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ʻO Javier Fidalgo, * Pierre-Antoine Deglesne, * Rodrigo Arroyo, * Lilian Sepúlveda, * Evgeniya Ranneva, Philippe Deprez Department of Science, Skin Tech Pharma Group, Castello D'Empúries, Catalonia, Sepania * Loaʻa i kēia mau mea kākau kekahi mau ʻike i kēia hana Equal kāʻei kua hāʻawi: Hyaluronic acid (HA) he polysaccharide kūlohelohe i hoʻohana ʻia i ka hana ʻana i nā mea hoʻopiha dermal no nā kumu aesthetic.No ka mea he hapalua kona mau lā i loko o nā ʻiʻo kanaka, ua hoʻololi ʻia nā mea hoʻopihapiha dermal HA-based e hoʻolōʻihi i ko lākou ola i ke kino.ʻO ka hoʻololi maʻamau i nā mea hoʻopihapiha hoʻopihapiha HA kūʻai aku ʻo ia ka hoʻohana ʻana i ka 1,4-butanediol diglycidyl ether (BDDE) ma ke ʻano he crosslinking agent e crosslink i nā kaulahao HA.Manaʻo ʻia ʻo BDDE koena a i ʻole i hoʻokō ʻia he mea ʻawaʻawa ʻole ma <2 ʻāpana o ka miliona (ppm);no laila, pono e helu ʻia ke koena BDDE i ka hoʻopiha dermal hope e hōʻoia i ka palekana o ka maʻi.Mea a me nā ʻano hana: Hōʻike kēia haʻawina i ka ʻike ʻana a me ke ʻano o kahi huahana o ka hopena hoʻohui kea ma waena o BDDE a me HA ma lalo o nā kūlana alkaline ma o ka hoʻohui ʻana i ka chromatography wai a me ka spectrometry nuipa (LC-MS).Nā hualoaʻa: Ma hope o nā loiloi like ʻole, ua ʻike ʻia ʻo nā kūlana alkaline a me ka wela kiʻekiʻe i hoʻohana ʻia e disinfect i ka HA-BDDE hydrogel i hāpai i ka hoʻokumu ʻana o kēia huahana hou, ʻo ka "propylene glycol-like" compound.Ua hōʻoia ka loiloi LC-MS he like ka nui monoisotopic o ka huahana me BDDE, kahi manawa paʻa ʻokoʻa (tR), a me kahi ʻano ʻokoʻa o ka UV absorbance (λ=200 nm).ʻAʻole like me BDDE, ua ʻike ʻia ma ka loiloi LC-MS ma lalo o nā kūlana ana like, ʻoi aku ka kiʻekiʻe o kēia huahana ma ka 200 nm.Ka hopena: Hōʻike kēia mau hopena ʻaʻohe epoxide i loko o ke ʻano o kēia pūhui hou.Ua wehe ʻia ke kūkākūkā e loiloi i ka pilikia o kēia huahana hou i loaʻa i ka hana ʻana o HA-BDDE hydrogel (HA dermal filler) no nā kumu kūʻai.Hua'ōlelo: hyaluronic acid, HA dermal filler, cross-linked hyaluronic acid, BDDE, LC-MS analysis, BDDE by-product.
ʻO nā mea hoʻopihapiha e pili ana i ka hyaluronic acid (HA) ka mea maʻamau a kaulana hoʻi i hoʻohana ʻia no nā kumu hoʻonaninani.1 He hydrogel kēia hoʻopiha dermal, maʻamau i haku ʻia he> 95% wai a me 0.5-3% HA, e hāʻawi iā lākou i kahi ʻano gel-like.ʻO 2 HA he polysaccharide a me ka mea nui o ka matrix extracellular o nā vertebrates.ʻO kekahi o nā meaʻai.Aia ia me (1,4)-glucuronic acid-β (1,3)-N-acetylglucosamine (GlcNAc) e hana hou ana i nā ʻāpana disaccharide i hoʻopili ʻia e nā paʻa glycosidic.Ua like kēia ʻano disaccharide i nā mea ola a pau.Ke hoʻohālikelike ʻia me kekahi mau mea hoʻopihapiha hoʻopihapiha hoʻopihapiha (e like me ka collagen), hana kēia waiwai iā HA he moleke biocompatible loa.Hiki i kēia mau mea hoʻopihapiha ke hōʻike i kahi kikoʻī kikoʻī o ke kaʻina amino acid i ʻike ʻia e ka ʻōnaehana pale o ka mea maʻi.
