IJavascript ivaliwe okwangoku kwibrawuza yakho.Xa i-javascript ivaliwe, eminye imisebenzi yale webhusayithi ayiyi kusebenza.
Javier Fidalgo, * Pierre-Antoine Deglesne, * Rodrigo Arroyo, * Lilian Sepúlveda, * Evgeniya Ranneva, Philippe Deprez Department of Science, Skin Tech Pharma Group, Castello D'Empúries, Catalonia, Spain * Aba babhali baneembono ezithile kulo msebenzi Equal imvelaphi yegalelo: Iasidi yeHyaluroniki (HA) yipolysaccharide eyenzeka ngokwendalo esetyenziswa kwimveliso yedermal fillers ngeenjongo zobuhle.Ekubeni inesiqingatha sobomi beentsuku ezininzi kwizicubu zomntu, i-HA-based dermal fillers iguqulwa ngokwekhemikhali ukwandisa ubomi babo emzimbeni.Ukuguqulwa okuqhelekileyo kwiifayili ze-HA-based based fillers kukusetyenziswa kwe-1,4-butanediol diglycidyl ether (BDDE) njenge-agent edibeneyo yokunqumla i-HA chain.I-BDDE eshiyekileyo okanye engaphendulwanga ithathwa njengeyona tyhefu kwi-<2 iinxalenye ngesigidi (ppm);ngoko ke, i-BDDE eseleyo kwi-dermal filler yokugqibela kufuneka ilinganiswe ukuqinisekisa ukhuseleko lwesigulane.Izinto kunye neendlela: Olu pho nonongo luchaza ukufunyanwa kunye nokubonakaliswa kwemveliso ye-cross-linking reaction phakathi kwe-BDDE kunye ne-HA phantsi kweemeko ze-alkali ngokudibanisa i-chromatography yamanzi kunye ne-mass spectrometry (LC-MS).Iziphumo: Emva kokuhlalutya okuhlukeneyo, kwafunyaniswa ukuba iimeko ze-alkaline kunye nokushisa okuphezulu okusetyenziselwa ukutshabalalisa i-HA-BDDE i-hydrogel yakhuthaza ukubunjwa kwale mveliso entsha, i-compound "propylene glycol-like".Uhlalutyo lwe-LC-MS luqinisekisile ukuba imveliso yemveliso inobunzima obufanayo be-monoisotopic njenge-BDDE, ixesha lokugcinwa okwahlukileyo (tR), kunye ne-Absorbent ye-UV eyahlukileyo (λ=200 nm) imo.Ngokungafani ne-BDDE, kwabonwa kuhlalutyo lwe-LC-MS ukuba phantsi kweemeko ezifanayo zokulinganisa, le mveliso inezinga eliphezulu lokufumanisa kwi-200 nm.Isiphelo: Ezi ziphumo zibonisa ukuba akukho epoxide kwisakhiwo sale khompawundi entsha.Ingxoxo ivulelekile ukuvavanya umngcipheko wale mveliso entsha efunyenwe kwimveliso ye-HA-BDDE hydrogel (HA dermal filler) ngeenjongo zorhwebo.Amagama angundoqo: i-hyaluronic acid, i-HA dermal filler, i-hyaluronic acid edibeneyo, i-BDDE, uhlalutyo lwe-LC-MS, i-BDDE ngemveliso.
Iifayili ezisekelwe kwi-hyaluronic acid (HA) zizona zixhaphakileyo kunye nezithandwayo zedermal fillers ezisetyenziselwa iinjongo zokuthambisa.I-1 Le dermal filler yi-hydrogel, ngokuqhelekileyo iqulunqwe>>95% yamanzi kunye ne-0.5-3% HA, ebanika isakhiwo esifana nejel.I-2 HA i-polysaccharide kunye neyona nto iphambili ye-matrix ye-extracellular ye-vertebrates.Enye yezithako.Iqukethe (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) ephindaphinda iiyunithi ze-disaccharide ezixhunywe ngamabhondi e-glycosidic.Le pateni ye-disaccharide iyafana kuzo zonke izinto eziphilayo.Xa kuthelekiswa nezinye iiprotein-based fillers (ezifana ne-collagen), le propati yenza i-HA i-molecule ehambelana kakhulu ne-biocompatible.Ezi zihluzi zinokubonisa ukulandelelana kwe-amino acid enokuthi ibonwe ngumzimba wesigulana.
