| Objectives:1. To establish a method for determination of metabolic profiling of urinary unsulfated and sulfated bile acids by ultra performance liquid chromatography-triple quadrupole time-of-flight mass spectroscopy, (UPLC-Triple-TOF/MS).2. To detect the profiling of urinary bile acids in women with normal pregnancy, mild and severe ICP by UPLC-Triple-TOF/MS, which to obtain the optimal model and explore potential biomarkers for ICP diagnosis and grading by classical discrimination analysis.Mehods:1. The separations of bile acids were achieved on a Phenomenex Kinetex XB-C18 column (50 × 2.1 mm,1.7 μm) with gradient elution of mobile phase A (15 mmol/L ammonium acetate) and mobile phase B (acetonitrile). The column was kept at 30 ℃ and the flow was 0.2 ml/min. The electrospray ionisaton (ESI) was operated in negative mode. Parent ions were quantitative ions under product ion scan which objective ions were mass±0.05Da. The injection was 5 μl and the elutions were quantitated by internal standard curves in 20 min.2. Urinary analytes were extracted by Bond Elut C18 cartridges.3. Sulfated bile acids without standards and isomers of known unsulfated bile acids were identified through accurate mass of MS and MS/MS, characteristic fragment ions and isotopic information by Peakview software.4. Profiling of urinary bile acids of 26 normal pregnancies,14 patients with mild ICP and 14 patients with severe ICP were determinated by UPLC-Triple-TOF/MS. And classic discriminant analysis was performed for obtaining the optimal diagnosis and grading model of ICP and choosing specific and sensitive biomarkers.Results:1. We optimized the LC condition to achieve baseline separation of 28 unsulfated and 1 sulfated bile acid standards.40 possible sulfated bile acids and 17 metabolites which isomeric with known unsulfated bile acids were identified.2. Urinary bile acids were linear in the range 0.020-20 umol/L, correlation coeffients of linear regress ranged from 0.9925-1.0000. The detection limits were 0.001 umol/L. The precisions of bile acids were CVs<17.8%for within-day and CVs<25.9%for between-day. All analytes achieved relatively satisfied recoveries under different concentrations.3. Compared with normal pregnancy, both unsulfated and sulfated bile acids were increased in three groups, while the increases in sulfated form were more significant. Increases in taurine-conjugated bile acids were more significant than glycine-conjugated bile acids.4. Compared with normal pregnancy, the levels of GDCA, ω-TMCA and TCA in urine of patients with mild ICP were significantly higher, and GCDCA and GCA were higher exceedingly. The levels of a-TMCA, THCA, GHCA, GCA and TLCA-S in urine of patients with severe ICP were significantly higher, and TUDCA and co-TMCA were higher exceedingly.5.27 possible sulfated and 15 possible unsulfated bile acids were determinated in urine. While TBA-S, GBA-S, TCA-S, GCA-S, GLCA-S and isomers of TBA, GBA and TCA were different in three groups.6. The correct rates for diagnosis of normal pregnancy, mild ICP and severe ICP reached 96.2%,92.9%,92.9%, and 94.4%cases were classified correctly in classical discrimination analysis model when the predicted varies were 40 different urinary bile acids.7. Combined VIP value, p(corr) value with different bile acids to choose possible biomarkers. TBA3, TBA-S2, TUDCA, TCA5, TCA4 and co-TMCA played important roles in ICP diagnosis and grading.Conclusions:1. A method for simultaneous determination of profiling of unsulfated and sulfated bile acids in urine by UPLC-Triple-TOF/MS was developped and validated.2. The three groups could be classified correctly by classical discrimination analysis based on metabolic profiling analysis of urinary bile acids. And some potential biomarkers were found in urine for ICP diagnosis and grading. |
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