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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. asalka wax ku biirinta: Hyaluronic acid (HA) waa polysaccharide si dabiici ah u soo baxa oo loo isticmaalo soo saarista buuxinta maqaarka ujeedooyin qurxoon.Maadaama ay ku leedahay nolol nus ah oo maalmo dhowr ah unugyada bini'aadamka, buuxinta maqaarka ee HA-ku salaysan ayaa kiimiko ahaan wax laga beddelay si ay u kordhiyaan noloshooda jidhka.Wax ka beddelka ugu badan ee buuxinta HA-ku-salaysan ganacsiga waa isticmaalka 1,4-butanediol diglycidyl ether (BDDE) oo ah wakiilka iskutallaabta si ay uga gudubto silsiladaha HA.BDDE haraaga ah ama aan la falgelin waxa loo arkaa mid aan sun ahayn <2 qaybood halkii milyan (ppm);sidaas darteed, haraaga BDDE ee buuxinta maqaarka u dambeeya waa in la xisaabiyaa si loo xaqiijiyo badbaadada bukaanka.Qalabka iyo hababka: Daraasadani waxay qeexaysaa ogaanshaha iyo sifada ka-soo-saarka falcelinta isku-xidhka ah ee u dhexeeya BDDE iyo HA ee xaaladaha alkaline iyadoo la isku darayo chromatography dareeraha ah iyo spectrometry mass (LC-MS).Natiijooyinka: Falanqaynta kala duwan ka dib, waxaa la ogaaday in xaaladaha alkaline-ka iyo heerkulka sare ee loo isticmaalo in lagu jeermiyo HA-BDDE hydrogel ay kor u qaadeen samaynta alaabtan cusub, "propylene glycol-like".Falanqaynta LC-MS waxay xaqiijisay in alaabtu leedahay cufnaanta monoisotopic sida BDDE, waqti haynta oo ka duwan (tR), iyo nuujinta UV ee kala duwan (λ=200 nm).Si ka duwan BDDE, waxaa lagu arkay falanqaynta LC-MS in isla xaaladaha cabbirka, badeecadan ay leedahay heerka ogaanshaha sare ee 200 nm.Gabagabo: Natiijooyinkani waxay muujinayaan in aysan jirin epoksaydh ku jira qaabka dhismahan cusub.Wadahadalku wuxuu u furan yahay in lagu qiimeeyo khatarta badeecadan cusub ee laga helay soo saarista HA-BDDE hydrogel (HA dermal filler) ujeeddooyin ganacsi.Erayada furaha: hyaluronic acid, filler HA dermal, hyaluronic acid cross-linked, BDDE, LC-MS falanqaynta, BDDE by-product.
Buuxiyeyaasha ku salaysan hyaluronic acid (HA) ayaa ah kuwa ugu caansan uguna caansan maqaarka maqaarka loo isticmaalo ujeedooyinka qurxinta.1 Buuxiyaha maqaarkan waa hydrogel, badanaa wuxuu ka kooban yahay> 95% biyo iyo 0.5-3% HA, kaas oo siinaya qaab-dhismeedka jel u eg.2 HA waa polysaccharide iyo qaybta ugu muhiimsan ee unugga ka baxsan ee laf dhabarta.Mid ka mid ah maaddooyinka.Waxay ka kooban tahay (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) soo noqnoqda unugyada disaccharide ee ku xiran curaarta glycosidic.Habkan disaccharide waa isku mid dhammaan noolaha.Marka la barbar dhigo qaar ka mid ah buuxinta borotiinka ku salaysan (sida kolajka), hantidan ayaa HA ka dhigaysa unug noole aad u habboon.Buuxiyeyaashani waxay soo bandhigi karaan gaar ahaan isku xigxiga amino acid kaas oo laga yaabo inuu aqoonsado habka difaaca bukaanka.
