Specific Distribution of Sialic Acids in Animal Tissues As Examined by LC−ESI-MS after Derivatization with 1,2-Diamino-4,5-Methylenedioxybenzene

Abstract

Simultaneous analysis of sialic acids has been a challenging target, because sialic acids having N- or O-acetyl, glycolyl, and sulfonic acid ester groups are labile during their release from carbohydrate chains and analytical procedures. In the present paper, we propose a method using high-performance liquid chromatography coupled with eletrospray ionization mass spectrometry (HPLC-ESI-MS). The method was evaluated by applying to the analysis of sialic acids in various tissues, especially digestive organs in mice and rats. The method was based on the in situ precolumn derivatization of sialic acids after releasing them by hydrolysis. The sialic acids were derivatized with 1,2-diamino-4,5-methylenedioxybenzene to form highly fluorescent quinoxaline derivatives. By using two different hydrolysis conditions (i.e., with 2 M acetic acid and with 0.1 M hydrochloric acid), both total sialic acids and sialic acid distributions were easily determined. We found that sialic acids showed characteristic distributions in the tissues of mice and rats. Further, HPLC-ESI-MS revealed that all the tissues examined in mice and rats commonly contained highly acetylated sialic acids and 8-O-sulfated N-acetylneuraminic acid.

Quantification of Free Sialic Acid in Urine by HPLC–Electrospray Tandem Mass Spectrometry: A Tool for the Diagnosis of Sialic Acid Storage Disease

“…For example, enzymatic methods appear to be more accurate than colorimetric methods (16,21 ), but neither method can differentiate among different neuraminic acids; HPAE-PAD, used mainly for the analysis of sialic acids released from glycoconjugates, can reduce the possibility of interferences by relatively long gradient separations. 20 ) are more selective and sensitive, but they are still time-consuming, multistep methods that have not been validated as a rapid diagnostic tool.In this report we describe and validate a simple, rapid, selective, and sensitive method for the analysis of NANA in urine samples based on HPLC combined with tandem MS (HPLC-tMS). The deaminated sialic acid 2-keto-3-deoxy-d-glycero-d-galactonononic acid (KDN) was used as an internal standard (IS) (17, 22 ) (see Fig.…”

“…Several types of analysis are available to quantify NANA, including colorimetric methods (10,11 ), thin-layer chromatography (12 ), gas chromatography-mass spectrometry (12,13 ), HPLC with fluorescence detection (14 ), HPLC with ultraviolet detection (15 ), enzymatic assays (14,16 ), high-performance anionexchange chromatography with pulsed amperometric detection (HPAE-PAD) (17,18 ), and HPLC with mass spectrometry (HPLC-MS) (19,20 ). The main disadvantages of these methods are a lack of selectivity, a lack of speed, or both.…”

“…For example, enzymatic methods appear to be more accurate than colorimetric methods (16,21 ), but neither method can differentiate among different neuraminic acids; HPAE-PAD, used mainly for the analysis of sialic acids released from glycoconjugates, can reduce the possibility of interferences by relatively long gradient separations. HPLC-MS approaches (19,20 ) are more selective and sensitive, but they are still time-consuming, multistep methods that have not been validated as a rapid diagnostic tool.…”

Capillary Electrophoresis of Glycans: Validation of Antibody Pharmaceuticals Based on Glycan Profiles

“…Complete separation between NeuAc-type and NeuGc-type N-glycan is not achieved even if using CE-LIF, because the structural difference between NeuAc and NeuGc is much smaller (Dmass 16) than those of total mass of N-glycans. Therefore, sialic acid species can be alternatively assessed by HPLC analysis after hydrolysis with diluted HCl followed by derivatization with 1,2-diamino-4,5-methylendioxybenzene (DMB) (Morimoto et al 2001). A portion of the mixture of total sialic acids is analyzed on an ODS column using a HPLC apparatus with a fluorescent detector.…”

Nidovirus Sialate-O-Acetylesterases

“…These variants differ with respect to substrate preference of their HE proteins, implying that they use different Sia receptors as attachment factors to initiate their infections in the mouse. It is of note that both 4-O-and 9-O-acetylated Sias as well as less common Sias like 5-N-acetyl-9-O-lactylneuraminic acid are present in mouse colon and trachea and in virtually all other target organs of MHV (58). 5 The consequences of Sia receptor specificity for host cell tropism and disease outcome may be considerable (59) and remain to be explored.…”