(M1430-09-61) Improvement upon a Multi-residue Method for Nitrosamine Analysis in Losartan Drug Product Using an Enhanced LC/MS/MS System

Purpose: Nitrosamine impurities are byproducts produced in trace amounts during manufacture of pharmaceutical drugs. They are a member of the “cohort of concern” classifying them as potentially genotoxic impurities and probable carcinogens if they exceeded the maximum allowable intake of 96 ng/kg for a treatment with long-term intake. Therefore, it is important to determine their levels in final drug products with a high level of sensitivity and confidence. In this study, we further improved upon an existing nitrosamine quantification method for pharmaceutical drug impurity analysis using an LC-MS/MS system. We compared various LC and MS source conditions along with different LC-MS/MS instrument platforms to improve analytical sensitivity. Our results show increased analytical sensitivity compared to the original method.

Methods: A C-18 column was used with mobile phases consisting of water and methanol with formic acid. Standards containing eight nitrosamines NDMA, NDEA, NMOR, NMEA, NPYR, NPIP, NDPA and NDBA were purchased from Agilent. Sample preparation was carried out following these steps: 100 mg Losartan potassium drug product were dissolved in 2 mL of MeOH / H2O (50%/50%), followed by sonication for 30 min. Then samples were centrifuged at 12,000 rpm for 10 min. Supernatants were collected and then 1:5 diluted with 100% milliQ water. Nitrosamine standards were spiked into solvent blank and API matrix respectively at concentrations ranging from 25 ppt to 10 ppb. The samples were analyzed using different LC/TQ systems with an APCI source.

Results: As of 2021, FDA guidance requires an LOQ of ≤0.03 ppm for analytical methods testing for the presence of nitrosamines in active pharmaceutical ingredients or drug products. By achieving a lower LOQ and LOD, drug products with lower dosages will be able to be assessed for the presence of nitrosamines without complicated sample prep procedures. To do this we first transferred an existing LC-MS/MS method for nitrosamine analysis to a 6475 LC-MS/MS system and tested the quantification performance. To further improve the analytical sensitivity, we compared various source conditions, including nebulizer gas type (Nitrogen or Zero-air), drying gas flow, Capillary voltage, etc. We compared several different LC-MS/MS models available in the lab and implemented multifactorial particle swarm optimization tuning into the procedure to ensure the instruments were performing with a high level of confidence. The preliminary results were summarized as 1) NDMA in API matrix can be quantified at 0.05 ng/mL in API extract using 6475 triple quadrupole LC/MS system with nitrogen as nebulizer gas, 2) The use of Zero-air (hydrocarbon free) as nebulizer gas in the APCI source further improved nitrosamine response when compared to Nitrogen gas. Preliminary tests have shown that switching from Nitrogen to hydrocarbon-free air has shown an increase in ionization efficiency for the compounds of interest and 3) Latest hardware design of LC-MS/MS instrument further decrease the limit of quantitation and limit of detection.

Conclusion: Improvement upon a LC-MS/MS method limit of detection and limit of quantitation for nitrosamines analysis in a sartan drug product using an enhanced LC-MS/MS system.