Marka loo isticmaalo buuxinta maqaarka, xaddidaadda ugu weyn ee HA waa isbeddelkeeda degdega ah ee unugyada sababtoo ah joogitaanka qoys gaar ah oo enzymes ah oo loo yaqaan hyaluronidases.Ilaa hadda, dhowr isbeddel oo kiimiko ah oo lagu sameeyay qaab dhismeedka HA ayaa lagu sharaxay si loo kordhiyo nolosha nuska HA ee unugyada.3 Inta badan isbeddelladan waxay isku dayaan inay yareeyaan gelitaanka hyaluronidase ee polymers-ka polysaccharide iyagoo isku xiraya silsiladaha HA.Sidaa darteed, iyada oo ay ugu wacan tahay samaynta buundooyinka iyo isku-xidhka isku-dhafka ah ee isku-dhafka ah ee u dhexeeya qaab-dhismeedka HA iyo wakiilka isku-dhafan, HA hydrogel-ku-xiran ayaa soo saara waxyaabo badan oo ka-hortagga enzyme-ka marka loo eego dabiiciga dabiiciga ah ee HA.4-6
Ilaa hadda, walxaha kiimikaad ee isku xidha isku xidhka loo isticmaalo in lagu soo saaro isku xidhka HA waxaa ka mid ah methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE)iyo poly(ethylene glycol) diglycidyl ether.10 ,11 BDDE hadda waa wakiilka ugu badan ee la isticmaalo.Inkasta oo noocyadan hydrogels-ka la xaqiijiyay inay badbaado qabaan tobannaan sano, wakiilada is-goysyada ee la isticmaalay waa reagents fal-celin ah oo laga yaabo inay noqdaan cytotoxic iyo, xaaladaha qaarkood, mutagenic.12 Sidaa darteed, waxa ay ku jiraan hadhaaga hadhaaga ah ee hydrogelka ugu dambeeya waa in ay ahaadaan kuwo sare.BDDE waxa loo tixgaliyaa in ay badbaado tahay marka fiirsashada hadhaagu ay ka yar tahay 2 qaybood halkii milyan (ppm).4
Waxaa jira dhowr habab oo lagu ogaanayo feejignaanta BDDE-ka-soo hartay, shahaadada isku-tallaabta iyo booska beddelka ee HA hydrogels, sida chromatography gaaska, cabbirka ka saarida chromatography oo ay weheliso spectrometry mass (MS), hababka cabbiraadda magnetic resonance (NMR) hababka cabbiraadda fluorescence, iyo Diode array oo ay ku lammaan tahay chromatography dareeraha waxqabadka sare (HPLC).13-17 Daraasadani waxay qeexaysaa ogaanshaha iyo sifaynta alaabta ka soo baxda HA hydrogel ee ugu dambaysa ee iskutallaabta ah ee ay soo saarto falcelinta BDDE iyo HA ee hoos yimaada xaaladaha alkaline.HPLC iyo chromatography dareere-mass spectrometry ( falanqaynta LC-MS).Maadaama sunta badeecadan BDDE aan la garanayn, waxaanu ku talinaynaa in qiyaasteeda hadhaaga la go'aamiyo si la mid ah habka caadiga ah ee lagu sameeyo BDDE ee alaabta u dambaysa.
Cusbada sodium ee la helay ee HA (Shiseido Co., Ltd., Tokyo, Japan) waxay leedahay miisaan kelli ah ~ 1,368,000 Da (habka Laurent) 18 iyo mawjadaha gudaha ee 2.20 m3/kg.Falcelinta isku xidhka, BDDE (≥95%) waxa laga soo iibsaday Sigma-Aldrich Co. (St. Louis, MO, USA).Salineed buffered Phosphate oo leh pH 7.4 ayaa laga soo iibsaday Shirkadda Sigma-Aldrich.Dhammaan dareerayaasha, acetonitrile iyo biyaha loo isticmaalo falanqaynta LC-MS waxaa laga soo iibsaday tayada heerka HPLC.Formic acid (98%) waxa loo iibsadaa sidii darajada reagent.
Dhammaan tijaabooyinka waxaa lagu sameeyay nidaamka UPLC Acquity (Waters, Milford, MA, USA) waxaana lagu xiray API 3000 triple quadrupole mass spectrometer qalabaysan isha ionization electrospray (AB SCIEX, Framingham, MA, USA).
Isku-dubbaridka HA hydrogels-ku-xiran ayaa lagu bilaabay iyadoo lagu daray 198 mg ee BDDE ilaa 10% (w / w) sodium hyaluronate (NaHA) xalinta iyadoo ay jirto 1% alkali (sodium hydroxide, NaOH).Xoojinta BDDE ee u dambaysay ee isku darka falcelinta waxay ahayd 9.9 mg/mL (0.049 mM).Kadibna, isku darka falcelinta ayaa si fiican loo qasay oo isku mid ah waxaana loo oggolaaday inay sii socoto 45 ° C ilaa 4 saacadood.19 pH falcelinta waxaa lagu hayaa ~12.
