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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 * Ava vanyori vane zvimwe zvavanoziva mubasa iri mupiro wekumashure: Hyaluronic acid (HA) i polysaccharide inongoitika inoshandiswa mukugadzira dermal fillers nekuda kwekunakisa.Sezvo iine hafu yeupenyu hwemazuva akati wandei munyama yemunhu, HA-based dermal fillers inogadziriswa kemikari kuti iwedzere hupenyu hwavo mumuviri.Iyo inonyanya kugadziriswa mune zvekutengesa HA-based fillers ndeye kushandiswa kwe1,4-butanediol diglycidyl ether (BDDE) semuchinjiro wekubatanidza kuyambuka HA maketani.Kusara kana kusagadziriswa BDDE inoonekwa seisina chepfu pa <2 zvikamu pamiriyoni (ppm);saka, iyo yakasara BDDE mune yekupedzisira dermal filler inofanirwa kuverengerwa kuve nechokwadi chekuchengetedzwa kwemurwere.Zvishandiso uye nzira: Ichi chidzidzo chinotsanangura kucherechedzwa uye hunhu hwechigadzirwa chemuchinjiko-chinosanganisa chekuita pakati peBDDE neHA pasi pemamiriro ealkaline nekubatanidza liquid chromatography uye mass spectrometry (LC-MS).Migumisiro: Mushure mekuongororwa kwakasiyana-siyana, zvakaonekwa kuti mamiriro e-alkaline uye kupisa kwepamusoro kunoshandiswa kuuraya utachiona hweHA-BDDE hydrogel yakakurudzira kuumbwa kwechigadzirwa ichi chitsva, "propylene glycol-like" compound.Ongororo yeLC-MS yakasimbisa kuti iyo-chigadzirwa ine yakafanana monoisotopic mass seBDDE, imwe yekuchengeta nguva (tR), uye yakasiyana UV absorbance (λ=200 nm) modhi.Kusiyana neBDDE, yakaonekwa muLC-MS kuongororwa kuti pasi pemamiriro akafanana ekuyerwa, ichi ne-chigadzirwa chine chiyero chepamusoro chekuonekwa pa200 nm.Mhedziso: Mhedzisiro iyi inoratidza kuti hapana epoxide muchimiro chekomboni itsva iyi.Nhaurirano yakazaruka kuti iongorore njodzi yeiyi itsva-chigadzirwa chinowanikwa mukugadzirwa kweHA-BDDE hydrogel (HA dermal filler) nokuda kwezvinangwa zvekutengesa.Keywords: hyaluronic acid, HA dermal filler, cross-linked hyaluronic acid, BDDE, LC-MS kuongorora, BDDE ne-chigadzirwa.
Mafirita akavakirwa pahyaluronic acid (HA) ndiwo anonyanya kufarirwa uye anozivikanwa dermal mafirita anoshandiswa mukushongedza.1 Iyi dermal filler is hydrogel, kazhinji inoumbwa > 95% mvura uye 0.5-3% HA, iyo inovapa gel-yakafanana chimiro.2 HA inonzi polysaccharide uye chikamu chikuru che extracellular matrix ye vertebrates.Imwe yezvigadzirwa.Inosanganisira (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) inodzokorora zvikamu zve disaccharide zvakabatanidzwa ne glycosidic bonds.Iyi disaccharide pateni yakafanana mune zvese zvipenyu.Kuenzaniswa nemamwe maprotein-based fillers (senge collagen), ichi chivakwa chinoita HA ive yakanyanya biocompatible molecule.Aya mafirita anogona kuratidza amino acid kutevedzana kweiyo inogona kuzivikanwa nemurwere immune system.
