Javascript nwere nkwarụ ugbu a na ihe nchọgharị gị.Mgbe Javascript kwụsịrị, ụfọdụ ọrụ nke webụsaịtị a agaghị arụ ọrụ.
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 * Ndị edemede a nwere nghọta ụfọdụ banyere ọrụ a Ha nhata. nkwado ndabere: Hyaluronic acid (HA) bụ polysaccharide na-emekarị nke a na-eji na-emepụta ihe na-emepụta ihe na-eme ka akpụkpọ ahụ dị mma maka ebumnuche ịchọ mma.Ebe ọ bụ na ọ nwere ọkara ndụ nke ọtụtụ ụbọchị n'ime anụ ahụ mmadụ, ihe ndị na-emepụta ihe na-emepụta ihe na-emepụta HA na-emezi ka ha gbasaa ndụ ha n'ime ahụ.Mgbanwe a na-emekarị na ndị na-emepụta HA na-azụ ahịa bụ iji 1,4-butanediol diglycidyl ether (BDDE) dị ka onye na-ahụ maka crosslinking iji jikọta ụdọ HA.A na-ewere BDDE fọdụrụnụ ma ọ bụ nke na-emeghị ihe ọ bụla na-adịghị egbu egbu na <2 akụkụ kwa nde (ppm);ya mere, BDDE fọdụrụ na nke ikpeazụ dermal filler ga-agụrịrị iji hụ na nchekwa onye ọrịa.Akụrụngwa na ụzọ: Ọmụmụ ihe a na-akọwa nchọpụta na njirimara nke ngwaahịa nke mmeghachi omume na-ejikọta ọnụ n'etiti BDDE na HA n'okpuru ọnọdụ alkaline site na ijikọta chromatography mmiri na spectrometry (LC-MS).Nsonaazụ: Mgbe nyochachara dị iche iche, achọpụtara na ọnọdụ alkaline na oke okpomọkụ a na-eji kpochapụ HA-BDDE hydrogel kwalitere nguzobe nke ngwaahịa ọhụrụ a, ihe “propylene glycol-like”.Nyocha LC-MS kwadoro na site-ngwaahịa nwere otu monoisotopic uka dị ka BDDE, oge njigide dị iche (tR), na ụdị nnabata UV dị iche (λ=200 nm).N'adịghị ka BDDE, a hụrụ ya na nyocha LC-MS na n'okpuru otu ọnọdụ nha, ngwaahịa a nwere ọnụọgụ nchọpụta dị elu na 200 nm.Mkpebi: Nsonaazụ ndị a na-egosi na ọ nweghị epoxide na nhazi nke ogige ọhụrụ a.Mkparịta ụka a ghere oghe iji nyochaa ihe ize ndụ nke ngwaahịa ọhụrụ a dị na mmepụta nke HA-BDDE hydrogel (HA dermal filler) maka ebumnuche azụmahịa.Keywords: hyaluronic acid, HA dermal filler, cross-linked hyaluronic acid, BDDE, LC-MS analysis, BDDE by-product.
Ihe ndochi dabere na hyaluronic acid (HA) bụ ihe nchacha akpụkpọ anụ na-ewu ewu na-ejikarị eme ihe ịchọ mma.1 Nke a dermal filler bụ hydrogel, nke na-emekarị> 95% mmiri na 0.5-3% HA, nke na-enye ha usoro dị ka gel.2 HA bụ polysaccharide na akụkụ bụ isi nke matrix extracellular nke vertebrates.Otu n'ime ihe ndị ahụ.Ọ mejupụtara (1,4) -glucuronic acid-β (1,3) -N-acetylglucosamine (GlcNAc) nkeji disaccharide na-emegharị site na njikọ glycosidic.Ụdị disaccharide a bụ otu n'ime ihe niile dị ndụ.E jiri ya tụnyere ụfọdụ ihe na-eme protein (dị ka collagen), ihe onwunwe a na-eme HA ka ọ bụrụ molekul dakọtara nke ukwuu.Ihe ndochi ndị a nwere ike igosipụta nkọwapụta usoro amino acid nke usoro ahụ ji alụso ọrịa ọgụ nwere ike mata.