Intaa ka dib, isku darka falcelinta waxaa lagu maydhay biyo, iyo HA-BDDE hydrogel ugu dambeeya ayaa la sifeeyay oo lagu qasi jiray PBS baffer si loo gaaro hagida HA ee 10 ilaa 25 mg / mL, iyo pH ugu dambeysa ee 7.4.Si loo jeermiyo HA hydrogels-ku-xiran ee la soo saaray, dhammaan hydrogels-yadan ayaa si toos ah loo daboolay (120 ° C ilaa 20 daqiiqo).Hydrogel-ka la safeeyey ee BDDE-HA waxa lagu kaydiyaa 4°C ilaa la falanqaynayo.
Si loo falanqeeyo BDDE ee ku jirta sheyga HA iskutallaabta, muunad 240 mg ah ayaa la miisaamay oo la galiyay daloolka dhexe (Microcon®; Merck Millpore, Billerica, MA, USA; mugga 0.5 mL) waxaana lagu qiyaasaa 10,000 rpm heerkulka qolka 10 daqiiqoWadar dhan 20 µL oo dareere ah hoos-u-jiid ayaa la ururiyay oo la falanqeeyay.
Si loo falanqeeyo heerka BDDE (Sigma-Aldrich Co) ee xaaladaha alkaline (1%, 0.1% iyo 0.01% NaOH), haddii shuruudaha soo socda la buuxiyo, muunadda dareeraha ah waa 1:10, 1:100, ama ilaa 1: 1,000,000 Haddii loo baahdo, isticmaal MilliQ biyaha deionized si loo gorfeeyo.
Qalabka bilawga ah ee loo isticmaalo falcelinta isku-dhafan (HA 2%, H2O, 1% NaOH, iyo 0.049 mM BDDE), 1 mL ee muunad kasta oo laga soo diyaariyey alaabtan ayaa la falanqeeyay iyadoo la adeegsanayo isla xaaladaha falanqaynta.
Si loo go'aamiyo gaar ahaan meelaha ugu sarreeya ee ka muuqda khariidadda ion, 10 µL ee 100 ppb BDDE xalka caadiga ah (Sigma-Aldrich Co) ayaa lagu daray muunadda 20 µL.Xaaladdan oo kale, xoojinta ugu dambeysa ee heerka muunad kasta waa 37 ppb.
Marka hore, diyaari xalka kaydka BDDE oo leh 11,000 mg/L (11,000 ppm) adoo ku milmaya 10 μL ee heerka BDDE (Sigma-Aldrich Co) oo leh 990 μL MilliQ (cufnaanta 1.1 g/ml).Isticmaal xalkan si aad ugu diyaarisid 110 µg/L (110 ppb) BDDE xalka sidii dhexdhexaadiye heer dhexe ah.Kadibna, isticmaal dhexdhexaadiyaha caadiga ah ee BDDE (110 ppb) si aad u diyaarisid qalooca caadiga ah adiga oo ku milmaya dareeraha dhexdhexaadka ah dhowr jeer si loo gaaro diiradda la rabo ee 75, 50, 25, 10, iyo 1 ppb.Sida ku cad Jaantuska 1, waxaa la ogaaday in qalooca caadiga ah ee BDDE laga bilaabo 1.1 ilaa 110 ppb uu leeyahay khad wanaagsan (R2>0.99).Qalooca caadiga ah ayaa lagu soo celiyay afar tijaabo oo madax banaan.
Jaantuska 1 BDDE qalooca cabbirka caadiga ah ee lagu helay falanqaynta LC-MS, kaas oo lagu arkay xidhiidh wanaagsan (R2>0.99).
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography dareere ah iyo spectrometry mass.
Si loo aqoonsado loona qiyaaso halbeegyada BDDE ee ku jira iskutallaabta HA iyo heerarka BDDE ee xalka saldhiga, falanqaynta LC-MS ayaa la isticmaalay.
