Development Of A High Performance Liquid Chromatography Fluorescence Detection Method For Plasma Indoles And Its Applications

Objective:1.To develop a simple,sensitive and accurate high performance liquid chromatography with fluorescence detection（HPLC-FLD）method for simultaneous determination of plasma indoles,including indoxyl sulfate（3-INDS）,indole-3-acetic acid（IAA）,indole-3-propionate（IPA）,indole（IND）and 3-methylindole（3-MI）.2.The plasma levels of indoles in polycystic ovary syndrome（PCOS）group and the control group were determined by the developed method to explore the relationship between indoles and glucose metabolism,as well as the role of indoles in depression of PCOS.Method:1.The plasma samples were prepared by liquid-liquid extraction,and then detected by HPLC-FLD method.The analytes were separated by Shim-Pack VP-ODS column（4.6 mm×150 mm,4.6μm）.Separation was performed with 10 mmol/L sodium dihydrogen phosphate/methanol（40/60,v/v）at a flow rate of 0.80 m L/min.The column temperature was 35°C.The injection volume was 50μL.The excitation wavelength(l_ex)and emission wavelength(l_em)were 280 nm and 355 nm,respectively.2.The developed method was applied to determine the concentrations of plasma indoles in the PCOS group and the control group.Correlation analysis,nonparametric tests and other statistical methods were adopted to explore the correlation between plasma indoles and glucose metabolism as well as depression.Results:1.The linearities in plasma were obtained in the range of 1.56-400.00μmol/L for 3-INDS,312.50-10000.00 nmol/L for IAA,125.00-6000.00nmol/L for IPA,6.25-400.00 nmol/L for IND and 1.56-400.00 nmol/L for 3-MI,respectively.The coefficients of variation（CVs）for each analyte of the intra-day and inter-day precisions were within 4.0%and 5.2%,respectively.The recoveries were in the range of 90.1%～109.3%with CVs ranging from 1.6%to 8.2%.2.The plasma levels of 3-INDS,IAA and IND were significantly elevated in women with PCOS compared to those in the control group.Increased levels of 3-INDS,IAA and IND were significantly correlated with glucose metabolism parameters,including fasting glucose,fasting insulin and HOMA-IR.3-INDS,IAA,IND and 3-MI correlated negatively with GLP-1.3-INDS（OR=1.208,95%CI:1.100～1.327,p=0.000）,IAA（OR=1.437,95%CI:1.095～1.885,p=0.009）and IND（OR=1.051,95%CI:1.023～1.080,p=0.000）were risk factors for PCOS.3.The concentration of IND was significantly higher of the depression group than that of the non-depression group in patients with PCOS.Besides,IND（OR=1.144,95%CI:1.028～1.272,p=0.014）was a risk factor for depression in patients with PCOS.The area under the curve（AUC）for IND was 0.873（p<0.001）,and the cut-off value of IND was 53.64 nmol/L（Youden index=0.623,Sensitivity=90.9%,Specificity=71.4%）,which indicated that IND had a good diagnostic value for depression in patients with PCOS.Conclusions:1.In this study,a HPLC-FLD method was established for the simultaneous detection of indoles in plasma,including 3-INDS,IAA,IPA,IND and 3-MI.The method was simple,sensitive,and suitable for clinical and scientific research.2.Indoles were related to the occurrence of insulin resistance in PCOS.3-INDS,IAA and IND were risk factors for PCOS,and IND was a risk factor for depression in PCOS.