Mgbe a na-eji ya dị ka ihe mkpuchi anụ ahụ, njedebe bụ isi nke HA bụ ntụgharị ngwa ngwa n'ime anụ ahụ n'ihi ọnụnọ nke otu ezinụlọ nke enzymes a na-akpọ hyaluronidases.Ka ọ dị ugbu a, a kọwawo ọtụtụ mgbanwe kemịkalụ na nhazi HA iji mee ka ọkara ndụ HA dị na anụ ahụ.3 Ọtụtụ n'ime mgbanwe ndị a na-anwa ibelata ohere nke hyaluronidase na polysaccharide polymers site na njikọ njikọ HA.Ya mere, n'ihi nguzobe nke àkwà mmiri na intermolecular covalent bonds n'etiti ihe owuwu HA na cross-linking agents, cross-linked HA hydrogel na-emepụta ihe ndị na-emebi emebi enzyme karịa ihe okike HA.4-6
Ka ọ dị ugbu a, ihe ndị na-eme ka ndị na-eme ihe na-emekọrịta ihe na-emepụta HA gụnyere methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) na poly (ethylene glycol) diglycidyl ether.10,11 BDDE bụ onye na-emekọrịta ihe na-ejikarị eme ihe ugbu a.Ọ bụ ezie na ụdị hydrogels ndị a egosipụtara na ọ dị mma ruo ọtụtụ iri afọ, ndị ọrụ na-emekọrịta ihe eji eme ihe bụ reactive reactive nke nwere ike ịbụ cytotoxic na, n'ọnọdụ ụfọdụ, mutagenic.12 Ya mere, ọdịnaya ha fọdụrụ na hydrogel ikpeazụ ga-adị elu.A na-ahụta BDDE dị ka nchekwa ma ọ bụrụ na ntinye ihe fọdụrụ erughị akụkụ 2 kwa nde (ppm).4
Enwere ụzọ dị iche iche iji chọpụta ntinye BDDE dị ala, ogo njikọ njikọ na ọnọdụ nnọchi na HA hydrogels, dị ka chromatography gas, chromatography ewepu nha yana yana spectrometry mass spectrometry (MS), resonance magnetik (NMR) ụzọ nha fluorescence, na Array diode jikọtara chromatography mmiri mmiri dị elu (HPLC).13-17 Ọmụmụ ihe a na-akọwa nchọpụta na njirimara nke ngwaahịa na njedebe ikpeazụ njikọ HA hydrogel nke mmeghachi omume nke BDDE na HA na-emepụta n'okpuru ọnọdụ alkaline.HPLC na mmiri chromatography-mass spectrometry (LC-MS analysis).Ebe ọ bụ na amabeghị nsi nke ngwaahịa BDDE a, anyị na-akwado ka ekpebie ngụpụta nke fọdụrụ ya n'ụzọ yiri usoro a na-emekarị na BDDE na ngwaahịa ikpeazụ.
Nnu sodium enwetara nke HA (Shiseido Co., Ltd., Tokyo, Japan) nwere ihe dị arọ nke ~ 1,368,000 Da (usoro Laurent) 18 na viscosity dị n'ime 2.20 m3 / kg.Maka mmeghachi omume na-agafe agafe, BDDE (≥95%) zụtara n'aka Sigma-Aldrich Co. (St. Louis, MO, USA).Azụtara saline phosphate nwere pH 7.4 n'aka Sigma-Aldrich Company.Ihe mgbaze niile, acetonitrile na mmiri ejiri na nyocha LC-MS zụtara site na ogo ọkwa HPLC.A na-azụta formic acid (98%) dị ka ọkwa reagent.
Emere nnwale niile na sistemu UPLC Acquity (Waters, Milford, MA, USA) wee jikọọ na API 3000 triple quadrupole mass spectrometer nke nwere isi iyi ionization electrospray (AB SCIEX, Framingham, MA, USA).
A malitere njikọ nke cross-linked HA hydrogels site n'ịgbakwunye 198 mg nke BDDE na 10% (w / w) sodium hyaluronate (NaHA) ngwọta na ọnụnọ nke 1% alkali (sodium hydroxide, NaOH).Ntinye BDDE ikpeazụ na ngwakọta mmeghachi omume bụ 9.9 mg/mL (0.049 mM).Mgbe ahụ, ngwakọta mmeghachi omume na-agwakọta nke ọma na homogenized ma kwe ka ọ gaa n'ihu na 45 Celsius maka awa 4.19 A na-edobe pH nke mmeghachi omume na ~ 12.
