Studies On Rapid Qualitative And Quantitative Determination Of VB2 And VB9 By HPTLC-FLD-SERS

As important members of Vitamin B,Vitamin B2(VB2)and Vitamin B9(VB9)play irreplaceable roles in many physiological reactions of normal metabolism.Currently,the use of VB2 and VB9 in dietary supplements,fortified foods and animal feeds is rapidly increasing.In order to achieve effective supervision and ensure product quality,a facile analytical method is primarily importance to ensure the sufficiency of VB2 and VB9.In the last years,high-performance thin layer chromatography(HPTLC)as a flexible platform effectively has linked to advanced detectors.HPTLC technology has shown great advantages in food and drug analysis.The aim of this work was to establish a method based on the combination of HPTLC,fluorescence densitometry(FLD)and surface enhanced Raman spectroscopy(SERS)for sensitive and confirmative screening of VB2 and VB9 in food,drug and animal feed,and the method was applied to the determination of VB2 and VB9 in actual samples.The main contents were as follows:Firstly,the method based on HPTLC-FLD for separation and quantification of VB2 and VB9 was established.The mobile phase was optimized,the mobile phase of VB2 was methanol/ethyl acetate/ammonia(2:8:1,v/v/v),the mobile phase of VB9 consisted of methanol/ethyl acetate/ammonia(6:4:1,v/v/v).In-situ gas phase oxidation derivatization method of VB9 based on thin layer chromatography plate was proposed,the optimum oxidation time was 3 min by continuous ozone oxidation experiments.FLD scanning conditions were optimized,360 nm was selected as the excitation wavelength,with K400 optical filter.The result showed the concentration of VB2 and VB9 in the range of 10~80 ng/zone had a good linear correlation in HPTLC plates,and R~2 was 0.999.The detection limits of VB2 and VB9are both 7 ng/zone.Secondly,the method of confirming VB2 and VB9 molecules by SERS on plates was established.The effects of different excitation wavelengths were compared on the SERS measurement,the optimal excitation wavelength of VB2 and VB9 was 532 nm.The properties of silver nanoparticles(AgNPs)obtained by different preparation methods were compared,showing that VB2 had the best SERS spectrum by using AgNPs which prepared by sodium borohydride method,VB9 had the best SERS spectrum by using AgNPs which prepared by sodium citrate method.The SERS spectra of VB2 and VB9 were analyzed,which showed that the aromatic rings of the VB2 was adsorbed on the silver surface,and the pteridine of the VB9was adsorbed on the silver surface.Lastly,the optimized HPTLC-FLD-SERS method was applied to the rapid screening of VB2 and VB9 in multi VB tablets,pig premixes and breakfast-fortified oatmeal.The quantitative capacity of the established method was assessed,the recovery of the three samples was 89%~116%,and the relative standard deviation(RSD%)was less than 13%.In addition,the method was good agreement with the data obtained by conventional high-performance liquid chromatography(HPLC-UV)as the benchmark.The results showed the method accurately detected VB2 and VB9 in samples,which proved the method was suitable for complex component samples.