B Y T TRNG I HC DC H NI O TH HUYNNH LNG CEFOPERAZON TRONG CH PHM BNG CC PH SNG VUNG KHA LUN TT NGHIP DC S I HC H NI - 2013 B Y T TRNG I HC DC H NI O TH HUYNNH LNG CEFOPERAZON TRONG CH PHM BNG CC PH SNG VUNGKHA LUN TT NGHIP DC S I HC Ngi hng dn: TS V NG HONG Ni thc hin:B mn ha phn tch c cht Trng i Hc Dc H Ni H NI 2013 Li cm n Li u tin, em xin gi li cm n chn thnh nht, su sc nht ti TS V NG HONG ngi trc tip giao ti v l ngi thy tn tnh hng dn, ging gii nhng vn thc mc v l thuyt cng nh thc nghim, gip em hon thnh ti ny.Tip , em xin gi li cm n n cc thy c b mn Ha Phn Tch, cc anhchkthutvingipemtrongvnhacht,dngcthnghim, phng th nghim v cc trang thit b khc trong qu trnh lm thc nghim. Emcngchnthnhcmnccbn,ccanhchlmticnglun ng h em v tinh thn, vt cht, kin thc em c th hon thin ti mt cch tt nht. Cuicngemxincmnnhtrngvccthyctrongbanlnho trng to iu kin cho em c c hi c thc hin ti, c c hi nghin cu,thchinammcamnh.Trongqutrnhlmthcnghimvvitbi chc chn cn c nhiu thiu st, em rt mong nhn c nhng kin ng gp em c th hon thin hn lun vn ca mnh. Em xin chn thnh cm n! H NI, 2013 o Th Huyn Mc lcTrang T VN ............................................................................. 01 CHNG I. TNG QUAN ..................................................... 02 1.1.Phng php cc ph...................................................... 02 1.1.1. Phng php cc ph sng vung (Square Wave Polarography) ............................................................................ 03 1.2.nh lng trong phn tch cc ph sng vung.......... 05 1.3.Cefoperazon .................................................................... 06 1.3.1. Cng thc cu to ............................................................ 06 1.4.Cc phng php nh lng cefoperazon .................. 06 CHNG II. I TNG V PHNG PHP NGHIN CU........................................................................... 16 2.1. i tng - nguyn liu v thit b ................................... 16 2.1.1. i tng nghin cu....................................................... 16 2.1.2. Nguyn liu v thit b ..................................................... 17 2.2. Phng php nghin cu................................................... 18 2.2.1.Xydngphngphpnhlngcefoperazon bng cc ph sng vung trong dung dch m................................. 19 2.2.2. ng dng phng php cc ph sng vung nh19 lng cefopearazon trong mt s ch phm trn th trng 2.2.3. X l kt qu thc nghim................................................19 CHNG III. THC NGHIM, KT QU V BN20 LUN................................................................................... 3.1. Kt qu thc nghim v nhn xt .....................................20 3.1.1. Chun b ha cht ............................................................20 3.1.2. Chun b mu....................................................................21 3.1.3.Nghincucchkhcccacefoperazonviin21 cc git thy ngn treo............................................................... 3.1.4.Xydngphngphpnhlngcefoperazon bng23 cc ph sng vung trong dung dch m................................. 3.1.4.1. Ti u ha iu kin phn tch ...................................23 3.1.4.2. Kho st khong tuyn tnh ........................................29 3.1.4.3. lp v ng.........................................................33 3.2.ngdngphngphpccphsngvungnh34 lng cefoperazon trongcc chphm trnth trng............................................................................................. 3.2.1. Chun b dung dch th.....................................................34 3.2.2. Kt qu nh