Xa isetyenziswe njenge-dermal filler, umda oyintloko we-HA ukuguqulwa kwayo ngokukhawuleza ngaphakathi kwezicubu ngenxa yobukho bentsapho ethile ye-enzyme ebizwa ngokuba yi-hyaluronidases.Ukuza kuthi ga ngoku, ukuguqulwa kweekhemikhali ezininzi kwisakhiwo se-HA kuye kwachazwa ukwandisa isiqingatha sobomi be-HA kwiithishu.I-3 Uninzi lwezi zilungiso zizama ukunciphisa ukufikelela kwe-hyaluronidase kwiipolymers ze-polysaccharide ngokudibanisa amakhonkco e-HA.Ngoko ke, ngenxa yokubunjwa kwamabhuloho kunye ne-intermolecular covalent bonds phakathi kwesakhiwo se-HA kunye ne-agent edibeneyo edibeneyo, i-HA hydrogel edibeneyo edibeneyo ivelisa iimveliso ezichasene ne-enzyme yokuchithwa kwe-enzyme kune-HA yendalo.4-6
Ukuza kuthi ga ngoku, iikhemikhali ze-crosslinking agents ezisetyenziselwa ukuvelisa i-HA edibeneyo ziquka i-methacrylamide, i-7 hydrazide, i-8 carbodiimide, i-9 divinyl sulfone, i-1,4-butanediol diglycidyl ether (BDDE) kunye ne-poly (ethylene glycol) i-diglycidyl ether.10 ,11 BDDE okwangoku yeyona arhente isetyenziswa ngokunqamlezayo.Nangona ezi ntlobo ze-hydrogel zibonakaliswe ukuba zikhuselekile amashumi eminyaka, i-agent e-crosslinking esetyenzisiweyo i-reagents esebenzayo enokuthi ibe yi-cytotoxic kwaye, kwezinye iimeko, i-mutagenic.12 Ke ngoko, umxholo wabo oshiyekileyo kwi-hydrogel yokugqibela kufuneka ube phezulu.I-BDDE ithathwa njengekhuselekile xa i-residual concentration ingaphantsi kweengxenye ze-2 ngesigidi (ppm).4
Kukho iindlela ezininzi zokubona ugxininiso lweBDDE olunentsalela ephantsi, idigri yokudibanisa okunqamlezayo kunye nesikhundla sokutshintsha kwii-HA hydrogels, ezifana nekhromatography yerhasi, ukukhutshelwa ngaphandle kobungakanani bechromatography edityaniswa ne-mass spectrometry (MS), i-nuclear magnetic resonance (NMR) iindlela zokulinganisa i-fluorescence, kunye Uluhlu lweDiode ludityaniswe nokusebenza okuphezulu kolwelo lwechromatography (HPLC).I-13-17 Olu pho nonongo luchaza ukufunyanwa kunye nokubonakaliswa kwemveliso kwi-HA hydrogel yokugqibela edibeneyo edibeneyo eveliswe ngokuphendula kwe-BDDE kunye ne-HA phantsi kweemeko ze-alkaline.I-HPLC kunye ne-liquid chromatography-mass spectrometry (uhlalutyo lwe-LC-MS).Ekubeni i-toxicity yale mveliso ye-BDDE ingaziwa, sincoma ukuba ukulinganisa kwayo okushiyekileyo kufuneka kunqunywe ngendlela efanayo kwindlela eqhelekileyo eyenziwa kwi-BDDE kwimveliso yokugqibela.
