Introduction
The "Statistical Compare" option available in ChromaTOF software allows the user to view statistical comparisons as a data processing step for groups of samples. The groups of samples are divided into different subsets or classes. ChromaTOF software aligns the data for the specified group of samples from the data processed peak tables. Upon completion of peak alignment, statistical information (such as minimum, maximum, average, relative standard deviation, etc.) on various peak properties (such as peak height, peak area, retention time, etc.) can be viewed in the Compound Table generated by Statistical Compare. The software will also compare statistical information from each class and between classes. Additionally, "Fisher Ratios" can be calculated from the Compound Table for each analyte. The Fisher ratio is a statistical calculation that can be used to discover the unknown chemical differences among known classes of complex samples. Statistical Compare results can also be exported as a .csv file and applied to third party software programs for supplemental data reduction such asmultivariate analysis.
In this paper, Statistical Compare is utilized to define the small metabolite profile with potentially significant class differences between trimethylsilyl (TMS) derivatized urine from diabetic and non-diabetic subjects analyzed by GCxGC-TOFMS. This research was designed to analyze TMS-derivatized urine samples for the small molecule metabolite profile with the intent to detect possible chemical variations between diabetic diseased state and normal control non-diabetic subjects. It is important to note that this research was conducted solely as a proof of concept study to test the validity of the Statistical Compare, Fisher Ratios, and .csv file export functions that are available in the ChromaTOF software. The data mining strategy used to distinguish analyte differences between diabetic and non-diabetic sample classes begins by aligning the analytes from the processed data files using the Statistical Compare feature. Following the Statistical Compare operation, Fisher Ratios are calculated to identify the compounds showing the highest variance. The resulting Compound Table is then exported as a .csv file to a third party multivariate analysis software package where PCA and clustering analysis was executed.
Experimental Conditions
Morning-fast urine samples were collected from four subjects, two non-diabetic normal controls, one type I diabetic, and one type II diabetic. Samples were stored under refrigeration prior to liquid/liquid extraction with methylene chloride and subsequent 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. The 10 mL aliquots were extracted with 2 mL of methlyene chloride into a 20 mL scintillation vial containing approximately 5 mg sodium sulfate. Derivatization was then carried out with BSTFA by placing 200 μL of extract into a sealed 2 mL auto sampler vial containing approximately 0.5 mg sodium sulfate followed by the addition of 30 μL of dry pyridine and 200 μL BSTFA to each vial. The samples were heated to 60°C for 1 hour and then analyzed by GCxGC-TOFMS. GCxGC-TOFMS results were generated with a LECO Pegasus 4D Time-of-Flight Mass Spectrometer (TOFMS). The Pegasus 4D GC-TOFMS instrument was equipped with an Agilent 7890 gas chromatograph featuring a LECO two stage cryogenic modulator and independently temperature controlled secondary oven. LECO ChromaTOF software was used for all acquisition control, data processing, Statistical Compare, and Fisher Ratio calculations. A 30 m x 0.25 mm x 0.25 μm film thickness, Rtx-5ms, (Restek Corp.) GC capillary column was used as the primary column for theGCxGC-TOFMS analysis. In the GCxGC configuration a 1.5 m x 0.18 mm id. x 0.18 μm film thickness, Rtx-200, (Restek Corp.) was placed inside the LECO secondary GC oven which follows the thermal modulator. The Helium carrier gas flow rate was set to 1.5mL/min at a corrected constant flow via pressure ramps. The primary column was programmed with an initial temperature of 40 C for 1.00 minute and ramped at 6 C/minute to 290 C for 10 minutes. The secondary column temperature program was set to an initial temperature of 50 C for 1.00 minute and then ramped at 6 C/minute to 300 C with a 10 minute hold time. The thermal modulator was set to +25 C relative to the primary oven and a modulation time of 5 seconds was used. The MS mass range was 45-800 m/z with an acquisition rate of 200 spectra per second. The ion source chamber was set to 230 C and the detector voltage was 1750V with an electron energy of -70eV. All of the data was processed with an identical data processing method and signal-to-noise ratio 100 prior to conducting Statistical Compare for the diabetic versus non-diabetic sample groups.
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