Comprehensive 2‐dimensional gas chromatography fast quadrupole mass spectrometry (GC × GC‐qMS) for urinary steroid profiling: Mass spectral characteristics with chemical ionization

Abstract

Comprehensive two dimensional GC (GC×GC), coupled to either a time of flight MS (TOF-MS) or a fast scanning quadrupole MS (qMS) has greatly increased the peak capacity and separation space compared to conventional GC-MS. However, commercial GC×GC-TOFMS systems are not equipped with chemical ionization (CI) and do not provide dominant molecular ions or enable single ion monitoring for maximal sensitivity. A GC×GC-qMS in mass scanning mode was investigated with EI and positive CI (PCI), using CH4 and NH3 as reagent gases. Compared to EI, PCI-NH3 produced more abundant molecular ions and high mass structure specific ions for steroid acetates. Chromatography in two dimensions was optimized with a mixture of 12 endogenous and 3 standard acetylated steroids (SM15-AC) relevant to doping control. Eleven endogenous target steroid acetates were identified in normal urine based on their two retention times, and EI and PCI-NH3 mass spectra; nine of these endogenous target steroid acetates were identified in congenital adrenal hyperplasia (CAH) patients. The difference between the urinary steroids profiles of normal individuals and from a CAH patient can easily be visually distinguished by their GC×GC-qMS chromatograms. We focus here on the comparison and interpretation of the various mass spectra of the targeted endogenous steroids. PCI-NH3 mass spectra were most useful for unambiguous molecular weight determination and for establishing the number of -OH by the losses of 1 or more acetate groups. We conclude that PCI-NH3 with GC×GC-qMS provides improved peak capacity and pseudomolecular ions with structural specificity.

The Human Urine Metabolome

“…Being an important and easily accessible biological fluid, urine has been the subject of detailed chemical analysis for more than 100 years [31], [32]. Extensive tables of normal reference ranges have been published for more than 100 urine ions and metabolites [33], [34], [35], [36], [37].…”

“…Extensive tables of normal reference ranges have been published for more than 100 urine ions and metabolites [33], [34], [35], [36], [37]. In addition to these referential clinical chemistry studies, several groups have applied various “global” metabolomic or metabolite profiling methods to urine, such as high resolution nuclear magnetic resolution (NMR) spectroscopy [38], [39], [40], high performance liquid chromatography (HPLC) [41], high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) [42], ultra high performance liquid chromatography-high resolution orbitrap mass spectrometry (UHPLC-HRMS) [43], ultra high performance liquid chromatography (UPLC-RP) [31], [44], fast atom bombardment ionization coupled with mass spectroscopy (FAB-MS) [35], two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GCxGC-MS) [32] or time-of-flight mass spectrometry (GCxGC-TOF-MS) [37], nanoflow liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) [33] and liquid chromatography coupled with electrospray quadruple time-of-flight mass spectrometry (LC/QTOF-MS) [36]. A combination of two or more profiling methods has also been used by many research groups to help improve metabolite coverage [34], [40].…”

Highly sensitive and selective analysis of urinary steroids by comprehensive two-dimensional gas chromatography combined with positive chemical ionization quadrupole mass spectrometry

“…GC×GC-qMS using PCI-NH 3 in combination with EI may provide useful information for identification of unknown compounds, such as designer steroids, in the urine matrices. GC×GC-MS has also been demonstrated for its suitability in metabolomic studies, such as clinical pathology for steroid dysfunction 12 , organic acid metabolite detection in human urine 41, 42 , metabolomic analysis of mouse tissue extracts 43, 44 , and metabolite analysis in plant studies 45, 46 . Future work will entail evaluation of GC×GC-qMS analyses of a range of human urines and informatics development to handle complex data sets.…”

Development of two‐dimensional gas chromatography (GC×GC) coupled with Orbitrap‐technology‐based mass spectrometry: Interest in the identification of biofuel composition

“…Generally, the rapid separation in the second column results in highly narrow peak widths ranging between 120 and 600 milliseconds at the baseline. This requires high‐data‐acquisition‐rate mass spectrometry (MS), such as time of flight MS (TOF–MS) or fast quadrupole MS (qMS) . Time‐of‐flight (TOF)‐MS is the main detection method for GC×GC because it enables data acquisition rates of more than 100 Hz for a wide scan range.…”