I-Javascript ivaliwe esipheqululini sakho okwamanje.Uma i-javascript ivaliwe, eminye imisebenzi yale webhusayithi ngeke isebenze.
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 * Laba babhali banemininingwane ethile ngalo msebenzi Equal isizinda somnikelo: I-Hyaluronic acid (HA) iyi-polysaccharide eyenzeka ngokwemvelo esetshenziswa ekukhiqizeni izigcwalisi zesikhumba ngezinjongo zobuhle.Njengoba inengxenye yempilo yezinsuku ezimbalwa ezicutshini zomuntu, ama-HA-based dermal fillers aguqulwa ngamakhemikhali ukuze andise impilo yawo emzimbeni.Ukuguqulwa okuvame kakhulu ku-HA-based fillers yezentengiselwano ukusetshenziswa kwe-1,4-butanediol diglycidyl ether (BDDE) njenge-agent exhumanisa ukuphambanisa amaketanga e-HA.I-BDDE eyinsalela noma engaphenduliwe ibhekwa njengengeyona into enobuthi ezingxenyeni ezingu-<2 ngesigidi (ppm);ngakho-ke, i-BDDE esele ku-dermal filler yokugcina kufanele ilinganiswe ukuze kuqinisekiswe ukuphepha kwesiguli.Izinto kanye nezindlela: Lolu cwaningo luchaza ukutholwa kanye nokuchazwa komkhiqizo womphumela wokuxhumanisa phakathi kwe-BDDE ne-HA ngaphansi kwezimo ze-alkaline ngokuhlanganisa i-chromatography ewuketshezi kanye ne-mass spectrometry (LC-MS).Imiphumela: Ngemva kokuhlaziywa okuhlukile, kwatholakala ukuthi izimo ze-alkaline kanye nokushisa okuphezulu okusetshenziselwa ukukhipha amagciwane i-HA-BDDE hydrogel kukhuthaze ukwakheka kwalo mkhiqizo omusha, i-compound "propylene glycol-like".Ukuhlaziywa kwe-LC-MS kuqinisekise ukuthi umkhiqizo womkhiqizo unesisindo esifanayo se-monoisotopic njenge-BDDE, isikhathi sokugcinwa esihlukile (tR), kanye nemodi ehlukile yokumunca i-UV (λ=200 nm).Ngokungafani ne-BDDE, kubonwe ekuhlaziyweni kwe-LC-MS ukuthi ngaphansi kwezimo ezifanayo zokulinganisa, lo mkhiqizo unenani eliphakeme lokutholwa ku-200 nm.Isiphetho: Le miphumela ibonisa ukuthi ayikho i-epoxide esakhiweni sale nhlanganisela entsha.Ingxoxo ivulekele ukuhlola ubungozi balo mkhiqizo omusha otholakala ekukhiqizweni kwe-HA-BDDE hydrogel (HA dermal filler) ngokwezinjongo zokuthengisa.Amagama angukhiye: i-hyaluronic acid, i-HA dermal filler, i-hyaluronic acid ehlangene, i-BDDE, i-LC-MS analysis, i-BDDE ngomkhiqizo.
Izigcwalisi ezisuselwe ku-hyaluronic acid (HA) zingama-dermal fillers ajwayelekile futhi adumile asetshenziselwa izinjongo zezimonyo.1 Le dermal filler iyi-hydrogel, ngokuvamile eyakhiwa > amanzi angama-95% kanye no-0.5-3% HA, okubanikeza isakhiwo esifana nejeli.I-2 HA iyi-polysaccharide futhi iyingxenye eyinhloko ye-matrix ye-extracellular yezilwane ezinomgogodla.Esinye sezithako.Iqukethe (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) ephindaphinda amayunithi e-disaccharide axhunywe ngamabhondi e-glycosidic.Le phethini ye-disaccharide iyafana kuzo zonke izinto eziphilayo.Uma kuqhathaniswa namanye ama-filler asekelwe kumaprotheni (njenge-collagen), lesi sakhiwo senza i-HA ibe yi-molecule ehambisana kakhulu ne-biocompatible.Lezi zigcwalisi zingabonisa ukulandelana kwe-amino acid okungase kuqashelwe amasosha omzimba esiguli.
