Cytisine (tseem hu ua baptitoxine) yog ib qho tseem ceeb alkaloid rho tawm los ntawm cov nroj tsuag leguminous xws li * Sophora alopecuroides * thiab * Thermopsis lanceolata *. Nws yog dav siv nyob rau hauv teb nrog rau cov kws kho mob, cog kev tiv thaiv, thiab cov kab mob lom; cov khoom purity ncaj qha txiav txim siab nws daim ntawv thov kev ua tau zoo thiab kev tswj hwm kev ua raws cai. Tam sim no, kev lag luam txoj hauv kev rau kev kuaj cytisine purity yog categorized rau hauv peb hom raws li precision, nqi, thiab daim ntawv thov scenario: ceev qualitative xeem, niaj hnub quantitative xeem, thiab siab -precision quantitative txiav txim. Cov txheej txheem sib txawv yog haum rau ntau hom qauv, xws li cov khoom siv raw, cov khoom tiav tiav, thiab cov qauv siv. Cov hauv qab no qhia txog cov txheej txheem kuaj kev lag luam thiab cov ntsiab lus tseem ceeb.
I. Nyias-Txheej Chromatography (TLC): Kev tshuaj ntsuam xyuas sai, tsim nyog rau kev sim ua ntej ntawm cov khoom nyoos
TLC yog txoj kev tshuaj ntsuam xyuas sai tshaj plaws thaum lub sijhawm tsim khoom thiab rho tawm ntawm cytisine. Nws yooj yim rau kev khiav lag luam thiab tus nqi - ua haujlwm tau zoo, ua haujlwm feem ntau los txiav txim siab muaj cytisine thiab ua qhov kev ntsuam xyuas ua ntej ntawm impurities. Nws yog qhov tsim nyog rau kev tshuaj xyuas thawj zaug purity ntawm cov khoom siv raw thiab cov khoom siv tsis zoo tab sis tsis tuaj yeem ua tiav qhov tseeb.
Cov txheej txheem ua haujlwm tseem ceeb: Ua ntej, npaj cov alkaline silica gel TLC phaj los ntawm kev sib xyaw Silica Gel G nrog sodium hydroxide tov rau hauv paste; kis nws tusyees, qhib nws ntawm 105℃rau ib teev, thiab cia nws txias rau kev siv. Tom ntej no, yaj cov qauv coj mus kuaj thiab cov qauv siv cytisine hauv methanol los npaj cov kev sim thiab cov kev daws teeb meem ntawm qhov tsim nyog, tom qab ntawd pom lawv mus rau TLC phaj. Siv cov txheej txheem hnyav ntawm benzene-acetone-ethyl acetate-methanol (4: 2: 1: 1) ua tus tsim tus neeg sawv cev thiab cia cov kuab tshuaj rau pem hauv ntej nce mus rau qhov siab preset. Thaum kawg, tshem lub phaj, cia nws huab cua- qhuav, thiab tshuaj tsuag nrog kev hloov kho Dragendorff's reagent (potassium bismuth iodide) rau kev pom.
Kev txhais cov txiaj ntsig: Tom qab pom qhov pom, saib txoj hauj lwm thiab qhov pom tseeb ntawm qhov chaw. Cov qauv ua raws cai qhia tau hais tias qhov chaw nrog cov xim tsis sib xws thiab tsis muaj qhov tseem ceeb tshaj plaws, nrog rau tus nqi Rf sib piv cov qauv siv (kev lag luam- tus qauv Rf tus nqi yog kwv yees li 0.23). Qhov tshwm sim ntawm qhov chaw txawv txav, tailing, los yog qhov sib txawv tseem ceeb hauv tus nqi Rf qhia tias muaj cov ntsiab lus tsis huv thiab tsis ua raws li cov qauv purity. Txoj kev no muaj cov kev txwv tseem ceeb: nws tsuas yog muaj txiaj ntsig zoo lossis ib nrab- kev tshuaj xyuas ntau, tsis tuaj yeem txiav txim siab qhov tseeb ntawm qhov tseeb, thiab ua rau cov txiaj ntsig tsis raug rau cov qauv uas muaj cov impurity siab.
II. High-Kev Ua Tau Zoo Liquid Chromatography (HPLC): Kev lag luam- qauv qauv tsim nyog rau cov khoom lag luam tiav
HPLC tam sim no yog tus qauv qauv rau kev ntsuas qhov purity ntawm kev lag luam -qib thiab kev lag luam raw khoom cytisine. Nws muaj qhov siab precision thiab rov tsim dua tshiab, tso cai rau kev txiav txim siab ntawm cov txiaj ntsig tshwj xeeb. Nws yog qhov tsim nyog rau cov khoom ua kom zoo dua qub nrog cov purity ntau dua 90% thiab ua tus thawj txoj hauv kev rau kev tswj hwm kev lag luam thiab thib peb -kev kuaj kev lag luam.