Ke hoʻohana ʻia ma ke ʻano he dermal filler, ʻo ka palena nui o HA ʻo ia kona huli wikiwiki ʻana i loko o nā ʻiʻo ma muli o ke alo o kahi ʻohana kikoʻī o nā enzyme i kapa ʻia he hyaluronidases.I kēia manawa, ua wehewehe ʻia kekahi mau hoʻololi kemika i ka hoʻolālā HA e hoʻonui i ka hapalua o ke ola o HA i loko o nā ʻiʻo.3 Ke ho'āʻo nei ka hapa nui o kēia mau hoʻololi e hōʻemi i ke komo ʻana o ka hyaluronidase i nā polysaccharide polymers ma o ka hoʻopili ʻana i nā kaulahao HA.No laila, ma muli o ka hoʻokumu ʻia ʻana o nā alahaka a me nā mea hoʻopaʻa covalent intermolecular ma waena o ka hale HA a me ka mea hoʻohui keʻa, ua hoʻopuka ka hydrogel HA i hoʻopili ʻia i nā huahana anti-enzyme degradation ma mua o ka HA maoli.4-6
I kēia manawa, ʻo nā mea hoʻohui kemika i hoʻohana ʻia no ka hana ʻana i ka crosslinked HA me ka methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) A me ka poly(ethylene glycol) diglycidyl ether.ʻO 10 ,11 BDDE i kēia manawa ka mea hoʻohana maʻamau i hoʻohana ʻia.ʻOiai ua hōʻoia ʻia kēia mau ʻano hydrogels i ka palekana no nā makahiki he nui, ʻo nā mea hoʻohui crosslinking i hoʻohana ʻia he reagent reactive i cytotoxic a, i kekahi mau hihia, mutagenic.12 No laila, ʻoi aku ka kiʻekiʻe o kā lākou koena i loko o ka hydrogel hope.Manaʻo ʻia ʻo BDDE he palekana ke emi ke koena o ke koena ma mua o 2 ʻāpana o ka miliona (ppm).4
Nui nā ʻano hana e ʻike ai i ka ʻike haʻahaʻa haʻahaʻa haʻahaʻa BDDE, cross-linking degere a me ke kūlana pani i loko o HA hydrogels, e like me ke kinoea chromatography, ka nui exclusion chromatography i hui pū ʻia me mass spectrometry (MS), nuclear magnetic resonance (NMR) fluorescence ana ana, a Ua hui pū ʻia nā ʻano diode kiʻekiʻe (HPLC).13-17 Hōʻike kēia haʻawina i ka ʻike ʻana a me ka ʻike ʻana o kahi huahana i loko o ka hydrogel HA i hoʻopaʻa ʻia ke keʻa hope i hana ʻia e ka hopena o BDDE a me HA ma lalo o nā kūlana alkaline.HPLC a me ka wai chromatography-mass spectrometry (LC-MS analysis).No ka mea, ʻaʻole ʻike ʻia ka ʻawaʻawa o kēia huahana o BDDE, manaʻo mākou e hoʻoholo ʻia kona helu ʻana i ke ʻano like me ke ʻano hana maʻamau ma BDDE i ka huahana hope.