Mgbe nke ahụ gasịrị, a na-asacha ngwakọta mmeghachi omume ahụ na mmiri, na HA-BDDE hydrogel ikpeazụ bụ nke a na-enyocha ma gbanye ya na PBS buffer iji nweta ntinye HA nke 10 ruo 25 mg / mL, na pH ikpeazụ nke 7.4.Iji mee ka mmiri ọgwụ HA hydrogels na-ejikọta ya, a na-ekpuchi hydrogels ndị a niile (120 Celsius C maka nkeji 20).A na-echekwa BDDE-HA hydrogel dị ọcha na 4 Celsius ruo mgbe nyocha.
Iji nyochaa BDDE dị na ngwaahịa HA jikọtara cross, a tụrụ atụ 240 mg ma weba ya n'ime oghere etiti (Microcon®; Merck Millipore, Billerica, MA, USA; olu 0.5 mL) na centrifuged na 10,000 rpm na ụlọ okpomọkụ. Nkeji 10.Ngụkọta nke 20µL nke mmiri mmiri na-agbadata ka anakọtara wee nyochaa ya.
Iji nyochaa ọkọlọtọ BDDE (Sigma-Aldrich Co) n'okpuru ọnọdụ alkaline (1%, 0.1% na 0.01% NaOH), ọ bụrụ na emezuru ọnọdụ ndị a, ihe nlele mmiri bụ 1:10, 1:100, ma ọ bụ ruo 1: 1,000,000 Ọ bụrụ na ọ dị mkpa, jiri MilliQ deionized mmiri maka nyocha.
Maka ihe mmalite ndị a na-eji eme ihe na-emekọrịta ihe (HA 2%, H2O, 1% NaOH, na 0.049 mM BDDE), 1 mL nke ihe nlele ọ bụla a kwadebere site na ihe ndị a na-enyocha site na iji otu ọnọdụ nyocha.
Iji chọpụta nkọwapụta nke ọnụ ọgụgụ kacha elu pụtara na maapụ ion, 10 µL nke 100 ppb BDDE ọkọlọtọ ngwọta (Sigma-Aldrich Co) ka agbakwunyere na nlele 20µL.N'okwu a, ntinye ikpeazụ nke ọkọlọtọ na nlele ọ bụla bụ 37 pb.
Nke mbụ, kwadebe ngwọta ngwaahịa BDDE na ntinye nke 11,000 mg/L (11,000 ppm) site na ịgbanye 10 μL nke ọkọlọtọ BDDE (Sigma-Aldrich Co) na mmiri 990 μL MilliQ (njupụta 1.1 g / ml).Jiri ngwọta a iji dozie ngwọta BDDE 110 μg/L (110 ppb) dị ka mgbaze ọkọlọtọ etiti.Mgbe ahụ, jiri ihe nrịbama ọkọlọtọ BDDE etiti (110 ppb) kwadebe usoro ọkọlọtọ site n'ịgbanye mgbaze etiti ọtụtụ oge iji nweta mkpokọta achọrọ nke 75, 50, 25, 10, na 1 ppb.Dịka e gosiri na eserese 1, a na-achọpụta na usoro ọkọlọtọ BDDE sitere na 1.1 ruo 110 ppb nwere ezigbo linearity (R2>0.99).A na-emegharị usoro ọkọlọtọ ahụ na nnwale anọ nọọrọ onwe ya.
Ọgụgụ 1 BDDE ọkọlọtọ calibration curve nwetara site na nyocha LC-MS, nke a na-ahụ ezigbo mmekọrịta (R2>0.99).
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography mmiri mmiri na spectrometry mass.
Iji chọpụta na ọnụ ọgụgụ BDDE ụkpụrụ dị na cross-linked HA na BDDE ụkpụrụ na isi ngwọta, LC-MS analysis jiri.
