To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis （2-D DIGE） and reversed phase high-performance liquid chromatography （RP-HPLC） followed by...To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis （2-D DIGE） and reversed phase high-performance liquid chromatography （RP-HPLC） followed by electrospray ionization mass spectrometry （ESI-MS） were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups： acute rejec- tion （AR）, stable renal function （SRF） and normal volunteer （N）. Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.展开更多
Ibandronate sodium monohydrate is a highly polar aliphatic compound that belongs to the bisphosphonate class,a widely used bone resorption inhibitor.The aliphatic nature of ibandronate precludes direct photometric det...Ibandronate sodium monohydrate is a highly polar aliphatic compound that belongs to the bisphosphonate class,a widely used bone resorption inhibitor.The aliphatic nature of ibandronate precludes direct photometric detection and its high polar-ity urged analysts to use ion-pairing agents in the mobile phase to promote retention in reversed-phase columns.In this work,a reversed-phase method for determination of ibandronate sodium monohydrate in pharmaceutical tablets is introduced by employing an online postcolumn ligand exchange reaction to enable photometric detection.The method offers for the first time the ability to separate the drug from its well-known impurities on conventional reversed-phase columns.The detection reac-tion depends on the ability of ibandronate and its degradation products to displace salicylate in the iron(III)salicylate complex,forming various colorless iron(III)complexes and showing a negative chromatographic signal for ibandronate and its degra-dants atλ_(max)=525 nm.The chromatographic separation was achieved using a Hypersil BDS C_(8) column(5μm particle size,150 mm×4.6 mm i.d.)and a mobile phase consisting of a mixture of aqueous formate buffer pH 3(pH 3,5 mM)and methanol in a ratio of 80:20%(v/v),delivered at a flow rate of 0.8 mL/min.The postcolumn iron(III)salicylate reagent was prepared at 250μM in aqueous formate buffer,delivered using a syringe pump at a flow rate of 0.5 mL/min.The proposed method was validated as per the guidelines of the International Conference on Harmonization Q2(R1)and was found linear over the range 40-120μg/mL(r=0.9995)with a limit of detection of 3.19μg/mL.Compared to currently reported methods,the proposed method is considerably simpler,faster,cheaper,and adequately sensitive and makes use of the most popular LC detectors.展开更多
基金supported by a grant from National Basic Research 973 Program of China (No.2009CB522407)
文摘To identify acute renal allograft rejection biomarkers in human serum, two-dimensional differential in-gel electrophoresis （2-D DIGE） and reversed phase high-performance liquid chromatography （RP-HPLC） followed by electrospray ionization mass spectrometry （ESI-MS） were used. Serum samples from renal allograft patients and normal volunteers were divided into three groups： acute rejec- tion （AR）, stable renal function （SRF） and normal volunteer （N）. Serum samples were firstly processed using Multiple Affinity Removal Column to selectively remove the highest abundance proteins. Differentially expressed proteins were analyzed using 2-D DIGE. These differential protein spots were excised, digested by trypsin, and identified by RP-HPLC-ESI/MS. Twenty-two differentially expressed proteins were identified in serum from AR group. These proteins included complement C9 precursor, apolipoprotein A-IV precursor, vitamin D-binding protein precursor, beta-2-glycoprotein 1 precursor, etc. Vitamin D-binding protein, one of these proteins, was confirmed by ELISA in the independent set of serum samples. In conclusion, the differentially expressed proteins as serum biomarker candidates may provide the basis of acute rejection noninvasive diagnosis. Confirmed vitamin D-binding protein may be one of serum biomarkers of acute rejection. Furthermore, it may provide great insights into understanding the mechanisms and potential treatment strategy of acute rejection.
文摘Ibandronate sodium monohydrate is a highly polar aliphatic compound that belongs to the bisphosphonate class,a widely used bone resorption inhibitor.The aliphatic nature of ibandronate precludes direct photometric detection and its high polar-ity urged analysts to use ion-pairing agents in the mobile phase to promote retention in reversed-phase columns.In this work,a reversed-phase method for determination of ibandronate sodium monohydrate in pharmaceutical tablets is introduced by employing an online postcolumn ligand exchange reaction to enable photometric detection.The method offers for the first time the ability to separate the drug from its well-known impurities on conventional reversed-phase columns.The detection reac-tion depends on the ability of ibandronate and its degradation products to displace salicylate in the iron(III)salicylate complex,forming various colorless iron(III)complexes and showing a negative chromatographic signal for ibandronate and its degra-dants atλ_(max)=525 nm.The chromatographic separation was achieved using a Hypersil BDS C_(8) column(5μm particle size,150 mm×4.6 mm i.d.)and a mobile phase consisting of a mixture of aqueous formate buffer pH 3(pH 3,5 mM)and methanol in a ratio of 80:20%(v/v),delivered at a flow rate of 0.8 mL/min.The postcolumn iron(III)salicylate reagent was prepared at 250μM in aqueous formate buffer,delivered using a syringe pump at a flow rate of 0.5 mL/min.The proposed method was validated as per the guidelines of the International Conference on Harmonization Q2(R1)and was found linear over the range 40-120μg/mL(r=0.9995)with a limit of detection of 3.19μg/mL.Compared to currently reported methods,the proposed method is considerably simpler,faster,cheaper,and adequately sensitive and makes use of the most popular LC detectors.