ʻO ka paʻakai sodium i loaʻa mai o HA (Shiseido Co., Ltd., Tokyo, Iapana) he molecular kaumaha o ~1,368,000 Da (Laurent method) 18 a me ka viscosity intrinsic o 2.20 m3/kg.No ka hopena crosslinking, ua kūʻai ʻia ʻo BDDE (≥95%) mai Sigma-Aldrich Co. (St. Louis, MO, USA).Ua kūʻai ʻia ʻo Phosphate buffered saline me ka pH 7.4 mai Sigma-Aldrich Company.Ua kūʻai ʻia nā mea hoʻoheheʻe a pau, acetonitrile a me ka wai i hoʻohana ʻia i ka loiloi LC-MS mai ka HPLC grade quality.Kūʻai ʻia ka waika Formic (98%) ma ke ʻano he reagent grade.
Ua hana ʻia nā hoʻokolohua āpau ma kahi ʻōnaehana UPLC Acquity (Waters, Milford, MA, USA) a pili i kahi API 3000 triple quadrupole mass spectrometer i hoʻolako ʻia me kahi kumu ionization electrospray (AB SCIEX, Framingham, MA, USA).
Ua hoʻomaka ʻia ka hoʻohui ʻana o nā hydrogels HA i hoʻopili ʻia me ka hoʻohui ʻana i ka 198 mg o BDDE i kahi solution 10% (w/w) sodium hyaluronate (NaHA) i mua o 1% alkali (sodium hydroxide, NaOH).ʻO ka manaʻo hope loa o BDDE i ka hui ʻana he 9.9 mg/mL (0.049 mM).A laila, hui maikaʻi ʻia ka hui ʻana a me ka homogenized a ʻae ʻia e hoʻomau ma 45 ° C no 4 mau hola.19 Mālama ʻia ka pH o ka hopena ma ~12.
Ma hope iho, holoi ʻia ka hui ʻana me ka wai, a ua kānana ʻia ka hydrogel HA-BDDE hope loa a hoʻoheheʻe ʻia me PBS buffer no ka loaʻa ʻana o kahi haʻahaʻa HA o 10 a 25 mg/mL, a me ka pH hope o 7.4.No ka hoʻomaʻemaʻe ʻana i nā hydrogels HA i hoʻopaʻa ʻia, ua hoʻopaʻa ʻia kēia mau hydrogel a pau (120°C no 20 mau minuke).Mālama ʻia ka BDDE-HA hydrogel i hoʻomaʻemaʻe ʻia ma 4 ° C a hiki i ka nānā ʻana.
No ke kālailai ʻana i ka BDDE i loaʻa i loko o ka huahana HA i hoʻopili ʻia, ua kaupaona ʻia kahi hāpana 240 mg a hoʻokomo ʻia i loko o ka lua waena (Microcon®; Merck Millipore, Billerica, MA, USA; volume 0.5 mL) a centrifuged ma 10,000 rpm i ka lumi wela. 10 minuke.He 20 µL o ka wai huki iho i hōʻiliʻili ʻia a nānā ʻia.
No ke kālailai ʻana i ka maʻamau BDDE (Sigma-Aldrich Co) ma lalo o nā kūlana alkaline (1%, 0.1% a me 0.01% NaOH), inā e hoʻokō ʻia kēia mau kūlana, ʻo ka hāpana wai ka 1:10, 1:100, a i ʻole. 1:1,000,000 Inā pono, e hoʻohana i ka wai deionized MilliQ no ka nānā ʻana.
No nā mea hoʻomaka i hoʻohana ʻia i ka hopena hoʻohui kea (HA 2%, H2O, 1% NaOH, a me 0.049 mM BDDE), 1 mL o kēlā me kēia hāpana i hoʻomākaukau ʻia mai kēia mau mea i kālai ʻia me ka hoʻohana ʻana i nā kūlana hoʻohālikelike like.
No ka hoʻoholo ʻana i ke ʻano kikoʻī o nā piko i ʻike ʻia ma ka palapala ion, 10 µL o 100 ppb BDDE solution standard (Sigma-Aldrich Co) i hoʻohui ʻia i ka laʻana 20 µL.I kēia hihia, ʻo 37 ppb ka pae hope loa o ka maʻamau i kēlā me kēia hāpana.
