Physicochemical, molecular and functional characteristics. Venom hyaluronidase hyase contributes to the diffusion of venom from the inoculation site. Rapid determination of hyaluronic acid concentration in. The unbranched chain forms a random coil with some stiffness. Determination of hyaluronic acid in pharmaceutical. For detection, modern instrumental tools, such as highly sensitive fluorescence. In this work, we purified and characterized hyase from the venom of vitalius dubius araneae, theraphosidae, a large theraphosid found in southeastern brazil.
Analysis of purified sodium hyaluronate ophthalmic solution. A liquid chromatography hplc method with uv detection was developed for determination of sodium hyaluronate in pharmaceutical formulation. Separation of hydrophobically modified hyaluronic acid. Youan laboratory of future nanomedicines and theoretical chronopharmaceutics, division of pharmaceutical sciences, school of pharmacy, university of missourikansas city, 2464 charlotte, kansas city, missouri 64108 usa. Jul 04, 2007 cytotoxic and antioxidant effects of unsaturated hyaluronic acid oligomers. Injectable dermal fillers are spacefilling gels and are available globally for replacing the hydrodynamic volume of the extracellular matrix, thus reducing the clinical effect of aging. Comprehensive analysis of modified hyaluronic acid by. Unlimited viewing of the articlechapter pdf and any associated supplements and figures. Effects of exogenous hyaluronic acid and serum on matrix.
Confarma has more than 10 years of experience on analyzing products based on this compound. Venom obtained by electrical stimulation of adult male and female v. Discussions about hplc, ce, tlc, sfc, and other liquid phase separation techniques. Hyaluronic acid is also an example of a commercially useful biopolymer with many existing and potential uses as a biomedical material. A comparison of techniques for the quantitative analysis of hyaluronic acid in equine synovial fluid. Rapid determination of hyaluronic acid concentration in fermentation broth with nearinfrared spectroscopy qin dong, hengchang zang, lixuan zang, aihua liu, yanli shi and hui zhang school of pharmaceutical sciences shandong university and. Sensitive and rapid hplc quantification of tenofovir from hyaluronic acid based nanomedicine vivek agrahari and bibotti c. Development of analytical method by rphplc technique for simultaneous determination of boric acid and chlorphenesin in medicinal powder rajan v. Determination of sodium hyaluronate in pharmaceutical formulations by hplcuv.
We offer a complete range of dedicated analytical services for the control of raw materials and finished products. Analysis of hyaluronic acid by reversed phase hplc. Gpcsec is the method of choice for measuring the molar mass distribution of. Determination of sodium hyaluronate in pharmaceutical formulations. Sensitive and rapid hplc quantification of tenofovir from.
Spectrophotometric method for the determination of sodium. A highperformance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine. Unlike other mucopolysaccharides, hyaluronic acid does not attach to proteins to form proteoglycans. List of suppliers of analytical solutions, instruments hplc, gc, standards and reagents related to hyaluronic acid. Hyaluronic acid analysis many health products like pharmaceuticals, medical devices and cosmetics are composed of hyaluronic acid. May 30, 2018 amphiphilic hyaluronic acid ha, synthesised by modifying ha to varying extents with acrylate groups, was successfully separated according to degree of substitution ds using solvent gradient high performance liquid chromatography hplc. Hplc separation of hyaluronic acid chromatography forum. A comparison of techniques for the quantitative analysis. Development of analytical method by rphplc technique for. Here, we would like to introduce examples of the use of sizeexclusion chromatography in the analysis of hyaluronic acid. Linhardt2 1department of biologia animale, university of modena and reggio emilia, modena, italy. Cytotoxic and antioxidant effects of unsaturated hyaluronic acid oligomers. The hadatx was shown to have the superior potential for the anticancer drug delivery.
Sodium hyaluronate is a polymer of disaccharides, composed of dglucuronic acid and dnacetylglucosamine, linked via alternating. Hyaluronic acid is an important biopolymer that bridges the gap between synthetic and naturally occurring polymers, i. Development of a validated hplc method for the determination of hyaluronic acid in dietary supplement formulations article in bulletin of the korean chemical society 364. Abstract a system capable of resolving all the known unsaturated nonsulfated, mono and disulfated disaccharides derived from chondroitin sulfate samples, dermatan sulfate, and hyaluronic acid after their derivatization with dansylhydrazine and separation by hplc and fluorimetric detection is reported.
