Semi-Targeted Profiling of Bile Acids by High-Resolution Mass Spectrometry in a Rat Model of Drug-Induced Liver Injury.

Using a semi-targeted approach, we have investigated the effect of acetaminophen on circulating bile acid profiles in rats, including many known bile acids and potential isomeric structures, as well as glucuronide and sulfate conjugates. The chromatographic separation was based on an optimized reverse-phase method exhibiting excellent resolution for a complex mix of bile acids using a solid-core C18 column, coupled to a high-resolution quadrupole time-of-flight system. The semi-targeted workflow consisted of first assigning all peaks detectable in samples from 46 known bile acids contained in a standard mix, as well as additional peaks for other bile acid isomers. The presence of glucuronide and sulfate conjugates was also examined based on their elemental formulae and detectable peaks with matching exact masses were added to the list of features for statistical analysis. In this study, rats were administered acetaminophen at four different doses, from 75 to 600 mg/kg, with the highest dose being a good model of drug-induced liver injury. Statistically significant changes were found by comparing bile acid profiles between dosing levels. Some tentatively assigned conjugates were further elucidated using in vitro metabolism incubations with rat liver fractions and standard bile acids. Overall, 13 identified bile acids, 23 tentatively assigned bile acid isomers, and 9 sulfate conjugates were found to increase significantly at the highest acetaminophen dose, and thus could be linked to drug-induced liver injury.

Targeted Analysis of 46 Bile Acids to Study the Effect of Acetaminophen in Rat by LC-MS/MS.

Bile acids represent a large class of steroid acids synthesized in the liver and further metabolized by many bacterial and mammalian enzymes. Variations in bile acid levels can be used as a measure of liver function. There still exists, however, a need to study the variation of individual circulating bile acids in the context of hepatotoxity or liver disease. Acetaminophen (APAP), a drug commonly taken to relieve pain and decrease fever, is known to cause acute liver failure at high doses. We have developed a targeted liquid chromatography-tandem mass spectrometry method to monitor the effects of different doses of APAP on the bile acid plasma profile in a rat model. The analysis method was optimized to ensure chromatographic resolution of isomeric species using a mixture of 46 standard bile acids, and 14 isotopically-labeled internal standard (IS) compounds detected in multiple reaction monitoring (MRM) mode on a triple quadrupole mass spectrometer. Four doses of acetaminophen were studied, the highest of which shows signs of hepatotoxicity in rats. This targeted method revealed that high dose APAP has an important effect on bile acid profiles. Changes were seen in several unconjugated bile acids as well as glycine conjugates; however, no obvious changes were apparent for taurine-conjugated species.