A METHOD FOR THE QUANTITATION OF CONJUGATED BILE-ACIDS IN DRIED BLOODSPOTS USING ELECTROSPRAY IONIZATION-MASS SPECTROMETRY

Bile acid concentrations are elevated in the blood of neonates with ch olestatic hepatobiliary disorders providing a possible means of screen ing for treatable conditions including biliary atresia. A method is de scribed for the determination of concentrations of conjugated bile aci ds in dried blood spots using electrospray ionization mass spectrometr y. Bile acids were eluted from the blood spots using methanol containi ng, as internal standards, the taurine and glycine conjugates of D-4-c henodeoxy-cholic acid and D-4-cholic acid. The samples were then recon stituted in acetonitrile/water and injected by autosampler into the el ectrospray source operating in negative ion mode. Optimal conditions w ere determined for both single quadrupole and tandem mass spectrometry analysis. Blood spot bile acid profiles were studied in two groups of infants (<1 y), a cholestatic group (conjugated bilirubin >25 mu mol/ L; n = 49), and a control group (n = 96). The best discrimination betw een the two groups was provided by measurements of taurodihydroxychola noates (normal <5 mu mol/L; cholestatic group 18-94 mu mol/L) and glyc odihydroxycholanoates (normal <5 mu mol/L; cholestatic group 11-66 mu moyl/L). The method can also be adapted to detect unusual bile acids w hich are diagnostic of inborn errors of bile acid synthesis and peroxi somal disorders. The method is fast, reliable, reproducible, and relat ively cheap; however, much more work is required to determine whether it can be used for mass screening for cholestasis. It will be necessar y to show that measurement of bile acid concentrations in blood spots obtained at 7-10 d can be used to detect infants who currently present with jaundice, pale stools, and dark urine during the first 6 mo of l ife.