SPE and GC–MS for Analyzing N–Nitrosamine Impurities in Cough Syrups

Scientists from the Mankind Research Centre in Haryana, India recently tested how effective solid-phase extraction (SPE) and gas chromatography–mass spectrometry (GC–MS) are for detecting small molecule N–nitrosamine impurities in antitussive syrups. Their findings were published in the Journal of Chromatography A (1).

Hand pouring medication or antipyretic syrup from bottle to spoon. healthcare, people and medicine concept. | Image Credit: © Siam - stock.adobe.com

Nitrosamines are organic compounds that stem from chemical reactions. They are organic compounds that existing at low levels in water and foods, but some can also form in drugs during manufacturing. These chemicals, called N-Nitrosodimethylamine (NDMA), N-Nitrosodiethylamine (NDEA), and N-Nitroso-N-methyl-4-aminobutyric acid (NMBA), may increase the risk of cancer if people are exposed to them above acceptable levels and over long time periods (2). Because of the highly toxic nature of these compounds, various regulatory agencies have recommended steps to establish control and limits regarding acceptable intake (AI) to monitor their presence in various drug substances and products.

Since nitrosamine impurities often have low AI, analysis is typically carried out with the help of instruments like liquid chromatography–mass spectrometry (LC–MS) and gas chromatography–mass spectrometry (GC–MS). In this article, the scientists present the application of solid phase extraction technique with GC–MS to enrich and analyze the nitrosamine impurities present in cough syrups. A quantitative testing method was developed for analyzing low molecular weight (small molecules) nitrosamine impurities in cough syrups using solid phase extraction (SPE) on strong cation-exchange functionalized polymeric sorbent cartridges followed by GC–MS.

Solid phase extraction is used to extract nitrosamine impurities from large sample volumes of cough syrup to achieve result recovery and reproducibility at lower standard concentrations. SPE is a non-equilibrium procedure that combines non-linear modes of chromatographic separation; these modes include sample loading/retention followed by stepwise sample desorption/elution or gradient dependent desorption for targeted analysis. Compared to liquid-liquid extraction (LLE), SPE is a targeted form of sample preparation that separates the analyte of interest from interfering compounds that may be present in samples. Altogether, SPE can help purify, fractionate, and concentrate targeted analytes with superior column loading and reduced mobile phase/solvent consumption.

The matrix spike recoveries of the nitrosamine impurities from the cough syrup samples were observed to be within the range of 90–120%. Limit of detection (LOD) achieved for N–Nitrosodimethylamine (NDMA) and N–Nitroso morpholine (NMOR) was about 0.1 ng/mL while the LOD for N–Nitrosodiethylamine (NDEA), N–Nitrosodiisopropylamine (NDIPA) and N–Nitrosoisopropylethylamine (NIPEA) impurities was about 0.02 ng/mL. The method was evaluated and found to meet the acceptable criteria as per the ICH Q2 guidelines for a working concentration range of 0.02 ng/mL to 1.2 ng/mL for the analyzed impurities. The selectivity of the nitrosamine impurities against the presence of drug product was established using multiple reaction monitoring (MRM) transitions during analysis.

Using SPE for sample clean up and quantitative analysis of nitrosamines was deemed suitable for its intended purpose, owing to limitations such as lower drug content, ppm level limit determinations and sample interference. This method met acceptable limits for the evaluated criteria, with the method’s sensitivity ensuring low-level determination of nitrosamine impurities in the sample. This approach could potentially be extended to various ranges of sample matrices, all while considering SPE developmental guidelines as shown in this study.

References

(1) Aggarwal, P.; Sharma, G.; Singh, V.; Dev, R.; Kumar, A. Solid-Phase Extraction Followed by Gas Chromatography-Mass Spectrometry for the Quantitative Analysis of Small Molecule N-Nitrosamine Impurities in Antitussive Syrups. J. Chromatogr. A 2024, 1732, 465148. DOI: 10.1016/j.chroma.2024.465148

(2) What to Know and Do About Possible Nitrosamines in Your Medication. U.S. FDA 2024. https://www.fda.gov/consumers/consumer-updates/what-know-and-do-about-possible-nitrosamines-your-medication (accessed 2024-8-6)