Selective and Accurate Quantification of N-Acetylglucosamine in Biotechnological Cell Samples via GC–MS/MS and GC–TOFMS

In the research article published in ACS Analytical Chemistry journal the researchers from the University of Natural Resources and Life Sciences, BOKU Vienna, and from Novartis Technical Operations Anti-Infectives, MS&T Laboratories reported a new approach towards the quantification of N-acetylglucosamine in biotechnological cell samples using GC–MS/MS and GC–TOFMS.

N-Acetylglucosamine is a crucial component of bacterial and fungal cell walls and the extracellular matrix in animals, playing roles in cell surface structure and signaling pathways. Given the challenges in distinguishing its stereoisomers, which share identical masses and similar fragmentation patterns, efficient separation techniques are essential. A gas chromatography method using automated online derivatization was developed for precise quantification of these stereoisomers. This approach, combined with ¹³C-labeled internal standards and mass spectrometric analysis, allows accurate detection of low-abundance stereoisomers, as demonstrated in Penicillium chrysogenum cultivations with strong consistency across different mass spectrometric techniques.

The original article

Selective and Accurate Quantification of N-Acetylglucosamine in Biotechnological Cell Samples via GC–MS/MS and GC–TOFMS

Teresa Mairinger, Michael Weiner, Stephan Hann, and Christina Troyer

Analytical Chemistry 2020 92 (7), 4875-4883

DOI: 10.1021/acs.analchem.9b04582

licensed under CC-BY 4.0

Selected sections from the article follow.

Abstract

N-Acetylglucosamine is a key component of bacterial and fungal cell walls and of the extracellular matrix of animal cells. It plays a variety of roles at the cell surface structure and is under discussion to be involved in signaling pathways. The presence of a number of N-acetylhexosamine stereoisomers in samples of biological or biotechnological origin demands for dedicated high efficiency separation methods, due to identical exact mass and similar fragmentation patterns of the stereoisomers. Gas chromatography offers high sample capacity, separation efficiency, and precision under repeatability conditions of measurement, which is a necessity for the analysis of low abundant stereoisomers in biological samples. Automated online derivatization facilitates to overcome the main obstacle for the use of gas chromatography in metabolomics, namely, the derivatization of polar metabolites prior to analysis. Using alkoximation and subsequent trimethylsilylation, carbohydrates and their derivatives are known to show several derivatives, since derivatization is incomplete as well as highly matrix dependent inherent to the high number of functional groups present in carbohydrates. A method based on efficient separation of ethoximated and trimethylsilylated N-acetylglucosamines was developed. Accurate absolute quantification is enabled using biologically derived ¹³C labeled internal standards eliminating systematic errors related to sample pretreatment and analysis. Due to the lack of certified reference materials, a methodological comparison between tandem and time-of-flight mass spectrometric instrumentation was performed for mass spectrometric assessment of trueness. Both methods showed limits of detection in the lower femtomol range. The methods were applied to biological samples of Penicillium chrysogenum cultivations with different matrices revealing excellent agreement of both mass spectrometric techniques.

Experimental Section

Automated Derivatization

The two-step derivatization of samples and standards with (1) ethylhydroxylamine or methylhydroxylamine hydrochloride and (2) MSTFA with 1% TMCS was performed automatically on a Gerstel MPS2 dual rail sample preparation robot (Gerstel GmbH, Muehlheim, Germany). The robot was equipped with two parallel rails, each carrying a robotic tower capable of performing independent liquid handling steps. One tower was equipped with a 10 μL syringe for sample injection; the second tower was equipped with a 100 μL syringe for the addition of the reagents. Both towers were also in use for transportation of the samples. Each rail was equipped with a heated agitator for derivatization. Underivatized dried samples were stored at 6 °C in a cooled stack until the onset of just-in-time derivatization. Reagents were stored at 6 °C in a Peltier cooled tray. Sample preparation as well as the injection were controlled by the Maestro software package 1.4.30.6. (Gerstel), which was linked with the MassHunter Acquisition software B07.06 (Agilent Technologies Inc.).

For derivatization and analysis, aliquots of 60 μL of standards, 50 μL of extracts, 80 μL of washing and quenching solutions, and 120 μL of the cultivation supernatant were used. The ratio of internal standard volume/sample volume was 0.2 for extracts, 0.15 for washing and quenching solutions, and 0.1 for supernatants.

20 μL of O-ethylhydroxylamine hydrochloride in pyridine (c(EtOX) = 20 mg mL−1) was added to the spiked samples and standards prior to drying in a vacuum centrifuge, in order to prevent the degradation of keto carbonyl group containing compounds. (23) The samples were evaporated to complete dryness at pressures of <1 mbar to ensure efficient and quick drying at low temperatures. Dried samples were immediately crimped with magnetic caps to enable transportation on the robot and were placed in the drawer cooled stack (6 °C) of the MPS 2 autosampler until derivatization. If dried samples were not analyzed within 24 h, they were redried before analysis. In the first derivatization step, the samples were dissolved in 18 μL of water-free pyridine containing 19 mg mL–1 EtOx. The samples were incubated (25 °C, 120 min) under agitation (250 rpm) to convert ketone and aldehyde carbonyl groups into ethylhydroxylamino groups. Subsequently, 42 μL of MSTFA with 1% TMCS were added and the samples were heated and agitated (250 rpm) at 40 °C for 50 min to replace acidic protons by trimethylsilyl groups. Syringes were washed with pyridine before and after each liquid dosage. After a cool down to room temperature for 1.5 min in an open tray of the MPS2, samples were cooled in the cooled stack at 6 °C for 5 min and were then injected for GC–MS analysis. After injection, samples were placed back into the cooled tray at 6 °C. Derivatization was controlled by Maestro software package (Gerstel). Sequences were scheduled in a way that samples were derivatized during the GC runs of the preceding samples, hence enabling injection of the derivatized samples immediately after sample preparation was finished.

