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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 * Cov kws sau ntawv no muaj qee qhov kev nkag siab txog txoj haujlwm no sib npaug. Kev pab cuam tom qab: Hyaluronic acid (HA) yog ib txwm tshwm sim polysaccharide siv hauv kev tsim cov dermal fillers rau kev zoo nkauj.Txij li thaum nws muaj ib nrab-lub neej ntawm ob peb hnub nyob rau hauv tib neeg cov ntaub so ntswg, HA-raws li dermal fillers yog chemically hloov kho lawv lub neej nyob rau hauv lub cev.Kev hloov pauv ntau tshaj plaws hauv kev lag luam HA-raws li cov khoom siv yog kev siv 1,4-butanediol diglycidyl ether (BDDE) ua tus neeg sawv cev sib txuas rau crosslink HA chains.Residual lossis unreacted BDDE yog suav tias yog tsis muaj tshuaj lom ntawm <2 qhov chaw ib lab (ppm);Yog li ntawd, BDDE residual nyob rau hauv qhov kawg dermal muab tub lim yuav tsum tau quantified kom tus neeg mob muaj kev nyab xeeb.Cov ntaub ntawv thiab cov hau kev: Txoj kev tshawb no piav qhia txog kev tshawb nrhiav thiab kev ua tus cwj pwm ntawm cov khoom siv los ntawm kev sib txuas ntawm cov tshuaj tiv thaiv kab mob sib kis ntawm BDDE thiab HA nyob rau hauv cov kab mob alkaline los ntawm kev sib xyaw ua kua chromatography thiab huab hwm coj spectrometry (LC-MS).Cov txiaj ntsig: Tom qab kev soj ntsuam sib txawv, nws tau pom tias cov kab mob alkaline thiab kub siab siv los tua kab mob HA-BDDE hydrogel txhawb kev tsim cov khoom siv tshiab no, "propylene glycol-like" compound.LC-MS tsom xam tau lees paub tias cov khoom los ntawm cov khoom muaj tib lub monoisotopic loj li BDDE, lub sijhawm sib txawv (tR), thiab qhov sib txawv UV absorbance (λ = 200 nm) hom.Tsis zoo li BDDE, nws tau pom nyob rau hauv LC-MS tsom xam tias nyob rau hauv tib yam kev ntsuas, cov khoom los ntawm cov khoom no muaj qhov ntsuas siab dua ntawm 200 nm.Xaus: Cov txiaj ntsig no qhia tau tias tsis muaj epoxide nyob rau hauv cov qauv ntawm cov khoom siv tshiab no.Kev sib tham qhib los ntsuas qhov kev pheej hmoo ntawm cov khoom lag luam tshiab no pom hauv kev tsim cov HA-BDDE hydrogel (HA dermal muab tub lim) rau kev lag luam.Keywords: hyaluronic acid, HA dermal muab tub lim, cross-linked hyaluronic acid, BDDE, LC-MS tsom xam, BDDE los ntawm cov khoom.
Fillers raws li hyaluronic acid (HA) yog cov feem ntau thiab nrov dermal fillers siv rau lub hom phiaj kom hniav zoo nkauj.1 Cov dermal muab tub lim no yog hydrogel, feem ntau yog tsim los ntawm> 95% dej thiab 0.5-3% HA, uas muab lawv cov qauv zoo li gel.2 HA yog ib qho polysaccharide thiab cov khoom tseem ceeb ntawm cov kab mob extracellular ntawm vertebrates.Ib qho ntawm cov khoom xyaw.Nws muaj (1,4)-glucuronic acid-β (1,3)-N-acetylglucosamine (GlcNAc) rov ua cov disaccharide units txuas nrog glycosidic bonds.Cov qauv disaccharide no zoo ib yam hauv txhua yam kab mob.Piv nrog rau qee cov protein-raws li cov fillers (xws li collagen), cov cuab yeej no ua rau HA cov khoom siv biocompatible molecule.Cov fillers tuaj yeem pom cov amino acid ib ntus tshwj xeeb uas tuaj yeem lees paub los ntawm tus neeg mob lub cev tiv thaiv kab mob.
