Laser‐induced fluorescence as a powerful detection tool for capillary electrophoretic analysis of heparin/heparan sulfate disaccharides

Abstract

In quest for high sensitivities necessary for determining the disaccharide composition of heparin/heparan sulfate present in trace amounts in biologic samples, an ultrahighly sensitive capillary electrophoresis (CE) method using laser-induced fluorescence (LIF) detection was developed. Heparin/heparan sulfate-derived Delta-disaccharides were derivatized with the fluorophore 2-aminoacridone and resolved by a reversed-polarity CE method. Estimation of the limit of detection in concentration term and limit of quantitation showed that LIF detection of AMAC-derivatives of Delta-disaccharides resulted in 27-744 times higher sensitivity as compared to those detected by UV at 255 nm. These data suggest that CE-LIF is a powerful tool to quantify minute amounts of heparin/heparan sulfate disaccharides.

Quantification of neutral human milk oligosaccharides by graphitic carbon high-performance liquid chromatography with tandem mass spectrometry

“…Moreover, MS is a very sensitive detector, and the lower limit of quantification of our method, 0.039 g/mL, is 2- to 3-orders of magnitude more sensitive than other human milk oligosaccharide instrumental analysis methods in the literature [14,17,21]. Moreover, our sensitivity for milk oligosaccharides is equivalent to the highly sensitive 2-aminoacridone (AMAC) ion-laser fluorescence method developed for analysis of the heparin sulfate disaccharides [46; 47]. Note that the LC-MS technique described herein does not require the derivatization and clean-up steps of fluorescence, but only a simple reduction.…”

Capillary electrophoresis as a simple and sensitive method to study polysaccharides of Sinorhizobium sp. NGR234

“…This kind of laser is quite expensive and not as robust as an Ar ion laser. 9-Aminoacridone (AMAC) [13] works nicely and allows detection at nM concentrations of oligosaccharides. Unfortunately, it is not charged making it difficult to separate the uncharged species.…”

Capillary electrophoresis of acidic oligosaccharides from human milk

“…For example, heparin/heparan sulfate-derived delta-disaccharides derivatized with the fluorophore AMAC and resolved by a reversed-polarity CE provide 10 times more sensitivity by UV absorption than the underivatized forms, and provide 100 times more sensitivity by LIF [35,36]. Such derivatization can cause dramatic changes in the electrophoretic properties of carbohydrates.…”

“…Reductive amination between the reducing end of the oligosaccharide and an amino group of the tag reagent is commonly used. Such tag reagents include 2-aminopyridine (2-AP) [33]; 2-aminoacridone (AMAC) [34][35][36][37]; 2-aminobenzoic acid (2-AA) [38]; 3-aminobenzoic acid [39]; p-aminobenzoic acid [40]; 3-(acetylamino)-6-aminoacridine (AA-Ac) [41]; 2-amino,6-amidobiotinyl-pyridine (BAP) [42]; 2-aminobenzamide [43]; 4-aminobenzonitrile [44]; 8-aminonaphthalene-1,3,6-trisulfonic acid (ANTS) [45]; 9-aminopyrene-1,4, 6-trisulfonate (APTS) [46][47][48]; 6-aminoquinoline (6-AQ) [49]; 5-carboxytetramethylrhodamine succinimidyl ester (TRSE) [50]; 1-maltohepaosyl-1,5-diaminonaphthalene [51]; 7-nitro-2,1,3-benzoxadiazole (NBD) [52]; 1-phenyl-3-methyl-5-pyrazolone (PMP) [53][54][55]; 1-(4-methoxy)phenyl-3-methyl-5-pyrazolone (PMPMP) [56]; and 3-(4-carboxybenzoyl)-2-quinolinecarboxyaldehyde (CBQCA) [57]. Acidic oligosaccharides can also be labeled through an amide bond between carboxylate groups present in the carbohydrate and an amino group of a tag reagent, such as 7-aminonaphthalene-1,3-disulfonic acid [58][59][60].…”