lng............................................................34 Bn lun.......................................................................................38 KT LUN V XUT........................................................41 TI LIU THAM KHO Danh mc ch ci vit tt HPLC-Highperformanceliquidchromatography (sc k lng hiu nng cao) UV-Vis:-Ultraviolet - visible (quang ph t ngoi kh kin) SWP-Square wave polarography (cc ph sng vung) HDME-Harging mercury drop electrode (in cc git thy ngn) B-R-Briston - Robinson (m vn nng) Current-Cng dng Amplitude-Bin xung Voltage step-Bc th Depossition time -Thi gian tch gp Frequency-Tn s TBC-Trung bnh cng SD- lch chun RSD- lch chun tng i Danh mc cc bngTrang Bng 3.1 -Ktquoccphsngvung cefoperazon0,03ppmtheomhnhthitkmt phchptrungtm(CentralCompositeFace- CCF) 26 Bng 3.2 -Phngtrnhbiudinmiquanhgia cngdnginviccthngsthigian tch gp, tn s, bin xung, bc th 29 Bng 3.3 -Ktqukhostkhongtuyntnh cefoperazon trong nn m B-R pH 4 30 Bng 3.4 -Ktqukhostkhongnngtuyn tnhcacefoperazon(0,010,07ppm)trong nn m B-R pH 4 khi c mt ca sulbactam 0,03 ppm 31 Bng 3.5 -Ktqukhostlpvngca dung dch cefoperazon 0,03 ppm 33 Bng 3.6 -Ktqukhostlpvngca dung dchcefoperazon0,03ppmkhic sulbactam 0,03 ppm 34 Bng 3.7 -Kt qu nh lng ccch phm ccha cefoperazon trn th trng 37 Bng 3.8 -Thng s qu trnh HPLC ca cefoperazon 20 ppm 38 Danh mc cc hnhTrang Hnh 1.1 -Cc thnh phn in p trong SWP 03 Hnh 1.2 -Dng tn hiu o ca phng php SWP 04 Hnh 1.3 -Cng thc cu to ca cefoperazon natri 06 Hnh 3.1a -ng cong vn-ampe vng ca dung dch cefoperazon0,03ppmtrongnnmvnnng pH 3,05 (a), 4,04 (b), 5,1 (c), 6,02 (d) 22 Hnh 3.1b - th s ph thuc v tr xut hin pc trn ngcongvn-ampevngcefoperazon0,03 ppm vo pH ca m B-R 23 Hnh 3.2a -Ccphsngvungcadungdch cefoperazon0,03ppmtrongccnnm phosphat pH 4 24 Hnh 3.2b -Ccphsngvungcadungdch cefoperazon0,03ppmtrongccnnmacetat pH 4 25 Hnh 3.2c -Ccphsngvungcadungdch cefoperazon 0,03 ppm trong cc nn m B-R pH 4 25 Hnh 3.3a -Mt pbiu din nhhngcabin xungvibcthncngdng(tns 80Hz, thi gian tch gp 120s) 28 Hnh 3.3b -Mt p biu din nh hng ca thi gian tchgpvitnsncngdng(bin xung 0,1 V, bc th 0,007 V) 28 Hnh 3.4 -Cc ph sng vung s ph thuc cng dng vo nng cefoperazon (0,01 0,06 ppm) trong nn m B-R 4 30 Hnh 3.5 - th biu din ng chun cefoperazon trong nn m B-R 4 31 Hnh 3.6 -Cc ph sng vung biu din s tng quan cngdnginvinng cefoperazon(0,010,07ppm)trongmB-R pH 4 khi c mt sulbactam 0,03 ppm 32 Hnh 3.7 -thbiudinngchuncefoperazon trong nnmB-R pH4khicmtca sulbactam 0,03 ppm 32 Hnh 3.8a -Ccphsngvungcacefoperazon trong ch phm Cefobid 36 Hnh 3.8b -Ccphsngvungcacefoperazon trong ch phm Sulperazon 36 Hnh 3.9 -Sckcadungdchcefoperazon20 ppm 38 Hnh 3.10 -Cng thc cu to ca cefoperazon 42 1 T VN Hin tng khng thuc ang l vn ton cu, c bit l cc nc ang pht trin. Chnh v l do ny, tc dng iu tr ca khng t cc thuc khng sinhgimingk.TiVitNam,cephalosporinvquinolonlhaidng khng sinh ang cn c s dng nhiu cc bnh vin. CefoperazonlmtcephalosporinthhIIIcchnhchotrnghp nhimkhunnng(nhimkhunngmt,ngh hptrnvdi,da vmmm,xngkhp,thnvngtitniu,nhimkhunhuyt)v nhim khun a khng thuc. Cefoperazon c tc dng dit khun do c ch thnh t bo vi khun ang phn chia v pht trin. Thuc bn vng vi men -lactamase do vi khun gram m tit ra nn c hot tnh i vi cc vi khun khng cc khng sinh khc. Cefoperazonkhnghpthuquangtiuha,nncdngdngtim bp hoc tim tnh mch. Trn th trng, cefoperazon c bo ch bt pha timmtthnhphnhoccskthpsulbactam,mtchtc ch men-lactamase lm tng hot tnh ca n. xc nh hm lng thuc khng sinh, HPLC v quang ph UV-Vis l hai phng php thng c s dng ti cc trung tm kim nghim thuc hin nay. Trong Dc in Anh, M, cefoperazon c nh lng bng