Thaum siv los ua dermal muab tub lim, qhov kev txwv tseem ceeb ntawm HA yog nws txoj kev hloov pauv sai hauv cov ntaub so ntswg vim muaj ib tsev neeg tshwj xeeb ntawm cov enzymes hu ua hyaluronidases.Txog tam sim no, ntau qhov kev hloov kho tshuaj hauv HA qauv tau piav qhia kom nce ib nrab-lub neej ntawm HA hauv cov ntaub so ntswg.3 Feem ntau ntawm cov kev hloov kho no sim txo qhov kev nkag mus ntawm hyaluronidase rau polysaccharide polymers los ntawm kev sib txuas HA chains.Yog li ntawd, vim yog kev tsim cov choj thiab cov kev sib txuas sib txuas ntawm cov khoom sib txuas ntawm cov qauv HA thiab tus neeg sawv cev sib txuas, tus ntoo khaub lig-txuas HA hydrogel tsim cov khoom tiv thaiv enzyme degradation ntau dua li lub ntuj HA.4-6
Txog tam sim no, cov tshuaj crosslinking agents siv los tsim crosslinked HA suav nrog methacrylamide, 7 hydrazide, 8 carbodiimide, 9 divinyl sulfone, 1,4-butanediol diglycidyl ether (BDDE) Thiab poly (ethylene glycol) diglycidyl ether.10 ,11 BDDE yog tam sim no feem ntau siv crosslinking tus neeg sawv cev.Txawm hais tias cov hom hydrogels no tau raug pov thawj tias muaj kev nyab xeeb rau ntau xyoo lawm, cov neeg ua haujlwm crosslinking siv yog cov reactive reagents uas tej zaum yuav cytotoxic thiab, qee zaum, mutagenic.12 Yog li ntawd, lawv cov ntsiab lus seem nyob rau hauv qhov kawg hydrogel yuav tsum siab.BDDE suav hais tias muaj kev nyab xeeb thaum qhov seem seem tsawg dua 2 feem ntawm ib lab (ppm).4
Muaj ntau txoj hauv kev txhawm rau txheeb xyuas qhov tsis tshua muaj BDDE concentration, kev sib txuas ntawm kev sib txuas thiab hloov txoj haujlwm hauv HA hydrogels, xws li roj chromatography, qhov loj me me chromatography txuas nrog huab hwm coj spectrometry (MS), nuclear magnetic resonance (NMR) fluorescence ntsuas txoj kev, thiab Diode array txuas nrog kev ua haujlwm siab ua kua chromatography (HPLC).13-17 Txoj kev tshawb no piav qhia txog kev tshawb nrhiav thiab kev ua tus cwj pwm ntawm cov khoom lag luam hauv qhov kawg sib txuas HA hydrogel uas tsim los ntawm cov tshuaj tiv thaiv ntawm BDDE thiab HA nyob rau hauv alkaline tej yam kev mob.HPLC thiab kua chromatography-mass spectrometry (LC-MS tsom xam).Txij li thaum lub toxicity ntawm no by-product ntawm BDDE tsis paub, peb pom zoo kom nws residue quantification yuav tsum tau txiav txim nyob rau hauv ib tug zoo xws li cov txheej txheem feem ntau ua nyob rau hauv BDDE nyob rau hauv cov khoom kawg.
Cov tau txais sodium ntsev ntawm HA (Shiseido Co., Ltd., Tokyo, Nyiv) muaj ib qho hnyav molecular ntawm ~ 1,368,000 Da (Laurent txoj kev) 18 thiab intrinsic viscosity ntawm 2.20 m3 / kg.Rau cov tshuaj tiv thaiv crosslinking, BDDE (≥95%) tau yuav los ntawm Sigma-Aldrich Co. (St. Louis, MO, USA).Phosphate buffered saline nrog pH 7.4 tau yuav los ntawm Sigma-Aldrich Company.Tag nrho cov kuab tshuaj, acetonitrile thiab dej siv hauv LC-MS tsom xam tau yuav los ntawm HPLC qib zoo.Formic acid (98%) yog muas raws li qib reagent.
Txhua qhov kev sim tau ua tiav ntawm UPLC Acquity system (Waters, Milford, MA, USA) thiab txuas nrog API 3000 triple quadrupole mass spectrometer nruab nrog lub tshuab hluav taws xob ionization (AB SCIEX, Framingham, MA, USA).