Ityuwa yesodium efunyenweyo ye-HA (Shiseido Co., Ltd., Tokyo, Japan) inobunzima be-molecular ~ 1,368,000 Da (indlela kaLaurent) 18 kunye ne-viscosity yangaphakathi ye-2.20 m3 / kg.Ukusabela kwe-crosslinking, i-BDDE (≥95%) yathengwa kwi-Sigma-Aldrich Co. (St. Louis, MO, USA).I-phosphate buffered saline ene-pH 7.4 yathengwa kwiNkampani yeSigma-Aldrich.Zonke i-solvents, i-acetonitrile kunye namanzi asetyenziswe kuhlalutyo lwe-LC-MS zathengwa kumgangatho webakala le-HPLC.I-asidi ye-Formic (98%) ithengwa njenge-reagent grade.
Zonke iimvavanyo zenziwa kwi-UPLC ye-Acquity system (Amanzi, i-Milford, MA, e-USA) kwaye iqhagamshelwe kwi-API 3000 ye-spektrometer ye-triple quadrupole mass exhotywe ngomthombo we-ionization we-electrospray (AB SCIEX, Framingham, MA, USA).
I-synthesis ye-HA hydrogels edibeneyo edibeneyo yaqalwa ngokongeza i-198 mg ye-BDDE kwi-10% (w / w) isisombululo se-sodium hyaluronate (NaHA) phambi kwe-1% ye-alkali (i-sodium hydroxide, i-NaOH).Ugxininiso lokugqibela lwe-BDDE kumxube wokuphendula ngu-9.9 mg / mL (0.049 mM).Emva koko, umxube wokuphendula uxutywe ngokucokisekileyo kwaye u-homogenized kwaye uvumeleke ukuba uqhubeke kwi-45 ° C kwiiyure ze-4.19 I-pH ye-reaction igcinwa kwi ~ 12.
Emva koko, umxube wokuphendula uhlanjwe ngamanzi, kwaye i-HA-BDDE yokugqibela i-hydrogel yahluzwa kwaye ihlanjululwe nge-PBS buffer ukufezekisa i-HA concentration ye-10 ukuya kwi-25 mg / mL, kunye ne-pH yokugqibela ye-7.4.Ukuze kufakwe inzalo kwi-HA hydrogels eveliswayo edibeneyo, zonke ezi hydrogel zi-autoclaved (120 ° C imizuzu engama-20).I-hydrogel ye-BDDE-HA ecocekileyo igcinwe kwi-4 ° C de kube kuhlalutya.
Ukuhlalutya i-BDDE ekhoyo kwimveliso ye-HA edibeneyo, isampuli ye-240 mg ilinganiswe kwaye yaziswa kwi-hole yeziko (Microcon®; Merck Millipore, Billerica, MA, USA; umthamo we-0.5 mL) kunye ne-centrifuged kwi-10,000 rpm kwiqondo lokushisa. 10 umzuzu.Itotali ye-20 µL yolwelo lokutsalela phantsi yaqokelelwa kwaye yahlalutywa.
Ukuze uhlalutye umgangatho weBDDE (Sigma-Aldrich Co) phantsi kweemeko ze-alkaline (1%, 0.1% kunye ne-0.01% NaOH), ukuba le miqathango ilandelayo idibene, isampuli yolwelo ngu-1: 10, 1: 100, okanye ukuya kuthi ga ngoku. I-1: 1,000,000 Ukuba kuyimfuneko, sebenzisa i-MilliQ deionized water ukuze uhlalutye.
Kwizinto zokuqalisa ezisetyenziswe kwi-reaction cross-linking reaction (HA 2%, H2O, 1% NaOH, kunye ne-0.049 mM BDDE), i-1 mL yesampula nganye elungiselelwe kwezi zinto yahlalutywa ngokusebenzisa iimeko ezifanayo zokuhlalutya.
Ukumisela ukuchana kweencopho ezibonakala kwimephu ye-ion, i-10 µL ye-100 ppb BDDE isisombululo esisemgangathweni (i-Sigma-Aldrich Co) yongezwa kwisampuli ye-20 µL.Kule meko, ukugxininiswa kokugqibela komgangatho kwisampuli nganye yi-37 ppb.
