Javascript ndiyoyimitsidwa pa msakatuli wanu pano.Javascript ikayimitsidwa, zina zatsambali sizigwira ntchito.
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 * Olemba awa ali ndi zidziwitso zina pa ntchitoyi Equal maziko operekera: Hyaluronic acid (HA) ndi polysaccharide yopezeka mwachilengedwe yomwe imagwiritsidwa ntchito popanga ma dermal fillers pofuna kukongoletsa.Popeza ili ndi theka la moyo wa masiku angapo m'matumbo aumunthu, HA-based dermal fillers amasinthidwa ndi mankhwala kuti atalikitse moyo wawo m'thupi.Kusintha kofala kwambiri pazamalonda za HA-based fillers ndikugwiritsa ntchito 1,4-butanediol diglycidyl ether (BDDE) ngati cholumikizira cholumikizira kuti chiwoloke unyolo wa HA.BDDE yotsalira kapena yosakhudzidwa imatengedwa kuti si ya poizoni pa <2 magawo pa milioni (ppm);Choncho, BDDE yotsalira mu dermal filler yomaliza iyenera kuwerengedwa kuti iwonetsetse chitetezo cha odwala.Zipangizo ndi Njira: Kafukufukuyu akufotokoza za kuzindikirika ndi kuzindikirika kwa njira yolumikizirana pakati pa BDDE ndi HA pansi pamikhalidwe yamchere mwa kuphatikiza chromatography yamadzi ndi ma spectrometry (LC-MS).Zotsatira: Pambuyo pa kusanthula kosiyana, zinapezeka kuti zinthu zamchere ndi kutentha kwakukulu komwe kumagwiritsidwa ntchito kupha tizilombo toyambitsa matenda a HA-BDDE hydrogel kumalimbikitsa kupanga mapangidwe atsopanowa, "propylene glycol-like" pawiri.Kusanthula kwa LC-MS kunatsimikizira kuti chopangidwacho chili ndi misa yofanana ya monoisotopic monga BDDE, nthawi yosungira yosiyana (tR), ndi njira yosiyana ya UV absorbance (λ=200 nm).Mosiyana ndi BDDE, zidawonedwa mu kusanthula kwa LC-MS kuti pansi pamiyeso yofananira, chopangira ichi chimakhala ndi chiwopsezo chapamwamba cha 200 nm.Kutsiliza: Zotsatirazi zikuwonetsa kuti palibe epoxide mu kapangidwe katsopano kameneka.Zokambiranazo ndi zotseguka kuti ziwone kuopsa kwa chinthu chatsopanochi chomwe chimapezeka popanga HA-BDDE hydrogel (HA dermal filler) pofuna malonda.Keywords: asidi hyaluronic, HA dermal filler, cross-linked hyaluronic acid, BDDE, LC-MS kusanthula, BDDE by-product.
Zodzaza zochokera ku hyaluronic acid (HA) ndizomwe zimagwiritsidwa ntchito popanga zodzikongoletsera.1 Izi dermal filler ndi hydrogel, kawirikawiri amakhala> 95% madzi ndi 0.5-3% HA, amene amawapatsa gel osakaniza kapangidwe.2 HA ndi polysaccharide ndipo chigawo chachikulu cha extracellular matrix of vertebrates.Chimodzi mwa zosakaniza.Amakhala ndi (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) yobwereza ma disaccharide unit omwe amagwirizanitsidwa ndi glycosidic bonds.Mtundu wa disaccharide uwu ndi wofanana m'zamoyo zonse.Poyerekeza ndi zodzaza ndi mapuloteni (monga collagen), katunduyu amapangitsa HA kukhala molekyulu yogwirizana kwambiri ndi biocompatible.Zodzaza izi zimatha kuwonetsa kutsatizana kwa amino acid komwe kungazindikiridwe ndi chitetezo chamthupi cha wodwalayo.