Lub synthesis ntawm cross-linked HA hydrogels tau pib los ntawm kev ntxiv 198 mg ntawm BDDE rau 10% (w/w) sodium hyaluronate (NaHA) tov nyob rau hauv muaj 1% alkali (sodium hydroxide, NaOH).Qhov kawg BDDE concentration nyob rau hauv cov tshuaj sib tov yog 9.9 mg / mL (0.049 mM).Tom qab ntawd, cov tshuaj tiv thaiv tau muab sib xyaw kom zoo thiab homogenized thiab tso cai mus rau ntawm 45 ° C rau 4 teev.19 Lub pH ntawm cov tshuaj tiv thaiv yog tswj ntawm ~ 12.
Tom qab ntawd, cov tshuaj tiv thaiv sib xyaw yog ntxuav nrog dej, thiab qhov kawg HA-BDDE hydrogel tau lim thiab diluted nrog PBS tsis tuaj yeem ua tiav HA concentration ntawm 10 txog 25 mg / mL, thiab qhov kawg pH ntawm 7.4.Txhawm rau kom tsis muaj menyuam cov khoom sib txuas HA hydrogels, tag nrho cov hydrogels no yog autoclaved (120 ° C rau 20 feeb).Cov purified BDDE-HA hydrogel khaws cia ntawm 4 ° C kom txog thaum kev soj ntsuam.
Txhawm rau txheeb xyuas BDDE tam sim no hauv cov khoom sib txuas HA, 240 mg qauv raug ntsuas thiab qhia rau hauv qhov chaw nruab nrab (Microcon®; Merck Millipore, Billerica, MA, USA; ntim 0.5 mL) thiab centrifuged ntawm 10,000 rpm ntawm chav tsev kub. 10 feeb.Tag nrho ntawm 20 µL ntawm cov kua rub-down tau sau thiab tshuaj xyuas.
Txhawm rau txheeb xyuas tus qauv BDDE (Sigma-Aldrich Co) nyob rau hauv cov xwm txheej alkaline (1%, 0.1% thiab 0.01% NaOH), yog tias muaj cov xwm txheej hauv qab no, cov qauv ua kua yog 1: 10, 1: 100, lossis nce mus txog 1: 1,000,000 Yog tias tsim nyog, siv MilliQ deionized dej rau kev tshuaj xyuas.
Rau cov khoom pib siv hauv kev sib txuas sib txuas (HA 2%, H2O, 1% NaOH, thiab 0.049 mM BDDE), 1 mL ntawm txhua tus qauv npaj los ntawm cov ntaub ntawv no tau txheeb xyuas siv tib yam kev soj ntsuam.
Txhawm rau txiav txim siab qhov tshwj xeeb ntawm cov peaks tshwm sim hauv daim ntawv qhia ion, 10 µL ntawm 100 ppb BDDE standard tov (Sigma-Aldrich Co) tau ntxiv rau 20 µL qauv.Hauv qhov no, qhov kawg concentration ntawm tus qauv hauv txhua tus qauv yog 37 ppb.
Ua ntej, npaj cov tshuaj BDDE nrog cov concentration ntawm 11,000 mg / L (11,000 ppm) los ntawm diluting 10 μL ntawm tus qauv BDDE (Sigma-Aldrich Co) nrog 990 μL MilliQ dej (density 1.1 g / mL).Siv cov tshuaj no los npaj 110 µg/L (110 ppb) BDDE tov raws li tus qauv nruab nrab dilution.Tom qab ntawd, siv qhov nruab nrab BDDE tus qauv diluent (110 ppb) los npaj cov qauv nkhaus los ntawm diluting intermediate diluent ob peb zaug kom ua tiav qhov xav tau ntawm 75, 50, 25, 10, thiab 1 ppb.Raws li pom hauv daim duab 1, nws pom tias BDDE tus qauv nkhaus ntawm 1.1 txog 110 ppb muaj qhov zoo linearity (R2> 0.99).Tus qauv nkhaus tau rov ua dua hauv plaub qhov kev sim ywj pheej.
Daim duab 1 BDDE tus qauv calibration nkhaus tau los ntawm LC-MS tsom xam, nyob rau hauv uas muaj kev sib raug zoo tau pom (R2> 0.99).
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, kua chromatography thiab huab hwm coj spectrometry.
Txhawm rau txheeb xyuas thiab txheeb xyuas cov qauv BDDE tam sim no nyob rau hauv cross-linked HA thiab BDDE cov qauv hauv cov kev daws teeb meem, LC-MS tsom xam tau siv.
