Mass spectrometry assay method for detection and quantitation of liver function metabolites

#The following information to describe the background of the invention is provided to assist the understanding of the invention and is not admitted to constitute or describe prior art to the invention.

#Liver function may be affected by various lifestyles, diseases, and disorders including, for example, diet, alcohol abuse, fatty liver disease, infections, medications, and cancer. Often, a significant amount of liver tissue may be damaged before a person experiences symptoms of liver dysfunction. Currently, the most common tests to assess liver function include measurements of specific enzymes and proteins in blood. These tests include alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), gamma-glutamyl transferase (GGT), and serum cholinesterase (ChE) total cholesterol, total triglycerides, albumin, albumin/globulin ratio, total protein, direct bilirubin, and total bilirubin.

#Current liver blood tests may provide a first indication of liver disease including Nonalcoholic Fatty Liver Disease (NAFLD), nonalcoholic steatohepatitis (NASH), fibrosis, and cirrhosis, but the tests do not detect early changes in liver function and are not able to distinguish between the diseases. When further evaluation shows no apparent reason for liver disease (such as medications, viral hepatitis, or excessive use of alcohol) and when x-rays or imaging studies of the liver show fat, NASH is suspected, but the only means of proving a diagnosis of NASH and separating it from simple fatty liver is a liver biopsy. A liver biopsy is also required to determine the presence and staging of liver fibrosis. Currently, no blood tests or scans can reliably provide this information. A liver biopsy which is an invasive procedure that requires a needle to be inserted through the skin and the removal of a small piece of the liver. The procedure is painful and associated with risk of excessive bleeding. Therefore, there exists a need for a less invasive method (i.e. a method that would not require a liver biopsy) to assess the function of the liver that supplements currently available clinical assays.

#Methods are described for measuring the amount of one or more analytes selected from the group of metabolites consisting of: 12,13-DiHOME, 3-hydroxybutyrate (BHBA), 3-hydroxyoctanoate, 3-methylglutarylcarnitine, 3-ureidopropionate, 7-alpha-hydroxy-4-cholesten-3-one (7-Hoca), citrate, fucose, fumarate, gamma-tocopherol, glutamate, glutarate, glycerol, glycochenodeoxycholate, glycocholate, hypoxanthine, maleate, malonate, mannose, orotate, 2,3-pyrdinedicarboxylate, ribose, serine, taurine, taurochenodeoxycholate, taurocholate, palmitoleate, linolenate, xanthine, xylitol, and combinations thereof, in a sample. Mass spectrometric methods are described for quantifying single and multiple analytes in a sample using a single injection method. The methods may use a liquid chromatography step such as UPLC or HILIC to perform a separation (purification, enrichment) of selected analytes combined with methods of mass spectrometry, thereby providing a high-throughput assay system that is amenable to automation for quantifying a plurality of analytes in a sample.

#The methods presented herein provide advantages over current methods to measure these analytes. The ability to quantifiably measure, in a single injection, a plurality of analytes in various combinations, reduces the time required to obtain analysis results, uses fewer resources in terms of laboratory disposables (e.g., tubes, pipette tips, reagents), laboratory instruments and human resources. These improvements lead to savings by decreasing the costs of the assays and increasing the instrument and laboratory capacity for sample analysis.

#Prior to describing this invention in further detail, the following terms are defined.