Ultrasound-assisted hydrolysis and chemical derivatization combined to lab-on-valve solid-phase extraction for the determination of sialic acids in human biofluids by µ-liquid chromatography-laser induced fluorescence.

The determination of sialic acids (SIAs) has recently gained interest because of their potential role as markers of inflammatory disorders or chronic diseases. Hydrolysis of conjugated derivatives, solid-phase extraction (SPE) and derivatization steps constitute sample preparation prior to insertion of the analytical sample into a µ-liquid chromatograph-laser induced fluorescence (µ-LC-LIF) detector in the present method for the determination of two representative SIAs of human metabolism. Ultrasound-accelerated hydrolysis released free SIAs, which were efficiently concentrated in a dynamic manner using a lab-on-valve (LOV) module that allows automation of SPE for preconcentration and cleanup. This step was on-line connected with DMB-labeling of SIAs (derivatization), which was shortened from 180 min required with the conventional heating method to 20min with ultrasound assistance. Individual separation of the target analytes was achieved within 20 min by µ-LC, while LIF detection endowed the overall method with high sensitivity. The LODs and LOQs provided by the method ranged 0.1-0.8 ng mL(-1) and 0.4-1.0 ng mL(-1) (between 0.1-0.8 pg and 0.4-1.0 pg expressed as on-column amount), respectively. High efficiency for interferents removal by SPE enabled the application of the method to four different biofluids-serum, urine, saliva and breast milk-for the determination of the target metabolites.

A comparative study of monosaccharide composition analysis as a carbohydrate test for biopharmaceuticals.

The various monosaccharide composition analysis methods were evaluated as monosaccharide test for glycoprotein-based pharmaceuticals. Neutral and amino sugars were released by hydrolysis with 4-7N trifluoroacetic acid. The monosaccharides were N-acetylated if necessary, and analyzed by high-performance liquid chromatography (HPLC) with fluorometric or UV detection after derivatization with 2-aminopyridine, ethyl 4-aminobenzoate, 2-aminobenzoic acid or 1-phenyl-3-methyl-5-pyrazolone, or high pH anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Sialic acids were released by mild acid hydrolysis or sialidase digestion, and analyzed by HPLC with fluorometric detection after derivatization with 1,2-diamino-4,5-methylenedioxybenzene, or HPAEC-PAD. These methods were verified for resolution, linearity, repeatability, and accuracy using a monosaccharide standard solution, a mixture of epoetin alfa and beta, and alteplase as models. It was confirmed that those methods were useful for ensuring the consistency of glycosylation. It is considered essential that the analytical conditions including desalting, selection of internal standards, release of monosaccharides, and gradient time course should be determined carefully to eliminate interference of sample matrix. Various HPLC-based monosaccharide analysis methods were evaluated as a carbohydrate test for glycoprotein pharmaceuticals by an inter-laboratory study.

Occurrence of sialic acids in healthy humans and different disorders.

Sialic acid (SA), N-acetylated derivatives of neuraminic acid, play a central role in the biomedical functioning of humans. The normal range of total sialic acid (TSA) level in serum/plasma is 1.58-2.22 mmol L-1, the free form of SA only constituting 0.5-3 mumol L-1 and the lipid-associated (LSA) forms 10-50 mumol L-1. Notably, considerably higher amounts of free SA are found in urine than in serum/plasma (approximately 50% of the total SA). In inherited SA storage diseases such as Salla's disease, SA levels are elevated many times over, and their determination during clinical investigation is well established. Furthermore, a number of reports describe elevated SA levels in various other diseases, tentatively suggesting broader clinical utility for SA markers. Increased SA concentrations have been reported during inflammatory processes, probably resulting from increased levels of richly sialylated acute-phase glycoproteins. A connection between increased SA levels and elevated stroke and cardiovascular mortality risk has also been reported. In addition, SA levels are slightly increased in cancer, positively correlating with the degree of metastasis, as well as in alcohol abuse, diabetes, chronic renal failure and chronic glomerulonephritis. Several different mechanisms are assumed to underlie the elevated SA concentrations in these disorders. The apparent non-specificity of SA to a given disease limits the potential clinical usefulness of SA determination. In addition, some non-pathological factors, such as aging, pregnancy and smoking, may cause changes in SA concentrations. The absolute increases in SA levels are also rather small (save those in inherited SA storage disorders); this further limits the clinical potential of SA as a marker. Tentatively, SA markers might serve as adjuncts, when combined with other markers, in disease screening, disease progression follow-up, and in the monitoring of treatment response. To become clinically useful, however, the existing SA determination assays need to be considerably refined to reduce interferences, to be specific for certain SA forms, and to be more easy to use.

Determination of N-acetylneuraminic acid by gas chromatography-mass spectrometry with a stable isotope as internal standard.

N-Acetylneuraminic acid was determined by gas chromatography-mass spectrometry using selected ion-monitoring technique with N-[2H3]acetylneuraminic acid as an internal standard. M-COOTMS fragments at m/z 624 of trimethylsilyl derivatives of N-acetylneuraminic acid and at m/z 627 of that of the internal standard were used as monitoring ions. The standard curve obtained was linear in the range of over 10(3), and the lower limit for quantitation was estimated to be a few hundred picograms. This method was used to measure total N-acetylneuraminic acid in the plasma of healthy humans and patients with lung cancer. The total N-acetylneuraminic acid level in the plasma was two to three times higher in the patients than in controls. A few hundred nanoliters of plasma was sufficient for the analysis. The mass fragmentogram of plasma gave a good signal/noise ratio, and measurements were very specific, accurate, and reproducible.