Kev sib cais chromatographic tau ua tiav ntawm LUNA 2.5 µm C18(2)-HST kem (50 × 2.0 mm2; Phenomenex, Torrance, CA, USA) thiab khaws cia ntawm chav tsev kub (25 ° C) thaum lub sijhawm tshuaj xyuas.Cov theem ntawm lub xov tooj ntawm tes muaj xws li acetonitrile (cov kuab tshuaj A) thiab dej (cov kuab tshuaj B) uas muaj 0.1% formic acid.Lub sijhawm mobile yog eluted los ntawm gradient elution.gradient yog raws li nram no: 0 feeb, 2% A;1 feeb, 2% A;6 feeb, 98% A;7 feeb, 98% A;7.1 feeb, 2% A;10 feeb , 2% A. Lub sij hawm khiav yog 10 feeb thiab qhov ntim txhaj tshuaj yog 20 µL.Lub sijhawm khaws cia ntawm BDDE yog kwv yees li 3.48 feeb (xws li 3.43 txog 4.14 feeb raws li kev sim).Lub sijhawm txawb tau muab tso rau ntawm tus nqi ntws ntawm 0.25 mL / min rau LC-MS tsom xam.
Rau BDDE tsom xam thiab kom muaj nuj nqis los ntawm MS, UPLC system (Dej) yog ua ke nrog API 3000 triple quadrupole mass spectrometer (AB SCIEX) nruab nrog ib qho electrospray ionization qhov chaw, thiab kev soj ntsuam yog ua nyob rau hauv zoo ion hom (ESI +).
Raws li cov ion fragment tsom xam ua nyob rau hauv BDDE, cov fragment nrog lub siab tshaj plaws siv tau txiav txim siab mus rau lub fragment sib raug rau 129.1 Da (Daim duab 6).Yog li ntawd, nyob rau hauv lub multi-ion saib xyuas hom (MIM) rau quantification, qhov loj hloov dua siab tshiab (mass-to-them ratio [m/z]) ntawm BDDE yog 203.3/129.1 Da.Nws kuj siv tag nrho scan (FS) hom thiab cov khoom ion scan (PIS) hom rau LC-MS tsom xam.
Txhawm rau txheeb xyuas qhov tshwj xeeb ntawm txoj kev, ib qho qauv dawb paug (thaum pib lub xov tooj ntawm tes) tau txheeb xyuas.Tsis muaj teeb liab raug kuaj pom hauv cov qauv dawb paug nrog kev hloov pauv loj ntawm 203.3/129.1 Da.Hais txog qhov rov ua dua ntawm qhov kev sim, 10 tus qauv txhaj tshuaj ntawm 55 ppb (hauv nruab nrab ntawm qhov ntsuas qhov nkhaus) tau txheeb xyuas, ua rau qhov seem ntawm tus qauv sib txawv (RSD) <5% (cov ntaub ntawv tsis qhia).
Cov ntsiab lus BDDE residual tau suav rau hauv yim qhov sib txawv autoclaved BDDE cross-linked HA hydrogels, sib xws rau plaub qhov kev sim ywj pheej.Raws li tau piav nyob rau hauv "Cov Khoom Siv thiab Cov Txheej Txheem" ntu, qhov ntau thiab tsawg yog ntsuas los ntawm qhov nruab nrab tus nqi ntawm qhov rov qab nkhaus ntawm BDDE tus qauv dilution, uas sib haum rau qhov tshwj xeeb ncov pom ntawm BDDE huab hwm coj hloov pauv ntawm 203.3/129.1 Da, nrog kev tuav cia. lub sij hawm ntawm 3.43 rau 4.14 feeb Tsis tos.Daim duab 2 qhia ib qho piv txwv chromatogram ntawm 10 ppb BDDE reference standard.Table 1 qhia txog cov ntsiab lus BDDE seem ntawm yim hydrogels sib txawv.Tus nqi ntau yog 1 txog 2.46 ppb.Yog li ntawd, qhov seem BDDE concentration nyob rau hauv cov qauv yog siv tau rau tib neeg siv (<2 ppm).
Daim duab 2 Ion chromatogram ntawm 10 ppb BDDE siv tus qauv (Sigma-Aldrich Co), MS (m/z) kev hloov pauv tau los ntawm LC-MS tsom xam ntawm 203.30/129.10 Da (hauv qhov zoo MRM hom).
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv kev soj ntsuam;MS, loj;m/z, huab hwm coj-rau-them ratio.