HPLC. Vi mong mun xut mt phng php c th thay th HPLC trong php nh lng cefoperazon trong ch phm, chng ti tin hnh ti ny vi 2 mc tiu: 1.Xy dng phng php nh lng cefoperazon bng cc ph sng vung vi in cc git thy ngn treo. 2.ngdngphngphpnynhlngcefoperazon trongmtsbtthucphatimcchacefoperazonhinanglu hnh trn th trng. 2 CHNG I. TNG QUAN 1.1. Phng php cc ph [1, 2] Phngphpccphlnhmccphngphpphntchdavovic nghin cu ng cong vn-ampe hay cn gi l ng cong phn cc. y lngcongbiudinsphthuccngdnginvointhkhi tin hnh in phn dung dch phn tch vi in cc git thu ngn. Phngphpny c Heyrovskyphtminhvonm1920vchonnay cngvisphttrincakhoahckthut,phngphpnyngycng c ci tin. C s ca phng php ny l da trn phn ng in ho ca cc cht phn tchtrongdung dchinlytrnin ccgitthu ngn theo phn ng: ox + ne kh Trong : ox: Dng oxy ho kh: Dng kh n: S e trao i Phng trnh nh lng l phng trnh Incovic: Id = 605 . n . D1/2 . m2/3 . t1/6 . C Id: Cng dng cc i gii hn (A) n: S electron tham gia vo phn ng in cc D: H s khuch tn (cm2/s) m: Tc chy git Hg (mg/s) 3 t: Chu k git ca in cc git (s) C: Nng cht phn tch (mM) Tuy nhindonhhngca dng tinnnnhyca php nhlng bng cc ph c in ch t c 10-4 10-5 M. Cng vi s pht trin ca khoa hc v k thut c nhiu ci tin c p dng vi mc ch tng nhy ca phng php cc ph. Mt trong nhng ci tin ny l s ra i k thut cc ph sng vung.1.1.1. Phng php cc ph sng vung (Square Wave Polarography)Hnh 1.1. Cc thnh phn in p trong SWP : in p mt chiu : in p xoay chiu 4 Hnh 1.2. Dng tn hiu o ca phng php SWP Phng phpcc phsngvung c a rabiBarkervJenkin(1582). Phngphpnyinccgitthungncphnccbngmtinp mtchiubinthintheothigian,ccngthmvomtinpxoay chiu dng vung gc c tn s 125 200 Hz v c bin c th thay i t 1 5 mV (hnh 1.1). Mc d in cc c phn cc thng xuyn bng in pxoaychiucngvoinpmtchiunhngnhmtthitbngb ngi ta ch ghi cng dng vo khong thi gian hp vo cui mi git, c th l 2 giy sau khi to thnh git, trong mt khong 100 200 giy ng vi cui na chu k trong iu kin . Trong thc t ngi ta thng o cng dng in hai thi im, sau khi np xung khong 17 ms v sau khi ngt xung 17 ms. Hiu ca hai gi tr dng in ny l tn hiu u ra. S ph thuc ca hiu dng in ny theo th in cccdngnhpc(hnh1.2).tinhpclEpctngngnhE1/2 trong cc ph c in. Bng phng php cc ph sng vung c th t nhy ti 10-7 M v chn lc l 104. 5 u im ca k thut ny l tc qut th nhanh nn mt t thi gian v nhy ln. Gi tr nh ipc c tnh theo phng trnh: ip = K . n2 . D1/2 . EA . CA Trong : K: Hng s n: S electron trao i trong phn ng D: H s khuch tn EA: Bin xung CA: Nng cht phn tch trong dung dch [2] 1.2. nh lng trong phn tch cc ph sng vung Vnguyn tc, vic xc nhnng cacht phn tchbngphngphp cc ph sng vung c th tin hnh theo cc k thut: So snh, ng chun, thm chun v thm ng chun.Trongkhalun ny,nng cacefoperazon c xc nhbngkthut ng chun. xy dng ng chun ngi ta chun b mt s dung dch chun c nng khcnhau,thngdng 58dung dchchun.ocngdngin ca tng dung dch chun v v th biu din mi quan h gia cng dngvnngcaccdungdchchun.Xcnhvngtuyntnhl khong nng cho on ng chun c h s xc nh R2 > 0,990. hn chphingoisuy,nng cc dung dchthphi nmtrongkhongnng ca dy chun. Nng ca dung dch th c xc nh da vo phng trnh hi qui tuyn tnh biu din mi quan h gia cng dng v nng cc dung dch chun [3]. 