Studying An The Determination Of Organic Active Components In Honey By Chromatographic Methodology

Honey contains a variety of nutrients and is very popular because of its strong effect on the health care.Many components in honey are capable of affecting its color,fragrance,flavor and function while nutritional value and biological activity,including antibacterial and antioxidant activity benefiting to health,are influenced by the natural active constituents.The combination of technologies of high performance capillary electrophoresis(HPCE)and high performance liquid chromatography(HPLC)combined with electrochemistry have more and more advantages in the analysis of small molecules such as high efficiency,wide application,reducing materials and alleviating the pollution.To better evaluate the quality of honey,we established the methods of HPCE and HPLC for separating the low molecular weight flavonoids,organic acids and water-soluble vitamines in honey.The main results were listed as follows:1.Flavonoid can be as one of the important constituents.We firstly develop the analysis method of flavonoids in honey,from different districts,through the capillary zone electrophoresis with UV detection.The compounds of rutin,quercetin,kaempferol and poplar ketone,as the typical flavonoids,can be separated completely in 5 minutes with the running buffer(pH=8.4)of 20 mM borate at 25 KV,which a fine liner correlation ranging from 2.0-500 mg/mL was obtainedwith the limit of detection from 1.17 mg/mL to 1.76 mg/mL and the recoveries are from 80% ~ 107%.The flavonoids in the real honey samples from 12 local apiaries in Henan Province,China were determined through this method,and only rutin and quercetin can be detected in most samples,which suggested both can be potentially used as the marker for identification of honey from different locations.2.The organic acids in honey are determined by capillary zone electrophoresis in this chapter.Determinations of seven organic acidsin honeywere developed through field amplification sample injection technology(FASI).We also investigated the parameters affecting FASI systematically,including pH,injection time,and water column length and buffer concentration.It wasshowed that the detection sensitivity had been significantly enhanced and the concentration enrichment factorswere increased in the range of 7-72-fold under the optimal conditions.And the limits of detection for tartaric acid,protocatechuic acid,chlorogenic acid,oxalic acid,gallic acid,citric acid and vanillic acid were 0.56,0.26,0.85,0.90,0.65,0.52 and 0.55 mg /L,respectively.This method was employed successfully to detect the organic acids in actual honey samples.3.Vitamins B1,B3,B5,B6 and vitamin C were separated from honey by using CZE.The separation conditions including the concentrations of buffer,separation voltage and column temperature were optimized by response surface methodology using the three factors and three levels through the software of Box-Behnken design.The optimum separation conditions were obtained as follows: buffer containing 17 mmol/L borax,15 kV of voltage,and column temperatureof19?.Using the selected optimal conditions,the water-soluble vitamin can be separated within 3 minutes from the honey samples.4.The detection of vitamin C and dopamine were established by HPLC-ECD.The samples were detected by Diamonsil C18(2)5? 150×4.6mm chromatographic column with the mobile phase containing 0.05 mol/L sodium dihydrogen phosphate,0.02 mol/L citric acid and methanol at the volume ratio 74: 24:2 and 280 nm as the detection wavelength.The linear ranges of vitamin C from HPLC and ECD were 2-200 ?mol/L and 0.05-200 ?mol/L,respectively,while the dopamine was 2-200 ?mol/L and 0.05-200 ?mol/L.And their limits of detection were 1.68 ?mol/L,0.03 ?mol/L(from HPLC and ECD for vitamin C)and 1.25 ?mol/L,0.01 ?mol/L(from HPLC and ECD for dopamine).HPLC-ECD can be used as a simple,sensitive and accurate method to determine the vitamin C and dopamine from honey.5.Reversed phase high performance liquid chromatography-electrochemical detection(RP-HPLC-ECD),as a reliable and sensitive method,was developed to determine three organic acids(gallic acid,chlorogenic acid and caffeic acid)in honey.The separation was carried out with a reversed-phase C18 column.And the mobile phases were consisted of 2% aqueous acetic acid solution(pH)and methanol,and samples were subjected to a gradient elution using a glassy electrode at + 0.80 V for detection.The linearity,accuracy,quantification and detection limits of this method were validated.The linear regression analysis of the calibration curve shows good linearity in the range from 0.005mg/L to 6.0mg/L,and the regression coefficient between the peak area and the concentration is more than 0.99 with the detection limit of 10-5 to 3*10-5 ng/mL.This proposed method was successfully applied to analyze the three organic acid compounds in real honey samples.