lcmsms analysis of uncommon paracetamol metabolites derived through in vitro polymerization and nitration reactions in liquid nitrogen paracetamol acetaminophen apap is a commonly used analgesic drug known paracetamol metabolites include the glucuronide sulfate and mercapturate nacetylbenzoquinonimine napqi is considered the toxic intermediate metabolite of paracetamol in vitro and in vivo studies indicate that paracetamol is also metabolized to additional poorly characterized metabolites for example metabolomic studies in urine samples of apaptreated mice revealed metabolites such as apapsulfateapap and apapssapap in addition to the classical phase ii metabolites here we report on the development and application of lcms and lcmsms approaches to study reactions of unlabelled and 2hlabelled apap with unlabelled and 15nlabelled nitrite in aqueous phosphate buffers ph 74 upon their immersion into liquid nitrogen 196c in mechanistic studies these reactions were also studied in aqueous buffer prepared in 18olabelled water lcms and lcmsms analyses were performed on a reversephase material c18 using gradient elution 2mm ammonium acetateacetonitrile in positive and negative electrospray mode we identified a series of apap metabolites including di tri and tetraapap mono and dinitroapap and nitric ester of diapap our study indicates that nitrite induces oxidation ie polymerization and nitration of apap when buffered apapnitrite solutions are immersed into liquid nitrogen these reactions are specific for nitrite with respect to nitrate and do not proceed via intermediate formation of napqi potassium ions and physiological saline but not thiols inhibit nitrite and shockfreezeinduced reactions of paracetamol the underlying mechanism likely involves in situ formation of no2 radicals from nitrite secondary to profound ph reduction down to ph 1 and disproportionation polymeric paracetamol species can be analyzed as pentafluorobenzyl derivatives by lcms but not by gcms