Simultaneous Determination and Quantification of Nine Nitrosamine Impurities in Semi-Solid Forms Using a GC–MS/MS Method

Abstract

Many studies are being conducted on the detection of nitrosamine impurities in solid formulations. However, research on semi-solid formulations such as gels, ointments and creams is not common. In particular, excipients used to increase viscosity and add fragrance can significantly impact the sample preparation. Volatile compounds derived from natural fragrances are composed of a wide variety of complex components, making them very difficult to handle and completely separate from the analytes. Due to the complex composition of these formulations, an analytical method was developed to accurately separate and analyze nine nitrosamine impurities (NDMA, NDEA, NMEA, NDPA, NDBA, NPIP, NMOR, DIPNA and EIPNA) simultaneously. To overcome challenges in the sample preparation of excipients with physical and chemical properties, the sample was prepared using solvents such as methanol, hexane, water and dichloromethane. The target analytes were extracted with dichloromethane for the final preparation for GC–MS/MS and the optimal conditions were established. While multiple GC columns were tested, peak overlapping interferences were observed, leading to the use of a 60m-long column to overcome peak overlap. The GC–MS/MS condition was set for optimal performance and ionization energy, with parameters adjusted for each analyte. The developed method was validated in accordance with guidelines to ensure its reliability and suitability. As a result, all nine nitrosamine impurities were simultaneously analyzed, confirming excellent performance. The sample preparation method and procedure, column specification and GC–MS/MS conditions have the potential to be adapted not only for semi-solid formulations of pharmaceuticals and cosmetics but also for other formulations such as solid and liquid samples, rendering them suitable for the analysis of nitrosamine impurities.

Introduction

Nitrosamine impurities are found in many fields such as food, cosmetics, pharmaceuticals and household products. In particular, trace amounts of impurities found in pharmaceuticals have become a major concern for many regulatory authorities. N-nitrosamines have been classified as “probable human carcinogens” by the International Agency for Research on Cancer [1] and were listed as a representative “cohort of concern” of mutagenic carcinogens (in addition to aflatoxin-like compounds and alkyl azoxy compounds) in the International Council for Harmonization (ICH) of Technical Requirements for Pharmaceuticals for Human Use Guidance for Industry M7 (R1) [2].

The nitrosamine impurity analytical methods published by regulatory authorities are primarily focused on raw materials, tablets, capsules and liquid formulations [3,4,5,6]. Most of these methods are single-component analyses, reflecting the difficulties of simultaneous analysis due to the characteristics of the matrix. No official analytical methods have been announced for semi-solid formulations, such as ointments, creams and gels. Methods for analyzing nitrosamine impurities in the form of highly viscous semi-solids are not common. Hydroxypropylmethylcellulose or carbomer swells to form a white colloidal solution with a certain viscosity. It can undergo a sol–gel phase transition due to temperature changes in a specific concentration of the solution. Polyethyleneglycol (PEG) was used as a non-volatile sticky liquid for viscosity as well as lubrication. Also, volatile compounds derived from natural fragrances are composed of a wide variety of complex components, making them very difficult to handle and completely separate them from the analytes.

In cases where preparation is not adequate, column clogging may occur [7]. In particular, the analysis of nitrosamine impurities with amphiphilic properties that dissolve in both specific organic solvents and aqueous solvents is very challenging due to the difficulty in extraction. When various excipients such as fragrance and colorants are added, it significantly influences the separation of each component. Particularly in simultaneous analysis methods, issues may arise, such as poor peak separation and asymmetry. Among similar types of cosmetics, such as cream, gel, lotion and shampoo, there is some information on the analysis of nitrosamines but it is from a paper [8] published a long time ago. Since then, with the advancement of many analytical techniques, a new method was sought to find a new, cutting-edge analysis. In addition, simultaneous analysis methods [9] for nitrosamine impurities found in specific reagents have been introduced but many reports introduced analyses of relatively simple matrices or liquid samples. Developing an analytical method that can simultaneously analyze nine nitrosamine impurities (NDMA, NDEA, NMEA, NDPA, NDBA, NPIP, NMOR, DIPNA and EIPNA) from semi-solid pharmaceuticals with complex compositions will greatly contribute to the improvement of pharmaceutical quality, and enable rapid and accurate analysis.

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Lee, N.; Go, H.; Park, Y.-j. Simultaneous Determination and Quantification of NineNitrosamine Impurities in Semi-Solid Forms Using a GC–MS/MS Method. Separations 2025, 12, 120. https://doi.org/10.3390/separations12050120