Development Of Three Flavonoid Certified Reference Materials

Nobiletin,hesperetin and tangeretin are flavonoids and have many pharmacological functions and biological activities,such as enhancing human immunity,anti-cancer,antibacterial and anti-inflammatory,and preventing diabetes and cardiac-cerebral vascular disease.They are widely used in the field of medicines,food and cosmetic.Certified reference material(CRM)is a substance or material having one or more sufficiently uniform characteristic values which have been identified,and can be used for the calibration of the equipment,the assignment values of materials and evaluation of analytical test methods.But at present,the number of related reference substances of flavonoids is small and the variety is few in our country.Technology for preparating and evaluating of related reference substances still needs to be improved.In this study,three flavonoid certified reference materials were developed,which could provide technical support for the quality control of related to citrus products of health products,cosmetics and medical products.In the study,the commerical raw materials were used as experimental materials.Hesperetin and tangeretin were purified by reverse phase C18 silica gel.Qualitative analysis of three flavonoids was carried out by mass spectrometry(MS),~1H nuclear magnetic resonance(~1HNMR)or infrared spectroscopy(IR)according to the requirements of technical specifications of certified reference material.The mass fraction of moisture in flavonoids was measured by a Karl Fischer titration.ICP-MS and ICP-OES were used to measure the mass fractions of nonvolatile inorganic impurities.The headspace gas chromatography coupled with FID detector was utilized to measure the mass fractions of volatile impurities.Purity of three flavonoids was determined by high performance liquid chromatography(HPLC)area normalization method,quantitative nuclear magnetic resonance(qNMR)and differential scanning calorimetry(DSC).Homogeneity testing and stability monitoring of three flavonoids reference material were examined by HPLC.The main research contents and conclusions were as follows:1.Hesperetin and tangeretin were purified by column chromatography.Through many experiments,the optimization method of purification for hesperetin and tangeretin was developed.In accordance with the purification methods,the value of the reference material of hesperetin can be promoted from 98.66%to 99.87%.The value of the reference material of tangeretin can be imporved from 98.29%to 99.98%.The results meet the requirements of development of national secondary reference materials.According to the established method,a large number of repeated experiments were carried out,and a lot of purified products of hesperetin and tangeretin were obtained.These products could be used for developing reference materials.The qualitative confirmations were made for the 3 reference materials by using MS,~1HNMR or IR.2.The purities of nobiletin CRM and hesperetin CRM were determined by HPLC area normalization method and ~1H qNMR respectively.The liquid phase quantitative analysis method of nobiletin and hesperetin was optimized by selecting chromatographic column,wavelength,sample concentration,mobile phase ratio and elution mode.The established liquid conditions of nobiletin were as follows:chromatographic column:Agilent Zorbax SB-C18(250 mm×4.6 mm,5μm),mobile phase:acetonitrile and water adjusted with 0.2%acetic acid at a ratio of 40:60,detection wavelength:334 nm,solution concentration:300 mg/L,flow rate:1.0mL/min,column temperature:25℃,injection volume:10μL.Established liquid conditions of hesperetin were as follows:chromatographic column:Agilent Zorbax SB-C18(250 mm×4.6 mm,5μm),mobile phase:methanol and water adjusted with0.2%acetic acid at a ratio of 55:45,detection wavelength:287 nm,solution concentration:300 mg/L,flow rate:1.0 mL/min,column temperature:25℃,injection volume:10μL.Mass fraction measurement of nobiletin and hesperetin using ~1H qNMR was performed on Bruker Avance III 400 MHz spectrometer equipped with pulsed field gradient probe at temperature of 293.4 K.Benzoic acid was the internal standard.The internal standard and sample material were dissolved in DMSO-d6 and analyzed by qNMR.The determination results of the HPLC-area normalization method and ~1H qNMR of nobiletin CRM were 99.58%and 99.28%respectively,and the average value was 99.43%.The determination results of the HPLC-area normalization method and ~1H qNMR of hesperetin CRM were 99.62%and 99.60%respectively,and the average value was 99.61%.The national secondary standard substance certificate numbers of nobiletin CRM and hesperetin CRM are GBW(E)100456 and GBW(E)100457,respectively.3.The purity of tangeretin CRM was determined by HPLC area normalization method and DSC respectively.The established liquid conditions of tangeretin were as follows:chromatographic column:Agilent Zorbax SB-C18(250 mm×4.6 mm,5μm),mobile phase:acetonitrile and water adjusted with 0.2%acetic acid at a ratio of 55:45, detection wavelength:375 nm,solution concentration:1000 mg/L,flow rate:1.0mL/min,column temperature:25℃,injection volume:10μL.Temperature program of differential scanning calorimetry(DSC)was as follows:from 148℃to158℃,the heating rate of 0.5℃/min.The determination results of the HPLC-area normalization method and DSC of tangeretin CRM were 99.82%and 99.81%respectively,and the average value was 99.82%.4.The homogeneity and stability of nobiletin,hesperetin and tangeretin were tested by using optimized HPLC conditions.The data of homogeneity was tested by one-way analysis of variance(F-test).The data of long-term and short-term stability measurements was examined by trend analysis(T-test).The results showed that nobiletin CRM and hesperetin CRM were stable over a period of at least 6 months at4℃,and tangeretin CRM were stable over a period of at least 12 months at 4℃.Three flavonoids CRM were sufficiently stable for dispatch under the transport conditions.The results were that the mass fraction of nobiletin CRM was 99.4%with an expanded uncertainty of 0.4%,the mass fraction of hesperetin CRM was 99.6%with an expanded uncertainty of 0.4%,the mass fraction of tangeretin CRM was 99.8%with an expanded uncertainty of 0.2%.