Uma isetshenziswa njenge-dermal filler, umkhawulo oyinhloko we-HA ukuguqulwa kwayo ngokushesha ngaphakathi kwezicubu ngenxa yokuba khona komndeni othize wama-enzyme okuthiwa ama-hyaluronidases.Kuze kube manje, ukuguqulwa kwamakhemikhali amaningana esakhiweni se-HA kuye kwachazwa ukwandisa isigamu sempilo ye-HA ezicutshini.3 Iningi lalezi zinguquko zizama ukunciphisa ukufinyelela kwe-hyaluronidase kuma-polysaccharide polymers ngokuxhumanisa amaketanga e-HA.Ngakho-ke, ngenxa yokwakhiwa kwamabhuloho kanye nezibopho ze-intermolecular covalent phakathi kwesakhiwo se-HA kanye ne-agent yokuxhumanisa, i-HA hydrogel exhunywe ngokuphambana ikhiqiza imikhiqizo eminingi yokuwohloka kwe-enzyme kune-HA yemvelo.4-6
Kuze kube manje, amakhemikhali e-crosslinking asetshenziswa ukukhiqiza i-HA exhumene ahlanganisa i-methacrylamide, i-7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) Kanye ne-poly(ethylene glycol) diglycidyl ether.10 ,11 I-BDDE okwamanje iyi-ejenti yokuxhumanisa esetshenziswa kakhulu.Nakuba lezi zinhlobo zama-hydrogel ziye zafakazelwa ukuthi ziphephile amashumi eminyaka, ama-crosslinking agents asetshenziswayo ama-reagents asebenzayo angase abe i-cytotoxic futhi, kwezinye izimo, i-mutagenic.12 Ngakho-ke, okuqukethwe kwabo okuyinsalela ku-hydrogel yokugcina kufanele kube phezulu.I-BDDE ibhekwa njengephephile uma ukugxiliswa kwensalela kungaphansi kwezingxenye ezingu-2 ngesigidi (ppm).4
Kunezindlela ezimbalwa zokubona ukugxilisa kwe-BDDE enensalela ephansi, idigri yokuxhumanisa kanye nendawo yokushintsha kuma-HA hydrogels, njenge-gas chromatography, i-chromatography engabandakanyi usayizi ehlanganiswe ne-mass spectrometry (MS), izindlela zokulinganisa i-nuclear magnetic resonance (NMR) fluorescence, kanye I-Diode array ihlanganiswe ne-high performance liquid chromatography (HPLC).I-13-17 Lolu cwaningo luchaza ukutholwa kanye nokubonakaliswa komkhiqizo ku-HA hydrogel yokugcina exhunywe ngokuphambana ekhiqizwa ukusabela kwe-BDDE ne-HA ngaphansi kwezimo ze-alkali.I-HPLC kanye ne-liquid chromatography-mass spectrometry (ukuhlaziywa kwe-LC-MS).Njengoba ubuthi balo mkhiqizo we-BDDE bungaziwa, sincoma ukuthi ukulinganisa kwayo okusele kufanele kunqunywe ngendlela efanayo nendlela evame ukwenziwa ku-BDDE emkhiqizweni wokugcina.
Usawoti we-sodium otholiwe we-HA (Shiseido Co., Ltd., Tokyo, Japan) unesisindo se-molecular ~ 1,368,000 Da (indlela kaLaurent) 18 kanye ne-viscosity yangaphakathi engu-2.20 m3 / kg.Ngokusabela kokuxhumanisa, i-BDDE (≥95%) ithengwe kwa-Sigma-Aldrich Co. (St. Louis, MO, USA).I-Phosphate buffered saline ene-pH 7.4 ithengwe kwa-Sigma-Aldrich Company.Zonke izincibilikisi, i-acetonitrile namanzi asetshenziswe ekuhlaziyeni kwe-LC-MS kwathengwa ngekhwalithi yebanga le-HPLC.I-Formic acid (98%) ithengwa njenge-reagent grade.
