GC-based Detection of Aldononitrile Acetate Derivatized Glucosamine and Muramic Acid for Microbial Residue Determination in Soil

Summary

We describe a method protocol for the GC-based analysis of the aldonitrile acetate derivatives of glucosamine and muramic acid extracted from soil. For elucidation of the chemical mechanism, we also present a strategy to confirm the structure of the derivative and the ion fragments formed upon electron ionization.

Abstract

Quantitative approaches to characterizing microorganisms are crucial for a broader understanding of the microbial status and function within ecosystems. Current strategies for microbial analysis include both traditional laboratory culture-dependent techniques and those based on direct extraction and determination of certain biomarkers^{1, 2}. Few among the diversity of microbial species inhabiting soil can be cultured, so culture-dependent methods introduce significant biases, a limitation absent in biomarker analysis.

The glucosamine, mannosamine, galactosamine and muramic acid have been well served as measures of both the living and dead microbial mass, of these the glucosamine (most abundant) and muramic acid (uniquely from bacterial cell) are most important constituents in the soil systems^{3, 4}. However, the lack of knowledge on the analysis restricts the wide popularization among scientific peers. Among all existing analytical methods, derivatization to aldononitrile acetates followed by GC-based analysis has emerged as a good option with respect to optimally balancing precision, sensitivity, simplicity, good chromatographic separation, and stability upon sample storage⁵.

Here, we present a detailed protocol for a reliable and relatively simple analysis of glucosamine and muramic acid from soil after their conversion to aldononitrile acetates. The protocol mainly comprises four steps: acid digestion, sample purification, derivatization and GC determination. The step-by-step procedure is modified according to former publications^{6, 7}. In addition, we present a strategy to structurally validate the molecular ion of the derivative and its ion fragments formed upon electron ionization. We applied GC-EI-MS-SIM, LC-ESI-TOF-MS and isotopically labeled reagents to determine the molecular weight of aldononitrile acetate derivatized glucosamine and muramic acid; we used the mass shift of isotope-labeled derivatives in the ion spectrum to investigate ion fragments of each derivatives⁸. In addition to the theoretical elucidation, the validation of molecular ion of the derivative and its ion fragments will be useful to researchers using δ¹³C or ion fragments of these biomarkers in biogeochemical studies^{9, 10}.

Protocol

1. Sample Preparation and Acid Extraction

1. Freeze-dry soil samples after field collection.
2. Grind and homogenize soil samples using a ball mill, soil grinder, or a mortar and pestle.
3. Weigh soil samples (containing > 0.3 mg N) into a 25 mL hydrolysis flask.
4. Add 10 mL 6M HCl into each hydrolysis flask, fill N₂ gas in the flasks, and cap tightly.
5. Hydrolyze at 105 °C in an incubator for 8 hours using an auto timer switch.

2. Sample Purification

1. Remove the flasks from the incubator; cool to room temperature.
2. Add 100 μL internal standard myo-inositol (1 mg m....

Discussion

The presented GC-based method for the analysis of aldononitrile acetate derivatized glucosamine and muramic acid provides a relatively rapid method to quantify these amino sugars, extracted from soil. The derivatives are chemically stable, and can be determined in one analysis. The method is not restricted to soil samples, and can be simplified for samples from non-soil matrix.

The vacuum pump used in this method is built to be resistant to acid. We further suggest setting up a base trap to p.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Muramic acid	Sigma-Aldrich	M2503-25MG
D-(+)-glucosamine hydrochloride	Sigma-Aldrich	G1514-100G
N-methyl-D-glucamine	Sigma-Aldrich	M2004-500G
Myo-inositol	Fisher Scientific	A307003G025
Methanol (dry)	Acros Organics	AC326950010
4-dimethylamino-pyridine	Acros Organics	AC148270050
Ethyl acetate	VWR international	BJGC100-4
Hydroxlamine hydrochloride	Fisher Scientific	H330-100
Pyridine	Fisher Scientific	P368-500
Acetic anhydride	Fisher Scientific	A10-100
Dichloromethane (Methylene chloride)	Fisher Scientific	D37-500
Hexane	Fisher Scientific	H303-4
Hydrochloric acid (6M)	Fisher Scientific	S456-4
Hydroxylamine hydrochloride-15N	Icon services	IN5280
Acetic anhydride-2H (D6C4O3)	Acros Organics	AC174670050
D-glucose-U-13C	Cambridge Isotope Laboratories	CLM-1396-1
Ammonium sufate-15N	Cambridge Isotope Laboratories	NLM-713-1
Rapid-Vap	Labconco Corp.	790002
Vacum pump	KNF Neuberger	D-79112
Hydrolysis flask	Fisher Scientific	06 423A
Derivatization microvial	Fisher Scientific	06-100E
GC	Hewlett-Packard	6890
MS	Hewlett-Packard	5972
LC-ESI-TOF-MS	Agilent Technologies	An Agilent 1200 series HPLC system coupled to an Agilent LC/MSD-TOF