Fluorophore-assisted carbohydrate electrophoresis for the determination of molecular mass of heparins and low-molecular-weight (LMW) heparins.
We report the use of fluorophore-assisted carbohydrate electrophoresis (FACE) to determine the molecular mass (M) values of heparins (Heps) and low-molecular-weight (LMW)-Hep derivatives. Hep are labeled with 8-aminonaphthalene-1,3,6-trisulfonic acid and FACE is able to resolve each fraction as a discrete band depending on their M. After densitometric acquisition, the migration distance of each Hep standard is acquired and the third-grade polynomial calibration standard curve is determined by plotting the logarithms of the M values as a function of migration ratio. Purified Hep samples having different properties, pharmaceutical Heps and various LMW-Heps were analyzed by both FACE and conventional high-performance size-exclusion liquid chromatography (HPSEC) methods. The molecular weight value on the top of the chromatographic peak (Mp), the number-average Mn, weight-average Mw and polydispersity (Mw/Mn) were examined by both techniques and found to be similar. This approach offers certain advantages over the HPSEC method. The derivatization process with 8-aminonaphthalene-1,3,6-trisulfonic acid is complete after 4 h so that many samples may be analyzed in a day also considering that multiple samples can be run simultaneously and in parallel and that a single FACE analysis requires approx. 15 min. Furthermore, FACE is a very sensitive method as it requires approx. 5-10 microg of Heps, about 10-100-fold lower than samples and standards used in HPSEC evaluation. Finally, the utilization of mini-gels allows the use of very low amounts of reagents with neither expensive equipment nor any complicated procedures having to be applied. This study demonstrates that FACE analysis is a sensitive method for the determination of the M values of Heps and LMW-Heps with possible utilization in virtually any kind of research and development such as quality control laboratories due to its rapid, parallel analysis of multiple samples by means of common and simple largely used analytical laboratory equipment.
Department of Biologia Animale, University of Modena and Reggio Emilia, Modena, Italy.
Dania
D
Buzzega
Francesca
F
Maccari
Nicola
N
Volpi
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004586:Electrophoresis / Q000379:methods*; D005456:Fluorescent Dyes; D006493:Heparin / Q000737:chemistry*; D006495:Heparin, Low-Molecular-Weight / Q000031:analogs & derivatives / Q000737:chemistry*; D008970:Molecular Weight; D009281:Naphthalenes / Q000737:chemistry; D012015:Reference Standards; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D006495:Heparin, Low-Molecular-Weight; D009281:Naphthalenes; D006493:Heparin; C046465:8-amino-1,3,6-naphthalenetrisulfonic acid