Lus Cim: Cov Qauv 1-8 yog autoclaved BDDE cross-linked HA hydrogels.Cov seem ntawm BDDE nyob rau hauv hydrogel thiab lub ncov ntawm BDDE tuav lub sij hawm kuj qhia.Thaum kawg, qhov muaj nyob ntawm cov ncov tshiab nrog lub sijhawm khaws cia sib txawv kuj tau tshaj tawm.
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;MRM, ntau yam tshuaj tiv thaiv kev soj ntsuam;tR, tuav lub sijhawm;LC-MS, kua chromatography thiab loj spectrometry;RRT, txheeb ze tuav lub sijhawm.
Kuj ceeb tias, kev soj ntsuam ntawm LC-MS ion chromatogram qhia tau hais tias raws li tag nrho cov autoclaved cross-linked HA hydrogel cov qauv soj ntsuam, muaj qhov ncov ntxiv ntawm lub sijhawm tuav luv ntawm 2.73 txog 3.29 feeb.Piv txwv li, Daim duab 3 qhia txog ion chromatogram ntawm tus qauv HA cross-linked, qhov twg lub ncov ntxiv tshwm sim ntawm lub sijhawm khaws cia sib txawv ntawm kwv yees li 2.71 feeb.Lub sijhawm soj ntsuam txheeb ze khaws cia (RRT) nruab nrab ntawm qhov pom tshiab thiab qhov siab tshaj plaws los ntawm BDDE tau pom tias yog 0.79 (Table 1).Txij li thaum peb paub tias lub ncov tshiab tau pom tsawg dua nyob rau hauv C18 kem siv hauv LC-MS tsom xam, lub ncov tshiab yuav sib haum mus rau lub ntsiab lus ntau dua li BDDE.
Daim duab 3 Ion chromatogram ntawm cross-linked HA hydrogel qauv tau txais los ntawm LC-MS (MRM loj hloov pauv 203.3/129.0 Da).
Cov ntawv luv: HA, hyaluronic acid;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv kev soj ntsuam;RRT, txheeb ze tuav lub sijhawm;tR, lub sijhawm khaws cia.
Txhawm rau txiav txim siab qhov muaj peev xwm ntawm cov peaks tshiab tau pom tuaj yeem yog cov kab mob uas muaj nyob hauv cov khoom siv raw, cov ntaub ntawv nyoos no kuj tau txheeb xyuas siv tib txoj kev tshuaj xyuas LC-MS.Cov ntaub ntawv pib soj ntsuam suav nrog dej, 2% NaHA hauv dej, 1% NaOH hauv dej, thiab BDDE ntawm tib qhov kev xav tau siv hauv kev sib txuas sib txuas.Lub ion chromatogram ntawm cov khoom siv pib siv tsis pom muaj qhov sib xyaw lossis qhov siab tshaj plaws, thiab nws lub sijhawm khaws cia sib raug rau qhov ncov tshiab tau pom.Qhov tseeb no pov tseg lub tswv yim hais tias tsis yog cov khoom pib xwb tuaj yeem muaj cov khoom sib txuas lossis cov khoom siv uas tuaj yeem cuam tshuam nrog cov txheej txheem kev tshuaj ntsuam, tab sis tsis muaj cov cim qhia tias muaj peev xwm sib kis nrog lwm cov khoom kuaj.Cov txiaj ntsig concentration tau txais tom qab LC-MS tsom xam ntawm BDDE thiab cov peaks tshiab tau qhia hauv Table 2 (piv txwv 1-4) thiab ion chromatogram hauv daim duab 4.
Lus Cim: Cov qauv 1-4 sib haum rau cov khoom siv raw siv los tsim autoclaved BDDE cross-linked HA hydrogels.Cov qauv no tsis yog autoclaved.
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv.
Daim duab 4 sib raug rau LC-MS chromatogram ntawm ib qho piv txwv ntawm cov khoom siv raw siv hauv kev sib txuas sib txuas ntawm HA thiab BDDE.
Nco tseg: Tag nrho cov no yog ntsuas ntawm tib qhov concentration thiab qhov sib piv siv los ua cov tshuaj tiv thaiv kev sib txuas.Cov lej rau cov khoom siv raw tshuaj ntsuam xyuas los ntawm chromatogram sib haum rau: (1) dej, (2) 2% HA aqueous tov, (3) 1% NaOH aqueous tov.Kev tshuaj xyuas LC-MS yog ua rau kev hloov pauv loj ntawm 203.30 / 129.10 Da (hauv hom MRM zoo).