Akagwiritsidwa ntchito ngati dermal filler, cholepheretsa chachikulu cha HA ndikusintha kwake mwachangu mkati mwa minyewa chifukwa cha kupezeka kwa gulu linalake la michere yotchedwa hyaluronidases.Pakadali pano, zosintha zingapo zamakina mu kapangidwe ka HA zafotokozedwa kuti zikuwonjezera theka la moyo wa HA m'matenda.3 Zambiri mwazosinthazi zimayesa kuchepetsa kupezeka kwa hyaluronidase ku ma polima a polysaccharide polumikizana ndi unyolo wa HA.Chifukwa chake, chifukwa cha mapangidwe a milatho ndi ma intermolecular covalent bond pakati pa HA kapangidwe ndi njira yolumikizira mtanda, HA hydrogel yolumikizana ndi mtanda imapanga zinthu zambiri zowononga anti-enzyme kuposa za HA zachilengedwe.4-6
Pakalipano, mankhwala ophatikizira omwe amagwiritsidwa ntchito popanga HA crosslinked ndi methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) Ndi poly(ethylene glycol) diglycidyl ether.10 ,11 BDDE pakadali pano ndiyomwe imagwiritsidwa ntchito kwambiri polumikizirana.Ngakhale mitundu iyi ya ma hydrogel yatsimikiziridwa kuti ndi yotetezeka kwazaka zambiri, zolumikizira zomwe zimagwiritsidwa ntchito ndi zotulutsa zomwe zitha kukhala cytotoxic ndipo, nthawi zina, mutagenic.12 Chifukwa chake, zotsalira zawo mu hydrogel yomaliza ziyenera kukhala zapamwamba.BDDE imatengedwa kuti ndi yotetezeka pamene ndende yotsalirayo ili yochepa kuposa magawo awiri pa milioni (ppm).4
Pali njira zingapo zodziwira ndende ya BDDE yotsalira, digiri yolumikizirana ndikulowa m'malo mu ma hydrogel a HA, monga gasi chromatography, kukula kwa chromatography kuphatikiza ndi mass spectrometry (MS), nyukiliya maginito resonance (NMR) njira zoyezera fluorescence, ndi Diode array kuphatikiza high performance liquid chromatography (HPLC).13-17 Kafukufukuyu akufotokoza za kudziwika ndi maonekedwe a chinthu chopangidwa ndi HA hydrogel yomaliza yopangidwa ndi machitidwe a BDDE ndi HA pansi pa zinthu zamchere.HPLC ndi liquid chromatography-mass spectrometry (LC-MS analysis).Popeza kuti kawopsedwe ka mankhwalawa a BDDE sikudziwika, timalimbikitsa kuti zotsalira zake zitsimikizidwe mofanana ndi njira yomwe nthawi zambiri imachitidwa pa BDDE pomaliza.
Mchere wa sodium wopezedwa wa HA (Shiseido Co., Ltd., Tokyo, Japan) uli ndi molekyulu yolemera ~ 1,368,000 Da (njira ya Laurent) 18 ndi kukhuthala kwamkati kwa 2.20 m3 / kg.Chifukwa cha crosslinking reaction, BDDE (≥95%) inagulidwa ku Sigma-Aldrich Co. (St. Louis, MO, USA).Phosphate wokhala ndi mchere wokhala ndi pH 7.4 adagulidwa ku Sigma-Aldrich Company.Zosungunulira zonse, acetonitrile ndi madzi omwe amagwiritsidwa ntchito pakuwunika kwa LC-MS adagulidwa kuchokera kugulu la HPLC.Formic acid (98%) imagulidwa ngati kalasi ya reagent.
Zoyeserera zonse zidachitika pa UPLC Acquity system (Waters, Milford, MA, USA) ndikulumikizidwa ndi API 3000 triple quadrupole mass spectrometer yokhala ndi gwero la electrospray ionization (AB SCIEX, Framingham, MA, USA).
Kaphatikizidwe ka ma HA hydrogel olumikizana ndi mtanda adayambika ndikuwonjezera 198 mg ya BDDE ku 10% (w/w) sodium hyaluronate (NaHA) yankho pamaso pa 1% alkali (sodium hydroxide, NaOH).Chomaliza BDDE ndende mu anachita osakaniza anali 9.9 mg/mL (0.049 mM).Kenako, zomwe osakaniza anali bwinobwino osakaniza ndi homogenized ndi kuloledwa chitani pa 45 ° C kwa 4 hours.19 pH ya zomwe zimachitika imasungidwa pa ~ 12.
