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In our previous blogs we discussed the need for a more comprehensive approach for monitoring contaminants in finished drug products.^1,2 Here we cover a generalized approach for the targeted, quantitative LC-MS/MS analysis of several commonly encountered nitrosamines in pharmaceuticals and ways to address specific challenges with their analysis.

Sample Preparation

While using pure water as an extraction solvent may seem attractive in some cases, many formulations will yield a gel-like slurry that is difficult to work with. Instead, a current approach used by the US FDA is to simply dissolve the drug product or substance in pure methanol and proceed with the analysis.³ Because nitrosamines are semi-volatile compounds, any vacuum or drying steps should be avoided to eliminate losses that could occur from these procedures. And although cleanup and concentration could be accomplished using SPE and centrifugation or positive pressure (as opposed to a vacuum), these steps can be avoided by simply using a more sensitive mass spectrometer.

Chromatography

Nitrosamines should be well separated from the active pharmaceutical ingredient in order to avoid any suppression effects that can result from co-elution with the highly concentrated API. Additionally, some nitrosamines can be quite hydrophilic, such as NDMA, and elute early in a standard gradient. Separations for a common group of nitrosamines have been developed by scientists in the applications group at Phenomenex on several different column phases.4 To avoid contamination of the instrument, a divert valve is used in-line with the LC so that the API peak can be directed to waste instead of the mass spectrometer.

Mass Spectrometry

When considering the type of ionization for this class of compounds, atmospheric pressure chemical ionization (APCI) provides good sensitivity and is less prone to matrix-based ionization effects than electrospray ionization (ESI). For detection and quantitation, targeted multiple reaction monitoring (MRM) experiments provide high sensitivity and high specificity. Interferences that are encountered using HRMS methods, such as the 15N isotope of the common solvent DMA co-eluting with NDMA, are simply avoided with MRM methods. All MRM transitions and source conditions for the quantitation of nitrosamines using SCIEX triple quadrupole systems have been carefully optimized and are freely available. Because of the lack of a blank matrix for matrix matching of calibration standards to samples, matrix spikes or standard addition quantitation can be used. This feature is provided in SCIEX OS Software data processing, and can increase confidence in the final result even from standards prepared in pure methanol.

Sensitivity of the Assay

Using the preparation method discussed above, the sample will be in pure methanol. In order to avoid problems with column retention and poor peak shape in reverse phase separations, small injection volumes are recommended (~1 µL) and the use of high sensitivity instruments. Currently, the US FDA has specified maximum allowable daily exposure amounts for these compounds,5 for example, 0.096 µg per day for NDMA. Thus, the required lower limit of quantitation will be based on the daily dosage of the pharmaceutical.

As an example, if the daily dosage of a drug is 500 mg per day, the required LLOQ would be:
LLOQ ≤ 0.096 µg/0.5g or 0.192 µg/g of NDMA in the API.

Assuming a sample preparation of 100 mg API in 1 mL MeOH, this equates to:
0.192 µg/g* 0.1 g/mL = 0.0192 µg/mL in the final extract, or just under 20 ng/mL.

Higher dosage pharmaceuticals will require even lower limits of quantitation to meet regulatory specifications. Additionally, starting in 2021, the FDA will implement lower allowable daily exposure limits. Thus, while a less sensitive instrument may meet current needs, more sensitive assays will be required in the future. SCIEX provides a suite of instruments that easily meet the sensitivity needs for today and for the future. To learn more about SCIEX solutions for the high sensitivity quantitation of nitrosamines visit: https://info.sciex.com/genotoxic-impurities

References

What have we learned from the nitrosamine crisis?, Neil Walsh, March 5, 2020. Are we proactively solving the nitrosamine crisis?, Neil Walsh, May 8, 2020.

https://www.fda.gov/media/138617/download

https://www.phenomenex.com/ViewDocument/?id=tn-1259%3A+rapid+analysis+of+genotoxic+nitrosamines+by+hplc-ms_ms

https://www.fda.gov/drugs/drug-safety-and-availability/fda-updates-and-press-announcements-angiotensin-ii-receptor-blocker-arb-recalls-valsartan-losartan

This blog is part 3 of a 3-part series on nitrosamine analysis. Read part 1 (What have we learned from the nitrosamine crisis?), and part 2 (Are we proactively solving the nitrosamine crisis?).