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Javier Fidalgo, * Pierre-Antoine Deglesne, * Rodrigo Arroyo, * Lilian Sepúlveda, * Evgeniya Ranneva, Philippe Deprez Sashen Kimiyya, Skin Tech Pharma Group, Castello D'Empúries, Catalonia, Spain * Waɗannan marubutan suna da ɗan haske game da wannan aikin Daidai. Bayanin gudummawa: Hyaluronic acid (HA) polysaccharide ne da ke faruwa ta halitta wanda aka yi amfani da shi wajen samar da filaye na dermal don dalilai na ado.Tun da yake yana da rabin rayuwa na kwanaki da yawa a cikin kyallen jikin ɗan adam, HA-based dermal fillers an gyare-gyare ta hanyar sinadarai don tsawaita rayuwarsu a cikin jiki.Mafi yawan gyare-gyaren da aka saba yi a cikin masu samar da tushen HA na kasuwanci shine amfani da 1,4-butanediol diglycidyl ether (BDDE) a matsayin wakili mai haɗin kai don ƙetare sarƙoƙin HA.BDDE saura ko ba a yi ba ana ɗaukarsa mara guba a <2 sassa a kowace miliyan (ppm);don haka, ragowar BDDE a cikin filler na ƙarshe dole ne a ƙididdige shi don tabbatar da amincin haƙuri.Kayayyaki da hanyoyin: Wannan binciken yana bayyana ganowa da kuma siffanta samfur ta hanyar haɗin kai tsakanin BDDE da HA a ƙarƙashin yanayin alkaline ta hanyar haɗa chromatography na ruwa da ƙididdigar taro (LC-MS).Sakamako: Bayan bincike daban-daban, an gano cewa yanayin alkaline da zafin jiki da ake amfani da su don lalata HA-BDDE hydrogel sun inganta samuwar wannan sabon samfurin, mahallin "propylene glycol-like".Binciken LC-MS ya tabbatar da cewa samfurin yana da nau'in monoisotopic iri ɗaya kamar BDDE, lokacin riƙewa daban (tR), da yanayin ɗaukar UV daban-daban (λ=200 nm).Ba kamar BDDE ba, an lura da shi a cikin nazarin LC-MS cewa a ƙarƙashin yanayin ma'auni iri ɗaya, wannan samfurin yana da ƙimar ganowa mafi girma a 200 nm.Kammalawa: Waɗannan sakamakon sun nuna cewa babu epoxide a cikin tsarin wannan sabon fili.An buɗe tattaunawar don tantance haɗarin wannan sabon samfurin da aka samu a cikin samar da HA-BDDE hydrogel (HA dermal filler) don dalilai na kasuwanci.Mahimman kalmomi: hyaluronic acid, HA dermal filler, giciye hyaluronic acid, BDDE, LC-MS bincike, BDDE ta-samfurin.
Fillers dangane da hyaluronic acid (HA) sune mafi yawan gama-gari kuma shahararrun masu gyaran fata da ake amfani da su don dalilai na kwaskwarima.1 Wannan dermal filler shine hydrogel, yawanci ya ƙunshi> 95% ruwa da 0.5-3% HA, wanda ke ba su tsari mai kama da gel.2 HA shine polysaccharide kuma babban bangaren matrix extracellular na vertebrates.Daya daga cikin sinadaran.Ya ƙunshi (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) maimaita raka'a disaccharide da aka haɗa ta hanyar haɗin glycosidic.Wannan ƙirar disaccharide iri ɗaya ce a cikin dukkan halittu.Idan aka kwatanta da wasu abubuwan da ke tushen furotin (kamar collagen), wannan kadarar ta sa HA ta zama kwayar halitta mai dacewa sosai.Waɗannan filaye na iya nuna ƙayyadaddun jerin amino acid waɗanda tsarin rigakafi na majiyyaci zai iya gane su.