Kana ichishandiswa sedermal filler, iyo yakanyanya kuganhurirwa yeHA ndeye kukurumidza kushanduka mukati mematishu nekuda kwekuvapo kweimwe mhuri yema enzyme inonzi hyaluronidases.Parizvino, magadzirirwo emakemikari akati wandei muHA chimiro akatsanangurwa kuwedzera hafu yehupenyu hweHA mumatishu.3 Zvizhinji zvezvigadziriso izvi zvinoedza kuderedza kupinda kwehyaluronidase kumapolysaccharide mapolymer kuburikidza ne-cross-linking HA cheni.Nokudaro, nekuda kwekuumbwa kwemabhiriji uye intermolecular covalent bonds pakati peHA chimiro uye muchinjiko-muchinjika agent, iyo cross-linked HA hydrogel inogadzira zvinhu zvakawanda zvinorwisa-enzyme zvinosvibisa kupfuura HA.4-6
Kusvika pari zvino, kemikari crosslinking agents inoshandiswa kugadzira crosslinked HA inosanganisira methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) Uye poly(ethylene glycol) diglycidyl ether.10 ,11 BDDE parizvino ndiyo inonyanya kushandiswa crosslinking agent.Kunyangwe idzi mhando dzemahydrogel dzakaratidzwa kuve dzakachengeteka kwemakumi emakore, iyo crosslinking agents inoshandiswa ndeye reactive reagents inogona kunge iri cytotoxic uye, mune dzimwe nguva, mutagenic.12 Naizvozvo, yavo yasara yemukati muyekupedzisira hydrogel inofanira kunge yakakwira.BDDE inoonekwa seyakachengeteka kana iyo yakasara yakasungwa isingasviki 2 zvikamu pamiriyoni (ppm).4
Pane nzira dzakati wandei dzekuona yakaderera-yakasara BDDE kusungwa, kuyambuka-inobatanidza dhigirii uye nzvimbo yekutsiva muHA hydrogel, senge gasi chromatography, saizi yekusaiswa chromatography yakasanganiswa ne mass spectrometry (MS), nuclear magnetic resonance (NMR) fluorescence yekuyera nzira, uye Diode array yakasanganiswa yakakwira performance liquid chromatography (HPLC).13-17 Ichi chidzidzo chinotsanangura kuonekwa uye kuonekwa kwechigadzirwa mukupedzisira-yakabatanidzwa HA hydrogel yakagadzirwa nekuita kweBDDE uye HA pasi pemamiriro e-alkaline.HPLC uye liquid chromatography-mass spectrometry (LC-MS kuongorora).Sezvo huturu hweiyi-chigadzirwa cheBDDE husingazivikanwe, isu tinokurudzira kuti iyo yasara quantification inofanirwa kutariswa nenzira yakafanana kune nzira inowanzoitwa paBDDE mune yekupedzisira chigadzirwa.
Iyo yakawanikwa sodium munyu weHA (Shiseido Co., Ltd., Tokyo, Japan) ine molecular uremu ~ 1,368,000 Da (Laurent nzira) 18 uye intrinsic viscosity ye2.20 m3 / kg.Nokuda kwekuita kwekubatanidza, BDDE (≥95%) yakatengwa kubva kuSigma-Aldrich Co. (St. Louis, MO, USA).Phosphate yakavharwa nesaline ine pH 7.4 yakatengwa kubva kuSigma-Aldrich Company.Zvese zvinonyungudutsa, acetonitrile uye mvura zvakashandiswa muLC-MS ongororo zvakatengwa kubva kuHPLC giredhi mhando.Formic acid (98%) inotengwa segiredhi reagent.
Zvese zviedzo zvakaitwa paUPLC Acquity system (Mvura, Milford, MA, USA) uye yakabatana neAPI 3000 katatu quadrupole mass spectrometer ine electrospray ionization sosi (AB SCIEX, Framingham, MA, USA).
Kuumbwa kwemuchinjika-yakabatana HA hydrogels kwakatangwa nekuwedzera 198 mg yeBDDE kune 10% (w/w) sodium hyaluronate (NaHA) mhinduro pamberi pe1% alkali (sodium hydroxide, NaOH).Mhedzisiro yeBDDE musanganiswa wekuita musanganiswa waiva 9.9 mg/mL (0.049 mM).Zvadaro, musanganiswa wekuita wakanyatsosanganiswa uye wakagadziriswa uye wakabvumirwa kupfuurira pa45 ° C kwemaawa mana.19 Iyo pH yemaitiro inochengetwa pa ~ 12.