Okokuqala, lungiselela isisombululo se-stock BDDE kunye ne-concentration ye-11,000 mg / L (11,000 ppm) ngokunciphisa i-10 μL ye-BDDE eqhelekileyo (i-Sigma-Aldrich Co) kunye ne-990 μL MilliQ yamanzi (ubuninzi be-1.1 g / mL).Sebenzisa esi sisombululo ukulungisa i-110 µg/L (110 ppb) isisombululo se-BDDE njengolunyulo oluphakathi oluphakathi.Emva koko, sebenzisa i-diluent ephakathi ye-BDDE ephakathi (110 ppb) ukulungisa igophe elisezantsi ngokuhlambulula i-diluent ephakathi amatyeli amaninzi ukuphumeza uxinaniso olufunekayo lwe-75, 50, 25, 10, kunye ne-1 ppb.Njengoko kuboniswe kwi-Figure 1, kufunyenwe ukuba i-curve ye-BDDE esemgangathweni ukusuka kwi-1.1 ukuya kwi-110 ppb inomgca olungileyo (R2> 0.99).Umgangatho oqhelekileyo waphindwa kwiimvavanyo ezine ezizimeleyo.
Umzobo we-1 BDDE ijika eliqhelekileyo lokulinganisa elifunyenwe ngohlalutyo lwe-LC-MS, apho ulungelelwaniso oluhle lubonwa (R2> 0.99).
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry.
Ukuze uchonge kwaye ulinganise imigangatho ye-BDDE ekhoyo kwi-HA edibeneyo kunye nemigangatho ye-BDDE kwisisombululo esisisiseko, uhlalutyo lwe-LC-MS lusetyenziswe.
Ukuhlukana kwe-chromatographic kwaphunyezwa kwikholamu ye-LUNA 2.5 µm C18 (2) -HST (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) kwaye igcinwe kwiqondo lokushisa (25 ° C) ngexesha lohlalutyo.Isigaba esihambahambayo siquka i-acetonitrile (i-solvent A) kunye namanzi (i-solvent B) equkethe i-0.1% ye-asidi ye-formic.Inqanaba elihambahambayo likhunjulwe yi-gradient elution.Ithambeka ngolu hlobo lulandelayo: 0 imizuzu, 2% A;1 umzuzu, 2% A;imizuzu emi-6, 98% A;imizuzu esi-7, 98% A;7.1 imizuzu, 2% A;Imizuzu eli-10 , 2% A. Ixesha lokubaleka yimizuzu eli-10 kwaye umthamo wenaliti yi-20 µL.Ixesha lokugcinwa kwe-BDDE li malunga nemizuzu ye-3.48 (ukusuka kwi-3.43 ukuya kwi-4.14 imizuzu esekelwe kwiimvavanyo).Isigaba esihambahambayo sakhutshwa ngesantya sokuhamba kwe-0.25 mL / min kuhlalutyo lwe-LC-MS.
Uhlalutyo lwe-BDDE kunye nobungakanani be-MS, inkqubo ye-UPLC (Amanzi) idibene ne-API 3000 i-triple quadrupole mass spectrometer (AB SCIEX) exhotywe ngomthombo we-ionization we-electrospray, kwaye uhlalutyo lwenziwa kwimodi ye-ion enhle (ESI +).
Ngokutsho kohlalutyo lwe-ion fragment olwenziwa kwi-BDDE, isiqwenga esinamandla aphezulu sinqunywe ukuba sibe yiqhekeza elihambelana ne-129.1 Da (Umfanekiso 6).Ngoko ke, kwimodi yokubeka iliso ye-multi-ion (MIM) yokulinganisa, ukuguqulwa kobunzima (i-mass-to-charge ratio [m / z]) ye-BDDE yi-203.3 / 129.1 Da.Ikwasebenzisa imowudi epheleleyo yokuskena (FS) kunye nemowudi ye-ion yemveliso (PIS) yohlalutyo lwe-LC-MS.