Konke ukuhlola kwenziwa kuhlelo lwe-UPLC Acquity (Amanzi, Milford, MA, USA) futhi kwaxhunywa ku-API 3000 triple quadrupole mass spectrometer efakwe umthombo we-ionization we-electrospray (AB SCIEX, Framingham, MA, USA).
Ukuhlanganiswa kwama-HA hydrogel axhumene nesiphambano kwaqalwa ngokungeza i-198 mg ye-BDDE kusixazululo esingu-10% (w/w) se-sodium hyaluronate (NaHA) phambi kwe-1% alkali (sodium hydroxide, NaOH).Ukugxila kokugcina kwe-BDDE kungxube yokusabela kube ngu-9.9 mg/mL (0.049 mM).Khona-ke, ingxube yokusabela ixutshwe kahle futhi i-homogenized futhi ivunyelwe ukuqhubeka ku-45 ° C amahora angu-4.19 I-pH yokusabela igcinwa ku-~12.
Ngemva kwalokho, ingxube yokusabela yagezwa ngamanzi, futhi i-HA-BDDE hydrogel yokugcina yahlungwa futhi yahlanjululwa nge-PBS buffer ukuze kuzuzwe ukuhlushwa kwe-HA kwe-10 kuya ku-25 mg / mL, kanye ne-pH yokugcina ye-7.4.Ukuze kufakwe amagciwane ama-HA hydrogel akhiqiziwe axhumene, wonke lawa ma-hydrogel afakwa ngokuzenzakalelayo (120°C imizuzu engu-20).I-hydrogel ye-BDDE-HA ehlanziwe igcinwa ku-4°C kuze kube yilapho ihlaziywa.
Ukuhlaziya i-BDDE ekhona emkhiqizweni we-HA oxhumene ngokuphambana, isampula engu-240 mg yalinganiswa futhi yafakwa emgodini ophakathi nendawo (Microcon®; Merck Millipore, Billerica, MA, USA; ivolumu 0.5 mL) futhi yafakwa i-centrifuged ku-10,000 rpm ekamelweni lokushisa. Imizuzu eyi-10.Isamba esingu-20 µL soketshezi lokudonsela phansi saqoqwa futhi sahlaziywa.
Ukuze uhlaziye izinga le-BDDE (Sigma-Aldrich Co) ngaphansi kwezimo ze-alkaline (1%, 0.1% kanye no-0.01% NaOH), uma izimo ezilandelayo zihlangatshezwana nazo, isampula yoketshezi ngu-1:10, 1:100, noma kufika ku- 1:1,000,000 Uma kudingeka, sebenzisa amanzi enziwe nge-MilliQ deionized ukuze uhlaziye.
Kuzinto zokuqala ezisetshenziswa ekuphenduleni kokuxhumanisa (HA 2%, H2O, 1% NaOH, kanye ne-0.049 mM BDDE), u-1 mL wesampula ngayinye elungiselelwe kusukela kulezi zinsiza iye yahlaziywa kusetshenziswa izimo ezifanayo zokuhlaziya.
Ukuze unqume ukucaciswa kweziqongo ezivela kumephu ye-ion, u-10 µL wesixazululo esijwayelekile esingu-100 ppb BDDE (Sigma-Aldrich Co) sengezwe kusampula engu-20 µL.Kulokhu, ukugxila kokugcina kwezinga kusampula ngayinye kungu-37 ppb.