Pashure pacho, musanganiswa wekuita wakashambidzwa nemvura, uye iyo yekupedzisira HA-BDDE hydrogel yakasvibiswa uye yakasvibiswa nePBS buffer kuti iwane HA kusanganiswa kwe10 kusvika 25 mg / mL, uye pH yekupedzisira ye7.4.Kuitira kuomesa iyo inogadzirwa muchinjika-yakabatana HA hydrogels, ese mahydrogel aya anovharirwa (120 ° C kwemaminetsi makumi maviri).Iyo yakacheneswa BDDE-HA hydrogel inochengetwa pa4 ° C kusvika kuongororwa.
Kuongorora iyo BDDE iripo muchinjika-yakabatana HA chigadzirwa, 240 mg sampu yakayerwa uye yakaunzwa mukati megomba (Microcon®; Merck Millipore, Billerica, MA, USA; vhoriyamu 0.5 mL) uye centrifuged pa10,000 rpm pane tembiricha yekamuri. Maminitsi gumi.Huwandu hwe20 µL hwemvura yekudhonza-pasi yakaunganidzwa uye yakaongororwa.
Kuti uongorore chiyero cheBDDE (Sigma-Aldrich Co) pasi pemamiriro ealkaline (1%, 0.1% uye 0.01% NaOH), kana zvinotevera zvakasangana, sampu yemvura ndeye 1:10, 1:100, kana kusvika 1:1,000,000 Kana zvichidikanwa, shandisa MilliQ deionized water kuti uongororwe.
Pazvinhu zvekutanga zvakashandiswa mumuchinjikwa-kubatanidza maitiro (HA 2%, H2O, 1% NaOH, uye 0.049 mM BDDE), 1 mL yemuenzaniso wega wega wakagadzirirwa kubva muzvinhu izvi wakaongororwa uchishandisa maitiro akafanana ekuongorora.
Kuti uone iwo chaiwo epamusoro anoonekwa muion mepu, 10 µL ye100 ppb BDDE yakajairwa mhinduro (Sigma-Aldrich Co) yakawedzerwa kune makumi maviri µL sampuro.Muchiitiko ichi, iyo yekupedzisira kusungirirwa kweyero mune yega yega sampu ndeye 37 ppb.
Chekutanga, gadzirira BDDE stock solution ine 11,000 mg/L (11,000 ppm) nekudzikisa 10 μL yestandard BDDE (Sigma-Aldrich Co) ne990 μL MilliQ mvura (density 1.1 g/mL).Shandisa mhinduro iyi kugadzirira 110 µg/L (110 ppb) BDDE mhinduro seyepakati chiyero dilution.Zvadaro, shandisa yepakati BDDE yakajairika diluent (110 ppb) kugadzirira yakajairwa curve nekunyungudutsa yepakati diluent kakawanda kuti uwane iyo yaunoda kusungwa ye75, 50, 25, 10, uye 1 ppb.Sezvinoratidzwa muMufananidzo 1, inowanikwa kuti BDDE yakajairwa curve kubva 1.1 kusvika 110 ppb ine mutsara wakanaka (R2> 0.99).The standard curve yakadzokororwa mune ina yakazvimirira kuedza.
Mufananidzo 1 BDDE yakajairika calibration curve yakawanikwa neLC-MS kuongororwa, umo kuwirirana kwakanaka kunoonekwa (R2> 0.99).
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, liquid chromatography uye mass spectrometry.
Kuti uone uye kuenzanisa zviyero zveBDDE zviripo mumuchinjikwa-yakabatanidzwa HA uye zviyero zveBDDE mukugadzirisa kwekugadzirisa, LC-MS kuongorora yakashandiswa.
