(M1430-05-26) Rapid and Reliable Nitrite Content Analysis in Pharmaceutical Excipients with the ACQUITY^TM QDa^TM II Mass Detector

Purpose: Nitrite is the most critical risk factor in N-Nitrosamine formation, present as an impurity in pharmaceutical excipients typically in parts per million (ppm). Screening for total nitrite content as a single underlying precursor to N-Nitrosamine formation can be a simple and pragmatic approach to support risk assessment in the pharmaceutical manufacturing process. A reliable solution is proposed to manage high-throughput routine workflows in excipient testing, while maintaining a low cost of analysis per sample. These results describe a UHPLC coupled to QDa II Mass Detector employed to assess the suitability of this analytical configuration for long term analysis of pharmaceutical excipients over the course of 29 days of analysis, demonstrating the performance, robustness, and reliability required for effective analysis.

Methods: Selected representative pharmaceutical excipients were extracted following a modified protocol from Jireš & Douša, where powdered excipients were taken through a 2,3-diaminonaphthalene (DAN) derivatisation method. The principle of this method is reaction of nitrite ions with DAN to form 1H-Naphtho(2,3-d)triazole (NAT) which can then be separated and detected as a proxy for total nitrite content, using mass detection. Unspiked excipient samples were quantified against a calibration curve of known nitrite concentration, the recoveries of spiked excipient samples were also assessed. Instrument QC standards of acetaminophen and caffeine were analysed throughout the analysis, to monitor system stability for the purposes of robustness testing. Samples were prepared in four sequential batches and assessed over 29 days of analysis.

Results: Determination of nitrite content in selected excipients was achieved by quantifying NAT (m/z 170) with the QDa II Mass Detector operated in positive electrospray ionization (ESI+). The derivatized end compound had a retention time (RT) of 2.73 mins in a 6-minute method. Nitrite was easily detected in maize starch excipients at 0.14 µg/g, in line with mean levels reported in excipients databases. The %RSD for n=27 injection of maize starch samples containing this amount of nitrite was 8.7%. Excipients samples of sorbitol, lactose monohydrate, maltodextrin, and calcium carbonate did not exhibit detectable levels of nitrite during the analysis. All excipient samples were spiked at 0.2, 2.0 and 5.0 µg/g, and recoveries measured across a 190-hour sample batch ranged from 70 - 129%. %RSDs across the spike levels of the excipients analysed did not exceed 18.5%, 11.1%, and 6.0% respectively. The total average recovery across all samples and spikes was 94%. Over the course of 29 days of analysis, ~6000 injections were performed on the QDa II with only 2 hours of instrument downtime required for source component cleaning. Throughout 520 hours of continuous analysis following the 2 hours of downtime, the %RSD of absolute peak area for any given QC level remained below 12.5%, demonstrating stability of the system throughout the analysis. The %RSDs of absolute peak area for all nitrite standard injections was consistently less than 14.7% for any given sample batch analysed. The %RSD of instrument QC injections in each sample batch did not exceed 9.8%, demonstrating consistent performance. The results from the experiments performed indicate that the ACQUITY QDa II Mass Detector is a robust and reliable instrument suitable for routine high throughput applications.

Conclusion: The results from the experiments performed indicate that the ACQUITY QDa II Mass Detector is a robust and reliable instrument suitable for routine high throughput applications, such as the analysis of nitrite in pharmaceutical excipients. The instrument successfully analysed ~6000 injections over 676 hours of analysis, experiencing only 2 hours of downtime.The %RSD of instrument QC injections in a given sample batch did not exceed 9.8%, demonstrating consistent performance. Throughout 520 hours of continuous analysis, the %RSD of absolute peak area for any given QC level remained below 12.5%, demonstrating excellent stability of the system throughout the analysis. The %RSDs of absolute peak area for all nitrite standard injections was consistently less than 14.7% for any given sample batch analysed. Nitrite levels were successfully detected in maize starch excipients at 0.14 µg/g, in line with mean levels reported in excipients databases, indicating the quantitative accuracy of the method. Other excipients did not exhibit detectable levels of nitrite during the analysis. This 6-minute method using the ACQUITY Arc UHPLC and ACQUITY QDa II Mass Detector is efficient and cost-effective approach for µg/g level quantitation of nitrite in pharmaceutical excipient analysis.

References: Jireš J, Douša M. Nitrites as precursors of N-nitrosation in pharmaceutical samples - A trace level analysis. J Pharm Biomed Anal. 2022 May 10;213:114677. doi: 10.1016/j.jpba.2022.114677. Epub 2022 Feb 18. PMID: 35248987.


Nitrite content (µg/g) in unspiked excipient samples analysed over 190 hours of continuous acquisition. Nitrite content in the maize starch sample exceeded the literature reported 0.5 x mean value of 0.105 µg/g. The precision of measurement of nitrite in this sample over n=27 injections indicate an %RSD of 8.7%


Absolute peak area of QC components (caffeine & acetaminophen) over 520 hours of continuous analysis


Average %RSDs of all excipient spike levels throughout the analysis, where each of the four datapoints per excipient spike level represents an average value for a spike level in a given sample batch