title: Quantitative determination and confirmatory analysis of N-acetylneuraminic and N-glycolylneuraminic acids in serum and urine by solid-phase extraction on-line coupled to liquid chromatography-tandem mass spectrometry.
abstract: N-acetylneuraminic acid (Neu5Ac) and N-acetylglycolylneuraminic acid (Neu5Gc), two acylated derivatives of 9-C carboxylated monosaccharides, are involved in a number of biological processes as modulators of glycoconjugates. A partially automated method is here presented for determination of these sialic acids in the two most important biofluids for clinical analysis: serum and urine. For this purpose, a solid-phase extraction (SPE) workstation was on-line connected to an LC-MS/MS triple quadrupole mass detector. Hydrolysis to release sialic acids bound to glycoconjugates and derivatization were the two steps implemented as sample preparation prior to SPE-LC-MS/MS analysis. Following thorough optimization of the SPE and LC-MS/MS conditions, the analytical method was validated using the standard addition approach to assess the presence of matrix effects. The proposed method affords detection limits of 0.03ng/mL and 0.04ng/mL for Neu5Ac and Neu5Gc, respectively. The precision (expressed as relative standard deviation) was 1.7 and 4.6% for within-day variability, and 4.8 and 7.2% for between-days variability. Accuracy, estimated using spiked (between 1 and 50ng/mL) and non-spiked samples of both biofluids, ranged from 95.2 to 99.6%. The method was applied to human serum and urine of healthy volunteers, thus showing its suitability for application in both clinical and research laboratories.
authors.affiliation: Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba E-14071, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía Hospital, University of Córdoba, Córdoba E-14071, Spain. Electronic address: q72prcaf@uco.es.
authors.forename: F
authors.initials: F
authors.lastname: Priego-Capote
authors.affiliation: Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba E-14071, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía Hospital, University of Córdoba, Córdoba E-14071, Spain.
authors.forename: M I
authors.initials: MI
authors.lastname: Orozco-Solano
authors.affiliation: Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba E-14071, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía Hospital, University of Córdoba, Córdoba E-14071, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Calderón-Santiago
authors.affiliation: Department of Analytical Chemistry, Annex Marie Curie Building, Campus of Rabanales, University of Córdoba, Córdoba E-14071, Spain; Maimónides Institute of Biomedical Research (IMIBIC), Reina Sofía Hospital, University of Córdoba, Córdoba E-14071, Spain.
authors.forename: M D
authors.initials: MD
authors.lastname: Luque de Castro
mesh_terms: D001774:Blood Chemical Analysis / Q000379:methods*; D002853:Chromatography, Liquid; D005260:Female; D006801:Humans; D019158:N-Acetylneuraminic Acid / Q000097:blood / Q000652:urine; D009438:Neuraminic Acids / Q000097:blood / Q000652:urine; D012680:Sensitivity and Specificity; D052616:Solid Phase Extraction; D053719:Tandem Mass Spectrometry; D016482:Urinalysis / Q000379:methods*
chemical_list: D009438:Neuraminic Acids; C032592:N-glycolylneuraminic acid; D019158:N-Acetylneuraminic Acid
keywords: Acetylneuraminic acid; Glycolylneuraminic acid; LC–MS/MS; Methyl ester acetylneuraminic acid; On-line SPE; Serum