title: Simultaneous determination of intracellular UDP-sugars in hyaluronic acid-producing Streptococcus zooepidemicus.
abstract: Two chromatographic methods for the quantitative analysis of uridine diphosphate (UDP) sugars involved in hyaluronan pathway of Streptococcus zooepidemicus (SEZ) were developed and compared. The sample preparation protocol using centrifugation and extraction in hot ethanol was employed prior to the analyses. Separation was achieved using an anion exchange Spherisorb SAX column or a Shodex QA-825 column connected with a photodiode array (PDA) detector. To increase the throughput of the chromatography method employing the Spherisorb SAX column, the solid phase extraction (SPE) procedure was introduced. Method validation results displayed that limits of detection (LODs) of UDP-glucose (UDP-Glc), UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-glucuronic acid (UDP-GlcA) calculated according to QC Expert software were in the low micromolar range and the coefficient of correlation (R(2)) was above 0.997. However, the analytical technique using the Spherisorb SAX column resulted in 80-90% recoveries and low LODs (≤6.19μM), the Shodex QA-825 column showed better long-term stability and reproducible chromatographic properties (RSD≤5.60%). The Shodex QA-825 column was successfully used to monitor UDP-sugar levels during the growth rate of SEZ cells.
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.
authors.forename: Lukáš
authors.initials: L
authors.lastname: Franke
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic. Electronic address: Dagmar.Cozikova@contipro.com.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Čožíková
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.
authors.forename: Dzianis
authors.initials: D
authors.lastname: Smirnou
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.
authors.forename: Martina
authors.initials: M
authors.lastname: Hermannová
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.
authors.forename: Tereza
authors.initials: T
authors.lastname: Hanová
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.
authors.forename: Andrea
authors.initials: A
authors.lastname: Růžičková
authors.affiliation: Contipro Pharma, Dolní Dobrouč 401, 561 02 Dolní Dobrouč, Czech Republic.
authors.forename: Vladimír
authors.initials: V
authors.lastname: Velebný
mesh_terms: D002852:Chromatography, Ion Exchange; D006820:Hyaluronic Acid / Q000378:metabolism; D042541:Intracellular Space / Q000737:chemistry; D016014:Linear Models; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D018502:Streptococcus equi / Q000737:chemistry* / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000032:analysis*
chemical_list: D014539:Uridine Diphosphate Sugars; D006820:Hyaluronic Acid
keywords: Hyaluronan; Ion exchange chromatography; UDP-sugars