Ukuze kuqinisekiswe ukuchaneka kwendlela, isampuli engenanto (isigaba sokuqala seselula) yahlalutywa.Akukho mqondiso ufunyenwe kwisampuli engenanto kunye nokuguqulwa kobuninzi be-203.3 / 129.1 Da.Ngokuphathelele ukuphindaphinda kovavanyo, ii-injection ze-10 eziqhelekileyo ze-55 ppb (embindini we-curve yokulinganisa) zahlaziywa, ezibangele ukuphambuka komgangatho oseleyo (RSD) <5% (idatha engaboniswa).
Umxholo oseleyo we-BDDE ulinganiswe kwii-hydrogels ezisibhozo ezihlukeneyo ezi-autoclaved ze-BDDE cross-linked HA, ezihambelana nezilingo ezine ezizimeleyo.Njengoko kuchazwe kwicandelo elithi "Izinto kunye neendlela", ubungakanani buvandlakanywa ngexabiso eliqhelekileyo le-curve yokubuyisela kwi-BDDE standard dilution, ehambelana nencopho ekhethekileyo efunyenwe kwi-BDDE yokuguqulwa kobunzima be-203.3 / 129.1 Da, kunye nokugcinwa ixesha 3.43 ukuya 4.14 imizuzu Ayilindanga.Umzobo 2 ubonisa umzekelo wechromatogram ye-10 ppb BDDE ireferensi yomgangatho.Itheyibhile yoku-1 ishwankathela umxholo we-BDDE oshiyekileyo wee-hydrogel ezisibhozo ezahlukeneyo.Uluhlu lwexabiso yi-1 ukuya kwi-2.46 ppb.Ngoko ke, i-BDDE i-concentration eseleyo kwisampuli iyamkeleka ukusetyenziswa komntu (<2 ppm).
Umzobo we-2 Ion chromatogram ye-10 ppb BDDE ireferensi yomgangatho (Sigma-Aldrich Co), i-MS (m / z) inguqu efunyenwe yi-LC-MS uhlalutyo lwe-203.30 / 129.10 Da (kwimodi ye-MRM enhle).
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;I-MRM, ukujongwa kweempendulo ezininzi;MS, ubunzima;m/z, umlinganiselo wobunzima ukuya kwintlawulo.
Qaphela: Iisampuli ze-1-8 zi-autoclaved BDDE cross-linked HA hydrogels.Isixa esishiyekileyo se-BDDE kwi-hydrogel kunye nencopho ye-BDDE yokugcina ixesha nayo ixeliwe.Okokugqibela, ubukho beencopho ezintsha ezinamaxesha ahlukeneyo okugcina nawo kuxeliwe.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;HA, iasidi ye-hyaluronic;I-MRM, ukujongwa kweempendulo ezininzi;tR, ixesha lokugcina;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;I-RRT, ixesha lokugcina elihambelanayo.
Okumangalisa kukuba, uhlalutyo lwe-LC-MS ion chromatogram lubonise ukuba ngokusekelwe kuzo zonke iisampuli ze-HA hydrogel ze-autoclaved cross-linked cross, kukho i-peak eyongezelelweyo kwixesha elifutshane lokugcinwa kwe-2.73 ukuya kwi-3.29 imizuzu.Ngokomzekelo, i-Figure 3 ibonisa i-ion chromatogram yesampuli ye-HA edibeneyo, apho i-peak eyongezelelweyo ibonakala ngexesha elihlukileyo lokugcinwa malunga nemizuzu ye-2.71.Ixesha lokugcinwa kwesalamane (RRT) phakathi kwencopho esanda kubonwa kunye nencopho evela kwi-BDDE ifunyenwe yi-0.79 (Itheyibhile 1).Ekubeni siyazi ukuba i-peak esanda kubonwa igcinwe ngaphantsi kwikholamu ye-C18 esetyenziswe kuhlalutyo lwe-LC-MS, i-peak entsha inokuhambelana ne-polar compound kune-BDDE.
Umzobo we-3 Ion chromatogram ye-HA hydrogel isampuli edibeneyo edibeneyo efunyenwe yi-LC-MS (ukuguqulwa kobunzima be-MRM 203.3 / 129.0 Da).
