Liquid chromatography-tandem mass spectrometry assay for the quantification of free and total sialic acid in human cerebrospinal fluid.
BACKGROUND
Analysis of sialic acid (SA) metabolites in cerebrospinal fluid (CSF) is important for clinical diagnosis. In the present study, a high-performance liquid chromatography-tandem mass spectrometry (HPLC/MS/MS) method for free sialic acid (FSA) and total sialic acid (TSA) in human CSF was validated.


METHODS
The method utilized a simple sample-preparation procedure of protein precipitation for FSA and acid hydrolysis for TSA. Negative electrospray ionisation was used to monitor the transitions m/z 308.2-->87.0 (SA) and m/z 311.2--> 90.0 ((13)C(3)-SA). Conjugated sialic acid (CSA) was calculated by subtracting FSA from TSA. We established reference intervals for FSA, TSA and CSA in CSF in 217 control subjects. The method has been applied to patients' samples with known differences in SA metabolites like meningitis (n=6), brain tumour (n=2), leukaemia (n=5), and Salla disease (n=1).


RESULTS
Limit of detection (LOD) was 0.54 microM for FSA and 0.45 mM for TSA. Intra- and inter-assay variation for FSA (21.8 microM) were 4.8% (n=10) and 10.4% (n=40) respectively. Intra- and inter-assay variation for TSA (35.6 microM) were 9.7% (n=10) and 12.8% (n=40) respectively. Tested patients showed values of TSA above established reference value.


CONCLUSIONS
The validated method allows sensitive and specific measurement of SA metabolites in CSF and can be applied for clinical diagnoses.
van der Ham
Maria
M
Department of Metabolic and Endocrine Diseases, University Medical Centre Utrecht, WKZ and Netherlands Metabolomics Centre, The Netherlands.
de Koning
Tom J
TJ
Lefeber
Dirk
D
Fleer
André
A
Prinsen
Berthil H C M T
BH
de Sain-van der Velden
Monique G M
MG
D001932:Brain Neoplasms / Q000134:cerebrospinal fluid; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D006801:Humans; D007938:Leukemia / Q000134:cerebrospinal fluid; D008581:Meningitis / Q000134:cerebrospinal fluid; D019158:N-Acetylneuraminic Acid / Q000134:cerebrospinal fluid*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D029461:Sialic Acid Storage Disease / Q000134:cerebrospinal fluid; D053719:Tandem Mass Spectrometry / Q000379:methods*
D019158:N-Acetylneuraminic Acid