Pambuyo pake, kusakaniza kotereku kunatsukidwa ndi madzi, ndipo chomaliza cha HA-BDDE hydrogel chinasefedwa ndi kuchepetsedwa ndi PBS buffer kuti akwaniritse HA ndende ya 10 mpaka 25 mg / mL, ndi pH yomaliza ya 7.4.Pofuna kuthira ma hydrogel opangidwa ndi HA, ma hydrogel onsewa amapangidwa ndi autoclaved (120 ° C kwa mphindi 20).BDDE-HA hydrogel yoyeretsedwa imasungidwa pa 4 ° C mpaka kusanthula.
Kuti muwunike BDDE yomwe ilipo muzogulitsa za HA, chitsanzo cha 240 mg chinayesedwa ndikulowetsedwa mu dzenje lapakati (Microcon®; Merck Millipore, Billerica, MA, USA; voliyumu 0.5 mL) ndi centrifuged pa 10,000 rpm kutentha kwapakati. 10 miniti.Zonse za 20 µL zamadzimadzi zokokera pansi zidasonkhanitsidwa ndikuwunikidwa.
Pofuna kusanthula muyezo wa BDDE (Sigma-Aldrich Co) pansi pa zinthu zamchere (1%, 0.1% ndi 0.01% NaOH), ngati zotsatirazi zikwaniritsidwa, chitsanzo chamadzimadzi ndi 1:10, 1:100, kapena mpaka 1: 1,000,000 Ngati kuli kofunikira, gwiritsani ntchito madzi a MilliQ deionized kuti muwunike.
Pazinthu zoyambira zomwe zimagwiritsidwa ntchito polumikizirana (HA 2%, H2O, 1% NaOH, ndi 0.049 mm BDDE), 1 mL yachitsanzo chilichonse chokonzedwa kuchokera kuzinthu izi idawunikidwa pogwiritsa ntchito njira zomwezo.
Kuti mudziwe zenizeni za nsonga zomwe zikuwonekera pamapu a ion, 10 µL ya 100 ppb BDDE njira yokhazikika (Sigma-Aldrich Co) idawonjezedwa ku chitsanzo cha 20 µL.Pachifukwa ichi, chiwerengero chomaliza cha muyeso uliwonse ndi 37 ppb.
Choyamba, konzani njira yothetsera BDDE yokhala ndi 11,000 mg/L (11,000 ppm) pochepetsa 10 μL ya BDDE wamba (Sigma-Aldrich Co) ndi 990 μL MilliQ madzi (kachulukidwe 1.1 g/mL).Gwiritsani ntchito yankho ili pokonzekera yankho la 110 µg/L (110 ppb) BDDE ngati njira yapakatikati yoyezera.Kenako, gwiritsani ntchito njira yapakatikati ya BDDE diluent (110 ppb) kuti mukonze mapindikidwe okhazikika pochepetsa kusungunuka kwapakatikati kangapo kuti mukwaniritse kuchuluka komwe mukufuna kwa 75, 50, 25, 10, ndi 1 ppb.Monga momwe tawonetsera pa Chithunzi 1, zikuwoneka kuti BDDE curve kuchokera ku 1.1 mpaka 110 ppb ili ndi mzere wabwino (R2> 0.99).Mpiringidzo wokhazikika unabwerezedwa muzoyesera zinayi zodziimira.
Chithunzi cha 1 BDDE curve calibration calibration yopezedwa ndi LC-MS kusanthula, momwe kulumikizana kwabwino kumawonedwa (R2> 0.99).
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, madzi chromatography ndi misa spectrometry.
Pofuna kuzindikira ndi kuwerengera miyezo ya BDDE yomwe ilipo mu HA yolumikizidwa ndi BDDE muzitsulo zoyambira, kusanthula kwa LC-MS kunagwiritsidwa ntchito.
