A novel LC-TQ-MS/MS method for quantifying mefenamic acid-NDSRI (N-nitroso drug substance-related impurity) in mefenamic acid tablet and pediatric suspension dosage forms: a comparative study with a cost-effective white, green, and blue UPLC method

Abstract

The current research presents novel LC-TQ-MS/MS and cost-effective UPLC methods intended for the accurate quantification of mefenamic acid-N-nitroso drug substance-related impurity-NDSRI (N-MFA) in mefenamic acid (MFA) tablet and pediatric suspension dosage forms. The acceptable intake of N-MFA is derived from the TD50 (Median Toxic Dose-50%) value of N-nitroso diphenylamine. The analytical separation was achieved for the UPLC method using an XBridge BEH Shield RP18 Column (150 × 3.0 mm, 3.5 μm), employing 0.1% formic acid and acetonitrile as mobile phases in a gradient mode. A flow rate of 0.7 mL min⁻¹ was set for the pump, and the detection wavelength was 230 nm. LC-TQ-MS-MS analysis was accomplished utilizing a Xevo TQ-XS mass spectrometer with electrospray ionization in the positive mode. N-MFA quantification was carried out using multiple reaction monitoring (MRM) as the mass transition: m/z 271.13 → 241.14. The proposed methods were successfully validated following ICH guidelines and are capable of quantifying at 0.01 ng mL⁻¹ at an MFA concentration of 1 mg mL⁻¹ for the LC-MS/MS method and at 0.036 μg mL⁻¹ at an MFA concentration of 10 mg mL⁻¹ for UPLC. The validated methods exhibited excellent linearity over the concentration ranges of 0.01 to 125 ppm for LC-MS/MS and 0.036 μg mL⁻¹ to 1.250 μg mL⁻¹ (3.6 to 125 ppm) for UPLC, with a Pearson correlation coefficient of 0.999. The method's trueness, in terms of accuracy recoveries, ranged from 90.43% to 101.34% for LC-MS/MS and 92.12% to 101.21% for UPLC. The applicability of these methods was successfully verified through the analysis of commercial samples of MFA formulations. Additionally, the sustainability and eco-friendliness of the method were assessed using whiteness and greenness metrics, including RGB12 (Red-Gree-Blue) tools, GAPI (Green Analytical Procedure Index), analytical eco-scale, AGREE (Analytical GREEnness), AGREEprep (Analytical GREEnnesspreparation), and BAGI (Blue Applicability Grade Index). Both methods can be used to determine N-MFA content in mefenamic acid formulation dosage based on the availability of instruments LC-TQ-MS/MS and UPLC in R&D (Research and Development) and quality control laboratories.