Kuparadzaniswa kwechromatographic kwakawanikwa pane LUNA 2.5 µm C18 (2) -HST column (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) uye yakachengetwa pakushambidzika kwekamuri (25 ° C) panguva yekuongorora.Iyo mobile phase ine acetonitrile (solvent A) uye mvura (solvent B) ine 0.1% formic acid.Iyo mobile phase inoburitswa ne gradient elution.The gradient iri sezvinotevera: maminitsi 0, 2% A;1 miniti, 2% A;6 maminitsi, 98% A;7 maminitsi, 98% A;7.1 maminitsi, 2% A;Maminitsi gumi , 2% A. Nguva yekumhanya imaminitsi gumi uye vhoriyamu yejekiseni i20 µL.Nguva yekuchengetedza yeBDDE inenge 3.48 maminetsi (kubva pa3.43 kusvika ku4.14 maminetsi zvichienderana nemiedzo).Chikamu chekufambisa chakapombwa pachiyero chekuyerera che 0.25 mL / min yeLC-MS kuongororwa.
Kuongororwa kweBDDE uye quantification neMS, iyo UPLC system (Mvura) inosanganiswa neAPI 3000 katatu quadrupole mass spectrometer (AB SCIEX) ine electrospray ionization sosi, uye kuongororwa kunoitwa mune yakanaka ion mode (ESI+).
Maererano neion fragment analysis yakaitwa paBDDE, chidimbu chine simba rakakwirira chakasarudzwa kuva chidimbu chinoenderana ne129.1 Da (Mufananidzo 6).Nokudaro, mu-multi-ion monitoring mode (MIM) yehuwandu hwehuwandu, huwandu hwekushandura (mass-to-charge ratio [m / z]) yeBDDE ndeye 203.3 / 129.1 Da.Iyo inoshandisawo yakazara scan (FS) modhi uye chigadzirwa ion scan (PIS) modhi yeLC-MS yekuongorora.
Kuti uone kujeka kweiyo nzira, sampuli isina chinhu (yekutanga mobile phase) yakaongororwa.Hapana chiratidzo chakaonekwa mumuenzaniso usina chinhu nekushandurwa kukuru kwe203.3 / 129.1 Da.Nezve kudzokorora kwekuedza, 10 majekiseni akajairwa e55 ppb (pakati peiyo calibration curve) akaongororwa, zvichikonzera kusara kwechiyero kutsauka (RSD) <5% (data isina kuratidzwa).
Izvo zvakasara zveBDDE zvemukati zvakaverengerwa mumasere akasiyana-siyana eBDDE akayambuka-akabatanidzwa HA hydrogels, inoenderana neina yakazvimirira kuyedza.Sezvinotsanangurwa muchikamu che "Zvishandiso uye Nzira", iyo quantification inoongororwa nehuwandu hwehuwandu hwehuwandu hwekudzoka kweBDDE standard dilution, iyo inofananidzwa nepamusoro pepamusoro yakaonekwa paBDDE mass transition ye 203.3 / 129.1 Da, nekuchengetedza. nguva ye3.43 kusvika 4.14 maminitsi Kwete kumirira.Mufananidzo 2 unoratidza muenzaniso chromatogram yegumi ppb BDDE referensi standard.Tafura 1 inopfupikisa zvakasara zveBDDE zvemukati ma hydrogel masere akasiyana.Huwandu hwekukosha ndeye 1 kusvika 2.46 ppb.Nokudaro, iyo yakasara yeBDDE yekuenzanisa mumuenzaniso inogamuchirwa kushandiswa kwevanhu (<2 ppm).
Mufananidzo 2 Ion chromatogram ye10 ppb BDDE reference standard (Sigma-Aldrich Co), MS (m/z) shanduko yakawanikwa neLC-MS kuongororwa kwe203.30 / 129.10 Da (mune MRM yakanaka mode).
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, liquid chromatography uye mass spectrometry;MRM, maitiro akawanda ekutarisa;MS, misa;m/z, huwandu-kuchaja reshiyo.
Ongorora: Samples 1-8 ndeye autoclaved BDDE cross-yakabatana HA hydrogels.Huwandu hwasara hweBDDE muiyo hydrogel uye iyo yepamusoro yeBDDE yekuchengetedza nguva inoshumwa zvakare.Pakupedzisira, kuvapo kwezvikwiriso zvitsva zvine nguva dzakasiyana dzekuchengeta kunoshumwawo.
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;MRM, maitiro akawanda ekutarisa;tR, nguva yekuchengetedza;LC-MS, liquid chromatography uye mass spectrometry;RRT, nguva yekuchengetedza.
