Analysis of lipopolysaccharides by methanolysis, trifluoroacetylation, and gas chromatography on a fused-silica capillary column

doi:10.1016/S0021-9673(00)99620-1

Journal of Chromatography A

Volume 240, Issue 2, 21 May 1982, Pages 405-413

https://doi.org/10.1016/S0021-9673(00)99620-1 Get rights and content

Abstract

A gas chromatographic method for simultaneous analysis of fatty acids and sugars of lipopolysaccharides (LPS) has been developed. The sample (1 mg or less) is methanolyzed at 85°C overnight in 2 M HCl in methanol. The released methyl esters and methyl glycosides are trifluoroacetylated and chromatographed on a methylsilicone-impregnated fused-silica capillary column. This column resolves all ordinary LPS sugars and fatty acids, and quantitative analysis is possible, including 2-deto-3-deoxyoctanoic acid (KDO), glucosamine and heptoses. 3,6-Dideoxyhexoses show some thermal degradation at 85°C during methanolysis, but this can be overcome by lowering the temperature to 37°C. For KDO the higher temperature similarly causes some degradation, but a reproducile response factor was found. The method appears to be useful for analysis of purified LPS as well as a means for monitoring for LPS content during purification of bacterial antigents of different kinds.

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Cited by (79)

The baseline resolution of Aldo-monosaccharide enantiomers: Simplified GC–MS analyses using acetal-trifluoroacetyl derivatives for complex samples
2019, Journal of Chromatography B: Analytical Technologies in the Biomedical and Life Sciences
Citation Excerpt :
This mixture was dried, and an appropriate amount of solvent (usually ethyl acetate) was added before injection into the GC (below): injected amounts were ~0.1–1.0 nmole in volumes of 1–2 μl. In some cases after TFAA derivatization the mixtures were dried and carbon tetrachloride was added, mixed, then the mixtures were re-dried before final solvent addition: this procedure removes a portion of reaction by-products such as trifluoroacetic acid [26,27]. GC–MS analyses were performed with an Agilent 6890 N GC (splitless mode) interfaced to an Agilent 5975B MS (Mass Selective Detector, a quadrupole mass spectrometer in EI mode, 70 eV).
A variety of scientific and technical fields rely on the analysis of monosaccharides (sugars). However, the multiple isomer forms of individual monosaccharides have long hampered their analyses in complex samples: significant chromatographic co-elution often leads to the loss of information on individual compounds. In addition, the chromatographic enantiomer resolution of monosaccharides has of course received less attention than molecular resolution. This would be expected in the case of rare monosaccharides, but enantiomer resolution of erythrose and threose have also received relatively little attention in the literature. Methods that are capable of the simultaneous molecular and enantiomer resolution/identification of suites of monosaccharides in single complicated mixtures are desirable. Here we report the results of attempts to resolve mixtures of the enantiomers of all three‑carbon (3C) to six‑carbon (6C) aldehyde (aldose) monosaccharides as acetal-trifluoroacetyl (acetal-TFA) derivatives by gas chromatography–mass spectrometry (GC–MS); glyceraldehyde (3C) is included as its TFA-only derivative. After a relatively simple derivatization procedure, the analyzed acetals are in the ethyl-, 2-propyl- and 2-butyl-TFA forms. Using chiral (cyclodextrin) and non-chiral (DB-17) GC columns we show that these enantiomer pairs can be baseline resolved - depending on the derivative/GC column combination - and that any single acetal/TFA combination can resolve the majority of enantiomers in a single run. Importantly, ribose and lyxose form only one significant (in abundance) stereoisomer with all derivative combinations while other monosaccharides form a maximum of only two significant stereoisomers: this greatly reduces obfuscation due to crowding on a given chromatogram. We conclude that GC–MS of acetal-TFA derivatives is the best (overall) analytical technique, to date, for the analysis of aldose-monosaccharide enantiomer ratios and reduces chromatogram clutter by favorably restricting the proliferation of monosaccharide isomers.
Occurrence and composition of extracellular lipids and polysaccharides in a full-scale membrane bioreactor
2009, Water Research
Citation Excerpt :
Extracellular DNA could be one source for this sugar because DNA accounted for approx. 2% (w/w) in EPS extracted from sludge flocs (data not shown). In general pentoses are relatively rare in microbial EPS (Guezennec et al., 1994) although ribose was found in E. coli K-13 polysaccharide (Bryn and Jantzen, 1982) and in EPS of methanogens (Veiga et al., 1997). It was shown in the present study that ribose is obviously a major component of the low MW fraction of EPS (<10 kDa) in sludge flocs and supernatant but it is not part of the membrane foulants.
The aim of this study was to characterize the polysaccharides and lipid fractions of membrane foulants in a full-scale membrane bioreactor (MBR) treating municipal wastewater. Both of these polymeric compounds are major components of bacterial lipopolysaccharides and are impacting membrane fouling; however most of the data so far have been collected by determining sum parameters rather than the detailed composition of these polymers.
