LC/ESI-MS/MS analysis of urinary 3β-sulfooxy-7β-N-acetylglucosaminyl-5-cholen-24-oic acid and its amides: new biomarkers for the detection of Niemann-Pick type C disease.

Abstract We developed a sensitive, reliable, and accurate LC/ESI-MS/MS method for measurement of 3β-sulfooxy-7β- N -acetylglucosaminyl-5-cholen-24-oic acid and its glycine and taurine amides in urine. This atypical C 24 bile acid has been reported previously to be present in the urine of patients with Niemann–Pick Type C (NPC) disease. In the method, targeted analytes are concentrated at the front edge of a trapping column, Shim-pack MAYI-C8, which permits elimination of contaminating molecules in the urinary matrix. The trapped analytes are then eluted, separated on a YMC-Pack Pro C18, and quantified with MS/MS using selected reaction monitoring. The method could detect (as amount injected) 2 pg of nonamidated 3β-sulfooxy-7β- N -acetylglucosaminyl-5-cholen-24-oic acid, 2 pg of its glycine-amide, and 0.6 pg of its taurine-amide, and is linear up to 300 pg. The method was then used to measure the three analytes in the urine of NPC patients ( N  = 2), 3β-hydroxysteroid dehydrogenase deficiency patients ( N  = 2), and healthy volunteers ( N  = 8). Measurable concentrations of all three analytes were present in all subjects. The urinary concentration of the sum of all three analytes was four hundred times greater in the 3 month NPC patient and 40 times greater in the adult patient than that of healthy volunteers. The markedly elevated urinary concentration of 3β-sulfooxy-7β- N -acetylglucosaminyl-5-cholen-24-oic acid and its amides in NPC patients suggests that these compounds may be valuable biomarkers for detection of NPC disease.