N-acetyl-D-mannosamine

Streptococcus pneumoniae is a major pathogen of bacterial pneumonia, meningitis, sepsis, and otitis media. The pathogenicity of this bacterium is largely attributed to its polysaccharide capsule, a protective layer around bacterial cell that enables bacteria to resist against host defense. Capsular polysaccharides (CPSs) of S. pneumoniae have been used as antigens to develop a variety of pneumococcal vaccines against invasive pneumococcal disease (IPD). These vaccines have been proven to be effective in reducing the incidence of IPD cases that are caused by vaccine-covered serotypes at the global scale. A crucial step in the manufacture of pneumococcal polysaccharide and conjugate vaccines is to purify native and depolymerized CPSs to meet strict quality standards in purity and structural integrity. The major impurities comprise proteins, nucleic acids and cell wall polysaccharides (CWPS). Traditionally, the removal of impurities to obtain purified native CPSs involves a complex process of purification, after which purified CPSs need to be further size-reduced to obtain depolymerized CPSs by multi-step approaches. In this study, we streamlined the process of CPS purification, which involves firstly ultrafiltration, followed by one-step acid precipitation, and finally diafiltration to obtain pure native CPSs. Furthermore, hydrolysis using trifluoroacetic acid (TFA) was integrated into the process to obtain purified depolymerized CPSs. The native and depolymerized CPSs produced by this optimized process were comparable to the materials obtained by the traditional approaches in purity and structural integrity, which would meet the quality standards of CPSs for vaccine production in the current edition of the European Pharmacopeia.

Glufosinate (GLUF) and glyphosate (GLY) are nonselective phosphorus-containing amino acid herbicides that are widely used in agricultural gardens and noncultivated areas. These herbicides give rise to a number of key metabolites, with 3-methyl phosphinicopropionic acid (MPPA), N-acetyl glufosinate (N-acetyl GLUF), aminomethyl phosphonic acid (AMPA), N-acetyl aminomethyl phosphonic acid (N-acetyl AMPA), N-acetyl glyphosate (N-acetyl GLY), N-methyl glyphosate (N-methyl GLY) as the major metabolites obtained from GLUF and GLY. Extensive use of these herbicides may lead to their increased presence in the environment, especially aquatic ecosystems. An increasing number of research studies into the toxicities of GLUF, GLY, and their metabolites have shown that these herbicides are potentially toxic to aquatic biota. GLUF and GLY, as well as their metabolites, are extremely polar and water-soluble, and they lack chromogenic and fluorescent groups; therefore, their concentrations are difficult to determine using conventional methods. Most analytical methods used to date have largely depended on derivatization procedures, leading to overall determination processes that are tedious and time-consuming. Therefore, establishing a quick and sensitive method that does not require derivatives for determining GLUF, GLY, and their metabolites in water environments, including surface water, sediments, and aquatic organisms, is an important endeavor. In this study, a new approach was developed based on pass-through solid-phase extraction coupled with ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) to determine GLUF, GLY, and their metabolites, including MPPA, N-acetyl GLUF, AMPA, N-acetyl AMPA, N-acetyl GLY, and N-methyl GLY, in sediments. Samples were extracted with 4% (v/v) ammonia water and purified using PRiME HLB pass-through solid-phase extraction columns. The extracts were filtered through a polyethersulfone microfiltration membrane and analyzed by UHPLC-MS/MS. Compounds were separated on a Metrosep A Supp 5 column (150 mm×4.0 mm, 5 μm) using gradient elution with water and 200 mmol/L ammonium hydrogen carbonate solution containing 0.05% (v/v) ammonia water as the mobile phases. Analytes were detected using MS/MS with a negative electrospray ionization (ESI^-) source in the multiple reaction monitoring (MRM) mode. A matrix-matched external-standard approach was used for quantitative analysis. GLUF, GLY, and their metabolites were detected within 15 min with good peak shapes and high responses. Calibration curves were linear in the range of 2.0-200 μg/L, with correlation coefficients exceeding 0.995. This method delivered limits of detection (LODs) and limits of quantification (LOQs) of 5 μg/kg and 20 μg/kg, respectively, for GLUF, MPPA, N-acetyl-GLUF, N-acetyl AMPA, N-acetyl GLY, and N-methyl GLY, and 10 μg/kg and 30 μg/kg, for GLY and AMPA, respectively. Average spiked recoveries at three levels (LOQ, 5LOQ, 10LOQ) in sediment with low organic matter content were in the range of 78.5%-107%, and the relative standard deviations (RSDs) were in the range of 1.32%-14.7% (n=6). Average spiked recoveries of 76.4%-113% were determined for three levels (LOQ, 5LOQ, and 10LOQ) in sediments with high organic matter contents, with RSDs of 2.60%-11.2% (n=6). The developed method was used to analyze GLUF, GLY, and their metabolites in ponds, lakes, reservoirs, and river sediments. No target compounds were detected in any sediment sample obtained from a lake, reservoir, or river; however, GLY and AMPA were detected in one pond-sediment sample at levels of 31.7 and 52.3 μg/kg, respectively. The developed approach is simple, fast, and green; moreover, it offers advantages, including high accuracies, high sensitivities, and good reproducibilities. Accordingly, the developed method is suitable for determining GLUF, GLY, and their metabolites in sediments and can provide technical support for studying residue characteristics and environmental behavior in sediments.