Lokacin amfani dashi azaman filler dermal, babban iyakancewar HA shine saurin saurinsa a cikin kyallen takarda saboda kasancewar takamaiman dangin enzymes da ake kira hyaluronidases.Ya zuwa yanzu, an bayyana gyare-gyaren sinadarai da yawa a cikin tsarin HA don ƙara rabin rayuwar HA a cikin kyallen takarda.3 Yawancin waɗannan gyare-gyaren gyare-gyare suna ƙoƙari don rage damar yin amfani da hyaluronidase zuwa polysaccharide polymers ta hanyar haɗin haɗin HA.Sabili da haka, saboda samuwar gadoji da haɗin gwiwar haɗin gwiwar haɗin gwiwar tsakanin tsarin HA da ma'auni mai haɗin gwiwa, haɗin haɗin HA hydrogel yana samar da ƙarin kayan lalata enzyme fiye da na HA.4-6
Ya zuwa yanzu, sinadarai da ake amfani da su don samar da haɗin gwiwar HA sun haɗa da methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) da poly (ethylene glycol) diglycidyl ether.10 ,11 BDDE a halin yanzu shine mafi yawan abin da ake amfani da shi na haɗin kai.Ko da yake an tabbatar da cewa waɗannan nau'ikan hydrogels suna da aminci shekaru da yawa, abubuwan haɗin gwiwar da aka yi amfani da su sune reactive reactive waɗanda ke iya zama cytotoxic kuma, a wasu lokuta, mutagenic.12 Don haka, abubuwan da suka rage a cikin hydrogel na ƙarshe dole ne su kasance babba.Ana ɗaukar BDDE lafiya lokacin da ragowar taro bai wuce sassa 2 a kowace miliyan (ppm).4
Akwai hanyoyi da yawa don gano ƙananan raguwa na BDDE, digiri na haɗin kai da matsayi na maye gurbin a cikin HA hydrogels, kamar gas chromatography, girman keɓance chromatography haɗe tare da ma'aunin spectrometry (MS), hanyoyin auna ma'aunin maganadisu na nukiliya (NMR), da kuma hanyoyin auna fluorescence. Diode array haɗe da babban aikin ruwa chromatography (HPLC).13-17 Wannan binciken ya kwatanta ganowa da kuma kwatanta samfurin samfurin a cikin HA hydrogel na ƙarshe da aka haɗa ta hanyar amsawar BDDE da HA a ƙarƙashin yanayin alkaline.HPLC da ruwa chromatography-mass spectrometry (LC-MS bincike).Tun da ba a san gubar wannan samfurin na BDDE ba, muna ba da shawarar cewa ya kamata a ƙayyade ƙimar ragowar ta ta hanyar da aka saba yi akan BDDE a cikin samfurin ƙarshe.
Gishirin sodium da aka samu na HA (Shiseido Co., Ltd., Tokyo, Japan) yana da nauyin kwayoyin nauyin ~ 1,368,000 Da (Hanyar Laurent) 18 da danko mai mahimmanci na 2.20 m3/kg.Don amsawar haɗin kai, BDDE (≥95%) an saya daga Sigma-Aldrich Co. (St. Louis, MO, Amurka).Saline buffered Phosphate tare da pH 7.4 an saya daga Kamfanin Sigma-Aldrich.Duk abubuwan kaushi, acetonitrile da ruwa da aka yi amfani da su a cikin binciken LC-MS an siyi su daga ingancin darajar HPLC.Formic acid (98%) ana siyan shi azaman reagent sa.
Dukkan gwaje-gwajen an yi su ne akan tsarin UPLC Acquity (Waters, Milford, MA, Amurka) kuma an haɗa su da API 3000 sau uku quadrupole mass spectrometer sanye take da tushen ionization na electrospray (AB SCIEX, Framingham, MA, Amurka).