You might be able to perform a reversed phase separation using the nonporous particle column you mention, but youll need to go to much higher acn content than 310%. Mass spectrometry for the characterization of unsulfated. According to the jp method, it is necessary for the system suitability to satisfy resolution rs of hyaluronic acid. Abstract a liquid chromatography hplc method with uv detection was.
Wook park department of food and drug, chosun university graduate school, gwangju 501. Hyaluronic acid, an important constituent of the connective tissue of animals, is a polysaccharide containing equivalent amounts of nacetyl glucosamine and glucuronic acid. Hyaluronic acid ha is the simplest glycosaminoglycan a class of negatively charged polysaccharides and a major constituent of the extracellular matrix ecm. It is found in most connective tissues and is particularly concentrated in synovial fluid, the vitreous fluid of the eye, umbilical cords. Mass spectrometry for the characterization of unsulfated chondroitin oligosaccharides from 2mers to 16mers. Currently, carbazole assay is widely used for ha content determination in routine analysis. Stability determination of the formulations containing.
A liquid chromatography hplc method with uv detection was developed for. A simple method for assessing hyaluronic acid production by. Purified sodium hyaluronate ophthalmic solution was added to the 17 th edition of the jp. Conformer generation is disallowed since too many atoms, too flexible, too many undefined stereo centers.
Being a polymer compound it lacks a uv absorbing chromophore. Background hyaluronic acid ha polymers have a high biocompatibility, which makes them very useful in creating hydrogels for medical research problems arise in the use of this polymer, as it easily degrades into low molecular weight species the ha hydrogels are chemically modified for medical applications. Purification and characterization of hyaluronic acid from. It is the main necessarily solved by meyer and palmer 1, kaur and jayaraman 2 and chauhan et al.
March 2019 720006499en tc pdf the chromatogram in figure 1 was created using the ri data from the analysis of two low molecular weight reference sugar samples. Studies have shown that the hyaluronic acid is not only a lubricant with dermatologic and ophthalmologic applications but also can be used in a. Amphiphilic hyaluronic acid ha, synthesised by modifying ha to varying extents with acrylate groups, was successfully separated according to degree of substitution ds using solvent gradient high performance liquid chromatography hplc. Determination of sodium hyaluronate in pharmaceutical. Determination of the unsaturated disaccharides of hyaluronic acid in. Sec analysis of hyaluronic acid, raffinose, and rhamnose.
Analysis of purified sodium hyaluronate ophthalmic. Comparison with hyaluronic acid oligomers nicola volpi1, zhenqing zhang2 and robert j. The molar mass range is from 5000 da to 20 000 000 da. Detection of a new reaction byproduct in bdde cross. Hyaluronic acid ha concentration is an important parameter in fermentation process. The anticancer activity of hadatx was investigated by its cytotoxicity against kb cell in vitro. Jan 19, 2005 hyaluronic acid is normally analyzed by sec but the mobile phase conditions buffer, salt would prevent the use of an elsd. Determination of hyaluronic acid in pharmaceutical products by spectrophotometry and hplc coupled to fluorescence or mass spectrometric detection. The bodys hyaluronic acid levels decrease with age and around 80 years of age it completely disappears 6, 7.
Detection of a new reaction byproduct in bdde crosslinked. Development of a validated hplc method for the determination. Sensitive and rapid hplc quantification of tenofovir from hyaluronic acidbased nanomedicine vivek agrahari and bibotti c. Pdf determination of sodium hyaluronate in pharmaceutical. Exclusion hplc have been used for determination of molecular weight and degradation percentage of ha from the retention times obtained by hplc 6,7. Development of a validated hplc method for the determination of. Gpcsec is the method of choice for measuring the molar mass distribution of hyaluronic acid. Three kinds of hyaluronic acid with mw of one million or more was analyzed with ohpak sb807 hq.
