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 C24 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 3b-sulfooxy- 7b-N-acetylglucosaminyl-5-cholen-24-oic acid, 2 pg of its glycine-amide, and 0.6 pg of its taurineamide, 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), 3b-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 3b-sulfooxy-7b-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.
- Multiple conjugate
- Niemann-Pick type C disease