Okokuqala, lungiselela isisombululo sesitoko se-BDDE esine-concentration ye-11,000 mg/L (11,000 ppm) ngokuhlambulula u-10 μL we-BDDE evamile (Sigma-Aldrich Co) ngamanzi angu-990 μL MilliQ (ukuminyana okungu-1.1 g/mL).Sebenzisa lesi sixazululo ukuze ulungiselele isisombululo se-BDDE esingu-110 µg/L (110 ppb) njengokuhlanjululwa okujwayelekile okumaphakathi.Bese, sebenzisa i-diluent evamile ye-BDDE emaphakathi (110 ppb) ukuze ulungise ijika elijwayelekile ngokuhlambulula i-diluent emaphakathi izikhathi ezimbalwa ukuze ufinyelele ukugxilisa okufiselekayo okungu-75, 50, 25, 10, kanye no-1 ppb.Njengoba kuboniswe kuMfanekiso 1, kutholakala ukuthi ijika elijwayelekile le-BDDE ukusuka ku-1.1 ukuya ku-110 ppb linomugqa omuhle (R2>0.99).Ijika elijwayelekile liphindwe ezivivinyweni ezine ezizimele.
Umfanekiso 1 Ijika lokulinganisa elijwayelekile le-BDDE elitholwe ukuhlaziywa kwe-LC-MS, lapho kuqashelwa khona ukuhlobana okuhle (R2>0.99).
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry.
Ukuze kuhlonzwe futhi kulinganise amazinga e-BDDE akhona ku-HA exhunywe ngokuphambana namazinga e-BDDE esixazululweni sesisekelo, kusetshenziswe ukuhlaziywa kwe-LC-MS.
Ukuhlukaniswa kwe-chromatographic kufinyelelwe kukholomu ye-LUNA 2.5 µm C18(2)-HST (50×2.0 mm2; Phenomenex, Torrance, CA, USA) futhi igcinwe ekamelweni lokushisa (25°C) phakathi nokuhlaziywa.Isigaba seselula siqukethe i-acetonitrile (i-solvent A) namanzi (i-solvent B) equkethe u-0.1% we-formic acid.Isigaba seselula sivezwa ukugqama kwe-gradient.I-gradient imi kanje: imizuzu engu-0, 2% A;1 iminithi, 2% A;6 amaminithi, 98% A;7 amaminithi, 98% A;7.1 imizuzu, 2% A;Imizuzu eyi-10 , 2% A. Isikhathi sokusebenza imizuzu eyi-10 kanti umthamo womjovo ngu-20 µL.Isikhathi sokugcina se-BDDE cishe imizuzu engu-3.48 (kusukela kumaminithi angu-3.43 kuye kwangu-4.14 ngokusekelwe ekuhlolweni).Isigaba seselula sampontshwa ngezinga lokugeleza elingu-0.25 mL/min ukuze kuhlaziywe i-LC-MS.
Ukuhlaziywa kwe-BDDE nokulinganisa nge-MS, uhlelo lwe-UPLC (Amanzi) luhlanganiswe ne-API 3000 triple quadrupole mass spectrometer (AB SCIEX) efakwe umthombo we-ionization we-electrospray, futhi ukuhlaziya kwenziwa ngemodi ye-ion enhle (ESI+).
Ngokusho kokuhlaziywa kwe-ion fragment okwenziwa ku-BDDE, ucezu olunomfutho ophakeme kakhulu lunqunywe ukuthi luyingxenye ehambisana ne-129.1 Da (Umfanekiso 6).Ngakho-ke, kumodi yokuqapha i-multi-ion (MIM) yokulinganisa, ukuguqulwa kwenqwaba (isilinganiso sobuningi bokushaja [m/z]) se-BDDE ngu-203.3/129.1 Da.Iphinde isebenzisa imodi yokuskena okugcwele (FS) kanye nemodi ye-ion yomkhiqizo (PIS) yokuhlaziya i-LC-MS.
Ukuze kuqinisekiswe ukucaciswa kwendlela, isampuli engenalutho (isigaba sokuqala seselula) sahlaziywa.Ayikho isignali etholiwe kusampula engenalutho ngokuguqulwa okukhulu kwe-203.3 / 129.1 Da.Ngokuphathelene nokuphindaphinda kokuhlolwa, imijovo evamile ye-10 ye-55 ppb (maphakathi nejika lokulinganisa) yahlaziywa, okuholele ekuphambukeni okujwayelekile okusele (RSD) <5% (idatha engaboniswa).