Zvinotoshamisa ndezvokuti kuongororwa kweLC-MS ion chromatogram kwakaratidza kuti zvichienderana neautoclaved cross-linked HA hydrogel samples yakaongororwa, pane imwe nzvimbo yepamusoro panguva pfupi yekuchengetedza nguva ye2.73 kusvika ku3.29 maminitsi.Semuyenzaniso, Mufananidzo 3 unoratidza ion chromatogram yemuchinjiko-yakabatana HA sampuro, apo imwe yepamusoro peak inoonekwa pane imwe nguva yekuchengetedza yakasiyana yeanenge maminitsi 2.71.Iyo yakacherechedzwa yekuchengetedza nguva (RRT) pakati peiyo ichangobva kucherechedzwa peak uye peak kubva kuBDDE yakawanikwa iri 0.79 (Tafura 1).Sezvo isu tichiziva kuti iyo ichangobva kucherechedzwa peak haina kuchengetwa muC18 column inoshandiswa muLC-MS kuongororwa, iyo itsva peak inogona kuenderana neakawanda polar compound kupfuura BDDE.
Mufananidzo 3 Ion chromatogram ye-cross-linked HA hydrogel sample yakawanikwa neLC-MS (MRM mass conversion 203.3 / 129.0 Da).
Mhedziso: HA, hyaluronic acid;LC-MS, liquid chromatography uye mass spectrometry;MRM, maitiro akawanda ekutarisa;RRT, nguva yekuchengetedza hama;tR, nguva yekuchengetedza.
Kuti tibvise mukana wekuti nhongonya nyowani dzakaonekwa dzinogona kunge dzakasvibiswa pakutanga dziripo mumidziyo yakashandiswa, zvinhu izvi zvakaongororwa zvakare zvichishandiswa nzira imwechete yeLC-MS yekuongorora.Zvinhu zvekutanga zvakaongororwa zvinosanganisira mvura, 2% NaHA mumvura, 1% NaOH mumvura, uye BDDE panguva imwechete yakashandiswa mumuchinjikwa-linking reaction.Iyo ion chromatogram yechinhu chekutanga chakashandiswa haina kuratidza chero komputa kana peak, uye nguva yekuchengeta kwayo inoenderana nepamusoro mutsva wakaonekwa.Chokwadi ichi chinorasa pfungwa yekuti hazvisi izvo zvekutanga chete zvinogona kunge zvine chero makomisheni kana zvinhu zvinogona kukanganisa maitiro ekuongorora, asi hapana chiratidzo chekugona kusvibiswa nezvimwe zvigadzirwa zvemurabhoritari.Huwandu hwekutsikisa hunowanikwa mushure meLC-MS kuongororwa kweBDDE uye mitsva mitsva inoratidzwa muTafura 2 (samples 1-4) uye ion chromatogram mumufananidzo 4.
Ongorora: Samples 1-4 inoenderana neyakagadzirwa zvinhu zvinoshandiswa kugadzira autoclaved BDDE cross-linked HA hydrogels.Aya masampuli haana autoclaved.
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, liquid chromatography uye mass spectrometry;MRM, akawanda maitiro ekutarisa.
Mufananidzo 4 unoenderana neLC-MS chromatogram yemuenzaniso wezvakagadzirwa zvakashandiswa muchinjika-inobatanidza kuita kweHA neBDDE.
Ongorora: Zvese izvi zvinoyerwa pachiyero chakafanana uye reshiyo inoshandiswa kuita muchinjiko-yekubatanidza maitiro.Nhamba dzezvakagadzirwa zvakaongororwa nechromatogram zvinoenderana ne: (1) mvura, (2) 2% HA aqueous solution, (3) 1% NaOH aqueous solution.Kuongororwa kweLC-MS kunoitwa pakushandurwa kwehuwandu hwe203.30 / 129.10 Da (mune MRM mode).
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, liquid chromatography uye mass spectrometry;MRM, akawanda maitiro ekutarisa.