Txheem kev lag luam chromatographic tej yam kev mob: A C18 kem (250 mm × 4.6 mm, 5 μm) yog siv. Cov theem ntawm lub xov tooj ntawm tes muaj xws li 0.05 mol / L sodium dihydrogen phosphate tov, methanol, thiab ammonium perchlorate (15 mL: 85 mL: 0.5 g). Kev ua haujlwm tsis tu ncua suav nrog tus nqi ntws tsis tu ncua ntawm 1.0 mL / min, kev tshawb pom wavelength ntawm 305 nm, kab ke kub ntawm 25 degree, thiab txhaj tshuaj ntim ntawm 10 μL. Nyob rau hauv cov xwm txheej no, lub ncov cytisine yog symmetrical nrog kev daws teeb meem zoo, zoo sib cais cov alkaloid impurities.
Cov txheej txheem kev sim ua haujlwm: Ua ntej, ua qauv ua ntej- cov qauv khoom yuav tsum tau qhuav ntawm 105℃rau 4 teev kom qhov hnyav tas li kom tshem tawm cov kev cuam tshuam ntawm noo noo; Tom qab qhov hnyav hnyav, cov qauv yog yaj hauv methanol ntawm ultrasonication, ua kom ntim, thiab lim los ntawm 0.45 μm organic membrane. Thib ob, kev npaj nkhaus txheem-cytisine txheem kev daws teeb meem ntawm ntau qhov sib txawv yog npaj thiab txhaj tshuaj; tus qauv nkhaus yog npaj nrog thaj tsam ncov ntawm y-axis thiab concentration ntawm x-axis, ua kom pom zoo linearity nyob rau hauv 0.04–1.2 mg / mL ntau. Thib peb, cov qauv kev soj ntsuam- cov tshuaj ntsuam xyuas tau txhaj tshuaj, thaj chaw ncov tau sau tseg, thiab cov ntsiab lus cytisine suav nrog tus qauv nkhaus los txiav txim siab qhov khoom kawg purity.
Txoj kev no zoo tiv thaiv kev cuam tshuam los ntawm cov impurities thiab cov txiaj ntsig tau nrog qhov yuam kev tsawg, ua tau raws li txhua qhov yuav tsum tau muaj rau kev tsim khoom, kev tswj kom zoo, thiab kev kuaj batch; nws ua lub hauv paus rau kev kuaj purity ntawm cytisine tiav cov khoom (purity Ntau dua los yog sib npaug rau 98%) thoob plaws hauv kev lag luam.
III. Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS): Kev tshuaj xyuas meej meej haum rau cov matrices nyuaj
LC-MS/MS yog ib tug siab- txoj kev kuaj xyuas qhov tseeb uas feem ntau siv los txiav txim siab purity thiab cov ntsiab lus ntawm cytisine hauv cov qauv nyuaj xws li cov khoom siv cog qoob loo, kev npaj lom neeg, thiab cov khoom noj khoom haus matrices. Nws muaj kev sib cais thiab kev tshawb pom muaj peev xwm tshaj li tus qauv HPLC rau cov qauv uas muaj cov impurities nyuaj lossis cov lej ntawm cov tshuaj ntsuam xyuas. Nws nquag ua haujlwm hauv kev tshawb fawb txog kev tshawb fawb, kev tsim txiaj ntsig ntawm cov khoom siab - kawg, thiab kev txheeb xyuas qhov tseeb.
Cov haujlwm tseem ceeb suav nrog kev rho tawm cov qauv siv acidified acetonitrile, ua raws li kev ua kom huv ntawm cov khoom-theem rho tawm (SPE) kab kom tshem tawm cov khoom cuam tshuam xws li pigments, polysaccharides, thiab lwm yam alkaloids. Kev kuaj xyuas yog ua los ntawm kev siv ntau hom Kev Tiv Thaiv Kev Tiv Thaiv (MRM), ua kom muaj kev sib txawv ntawm cov cytisine thiab cov qauv zoo sib xws nrog cov impurities. Nrog rau qhov tsis tshua muaj kev txwv ntawm kev tshawb pom, nws raug ntsuas qhov purity ntawm cov qauv nrog cov ntsiab lus cytisine tsawg, ua tau zoo kov yeej qhov cuam tshuam rau impurity cuam tshuam nrog UV spectrophotometric thiab titration txoj kev. Txawm li cas los xij, vim yog cov khoom siv siab thiab kev sim ua haujlwm nyuaj, txoj kev no tsis tsim nyog rau kev sim batch niaj hnub hauv cov chaw tsim khoom.