6 1.3. Cefoperazon1.3.1. Cng thc cu to [28] Hnh 1.3. Cng thc cu to ca cefoperazon Natri-Tn khoa hc: Sodium (6R,7R) - 7 - [[(2R) - 2 - [[(4 - ethyl - 2,3 -dioxopiperazin-1-yl)carbonyl]amino]-2-(4-hydroxyphenyl) acetyl]amino]-3-[[(1-methyl-1Htetrazol-5-yl)sulphanyl] methyl]-8-oxo-5-thia-1azabicyclo[4,2]oct-2-ene-2- carboxylate -Cng thc phn t: C25H27N9O8S2Na -Khi lng phn t: 668 - tan: Tan tt trong nc, methanol, t tan trong ethanol 96% 1.4. Cc phng php nh lng cefoperazon Phng phpc im Ti liu tham kho Sc k lng hiu nng cao nh lng cefoperazon v sulbactam trong ch phm -Chun ni: Ornidazol -Phang:mphosphatpH [14] 7 3,5 : acetonitril (35 : 65) -Ct:KromasilC85m,150x 4,6 mm -Tc dng: 1ml/pht -Detector: 215 nm nhlngcefoperazontrongchphmv huyt tng -Chun ni: Ornidazol -Phang:mphosphat20 mM pH 3,5 : acetonitril (35 : 65) -Ct:Phenomenex phenylhexyl 5 m, 250 x 4,6 mm -Tc dng: 1ml/pht -=190 nm [15] nhlngngthisulbactamv cefoperazon trong ch phm -Chun ni: Captopril -Phang:ncpH3,2: acetonitril (40 : 60) -Ct:PhenomenexGeminiC18 5 m, 250 x 4,6 mm -Tc dng: 0,5 ml/pht -Detector: 210 nm [21] 8 nhlngngthisulbactam, sultamicilintosylat,cefaclor,ampicillinv cefoperazon trong ch phm -Chun ni: Salicylamid -Phang:MeOH25%trong tetramethylammonium0,005Miu chnh ti pH 3,4 bng acid phosphoric -CtSpherisorbODS2,5m, 120 x 4,0 mm -Detector: 230 nm -Tc dng: 1 ml/pht [4] nh lng cefoperazon trong ch phm -Pha ng: Amoni acetat pH 5,5 : MeOH (30 : 70) -Ct:SupelcoC185m,250x 4,6 mm -Tc dng: 0,5 ml/pht -Detector: 250 nm [26] nhlngngthiccpenicillinv cefoperazontrongchphmvdchsinh hc -Pha ng: MeOH : NaCl 0,2 M (35 : 65 65 : 35) [13] 9 -Ct: Alltech C18 10 m, 250 x 4,6 mm -Tc dng 1,5 2,2 ml/pht -Detector:inhasauphn ngphnhydongunbcx Photronix Model 816 UV nhlngngthiampicillin, cefoperazon v sulbactam trong ch phm -Phang:MeOH:m tetraethylammoniumacetat5mM pH 4,5 (35 : 65) -Ct: pha lin kt |-cyclodextrin cyclobond I, 5 m, 250 x 4,6 mm -Tc dng: 0,8 ml/pht -Detector: 280 nm [7] nhlngngthicefalexin, cefoperazon,ceftriaxon,ceftazidim, cefepim,cefoperazonvsulbactamtrong ch phm -Phang:KH2PO4(50mM, pH 4,6) : acetonitril (80 : 20) -Ct:WatersBondapakC18 10 m, 250 x 4,6 mm -Tc dng: 1 ml/pht - Detector: 230 nm [17] 10 Scklp mng nh lng cefoperazon trong ch phm -Pha tnh: Silica gel G60 F254 -Phang:Ethylacetat: methanol : nc : aceton (3:1:1:2) -Phthin:Quanstdin UV 220 nm 50 Hz sau khi phun thuc th Dragendorff [9] Sckin ng mixen nhlngngthicephazolin, cefuroxym,ceftriaxon,cefoperazonv ceftazidim trong hn hp -Ct: Mao qun silica nung chy c lp bao polyimid 60 cm 75 m -Phang:mphosphat: boratpH6,5ccha10g/lnatri dodecylsulfatv17,4g/l pentanesulfonic acid - Detector: 214 nm [20] nh lng cefoperazon trong ch phm -Dung mi: MeOH -o quang ti = 230 nm - odintchdingcongph hp th trong khong 225 235 nm [16] nhlngngthicefoperazonv 11 sulbactam trong ch phm -Dung mi: Nc -Phohm(A=4nm)bc 1,bc2:theophpogiaoim khng 277,5; 267,5, 272,5; 290 nm v phpoth267,5nm;290nm; 272,5 nm; 290 nm [6] nh lng cefoperazon trong ch phm -Tophnngvifolin-ciocalteautrongmitrngkim (Na2CO3)voquangtimaxca hp cht mu xanh 652 nm. [24] nh lng cefoperazon trong ch phm -Tophnngvifolin- ciocalteauphenoltrongmitrng Na2CO3 v o quang ti max ca hp cht mu xanh 668 nm. [27] Quang ph UV nh lng cefoperazon trong ch phm o quang ti -Nitrathavtophcvitc nhn i nhn (I): 390 nm -Nitrathavtochelatkim loi (II): 520 nm -To lin kt vi diazo (III): 435 nm [23] 12 -Phnngvimuingv chitphcchelattothnhvo cloroform (IV): 415 nm nh lng cefoperazon trong ch phm -oquangtrong1,2-dicloroethan ti 364 nm sn phm ca phn ng vi iod. -oquangtrongmethanolti 460nmsnphmcaphnngvi 2,3-dicloro-5,6-dicyano-p-benzo-quinon. -oquangtrongacetonitrilti 843nmsnphmcaphnngvi 7,7,8,8-tetracyanoquinodimethan. [19] nh lng cefoperazon trong ch phm -oquangti=510nmsau 30phtphnngviquercetin coxyhabiN-B-Romosuccinimide [10] Quang ph hunh quang nh lng cefoperazon trong ch phm -oquangphcbcbato thnhnhphnngviTb3+trong [25] 13 m Tris vi pH 8 - kch thch = 240 nm, pht x = 485 nm nh lng cefoperazon trong ch phm -oquangdnxutcoumarin phtquangmuvngctothnh nhphnngviethylacetattrong mi trng acid sulfuric -kchthch =412, phtx=465 nm [5] Somuv ph hp th nguyn t nh lng cefoperazon trong ch phm -Somutibcsng525nm hocoquangtibcsng358,6 nmcpiontothnhgiachtphn tchvmuiamonireineckattrong mi trng acid. -Dung mi: Aceton [22] Vn-ampe nhlngcefoperazontrongchphmv dch sinh hc Vn-ampe sng vung -incccngtc:Gitthy ngn treo -Thi gian tch gp: 60 150 s -Th tch gp: 0,3 V -Bc th: 8 mV [12] 14 -Bin xung: 25 mV -Tn s xung: 60 Hz -Chtinlynn:macetat pH 4,2 nhlngcefoperazontrongchphm,t bo vi khun, sa v nc tiu. -Vn-ampe sng vung -incccngtc:Gitthy ngn treo -Th bt u: 0,0 V -Thi gian tch gp: 1 s -Bc th: 5 mV -Bin xung: 25 mV -Tn s xung: 500 Hz -Cht in ly nn: m Britton Robinson pH 4,4 [18] nh lng cefoperazon trong nc tiu. -Vn-ampe o dng trc tip v xung vi phn -incccngtc:Gitthy ngn treo -Th tch gp: 0,4 V -Tcqut:100mV/s(o dngtrctip),5mV/s(xungvi phn) [11] 15 -Khong th: 0,4 1,0 V -Bin xung: 50 mV - lp ca xung: 0,10 s -Chtinlynn:HClO40,03 M, pH 2,0 nhlngcefoperazontrongchphmv huyt tng -in cc cng tc: Cacbon than ch -in ly nn: m phosphat 0,2 M pH 2 Vn-ampe xung vi phn -Tc qut: 20 mV/s -Bin xung: 50 mV - rng xung: 50 ms Vn-ampe sng vung -Bin xung: 25 mV -Tn s: 15 Hz -Bc th: 4 mV [8] 16 CHNG II I TNG V PHNG PHP NGHIN CU 2.1. i tng nguyn liu v thit b2.1.1. i tng nghin cu Bit dcc im Cefobid Cefoperazon 1g Nh sn xut: Haupt Pharma Latina S.r.l, Italy S l:11600606 S ng k: VN 13299 11 Ngy sn xut: 09/ 09/ 2011 Hn s dng: 07/ 09/ 2013 Neoaxon Cefoperazon 1g Nh sn xut: Samchundang Pharm, Hn Quc S l: 11002 S ng k: VN 4068 07 Ngy sn xut: 14/ 04/ 2011 Hn s dng: 13/ 04/ 2013 Bacamp Cefoperazon 500 mg Sulbactam 500 mg Nh sn xut: Hanlim Pharmaceutical, Hn Quc S l: 10701 S ng k: VN 5179 10 Ngy sn xut: 04/ 07/ 2011 Hn s dng: 03/ 07/ 2014 17 Cefactam Cefoperazon 1 g Sulbactam 1 g Nh sn xut: Laboratorio Libra S.A, Uruguay S l: 04120034 S ng k: VN 4852 07 Ngy sn xut: 10/ 2012 Hn s dng: 10/ 2014 Jincetam Cefoperazon 500 mg Sulbactam 500 mg Nh sn xut: Hankook Korus Pharm, Hn Quc S l: A01709 S ng k: VN 8024 09 Ngy sn xut: 21/ 06/ 2012 Hn s dng: 20/ 06/ 2015 Sulperazon Cefoperazon 500 mg Sulbactam 500 mg Nh sn xut: Pfizer, M S l: 12902400 Ngy sn xut: 10/ 02/ 2012 Hn s dng: 08/ 02/ 2014 2.1.2. Nguyn liu v thit b -Nguyn liu: + Cht chun: Cefoperazon (Vin kim nghim trung ng) 89,82% C25H26N9O8S2 SKS: WS.0211230.01 (08.07) Bo qun: 2 8oC, trnh nh sng + Cht chun: sulbactam (Vin kim nghim trung ng) 899,1 g C8H11NO5S/ mg (khan) 18 m: 0,45% SKS: 0103159 Bo qun: 2 8oC, trnh nh sng + Nc ct 2 ln lc loi b ion +DungdchCH3COOHc(98%khilng/khilng),dungdch H3PO4c(84%khilng/khilng),dungdchHClc(ttrng 20oC = 1,18, nng khong 11,5 M), NaOH, acid boric, acid acetic c (t tiu chun phn tch) -Dng c: + Bnh nh mc: 25 ml, 50 ml, 100 ml + Cc c m: 100 ml, 250 ml, 500 ml + Pipet: 1 ml, 5 ml, 10 100 l, 100 1000 l -My mc thit b: +MyccphComputrate797VAviphnmmchuyndng797VA ComputraceSoftware1.3(MetrohmAG,ThyS)cchytrnhiu hnh Windows XP. H 3 in cc ca my gm c: in cc cng tc: Cc git Hg treo (HDME) in c so snh: Ag,AgCl,KCl in cc b tr: Pt Loi O2 ha tan bng N2 99,99% + My o pH: EUTECH INSTRUMENTS pH 510 + My ct nc 2 ln (HAMILTON) + My lc nc: Maxima Ultra pure water (ELGA) + My siu m: Ultrasonic LC 60 H (ELMA, c) + Cn phn tch TE214S (Sartorius) 2.2. Phng php nghin cu 19 2.2.1.Xy dng phng php nhlngcefoperazonbngccphsng vung-Ti u ha iu kin phn tch -Chn khongnngcsphthuctuyntnh gianng v gi tr cng dng in tng ng -Kim tra lp li ca phng php -Kim tra ng ca phng php 2.2.2.ngdngphngphpccphsngvungnhlng cefoperazon trong