Quantification of free and total sialic acid excretion by LC-MS/MS

Background: The main purpose for measuring urinary free sialic acid (FSA) is to diagnose sialic acid (SA) storage diseases. Elevated amounts of conjugated sialic acid (CSA) are observed in several diseases indicating the need to quantify CSA as well. A LC-MS/MS method for quantification of FSA and total sialic acid (TSA) in urine is developed and validated. Methods: FSA is analyzed directly after filtration of urine samples. For determination of TSA an enzymatic (neuraminidase) and a chemical (acid) hydrolysis were compared.13C3-sialic acid was used as internal standard. LC-MS/MS was performed in negative electrospray ionisation mode with multiple reaction monitoring of transitions m/z 308.2 → 87.0 (SA) and m/z 311.2 → 90.0 (13C3-SA). CSA was calculated by subtracting FSA from TSA. Results: Limit of detection for FSA and TSA was 0.3 and 1.7 μmol/L, respectively. Limit of quantification for FSA and TSA was 1.0 and 5.0 μmol/L. Intra- and inter-assay variations of FSA were 4.6% and 6.6% (n = 10) for FSA and 6.5% and 3.6% (n = 10) for TSA. Linearity was tested till 7800 μmol/L (r2= 0.9998). Values of SA analyzed after neuraminidase- or acid hydrolysis treatment were comparable. Urine samples from patients with inborn errors of SA (related) metabolism were analyzed and compared with age-related reference values. Conclusion: A method has been developed for routine determination of urinary FSA and TSA. The method is rapid, specific, robust and sensitive. Age-related reference values for FSA, TSA and CSA were determined and improved diagnostic efficacy.

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