A Simple and Sensitive UPLC–MS/MS Method for the Simultaneous and Trace Level Quantification of Two Potential Genotoxic Impurities in Midazolam Drug Substance.

To promote the quality control of midazolam and ensure the life safety of patients, a sensitive and simple ultra-high performance liquid chromatography–mass spectrometry (UPLC–MS/MS) method was developed for the trace determination of two potential genotoxic impurities (PGIs) in midazolam raw materials under the positive electrospray ionization (ESI) mode with multiple reaction monitoring (MRM). The two PGIs were well separated in a gradient elution mode at a 0.4 mL/min flow rate on an ACQUITY UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 µm) that was maintained at 40 °C. 0.01 mol/L ammonium formate aqueous solution (0.1% formic acid) and acetonitrile (0.1% formic acid) were mobile phase A and mobile phase B, respectively. The m/z of 343.00/123.00 for PGI-1and m/z of 331.20/301.20 for PGI-2 were detected in a triple quadrupole mass detector. The calibration curves showed that the two PGIs have good linearity between the concentration range of 0.004 and 0.8 ppm. The correlation coefficients of PGI-1 and PGI-2 were 0.9996 and 0.9998, respectively. The recoveries of the two PGIs at three concentration levels were in the range of 81–103%. The limits of quantification (LOQ) for PGI-1 and PGI-2 were 0.0545 and 0.0111 ng/mL respectively. The precision relative standard deviation (RSD) of each impurity was observed less than 3.0%. Method validation results indicated that the linearity, accuracy, precision, and sensitivity of the developed UPLC–MS/MS method are satisfactory. Thus, the method can quantitatively detect the two trace PGIs in midazolam. This study can provide a reference for manufacturers and relevant researchers of midazolam, reduce the quality risk of midazolam drugs and ensure the safety of patients. [ABSTRACT FROM AUTHOR]