GC×GC–TOF-MS with Fisher Ratios for Metabolic Profiling in Urine

Introduction

Comprehensive multidimensional gas chromatography time-of-flight mass spectrometry has emerged as an excellent instrumental option for the characterization of small metabolite profiles. This application note presents a data mining strategy from results obtained from a diabetic and non-diabetic trimethylsilyl (TMS) derivatized urine study analysed by GC×GC–TOF-MS. A Fisher ratio plot was also generated from grouped sample comparison results.

Experimental Conditions

Sample preparation

4 urine samples

2 non-diabetic control subjects

1 type I diabetic

1 type II diabetic

Sample extraction procedure

10 mL aliquots acidified to pH₂ with concentrated H₂SO₄. 6 samples from each subject were extracted with 2 mL methylene chloride.

Approximately 2 mg of sodium sulphate was added to each extract.

BSTFA derivatization procedure

200 uL extract was placed in a 2 mL amber glass autosampler vial containing 0.5 mg of sodium sulphate.

30 uL of pyridine was added to the vial.

100 uL of BSTFA was placed in the vial.

Samples were heated at 60 °C for 1 hour.

Derivatized samples were then analysed by GC×GC–TOF-MS on the same day as prepared.

GC × GC–TOF-MS analysis parameters

GC: Agilent 7890 with LECO quad jet dual stage cryogenic modulator and secondary oven

Primary column: 30 m × 0.25 mm i.d. × 0.25 μm film thickness Rtx-5ms (Restek Corp., Bellefonte, Pennsylvania, USA)

Secondary column: 1.5 m × 0.18 mm i.d. × 0.20 μm film thickness Rtx-200 (Restek Corp., Bellefonte, Pennsylvania, USA)

Carrier: He, set @ 1.5 mL/min

Injection mode: Splitless injection volume: 3 uL

Primary column temperature programme: Initial temperature set @ 40 °C for 1 min ramped @ 6 °C/min to 290 °C, final hold time 10 min

Secondary column temperature programme: Initial temperature set @ 50 °C for 1 min ramped @ 6 °C/min to 300 °C, final hold time 10 min

Total run time: 52.67 minutes

GC × GC parameters

Column temperature offset: 10 °C

Modulator temperature offset: 25 °C

Modulation period: 5 s; Hot pulse time: 0.8 s

Cool time between stages: 1.7 s

Mass spectrometer: LECO Pegasus 4D

Acquisition rate: 200 spectra/s

Electron energy: –70 eV

Contact LECO for complete parameters.

Figure 1: Fisher ratio plot showing unknown chemical differences between non-diabetic and diabetic sample groups.

Results and Conclusions

The results demonstrate that significantly increased analytical performance is achieved by using GC×GC–TOF-MS for the characterization of small molecule metabolite profiles. TOF-MS provides non-skewed mass spectra and fast acquisition needed to deconvolute complex overlapping peaks as well as the data density required to characterize GC×GC analysis. The data was refined through background elimination of erroneous peaks produced by column bleed and derivatization reagents. Data alignment was then performed by the statistical compare feature contained in LECO's ChromaTOF software and a Fisher ratio plot (Figure 1) calculated for each analyte of the compound table was used to identify metabolites with the highest variance. The high variance data was subsequently exported as a spreadsheet and applied to multivariate principal component and clustering analysis.

LECO ETC (European Technical Centre)

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E-mail: Sepsci@leco.de

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