An fara haɗin haɗin haɗin HA hydrogels ta hanyar ƙara 198 MG na BDDE zuwa 10% (w / w) sodium hyaluronate (NaHA) bayani a gaban 1% alkali (sodium hydroxide, NaOH).Matsakaicin BDDE na ƙarshe a cikin cakuda dauki shine 9.9 mg/mL (0.049 mM).Sa'an nan, da dauki cakuda da aka hade sosai da kuma homogenized da kuma barin ci gaba a 45 ° C na 4 hours.19 Ana kiyaye pH na amsawa a ~ 12.
Bayan haka, an wanke cakuda da ruwa da ruwa, kuma an tace HA-BDDE hydrogel na ƙarshe kuma an diluted tare da buffer PBS don cimma ƙimar HA na 10 zuwa 25 mg / mL, da pH na ƙarshe na 7.4.Domin bakara da samar da giciye-linked HA hydrogels, duk wadannan hydrogels an autoclaved (120 ° C na 20 minutes).Ana adana BDDE-HA hydrogel mai tsabta a 4 ° C har sai an bincika.
Don nazarin BDDE da ke cikin samfurin HA mai haɗin giciye, an auna samfurin 240 MG kuma an gabatar da shi a cikin rami na tsakiya (Microcon®; Merck Millipore, Billerica, MA, Amurka; ƙarar 0.5 mL) kuma a tsakiya a 10,000 rpm a dakin da zafin jiki. Minti 10.An tattara jimlar 20 µL na ruwa da aka cire kuma an bincika.
Domin yin nazarin ma'auni na BDDE (Sigma-Aldrich Co) a ƙarƙashin yanayin alkaline (1%, 0.1% da 0.01% NaOH), idan waɗannan sharuɗɗan sun cika, samfurin ruwa shine 1:10, 1:100, ko har zuwa 1: 1,000,000 Idan ya cancanta, yi amfani da ruwan da aka lalatar da MilliQ don bincike.
Don kayan farawa da aka yi amfani da su a cikin haɗin kai (HA 2%, H2O, 1% NaOH, da 0.049 mM BDDE), 1 ml na kowane samfurin da aka shirya daga waɗannan kayan an yi nazari ta amfani da yanayin bincike iri ɗaya.
Don tantance takamaiman kololuwar da ke bayyana a cikin taswirar ion, 10 µL na 100 ppb BDDE daidaitaccen bayani (Sigma-Aldrich Co) an ƙara zuwa samfurin 20 µL.A wannan yanayin, ƙaddamarwar ƙarshe na daidaitattun a cikin kowane samfurin shine 37 ppb.
Da farko, shirya wani bayani na BDDE tare da maida hankali na 11,000 mg/L (11,000 ppm) ta hanyar diluting 10 μL na daidaitaccen BDDE (Sigma-Aldrich Co) tare da ruwa 990 μL MilliQ (yawan 1.1 g/ml).Yi amfani da wannan maganin don shirya 110 μg/L (110 ppb) BDDE bayani a matsayin matsakaicin matsakaicin dilution.Sa'an nan, yi amfani da tsaka-tsakin ma'auni na BDDE (110 ppb) don shirya daidaitaccen lanƙwasa ta hanyar diluting na tsaka-tsakin sau da yawa don cimma burin da ake so na 75, 50, 25, 10, da 1 ppb.Kamar yadda aka nuna a Hoto 1, an gano cewa ma'aunin BDDE daga 1.1 zuwa 110 ppb yana da kyakkyawan layi (R2>0.99).An maimaita daidaitaccen lanƙwasa a cikin gwaje-gwaje masu zaman kansu guda huɗu.
Hoto 1 BDDE daidaitaccen ma'auni na daidaitawa da aka samu ta hanyar bincike na LC-MS, wanda aka lura da kyakkyawar dangantaka (R2> 0.99).
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, ruwa chromatography da taro spectrometry.
Don ganowa da ƙididdige ƙa'idodin BDDE da ke cikin haɗe-haɗe da haɗe-haɗe da ka'idodin BDDE a cikin tushen bayani, an yi amfani da bincike na LC-MS.
