β-煙酰胺單核苷酸 純度和含量測定

4β-煙酰胺單核苷酸純度和含量的測定高效液相色譜法NMN（Nicotinamidemononucleotide全稱“β11H15N2O8P生物法是指以發(fā)酵、生物轉化、酶法、細胞培養(yǎng)和生物提取為一種或多種組合手段為技54.1高效液相色譜儀：配有紫外檢測器或DA4.5微孔過濾器。4.7粉碎機。6），樣瓶中，待測。若樣品測試濃度超出工作曲線范圍，可適當稀釋后再00600積和保留時間。根據(jù)色譜峰的面積，以面積歸一化法計算產品純度，以單點外標定量法計算產品7 A0——原料樣品溶液（6.1）中所有峰的面積和。X=CT根V..........................................m——試樣的質量,單位為克(g)。 ）；C——樣品溶液（6.2）中β-煙酰胺單核苷酸濃度（mg/L8V——定容體積（mLm——成品樣品的稱樣量（g）；9Determinationforβ-nicotinamideMononucleotideContentHighPerformanceLiquidChromatography2.1β-NicotinamideMononucleNMN（Nicotinamidemononucleotide）:Thefullnameis“β-NicotinamideMononucleotide”;itisanaturallyexistedbiologicalactivenucformofNMN.2.2Molecularformula,CASNO.andmolecularmassofβ-NicotinamideMononucleotide2.2.1Molecularformula:C11H15N2O8P2.2.3Relativemolecularmass:334.22(aspertheinternationalrelativeatomicmassof2016)Biologicalmethodreferstotheproductionprocesswithfermentation,biotransformation,enmethod,cellcultureandbiologicalextractionasoneormorecombiThebelowusedreagentsareallanalyticalreagentsunlessotherwisem3.1Referenceproductofβ-NicotinamideMononucleotide:Thecontentisnotlowerthan95%.3.2Methanol:Chromatogr3.3Potassiumdihydrogenph3.4Tetrabutylammoniumhydrogens4.1Highperformanceliquidchromatography:equippedwithultravioletdetectororDADdetectorand4.2Chromatographiccolumn:C18ODS-BP5um4.6mmx250mm(orchromatographiccolumnwiththeequivalentanalysiseffect).4.3Analysisbalance:withtheminimalgraduationvalueas0.01mg4.4Ultrasonicdissol4.6Microporefilteringmembrane:0.45μm4.7Grinder5.Preparationofstandardsolution5.1PreparationofstandardAccuratelyweigh100mg(preciseto0.01mg)standardproductofβ-NicotinamideMondissolveitintoabottleof100mLcompletelythroughultrasonictreatment,makthesolutionwith0.45ummicroporefilteringmembraneforwate5.2Preparationofstandardintermediatesolution：250mg/LAccuratelyweigh25mLstandardstocksolution(A.primarywatertothescale,andprepareitintostandardintermediatesolut5.3Preparationofstandardworkingsolutionrespectivelyanddilutethemto6.Pre-treatmentofsample6.1SampleofrawmaterialWeight100mg(preciseto0.01watercompletelythroughultrasonictreatm0.45ummicroporefilteringmembraneforwatersystem.Theobtainedfiltrat6.2Sampleoffinishedproducteightabout50mgofthefinisheddissolveitintoabottleof100mLwith50mLofprimarywatemakethevolumeconstanttothescalewithprimarywater,filterthesolutionwith0.45ummicroporefmembraneforwatersystem,movethefiltrateintothesamplebottleofliquidchromatographyfcasethesampletestingconcentrationexceedstheworkingcurves7.Chromatographanalysisco7.1Columntemperature:30℃.7.2Testingwavelength7.3MobilephaseA:Weight13.6gpotassiumdihydrogenphosphateand2.7gtetrabutylammoniusulfateanddissolvetheminto800mLofprimarywater,mixupotassiumhydroxidesolutiontoregulatethepHvalueoftwithwaterto1000mL,add64mLofmethanol,mixitevenly,leachwmembrane,andfinallyhaveultrasonicdegassing.7.4MobilephaseB:Usethemeasuringcylindertoweigh700mLofmethanolandmakeitsvolumeconstantat1000mLwithprimarywater,andkeepitforlaterafterultrasonictreatment.006008.1SampleofrawmateriUnderchromatographcondisolution(6.1)respectively,recordtheareaandpreservationtimeofchromatographpeak.Aspertheaofthechromatographpeak,calculatetheproductcontentwithareanormalizationmethod.8.2Sampleoffinishedproductrespectively,recordtheareaandpreservationtimeofchromatographpeak.Aspertheareaofthechromatographpeak,calculatetheproductcontentwithexternalstandardm9.1Sampleofrawmate9.1.1Theβ-NicotinamideMononucleotidecontentinthesamA0Inwhich,P——thecontentofβ-NicotinamideMononucleotideintherawmaterial,withtheunitaspercentage(%)。AT——Mainpeakareaofβ-NicotinamideMononucleotideinthestandardstocksolutionA0——Theareasumofallthepeaksinsamplesolutionofrawmaterial.9.1.2Theβ-NicotinamideMononucleotidecontentinthesamplemWhere:X--inthetestsampleβ-Thecontentofnicotinamidemononucleotidewapercentage(%);CT--concentrationofNMNinrawmaterquantitativeexternalstandardV--constantvolumeofsample(mLm--massofsample,ingram(g).9.2Sampleoffinishedproduct9.2.1ThenetcontentofeachfinishedproductsampleshallbecalculatW=NOnecapsule:W-netcontentofeachfinishedproductsample(g/caMtotal--weighthetotalmassofallfinishedproducts(g)N--thequantityofallfinishedproducts(grains)9.2.2Theβ-NicotinamideMononucleotidecontentinthesX=.........................................Inwhich:X——Thecontentofβ-NicotinamideMononucleotideinthesampleoffinishedproduct,withth