A Study On The HPLC Fingerprint Of Robinia Honeys Produced In China

Honey is a traditional and inartificial health food.It contain carbohydrate,protein,amino acid,vitamine,microelement,organic acid,pigment,pollen,hormone,trace-active component and so on.No honey is completely the same as another one.Scientifically,in most cases this variability is in connection with the floral and geographical origin of honey,and in respect of climate,soil and other technique.As the composites of honey from different place or in different season vary so obviously that it is understandable to develop multi-techniques in quality control of honey.In this paper,the author intent to develop a feasible,efficient and precise method of rationally controlling and evaluating the quality of honey for Robinia honeys and detecting adulterations,using HPLC fingerprint technology in the analysis of carbohydrate and flavonoid of Robinia honeys produced in China.In this work of the HPLC fingerprint analysis of carbohydrate of Robinia honeys,a NH₂ column,refractive index detector,acetonitrile-water(75:25)as mobile phase were used.Flow rate was 1.0ml/min,the temperature of column and detector were respectively 37℃and 40℃.The injection volume was 25μL.The HPLC analysis of isolated Robinia honeys had 4 collective peaks and 1 significant peak.Fructose,glucose,sucrose and maltose were identified.The share of fructose and glucose in total component was 65%,the share of sucrose in total component was under 2%; Comparison of honey from 6 provinces(Shanxi,Zhejiang,Guangxi,Shandong,Gansu and Hubei) in two production seasons,total content of fructose and glucose were higher in the samples produced in the year 2007.The content of carbohydrate of samples from Shanxi,Gansu and Hubei were higher and Zhejiang,Guangxi were lower than other.Base on the result of fake honey and data in the literature,and relate on GB18796-2005,we suggest that the honey would be considered an unqualified product when the reserve time of a marked peak was 18.189 or the rate of maltose to total component was more than 6%,with application of HPLC fingerprint technology in the analysis of carbohydrate of Robinia honeys produced in China.The result of extraction of flavonoid on Robinia honeys showed that XAD-2 macroporous resion was better than DM130 and NAK-9 macro resions by large adsorption and easy-eluting the flavonoid of Robinia honeys.The optimum technical operations were as follows:the pH of solution was farthest 3;the velocity of adsorbing and eluting were 1ml/min;with 70%ethanol and lg XAD-2 macroporous resion,the capacity of adsorption was 20.12mg.In the test of HPLC fingerprint of 25 Robinia honeys from two production seasons,the optimum operations were as follows:Hypersil BDS C¹⁸(4.6×250mm,5μm)column was used.The mobile phase consisted of a mixture of water and formic acid(95:5)(solvent A)and methanol(solvent B)at a flow rate of 1mL/min.To achieve better separation gradient elution was used starting with 30%of methanol for the first 15 min,and then increasing to 40%of methanol at 20 min,45%of methanol at 30 min,60%of methanol at 50 min,80%of methanol at 52 min,and which then maintaining until the end of analysis in the 60 min.Chromatograms were recorded at one wavelengths(340nm).The injection volume was 25μL.The HPLC analysis of Chromatograms showed that all the samples had different flavonoid profile of flavone component and its content.7 collective peaks and 1 significant peak were detected at 340nm.Rutin,quercetin and kaempferol were found in any of analysed samples.Comparison of honey from 6 provinces(Shanxi,Zhejiang, Guangxi,Shandong,Gansu and Hubei)in two production seasons,the difference was presented on variety and content of flavonoid.The contents of rutin,quercetin and kaempferol were respectively 7.29～42.31μg/100g,2.61～70.51μg/100g and 2.97～110.63μg/100g.Total content of rutin,quercetin and kaempferol of samples from Shandong was the highest and Hubei was the lowest.Because of the data available in(?),the difference of(?)and content of flavonoids of Robinia honeys depend on climate conditions(temperature and rainfall)and environment.According to the report of China Meteorological Service in 2006(March to May),it was extremely higher hot and much sunshine in contrast to 2007.It was showed carbohydrate content were higher in the samples produced in the year 2007.Bell proposed that flavonoids synthanism should be considered as a plant defence mechanism against stress.Similarly,it was showed particular or total flavonoids content were higher in the samples produced in the year 2006.At the same time,the diversification of regionally sunlight brings out the fact that the change of flavonoid content that Shandong samples was the highest in 6 province samples.The unknown peak laying between Sucrose and Maltose chromatographic peaks need to be identified in the future study:To those 4 unknown chromatographic peaks of flavone component virtue of Inner-Mark or HPLC-MS can be used to determine.In combination with obtained chromatographic fingerprint of samples,they could be used as a reference method of controlling and evaluating the quality of honey for Robinia honeys and detecting adulterations.