mt s ch phm trn th trngnh lng cefoperazon trong mt s ch phm bng hai phng php: - Cc ph sng vung (SWP) - Sc k lng hiu nng cao (HPLC) theo quy nh ca Dc in M2.2.3. X l kt qu thc nghim Sphthuctuyntnh ca nng vi cng dng incthitlp bngphngphpbnhphngtithiuvihstngquancang chun R2 > 0,990. So snh kt qu ca SWP vi HPLC bng kim nh t-cp vi mc tin cy 95%. Gi tr trung bnh:nXXnii ==1 lch chun: SD = ) 1 () (12=nX Xnii lch chun tng i:100 = RSD XSD 20 CHNG III THC NGHIM, KT QU V BN LUN 3.1. Kt qu thc nghim v nhn xt 3.1.1. Chun b ha cht 1) Pha m vn nng (m Britton Robinson) Hn hp gm 3 acid: CH3COOH 0,04 M + H3BO3 0,04 M + H3PO4 0,04 M. - Ly 0,24 ml dung dch CH3COOH c ha tan vo khong 100 ml c dung dch CH3COOH 0,04 M. - Ly 0,25 ml dung dch H3PO4 c ha tan vo khong 100 ml c dung dch H3PO4 0,04 M. - Cn 0,27 g H3BO3 ha tan vo khong 100 ml c dung dch H3BO3 0,04 M. Dung dch NaOH 0,2 M: Cn 0,8 g NaOH ha tan vo khong 100 ml. Trnhnhp3 acid vidungdchNaOH0,2 Mtheocctlthchhp c cc pH khc nhau. 2) Pha m acetat pH 4 - PhadungdchCH3COOH:Lykhong1,15mldungdch CH3COOH c ha tan vo khong 100 ml. - Pha dung dch CH3COONa: Cn 2,72 g CH3COONa.3H2O ha tan vo khong 100 ml. - TrnccdungdichCH3COOHvCH3COONatheomttl thch hp c dung dch m pH 4 3) Pha m phosphat pH 4 - Cn 2,78 g NaH2PO4 ha tan vo khong 100 ml - Cn 5,3 g Na2HPO4 ha tan vo khong 100 ml - Trn cc dung dch NaH2PO4 v dung dch Na2HPO4 theo t l thch hp c dung dch m phosphat pH 4. 21 3.1.2. Chun b mu Pha mu chun: Pha dung dch chun gc cefoperazon 500 ppm: - Cnchnhxckhong0,0560gchtchuncefoperazonnh mc vi nc thnh 100 ml. Bo qun dung dch ny trong l ti mu trong t lnh khong mt tun. - Ccdungdchcefoperazonlmviccphalongtdung dch chun gc trong bnh nh mc 100 ml. 3.1.3.Nghincucchkhcccacefoperazonviincc gitthy ngn treo - Tinhnhongcongvn-ampevngviinccgit thyngntreodungdchcefoperazon0,03ppmtrongnnmvn nng(BrittonRobinson)vipH210,viccthngsmynh sau: Th tch gp 0,3 V Thi gian tch gp100 s Thi gian cn bng10 s Thi gian sc kh100 s Khong th 0,3 1,2 V Bc th0,005 V Tc qut0,3 V/s Kch thc git4 Tc khuy2000 vng/pht - KtquchothykhipHtng,pcngycngthnvdch chuyndntheo chiumcath. Vi pH 7,khngc pcxuthin 22 trnngcongvn-ampecadungdchcefoperazon0,03ppm(hnh 3.1a). S ph thuc ca v tr xut hin pc vo pH c biu din bng phngtrnhhiquytuyntnhE=0,0537pH0,792,viR2= 0,9994 (hnh 3.1b) Hnh 3.1a. ng cong vn-ampe vng ca dung dch cefoperazon 0,03 ppm trong nn m vn nng pH 3,05 (a), 4,04 (b), 5,1 (c), 6,02 (d) 23 Hnh 3.1b. th s ph thuc v tr xut hin pc trn ng cong vn-ampe vng cefoperazon 0,03 ppm vo pH ca B-R3.1.4.Xy dng phng php nhlngcefoperazon bngccphsng vung3.1.4.1. Ti u ha iu kin phn tch - Kho st s b - Kho st cc loi m Tin hnh kho st s xut hin pc ca cefoperazon 0,03 ppm trong cc nn m khc nhau pH 4 (hnh 3.2a, b, c) bng phng php cc ph sng vung vi cng thng s my nh sau: 24 Th tch gp 0,3 V Thi gian tch gp100 s Thi gian cn bng10 s Thi gian sc kh100 s Khong th 0,3 1,2 V Bc th0,005 V Tc qut0,3 V/s Tc khuy2000 vng/pht Kch c git4 Hnh 3.2a. Cc ph sng vung ca dung dch cefoperazon 0,03 ppm trong nn m phosphat pH 4 100 nA 25 Hnh 3.2b. Cc ph sng vung ca dung dch cefoperazon 0,03 ppm trong nn m acetat pH 4 Hnh 3.2c. Cc ph sng vung ca dung dch cefoperazon 0,03 ppm trong nn m B-R pH 4 100 nA 100 nA 26 Hnh3.2cchothysngkhcacefoperazonchnhnhnu,cng dngcaonhttrongnnmB-Rvthphntrongnnmacetatv phosphat. Do vy, m B-R pH 4 c la chn cho cc kho st tip theo. - Ti u ha iu kin phn tch Qua kho st s b 4 yu t nh hng nht c chn ra l tn s (60 80 Hz), thi gian tch gp (100 120 s), bin xung (0,05 0,1) v bc th (0,005 0,007 V), m hnh th nghim c b tr nh trong bng 3.1 v 3.2. Bng 