ʻO ka mua, e hoʻomākaukau i kahi solution stock BDDE me ka hoʻonui ʻana o 11,000 mg/L (11,000 ppm) ma ka diluting 10 μL o BDDE maʻamau (Sigma-Aldrich Co) me 990 μL MilliQ wai (density 1.1 g/mL).E hoʻohana i kēia hoʻonā no ka hoʻomākaukau ʻana i kahi solution BDDE 110 µg/L (110 ppb) ma ke ʻano he dilution maʻamau waena.A laila, e hoʻohana i ka mea hoʻoheheʻe maʻamau BDDE waena (110 ppb) e hoʻomākaukau i ka pihi maʻamau ma o ka hoʻoheheʻe ʻana i ka diluent waena i mau manawa e loaʻa ai ka manaʻo i makemake ʻia o 75, 50, 25, 10, a me 1 ppb.E like me ka mea i hōʻike ʻia ma ka Figure 1, ua ʻike ʻia he laina laina maikaʻi ka BDDE maʻamau mai 1.1 a 110 ppb (R2>0.99).Ua hana houʻia ka'ōkuhi maʻamau i nā hoʻokolohua kūʻokoʻaʻehā.
Kiʻi 1 BDDE maʻamau calibration curve i loaʻa ma ka LC-MS analysis, kahi i ʻike ʻia ai kahi pilina maikaʻi (R2>0.99).
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography wai a me ka spectrometry lehulehu.
No ka ʻike ʻana a me ka helu ʻana i nā kūlana BDDE i loaʻa i ka HA i hoʻopili ʻia a me nā kūlana BDDE i ka hopena kumu, ua hoʻohana ʻia ka loiloi LC-MS.
Loaʻa ka hoʻokaʻawale chromatographic ma kahi kolamu LUNA 2.5 µm C18 (2) -HST (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) a mālama ʻia ma ka lumi wela (25°C) i ka wā o ka nānā ʻana.ʻO ka māhele paʻa he acetonitrile (solvent A) a me ka wai (solvent B) i loaʻa ka 0.1% formic acid.Hoʻopau ʻia ka pae paʻa e ka elution gradient.Penei ka gradient: 0 minuke, 2% A;1 minuke, 2% A;6 minuke, 98% A;7 minuke, 98% A;7.1 minuke, 2% A;10 minuke, 2% A. ʻO ka manawa holo he 10 mau minuke a ʻo ka nui o ka injection he 20 µL.ʻO ka manawa paʻa o BDDE ma kahi o 3.48 mau minuke (mai 3.43 a 4.14 mau minuke ma muli o nā hoʻokolohua).Ua hoʻomoʻi ʻia ka pae kelepona ma kahi kahe kahe o 0.25 mL/min no ka loiloi LC-MS.
No ka BDDE kālailai a me ka quantification e MS, ka UPLC nenoaiu (Waters) ua hui me ka API 3000 triple quadrupole mass spectrometer (AB SCIEX) lako me ka electrospray ionization kumu, a me ka anamanaʻo i loko o ka maikai ion mode (ESI+).
Wahi a ka ʻāpana ʻāpana ion i hana ʻia ma BDDE, ua hoʻoholo ʻia ka ʻāpana me ka ikaika kiʻekiʻe loa e like me ka 129.1 Da (Figure 6).No laila, i ka multi-ion monitor mode (MIM) no ka helu ʻana, ʻo ka hoʻololi nui (mass-to-charge ratio [m/z]) o BDDE he 203.3/129.1 Da.Hoʻohana pū ia i ke ʻano scan piha (FS) a me ke ʻano huahana ion scan (PIS) no ka loiloi LC-MS.
