the fully porous Luna 5 µm C18(2) column to a core-shell Kinetex 2.6 µm C18 150 x 4.6 mm column (Figure 4).  When switching to the core-shell based column, you will immediately notice that the retention time decreases from 13 min to about 7.3 min AND because of the high efficiency of the Kinetex core-shell media, we INCREASE our resolution to 11.82.  So, we are getting better resolution and a ~40% decrease in analysis time simply by switching from the fully porous 5 µm packed column to a column of the same size and dimension packed with 2.6 µm Kinetex core-shell media.  That change in particle size is within the acceptable allowable adjustment in particle size for isocratic Ph. Eur. method (up to a -50% in particle size). Logically, with the same column ID and flow rate are the same, you would expect Kinetex and Luna to have similar retention times, however we have a lower surface area with Kinetex, therefore less retention.

Figure 4.  Metoprolol and related substances using the core-shell Kinetex 5 µm C18 150 x 4.6 mm

European Pharmacopeia

But wait!  We’re not done yet.  We can even further improve our productivity moving to a shorter column length (100 x 4.6 mm) and increasing the flow rate from 1 mL/min to 1.5 mL/min.  Both of these changes are within the acceptable range of modifications as indicated in Figure 2 (± 70% change in column length and ± 50% change in flow rate).  The resulting chromatogram (Figure 5) shows that our run time has been decreased to just ~3.3min.  That is almost a 75% decrease in analysis time from our original method, and we still are meeting our minimum resolution value for system suitability with an Rs of 9.21 (minimum Rs = 6.0).  That’s almost a 4-fold increase in productivity, all by making a couple of very simple changes to column format and flow rate.  Of course, the best part is that these adjustments fall within the acceptable range of method adjustments specified by the European Pharmacopeia so you should not even be required to perform a method re-validation.

Figure 5.  Metoprolol and related substances using the core-shell Kinetex 5 µm C18 100 x 4.6 mm column at a flow rate of 1.5 mL/min

European Pharmacopeia

So, there you have it. Another example of how using Kinetex core-shell technology with some simple (and allowable) method adjustments to compendial methods can allow improvement in your laboratory productivity without sacrificing chromatographic performance. If you want more information, please contact your local Phenomenex representative!

Back to the Lab!


Related Articles:

“USING CORE-SHELL TECHNOLOGY TO IMPROVE HPLC METHODS WITHIN USP: PART 1”

“WHAT IS USP CHAPTER 621?”

“REVISION OF EUROPEAN PHARMACOPEIA (EP) CHAPTER 2.2.46”

Summary
Using Core-Shell Technology to Improve HPLC Methods within USP: Part 2
Article Name
Using Core-Shell Technology to Improve HPLC Methods within USP: Part 2
Description
Dr. Jeff Layne continues his series on harnessing the power of core-shell technology to improve HPLC methods within acceptable EP guidelines.

Leave a Reply