Izifinyezo: HA, i-hyaluronic acid;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;I-MRM, ukujongwa kweempendulo ezininzi;I-RRT, ixesha lokugcinwa ngokunxulumene;tR, ixesha lokugcina.
Ukuze kuthintelwe ukuba kunokwenzeka ukuba iincopho ezintsha ezibonwayo zinokuba zingcoliseko zikhoyo ekuqaleni kwizinto ezisetyenzisiweyo, ezi zinto zisetyenzisiweyo nazo zahlalutywa kusetyenziswa indlela yohlalutyo lwe-LC-MS efanayo.Izinto zokuqalisa ezihlalutywayo ziquka amanzi, i-2% ye-NaHA emanzini, i-1% ye-NaOH emanzini, kunye ne-BDDE kwi-concentration efanayo esetyenziswe kwi-cross-linking reaction.I-ion chromatogram yezinto zokuqala ezisetyenzisiweyo ayizange ibonise nayiphi na i-compound okanye i-peak, kwaye ixesha lokugcinwa kwayo lihambelana nencopho entsha eboniweyo.Le nyaniso ilahla ingcamango yokuba kungekuphela nje into yokuqala enokuthi iqulethe nayiphi na i-compounds okanye izinto ezinokuphazamisa inkqubo yokuhlalutya, kodwa akukho phawu lokungcoliswa okunokwenzeka kunye nezinye iimveliso zebhubhoratri.Amaxabiso okuxininisa afunyenwe emva kohlalutyo lwe-LC-MS ye-BDDE kunye neencopho ezintsha ziboniswa kwiThebhile 2 (iisampulu 1-4) kunye ne-ion chromatogram kuMfanekiso 4.
Qaphela: Iisampuli ze-1-4 zihambelana nezinto ezisetyenziselwa ukuvelisa i-autoclaved BDDE cross-linked HA hydrogels.Ezi sampuli azizange zifakwe ngokuzenzekelayo.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;HA, iasidi ye-hyaluronic;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;MRM, iliso lokusabela ngeendlela ezininzi.
Umzobo we-4 uhambelana ne-chromatogram ye-LC-MS yesampuli yezinto ezisetyenziswayo kwi-cross-linking reaction ye-HA kunye ne-BDDE.
Qaphela: Zonke ezi zilinganiselwa kugxininiso olufanayo kunye nomlinganiselo osetyenziswayo ukwenza ukusabela kokuhlangana.Amanani ezinto ezibonakalayo ezihlalutywe yichromatogram zihambelana ne: (1) amanzi, (2) 2% HA Isisombululo se-aqueous, (3) 1% i-NaOH isisombululo se-aqueous.Uhlalutyo lwe-LC-MS lwenziwa ukuguqulwa kobuninzi be-203.30 / 129.10 Da (kwimodi ye-MRM enhle).
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;HA, iasidi ye-hyaluronic;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;MRM, iliso lokusabela ngeendlela ezininzi.