No ka hōʻoia ʻana i ke ʻano kikoʻī o ke ʻano, ua ʻimi ʻia kahi laʻana blank (hoʻomaka mua o ka mobile phase).ʻAʻohe hōʻailona i ʻike ʻia ma ka hāpana hakahaka me ka hoʻololi nui ʻana o 203.3/129.1 Da.E pili ana i ka hoʻihoʻi hou ʻana o ka hoʻokolohua, ua loiloi ʻia nā ʻōkuhi maʻamau 10 o 55 ppb (i ka waena o ka curve calibration), e hopena i kahi koena maʻamau (RSD) <5% (ʻike ʻole i hōʻike ʻia).
Ua helu 'ia ke koena BDDE ma loko o 'ewalu mau 'ano like 'ole autoclaved BDDE cross-linked HA hydrogels, e pili ana i 'ehā mau ho'okolohua kū'oko'a.E like me ka mea i wehewehe ʻia ma ka ʻāpana "Materials and Methods", loiloi ʻia ka quantification e ka awelika waiwai o ka regression curve o ka dilution maʻamau BDDE, e pili ana i ka piko kūʻokoʻa i ʻike ʻia ma ka BDDE mass transition o 203.3/129.1 Da, me kahi paʻa. manawa o 3.43 a 4.14 minuke ʻAʻole kali.Hōʻike ka Figure 2 i kahi laʻana chromatogram o ka maʻamau kuhikuhi 10 ppb BDDE.Hōʻuluʻulu ka Papa 1 i ke koena BDDE maʻiʻo o ʻewalu hydrogels like ʻole.ʻO ka laulā waiwai he 1 a 2.46 ppb.No laila, ʻae ʻia ke koena BDDE i loko o ka hāpana no ka hoʻohana kanaka (<2 ppm).
Kiʻi 2 Ion chromatogram o 10 ppb BDDE kuhikuhi maʻamau (Sigma-Aldrich Co), MS (m / z) hoʻololi i loaʻa ma ka LC-MS anamanaʻo o 203.30/129.10 Da (ma ka maikaʻi MRM mode).
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui;MS, nuipa;m/z, lākiō nui-a-kaumaha.
'Ōlelo Aʻo: ʻO nā laʻana 1-8 he mau BDDE i hoʻopaʻa ʻia i ka hau hydrogels.Hōʻike pū ʻia ke koena o ka BDDE i ka hydrogel a me ka piko o ka manawa paʻa BDDE.ʻO ka hope, hōʻike pū ʻia ke ola ʻana o nā piko hou me nā manawa paʻa like ʻole.
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;MRM, ka nānā 'ana i nā pane he nui;tR, manawa paa;LC-MS, chromatography wai a me ka spectrometry lehulehu;RRT, ka manawa paʻa pili.
ʻO ka mea kupanaha, ua hōʻike ʻia ka nānā ʻana o ka LC-MS ion chromatogram ma muli o nā hōʻailona HA hydrogel i hoʻopaʻa ʻia i autoclaved i hoʻopaʻa ʻia, aia kahi kiʻekiʻe ma ka manawa paʻa pōkole o 2.73 a 3.29 mau minuke.No ka laʻana, hōʻike ka Figure 3 i ka chromatogram ion o kahi hāpana HA i hoʻopili ʻia, kahi e ʻike ʻia ai kahi piko hou ma kahi manawa paʻa ʻokoʻa ma kahi o 2.71 mau minuke.ʻO ka manawa paʻa pili i ʻike ʻia (RRT) ma waena o ke kiʻekiʻe hou i ʻike ʻia a me ka piko mai BDDE i ʻike ʻia ʻo 0.79 (Table 1).No ka mea, ʻike mākou ʻaʻole i paʻa ka piko hou i ka kolamu C18 i hoʻohana ʻia i ka loiloi LC-MS, pili paha ka piko hou i kahi hui polar ma mua o BDDE.