GC–MS/MS and GC–TOFMS Analysis

GC separation was carried out on Agilent 7890B gas chromatographs coupled either with an Agilent Technologies 7200B Q-TOF (GC–TOFMS) mass spectrometer or with an Agilent Technologies 7010B Triple Quadrupole (GC–MS/MS) mass spectrometer (Agilent Technologies Inc.). Both GC–MS systems were equipped with (1) a nonpolar deactivated precolumn (3 m × 0.25 μm i.d., Phenomenex), which was connected to the analytical column via a backflush unit (purged ultimate union), and (2) a nonpolar deactivated postcolumn (3 m × 0.18 μm i.d., Phenomenex), which was connected with the analytical column via a second ultimate union T-piece serving as a restrictor column to the MS. The following analytical columns were tested: ZORBAX DB-5MS + 10 m Duragard capillary column (5% phenyl–95% dimethylpolysiloxane, 30 m × 250 μm × 0.25 μm) from Agilent Technologies Inc., and Optima 5 MS Accent (5% phenyl–95% dimethylpolysiloxane, 60 m × 250 μm × 0.25 μm) and Optima 1 MS Accent (100% dimethylpolysiloxane, 30 m × 250 μm × 0.25 μm), both from Macherey Nagel.
The GC–QTOFMS instrument was equipped with an Agilent split/splitless injector, whereas the GC–MS/MS was equipped with an Agilent Multi Mode Inlet. Agilent Ultra Inert splitless liners with a single taper were used. Sample aliquots of 1 μL were injected in the splitless mode (220 °C, splitless time 2 min, septum purge flow 3 mL min–1). The following GC-temperature program was used: 70 °C (hold for 1 min), 30 °C min–1 to 240 °C, 2 °C min–1 to 260 °C, with 30 °C min–1 to 310 °C (hold for 3 min). The transfer line to the MS was kept at 280 °C. Helium 5.0 was used as carrier gas with a constant flow of 1.1 mL min–1 for the precolumn, 1.2 mL min–1 for the analytical column, and 1.4 mL min–1 for the post column. The electron ionization (EI) source of the instruments was operated with an electron energy of 70 eV and an emission current of 35 μA for the QTOF and 100 μA for the MS/MS at a temperature of 230 °C. For both instruments, the quadrupole was set at 150 °C. For chemical ionization (CI), an emission current of 10 μA was used, the ion source was operated at 150 °C, and a reagent gas flow of 20% CH4 (corresponding to 1 mL min–1) was employed. (24) The QTOF was operated in TOF-mode with a scan speed of 3.33 Hz and in 2 GHz Extended Dynamic Range mode. The mass accuracy was <2 ppm for the calibration ions and resolution at approximately 8000 RFWHM at m/z 501.9706. Mass spectra were acquired in profile mode and were converted postacquisition to centroid. The GC–MS/MS system was operated with 3.5 Hz, and the resolution was set to widest (corresponding to a mass isolation window of 2.5 m/z) for both mass selective quadrupoles. For data evaluation, MassHunter Quantitative analysis B.08.00 (Agilent Technologies Inc.) was used.

Anal. Chem. 2020, 92, 7, 4875-4883: Figure 1. Extracted ion chromatograms (GC–CI-TOF) of the three biologically relevant N-acetylated hexosamines, i.e., N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), andN-acetylmannosamine (ManNAc), using two different oximation reagents, namely, MOX and ETOX (dotted and dashed lines, respectively). For this purpose, the [M – CH₃]⁺ of the respective cis and trans 4TMS derivative was extracted. (a) Separation using a 60 m Optima 1 MS column is depicted. (b) Separation using a 60 m Optima 5 MS is shown.

Conclusions

A robust and highly selective GC based separation method with mass spectrometric detection for GlcNAc was validated and successfully employed for the analysis of biotechnologically relevant Penicillium chrysogenum samples. GlcNAc was detectable in all biological sample types. The presence of GlcNAc in the cultivation supernatant underlines the relevance of a separate sampling/preparation procedure for quenching supernatant, washing supernatant, cultivation supernatant, and cell-extract in combination with a robust quantification method for accurate intracellular balancing of GlcNAc in biological samples. In contrast to matrices like urine or blood plasma, the matrix of biotechnological samples can change vastly in the course of a batch or fed-batch experiment. This highly concentrated matrix clearly leads to a rapid degradation of the performance of the GC–MS system, more specifically S/N ratio and sensitivity. This heavy matrix, together with the strong matrix dependence of the derivatization efficiency, renders internal standardization with stable isotope labeling indispensable.

Nevertheless, the present method proved to be robust, since even after more than 50 injections, concentrations of less than 30 nmol L⁻¹ were still possible to be quantified. GC–MS instruments with automated liner exchange and ion source cleaning systems could restore the performance of the system within sequences.

A comparison of MS/MS and TOFMS shows comparable validation results with the exception of the linear range, which is more limited for TOFMS. However, TOFMS shows the advantage of changing data evaluation strategies retrospectively, such as the use of lower abundant isotopologues for peak integration in case of detector saturation. By this means, the full potential of TOFMS for accurate quantitation can be exploited.