Emechara nkewa nke chromatographic na kọlụm LUNA 2.5 µm C18 (2) -HST (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) ma debe ya na ụlọ okpomọkụ (25 Celsius) n'oge nyocha.Usoro mkpanaka nwere acetonitrile (ihe mgbaze A) na mmiri (ihe mgbaze B) nwere 0.1% formic acid.A na-ewepụ usoro mkpanaka site na gradient elution.The gradient bụ dị ka ndị a: 0 nkeji, 2% A;1 nkeji, 2% A;Nkeji 6, 98% A;Nkeji 7, 98% A;7.1 nkeji, 2% A;Nkeji 10, 2% A. Oge ịgba ọsọ bụ nkeji iri yana olu injection bụ 20µL.Oge njide nke BDDE bụ ihe dịka nkeji 3.48 (site na nkeji 3.43 ruo 4.14 dabere na nnwale).A na-agbanye usoro mkpanaaka na ọsọ ọsọ nke 0.25 mL / min maka nyocha LC-MS.
N'ihi na BDDE analysis na quantification site MS, UPLC usoro (Mmiri) na-ejikọta na API 3000 triple quadrupole mass spectrometer (AB SCIEX) nke nwere ihe electrospray ionization isi iyi, na nyocha na-eme na nti ion mode (ESI +).
Dị ka nyocha nke ion nke a na-eme na BDDE, a na-ekpebi nkebi nke nwere ike kachasị elu ka ọ bụrụ mpempe akwụkwọ kwekọrọ na 129.1 Da (Nyocha 6).Ya mere, na multi-ion nlekota mode (MIM) maka quantification, ntughari uka (mass-to-charge ratio [m/z]) nke BDDE bụ 203.3/129.1 Da.Ọ na-ejikwa ọnọdụ nyocha zuru oke (FS) yana ọnọdụ nyocha ion ngwaahịa (PIS) maka nyocha LC-MS.
Iji nyochaa nkọwapụta nke usoro ahụ, a nyochara ihe nlele oghere (akụkụ mbụ mkpanaka).Enweghị akara ngosi achọpụtara na nlele oghere nwere ntughari oke nke 203.3/129.1 Da.Banyere nkwughachi nke nnwale ahụ, a nyochara 10 ọkọlọtọ injections nke 55 ppb (n'etiti etiti calibration curve), na-ebute mgbanwe ọkọlọtọ fọdụrụ (RSD) <5% (data egosighi).
Agụpụtara ọdịnaya BDDE fọdụrụnụ n'ime ọkụ HA hydrogels nwere njikọ BDDE asatọ dị iche iche, dabara na nnwale anọ nọọrọ onwe ya.Dị ka akọwara na ngalaba " Akụrụngwa na Ụzọ ", a na-enyocha ọnụọgụgụ site na nkezi uru nke regression curve nke BDDE ọkọlọtọ dilution, nke kwekọrọ na ọnụ ọgụgụ kasị elu nke achọpụtara na BDDE mass mgbanwe nke 203.3 / 129.1 Da, na njigide. oge nke 3.43 ruo 4.14 nkeji anaghị echere.Ọgụgụ 2 na-egosi ihe atụ chromatogram nke ọkọlọtọ ntụnye aka 10ppb BDDE.Tebụl 1 na-achịkọta ọdịnaya BDDE fọdụrụnụ nke hydrogels asatọ dị iche iche.Ọnụ ahịa dị nso bụ 1 ruo 2.46 ppb.Ya mere, ntinye BDDE fọdụrụnụ na nlele ahụ bụ nke a na-anabata maka ojiji mmadụ (<2 ppm).
Ọgụgụ 2 Ion chromatogram nke 10 ppb BDDE ụkpụrụ ntụaka (Sigma-Aldrich Co), mgbanwe MS (m/z) nwetara site na nyocha LC-MS nke 203.30/129.10 Da (na ọnọdụ MRM dị mma).
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, otutu mmeghachi omume nlekota oru;MS, oke;m/z, oke-na-ụcha.
Mara: Ihe nlele 1-8 bụ BDDE nwere njikọ chiri anya HA hydrogels.Ọnụ ego BDDE fọdụrụnụ na hydrogel na ọnụ ọgụgụ kacha elu nke oge njide BDDE ka na-akọkwa.N'ikpeazụ, a na-akọkwa ịdị adị nke elu ọhụrụ nwere oge njide dị iche iche.
