Currently we are facing a Development issue with N-Nitroso Imidazole content in Clotrimazole Durg substance and Drug product. When we perform mannual MS tunning through syringe we got the Precursor and Product Ions respectively but when we run the MRM method we get higher side baseline and the response is also not linear as per concentrations . And while performing with GC-MS methodology we are only detecting Imidazole mass , from N-Nitroso imidazole Nitrogen & Oxygen will be removed as per reaction mechanism , hence we will only get Imidazole mass . Wich can not be quantified for intend use of method for analysis .
if anyone has performed the development kindly please help me to resolve the issue.
When you did the manual tunnig, did prepare de standard solution in the same solvent mix of the mobile phase? Or aclose mixture of organic/aqueous solvent of when the peak is eluted (in case of gradient).
Yes , I had used same solvent for optimization and for peak elution , My concern is that, i am getting precursor of 98.1 m/z of N-Nitroso Imidazole and the outcome product ion is 68.1 m/z and 82.1 m/z . The product ion 68.1 is present there wich is a m/z of Imidazole. I had taken trials on ESI and APCI methodology. By performing MRM scan using above parameters its also detecting Imidazole,due to its precursor ion Mass is confirming product ion scan for MRM parameters of N-Nitroso Imidazole. Hence my method is not selective for quantition of N-Nitroso Imidazole.
Hi Viraj, I think you can optimize the HPLC condition to sperate the N-Nitroso Imidazole and Imidazole peak in order to avoid false positive from imidazole. Based on reversed HPLC, the polarity of N-Nitroso imidazole is less than imidazole.
Has your issue been resolved？ We are facing this development issue as well. We have tried both LC-MS and GC-MS, and only got Imidazole mass. Hope i can get some suggestions from you.
I believe the analytical separation of imidazole and N-nitroso imidazole is the main challenge here. The following can be attempted
Considering the polarity of both the molecules, HILIC, Amino or other similar columns can be used to separate the two compounds.
Alternatively, Imidazole interference can be removed by trying to derivatize it using the available secondary amine. (In this case N-Nitroso Imidazole cannot be derivatized as the secondary amine is already bonded). This may help in the resolution of the compounds. Or use of LLE to remove interference of imidazole using its basic nature.
If in source fragmentation is the concern, (breakage of N-Nitroso imidazole to imidazole) mass parameters need to be optimized.
Fragment at m/z 82 can be used for quantification if m/z 68 is a concern due to overlapping mass of imidazole. Alternatively HRMS system can also be attempted.
If in source fragmentation remains a concern, then an HPLC method can also be attempted (provided that the limit allows its use). Imidazole does show some UV absorbance.