Kiʻi 3 Ion chromatogram o kea-pili HA hydrogel hāpana i loaʻa e LC-MS (MRM hoʻololi nuipa 203.3/129.0 Da).
Nā pōkole: HA, hyaluronic acid;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui;RRT, ka manawa paʻa pili;tR, manawa paa.
I mea e pale aku ai i ka hiki ʻana o nā peaks hou i ʻike ʻia he mea haumia i loaʻa mua i nā mea maka i hoʻohana ʻia, ua ʻimi ʻia kēia mau mea maka me ka hoʻohana ʻana i ke ʻano loiloi LC-MS like.ʻO nā mea hoʻomaka i kālailai ʻia, ʻo ia ka wai, 2% NaHA i ka wai, 1% NaOH i ka wai, a me BDDE ma ka ʻike like i hoʻohana ʻia i ka hopena hoʻohui kea.ʻAʻole i hōʻike ʻia ka chromatogram ion o ka mea hoʻomaka i hoʻohana ʻia i kahi hui a i ʻole ka piko, a ʻo kona manawa paʻa e pili ana i ka piko hou i ʻike ʻia.Hōʻole kēia ʻoiaʻiʻo i ka manaʻo ʻaʻole i ka mea hoʻomaka wale nō ke komo i nā pūhui a i ʻole nā mea e hoʻopilikia ai i ke kaʻina hana loiloi, akā ʻaʻohe hōʻailona o ka hiki ke hoʻopaʻa ʻia me nā huahana ʻē aʻe.Hōʻike ʻia nā koina koʻikoʻi ma hope o ka nānā ʻana o LC-MS o BDDE a me nā peaks hou ma ka Papa 2 (nā laʻana 1-4) a me ka chromatogram ion ma ke Kiʻi 4.
'Ōlelo Aʻo: ʻO nā laʻana 1-4 e pili ana i nā mea maka i hoʻohana ʻia no ka hana ʻana i nā BDDE cross-linked HA hydrogels autoclaved.ʻAʻole i autoclaved kēia mau laʻana.
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui.
Kiʻi 4 pili i ka LC-MS chromatogram o kahi laʻana o ka mea maka i hoʻohana ʻia i ka hoʻololi kea o HA a me BDDE.
'Ōlelo Aʻo: Ua ana ʻia kēia mau mea a pau ma ka ʻike like ʻana a me ka ratio i hoʻohana ʻia no ka hoʻokō ʻana i ka hopena hoʻopili kea.ʻO nā helu no nā mea maka i kālailai ʻia e ka chromatogram e like me: (1) wai, (2) 2% HA wai wai, (3) 1% NaOH wai wai.Hana ʻia ka loiloi LC-MS no ka hoʻohuli nui ʻana o 203.30/129.10 Da (ma ke ʻano MRM maikaʻi).
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui.
Ua aʻo ʻia nā kūlana i alakaʻi ai i ka hoʻokumu ʻana i nā piko hou.I mea e aʻo ai i ke ʻano o ka hopena i hoʻohana ʻia no ka hana ʻana i ka hydrogel HA i hoʻopaʻa ʻia i ke keʻa i ka reactivity o ka BDDE cross-linking agent, e alakaʻi ana i ke kūkulu ʻana i nā piko hou (hiki i nā huahana), ua hana ʻia nā ana ʻokoʻa.Ma kēia mau hoʻoholo ʻana, ua aʻo mākou a hoʻopaʻa ʻia i ka crosslinker BDDE hope loa, i mālama ʻia me nā ʻano ʻokoʻa o NaOH (0%, 1%, 0.1%, a me 0.01%) i loko o kahi ʻano wai, i ukali ʻia e ka autoclaving a i ʻole.ʻO ke kaʻina hana bacteria e hoʻohālikelike i nā kūlana like me ke ʻano i hoʻohana ʻia e hana i ka hydrogel HA i hoʻopili ʻia.E like me ka mea i wehewehe ʻia ma ka ʻāpana "Materials and Methods", ua nānā ʻia ka hoʻololi nui o ka hāpana e LC-MS i 203.30 / 129.10 Da.Hoʻopiliʻia ka BDDE a me ka noʻonoʻo o ka piko hou, a ua hōʻikeʻia nā hualoaʻa ma ka Papa 3. ʻAʻole iʻikeʻia nā piko hou i nā hōʻailonaʻaʻole i autoclaved, me ka nānāʻole i ka loaʻaʻana o ka NaOH i loko o ka hopena (nā laʻana 1-4, Table. 3).No nā laʻana autoclaved, ʻike wale ʻia nā piko hou ma ke alo o ka NaOH i loko o ka hopena, a ʻo ka hoʻokumu ʻana o ka piko me he mea lā e hilinaʻi ʻia ma ka hoʻopaʻa ʻana o NaOH i ka hopena (nā laʻana 5-8, Papa 3) (RRT = 0.79).Hōʻike ka Figure 5 i kahi laʻana o kahi chromatogram ion, e hōʻike ana i ʻelua mau laʻana autoclaved ma ke alo a i ʻole ka nele o NAOH.
