Determination Of Glucosamine And Galactosamine In Food By Liquid Chromatography

Glucosamine and galactose are c-4 differential isomers,formed by the substitution of an amino group for a hydroxyl group at the C2 position,and have a variety of biological activities.Glucosamine is an intermediate material for the synthesis of mucopolysaccharides,glycoproteins,proteoglycans and other macromolecules,especially for the synthesis of articular cartilage and synovial fluid molecules,which can stimulate the growth of chondrocytes.Glucosamine plays a certain role in osteoarthritis and immune regulation.Galactose aminophosphate is an interference agent for hepatocyte uracil nucleoside.After entering the body,the uracil phosphate is exhausted,leading to a series of damaged organelles and protein synthesis disorder,resulting in hepatocyte damage.In view of the superior effect of glucosamine and the toxic effect of galactose,it is very important to monitor the content of glucosamine and galactose in food to protect human health.At present,the methods for simultaneous determination of glucosamine and galactose include spectrophotometry,ion chromatography,etc.,but the method has weak stability,low accuracy and poor reproducibility.Therefore,this paper established a pre-column derivation-HIGH performance liquid chromatography UV detection method that can simultaneously determine and analyze glucosamine and galactose.The results are as follows:1.Optimization of derivatization reaction conditions.Use of derivative reagent fluorene methoxyl carbonyl chloride glucosamine hydrochloride and galactosamine hydrochloride mixed standard solution reaction,generate compound with ultraviolet absorption,based on the chemical reaction mechanism of the compounds,using Waters2695 joint diode array detector,high performance liquid chromatograph analyzing derivative compounds for testing,And the derivative conditions are optimized,including the derivative temperature,derivative time,and the p H value of the buffer solution,and finally determine the best conditions,that is,the reaction temperature: room temperature;Reaction time: 10 min;Buffer solution p H value: 10,under this test conditions obtained the maximum UV absorption value.2.Optimization of liquid chromatography conditions.Based on the best value of derivative reaction conditions,further optimization was carried out for the liquid phase conditions.Using Waters2695 high performance liquid chromatography combined with diode array detector,the derivative compounds were detected and analyzed,including determination of detection wavelength,determination of type of liquid phase mobile phase,optimization of mobile phase flow rate and determination of elution mode.Based on the basic conditions of liquid chromatography determination,the final test scheme was determined.The chromatographic column was Eclipse XDB-C18 5 μm 4.6×150 mm.Acetonitrile was used as the mobile phase,and 0.1% phosphoric acid aqueous solution was used as the water phase.The gradient elution method was used for analysis.Galactose and glucosamine peaked at 10:75min and 11:17min respectively.In addition,amino acid interference test was carried out,using amino acid standard solution and glucosamine-galactose amino mixed standard solution derivatization reaction together,in the optimized derivatization conditions and chromatographic conditions for determination and analysis,the test proved that under the detection conditions of amino acids to the test results no interference.3.Actual sample testing.Using the above test to establish HPLC ultraviolet derivative method for bird’s nest,milk,starch,three kinds of tested food matrix analysis,in order to confirm the feasibility of test method,test methodology verification analysis,including the target compounds,detection limit,the linear relationship between the quantitative limit,standard addition recovery and precision of test,the final result shows that,When the linear range of derivative compounds was0-100 μg/m L,the correlation coefficient of glucosamine and galactose was 0.9994 and 0.9993,respectively.The limits of detection and quantification of the target compounds were 1 mg/kg and 4 mg/kg,respectively.The recoveries of standard addition ranged from 94.09% to 114.15% at three different concentrations.The relative standard deviation was in the range of 0.38% ～ 5.29%.The stability analysis showed that the target compound was stable within 10 h,and the detection method was highly sensitive and specific,which was suitable for the determination of glucosamine and galactose in food.