An sami rabuwar chromatographic akan wani LUNA 2.5 µm C18 (2) -HST shafi (50 × 2.0 mm2; Phenomenex, Torrance, CA, Amurka) kuma an kiyaye shi a dakin da zafin jiki (25 ° C) yayin bincike.Tsarin wayar hannu ya ƙunshi acetonitrile (mai narkewa A) da ruwa (mai narkewa B) mai ɗauke da 0.1% formic acid.Sashin wayar hannu yana kurewa ta hanyar gradient elution.Matsakaicin matakin shine kamar haka: Minti 0, 2% A;Minti 1, 2% A;Minti 6, 98% A;Minti 7, 98% A;Minti 7.1, 2% A;Minti 10, 2% A. Lokacin gudu shine mintuna 10 kuma ƙarar allurar shine 20µL.Lokacin riƙewa na BDDE kusan mintuna 3.48 ne (daga 3.43 zuwa mintuna 4.14 dangane da gwaji).An fitar da tsarin wayar hannu a cikin adadin 0.25 mL/min don nazarin LC-MS.
Don nazarin BDDE da ƙididdigewa ta MS, an haɗa tsarin UPLC (Waters) tare da API 3000 sau uku quadrupole mass spectrometer (AB SCIEX) sanye take da tushen ionization na electrospray, kuma ana yin binciken a cikin yanayin ion mai kyau (ESI +).
Dangane da binciken gutsuttsarin ion da aka yi akan BDDE, an ƙaddara juzu'in da mafi girman ƙarfi ya zama guntun da ya dace da 129.1 Da (Hoto 6).Sabili da haka, a cikin yanayin saka idanu da yawa (MIM) don ƙididdigewa, yawan jujjuyawar taro (m/z) na BDDE shine 203.3/129.1 Da.Hakanan yana amfani da yanayin cikakken sikanin (FS) da yanayin sikanin samfurin (PIS) don nazarin LC-MS.
Domin tabbatar da ƙayyadaddun hanyar, an bincika samfurin mara kyau (lokacin farko na wayar hannu).Ba a gano sigina ba a cikin samfurin mara kyau tare da jujjuyawar taro na 203.3 / 129.1 Da.Game da maimaita gwajin gwajin, an yi nazarin allurai guda 10 na 55 ppb (a tsakiyar madaidaicin daidaitawa), wanda ya haifar da raguwar daidaitattun daidaito (RSD) <5% (bayanan da ba a nuna ba).
Sauran abubuwan BDDE an ƙididdige su a cikin BDDE masu haɗin haɗe-haɗe na HA hydrogels daban-daban guda takwas, daidai da gwaje-gwaje masu zaman kansu guda huɗu.Kamar yadda aka bayyana a cikin sashin "Kayan Kayayyaki da Hanyoyi", ana ƙididdige ƙididdigewa ta matsakaicin ƙimar madaidaicin juzu'i na BDDE daidaitaccen dilution, wanda yayi daidai da ƙaƙƙarfan kololuwar da aka gano a BDDE taro na 203.3 / 129.1 Da, tare da riƙewa. lokacin 3.43 zuwa 4.14 mintuna Ba jira ba.Hoto 2 yana nuna misalin chromatogram na ma'auni na 10ppb BDDE.Tebu na 1 ya taƙaita ragowar abun ciki na BDDE na hydrogels takwas daban-daban.Matsakaicin ƙimar shine 1 zuwa 2.46 pb.Saboda haka, ragowar BDDE maida hankali a cikin samfurin abin karɓa ne don amfanin ɗan adam (<2 ppm).
Hoto 2 Ion chromatogram na 10 ppb BDDE misali misali (Sigma-Aldrich Co), MS (m/z) miƙa mulki samu ta LC-MS bincike na 203.30/129.10 Da (a cikin m MRM yanayin).
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa;MS, taro;m/z, rabon taro-zuwa-caji.