Kala soocida chromatographic waxaa lagu gaaray LUNA 2.5 µm C18 (2) -HST tiir (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) waxaana lagu hayaa heerkulka qolka (25 ° C) inta lagu jiro falanqaynta.Marxaladda guurguura waxay ka kooban tahay acetonitrile (millaha A) iyo biyo (millaha B) oo ka kooban 0.1% formic acid.Marxaladda guurguura waxa meesha ka saaray ilbaxnimo gradient ah.Jaangooyuhu waa sida soo socota: 0 daqiiqo, 2% A;1 daqiiqo, 2% A;6 daqiiqo, 98% A;7 daqiiqo, 98% A;7.1 daqiiqo, 2% A;10 daqiiqo , 2% A. Wakhtiga oroduhu waa 10 daqiiqo mugga cirbadduna waa 20 µL.Waqtiga haynta BDDE waa ilaa 3.48 daqiiqo (oo u dhaxaysa 3.43 ilaa 4.14 daqiiqo oo ku salaysan tijaabooyin).Marxaladda mobaylka waxaa lagu shubay xadiga socodka 0.25 mL/min ee falanqaynta LC-MS.
Falanqaynta BDDE iyo qiyaasidda MS, nidaamka UPLC (Biyaha) waxaa lagu daraa API 3000 triple square mass spectrometer (AB SCIEX) oo ku qalabaysan isha ionization electrospray, falanqaynta waxaa lagu sameeyaa habka ion togan (ESI +).
Marka loo eego falanqaynta jajabka ion ee lagu sameeyay BDDE, jajabka leh xoojinta ugu sareysa ayaa lagu go'aamiyay inuu yahay jajab u dhigma 129.1 Da (Jaantus 6).Sidaa darteed, habka la socodka multi-ion (MIM) ee qiyaasidda, beddelka tirada badan (m/z) ee BDDE waa 203.3/129.1 Da.Waxa kale oo ay isticmaashaa qaabka sawirka buuxa (FS) iyo sawirka ion alaabta (PIS) ee falanqaynta LC-MS.
Si loo xaqiijiyo gaar ahaan habka, muunad maran (wajiga hore ee wareega) ayaa la falanqeeyay.Wax calaamad ah laguma helin muunadda bannaan oo leh beddelaad ballaaran oo ah 203.3/129.1 Da.Marka laga hadlayo dib-u-celinta tijaabada, 10 irbadood oo caadi ah oo ah 55 ppb (dhexda qalooca cabbirka) ayaa la falanqeeyay, taasoo keentay leexinta heerka hadhaaga (RSD) <5% (xogta aan la muujin).
Waxyaabaha hadhaaga ah ee BDDE waxa lagu qiyaasey sideed kala duwan oo iskood isku xidhan oo BDDE iskutallaabta HA hydrogels, oo u dhiganta afar tijaabo oo madax banaan.Sida lagu qeexay qaybta "Qalabyada iyo Hababka", qiyaasitaanka waxaa lagu qiimeeyaa celceliska qiimaha qalooca dib u celinta ee BDDE heerka qasin, kaas oo u dhigma heerka ugu sarreeya ee lagu ogaaday kala-guurka ballaaran ee BDDE ee 203.3 / 129.1 Da, oo leh xajin. wakhtiga 3.43 ilaa 4.14 daqiiqo Ma sugin.Jaantuska 2 wuxuu muujinayaa tusaale chromatogram-ka 10 ppb BDDE heerka tixraaca.Shaxda 1 ayaa soo koobaysa hadhaaga BDDE ee ka kooban sideed hydrogel oo kala duwan.Qiyaasta qiimaheedu waa 1 ilaa 2.46 pb.Sidaa darteed, fiirsashada BDDE ee hadhay ee muunada waa la aqbali karaa isticmaalka aadanaha (<2 ppm).
Jaantuska 2 Ion chromatogram ee 10 ppb BDDE heerka tixraaca (Sigma-Aldrich Co), MS (m/z) kala guurka oo lagu helay falanqaynta LC-MS ee 203.30/129.10 Da (oo ku jira qaabka MRM togan).
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan;MS, cuf;m/z, saamiga cuf-ku-dallacsiinta.
Fiiro gaar ah: Tusaalooyinka 1-8 waa iskutallaabta BDDE ee HA hydrogels oo iskutallaabta.Qadarka hadhaaga BDDE ee ku jira hydrogel iyo wakhtiga ugu sarreeya ee BDDE haynta ayaa sidoo kale la soo sheegay.Ugu dambeyntii, jiritaanka heerar cusub oo leh waqtiyo haynta kala duwan ayaa sidoo kale la soo sheegay.