Kupatukana kwa chromatographic kunapezedwa pa LUNA 2.5 µm C18 (2) -HST (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) ndikusungidwa kutentha (25 ° C) panthawi yofufuza.Gawo la mafoni limapangidwa ndi acetonitrile (zosungunulira A) ndi madzi (zosungunulira B) okhala ndi 0.1% formic acid.Gawo la mafoni limasinthidwa ndi gradient elution.The gradient ndi motere: Mphindi 0, 2% A;1 mphindi, 2% A;Mphindi 6, 98% A;Mphindi 7, 98% A;Mphindi 7.1, 2% A;Mphindi 10 , 2% A. Nthawi yothamanga ndi mphindi 10 ndipo voliyumu ya jekeseni ndi 20 µL.Nthawi yosungira BDDE ndi pafupi maminiti a 3.48 (kuchokera ku 3.43 mpaka maminiti a 4.14 kutengera zoyesera).Gawo loyendetsa galimoto linaponyedwa pamtunda wa 0.25 mL / min pakuwunika kwa LC-MS.
Pakuwunika kwa BDDE ndi kuchuluka kwa MS, dongosolo la UPLC (Waters) limaphatikizidwa ndi API 3000 triple quadrupole mass spectrometer (AB SCIEX) yokhala ndi gwero la electrospray ionization, ndipo kusanthula kumachitika munjira yabwino ya ion (ESI +).
Malingana ndi kusanthula kwachidutswa cha ion chomwe chinachitidwa pa BDDE, chidutswacho chokhala ndi mphamvu kwambiri chinatsimikiziridwa kukhala chidutswa chofanana ndi 129.1 Da (Chithunzi 6).Choncho, mu multi-ion monitoring mode (MIM) ya quantification, kutembenuka kwakukulu (mass-to-charge ratio [m / z]) kwa BDDE ndi 203.3 / 129.1 Da.Imagwiritsanso ntchito mawonekedwe a scan (FS) ndi mawonekedwe a ion scan (PIS) pakuwunika kwa LC-MS.
Pofuna kutsimikizira tsatanetsatane wa njirayo, chitsanzo chopanda kanthu (gawo loyamba la mafoni) linawunikidwa.Palibe chizindikiro chomwe chinapezeka mu chitsanzo chopanda kanthu ndi kutembenuka kwakukulu kwa 203.3 / 129.1 Da.Ponena za kubwereza kuyesera, jekeseni wa 10 wa 55 ppb (pakati pa curve calibration) adawunikidwa, zomwe zinachititsa kuti pakhale kusiyana kotsalira (RSD) <5% (deta yosasonyezedwa).
Zotsalira za BDDE zidawerengedwa m'ma hydrogel asanu ndi atatu osiyana a BDDE olumikizidwa ndi HA, olingana ndi zoyeserera zinayi zodziyimira pawokha.Monga tafotokozera mu gawo la "Zida ndi Njira", kuchuluka kwake kumawunikidwa ndi mtengo wapakati wa regression curve wa BDDE dilution standard, zomwe zimagwirizana ndi nsonga yapadera yomwe imapezeka pa BDDE mass transition of 203.3 / 129.1 Da, ndi kusungirako nthawi ya 3.43 kuti 4.14 mphindi Osadikirira.Chithunzi 2 chikuwonetsa chitsanzo cha chromatogram cha 10 ppb BDDE reference standard.Table 1 ikufotokoza mwachidule za BDDE zotsalira za ma hydrogel asanu ndi atatu.Mtengo wake ndi 1 mpaka 2.46 ppb.Choncho, zotsalira za BDDE mu chitsanzo ndizovomerezeka kuti zigwiritsidwe ntchito ndi anthu (<2 ppm).
Chithunzi 2 Ion chromatogram ya 10 ppb BDDE reference standard (Sigma-Aldrich Co), MS (m/z) kusintha kopezedwa ndi LC-MS kusanthula kwa 203.30 / 129.10 Da (mu njira yabwino ya MRM).
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kambiri kachitidwe;MS, misa;m/z, chiŵerengero cha misa-to-charge.
Zindikirani: Zitsanzo 1-8 ndi autoclaved BDDE cross-linked HA hydrogels.Kuchuluka kotsalira kwa BDDE mu hydrogel ndi nsonga ya nthawi yosungira ya BDDE imanenedwanso.Potsirizira pake, kukhalapo kwa nsonga zatsopano zokhala ndi nthawi zosungirako zosiyana kumanenedwanso.
