A novel LIF‐CE method for the separation of hyaluronan‐ and chondroitin sulfate‐derived disaccharides: Application to structural and quantitative analyses of human plasma low‐ and high‐charged chondroitin sulfate isomers

Abstract

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.

Intracellular adenosine 5′‐triphosphate, adenosine 5′‐diphosphate, and adenosine 5′‐monophosphate detection by short‐end injection capillary electrophoresis using methylcellulose as the effective electroosmostic flow suppressor

“…To reduce the migration time of AMP we attempted to minimize the EOF, which in the adopted electrophoretic conditions was opposite to the migration direction of analytes and though at pH 3.8 its intensity was low, it delayed the AMP migration. As previously described [25], we minimized the EOF by adding MC (0.01% final concentration) to the run buffer, thus decreasing the migration time of AMP to about 10 min (Fig. 1B).…”

Characterization of the quantitative relationship between signal-to-noise (S/N) ratio and sample amount on-target by MALDI-TOF MS: Determination of chondroitin sulfate subsequent to enzymatic digestion

“…Method based on Limit of detection Zaia and Costello [6] Electrospray ionization MS 0.2-0.1 g/0.4-0.2 nmol Barroso et al [7] LC/MS (ESI) 0.1 ng/0.2 pmol Laremore and Linhardt [8] Detection of cesium salts of CS by MALDI-TOF MS 0.4 pmol Imanari et al [9] HPLC detection of GAG subsequent to enzymatic digestion g to ng detection limits depending on detection after separation Zhang et al [10] Thin-layer chromatography (TLC) About 6 nmol Grimshaw [11] Capillary electrophoresis with direct UV detection 25 g mL −1 Zinellu et al [12] Capillary electrophoresis with laser-induced fluorescence detection 0.02 g mL −1 Volpi and Maccari [13] Electrophoresis of intact polysaccharides 1 g to 1 ng (depending on the applied dye) Whiteman [14] (review in [15])…”

Analysis of Glycosaminoglycans by Electrophoretic Approach

“…Capillary electrophoretic (CE) technique has been previously used with success for the separation of AMAC-derivatized -disaccharide obtained from different GAGs [41], as well as for the separation of AMAC-derivatized oligomeric domains obtained from chondroitin sulfate, using both UV detector and LIF detector. This method was optimized and improved by Zinellu et al [42], for the analysis of both HA and CS/DS disaccharides in human plasma. Similarly, twelve different unsaturated disaccharides obtained from HS/HE were analyzed with CE in a reversed polarity mode, giving higher sensitivity when the disaccharides were derivatized with AMAC and detected with LIF [43].…”

“…A related developed method including similar CZE conditions and AMAC derivatization was used to attempt higher sensitivity for the analysis of all known HE/HS-deriveddisaccharides [43]. When a laser-induced fluorescence (LIF) detector is used, the detection limits of the above methodologies may decrease up to 1000-fold [41,42,80]. The HS/HE analysis CE protocols, previously reviewed by Karamanos et al [81], include different conditions of polarity and applied voltage, as well as buffer composition, concentration and pH.…”