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;MRM, la socodka falcelinta badan;tR, wakhtiga haynta;LC-MS, chromatography dareere ah iyo spectrometry mass;RRT, waqti haynta qaraabo ah.
Waxaa la yaab leh, falanqaynta chromatogram-ka LC-MS ion chromatogram waxay muujisay in iyada oo ku saleysan dhammaan shaybaarada iskutallaabta iskutallaabta ee HA hydrogel ee la falanqeeyay, waxaa jiray heer sare oo dheeri ah waqtiga haynta gaaban ee 2.73 ilaa 3.29 daqiiqo.Tusaale ahaan, Jaantuska 3 wuxuu muujinayaa chromatogram-ka ion ee muunad HA iskutallaabta ah, halkaas oo ugu sarraysa ay u muuqato waqti haynta ka duwan oo ku dhow 2.71 daqiiqo.Wakhtiga haynta qaraabo la arkay (RRT) ee u dhexeeya heerka ugu sarreeya ee dhowaan la arkay iyo kan ugu sarreeya ee BDDE waxa la ogaaday inuu yahay 0.79 (Shaxda 1).Mar haddii aan ognahay in heerka ugu sarreeya ee dhowaan la arkay uu yar yahay in lagu hayo tiirka C18 ee lagu isticmaalo falanqaynta LC-MS, meesha ugu sarreysa ayaa laga yaabaa inay u dhigmato unug ka badan BDDE.
Jaantus 3 chromatogram Ion ee muunad HA hydrogel-ku-xiran oo ay heshay LC-MS (Beddelka baaxadda MRM 203.3/129.0 Da).
Soo gaabinta: HA, hyaluronic acid;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan;RRT, wakhtiga haynta qaraabo;tR, wakhtiga haynta
Si meesha looga saaro suurtogalnimada in meelaha ugu sarreeya ee cusub ee la arkay ay noqon karaan wasakh asal ahaan ku jira alaabta ceeriin ee la isticmaalo, alaabtan cayriin ayaa sidoo kale la falanqeeyay iyada oo la adeegsanayo habka falanqaynta LC-MS.Alaabta bilowga ah ee la falanqeeyay waxaa ka mid ah biyaha, 2% NAHA oo biyo ah, 1% NaOH oo biyo ah, iyo BDDE isla diirada loo isticmaalo falcelinta isku xidhka.Chromatogram-ka ion ee walxaha bilawga ah ee la isticmaalay ma muujin wax iskudhis ah ama ugu sarreeya, iyo wakhtiga sii hayntiisu waxay u dhigantaa heerka ugu sarreeya ee cusub ee la arkay.Xaqiiqadani waxay meesha ka saaraysaa fikradda ah in kaliya maaddada bilawga ah ay ku jiri karaan wax kasta oo isku dhafan ama walxaha laga yaabo inay farageliyaan habka falanqaynta, laakiin ma jirto calaamad muujinaysa suurtagalnimada is-dhaafsiga alaabada kale ee shaybaarka.Qiimaha fiirsashada ee la helay ka dib falanqaynta LC-MS ee BDDE iyo meelaha ugu sarreeya ayaa lagu muujiyey Shaxda 2 (muunado 1-4) iyo chromatogram-ka ion ee Jaantuska 4.
Fiiro gaar ah: Tusaalooyinka 1-4 waxay u dhigmaan alaabta ceeriin ee loo isticmaalo in lagu soo saaro BDDE iskutallaabta HA hydrogels.Tijaabooyinkan si toos ah looma xidhin.
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan.
Jaantuska 4 wuxuu u dhigmaa chromatogram-ka LC-MS ee muunadda alaabta ceeriin ee loo isticmaalo falcelinta isku-tallaabta ee HA iyo BDDE.
Fiiro gaar ah: Dhammaan kuwan waxaa lagu cabbiraa isla xoogga iyo saamiga loo isticmaalo in lagu fuliyo falcelinta isku-xiran.Nambarada alaabta ceeriin ee uu chromatogram-ku falanqeeyay waxay u dhigantaa: (1) biyo, (2) 2% HA xal aqueous ah, (3) 1% NaOH aqueous.Falanqaynta LC-MS waxaa loo sameeyaa beddelka tirada badan ee 203.30/129.10 Da (qaabka MRM ee togan).
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan.
