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

Abstract

Background: Sialic acid storage diseases (SSDs) are severe autosomal recessive neurodegenerative disorders caused by a transport defect across the lysosomal membrane, which leads to accumulation of sialic acid in tissues, fibroblasts, and urine. Defective free sialic acid transport can be established by quantification of free sialic acid in urine. Methods: Urine sample size was adjusted to the equivalent of 100 nmol of creatinine. After addition of 2-keto-3-deoxy-D-glycero-D-galactonononic acid as internal standard, samples were diluted with water to an end volume of 250 L. We used 10 L for HPLC-tandem mass spectrometric analysis in the negative electrospray ionization mode, monitoring transitions m/z 308.33m/z 86.9 (sialic acid) and m/z 267.23m/z 86.9 (internal standard). The overall method was validated and studied for ion suppression, interfering compounds, and pH effects. Samples from controls (n ‫؍‬ 72) and SSD patients (n ‫؍‬ 3) were analyzed. Results: The limit of detection was 3 mol/L. Intraassay imprecision (CV; n ‫؍‬ 10) was 6%, 3%, and 2% at 30, 130, and 1000 mmol/mol creatinine, respectively; corresponding interassay CV (n ‫؍‬ 10) were 5%, 5%, and 2%. Recovery was 109% (100 -1000 mmol/mol creatinine). (2, 4 -6 ).The known forms of SSD are primarily caused by a transport defect across the lysosomal membrane (3, 7 ), which leads to the accumulation of free sialic acid [Nacetylneuraminic acid (NANA); Fig. 1] in tissues, fibroblasts, and urine. The origin of the transport defect lies in a mutation in the SLC17A5 gene, which encodes for sialin protein (8, 9 ).Defective free sialic acid transport can be established by quantitative analysis of NANA in urine relative to the concentration of creatinine. 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. 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 ) (s...

Multiple-reaction monitoring liquid chromatography mass spectrometry for monosaccharide compositional analysis of glycoproteins

“…The LOD of sialic acids was determined to be 5 and 10 pg for NeuAc and NeuGc, respectively. These LODs are significantly lower than what has been previously reported [55,56].…”

Clinical, morphological, and molecular aspects of sialic acid storage disease manifesting in utero

“…Our experience confirms that prenatal diagnosis is reliable using measurement of free sialic acid in trophoblastic biopsies, avoiding the variability of free sialic acid concentration due to different subcultures (cases 5 and 8, table 3) and/or culture conditions. 20 New technologies such as HPLC electrospray tandem mass spectrometry 21 could facilitate the biochemical diagnosis.…”

Measurement of free and total sialic acid by isotopic dilution liquid chromatography tandem mass spectrometry method

“…Several methods have been developed to quantify Neu5Ac in biological samples including Warren method spectrophotometry [3][4][5][6][7][8][9], spectrofluorimetry [10], enzymology [11], gas chromatography (GC) [12] and high-performance liquid chromatography (HPLC) coupled with ultraviolet [13,14], fluorimetric [15][16][17][18], amperometric [19], and mass spectrometry detection [20][21][22][23][24][25]. The limit of the classical Waren method is its lack of specificity.…”

“…Tandem mass spectrometry methods have been proposed to achieve the required specificity. However, these methods [20][21][22][23][24] use reversephase liquid chromatography to analyze the Neu5Ac despite its high hydrophilic propriety. Consequently, Neu5Ac is eluted nearby the solvent front leading to a potential high matrix effect.…”