Lura: Samfuran 1-8 an haɗa BDDE masu haɗe-haɗe ta atomatik na HA hydrogels.Ragowar adadin BDDE a cikin hydrogel da kololuwar lokacin riƙewar BDDE kuma an ba da rahoton.A ƙarshe, an kuma ba da rahoton kasancewar sabbin kololuwa tare da lokutan riƙewa daban-daban.
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;MRM, saka idanu da yawa;tR, lokacin riƙewa;LC-MS, ruwa chromatography da taro spectrometry;RRT, lokacin riƙewa dangi.
Abin mamaki shine, nazarin chromatogram na LC-MS ion chromatogram ya nuna cewa bisa ga dukkanin samfurori na HA hydrogel da aka yi amfani da su ta atomatik da aka bincika, an sami ƙarin kololuwa a cikin ɗan gajeren lokacin riƙewa na 2.73 zuwa 3.29 mintuna.Misali, Hoto na 3 yana nuna chromatogram ion na samfurin HA mai haɗe-haɗe, inda ƙarin kololuwa ya bayyana a wani lokacin riƙewa daban na kusan mintuna 2.71.Lokacin riƙe dangi da aka lura (RRT) tsakanin sabon kololuwar gani da kololuwa daga BDDE an gano shine 0.79 (Table 1).Tun da mun san cewa sabon kololuwar da aka lura ba a riƙe shi a cikin ginshiƙi na C18 da aka yi amfani da shi a cikin binciken LC-MS, sabon kololuwar na iya yin daidai da mahaɗin polar fiye da BDDE.
Hoto 3 Ion chromatogram na haɗe-haɗe HA hydrogel samfurin samu ta LC-MS (MRM taro hira 203.3 / 129.0 Da).
Takaitacce: HA, hyaluronic acid;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa;RRT, lokacin riƙewa dangi;tR, lokacin riƙewa.
Domin kawar da yuwuwar cewa sabbin kololuwar da aka lura na iya zama gurɓatattun abubuwan da suka samo asali a cikin albarkatun da aka yi amfani da su, an kuma bincika waɗannan albarkatun ta hanyar amfani da hanyar bincike na LC-MS iri ɗaya.Abubuwan da aka fara nazarin sun haɗa da ruwa, 2% NaHA a cikin ruwa, 1% NaOH a cikin ruwa, da BDDE a daidai wannan taro da aka yi amfani da su a cikin haɗin kai.ion chromatogram na kayan farawa da aka yi amfani da shi bai nuna wani fili ko kololuwa ba, kuma lokacin riƙewarsa yayi daidai da sabon kololuwar da aka gani.Wannan gaskiyar ta watsar da ra'ayin cewa ba kawai kayan farawa na iya ƙunsar duk wani mahadi ko abubuwan da zasu iya tsoma baki tare da hanyar bincike ba, amma babu alamar yiwuwar ƙetare tare da sauran kayan aikin dakin gwaje-gwaje.Ana nuna ƙimar tattarawa da aka samu bayan nazarin LC-MS na BDDE da sabbin kololuwa a cikin Tebur 2 (samfurori 1-4) da chromatogram ion a cikin Hoto 4.
Lura: Samfuran 1-4 sun dace da albarkatun ƙasa da aka yi amfani da su don samar da autoclaved BDDE giciye HA hydrogels.Waɗannan samfuran ba a haɗa su ta atomatik ba.
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa.
Hoto 4 yayi daidai da LC-MS chromatogram na samfurin albarkatun albarkatun da aka yi amfani da su a cikin haɗin kai na HA da BDDE.
Lura: Duk waɗannan ana auna su ne a cikin taro iri ɗaya da rabon da aka yi amfani da su don aiwatar da halayen haɗin kai.Lambobi don albarkatun albarkatun da chromatogram ya bincika sun dace da: (1) ruwa, (2) 2% maganin ruwa na HA, (3) 1% NaOH maganin ruwa.Ana yin nazarin LC-MS don yawan jujjuyawar 203.30/129.10 Da (a cikin ingantaccen yanayin MRM).
