Efficient derivatization-free monitoring of glycosyltransferase reactions via flow injection analysis-mass spectrometry for rapid sugar analytics.

The widespread application of enzymes in industrial chemical synthesis requires efficient process control to maintain high yields and purity. Flow injection analysis-electrospray ionization-mass spectrometry (FIA-ESI-MS) offers a promising solution for real-time monitoring of these enzymatic processes, particularly when handling challenging compounds like sugars and glycans, which are difficult to quickly analyze using liquid chromatography-mass spectrometry due to their physical properties or the requirement for a derivatization step beforehand. This study compares the performance of FIA-MS with traditional hydrophilic interaction liquid chromatography (HILIC)-ultra high-performance liquid chromatography (UHPLC)-mass spectrometry (MS) setups for the monitoring of the enzymatic synthesis of N-acetyllactosamine (LacNAc) using beta-1,4-galactosyltransferase. Our results show that FIA-MS, without prior chromatographic separation or derivatization, can quickly generate accurate mass spectrometric data within minutes, contrasting with the lengthy separations required by LC-MS methods. The rapid data acquisition of FIA-MS enables effective real-time monitoring and adjustment of the enzymatic reactions. Furthermore, by eliminating the derivatization step, this method offers the possibility of being directly coupled to a continuously operated reactor, thus providing a rapid on-line methodology for glycan synthesis as well.

Derivatization-free determination of short-chain volatile amines in human plasma and urine by headspace gas chromatography-mass spectrometry.

BACKGROUND: Short-chain volatile amines (SCVA) are an interesting compound class playing crucial roles in physiological and toxicological human settings. Dimethylamine (DMA), trimethylamine (TMA), diethylamine (DEA), and triethylamine (TEA) were investigated in detail. METHODS: Headspace gas chromatography coupled to mass spectrometry (HS-GC-MS) was used for the simultaneous qualitative and quantitative determination of four SCVA in different human body fluids. Four hundred microliters of Li-heparin plasma and urine were analyzed after liberation of volatile amines under heated conditions in an aqueous alkaline and saline environment. Target analytes were separated on a volatile amine column and detected on a Thermo DSQ II mass spectrometer scheduled in single ion monitoring mode. RESULTS: Chromatographic separation of selected SCVA was done within 7.5 minutes. The method was developed and validated with respect to accuracy, precision, recovery and stability. Accuracy and precision criteria were below 12% for all target analytes at low and high levels. The selected extraction procedure provided recoveries of more than 92% from both matrices for TMA, DEA and TEA. The recovery of DMA from Li-heparin plasma was lower but still in the acceptable range (>75%). The newly validated method was successfully applied to plasma and urine samples from healthy volunteers. Detected concentrations of endogenous metabolites DMA and TMA are comparable to already known reference ranges. CONCLUSION: Herein, we describe the successful development and validation of a reliable and broadly applicable HS-GC-MS procedure for the simultaneous and quantitative determination of SCVA in human plasma and urine without relying on derivatization chemistry.

Towards a more complete glycome: Advances in ion chromatography-mass spectrometry (IC-MS) for improved separation and analysis of carbohydrates.

To date, few tools are available for the analysis of the glycome without derivatization, a process which is known to introduce issues such as differential loss of sialic acid and incomplete labeling. We have previously reported the use of ion chromatography-mass spectrometry (IC-MS) to analyze native sialylated and sulfated glycans. Here, we introduce improvements to IC column technology, enabling the separation of neutral glycans while maintaining charge separation capabilities. When implemented in an IC-MS workflow, this enables the structural characterization of a broad array of chemically distinct glycans. With the newly developed IC column and modified IC-MS instrumentation configuration, we qualitatively investigated O-glycome profiles in bovine fetuin and porcine gastric mucins. The improved chromatographic resolution in combination with high-resolution MS data present a powerful tool for glycan structural identification.

Modulating Linker Composition of Haptens Resulted in Improved Immunoassay for Histamine.

Histamine (HA) is an important food contaminant generated during food fermentation or spoilage. However, an immunoassay for direct (derivatization free) determination of HA has rarely been reported due to its small size to induce the desired antibodies by its current hapten-protein conjugates. In this work, despite violating the classical hapten design criteria which recommend introducing a linear aliphatic (phenyl free) linker into the immunizing hapten, a novel haptens, HA-245 designed and synthesized with a phenyl-contained linker, exhibited significantly enhanced immunological properties. Thus, a quality-improved monoclonal antibody (Mab) against HA was elicited by its hapten-carrier conjugates. Then, as the linear aliphatic linker contained haptens, Hapten B was used as linker-heterologous coating haptens to eliminate the recognition of linker antibodies. Indirect competitive ELISA (ic-ELISA) was developed with a 50% inhibition concentration (IC50) of 0.21 mg/L and a limit of detection (LOD) of 0.06 mg/L in buffer solution. The average recoveries of HA from spiked food samples for this ic-ELISA ranged from 84.1% and 108.5%, and the analysis results agreed well with those of referenced LC-MS/MS. This investigation not only realized derivatization-free immunoassay for HA, but also provided a valuable guidance for hapten design and development of immunoassay for small molecules.

Development and validation of a GC-FID method for quantitative analysis of oleic acid and related fatty acids.

Oleic acid is a common pharmaceutical excipient that has been widely used in various dosage forms. Gas chromatography (GC) has often been used as the quantitation method for fatty acids normally requiring a derivatization step. The aim of this study was to develop a simple, robust, and derivatization-free GC method that is suitable for routine analysis of all the major components in oleic acid USP-NF (United States Pharmacopeia-National Formulary) material. A gas chromatography-flame ionization detection (GC-FID) method was developed for direct quantitative analysis of oleic acid and related fatty acids in oleic acid USP-NF material. Fifteen fatty acids were separated using a DB-FFAP (nitroterephthalic acid modified polyethylene glycol) capillary GC column (30 m×0.32 mm i.d.) with a total run time of 20 min. The method was validated in terms of specificity, linearity, precision, accuracy, sensitivity, and robustness. The method can be routinely used for the purpose of oleic acid USP-NF material analysis.

Development and validation of a GC-FID method for quantitative analysis of oleic acid and related fatty acids☆ / 药物分析学报

Oleic acid is a common pharmaceutical excipient that has been widely used in various dosage forms. Gas chromatography (GC) has often been used as the quantitation method for fatty acids normally requiring a derivatization step. The aim of this study was to develop a simple, robust, and derivatization-free GC method that is suitable for routine analysis of all the major components in oleic acid USP-NF (United States Pharmacopeia-National Formulary) material. A gas chromatography-flame ionization detection (GC-FID) method was developed for direct quantitative analysis of oleic acid and related fatty acids in oleic acid USP-NF material. Fifteen fatty acids were separated using a DB-FFAP (nitroterephthalic acid modified polyethylene glycol) capillary GC column (30 m × 0.32 mm i.d.) with a total run time of 20 min. The method was validated in terms of specificity, linearity, precision, accuracy, sensitivity, and robustness. The method can be routinely used for the purpose of oleic acid USP-NF material analysis.