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;HA, asidi hyaluronic;MRM, kuwunika kambiri kachitidwe;tR, nthawi yosungira;LC-MS, madzi chromatography ndi misa spectrometry;RRT, nthawi yosungirako.
Chodabwitsa n'chakuti kusanthula kwa LC-MS ion chromatogram kunasonyeza kuti kutengera zitsanzo zonse za HA hydrogel zomwe zimagwirizanitsidwa ndi autoclaved cross-linked, panali chiwongoladzanja chowonjezera pa nthawi yochepa yosungiramo 2.73 mpaka 3.29 mphindi.Mwachitsanzo, Chithunzi 3 chikuwonetsa chromatogram ya ion ya chitsanzo cha HA cholumikizidwa, pomwe chiwongola dzanja chowonjezera chikuwonekera pa nthawi yosunga yosiyana pafupifupi mphindi 2.71.Nthawi yosungidwa yosungidwa (RRT) pakati pa nsonga yomwe idawonedwa kumene ndi nsonga yochokera ku BDDE idapezeka kuti ndi 0.79 (Table 1).Popeza tikudziwa kuti nsonga yomwe yangowonedwa kumene imasungidwa pang'ono mu gawo la C18 lomwe limagwiritsidwa ntchito mu kusanthula kwa LC-MS, nsonga yatsopanoyo imatha kufanana ndi gulu la polar kuposa BDDE.
Chithunzi cha 3 Ion chromatogram ya mtanda wa HA hydrogel chitsanzo chopezedwa ndi LC-MS (MRM mass conversion 203.3 / 129.0 Da).
Zidule: HA, asidi hyaluronic;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kambiri kachitidwe;RRT, nthawi yosunga wachibale;tR, nthawi yosungira.
Pofuna kuletsa kuthekera kwakuti nsonga zatsopano zomwe zawonedwa zingakhale zodetsa zomwe zidalipo kale muzopangira zogwiritsidwa ntchito, zida izi zidawunikidwanso pogwiritsa ntchito njira yowunikira ya LC-MS.Zida zoyambira zomwe zimawunikidwa zimaphatikizapo madzi, 2% NaHA m'madzi, 1% NaOH m'madzi, ndi BDDE pamlingo womwewo womwe umagwiritsidwa ntchito polumikizirana.Chromatogram ya ion yazinthu zoyambira zomwe idagwiritsidwa ntchito sinawonetse nsonga kapena nsonga, ndipo nthawi yosungira imafanana ndi nsonga yatsopano yomwe yawonedwa.Izi zimataya lingaliro lakuti osati zinthu zoyambira zokha zomwe zingakhale ndi mankhwala kapena zinthu zomwe zingasokoneze ndondomeko ya kusanthula, koma palibe chizindikiro cha kuipitsidwa ndi zinthu zina za labotale.Miyezo yomwe idapezedwa pambuyo pa kusanthula kwa LC-MS kwa BDDE ndi nsonga zatsopano zikuwonetsedwa mu Table 2 (zitsanzo 1-4) ndi ion chromatogram pa Chithunzi 4.
Zindikirani: Zitsanzo 1-4 zimagwirizana ndi zida zomwe zimagwiritsidwa ntchito popanga ma autoclaved BDDE cross-linked HA hydrogels.Zitsanzozi sizinali autoclaved.
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;HA, asidi hyaluronic;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kwamachitidwe angapo.
Chithunzi 4 chikugwirizana ndi LC-MS chromatogram ya chitsanzo cha zopangira zomwe zimagwiritsidwa ntchito polumikizana ndi HA ndi BDDE.
Zindikirani: Zonsezi zimayesedwa pamlingo womwewo komanso chiyerekezo chomwe chimagwiritsidwa ntchito pochita zomwe zimagwirizanitsa.Manambala a zinthu zopangira zomwe zafufuzidwa ndi chromatogram zimagwirizana ndi: (1) madzi, (2) 2% HA aqueous solution, (3) 1% NaOH aqueous solution.Kusanthula kwa LC-MS kumapangidwira kutembenuka kwakukulu kwa 203.30 / 129.10 Da (mu njira yabwino ya MRM).
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;HA, asidi hyaluronic;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kwamachitidwe angapo.