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa.
An yi nazarin yanayin da ya haifar da samuwar sababbin kololuwa.Don yin nazarin yadda yanayin halayen da aka yi amfani da su don samar da haɗin gwiwar HA hydrogel ya shafi mayar da martani na wakilin BDDE mai haɗin gwiwa, wanda ke haifar da sababbin kololuwa (samfuran da za a iya samu), an yi ma'auni daban-daban.A cikin waɗannan ƙayyadaddun ƙayyadaddun, mun yi nazari da kuma nazarin BDDE crosslinker na ƙarshe, wanda aka bi da shi tare da nau'i daban-daban na NaOH (0%, 1%, 0.1%, da 0.01%) a cikin matsakaici mai ruwa, wanda ya biyo baya ko ba tare da autoclaving ba.Hanyar ƙwayoyin cuta don kwaikwaya yanayi iri ɗaya ne da hanyar da ake amfani da ita don samar da haɗin giciye HA hydrogel.Kamar yadda aka bayyana a cikin sashin "Kayan Kayayyaki da Hanyoyi", LC-MS an yi nazari akan yawan canjin samfurin zuwa 203.30 / 129.10 Da.Ana ƙididdige BDDE da ƙaddamar da sabon kololuwa, kuma an nuna sakamakon a cikin Table 3. Babu sabon kololuwa da aka gano a cikin samfuran da ba a sarrafa su ba, ko da kuwa kasancewar NaOH a cikin maganin (samfurori 1-4, Tables). 3).Don samfurori na autoclaved, ana gano sababbin kololuwa kawai a gaban NaOH a cikin bayani, kuma samuwar kololuwar alama ya dogara da ƙaddamarwar NaOH a cikin maganin (samfurori 5-8, Table 3) (RRT = 0.79).Hoto na 5 yana nuna misali na chromatogram ion, yana nuna samfurori guda biyu da aka haɗa a gaban ko babu NAOH.
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa.
Lura: Babban chromatogram: An bi da samfurin tare da 0.1% NaOH bayani mai ruwa da kuma autoclaved (120 ° C na minti 20).Ƙarƙashin chromatogram na ƙasa: Ba a bi da samfurin tare da NaOH ba, amma an sanya shi ta atomatik a ƙarƙashin yanayi iri ɗaya.LC-MS yayi nazari akan yawan juzu'i na 203.30/129.10 Da (a cikin ingantaccen yanayin MRM).
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa.
A cikin duk samfurori da aka yi da autoclaved, tare da ko ba tare da NaOH ba, an rage yawan ƙwayar BDDE sosai (har zuwa sau 16.6) (samfurori 5-8, Table 2).Ragewar maida hankali na BDDE na iya kasancewa saboda gaskiyar cewa a yanayin zafi mai girma, ruwa na iya aiki azaman tushe (nucleophile) don buɗe zoben epoxide na BDDE don samar da fili mai 1,2-diol.Ingancin monoisotopic na wannan fili ya bambanta da na BDDE don haka ba zai shafa ba.LC-MS ya gano yawan motsi na 203.30/129.10 Da.
A ƙarshe, waɗannan gwaje-gwajen sun nuna cewa haɓakar sabbin kololuwa ya dogara da kasancewar BDDE, NAOH, da tsarin autoclaving, amma ba shi da alaƙa da HA.