IV. Differential Scanning Calorimetry (DSC): Purity assignment for reference materials and verification of high -cov khoom purity
DSC yog thawj txoj hauv kev los muab cov txiaj ntsig purity rau cov ntaub ntawv pov thawj hauv lub tebchaws thiab cov qauv siv siab - purity (Ntau dua lossis sib npaug li 99.5%) ntawm cytisine. Nws txiav txim siab purity raws li qhov sib txawv ntawm thermal zog thiab yog dav siv los xyuas qhov purity ntawm siab -kawg siv cov qauv thiab kev tshawb fawb-qib siab-purity cytisine, sawv cev rau ib qho ntawm cov txheej txheem tshawb pom tseeb tshaj plaws hauv kev lag luam.
Txoj kev no yuav tsum tsis muaj cov qauv ua ntej kho; nws yog xam cov ntsiab lus impurity thiab purity los ntawm kev saib xyuas cov kev hloov pauv thaum lub sij hawm tus qauv melting txheej txheem thiab sib piv cov txiaj ntsig tawm tsam tus qauv siv tus ntsuas thermogram, muab qhov tshwj xeeb ruaj khov thiab raug. Raws li lub teb chaws tus qauv rau cov ntaub ntawv pov thawj, cytisine siv cov ntaub ntawv yuav tsum tau ua purity nqi assignment siv ib tug ua ke ntawm HPLC thiab DSC txoj kev ua ntej ua raws li purity parameters yuav muab tau.
V. Cov txheej txheem tshawb xyuas cov txheej txheem ib txwm muaj (Tsib ntus raug txiav tawm; rau kev siv nkaus xwb)
UV absorption thiab acid-base titration feem ntau siv hauv kev lag luam yav dhau los los txiav txim siab cytisine purity; tam sim no, lawv tsuas yog ua haujlwm rau kev kwv yees ceev ceev ntawm purity nyob rau hauv qis - cov khoom siv roj av. UV spectrophotometric txiav txim siab cov ntsiab lus raws li absorbance ntawm cov wavelengths tshwj xeeb, thaum titration cia siab rau cov kua qaub-base neutralization rau quantification. Txawm hais tias ob txoj hauv kev yooj yim rau kev khiav lag luam thiab xav tau cov cuab yeej siv tsawg tsawg, lawv raug kev txom nyem los ntawm qhov tsis zoo: lawv muaj kev cuam tshuam los ntawm impurities xws li lwm yam alkaloids, organic acids, thiab pigments, uas ua rau muaj kev ntsuas tsis raug. Yog li ntawd, lawv tsuas yog tsim nyog rau cov qauv yooj yim nrog siab purity thiab qis impurity; Tsis muaj peev xwm ua tau raws li kev tswj hwm kev lag luam tam sim no thiab cov qauv kev ua raws cai, lawv tau raug hloov pauv ntau los ntawm HPLC.
VI. Kev txiav txim siab tseem ceeb rau kev xeem (Kev Lag Luam Txuj Ci)
1. Cov qauv ua ntej-kev kho mob: Ua ntej kuaj, tag nrho cov qauv cytisine yuav tsum tau qhuav ntawm 105℃rau 4 teev kom txog thaum qhov hnyav tas li kom tshem tawm cov dej noo noo; Qhov no tiv thaiv ya raws los ntawm diluting cov khoom xyaw nquag thiab ua rau kev nyeem ntawv tsis muaj purity tsawg. Tus qauv siv cov khoom siv ampoules yuav tsum tau siv tam sim tom qab qhib thiab tsis tuaj yeem muab rov qab siv dua.
2. Impurity Control: Nroj tsuag- cov qauv muab tau los yuav tsum tau ua kom huv huv kom tshem tawm cov residual polysaccharides, flavonoids, thiab lwm yam alkaloids, yog li tiv thaiv impurities los ntawm kev cuam tshuam nrog chromatographic ncov thiab cuam tshuam cov txiaj ntsig kev xeem.
3. Txoj Kev Xaiv: TLC yog siv rau kev tshuaj xyuas ua ntej ntawm cov khoom siv roj av; HPLC raug xaiv rau kev lag luam tiav cov khoom lag luam zoo tswj; LC-MS/MS raug xaiv rau cov qauv nrog cov matrices nyuaj; thiab kev sib koom ua ke ntawm HPLC thiab DSC yog ua haujlwm rau kev soj ntsuam ntawm siab - qauv purity.
Cov ntsiab lus
Cov txheej txheem kev lag luam theem rau cytisine purity kuaj tau raug tsim. HPLC tau dhau los ua tus qauv tseem ceeb rau kev tsim khoom lag luam thiab kev lag luam kev lag luam vim nws qhov tseeb, kev ua tau zoo, thiab tus nqi- ua tau zoo. TLC yog qhov tsim nyog rau kev soj ntsuam ua ntej sai, thaum LC-MS/MS thiab DSC ua tau raws li qhov siab- qhov yuav tsum tau kuaj xyuas qhov tseeb rau cov qauv nyuaj thiab siab - qauv purity, feem. Txhua txoj kev ua lub luag haujlwm tshwj xeeb, sib sau ua ib qho kev daws teeb meem rau cytisine purity test.
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