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;MRM, otutu mmeghachi omume nlekota oru;tR, oge njide;LC-MS, chromatography mmiri mmiri na spectrometry mass;RRT, oge njigide.
N'ụzọ dị ịtụnanya, nyocha nke LC-MS ion chromatogram gosiri na dabere na ihe nlele HA hydrogel nke jikọtara ya na autoclaved nke a na-enyocha, enwere mmụba ọzọ na obere oge njide nke 2.73 ruo 3.29 nkeji.Dịka ọmụmaatụ, eserese 3 na-egosi ion chromatogram nke ihe nlele HA na-ejikọta ya, ebe ọnụ ọgụgụ ọzọ na-apụta n'oge njide dị iche nke ihe dịka 2.71 nkeji.Achọpụtara oge njide ikwu (RRT) n'etiti ọnụ ọgụgụ kacha elu a hụrụ ọhụrụ na ọnụ ọgụgụ kacha elu sitere na BDDE bụ 0.79 (Table 1).Ebe ọ bụ na anyị maara na ọnụ ọgụgụ kasị elu a hụrụ ọhụrụ ejighị n'aka na kọlụm C18 e ji mee ihe na nyocha LC-MS, ọnụ ọgụgụ ọhụrụ ahụ nwere ike ịdabere na ngwakọta polar karịa BDDE.
Ọgụgụ 3 Ion chromatogram nke ihe atụ HA hydrogel jikọrọ cross-linked nwetara site na LC-MS (MRM mass ntughari 203.3 / 129.0 Da).
Mbiri: HA, hyaluronic acid;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, otutu mmeghachi omume nlekota oru;RRT, oge njide ikwu;tR, oge njide.
Iji wepụta ohere na ọnụ ọgụgụ ọhụrụ a hụrụ nwere ike bụrụ ihe mmetọ dị na ngwa ngwa eji eme ihe, a na-enyochakwa akụrụngwa ndị a site na iji otu usoro nyocha LC-MS.Ihe mmalite nke nyochara gụnyere mmiri, 2% NaHA n'ime mmiri, 1% NaOH na mmiri, na BDDE n'otu ntinye uche a na-eji na mmeghachi omume na-agafe agafe.ion chromatogram nke mmalite ihe eji eme ihe egosighi ihe ọ bụla ma ọ bụ ọnụ ọgụgụ dị elu, na oge njigide ya kwekọrọ na elu ọhụrụ a hụrụ.Eziokwu a na-atụfu echiche ahụ na ọ bụghị naanị ihe mmalite nwere ike ịnwe ogige ma ọ bụ ihe ọ bụla nwere ike igbochi usoro nyocha, ma ọ dịghị ihe ịrịba ama nke nwere ike ịmefe obe na ngwaahịa ụlọ nyocha ndị ọzọ.A na-egosi ụkpụrụ ntinye uche nke enwetara mgbe nyocha LC-MS nke BDDE na ọnụ ọgụgụ ọhụrụ dị na Tebụl 2 (ihe atụ 1-4) na ion chromatogram na Figure 4.
Mara: Nlereanya 1-4 kwekọrọ na akụrụngwa eji emepụta BDDE cross-linked HA hydrogels.Emebeghị ihe nlele ndị a n'onwe ha.
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, nlekota nzaghachi otutu.
Ọnụọgụ 4 kwekọrọ na LC-MS chromatogram nke ihe atụ nke akụrụngwa eji eme ihe na-emekọrịta ihe nke HA na BDDE.
Rịba ama: A na-atụ ihe ndị a niile n'otu ntinye uche na nha nke ejiri mee mmeghachi omume njikọ cross.Ọnụọgụ maka akụrụngwa nke chromatogram tụlere kwekọrọ na: (1) mmiri, (2) 2% HA ngwọta mmiri, (3) 1% NaOH mmiri mmiri.A na-eme nyocha LC-MS maka ngbanwe oke nke 203.30/129.10 Da (na ọnọdụ MRM dị mma).
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, nlekota nzaghachi otutu.