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;HA, hyaluronic acid;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv.
Cov xwm txheej uas ua rau tsim cov ncov tshiab tau kawm.Txhawm rau kawm yuav ua li cas cov tshuaj tiv thaiv kab mob siv los tsim cov cross-linked HA hydrogel cuam tshuam cov reactivity ntawm BDDE cross-linking tus neeg sawv cev, ua rau tsim cov peaks tshiab (muaj peev xwm los ntawm cov khoom), kev ntsuas sib txawv tau ua.Hauv cov kev txiav txim siab no, peb tau kawm thiab txheeb xyuas qhov kawg BDDE crosslinker, uas tau kho nrog ntau qhov sib txawv ntawm NaOH (0%, 1%, 0.1%, thiab 0.01%) hauv qhov nruab nrab aqueous, ua raws li los yog tsis muaj autoclaving.Cov txheej txheem kab mob los simulate tib yam kev mob yog tib yam li txoj kev siv los tsim cov kab sib txuas HA hydrogel.Raws li tau piav qhia hauv ntu "Cov Khoom Siv thiab Cov Txheej Txheem", qhov kev hloov pauv loj ntawm cov qauv tau txheeb xyuas los ntawm LC-MS rau 203.30 / 129.10 Da.Lub BDDE thiab cov concentration ntawm lub ncov tshiab yog xam, thiab cov txiaj ntsig tau pom nyob rau hauv Table 3. Tsis muaj cov peaks tshiab tau kuaj pom hauv cov qauv uas tsis yog autoclaved, tsis hais seb muaj NaOH nyob rau hauv cov kev daws teeb meem (piv txwv 1-4, Table 3).Rau cov qauv autoclaved, cov peaks tshiab tsuas yog kuaj pom nyob rau hauv lub xub ntiag ntawm NaOH hauv kev daws, thiab qhov tsim ntawm lub ncov zoo li nyob ntawm NaOH concentration hauv cov tshuaj (cov qauv 5-8, Table 3) (RRT = 0.79).Daim duab 5 qhia ib qho piv txwv ntawm ib qho ion chromatogram, qhia ob qho qauv autoclaved nyob rau hauv lub xub ntiag los yog tsis muaj NAOH.
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv.
Lus Cim: Sab saum toj chromatogram: Cov qauv raug kho nrog 0.1% NaOH aqueous tov thiab autoclaved (120 ° C rau 20 feeb).Hauv qab chromatogram: Cov qauv tsis tau kho nrog NaOH, tab sis autoclaved nyob rau hauv tib yam mob.Kev hloov pauv loj ntawm 203.30 / 129.10 Da (hauv hom MRM zoo) tau txheeb xyuas los ntawm LC-MS.
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv.
Hauv tag nrho cov qauv autoclaved, nrog lossis tsis muaj NaOH, BDDE concentration tau txo qis heev (mus txog 16.6 zaug) (cov qauv 5-8, Table 2).Qhov txo qis hauv BDDE concentration tuaj yeem yog vim qhov kub thiab txias, dej tuaj yeem ua lub hauv paus (nucleophile) los qhib lub nplhaib epoxide ntawm BDDE los ua ib qho 1,2-diol compound.Lub monoisotopic zoo ntawm cov compound no txawv ntawm BDDE thiab yog li ntawd yuav tsis cuam tshuam.LC-MS tau kuaj pom qhov hloov pauv loj ntawm 203.30 / 129.10 Da.
Thaum kawg, cov kev sim no qhia tau tias tiam ntawm cov ncov tshiab nyob ntawm qhov muaj BDDE, NAOH, thiab cov txheej txheem autoclaving, tab sis tsis muaj dab tsi ua rau HA.