Iimeko ezakhokelela ekusekweni kweencopho ezintsha zaphononongwa.Ukuze ufunde indlela iimeko zokuphendula ezisetyenziselwa ukuvelisa i-HA hydrogel edibeneyo edibeneyo ichaphazela ukuphinda kusebenze i-agent ye-BDDE yokudibanisa, ekhokelela ekubunjweni kweencopho ezintsha (imveliso enokwenzeka), imilinganiselo eyahlukeneyo yenziwa.Kule migqaliselo, sifunde kwaye sihlalutya i-crosslinker ye-BDDE yokugqibela, eyaphathwa ngeendlela ezahlukeneyo ze-NaOH (0%, 1%, 0.1%, kunye ne-0.01%) kwi-medium medium, elandelwa okanye ngaphandle kwe-autoclaving.Inkqubo yebhaktheriya yokulinganisa iimeko ezifanayo iyafana nendlela esetyenziselwa ukuvelisa i-HA hydrogel edibeneyo.Njengoko kuchazwe kwicandelo elithi "Izinto kunye neendlela", ukuguqulwa kobuninzi besampuli kwahlalutywa yi-LC-MS ukuya kwi-203.30 / 129.10 Da.I-BDDE kunye noxinzelelo lwencopho entsha ibalwa, kwaye iziphumo ziboniswe kwiThebhile 3. Akukho zincomo ezintsha zifunyenwe kwiisampuli ezingazange zifakwe kwi-autoclaved, kungakhathaliseki ukuba khona kwe-NaOH kwisisombululo (iisampulu 1-4, iThebhile 3).Kwiisampuli ze-autoclaved, iincopho ezintsha zibonwa kuphela phambi kwe-NaOH kwisisombululo, kwaye ukubunjwa kwe-peak kubonakala kuxhomekeke kwi-concentration ye-NaOH kwisisombululo (iisampulu 5-8, iThebhile 3) (RRT = 0.79).Umzobo we-5 ubonisa umzekelo we-chromatogram ye-ion, ebonisa iisampuli ezimbini ze-autoclaved phambi okanye ukungabikho kwe-NAOH.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;MRM, iliso lokusabela ngeendlela ezininzi.
Qaphela: I-chromatogram ephezulu: Isampuli yaphathwa nge-0.1% ye-NaOH isisombululo se-aqueous kunye ne-autoclaved (120 ° C imizuzu ye-20).Ikhromatogram esezantsi: Isampuli ayizange iphathwe nge-NaOH, kodwa i-autoclaved phantsi kweemeko ezifanayo.Ukuguqulwa kobuninzi be-203.30 / 129.10 Da (kwimodi ye-MRM encomekayo) yahlalutywa yi-LC-MS.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;MRM, iliso lokusabela ngeendlela ezininzi.
Kuzo zonke iisampuli ze-autoclaved, kunye okanye ngaphandle kwe-NaOH, i-BDDE igxininiso yancitshiswa kakhulu (ukuya kumaxesha e-16.6) (iisampulu 5-8, iThebhile 2).Ukuncipha kwi-concentration ye-BDDE ingaba ngenxa yokuba kwiqondo lokushisa eliphezulu, amanzi anokusebenza njengesiseko (nucleophile) ukuvula i-epoxide ring ye-BDDE ukwenza i-1,2-diol compound.Umgangatho we-monoisotopic wale khompawundi uhluke kwi-BDDE kwaye ngoko awuyi kuchaphazeleka.I-LC-MS ifumene utshintsho olukhulu lwe-203.30 / 129.10 Da.
Ekugqibeleni, ezi zilingo zibonisa ukuba ukuveliswa kweencopho ezintsha kuxhomekeke kubukho beBDDE, NAOH, kunye nenkqubo ye-autoclaving, kodwa ayinanto yokwenza ne-HA.
Incopho entsha efunyenwe ngexesha lokugcinwa malunga nemizuzu ye-2.71 emva koko ibonakaliswe yi-LC-MS.Ngenxa yale njongo, i-BDDE (9.9 mg / mL) ifakwe kwi-1% ye-NaOH isisombululo samanzi kunye ne-autoclaved.Kwitheyibhile ye-4, iimpawu zencopho entsha zifaniswa ne-BDDE yereferensi eyaziwayo (ixesha lokugcinwa malunga nemizuzu ye-3.47).Ngokusekelwe kuhlalutyo lwe-ion lokuqhekeka kweengqungquthela ezimbini, kunokugqitywa ukuba i-peak kunye nexesha lokugcinwa kwemizuzu ye-2.72 ibonisa iziqwenga ezifanayo kunye ne-BDDE peak, kodwa ngamandla ahlukeneyo (Umfanekiso 6).Kwincopho ehambelana nexesha lokugcinwa (PIS) yemizuzu ye-2.72, i-peak eqinile yabonwa emva kokuhlukana kwi-mass ye-147 Da.Kwi-concentration ye-BDDE (9.9 mg / mL) esetyenziswe kule sigqibo, iindlela ezahlukeneyo zokuxhamla (UV, λ = 200 nm) kwi-spectrum ye-ultraviolet nazo zabonwa emva kokuhlukana kwe-chromatographic (Umfanekiso 7).Incopho kunye nexesha lokugcinwa kwemizuzu ye-2.71 isabonakala kwi-200 nm, ngelixa i-BDDE i-peak ayinakubonwa kwi-chromatogram phantsi kweemeko ezifanayo.