A mụrụ ọnọdụ ndị mere ka e guzobe elu ọhụrụ.Iji mụọ otú ọnọdụ mmeghachi omume na-esi na-emepụta HA hydrogel na-ejikọta ya na-emetụta mmeghachi omume nke onye na-ahụ maka BDDE cross-linking, na-eduga n'ịmepụta ọnụ ọgụgụ ọhụrụ (ngwaahịa nwere ike ime), a na-eme nha dị iche iche.Na mkpebi ndị a, anyị mụrụ ma nyochaa BDDE crosslinker ikpeazụ, bụ nke a na-emeso ya dị iche iche nke NaOH (0%, 1%, 0.1%, na 0.01%) na mmiri mmiri, na-esote ma ọ bụ na-enweghị autoclaving.Usoro nje bacteria iji mee ka otu ọnọdụ ahụ dị ka usoro eji emepụta HA hydrogel na-ejikọta ya.Dị ka akọwara na ngalaba "Ihe na Ụzọ", LC-MS nyochaa ngbanwe nke ihe nlele ahụ na 203.30 / 129.10 Da.A na-agbakọ BDDE na ntinye uche nke ọnụ ọgụgụ ọhụrụ ahụ, a na-egosipụtakwa nsonaazụ ya na Tebụl 3. Achọpụtaghị ọnụ ọgụgụ ọhụrụ ọ bụla n'ime ihe atụ ndị a na-emeghị nke ọma, n'agbanyeghị ọnụnọ NaOH na ngwọta (ihe atụ 1-4, Tebụl). 3).Maka ihe atụ nke autoclaved, a na-achọpụta ọnụ ọgụgụ ọhụrụ naanị n'ihu NaOH na ngwọta, na nhazi nke elu ahụ yiri ka ọ dabere na ntinye uche NaOH na ngwọta (ihe atụ 5-8, Tebụl 3) (RRT = 0.79).Ọgụgụ 5 na-egosi ihe atụ nke ion chromatogram, na-egosi ihe atụ abụọ autoclaved na ọnụnọ ma ọ bụ enweghị NAOH.
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, nlekota nzaghachi otutu.
Rịba ama: chromatogram dị elu: A na-emeso ihe nlele ahụ na 0.1% NaOH aqueous solution na autoclaved (120 ° C maka nkeji 20).chromatogram dị n'okpuru: Ejighị NaOH emeso ihe nlele ahụ, mana ejiri ya mee ihe n'otu ọnọdụ ahụ.LC-MS tụlere ntughari oke nke 203.30/129.10 Da (na ọnọdụ MRM dị mma).
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, nlekota nzaghachi otutu.
N'ime ihe nlele autoclaved, yana ma ọ bụ na-enweghị NaOH, a na-ebelata mkpokọta BDDE nke ukwuu (ruo ugboro 16.6) (nlele 5-8, Tebụl 2).Mbelata nke ntinye uche BDDE nwere ike ịbụ n'ihi na na okpomọkụ dị elu, mmiri nwere ike ịrụ ọrụ dị ka ntọala (nucleophile) iji mepee mgbanaka epoxide nke BDDE iji mepụta ogige 1,2-diol.Ogo monoisotopic nke ogige a dị iche na nke BDDE ya mere agaghị emetụta ya.LC-MS chọpụtara ngbanwe oke nke 203.30/129.10 Da.
N'ikpeazụ, nyocha ndị a na-egosi na ọgbọ nke ọnụ ọgụgụ ọhụrụ na-adabere na ọnụnọ nke BDDE, NAOH, na usoro autoclaving, ma ọ nweghị ihe jikọrọ ya na HA.