Sabon kololuwar da aka samu a lokacin riƙewa na kusan mintuna 2.71 sannan an siffanta shi da LC-MS.Don wannan dalili, an sanya BDDE (9.9 mg/mL) a cikin maganin ruwa na 1% NaOH kuma an sanya shi ta atomatik.A cikin Tebur 4, ana kwatanta halayen sabon kololuwa tare da sanannen kololuwar tunani na BDDE (lokacin riƙewa kusan mintuna 3.47).Dangane da bincike na ion fragmentation na kololuwa biyu, ana iya kammala cewa kololuwar tare da lokacin riƙewa na mintuna 2.72 yana nuna guntu iri ɗaya kamar kololuwar BDDE, amma tare da ƙarfi daban-daban (Hoto 6).Don kololuwar daidai da lokacin riƙewa (PIS) na mintuna 2.72, an sami ƙarin kololuwa mai ƙarfi bayan rarrabuwa a taro na 147 Da.A BDDE maida hankali (9.9 mg / mL) da aka yi amfani da shi a cikin wannan ƙaddarar, nau'ikan shayarwa daban-daban (UV, λ = 200 nm) a cikin bakan ultraviolet kuma an lura da su bayan rabuwar chromatographic (Hoto 7).Kololuwa tare da lokacin riƙewa na mintuna 2.71 har yanzu ana iya gani a 200 nm, yayin da ba za a iya ganin kololuwar BDDE a cikin chromatogram ƙarƙashin yanayi iri ɗaya ba.
Tebur 4 Sakamakon Halayen sabon kololuwa tare da lokacin riƙewa na kusan mintuna 2.71 da kololuwar BDDE tare da lokacin riƙewa na mintuna 3.47
Lura: Don samun waɗannan sakamakon, an yi nazarin LC-MS da HPLC (MRM da PIS) akan kololuwar biyu.Don nazarin HPLC, ana amfani da ganowar UV tare da tsawon 200 nm.
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;HPLC, high yi ruwa chromatography;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa;m/z, rabon taro-zuwa-caji;PIS, samfurin ion dubawa;hasken ultraviolet, hasken ultraviolet.
Lura: Ana samun gutsuwar taro ta hanyar bincike na LC-MS (PIS).Babban chromatogram: babban bakan na BDDE daidaitaccen guntun samfurin.Ƙarƙashin chromatogram na ƙasa: Yawan adadin sabon kololuwar da aka gano (RRT mai alaƙa da kololuwar BDDE shine 0.79).An sarrafa BDDE a cikin 1% NaOH bayani kuma an sanya shi ta atomatik.
Takaitacce: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, ruwa chromatography da taro spectrometry;MRM, saka idanu da yawa;PIS, samfurin ion scan;RRT, lokacin riƙewa dangi.
Hoto 7 Ion chromatogram na 203.30 Da precursor ion, da (A) sabon kololuwa tare da lokacin riƙewa na mintuna 2.71 da (B) ganowar UV na daidaitaccen ma'auni na BDDE a mintuna 3.46 a 200 nm.
A cikin duk haɗin haɗin HA hydrogels da aka samar, an lura cewa ragowar BDDE maida hankali bayan ƙididdigar LC-MS shine <2 ppm, amma sabon kololuwar da ba a sani ba ya bayyana a cikin bincike.Wannan sabon kololuwar bai dace da daidaitaccen samfurin BDDE ba.Madaidaicin samfurin BDDE shima an yi juzu'i mai inganci iri ɗaya (Juyin MRM 203.30/129.10 Da) a cikin ingantaccen yanayin MRM.Gabaɗaya, ana amfani da wasu hanyoyin bincike kamar chromatography azaman iyaka gwaje-gwaje don gano BDDE a cikin hydrogels, amma matsakaicin iyakar ganowa (LOD) ya ɗan yi ƙasa da 2 ppm.A gefe guda, ya zuwa yanzu, an yi amfani da NMR da MS don kwatanta matakin haɗin kai da / ko gyare-gyare na HA a cikin sassan sukari na samfuran HA masu haɗin gwiwa.Manufar waɗannan fasahohin ba ta taɓa kasancewa don ƙididdige ganowar BDDE na saura ba a irin wannan ƙananan ƙima kamar yadda muka bayyana a cikin wannan labarin (LOD na hanyar LC-MS ɗin mu = 10 ppb).
Lokacin aikawa: Satumba-01-2021