Okuqukethwe kwe-BDDE okuyinsalela kwalinganiswa ngama-HA hydrogel ayisishiyagalombili ahlukene e-BDDE axhumene ngokuphambanayo, ahambisana nokuhlolwa okune okuzimele.Njengoba kuchazwe esigabeni "Izinto Nezindlela", ukulinganisa kuhlolwa inani elimaphakathi lejika lokuhlehla lokuhlanjululwa okujwayelekile kwe-BDDE, elihambisana nenani eliphakeme eliyingqayizivele elitholwe ekushintsheni kwenqwaba ye-BDDE ye-203.3/129.1 Da, enokugcinwa isikhathi esisuka ku-3.43 ukuya ku-4.14 amaminithi Alindile.Umfanekiso 2 ubonisa isibonelo sechromatogram yezinga eliyinkomba le-10 ppb BDDE.Ithebula 1 lifingqa okuqukethwe okusele kwe-BDDE kwama-hydrogel ayisishiyagalombili ahlukene.Ububanzi bevelu bungu-1 kuye ku-2.46 ppb.Ngakho-ke, ukugxila kwe-BDDE okuyinsalela kusampula kuyamukeleka ukuze kusetshenziswe abantu (<2 ppm).
Umfanekiso 2 I-chromatogram ye-Ion ye-10 ppb BDDE inkomba ejwayelekile (Sigma-Aldrich Co), inguquko ye-MS (m/z) etholwe ukuhlaziya kwe-LC-MS kwe-203.30/129.10 Da (ngemodi ye-MRM enhle).
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha ukusabela okuningi;MS, inqwaba;m/z, isilinganiso somthamo wokushaja.
Qaphela: Amasampula 1-8 angama-autoclaved BDDE cross-linked HA hydrogel.Inani eliyinsalela le-BDDE ku-hydrogel kanye nenani eliphakeme lesikhathi sokugcinwa kwe-BDDE nakho kuyabikwa.Okokugcina, ukuba khona kweziqongo ezintsha ezinezikhathi ezihlukene zokugcinwa nakho kuyabikwa.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-HA, i-hyaluronic acid;I-MRM, ukuqapha ukusabela okuningi;tR, isikhathi sokugcina;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-RRT, isikhathi sokugcina esihlobene.
Ngokumangalisayo, ukuhlaziya kwe-LC-MS ion chromatogram kubonise ukuthi ngokusekelwe kuwo wonke amasampula e-HA hydrogel axhumene ne-autoclaved cross-linked ahlaziywa, kube nenani eliphakeme elengeziwe ngesikhathi esifushane sokugcinwa kwemizuzu engu-2.73 kuya ku-3.29.Isibonelo, Umfanekiso 3 ubonisa i-ion chromatogram yesampula ye-HA exhunywe ngokuphambanayo, lapho ukuphakama okwengeziwe kuvela ngesikhathi esihlukile sokugcina cishe imizuzu engu-2.71.Isikhathi sokugcinwa esihlobene (i-RRT) esiphawuliwe phakathi kwesiqongo esisanda kubonwa kanye nesiqongo esivela ku-BDDE sitholwe siyi-0.79 (Ithebula 1).Njengoba sazi ukuthi inani eliphakeme elisanda kubukwa aligcinwa kancane kukholamu ye-C18 esetshenziswe ekuhlaziyeni kwe-LC-MS, ukuphakama okusha kungase kuhambisane nenhlanganisela ye-polar eyengeziwe kune-BDDE.
Umfanekiso we-3 Ion chromatogram yesampula ye-HA hydrogel exhunywe ngokuphambanayo etholwe yi-LC-MS (ukuguqulwa kwesisindo se-MRM 203.3/129.0 Da).
Izifinyezo: HA, i-hyaluronic acid;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha ukusabela okuningi;I-RRT, isikhathi sokugcina esihlobene;tR, isikhathi sokugcina.
