Agilent 7100 ce capillary electrophoresis system

Plasma metabolites were extracted and analyzed using the method recommended by Human Metabolome Technologies, Inc. (Tsuruoka, Japan). Briefly, methanol containing Internal Standard Solution 1 (H3304–1002, Human Metabolome Technologies, Inc.) was added to the plasma samples and mixed well. Subsequently, Milli-Q water and chloroform were added and centrifuged at 2300× g for 5 min at 4 °C. The upper aqueous layer was collected and filtered using a Millipore 5-kDa cutoff filter and dried. The metabolites were resuspended in 25 µL Milli-Q water containing Internal Standard Solution 3 (H3304–1004, Human Metabolome Technologies, Inc.) and used for capillary electrophoresis–mass spectrometry (CE–MS) analysis. All CE–MS experiments were performed using the Agilent 7100 CE capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) connected to the Agilent 6530 Accurate Quadrupole Time-of-Flight MS system (Agilent Technologies, Palo Alto, CA, USA). Each metabolite was identified and quantified based on the peak information, including mass-to-charge ratio, migration time, and peak area, using Profinder software, version B.08.00 (Agilent Technologies, Palo Alto, CA, USA). If a metabolite was undetected, one-half of the minimum concentration detected by the respective metabolite was used as the concentration.

Serum metabolites of the Control and DKD groups at 6 and 15 weeks of age were extracted and analyzed by the Human Metabolome Technologies (HMT Inc., Yamagata, Japan) method. Briefly, methanol containing Internal Standard Solution 1 (HMT Inc.) was added to the sera and mixed well. Subsequently, 200 µL of Milli‐Q water (Merck Millipore, Burlington, MA, USA) and 500 µL of chloroform were added and centrifuged at 2300 g for 10 min at 4 °C. The upper aqueous layer was collected and then filtrated with a Millipore 5‐kDa cutoff filter and dried. The metabolites were resuspended in 25 µL of Milli‐Q water containing Internal Standard Solution 3 (HMT Inc) and applied to CE‐MS. All CE‐MS experiments were performed using an Agilent 7100 CE capillary electrophoresis system (Agilent Technologies, Santa Clara, Waldbronn, Germany) connected to an Agilent 6530 accurate Q‐TOF‐MS system (Agilent Technologies Inc., Santa Clara, CA, USA).
The data obtained by CE‐MS analysis were preprocessed using masshunter Workstation Software Qualitative Analysis version B.06.00 (Agilent Technologies Inc.). Each metabolite was identified and quantified based on the peak information including m/z, migration time, and peak area. The metabolite peaks were analyzed statistically using the mass profiler professional software (Agilent Technologies Inc.).