A comparison of methods of preparing the hyaluronic acid of equine synovial fluid for quantitative spectrophotographic analysis is presented. Effects of exogenous hyaluronic acid and serum on matrix organization and stability in the mouse cumulus celloocyte complex received for publication, april 14, 1993 antonella camaioni, vincent c. The degree of polymerization of purified hyaluronic acid, as determined by viscosity measurements, depends upon. For its strong viscosity and elasticity, ha is an important content in many kinds of tissues. Determination of hyaluronic acid can be performed using different techniques, e. List of suppliers of analytical solutions, instruments, standards and reagents related to hyaluronic acid. A comparison of techniques for the quantitative analysis of. Sec analysis of hyaluronic acid application note introduction hyaluronic acid is a high molecular weight linear polymer with repeating disaccharide units, each containing one residue of dglucuronic acid and nacetyldglucosamine linked by glycosidic bonds. Because its poor watersoluble, we changed it as sodium hyaluranate sh. Hyaluronic acid ha has been successfully produced on an industrial scale with streptococcus species.
Purification and characterization of hyaluronic acid from chicken combs. Background hyaluronic acid ha polymers have a high biocompatibility, which makes. Determination of hyaluronic acid in pharmaceutical products. Algvereintravitreal injection of high molecular weight hyaluronic acid in. Get a printable copy pdf file of the complete article 679k.
Hyaluronic acid is mainly used in medical and cosmetic applications. Development of a validated hplc method for the determination of hyaluronic acid in dietary supplement formulations. Hyaluronic acid is a polym er of disaccharides and can have up to 25,000 disaccharide repeat units. To determine the ha content, the ha polymer was enzymatically digested by specific hyaluronidase from streptomyces. Development of a validated hplc method for the determination of hyaluronic acid in dietary supplement formulations sang. Hyaluronic acid has a greater capacity to hold water than any other natural or synthetic polymer. High performance liquid chromatographymass spectrometry for mapping and sequencing glycosaminoglycanderived oligosaccharides. Online hplcesims separation and characterization of. Detection of a new reaction byproduct in bdde crosslinked autoclaved hyaluronic acid hydrogels by lcms analysis javier fidalgo, pierreantoine deglesne, rodrigo arroyo, lilian sepulveda, evgeniya ranneva, philippe deprez scientific department, skin tech pharma group, castello dempuries, cataluna, spain these authors contributed equally to this work background. Anova manova, new man keuls test correlation have been.
Hyaluronic acid is an anionic mucopolysaccharide which has a chain alternating disaccharide units composed of nacetyldglucosamine and dglucuronic acid. Hyaluronic acid is a naturally occurring biopolymer, which has important biological functions in bacteria and higher animals including humans. Hyaluronic acid and chondroitin sulfate unsaturated. Two hplc methods based on the amphiphilic structure of the ha were developed. Hyaluronic acid ha is a high molecular weight biopolysacharide, discovered in 1934, by karl meyer and his assistant, john palmer in the vitreous of bovine eyes. A short run time in the hplc method always facilitate analysis of. Hyaluronic acid degradation with bupivacaine, in vitro study. Materials and methods instrumentation the hplc system, waters alliance 2695 hplc system equipped with separation module and refractive index detector 2414, was used. A new method is proposed which appears superior to the previous methods. Studies have shown that the hyaluronic acid is not only a lubricant with dermatologic and. Mar 25, 2015 development of a validated hplc method for the determination of hyaluronic acid in dietary supplement formulations sang.
Determination of ferulic acid content in cyperus rotundus by hplc. The content of hyaluronic acidderived disaccharide isthen analyzed by high perform ance liquid chromatography. The aim of the present work is to verify the applicability of our method for the simultaneous determination of iduronic acid, glucuronic acid, and galactosamine in galactosaminoglycans from purified human arterial pgs. Anova manova, new man keuls test correlation have been used to determinate statistical significance. Purification and characterization of a hyaluronidase from. Determination of hyaluronic acid in pharmaceutical products by. Determination of ferulic acid content in cyperus rotundus. However, this method is timeconsuming, environment polluting and has the risk of microbial contamination, as well as the results lag behind fermentation process. Hyaluronic acid is normally analyzed by sec but the mobile phase conditions buffer, salt would prevent the use of an elsd.