Photometric analysis of sugars showed that uronic acids (glucuronic, mannuronic and galacturonic acid) as common units of bacterial polysaccharides accounted for 8% (w/w) of extracellular polymeric substances (EPS) in activated sludge flocs. Further the so-called polysaccharide peak of EPS, with a molecular weight >10 kDa according to size exclusion chromatography, was proven to contain bacterial sugar units as shown by high resolution LC–MS. Interestingly, only traces of uronic acids could be detected in EPS of the membrane fouling layer.
A far more dramatic enrichment in the fouling layer was revealed for the lipid fraction of EPS, which was determined as fatty acid methyl esters by GC–MS. The weight percentage of fatty acids in EPS extracted from fouled ultrafiltration membranes was much higher (10%) than in the activated sludge itself (1–3%). The fatty acids accumulated on the membrane fouling layer were obviously not only of microbial origin (C16:0, C18:0) but also derived from the raw wastewater itself (C9:0). Hydrophobic interaction of lipids with the PVDF (polyvinylidene fluoride) membrane material therefore seems a plausible explanation for the observed fouling phenomenon. The results suggest that fatty acids from bacterial lipopolysaccharides as well as from synthetic sources are of much higher relevance to membrane fouling than previously assumed.
The influence of sanitizers on the lipopolysaccharide composition of Escherichia coli O111
2006, International Journal of Food Microbiology
Citation Excerpt :
Subsequent trifluoroacetylation of the LPS was performed according to the method of Bryn and Jantzen (1982).
This study focused on the influence of typical sanitizers on the composition of the lipopolysaccharides (LPS) produced by the verocytotoxin-producing (VTEC) Escherichia coli O111. We also aimed to cast light on the applicability of O-antigen-based serotyping and endotoxin based Limulus Amebocyte Lysate (LAL) assays applied in the food industry for the identification and quantification of Gram-negative bacteria. E. coli O111 was propagated in the presence of three typical commercially applied sanitizing solutions that included a Clean in Place (CIP) chlorinated sanitizer (bacteriocidal), heavy-duty alkaline sanitizer (bacteriocidal) and a phenolic hand wash solution (bacteriostatic). After the required growth phase was reached the LPS from both the intact cells and debris was extracted and methanolysed followed by trifluoroacetylation. Subsequently GC–MS analysis and the chromogenic LAL assay were applied to assess both the ultra-structure and the toxicity of the extracted LPS. The viability and debris formation during growth was also evaluated to verify the bacteriocidial and static effect of the applied sanitizers as well as to assess its relationship with LPS formation. The total LPS produced was quantified at 1.3 × 10⁶ [KDO] · OD_620 nm^− 1 for the control samples, 6.5 × 10³ [KDO] · OD_620 nm^− 1 for E. coli grown in the presence of CIP chlorinated sanitizer and 2.1 × 10⁵ and 2.85 × 10⁶ [KDO] · OD_620 nm^− 1 for the organisms grown in the presence of heavy-duty alkaline sanitizer and phenolic hand wash solution respectively (KDO = 2-keto-3-deoxy-octulosonic acid). A negative correlation (r² = − 0.880) between the [KDO] and Δviability was evident and indicated that E. coli O111 responds to factors that hinder viability by producing more LPS in its outer membrane. Subsequent assessment of the LPS ultra-structure revealed a definite change in both the total assessed saccharide and lipid fractions. The cumulative change of the LPS in response to the sanitizers further appeared to influence the toxicity of the LPS as the latter change could not be related to an individual compound within any of the assessed fractions. This emphasised the fact that the quantity of LPS obtained from E. coli O111 in this study, did not seem to determine the toxicity of the organism. From the results we further propose a coefficient that could be applied to describe the response of E. coli O111 LPS to sanitizers and caution against the application of serotyping (based on the O-antigen) and the LAL assay to quantify and identify E. coli O111 obtained from food strata where the possibility of sanitizer contamination exists.
An integrity assay for a meningococcal type B conjugate vaccine
2004, Analytical Biochemistry
Citation Excerpt :
Attempts to improve monomer production from various-sized poly- and oligosaccharides under more strenuous hydrolysis conditions led to more degradation and so were abandoned. To maximize liberation while minimizing the degradation to the sialic acid monomer during the depolymerization, an anhydrous hydrolysis with hydrochloric acid in methanol, known as methanolysis, an established procedure for dissociation of polysaccharides [28], was employed. Methanolysis treatment will depolymerize the polysaccharide and generate a stable methyl glycoside at the anomeric carbon.
