The Case of the Too Big Little Peak

Jun 01, 2011
Volume 24, Issue 6

What do you do when a peak grows over time?

I have been working with a woman, who attended one of my classes recently, to solve a problem with a liquid chromatography (LC) method in her laboratory. The problem makes a good case study for "LC Troubleshooting", because it allows us to review how to attack a problem that may not have an easy fix.

The method is for the analysis of a pharmaceutical product in a cream formulation. A preservative is added to the formulation at a 0.2% level, but has a puzzling behaviour in one of the container types in a 12-month stability study at 25 °C. In a 1 oz tube, the preservative peak increases by about 20% from the day 1 concentration, but in a 2 oz tube under the same storage conditions, the same formulation shows no increase in the preservative. At a higher temperature, accelerated stress condition, no change is seen in either container. The challenge is how to separate the preservative from the interference and/or identify the interference so that it can be eliminated, if necessary.

The method is run on a 150 × 4.6 mm, 5 μm particle C8 column thermostatted to 35 °C and uses a 35:65 methanol:buffer mobile phase; UV detection is at 270 nm. The buffer is prepared by adding 1 mL of triethylamine (TEA) to 1 L of water and adjusting the pH to 3.0 with concentrated phosphoric acid. Sample is prepared by dissolving an aliquot in methanol, then diluting to 50:50 methanol:water followed by injection of 50 μL of this solution. The preservative peak is eluted at about 7 min with a flow-rate of 1 mL/min.

When confronted with problems like this, I like to take a four-step approach:

1) Determine what the goal is.

2) Examine the existing method for potential problems.

3) Plan some experiments that might solve the problem.

4) See if there are any alternative approaches that make sense.

The goal of the current work is to isolate the impurity from the preservative. Once this is done, a determination can be made to decide whether the formulation needs to be modified to avoid the appearance of the impurity or if it can be ignored. I suspect that it can be ignored, because at ≈20% of a 0.2% peak, the concentration would be less than the 0.05% reporting level.

The Current Method

Let's next take a look at the existing method — is there anything that raises a possible red flag? The first concern I have is when I see triethylamine-phosphate (TEAP) used as a buffer. This was a very common buffer when the older, low-purity, type-A silica columns were in use. The addition of TEA to the mobile phase made sense, because it acted to help suppress active silanol groups on the silica-based column packing.

In these older columns, there was a significant amount of metal contamination, sometimes >100 ppm, of iron and aluminium. These metals served to "activate" silanol groups to form cation exchange sites on the surface. These were a primary cause of the significant peak tailing observed for bases when analysed on such columns. With today's higher-purity, type-B silicas used for the support in most columns, the metal content is reduced to less than 5 ppm in some cases, so the presence of ionized silanol groups is much less of a concern. This is especially true when the mobile phase pH is low, such as pH 3 used in the present method. For these reasons, TEA is seldom used today. Does it hurt? I doubt it, but I'm a strong believer in the KISS principle — Keep It Simple, Stupid. In other words, extra additives to the mobile phase that don't contribute positively to the separation just make the mobile phase more complicated. And more complicated means more chances for something to go wrong. So this suggests to me that the method may have been one that was based on something from the past and just extended to new conditions.

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