Ukuze kukhishwe ukuthi kungenzeka ukuthi iziqongo ezintsha ezibonwayo zingase zibe ukungcola okukhona ekuqaleni ezintweni ezingavuthiwe ezisetshenziswayo, lezi zinto zokusetshenziswa nazo zahlaziywa kusetshenziswa indlela efanayo yokuhlaziya i-LC-MS.Izinto zokuqala ezihlaziywayo zihlanganisa amanzi, i-2% NaHA emanzini, i-1% NaOH emanzini, kanye ne-BDDE ekugxilweni okufanayo okusetshenziselwa ukusabela kokuxhumanisa.I-ion chromatogram yento yokuqala esetshenzisiwe ayizange ibonise noma iyiphi inhlanganisela noma ukuphakama, futhi isikhathi sokugcinwa kwayo sihambisana nenani eliphakeme elisha eliphawuliwe.Leli qiniso liyawulahla umqondo wokuthi akuyona nje into yokuqala engase iqukethe noma yiziphi izinhlanganisela noma izinto ezingase ziphazamise inqubo yokuhlaziya, kodwa alukho uphawu lokungcoliswa okungaba khona kweminye imikhiqizo yaselabhorethri.Amanani okugxila atholwe ngemva kokuhlaziywa kwe-LC-MS ye-BDDE neziqongo ezintsha ziboniswa kuThebula 2 (amasampula 1-4) kanye ne-ion chromatogram kuMfanekiso 4.
Qaphela: Amasampula 1-4 ahambisana nezinto ezingavuthiwe ezisetshenziselwa ukukhiqiza ama-hydrogel e-HA axhumene ngokuzenzakalelayo e-BDDE.Lawa masampuli awazange afakwe ngokuzenzakalelayo.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-HA, i-hyaluronic acid;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha kokusabela okuningi.
Umfanekiso wesi-4 uhambisana ne-chromatogram ye-LC-MS yesampula yezinto ezingavuthiwe ezisetshenziswe ekuphenduleni kokuxhumanisa kwe-HA ne-BDDE.
Qaphela: Konke lokhu kukalwa ngesilinganiso esifanayo nesilinganiso esisetshenziselwa ukwenza ukusabela kokuxhumanisa.Izinombolo zezinto zokusetshenziswa ezihlaziywa yichromatogram zihambisana nalokhu: (1) amanzi, (2) 2% HA isixazululo esinamanzi, (3) 1% isixazululo esinamanzi se-NaOH.Ukuhlaziywa kwe-LC-MS kwenzelwa ukuguqulwa kwenqwaba ye-203.30/129.10 Da (ngemodi ye-MRM enhle).
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-HA, i-hyaluronic acid;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha kokusabela okuningi.
Izimo ezaholela ekwakhekeni kweziqongo ezintsha zacwaningwa.Ukuze ufunde ukuthi izimo zokusabela ezisetshenziselwa ukukhiqiza i-HA hydrogel exhunywe esiphambanweni zithinta kanjani ukusebenza kabusha kwe-agent ye-BDDE yokuxhumanisa, okuholela ekwakhekeni kweziqongo ezintsha (imikhiqizo engenzeka), izilinganiso ezahlukene zenziwa.Kulezi zinqumo, safunda futhi sahlaziya i-crosslinker yokugcina ye-BDDE, ephathwe ngokugxila okuhlukile kwe-NaOH (0%, 1%, 0.1%, kanye no-0.01%) endaweni enamanzi, elandelwa noma ngaphandle kwe-autoclaving.Inqubo yebhaktheriya yokulingisa izimo ezifanayo iyafana nendlela esetshenziswa ukukhiqiza i-HA hydrogel exhumene.Njengoba kuchazwe esigabeni esithi "Izinto Nezindlela", ukuguqulwa kwenqwaba yesampula kwahlaziywa yi-LC-MS kuya ku-203.30 / 129.10 Da.I-BDDE kanye nokugxila kwenani eliphakeme elisha kubalwa, futhi imiphumela ikhonjiswe kuThebula 3. Awekho amaqophelo amasha atholwe kumasampula angazange afakwe ngokuzenzakalelayo, kungakhathaliseki ukuba khona kwe-NaOH kusixazululo (amasampula 1-4, Ithebula. 3).Kumasampula e-autoclaved, iziqongo ezintsha zitholwa kuphela lapho kukhona i-NaOH esixazululweni, futhi ukwakheka kokuphakama kubonakala kuncike ekugxilweni kwe-NaOH esixazululweni (amasampuli 5-8, Ithebula 3) (RRT = 0.79).Umfanekiso wesi-5 ubonisa isibonelo se-ion chromatogram, ebonisa amasampula amabili e-autoclaved ekhona noma engekho i-NAOH.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha kokusabela okuningi.