The development of an analytical procedure for the evaluation of a conjugate vaccine’s structural wholeness or integrity is described. The principle component of the vaccine was the N-propionylated group B meningococcal polysaccharide (NPr-GBMP) covalently attached to a carrier protein. The goal of the procedure was to determine whether any whole polysaccharide, oligosaccharide, or monosaccharide, from minute to moderate levels, became detached off the conjugate. Free saccharide was isolated from the formulation, which included an aluminum hydroxide adjuvant for analysis. Due to its linkage, the NPr-GBMP did not release sialic acid efficiently with acid hydrolysis to the extent necessary for accurate quantitation. To accomplish depolymerization, the NPr-GBMP was subjected to methanolysis, 3 N hydrochloric acid in methanol for 16 h at 80 °C. The main product of the methanolysis reaction was a de-N-acylated methyl glycoside of sialic acid. N-acetylneuraminic acid oligomers and colominic acid were used to confirm the methanolysis depolymerization efficiency of the α(2→8) saccharides; with the treatment all oligomers produced a common methyl glycoside. For this determination anion exchange chromatography and size exclusion chromatography were both interfaced to an integrated pulsed amperometric detector. Sensitivity and linearity were demonstrated to be sufficient for the application with vaccine dose formulations with low total saccharide concentrations.
The obligate predatory Bdellovibrio bacteriovorus possesses a neutral lipid a containing α-D-mannoses that replace phosphate residues. Similarities and differences between the lipid As and the lipopolysaccharides of the wild type strain B. bacteriovorus HD100 and its host-independent derivative HI100
2003, Journal of Biological Chemistry
Citation Excerpt :
Electron impact was carried out at 70 eV and chemical ionization mass spectra (chemical ionization-MS) were recorded with ammonia as reactant gas (0.1 kilopascal). Sugar Analysis—Qualitative examination of the sugar portion of LPS was performed by methylation (0.5 m HCl/CH3OH, 45 min, 85 °C, and 2 m HCl/CH3OH, 16 h, 85 °C) followed by peracetylation (22). The absolute configuration of the mannose and 2,3-diamino-2,3-dideoxyglucose of the lipid A from B. bacteriovorus HD100 was determined with the acetylated (R)-2-butylglycosides (23).
Bdellovibrio bacteriovorus are predatory bacteria that penetrate Gram-negative bacteria and grow intraperiplasmically at the expense of the prey. It was suggested that B. bacteriovorus partially degrade and reutilize lipopolysaccharide (LPS) of the host, thus synthesizing an outer membrane containing structural elements of the prey. According to this hypothesis a host-independent mutant should possess a chemically different LPS. Therefore, the lipopolysaccharides of B. bacteriovorus HD100 and its host-independent derivative B. bacteriovorus HI100 were isolated and characterized by SDS-polyacrylamide gel electrophoresis, immunoblotting, and mass spectrometry. LPS of both strains were identified as smooth-form LPS with different repeating units. The lipid As were isolated after mild acid hydrolysis and their structures were determined by chemical analysis, by mass spectrometric methods, and by NMR spectroscopy. Both lipid As were characterized by an unusual chemical structure, consisting of a β-(1→6)-linked 2,3-diamino-2,3-dideoxy-d-glucopyranose disaccharide carrying six fatty acids that were all hydroxylated. Instead of phosphate groups substituting position O-1 of the reducing and O-4′ of the nonreducing end α-d-mannopyranose residues were found in these lipid As. Thus, they represent the first lipid As completely missing negatively charged groups. A reduced endotoxic activity as determined by cytokine induction from human macrophages was shown for this novel structure. Only minor differences with respect to fatty acids were detected between the lipid As of the host-dependent wild type strain HD100 and for its host-independent derivative HI100. From the results of the detailed analysis it can be concluded that the wild type strain HD100 synthesizes an innate LPS.
Quantification of lipopolysaccharides in outer membrane vesicle vaccines against meningococcal disease. High-performance liquid chromatographic determination of the constituent 3-hydroxy-lauric acid
2002, Biologicals
A high-performance liquid chromatographic (HPLC) assay for quantification of lipopolysaccharides (LPSs, endotoxins) in outer membrane vesicle vaccines against meningococcal disease has been developed. The LPS constituent, 3-hydroxy-lauric acid, served as marker substance for the quantification. LPS from the vaccine was precipitated by ethanol and the fatty acid constituents, including 3-hydroxy-lauric acid, were released by acidic hydrolysis, collected and purified by solid phase extraction on C18 disc-cartridges and converted into phenacyl esters for UV detection at 240 nm. Quantification of the derivatized 3-hydroxy-lauric acid was achieved by HPLC using a Brownlee RP-18 reversed phase column with acetonitrile/water (68:32, v/v) as mobile phase. The method was found to be linear over the range 3–49 μg LPS/ml with a sensitivity of 1·6 (μg/ml)⁻¹. The repeatability (within-day precision) of the method at three levels (3–49 μg LPS/ml) was 6–14% relative standard deviation and the intermediate (between-day) precision was 7% relative standard deviation (at level 15 μg LPS/ml). The method has been successfully used in the quality control of a meningococcal B outer membrane vesicle vaccine, containing 4–8% LPS relative to protein (w/w), in our laboratory for three years.

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Analysis of lipopolysaccharides by methanolysis, trifluoroacetylation, and gas chromatography on a fused-silica capillary column

Abstract

Carbohydr. Res.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

J. Chromatogr.

Bacterial. Rev.

Acta Paht. Microbial. Scand. Sect. B.