3.1. Kt qu o cc ph sng vung cefoperazon 0,03 ppm theo m hnh thit k mt phc hp trung tm (Central Composite Face - CCF) SttThi gian tch gp (s) Tn s (Hz) Bin xung (V) Bc th (V) Cng dng (nA) 1120700,0750,006 166 2110700,0750,006(*) 3120600,10,005 110 4120800,050,007 188 5120800,10,005 164 6100700,0750,006 142 7120600,10,007 161 8100600,050,005 101 9100800,10,005 144 10110700,0750,005 132 11100800,050,007 173 27 12110700,0750,006 155 13110700,10,006 157 14100600,050,007 132 15100600,10,005 110 16110700,0750,006 152 17120800,10,007 215 18120600,050,005 114 19110600,0750,006 133 20120600,050,007 149 21100800,050,005 131 22110700,0750,007 176 23120800,050,005 154 24100600,10,007 142 25110800,0750,006 173 26110700,050,006 143 27100800,10,007 187 Ch thch: (*) s liu khng c s dng cho ti uKtqutiucxcnhchoccyutsaukhisdngphnmm Modde 9.1 l: thi gian tch gp 120 s, bin xung 0,1 V, bc th 0,007 V, tn s 80 Hz. Hnh 3.3a v b biu din nh hng ca cc yu t kho st vi cng dng o c bng phng php cc ph sng vung. 28 Hnh 3.3a. Mt p biu din nh hng ca bin xung v bc th n cng dng (tn s 80 Hz, thi gian tch gp 120 s) Hnh 3.3b. Mt p biu din nh hng ca thi gian tch gp v tn s n cng dng (bin xung 0,1 V, bc th 0,007 V) Mi quan h gia cc yu t nh hng vi gi tr cng dng in c trnh by trong bng 3.2 29 Bng 3.2. Phng trnh biu din mi quan h gia cng dng in v cc thng s thi gian tch gp, tn s, bin xung, bc th Phng trnh I = 8,83 U1 20,94 U2 5,83 U3 20,17 U4 2,31 U1. U2 2,31 U2. U3 2,19 U3. U4 R2 = 0,991Q2 = 0,932 U1: Thi gian tch gpU2: Tn sU3: Bin xung U4: Bc th Ch thch R2: Tnh ph hp Q2: Kh nng d on 3.1.4.2. Kho st khong tuyn tnh - Miquanhgianngcefoperazon(0,01 0,06 ppm)vi cngdngincobngphngphpccphsngvung trong nn m B-R 4 biu din trong bng 3.3 v 3.4. 30 Bng 3.3. Kt qu kho st khong tuyn tnh cefoperazon trong nn m B-R pH 4 C (ppm)V tr pc E (V)I (nA) 0,01 0,749 112 0,02 0,763 172 0,03 0,756 230 0,04 0,756 288 0,05 0,749 348 0,06 0,742 393 Hnh 3.4. Cc ph sng vung s ph thuc cng dng vo nng cefoperazon (0,01 0,06 ppm) trong nn m B-R pH 4 31 Hnh 3.5. th biu din ng chun cefoperazon trong nn m B-R pH 4 Bng 3.4. Kt qu kho st khong nng tuyn tnh ca cefoperazon (0,01 0,07 ppm) trong nn m B-R pH 4 khi c mt ca sulbactam 0,03 ppm C (ppm)V tr pc E (V)I (nA) 0,01 0,819 83,4 0,02 0,819 143 0,03 0,819 205 0,04 0,819 267 0,05 0,819 329 0,06 0,819 385 0,07 0,819 448 32 Hnh 3.6. Cc ph sng vung biu din s tng quan cng dng in vi nng cefoperazon (0,01 0,07 ppm) trong m B-R pH 4 khi c mt sulbactam 0,03 ppm Hnh 3.7. th biu din ng chun cefoperazon trong nn m B-R pH 4 khi c mt sulbactam 0,03 ppm 33 3.1.4.3. lp v ng lp v ng ca phng php c xc nh da trn kt qu ca php o 6 dung dch chun cefoperazon 0,03 ppm pha t chun gc 500 ppm trong cng iu kin. Kt qu c trnh by trong bng 3.5 v bng 3.6 Bng 3.5. Kt qu kho st lp v ng ca dung dch cefoperazon 0,03 ppm Ln I (nA)V tr pc E (V)% Tm li1 233 0,749101,2 2 225 0,74997,9 3 232 0,749100,8 4 230 0,74299,9 5 231 0,742100,4 6 242 0,74298,3 SD1,4 TBC99,75 RSD1,4% 34 Bng 3.6 .Kt qu kho st lp v ng ca dung dch cefoperazon 0,03 ppm khi c sulbactam 0,03 ppm LnI (nA)V tr pc E (V)% Tm li1 200 0,81997,3 2 202 0,83398,4 3 203 0,81998,9 4 205 0,826100,0 5 206 0,826100,6 6 208 0,819101,6 TBC99,45 SD1,5 RSD1,5% 3.2.ngdngphngphpccphsngvungnhlng cefoperazon trong cc ch phm trn th trng 3.2.1. Chun b dung dch th - Thuc bt pha tim n thnh phn: Cefoperazon 1 g Cn chnh xc mt lng bt thuc tng ng khong 0,0500 g cefoperazon nh mc vi nc thnh 100 ml. Ly 0,5 ml dung dch ny cho vo bnh nh mc 100 ml, thm nc va c dung dch th. 35 - Thuc bt pha tim hai thnh phn: Cefoperazon