Qaphela: I-chromatogram ephezulu: Isampula liphathwe ngesisombululo se-aqueous se-NaOH esingu-0.1% kanye ne-autoclaved (120°C imizuzu engu-20).I-chromatogram engezansi: Isampula ayizange iphathwe nge-NaOH, kodwa i-autoclaved ngaphansi kwezimo ezifanayo.Ukuguqulwa kwenqwaba ye-203.30 / 129.10 Da (kwimodi ye-MRM enhle) kwahlaziywa yi-LC-MS.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha kokusabela okuningi.
Kuwo wonke amasampula e-autoclaved, nge-NaOH noma ngaphandle kwayo, ukugxila kwe-BDDE kuye kwancishiswa kakhulu (kufika ezikhathini ezingu-16.6) (amasampuli 5-8, Ithebula 2).Ukuncipha kokuhlushwa kwe-BDDE kungase kube ngenxa yokuthi emazingeni okushisa aphezulu, amanzi angenza njengesisekelo (i-nucleophile) ukuvula indandatho ye-epoxide ye-BDDE ukuze enze i-1,2-diol compound.Ikhwalithi ye-monoisotopic yale nhlanganisela ihlukile kuleyo ye-BDDE ngakho-ke ngeke ithinteke.I-LC-MS ithole ukushintshwa okukhulu kwe-203.30/129.10 Da.
Okokugcina, lezi zivivinyo zibonisa ukuthi ukukhiqizwa kweziqongo ezintsha kuncike ebukhoneni be-BDDE, NAOH, kanye nenqubo ye-autoclaving, kodwa akuhlangene ne-HA.
Inani eliphakeme elisha elitholwe ngesikhathi sokugcinwa cishe imizuzu eyi-2.71 labe selibonakala nge-LC-MS.Ngale njongo, i-BDDE (9.9 mg/mL) yafakwa kusixazululo samanzi se-NaOH esingu-1% futhi yafakwa ngokuzenzakalelayo.Kuthebula lesi-4, izici zenani eliphakeme elisha ziqhathaniswa nereferensi eyaziwayo ye-BDDE (isikhathi sokugcina cishe imizuzu engu-3.47).Ngokusekelwe ekuhlaziyweni kokuhlukaniswa kwe-ion kweziqongo ezimbili, kungaphethwa ngokuthi inani eliphakeme elinesikhathi sokugcina semizuzu engu-2.72 libonisa izingcezu ezifanayo nenani eliphakeme le-BDDE, kodwa ngokuqina okuhlukile (Umfanekiso 6).Ngenani eliphakeme elihambisana nesikhathi sokugcina (PIS) samaminithi angu-2.72, inani eliphakeme kakhulu labonwa ngemva kokuhlukaniswa ngenqwaba ye-147 Da.Ekugxilweni kwe-BDDE (9.9 mg/mL) okusetshenziswe kulokhu kunqunywa, izindlela zokumunca ezahlukene (UV, λ=200 nm) ku-ultraviolet spectrum nazo zabonwa ngemva kokuhlukaniswa kwe-chromatographic (Umfanekiso 7).Isilinganiso esiphezulu esinesikhathi sokugcina semizuzu engu-2.71 sisabonakala ku-200 nm, kuyilapho ukuphakama kwe-BDDE akukwazi ukubonwa ku-chromatogram ngaphansi kwezimo ezifanayo.
