Understanding In Vitro Nitrosamine Formation During Sample Preparation

Confirmatory Testing & Analytical Challenges
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Hello everyone,

I’ve observed an interesting and somewhat confusing behavior regarding nitrosamine formation in our API and would appreciate any input or similar experiences.

We initially used a simple ACN/H2O (1:1) solvent system and typically measured nitrosamine levels in the range of 0.2–0.4 ppm.

We then developed a modified method using:

- ACN/H2O + 5 g/L Vitamin C

- ACN/H2O + 5 g/L Vitamin C adjusted to pH 7

Both modified methods initially gave similar lower results. However:

- Only the pH 7 variant was stable enough over time for reliable measurement

- In both cases (even with pH 7), the nitrosamine level increased over time and eventually approached values similar to the original solvent

An interesting observation is the following:

Initial value (old method) → measured with new method (pH 7):

0.4 ppm → 0.2 ppm

0.2 ppm → 0.02 ppm

So the reduction factor is clearly not constant:

- at higher levels → roughly 50% reduction

- at lower levels → up to 90% reduction

This makes it difficult to assess the “true” nitrosamine content in the product.

Has anyone observed similar non-linear suppression effects when using scavengers like Vitamin C?

Any insights on how to better distinguish real content vs. sample preparation artefacts would be highly appreciated.

Best regards,

Philipp

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Have you tried adjusting the level of Vitamin C away from 5g/L - either higher or lower?

Just a thought, but if your amount of Vit C in your sample prep was sufficient to scavenge the nitrite responsible for the formation of 0.18 ppm of nitrosamine then your levels would drop from 0.2 to 0.02 ppm and 0.4 to 0.22 ppm - close to what is observed.

If this was the case then a higher level of Vit C could potentially drop the 0.2 ppm to nothing and the 0.4 ppm lower.

A lower level of Vit C would drop both of them by a lower amount.

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Can’t remember the exact amount off the top of my head, but our default was 1.5% or thereabouts

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Dear Phil,

can i ask the reason for using Ascorbic acid?

Is only for scaveging the nitrites from the diluent and stop further formation of nitrosamines?

I am asking this, as our feedback from the lab is that, avoiding sonication, we did not notice any strange trend in the level of nitrosamine we record and we do not use ascorbic acid as a scavenger in the diluent.

Have you try to test without ascorbic acid?

best regards

Christos

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Thanks for pointing this out . I originally focused on the ratios but overlooked that the reduction is actually quite consistent in absolute terms.

We investigated the effect of ascorbic acid (Vit-C) addition on the measured Nitrosamine levels.

  • Decreasing Vit-C leads to higher Nitrosamine values

  • Doubling the amount does not further reduce the levels

  • Higher concentrations are not feasible due to phase separation (ACN/water system)

We also checked both separated phases individually and summed up the Nitrosamine content. The total matches the result obtained using 5 g/L Vit-C.

Initially, this behavior was somewhat counterintuitive, but looking at it more closely:

  • All API batches tested so far show very similar nitrite levels (~0.1 ppm)

  • This corresponds theoretically to ~0.5 ppm Nitrosamine formation (assuming full conversion)

Experimentally, we observe a reduction of ~0.2 ppm when adding Vit-C, which is roughly 40% of the theoretical artefact level.

This is consistent with:

  • identical nitrite levels → same maximum artefact potential

  • constant Vit-C concentration → similar quenching efficiency
    → resulting in a fairly constant reduction across samples

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