Itheyibhile ye-4 yeempawu zeempawu zencopho entsha kunye nexesha lokugcinwa malunga nemizuzu ye-2.71 kunye ne-BDDE ephezulu kunye nexesha lokugcinwa kwemizuzu ye-3.47
Qaphela: Ukufumana ezi ziphumo, uhlalutyo lwe-LC-MS ne-HPLC (MRM nePIS) lwenziwa kwiincopho ezimbini.Uhlalutyo lwe-HPLC, ukubonwa kwe-UV kunye nobude be-200 nm kusetyenziswa.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-HPLC, ukusebenza okuphezulu kwechromatography yolwelo;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;I-MRM, ukujongwa kweempendulo ezininzi;m/z, umlinganiselo wobunzima ukuya kwintlawulo;PIS, imveliso Ion ukuskena;ukukhanya kwe-ultraviolet, ukukhanya kwe-ultraviolet.
Qaphela: Amaqhekeza obunzima afunyanwa ngohlalutyo lwe-LC-MS (PIS).Ikhromatogram ephezulu: i-spectrum yobunzima be-BDDE amaqhekeza eisampulu eziqhelekileyo.Ikhromatogram esezantsi: Ubume bobunzima bencopho entsha ichongiwe (i-RRT ehambelana nencopho yeBDDE yi-0.79).I-BDDE iqhutywe kwi-1% yesisombululo se-NaOH kunye ne-autoclaved.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography yolwelo kunye ne-mass spectrometry;I-MRM, ukujongwa kweempendulo ezininzi;PIS, imveliso ion scan;I-RRT, ixesha lokugcina elihambelanayo.
Umzobo we-7 Ion chromatogram ye-203.30 Da precursor ion, kunye (A) i-peak entsha kunye nexesha lokugcinwa kwemizuzu ye-2.71 kunye (B) ukufunyanwa kwe-UV ye-BDDE yereferensi yomgangatho ophezulu kwi-3.46 imizuzu kwi-200 nm.
Kuzo zonke i-HA hydrogels edibeneyo edibeneyo edibeneyo eveliswe, kwabonwa ukuba i-BDDE i-concentration eseleyo emva kokuba i-LC-MS ye-quantification yayingu- <2 ppm, kodwa i-peak entsha engaziwayo ibonakala kuhlalutyo.Le ncopho intsha ayihambelani nemveliso eqhelekileyo yeBDDE.Imveliso esemgangathweni ye-BDDE iphinde yafumana ukuguqulwa komgangatho ofanayo (ukuguqulwa kwe-MRM 203.30 / 129.10 Da) uhlalutyo kwimodi ye-MRM efanelekileyo.Ngokuqhelekileyo, ezinye iindlela zokuhlalutya ezifana ne-chromatography zisetyenziswa njengeemvavanyo zomda ukufumanisa i-BDDE kwi-hydrogels, kodwa umda wokufumanisa ubuninzi (LOD) ungaphantsi kancinci kune-2 ppm.Ngakolunye uhlangothi, ukuza kuthi ga ngoku, i-NMR kunye ne-MS zisetyenziselwe ukubonakalisa i-degree of cross-linking kunye / okanye ukuguqulwa kwe-HA kwiinqununu zeyunithi yeshukela yeemveliso ze-HA ezidibeneyo.Injongo yolu buchule ayizange ibe kukulinganisa ukufunyanwa kwe-BDDE okushiyekileyo kwiindawo eziphantsi njengoko sichaza kweli nqaku (LOD ye-LC-MS yethu indlela = 10 ppb).
Ixesha lokuposa: Sep-01-2021