Small-molecule metabolite analysis presents challenges that historically have relied heavily upon standard quadrupole gas chromatography–mass spectrometry (GC–MS) utilizing targeted methods of selected ion monitoring and MS-MS mass spectrometric techniques. The complex nature of metabolomic samples demands analytical solutions and instrumental methods that will identify the small molecule metabolite profile completely as well as discover significant key components of interest.

Following two-dimensional (GC×GC)–time of flight (TOF)-MS, the data processed diabetic and nondiabetic samples were analyzed by proprietary software.

Experimental

Morning fast urine samples were collected from four subjects: two nondiabetic normal controls, one type I diabetic, and one type II diabetic. Samples were stored under refrigeration at 4 °C before liquid–liquid extraction with methylene chloride and derivatization with N,O-bis-(trimethylsilyl)-trifluoroacetamide (BSTFA). Six 10-mL aliquots from each subject were prepared by acidification with concentrated sulfuric acid to pH 2. Aliquots (10 mL each) were extracted with 2 mL of methlyene chloride into a 20-mL scintillation vial containing approximately 5 mg of sodium sulfate. Derivatization was carried out with BSTFA by placing 200 μL of extract into a sealed 2-mL autosampler vial containing approximately 0.5 mg of sodium sulfate. A 30-μL aliquot of dry pyridine was added to the vial. A 200-μL aliquot of BSTFA was added to each vial. The samples were heated to 60 °C for 1 h and then analyzed.

Figure 1