Mamiriro ezvinhu akatungamirira pakuumbwa kwepamusoro pepamusoro akaongororwa.Kuti udzidze kuti maitiro ekugadzirisa anoshandiswa sei kubudisa muchinjika-yakabatanidzwa HA hydrogel inokanganisa reactivity yeBDDE cross-linking agent, zvichiita kuti pave nekugadzirwa kwepamusoro pepamusoro (zvinogoneka ne-zvigadzirwa), zviyero zvakasiyana zvakaitwa.Muzvisungo izvi, takadzidza uye takaongorora iyo yekupedzisira BDDE crosslinker, iyo yakabatwa neyakasiyana-siyana yeNaOH (0%, 1%, 0.1%, uye 0.01%) mune aqueous medium, inoteverwa kana isina autoclaving.Iyo mabhakitiriya maitiro ekutevedzera iwo akafanana mamiriro akafanana neiyo nzira inoshandiswa kugadzira muchinjiko-yakabatana HA hydrogel.Sezvakatsanangurwa muchikamu che "Zvinhu uye Nzira", shanduko yakawanda yemuenzaniso yakaongororwa neLC-MS kusvika ku203.30 / 129.10 Da.BDDE uye kusungirirwa kwepamusoro pepamusoro kuverengwa, uye zvigumisiro zvinoratidzwa muTebhu 3. Hapana mitsva mitsva yakaonekwa mumasampuli akanga asina autoclaved, pasinei nekuvapo kweNaOH mumhinduro (samples 1-4, Tafura. 3).Kune autoclaved samples, mitsva mitsva inongoonekwa muhupo hweNaOH mune mhinduro, uye kuumbwa kwepamusoro kunoratidzika kunoenderana neNaOH yekuisa mumhinduro (samples 5-8, Tafura 3) (RRT = 0.79).Mufananidzo 5 unoratidza muenzaniso weion chromatogram, inoratidza maviri autoclaved samples muhupo kana kusavapo kweNAOH.
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, liquid chromatography uye mass spectrometry;MRM, akawanda maitiro ekutarisa.
Cherechedza: Chromatogram yepamusoro: Sample yakabatwa ne 0.1% NaOH aqueous solution uye autoclaved (120 ° C kwemaminitsi makumi maviri).Pazasi chromatogram: Sample haina kurapwa neNaOH, asi autoclaved pasi pemamiriro akafanana.Iko kutendeuka kukuru kwe203.30 / 129.10 Da (mune MRM yakanaka mode) yakaongororwa neLC-MS.
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, liquid chromatography uye mass spectrometry;MRM, akawanda maitiro ekutarisa.
Mune ese autoclaved samples, ine kana isina NaOH, iyo BDDE yevasungwa yakaderedzwa zvakanyanya (kusvika ku16.6 nguva) (samples 5-8, Tafura 2).Kuderera kweBDDE kusungirirwa kunogona kunge kwakakonzerwa nekuti pakupisa kwakanyanya, mvura inogona kuita senheyo (nucleophile) kuvhura epoxide ring yeBDDE kuti iite 1,2-diol compound.Iyo monoisotopic mhando yekomboni iyi yakasiyana neiyo yeBDDE uye saka haizokanganiswe.LC-MS yakaona kuchinja kukuru kwe203.30 / 129.10 Da.
Pakupedzisira, zviedzo izvi zvinoratidza kuti chizvarwa chezvitsva zvitsva zvinoenderana nekuvapo kweBDDE, NAOH, uye autoclaving process, asi haina chekuita neHA.