Mikhalidwe yomwe inachititsa kuti pakhale nsonga zatsopano zinaphunziridwa.Kuti tiphunzire momwe zinthu zomwe zimagwiritsidwira ntchito popanga HA hydrogel yolumikizidwa ndi mtanda imakhudzanso reactivity ya BDDE yolumikizana ndi wothandizila, zomwe zimapangitsa kuti pakhale nsonga zatsopano (zotheka ndi zopangidwa), miyeso yosiyana idachitidwa.Pazidziwitso izi, tidaphunzira ndikusanthula cholumikizira chomaliza cha BDDE, chomwe chidathandizidwa ndi magawo osiyanasiyana a NaOH (0%, 1%, 0.1%, ndi 0.01%) mu sing'anga yamadzi, yotsatiridwa kapena popanda autoclaving.Njira ya mabakiteriya yotengera momwe zinthu ziliri ndizofanana ndi njira yomwe imagwiritsidwa ntchito popanga HA hydrogel yolumikizana.Monga tafotokozera mu gawo la "Zida ndi Njira", kusintha kwakukulu kwa chitsanzocho kunayesedwa ndi LC-MS ku 203.30 / 129.10 Da.BDDE ndi chiwerengero cha nsonga zatsopano zimawerengedwa, ndipo zotsatira zake zikuwonetsedwa mu Table 3. Palibe nsonga zatsopano zomwe zinapezedwa mu zitsanzo zomwe sizinali za autoclaved, mosasamala kanthu za kukhalapo kwa NaOH mu yankho (zitsanzo 1-4, Table. 3).Kwa zitsanzo za autoclaved, nsonga zatsopano zimangopezeka pamaso pa NaOH mu yankho, ndipo mapangidwe apamwamba akuwoneka kuti amadalira chiwerengero cha NaOH mu yankho (zitsanzo 5-8, Table 3) (RRT = 0.79).Chithunzi 5 chikuwonetsa chitsanzo cha chromatogram ya ion, yomwe ikuwonetsa zitsanzo ziwiri za autoclaved pamaso kapena kusakhalapo kwa NAOH.
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kwamachitidwe angapo.
Zindikirani: Chromatogram yapamwamba: Chitsanzocho chinachiritsidwa ndi 0.1% NaOH yamadzimadzi amadzimadzi ndi autoclaved (120 ° C kwa mphindi 20).Chromatogram yapansi: Chitsanzocho sichinachiritsidwe ndi NaOH, koma chopangidwa ndi autoclaved pansi pamikhalidwe yomweyi.Kutembenuka kwakukulu kwa 203.30 / 129.10 Da (mu njira yabwino ya MRM) kunafufuzidwa ndi LC-MS.
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kwamachitidwe angapo.
Mu zitsanzo zonse za autoclaved, kapena popanda NaOH, chiwerengero cha BDDE chinachepetsedwa kwambiri (mpaka nthawi 16.6) (zitsanzo 5-8, Table 2).Kuchepa kwa chiwerengero cha BDDE kungakhale chifukwa chakuti kutentha kwakukulu, madzi amatha kukhala maziko (nucleophile) kuti atsegule mphete ya epoxide ya BDDE kuti apange 1,2-diol compound.Ubwino wa monoisotopic wamtunduwu ndi wosiyana ndi wa BDDE ndipo chifukwa chake sudzakhudzidwa.LC-MS idazindikira kusintha kwakukulu kwa 203.30 / 129.10 Da.
Pomaliza, mayeserowa amasonyeza kuti kubadwa kwa nsonga zatsopano kumadalira kukhalapo kwa BDDE, NAOH, ndi ndondomeko ya autoclaving, koma ilibe kanthu kochita ndi HA.
