Synthesis and Trace-Level Quantification of Mutagenic and Cohort-of-Concern Ciprofloxacin Nitroso Drug Substance-Related Impurities (NDSRIs) and Other Nitroso Impurities Using UPLC-ESI-MS/MS-Method Optimization Using I-Optimal Mixture Design

Abstract

Globally, the pharmaceutical industry has been facing challenges from nitroso drug substance-related impurities (NDSRIs). In the current study, we synthesized and developed a rapid new UPLC-MS/MS method for the trace-level quantification of ciprofloxacin NDSRIs and a couple of N-nitroso impurities simultaneously. (Q)-SAR methodology was employed to assess and categorize the genotoxicity of all ciprofloxacin N-nitroso impurities. The projected results were positive, and the cohort of concern (CoC) for all three N-nitroso impurities indicates potential genotoxicity. AQbD-driven I-optimal mixture design was used to optimize the mixture of solvents in the method. The chromatographic resolution was accomplished using an Agilent Poroshell 120 Aq-C18 column (150 mm × 4.6 mm, 2.7 μm) in isocratic elution mode with 0.1% formic acid in a mixture of water, acetonitrile, and methanol in the ratio of 475:500:25 v/v/v at a flow rate of 0.5 mL/min. Quantification was carried out using triple quadrupole mass detection with electrospray ionization (ESI) in a multiple reaction monitoring technique. The finalized method was validated successfully, affording ICH guidelines. All N-nitroso impurities revealed excellent linearity over the concentration range of 0.00125-0.0250 ppm. The Pearson correlation coefficient of each N-nitroso impurity was >0.999. The method accuracy recoveries ranged from 93.98 to 108.08% for the aforementioned N-nitrosamine impurities. Furthermore, the method was effectively applied to quantify N-nitrosamine impurities simultaneously in commercially available formulated samples, with its efficiency recurring at trace levels. Thus, the current method is capable of determining the trace levels of three N-nitroso ciprofloxacin impurities simultaneously from the marketed tablet dosage forms for commercial release and stability testing.

Figure 1

Possible pathway of the N-nitroso group interaction with DNA via the cyclic alkyl diazonium ion.

Figure 2

Chemical Structures of (a) COX API, (b) COX NDSRI, (c) COX N-nitroso impurity-1, and (d) COX N-nitroso impurity-2.

Figure 3

Synthetic scheme of (A) COX impurities and corresponding N-nitroso impurities and (B) reaction mechanism of the formation N-N = O functional group.

Figure 4

Normal plots, predicted vs actual plots, trace plots, and triangle 2D contour graphs R1: resolution between COX and N-nitroso COX impurity-1, R2: resolution between N-nitroso COX impurity-1, and N-nitroso COX, and R3: retention time of N-nitroso COX impurity-2.

Figure 5

Numerical optimization plots.

Figure 6

Surface plots and overlay plot.

Figure 7

Typical chromatogram of separation of COX-N-nitroso impurities.

Figure 8

MRM chromatograms of (a) diluent blank, (b) placebo, (c) COX N-nitroso impurity-1, (d) COX-NDSRI, and (e) COX N-nitroso impurity-2.

Figure 9

Typical LOQ MRM chromatograms of (a) COX N-nitroso impurity-1, (b) COX-NDSRI, and (c) COX N-nitroso impurity-2.