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui.
Nānā: ʻO ka chromatogram kiʻekiʻe: Ua mālama ʻia ka hāpana me ka 0.1% NaOH aqueous solution a autoclaved (120°C no 20 mau minuke).Kromatogram lalo: ʻAʻole mālama ʻia ka hāpana me NaOH, akā ua hoʻopaʻa ʻia ma lalo o nā kūlana like.ʻO ka hoʻololi nui o 203.30/129.10 Da (ma ke ʻano MRM maikaʻi) ua nānā ʻia e LC-MS.
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui.
I loko o nā mea hoʻohālike autoclaved a pau, me ka NaOH a i ʻole, ua hoʻemi nui ʻia ka manaʻo BDDE (hiki i 16.6 manawa) (nā laʻana 5-8, Papa 2).ʻO ka emi ʻana o ka manaʻo BDDE ma muli o ka ʻoiaʻiʻo ma nā wela kiʻekiʻe, hiki i ka wai ke hana ma ke ʻano he kumu (nucleophile) e wehe i ke apo epoxide o BDDE e hana i kahi pūhui 1,2-diol.ʻOkoʻa ka maikaʻi monoisotopic o kēia pūhui mai ka BDDE a no laila ʻaʻole e pili.Ua ʻike ʻo LC-MS i kahi hoʻololi nui o 203.30/129.10 Da.
ʻO ka mea hope loa, hōʻike kēia mau hoʻokolohua e pili ana ka hanauna o nā piko hou ma ke alo o BDDE, NAOH, a me ke kaʻina autoclaving, akā ʻaʻohe mea pili me HA.
ʻO ka piko hou i loaʻa i kahi manawa paʻa ma kahi o 2.71 mau minuke i hōʻike ʻia e LC-MS.No kēia kumu, ua hoʻokomo ʻia ʻo BDDE (9.9 mg/mL) i loko o kahi 1% NaOH aqueous solution a autoclaved.Ma ka Papa 4, ua hoʻohālikelike ʻia nā ʻano o ka piko hou me ka piko kuhikuhi BDDE i ʻike ʻia (ka manawa paʻa ma kahi o 3.47 mau minuke).Ma muli o ka hoʻopili ʻana i ka ion fragmentation o nā piko ʻelua, hiki ke hoʻoholo ʻia ʻo ka piko me ka manawa paʻa o 2.72 mau minuke e hōʻike ana i nā ʻāpana like me ka BDDE peak, akā me nā ikaika like ʻole (Figure 6).No ka piko e pili ana i ka manawa hoʻopaʻa (PIS) o 2.72 mau minuke, ua ʻike ʻia kahi kiʻekiʻe ikaika ma hope o ka ʻāpana ʻana i ka nui o 147 Da.Ma ka helu BDDE (9.9 mg/mL) i hoʻohana ʻia i kēia hoʻoholo ʻana, ua ʻike pū ʻia nā ʻano ʻano absorbance like ʻole (UV, λ=200 nm) i ka spectrum ultraviolet ma hope o ka hoʻokaʻawale chromatographic (Figure 7).ʻIke ʻia ka piko me ka manawa paʻa o 2.71 mau minuke ma 200 nm, ʻoiai ʻaʻole hiki ke nānā ʻia ka piko BDDE i ka chromatogram ma lalo o nā kūlana like.