Ọhụụ ọhụrụ a chọtara n'oge njide dị ihe dị ka nkeji 2.71 bụ nke LC-MS mara ya.Maka nzube a, BDDE (9.9 mg/mL) ka etinyere na ngwọta mmiri 1% NaOH na autoclaved.Na Tebụl 4, a na-atụnyere njirimara nke ọnụ ọgụgụ ọhụrụ ahụ na ọnụ ọgụgụ ntụaka BDDE ama ama (oge njide ihe dịka nkeji 3.47).Dabere na nyocha nke nbibi nke ion nke ọnụ ọgụgụ abụọ ahụ, enwere ike ikwubi na ọnụ ọgụgụ kacha elu nwere oge njide nke nkeji 2.72 na-egosi otu iberibe dị ka ọnụ ọgụgụ BDDE, mana nwere ike dị iche iche (Nyocha 6).Maka ọnụ ọgụgụ kachasị elu nke kwekọrọ na oge njide (PIS) nke nkeji 2.72, a na-ahụ ọnụ ọgụgụ dị elu karị mgbe nkewasịrị na oke nke 147 Da.Na mkpokọta BDDE (9.9 mg / mL) ejiri mee mkpebi a, a na-ahụkwa ụdị nnabata dị iche iche (UV, λ = 200 nm) na ụdị ultraviolet mgbe nkewa chromatographic gasịrị (Nyocha 7).A ka na-ahụ ọnụ ọgụgụ kasị elu nke nwere oge njide nke nkeji 2.71 na 200 nm, ebe enweghị ike ịhụ ọnụ ọgụgụ BDDE na chromatogram n'okpuru otu ọnọdụ ahụ.
Tebụl 4 Ngosipụta njirimara nke ọnụ ọgụgụ ọhụrụ nwere oge njide ihe dị ka nkeji 2.71 yana ọnụ ọgụgụ BDDE nwere oge njide nke nkeji 3.47.
Mara: Iji nweta nsonaazụ ndị a, a na-eme nyocha LC-MS na HPLC (MRM na PIS) na elu abụọ ahụ.Maka nyocha HPLC, a na-eji nchọpụta UV nwere ogologo 200 nm.
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;HPLC, chromatography mmiri mmiri dị elu;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, otutu mmeghachi omume nlekota oru;m/z, oke-na-ụcha;PIS, nyocha ion ngwaahịa;ìhè ultraviolet, ìhè ultraviolet.
Mara: A na-enweta iberibe uka ndị ahụ site na nyocha LC-MS (PIS).chromatogram kacha elu: nnukwu ụdịdị dị iche iche nke iberibe ihe atụ ọkọlọtọ BDDE.chromatogram nke ala: Achọpụtara ụdị dị iche iche nke ọnụ ọgụgụ ọhụrụ (RRT jikọtara ya na ọnụ ọgụgụ BDDE bụ 0.79).A na-ahazi BDDE na 1% NaOH ngwọta na autoclaved.
Mbiri: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography mmiri mmiri na spectrometry mass;MRM, otutu mmeghachi omume nlekota oru;PIS, nyocha ion ngwaahịa;RRT, oge njigide.
Ọgụgụ 7 Ion chromatogram nke 203.30 Da precursor ion, na (A) ọnụ ọgụgụ ọhụrụ nwere oge njide nke nkeji 2.71 na (B) nchọpụta UV nke ụkpụrụ ntụaka BDDE na nkeji 3.46 na 200 nm.
N'ime njikọ HA hydrogels niile jikọtara ọnụ, a hụrụ na ntinye BDDE fọdụrụnụ mgbe ọnụọgụ LC-MS gachara bụ <2 ppm, mana ọnụ ọgụgụ ọhụrụ amaghi ama pụtara na nyocha.Ọnụọgụ ọhụrụ a adabaghị na ngwaahịa ọkọlọtọ BDDE.Ngwaahịa ọkọlọtọ BDDE abanyekwala n'otu ngbanwe dị mma (mgbanwe MRM 203.30/129.10 Da) na ọnọdụ MRM dị mma.N'ozuzu, a na-eji ụzọ nyocha ndị ọzọ dị ka chromatography dị ka ule oke iji chọpụta BDDE na hydrogels, mana njedebe nchọpụta kachasị (LOD) dị ntakịrị ala karịa 2 ppm.N'aka nke ọzọ, ka ọ dị ugbu a, a na-eji NMR na MS mee ihe iji kọwaa ogo nke njikọ njikọ na / ma ọ bụ mgbanwe nke HA na mpempe akwụkwọ sugar nke ngwaahịa HA.Ebumnuche nke usoro ndị a abụbeghị ịkọwapụta nchọpụta BDDE fọdụrụnụ na oke dị ala dị ka anyị kọwara n'isiokwu a (LOD nke usoro LC-MS anyị = 10 ppb).
Oge nzipu: Sep-01-2021