Iyo yepamusoro peak yakawanikwa panguva yekuchengetedza ingangoita 2.71 maminetsi yakabva yaratidzwa neLC-MS.Nechinangwa ichi, BDDE (9.9 mg/mL) yakaiswa mu 1% NaOH aqueous solution uye autoclaved.MuTafura 4, hunhu hwepamusoro mutsva hunofananidzwa neiyo inozivikanwa BDDE referensi peak (nguva yekuchengetedza ingangoita 3.47 maminetsi).Kubva pane ion kupatsanurwa kuongororwa kwepamusoro pepamusoro, zvinogona kugumiswa kuti iyo yepamusoro ine nguva yekuchengetedza ye2.72 maminitsi inoratidza zvidimbu zvakafanana neBDDE peak, asi nemasimba akasiyana (Mufananidzo 6).Pamusoro pepamusoro inoenderana nenguva yekuchengetedza (PIS) ye2.72 maminetsi, imwe yakanyanya kusimba yakaonekwa mushure mekupatsanurwa pahuwandu hwe147 Da.PaBDDE concentration (9.9 mg/mL) inoshandiswa mukutsunga uku, nzira dzakasiyana dzekunyura (UV, λ = 200 nm) mu ultraviolet spectrum dzakaonekwawo mushure mekuparadzaniswa kwechromatographic (Mufananidzo 7).Iyo yepamusoro ine nguva yekuchengetedza ye 2.71 maminitsi ichiri kuonekwa pa 200 nm, nepo BDDE peak haigoni kuonekwa muchromatogram pasi pemamiriro akafanana.
Tafura 4 Characterization mhedzisiro yepeak nyowani ine nguva yekuchengeta ingangoita 2.71 maminetsi uye iyo BDDE peak ine nguva yekuchengetedza ye3.47 maminetsi.
Ongorora: Kuti uwane mhedzisiro iyi, LC-MS uye HPLC inoongorora (MRM nePIS) yakaitwa panhongonya mbiri.Kuongorora kweHPLC, kutariswa kweUV ine wavelength ye200 nm inoshandiswa.
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;HPLC, yakakwira performance liquid chromatography;LC-MS, liquid chromatography uye mass spectrometry;MRM, maitiro akawanda ekutarisa;m/z, huwandu-kuchaja reshiyo;PIS, chigadzirwa Ion scanning;ultraviolet chiedza, ultraviolet chiedza.
Ongorora: Zvimedu zvakakura zvinowanikwa neLC-MS ongororo (PIS).Yepamusoro chromatogram: misa spectrum yeBDDE yakajairwa sampuli zvimedu.Pazasi chromatogram: Iyo yakawanda spectrum yenhongonya nyowani yaonekwa (RRT yakabatana neBDDE peak ndeye 0.79).BDDE yakagadziriswa mu1% NaOH mhinduro uye autoclaved.
Mhedziso: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, liquid chromatography uye mass spectrometry;MRM, maitiro akawanda ekutarisa;PIS, chigadzirwa ion scan;RRT, nguva yekuchengetedza.
Mufananidzo 7 Ion chromatogram ye203.30 Da precursor ion, uye (A) iyo yepamusoro peak ine nguva yekuchengeta ye2.71 maminetsi uye (B) iyo UV yekuona yeBDDE reference standard peak pa3.46 maminetsi pa200 nm.
Mumuchinjika-yakabatana HA hydrogels akagadzirwa, zvakaonekwa kuti yakasara BDDE kusungwa mushure meLC-MS quantification yaive <2 ppm, asi nyowani isingazivikanwe peak yakaonekwa mukuongorora.Iyi peak nyowani haienderane neBDDE yakajairwa chigadzirwa.Iyo BDDE yakajairwa chigadzirwa yakavewo neiyo yakafanana yemhando shanduko (MRM shanduko 203.30 / 129.10 Da) kuongororwa mune yakanaka MRM modhi.Kazhinji, dzimwe nzira dzekuongorora dzakadai sechromatography dzinoshandiswa semiyedzo yemuganhu kuona BDDE mumahydrogel, asi iyo yepamusoro yekuona muganho (LOD) yakaderera zvishoma pane 2 ppm.Kune rumwe rutivi, kusvika ikozvino, NMR neMS zvakashandiswa kuratidza dhigirii ye-cross-linking uye / kana kugadziriswa kweHA mune shuga unit zvimedu zve-cross-linked HA zvigadzirwa.Chinangwa chemaitiro aya hachina kumbobvira chave chekuenzanisa zvakasara BDDE kuonekwa panzvimbo dzakaderera sezvatinotsanangura muchinyorwa chino (LOD yedu LC-MS nzira = 10 ppb).
Nguva yekutumira: Sep-01-2021