Simultaneous analysis of carcinogenic N-nitrosamine impurities in metformin tablets using on-line in-tube solid-phase microextraction coupled with liquid chromatography-tandem mass spectrometry

Contamination of active pharmaceutical ingredients (APIs) and pharmaceutical preparations with carcinogenic N-nitrosamines has led to recalls of these products and supply shortages to patients. The present study describes the development of a highly sensitive method for simultaneous analysis of seven N-nitrosamines using on-line in-tube solid-phase microextraction (IT-SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS) to determine their actual contamination in metformin tablets. Using a Carboxen 1006 PLOT capillary as the extraction device for IT-SPME, these compounds were efficiently extracted and concentrated 6‒24-fold by subjecting 40 µL of sample to 25 repeated draw/eject cycles at a rate of 0.2 mL/min. The seven N-nitrosamines were separated within 11 min by gradient elution with 0.1 % formic acid solution and acetonitrile as the mobile phase using a CAPCELL PAK C18 MGII column and detected by multiple reaction monitoring in positive ion mode. The calibration curve showed linearity in the range 0.2‒50 ng/mL and detection limits (S/N = 3) in the range 3‒112 pg/mL. The intra-day and inter-day precisions were less than 5.5 % and 7.0 % (n = 6), respectively, with accuracies ranging from 93‒117 %. Following ultrasonic extraction with water, centrifugation and filtration of the supernatant liquid through a membrane filter, the N-nitrosamine impurities in metformin tablets could be analyzed by IT-SPME/LC‒MS/MS. Their limits of quantification (S/N = 10) were 0.1‒5.1 pg/mg API and recoveries ranged from 87‒102 %. Analysis of eight metformin tablets from eight manufacturers showed that 5.8‒7.5 pg/mg N-nitrosodimethylamine were present in three tablets, with no other N-nitrosamines detected in any of the eight tablets. This method may be useful in testing for N-nitrosamine impurities in pharmaceutical preparations.