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Tony Taylor BSc CChem FRSC Cert. Ed.

Tony has been doing, researching, teaching and training in analytical chemistry for the past 28 years.  He comes from a pharmaceutical and polymer analysis background and continues to work with both liquid and gas phase techniques at Crawford Scientific (UK).

His main interests are the use of LC-MS and GC-MS for structural characterisation and the quantitation of trace components in complex matrices.  He is professionally qualified trainer and is Technical Director of the CHROMacademy.

 

Allowable changes to Pharmacopeial HPLC Methods

I started my career working in a Quality Control laboratory.  I'm familiar with the constraints that must be imposed to ensure consistent global standards and the pace at which change can be implemented without loss of control either within a business or across an industry.  OK – scene set.

However, I really feel it's time for the Pharmacopeia to drag themselves into the 21st Century and realise the benefits of using smaller diameter stationary phase particles and superficially porous particle morphology.  Higher resolution and higher throughput can both be realised by these approaches, which can be very beneficial in a QC laboratory environment, yet many pharmacopeial methods still use long columns with large (5mm) fully porous particles.

Whilst I'm sure many of you will be aware of what changes are possible to pharmacopeial methods, I still see many old or under developed methods which could be updated and I'm often asked what changes can and can't be made – hence I've written this so I can point folks to it for reference!

The big change to allowable method adjustments came in August 2014 when USP37-NF32S1 was released and Chapter <621> Chromatography set-out a revised schedule of allowable changes to methods.  For HPLC methods I've outlined the changes in Table 1 below with some further explanation of how the changes have been interpreted.

Parameter

USP 34

USP37-NF32S1

 

Isocratic or Gradient

Isocratic Method

Gradient Method

Mobile phase pH +/- 0.2 pH units +/- 0.2 pH units +/- 0.2 pH units
Concentration of Salts +/- 10% providing pH limits not exceeded +/- 10% providing pH limits not exceeded +/- 10% providing pH limits not exceeded
Mobile phase ratio Minor component (<50%)
+/- 30% relative
(+/- 10% absolute)
Minor component (<50%)
+/- 30% relative
(+/- 10% absolute)
No changes allowed
Column i.d. Flexible  - maintain linear velocity Flexible  - maintain linear velocity No changes allowed
Column length +/- 70% L/dp No changes allowed
Particle Size - 50% L/dp (or N) : -25 to +50% No changes allowed
Flow rate Based on column dimensionsa or +/- 50% Based on particle sizeb or +/- 50% provided N decreases ≤20% No changes allowed
Detector wavelength No changes allowed No changes allowed No changes allowed
Column Temp. +/- 10oC +/- 10oC +/- 10oC
Injection Volume Unlimited reduction provided precision and detection limits met Unlimited change provided precision and detection limits met Unlimited change provided precision and detection limits met
[a]
[eqn. 1] F – flow rate, L- column length, d – column diameter
 
[b]

[eqn. 2] F – flow rate, dc- column internal diameter, dp – particle diameter

Some notes and comments on the new allowable changes to help interpret them and highlight issues and difficulties.

1.

Please note the (severe) restrictions on gradient HPLC methods.  Adjustments to methods which take advantage of smaller or superficially porous particles are restricted to isocratic methods only.  Again – whilst being sympathetic to the need for control, I do wonder why there have not been more efforts to specify allowable changes to gradient methods

 

2.

For clarification on the mobile phase strength guidelines;

An eluent containing 60:40 aqueous:organic can be changed in the following ways –
A 30% change to the 40% organic is 12%, however this exceeds the 10% absolute limit of change allowed – therefore the range of allowable change for this eluent system would be 20% - 50% i.e. +/-10% absolute.

An eluent containing 90:10 aqueous:organic can be changed in the following ways –
A 30% change to the 10% organic is 3.0%, which does not exceed the 10% absolute limit of change allowed – therefore the range of allowable change for this eluent system would be 7.0 – 13.0%  i.e. +/-30% of the original organic %.

3.

The L/dp ratio can be explained using the Equation 3;

[eqn. 3]

 

For small molecules separated using well packed columns with fully porous particles, the reduced plate height will have a value of around 2 [1].  This constant for the reduced plate height allows us to use the column length to particle size ratio (L/dp) within the range of -25% to +50% of the original value.

So an original isocratic method using a 150 x 4.6mm column with 5μm particles (L/dp = 30,000) with a flow rate of 1mL/min. and an injection volume of 10μL can be scaled to a column dimension of

 

50 x 2.1mm with 1.8μm particles   (L/dp = 27,778)

This represents a change in L/dp of approx. -7.4%, which is allowable under the USP37-NF32S1 guidelines.

Of course, scaling to this extent will need some adjustments in flow rate which can be calculated using Equation 2 above;

 
 

This change is within the allowable +/- 50% range from the original 1mL/min. flow rate but we need to also ensure that the efficiency of the peaks does not reduce by more than 20%.

With efficiency in mind, one must be very mindful of the requirements for lower extra column volume within the system, and it will be likely that a different HPLC system will be required with lower extra column and flow cell volumes and the ability to cope with higher back pressures.

We can also approximate the new injection volume using the equation;

 
 

It's important here to note that here we have used the calculations above to give us an 'equivalent' separation within approximately the same time frame.  One may wish to increase the flow rate in order to obtain a faster separation and we would therefore need to carefully check system back pressure is within the allowable range for the instrument and that the USP37-NF32S1 guidelines of F1 +/- 50% and N decreases ≤20% are met

4.

For superficially porous particles, the reduced plate height value can change to values between 1.4 and 1.6 and so we will obtain a marked increase in the value for N according to equation 3 above and in these circumstances we need to take care not to EXCEED the range of values given for 'equivalent N' in the USP37-NF32S1 guidelines (-25 to +50% of original N value).  An example for the method translation of a Naproxen and related substance assay are shown below, where various L/dp combinations of core-shell columns are tried without any other adjustments to the method conditions;
 

50:49:1 CH3CN:H2O:CH3COOH, 1.2 mL/min, All Pressures < 300 bar, Sample: Naproxen, Butyrophenone

Reproduced with permission of Agilent Technologies from publication 5991-5408EN and Poster Presentation 'Transferring and Scaling Methods amongst a variety of Superficially Porous Particles', Anne Mack, HPLC 2015.

  One should recognise that several of these chromatograms produce excellent separations but which are outside the allowable range USP37-NF32S1 – and these 'transgressions' in either L/dp ratio or efficiency (N) are highlighted in red.  However, it should also be obvious that by choosing a 100 x 4.6, 4μm core-shell column and changing no other parameters, the guidelines are met and the overall run time is halved.

So, I've shown here that we can take a pragmatic or empirical approach to changing isocratic HPLC methods from the USP to take advantage of modern particle technologies.  Let's hope to see a set of guidelines which highlight allowable changes to gradient HPLC methods soon!  As a foot note – please just be aware that these changes are for USP methods only and that other Pharmacopeial authorities may differ in the changes which are allowed.

[1] Snyder, L. R.; Kirkland, J.J.; Dolan, J.W., Introduction to Modern Liquid Chromatography, 3rd ed.; John Wiley & Sons:  U.S. , 2010, p205

 
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