Pachimake chatsopano chomwe chinapezeka pa nthawi yosungira pafupifupi mphindi 2.71 kenako chimadziwika ndi LC-MS.Pachifukwa ichi, BDDE (9.9 mg / mL) inayikidwa mu 1% NaOH yankho lamadzimadzi ndi autoclaved.Mu Table 4, mawonekedwe a nsonga yatsopano amafananizidwa ndi nsonga yodziwika ya BDDE (nthawi yosungira pafupifupi mphindi 3.47).Malingana ndi kusanthula kwa ion kugawanika kwa nsonga ziwiri, tingathe kunena kuti chiwongoladzanja chokhala ndi nthawi yosungiramo mphindi 2.72 chimasonyeza zidutswa zofanana ndi nsonga ya BDDE, koma ndi mphamvu zosiyana (Chithunzi 6).Pachimake chofanana ndi nthawi yosungira (PIS) ya maminiti a 2.72, chiwongoladzanja chowonjezereka chinawonedwa pambuyo pa kugawanika pa misa ya 147 Da.Pa BDDE ndende (9.9 mg/mL) yogwiritsidwa ntchito potsimikiza izi, mitundu yosiyanasiyana ya kuyamwa (UV, λ = 200 nm) mu ultraviolet spectrum inawonedwanso pambuyo pa kupatukana kwa chromatographic (Chithunzi 7).Pachimake chokhala ndi nthawi yosungiramo mphindi 2.71 chikuwonekerabe pa 200 nm, pomwe nsonga ya BDDE siyingawonekere mu chromatogram pansi pamikhalidwe yomweyi.
Table 4 Makhalidwe amatsatira pachimake chatsopano ndi nthawi yosungira pafupifupi mphindi 2.71 ndi BDDE pachimake ndi nthawi yosungira ya 3.47 mphindi
Zindikirani: Kuti mupeze zotsatira izi, kusanthula kwa LC-MS ndi HPLC (MRM ndi PIS) kunachitika pamapiri awiriwa.Pakuwunika kwa HPLC, kuzindikira kwa UV ndi kutalika kwa 200 nm kumagwiritsidwa ntchito.
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;HPLC, mkulu ntchito madzi chromatography;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kambiri kachitidwe;m/z, chiŵerengero cha misa-to-charge;PIS, mankhwala Ion kupanga sikani;kuwala kwa ultraviolet, kuwala kwa ultraviolet.
Chidziwitso: Zidutswa zazikuluzikulu zimapezedwa ndi LC-MS kusanthula (PIS).Chromatogram yapamwamba: ma sipekitiramu ambiri a zidutswa za zitsanzo za BDDE.Chromatogram yapansi: Kuchuluka kwa nsonga yatsopano yomwe yapezeka (RRT yolumikizidwa ndi nsonga ya BDDE ndi 0.79).BDDE idakonzedwa mu 1% NaOH yankho ndi autoclaved.
Mafupipafupi: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, madzi chromatography ndi misa spectrometry;MRM, kuwunika kambiri kachitidwe;PIS, mankhwala ion scan;RRT, nthawi yosungirako.
Chithunzi 7 Ion chromatogram ya 203.30 Da precursor ion, ndi (A) pachimake chatsopano chokhala ndi nthawi yosungira mphindi 2.71 ndi (B) kuzindikira kwa UV kwa BDDE reference standard pachimake pa mphindi 3.46 pa 200 nm.
M'magawo onse a HA hydrogel opangidwa ndi mtanda, adawona kuti BDDE yotsalira pambuyo pa LC-MS quantification inali <2 ppm, koma nsonga yatsopano yosadziwika idawonekera pakuwunika.Chiwongola dzanja chatsopanochi sichikufanana ndi mankhwala wamba a BDDE.Chogulitsa chokhazikika cha BDDE chakhalanso ndi kusinthika kwamtundu womwewo (MRM kutembenuka 203.30 / 129.10 Da) kusanthula mumayendedwe abwino a MRM.Nthawi zambiri, njira zina zowunikira monga chromatography zimagwiritsidwa ntchito ngati kuyesa malire kuti azindikire BDDE mu ma hydrogel, koma malire ozindikira (LOD) ndi otsika pang'ono kuposa 2 ppm.Kumbali ina, mpaka pano, NMR ndi MS zakhala zikugwiritsidwa ntchito kuwonetsa kuchuluka kwa kulumikizana ndi / kapena kusinthidwa kwa HA mu zidutswa za shuga za HA zopangidwa ndi HA.Cholinga cha njirazi sichinakhalepo kuwerengera zotsalira za BDDE pazigawo zochepa monga momwe tafotokozera m'nkhaniyi (LOD ya njira yathu ya LC-MS = 10 ppb).
Nthawi yotumiza: Sep-01-2021