Lub ncov tshiab pom ntawm lub sijhawm khaws cia ntawm kwv yees li 2.71 feeb tom qab ntawd tus cwj pwm los ntawm LC-MS.Rau lub hom phiaj no, BDDE (9.9 mg / mL) yog incubated nyob rau hauv ib tug 1% NaOH aqueous tov thiab autoclaved.Hauv Table 4, cov yam ntxwv ntawm lub ncov tshiab tau muab piv nrog cov paub txog BDDE siv ncov (lub sijhawm khaws cia kwv yees li 3.47 feeb).Raws li kev txheeb xyuas ion fragmentation ntawm ob lub ncov, nws tuaj yeem txiav txim siab tias lub ncov nrog lub sijhawm khaws cia ntawm 2.72 feeb qhia tau hais tias tib qhov tawg ntawm BDDE ncov, tab sis muaj qhov sib txawv (Daim duab 6).Rau lub ncov sib xws rau lub sijhawm khaws cia (PIS) ntawm 2.72 feeb, qhov siab tshaj plaws tau pom tom qab kev tawg ntawm qhov loj ntawm 147 Da.Ntawm BDDE concentration (9.9 mg/mL) siv nyob rau hauv qhov kev txiav txim siab no, txawv absorbance hom (UV, λ = 200 nm) nyob rau hauv lub ultraviolet spectrum kuj tau pom tom qab chromatographic sib cais (Daim duab 7).Lub ncov nrog lub sijhawm tuav ntawm 2.71 feeb tseem pom ntawm 200 nm, thaum lub ncov BDDE tsis tuaj yeem pom nyob rau hauv chromatogram raws li tib yam.
Table 4 Tus cwj pwm tshwm sim ntawm lub ncov tshiab nrog lub sijhawm khaws cia li ntawm 2.71 feeb thiab BDDE ncov nrog lub sijhawm tuav ntawm 3.47 feeb
Nco tseg: Txhawm rau kom tau txais cov txiaj ntsig no, LC-MS thiab HPLC kev txheeb xyuas (MRM thiab PIS) tau ua nyob rau ntawm ob lub ncov.Rau HPLC tsom xam, UV nrhiav pom nrog lub wavelength ntawm 200 nm yog siv.
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;HPLC, kev ua haujlwm siab ua kua chromatography;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv kev soj ntsuam;m/z, huab hwm coj-rau-them ratio;PIS, khoom Ion scanning;ultraviolet lub teeb, ultraviolet lub teeb.
Nco tseg: Cov khoom tawg yog tau los ntawm LC-MS tsom xam (PIS).Sab saum toj chromatogram: huab hwm coj spectrum ntawm BDDE tus qauv qauv tawg.Hauv qab chromatogram: Qhov loj spectrum ntawm lub ncov tshiab kuaj pom (RRT txuam nrog BDDE ncov yog 0.79).BDDE tau ua tiav hauv 1% NaOH tov thiab autoclaved.
Cov ntawv luv: BDDE, 1,4-butanediol diglycidyl ether;LC-MS, kua chromatography thiab loj spectrometry;MRM, ntau yam tshuaj tiv thaiv kev soj ntsuam;PIS, khoom ion scan;RRT, txheeb ze tuav lub sijhawm.
Daim duab 7 Ion chromatogram ntawm 203.30 Da precursor ion, thiab (A) lub ncov tshiab nrog lub sijhawm khaws cia ntawm 2.71 feeb thiab (B) UV nrhiav pom ntawm BDDE tus qauv siv lub ncov ntawm 3.46 feeb ntawm 200 nm.
Nyob rau hauv tag nrho cov cross-linked HA hydrogels tsim, nws tau pom tias qhov seem BDDE concentration tom qab LC-MS quantification yog <2 ppm, tab sis ib tug tshiab tsis paub ncov tshwm nyob rau hauv kev tshuaj ntsuam.Lub ncov tshiab no tsis phim BDDE tus qauv khoom.Cov khoom lag luam BDDE kuj tau dhau los ua qhov kev hloov pauv zoo ib yam (MRM hloov dua siab tshiab 203.30 / 129.10 Da) kev tshuaj xyuas hauv hom MRM zoo.Feem ntau, lwm txoj kev tshuaj ntsuam xyuas xws li chromatography yog siv los txwv kev ntsuas kom pom BDDE hauv hydrogels, tab sis qhov siab tshaj plaws kuaj pom (LOD) yog me ntsis qis dua 2 ppm.Ntawm qhov tod tes, txog tam sim no, NMR thiab MS tau siv los ua tus yam ntxwv ntawm kev sib txuas thiab / lossis kev hloov kho HA hauv cov khoom qab zib ntawm cov khoom sib txuas HA.Lub hom phiaj ntawm cov tswv yim no yeej tsis tau txheeb xyuas qhov seem ntawm BDDE nrhiav pom ntawm qhov tsis tshua muaj siab li peb tau piav qhia hauv tsab xov xwm no (LOD ntawm peb txoj kev LC-MS = 10 ppb).
Post lub sij hawm: Sep-01-2021