Developing a method for nitrosamine analysis in pharmaceutical products
Written by Kirsten Craven , Senior Global Marketing Manager for Pharma global strategic marketing at SCIEX
In our previous posts we discussed the need for a more comprehensive approach for monitoring contaminants in finished drug products.?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:
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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 implemented lower allowable daily exposure limits.
SCIEX provides a suite of instruments that easily meet the sensitivity needs for today and for the future. To learn more about our solutions for the high sensitivity quantitation of nitrosamines, you can visit our website here.
About the Author:?Kirsten Craven
Kirsten Craven is the Senior Global Marketing Manager for Pharma global strategic marketing at SCIEX. In this role, she manages strategic marketing for the pharmaceutical industry. Kirsten spent the first part of her career working in laboratories across multiple industries before moving into product management, and most recently pharma marketing.
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