Waxaa la darsay xaaladaha keenay samaynta meelaha ugu sarreeya.Si loo barto sida xaaladaha falcelinta loo isticmaalo in lagu soo saaro isku-xirnaanta HA hydrogel ay saameynayso fal-celinta wakiilka isku-dhafan ee BDDE, taasoo horseedaysa samaynta heerar cusub (alaabooyin suurtagal ah), cabbirro kala duwan ayaa la sameeyay.Go'aannadan, waxaan ku barannay oo aan ku falanqeynay crosslinker-ka BDDE ee ugu dambeeya, kaas oo lagu daaweeyay xaddi kala duwan oo NaOH ah (0%, 1%, 0.1%, iyo 0.01%) dhex dhexaad aqueous ah, oo ay ku xigto ama aan lahayn autoclaving.Habka bakteeriyadu si ay u ekaysiiyaan xaaladaha isku midka ah waxay la mid tahay habka loo isticmaalo in lagu soo saaro HA hydrogel-isku-xiran.Sida lagu qeexay qaybta "Qalabyada iyo Hababka", kala-guurka ballaaran ee muunada waxaa falanqeeyay LC-MS ilaa 203.30/129.10 Da.BDDE iyo xoojinta ugu sarreeya ee cusub ayaa la xisaabiyaa, natiijooyinkana waxaa lagu muujiyey Shaxda 3. Ma jiraan wax cusub oo cusub oo lagu ogaaday shaybaarrada aan la isku dhejin, iyada oo aan loo eegin joogitaanka NaOH ee xalka (muundooyinka 1-4, Shaxda 3).Shaybaarada autoclaved, meelaha ugu sarreeya ayaa kaliya lagu ogaadaa joogitaanka NaOH ee xalka, iyo samaynta ugu sarreysa waxay u muuqataa inay ku xiran tahay xoojinta NaOH ee xalka (muundooyinka 5-8, Shaxda 3) (RRT = 0.79).Jaantuska 5 wuxuu muujinayaa tusaale chromatogram-ka ion, oo muujinaya laba muunado oo iswada leh oo joogitaanka ama maqnaanshaha NAOH.
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan.
Fiiro gaar ah: chromatogram-ka sare: Muunada waxaa lagu daaweeyay 0.1% NaOH aqueous aqueous iyo autoclaved (120°C ilaa 20 daqiiqo).chromatogram-ga hoose: Muunada laguma daawayn NaOH, laakiin si iskeed ah ayaa loo jeexjeexay iyadoo lagu hoos jiro xaalado isku mid ah.Isbeddelka tirada badan ee 203.30 / 129.10 Da (qaabka saxda ah ee MRM) ayaa lagu falanqeeyay LC-MS.
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan.
Dhammaan shaybaarrada autoclaved, oo leh ama la'aanteed NaOH, fiirsashada BDDE si weyn ayaa loo dhimay (ilaa 16.6 jeer) (muunado 5-8, Shaxda 2).Hoos u dhaca xoogga BDDE waxaa laga yaabaa inay sabab u tahay xaqiiqda ah in heerkulka sare, biyuhu ay u dhaqmi karaan sidii saldhig (nucleophile) si ay u furaan giraanta epoxide ee BDDE si ay u sameeyaan xarun 1,2-diol ah.Tayada monoisotopic ee xaruntani way ka duwan tahay tan BDDE sidaa awgeedna ma saamayn doonto.LC-MS waxay ogaatay isbeddel ballaaran oo ah 203.30/129.10 Da.
Ugu dambeyntii, tijaabooyinkan ayaa muujinaya in jiilka ugu sarreeya ee cusub ay ku xiran tahay joogitaanka BDDE, NAOH, iyo habka autoclaving, laakiin wax shaqo ah kuma laha HA.