Ithebula lesi-4 Imiphumela yezinhlamvu zenani eliphakeme elisha elinesikhathi sokugcina cishe imizuzu engu-2.71 kanye nenani eliphakeme le-BDDE nesikhathi sokugcina esiyimizuzu engu-3.47
Qaphela: Ukuze uthole le miphumela, ukuhlaziya kwe-LC-MS ne-HPLC (MRM ne-PIS) kwenziwe eziqongweni ezimbili.Ukuze kuhlaziywe i-HPLC, kusetshenziswa ukutholwa kwe-UV enobude obungamaza obungama-200 nm.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-HPLC, ukusebenza okuphezulu kwe-chromatography ye-liquid;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha ukusabela okuningi;m/z, isilinganiso somthamo wokushaja;I-PIS, ukuskena kwe-Ion yomkhiqizo;ukukhanya kwe-ultraviolet, ukukhanya kwe-ultraviolet.
Qaphela: Izingcezu ezinkulu zitholwa ngokuhlaziywa kwe-LC-MS (PIS).I-chromatogram ephezulu: i-mass spectrum yezingcezu zesampula ezijwayelekile ze-BDDE.I-chromatogram engezansi: I-spectrum enkulu yenani eliphakeme elisha litholiwe (i-RRT ehlotshaniswa nokuphakama kwe-BDDE ngu-0.79).I-BDDE icutshungulwe ngesixazululo esingu-1% se-NaOH futhi yafakwa ngokuzenzakalelayo.
Izifinyezo: BDDE, 1,4-butanediol diglycidyl ether;I-LC-MS, i-chromatography ewuketshezi kanye ne-mass spectrometry;I-MRM, ukuqapha ukusabela okuningi;I-PIS, ukuskena kwe-ion yomkhiqizo;I-RRT, isikhathi sokugcina esihlobene.
Umfanekiso 7 Ion chromatogram ye-203.30 Da precursor ion, kanye (A) nenani eliphakeme elisha elinesikhathi sokugcina samaminithi angu-2.71 kanye (B) nokutholwa kwe-UV kwereferensi evamile ye-BDDE kumaminithi angu-3.46 ku-200 nm.
Kuwo wonke ama-HA hydrogel axhumene nesiphambano akhiqizwayo, kwaphawulwa ukuthi ukugxilwa kwe-BDDE okusele ngemuva kokulinganisa kwe-LC-MS kwaba <2 ppm, kodwa inani eliphakeme elisha elingaziwa livele ekuhlaziyeni.Lesi siqongo esisha asifani nomkhiqizo ojwayelekile we-BDDE.Umkhiqizo ojwayelekile we-BDDE uphinde wabhekana nokuguqulwa kwekhwalithi efanayo (ukuguqulwa kwe-MRM 203.30/129.10 Da) kumodi ye-MRM enhle.Ngokuvamile, ezinye izindlela zokuhlaziya ezifana ne-chromatography zisetshenziswa njengokuhlolwa komkhawulo ukuze kutholwe i-BDDE kuma-hydrogel, kodwa umkhawulo wokuthola omkhulu (LOD) ungaphansi kancane kuno-2 ppm.Ngakolunye uhlangothi, kuze kube manje, i-NMR ne-MS isetshenziselwe ukuveza izinga lokuxhumanisa kanye/noma ukuguqulwa kwe-HA ezicucu zeyunithi yoshukela zemikhiqizo ye-HA exhumene.Inhloso yalawa masu akukaze kube ukulinganisa ukutholwa kwe-BDDE eyinsalela ezindaweni eziphansi njengoba sichaza kulesi sihloko (LOD yendlela yethu ye-LC-MS = 10 ppb).
Isikhathi sokuthumela: Sep-01-2021