We all know that no HPLC method is perfect and that it may need to be “tweaked” once in a while to make it operate properly. But in today’s highly regulated environment, there’s always a question about whether or not such adjustments are allowed. For compendial methods in the pharmaceutical industry, the various pharmacopoeia give guidelines about method adjustment, for example, the European Pharmacopoeia (EP) states:
“The extent to which the various parameters of a chromatographic test may be adjusted to satisfy the system suitability criteria without fundamentally modifying the methods are listed” [in Table 1].
The United States Pharmacopoeia (USP) states:
“The specification of definitive parameters in a monograph does not preclude the use of other suitable operating conditions. Adjustments permitted only when… the adjustments improved the quality of the chromatography with respect to the system suitability requirements.”
The allowed adjustments are listed, as summarized in Table 1. I don’t have ready access to the pharmacopoeia of other jurisdictions (e.g., British or Japanese), but I expect that their guidelines are similar on this issue. If any of you readers wants to provide me with a pdf copy of the appropriate section of other pharmacopoeia, please send this to TechTips@sepscience.com.
Because of the global nature of the pharmaceutical industry, many of the pharmacopoeial organizations are trying to “harmonize” their regulations so that the same rules are used worldwide. The key factor in the present discussion is the system suitability test. Adjustments to a method are allowed within the prescribed limits as long as system suitability passes. If adjustments exceed the prescribed limits, the implication is that some kind of re-validation will be required.
pH and Buffer
Let’s first look at the allowable changes in the mobile-phase pH, as shown in Table 1. We can see that a change of up to ±0.2 pH units is listed. This means that if the method calls for a buffer adjusted to pH 2.5, any pH between 2.3 and 2.7 is allowed, provided system suitability passes. Considering normal laboratory error, this makes sense. If you adjust the pH of a solution using a pH meter, as most of us do, the normal variation is ≈±0.1 pH units. So if ±0.1 is the expected variation under normal circumstances, it makes sense that you should be able to make a little more adjustment than this. Also note that the EP has an additional guideline that if pH is controlled and the sample contains only neutral compounds, a change of up to ±1.0 pH units can be made. This also makes sense, because, although the mobile phase pH should not affect the retention or peak shape for neutrals, it also has other influences in the system, such as controlling ionization of the column. A more liberal adjustment interval should be allowed for neutrals than ionic compounds, and this is possible under the EP guidelines. And remember that the buffer pH should always be measured and adjusted in the aqueous solution before organic solvents are added – the presence of organic changes the way the pH meter reads and is not equivalent to the pH in an aqueous solution.
In a similar manner, the ionic strength of the buffer can be adjusted by ±10%. So if you are using a 25 mM phosphate buffer, you can adjust by ±10% x 25 mM = ±2.5 mM, or 22.5 – 27.5 mM. In my opinion, this is a bit restrictive. In my experience, reversed-phase methods are not very sensitive to buffer concentration. For example, my guess is that most methods with a 25 mM buffer would work just fine from 10 mM to 50 mM. When ionized compounds are present and ion exchange is the retention mechanism, then buffer molarity will be much more important.
Just as the American automobile advertisements always contain the statement, “your mileage may vary,” I need to include a little disclaimer here. Unfortunately, the regulations written by any agency are not always crystal clear. They leave some interpretation to the reader. In the case of this present discussion of adjustment vs modification, I’ve given my interpretations of the regulations, but these are by no means official interpretations. You will need to read the regulations yourself and decide how you will interpret them. It is a good idea to write down this interpretation as a standard operating procedure (SOP) so that everyone in your organization follows the same interpretation. In the next few weeks, we’ll look at the remaining variables in Table 1.
1. European Pharmacopoeia 6.0 (2010) Section 18.104.22.168.
2. United States Pharmacopoeia 34 (2011) Section 621.
This blog article series is produced in collaboration with John Dolan, best known as one of the world’s foremost HPLC troubleshooting authorities. He is also known for his research with Lloyd Snyder, which resulted in more than 100 technical publications and three books. If you have any questions about this article send them to TechTips@sepscience.com