Meesha ugu sarreysa ee la helay waqti haynta ku dhawaad 2.71 daqiiqo ayaa markaas lagu sifeeyay LC-MS.Ujeedadaas awgeed, BDDE (9.9 mg/mL) ayaa lagu dhex shubay 1% NaOH aqueous iyo autoclaved.Jadwalka 4, sifooyinka ugu sarreeya ee cusub ayaa la barbardhigay heerka ugu sarreeya ee tixraaca BDDE (waqtiga haynta qiyaastii 3.47 daqiiqo).Iyada oo ku saleysan falanqaynta kala-goynta ion ee labada ugu sarreysa, waxaa lagu soo gabagabeyn karaa in ugu sarreysa ee leh waqtiga haynta ee 2.72 daqiiqo ay muujinayso jajab la mid ah kan ugu sarreeya BDDE, laakiin leh xoogaa kala duwan (Jaantus 6).Marka la eego heerka ugu sarreeya ee u dhigma wakhtiga haynta (PIS) ee 2.72 daqiiqo, ugu sarreeya ayaa la arkay ka dib markii la kala qaybiyey tiro dhan 147 Da.Marka la eego fiirsashada BDDE (9.9 mg / mL) ee loo isticmaalo go'aaminta, hababka nuugista ee kala duwan (UV, λ = 200 nm) ee ultraviolet spectrum ayaa sidoo kale lagu arkay ka dib kala-soocidda chromatographic (Jaantus 7).Waqtiga ugu sarreeya ee haynta ee 2.71 daqiiqo ayaa weli la arki karaa 200 nm, halka BDDE ugu sarreysa aan lagu arki karin chromatogram isla xaalado isku mid ah.
Shaxda 4 Natiijooyinka sifada ee ugu sarraysa cusub oo leh wakhti haynta ku dhawaad 2.71 daqiiqo iyo BDDE ugu saraysa oo leh wakhti haynta 3.47 daqiiqo
Fiiro gaar ah: Si loo helo natiijooyinkan, LC-MS iyo falanqaynta HPLC (MRM iyo PIS) ayaa lagu sameeyay labada ugu sarreeya.Falanqaynta HPLC, ogaanshaha UV oo leh hirar dhererkiisu yahay 200 nm ayaa la isticmaalaa.
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;HPLC, chromatography dareere waxqabadka sare;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan;m/z, saamiga cuf-ku-dallacsiinta;PIS, iskaanka alaabta Ion;iftiinka ultraviolet, iftiinka ultraviolet.
Xusuusin: Qaybaha tirada badan waxaa lagu helay falanqaynta LC-MS (PIS).chromatogramka ugu sarreeya: spectrum mass of BDDE jajab muunad caadiga ah.chromatogram-ga hoose: Tirada guud ee ugu sarreysa ee cusub ee la ogaaday (RRT ee la xidhiidha BDDE ugu sarreeya waa 0.79).BDDE waxaa lagu farsameeyay 1% NaOH xal iyo autoclaved.
Soo gaabinta: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography dareere ah iyo spectrometry mass;MRM, la socodka falcelinta badan;PIS, iskaanka ion alaabta;RRT, waqti haynta qaraabo ah.
Jaantus 7 Ion chromatogram ee 203.30 Da precursor ion, iyo (A) ugu sarraysa ee leh wakhtiga haynta ee 2.71 daqiiqo iyo (B) ogaanshaha UV ee heerka ugu sarreeya ee tixraaca BDDE ee 3.46 daqiiqo at 200 nm.
Dhammaan HA hydrogels-ku-xiran ee la soo saaray, waxaa la arkay in haraaga BDDE ee hadhaaga ka dib qiyaasta LC-MS ay ahayd <2 ppm, laakiin ugu sarreeya oo aan la garanayn ayaa ka soo muuqday falanqaynta.Meeshan cusubi kuma habboona alaabta caadiga ah ee BDDE.Badeecada caadiga ah ee BDDE ayaa sidoo kale soo martay beddelaad tayo la mid ah ( beddelka MRM 203.30/129.10 Da) falanqaynta qaabka MRM ee togan.Guud ahaan, hababka kale ee gorfaynta sida chromatography ayaa loo isticmaalaa sida xaddidaadda tijaabooyinka lagu ogaanayo BDDE ee hydrogels, laakiin xadka ogaanshaha ugu badan (LOD) wax yar ayuu ka hooseeyaa 2 ppm.Dhanka kale, ilaa hadda, NMR iyo MS ayaa loo isticmaalay in lagu garto heerka isku-xirnaanta iyo / ama wax ka beddelka HA ee qaybaha sonkorta ee qaybaha isku-xidhan ee HA.Ujeedada farsamooyinkaan waligood may ahayn in lagu qiyaaso soo-gaadhista BDDE ee soo hadhay ee xad-dhaafka hooseeya sida aan ku qeexnay maqaalkan (LOD ee habkayaga LC-MS = 10 ppb).
Waqtiga boostada: Sebtembar-01-2021