Papa 4 Nā hopena hōʻikeʻike o ka piko hou me ka manawa paʻa ma kahi o 2.71 mau minuke a me ka piko BDDE me ka manawa paʻa o 3.47 mau minuke.
Nānā: No ka loaʻa ʻana o kēia mau hopena, ua hana ʻia nā loiloi LC-MS a me HPLC (MRM a me PIS) ma nā piko ʻelua.No ka nānā 'ana i ka HPLC, ho'ohana 'ia ka 'ike UV me ka nalu o 200 nm.
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;HPLC, chromatography wai hana kiʻekiʻe;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui;m/z, lākiō nui-a-kau;PIS, huahana Ion scanning;kukui ultraviolet, kukui ultraviolet.
'Ōlelo Aʻo: Loaʻa nā ʻāpana nui ma o ka LC-MS analysis (PIS).Kromatogram kiʻekiʻe: ʻano nui o nā ʻāpana laʻana maʻamau o BDDE.ʻO ka chromatogram lalo: ʻO ka nui o ke kiʻekiʻe hou i ʻike ʻia (RRT pili me ka BDDE peak ʻo 0.79).Ua hana ʻia ʻo BDDE ma 1% NaOH solution a autoclaved.
Nā pōkole: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, chromatography wai a me ka spectrometry lehulehu;MRM, ka nānā 'ana i nā pane he nui;PIS, scan ion huahana;RRT, ka manawa paʻa pili.
Kii 7 Ion chromatogram o ka 203.30 Da precursor ion, a me (A) ka hou peak me ka manawa paa o 2.71 minuke a me (B) ka UV ike o ka BDDE kuhikuhi maʻamau piko ma 3.46 minuke ma 200 nm.
I loko o nā hydrogels HA i hoʻopili ʻia a pau, ua ʻike ʻia ʻo ke koena o ka BDDE i hoʻopaʻa ʻia ma hope o ka helu LC-MS he <2 ppm, akā ua ʻike ʻia kahi kiʻekiʻe i ʻike ʻole ʻia i ka loiloi.ʻAʻole kūpono kēia piko hou i ka huahana maʻamau BDDE.ʻO ka huahana maʻamau BDDE ua loaʻa pū i ka hoʻololi maikaʻi like (MRM hoʻololi 203.30/129.10 Da) ka nānā ʻana ma ke ʻano MRM maikaʻi.ʻO ka maʻamau, hoʻohana ʻia nā ʻano analytical ʻē aʻe e like me ka chromatography e like me nā hoʻāʻo palena no ka ʻike ʻana i ka BDDE i loko o nā hydrogels, akā haʻahaʻa iki ka palena ʻike kiʻekiʻe (LOD) ma mua o 2 ppm.Ma kekahi ʻaoʻao, a hiki i kēia manawa, ua hoʻohana ʻia ʻo NMR a me MS e ʻike i ke degere o ka hoʻopili ʻana a/a i ʻole ka hoʻololi ʻana o HA i nā ʻāpana kō o nā huahana HA i hoʻopili ʻia.ʻAʻole ke kumu o kēia mau ʻenehana i ka helu ʻana i ke koena o ka ʻike BDDE i nā haʻahaʻa haʻahaʻa e like me kā mākou e wehewehe nei ma kēia ʻatikala (LOD o kā mākou ala LC-MS = 10 ppb).
Ka manawa hoʻouna: Sep-01-2021