kt hp vi sulbactam t l 1:1. Cn chnh xc mt lng thuc tng ng khong 0,0500 g cefoperazon nh mcvincthnh100ml.Lychnhxc0,5mldungdchnychovo bnh nh mc 100 ml, thm nc va c dung dch th. 3.2.2. Kt qu nh lng Tin hnh nh lng bng phng php ng chun, ly 0,3 ml dung dch th nh mc thnh 25 ml vi dung dch m B-R pH. o dng cc ph sng vung vi cc thng s sau: Th tch gp 0,3 V Thi gian tch gp120 s Thi gian cn bng10 s Bin xung0,1 V Tn s80 Hz Bc th0,007 V Tc khuy2000 vng/ pht Khong th 0,3 1,2 V Thi gian sc kh100 s Kch c git4 Kt qu nh lng c trnh by (hnh 3.6a v b) v (bng 3.7) (v d vi ch phm Cefobid v Sulperazon). Kim nh t ghp cp vi tin cy 95% cho thy khng c s khc bit c ngha thng k khi so snh kt qu nh lng cc ch phm kho st bng cc ph sng vung v HPLC. 36 Hnh 3.8a. Cc ph sng vung ca cefoperazon trong ch phm Cefobid Hnh 3.8b. Cc ph sng vung ca cefoperazon trong ch phm Sulperazon Bng 3.7. Kt qu nh lng cc ch phm c cha cefoperazon trn th trng 37 Ch phm Hm lng % so vi nhn (TBC SD, n = 6) Cc ph sng vungHPLC Cefobid101,2 1,2100,8 1,3 Neoaxon100,2 0,5100,7 0,7 Sulperazon101,1 1,2100,4 0,9 Jincetam100,8 1,199,8 0,7 Bacamp99,9 1,3100,9 1,1 Cefactam102,3 0,4100,1 1,1 Trongnghincuny,phng php HPLCcsdnglmphngphp i chiu.iu kin sc k:-Ct: Apollo C18 150 mm x 4,6 mm, 5 m -Detector UV: 210 nm -Tc d dng: 0,8 ml/pht -Phang:Hnhpacetonitril:acidphotphoricpH3,2 (60 : 40, tt/tt) -Th tch tim mu: 20 l Hnh3.9biudinsckcachuncefoperazon20ppm.Ccthngs ca qu trnh sc k c trnh by trong bngBng 3.8. Thng s qu trnh HPLC ca cefoperazon 20 ppm 38 S ttThng sCefoperazon 1 lpRSD < 2% 2 ng99 101% 3Khong tuyn tnh 10 35 mg/lR2 > 0,990 4H s bt iAF = 0,8 5S a l thuyt /m44000 6Thi gian lu (pht)4,4 Hnh 3.9. Sc k ca dung dch chun cefoperazon 20 ppm Bn lun: Ccphvn-ampevngcadungdchcefoperazon0,03 ppmtrongnn m vn nng (Britton Robinson) vi pH 3 6 cho thy c duy nht mt pc khng thun nghch tng ng vi qu trnh kh catod trong khong th 0,3 1,2 V (hnh 3.1a). 39 Hnh 3.10. Cng thc cu to ca cefoperazon Theo l thuyt, s xut hin ca pc ny c th l do s kh lin kt i v tr C3 C4 hoc nguyn t nit v tr lin kt vi C7 (hnh 3.10) [18]. y l hai v tr ngho in t c kh nng nhn in t trong iu kin pH < 6.Vi 2 v tr ny,khitin hnh kh cc phi xut hin hai pc trn cc ph . Tuy nhin do hiu ng lin hp e vo vng (C3 C4) v s n ng khng gian lm cho v tr C3 kh nhn in t hn so vi nit v tr C7. Do vy pc kho st c c th do s kh nit v tr C7 theo phng trnh sau: ThcapcckhuynhhngdchchuynvgitrmhnkhitngpH chngtcsthamgiacaprotontrongphnngincc.Miquanh tuyn tnh gia pc v pH ca m c biu din bng phng trnh hi quy tuyntnhE =0,0537 pH 0,792,vi R2=0,9994(hnh3.1b). Gitr 40 dccaphngtrnhny53,7mV=59/.(m/na).Trongmvnals proton v s electron tham gia phn ng kh cc; l h s chuyn i xng. Vi c ch phn ng nh xut trn, m = 1 v na = 2 = 0,55 chng t cefoperazon tham gia phn ng khng thun nghch trn in cc git thy ngn. Trongkhalun ny,vic ti uha iu kinphntchc btu bng lachnloidungdchm.Sxutpctrnccphsngvungca cefoperazon 0,03 ppm trong cc nn m phosphat, acetat, B-R pH 4 cho thy m B-R ph hp nht cho php nh lng (hnh 3.2c). Qui hoch ha thc nghim theo thit k mt phc hp trung tm s dng phn mm Modde 9.1 (bng3.1)xcnhiukintiucaphpnhlngbngccph sng vung trn in cc git thy ngn nh sau:Th tch gp 0,3 V Thi gian tch gp120 s Thi gian cn bng10 s Bin xung0,1 V Tn s80 Hz Bc th0,007 V Tc khuy2000 vng/pht Khong th 0,3 1,2 V Thi gian sc kh100 s Kch c git4 41 Trncscciukinphntchclachntrn,phpnhlng cefoperazontrongchphm cthmnhtheocctiuchkhongtuyn tnh lp v ng. KtquchothyccthngsclachntrnmyccphComputrate 797VAthamnyucuvchnhxccaphpnhlng cefoperazon(bng3.5,3.6).Ccphsngvungcaccdungdch cefoperazon chothymitng quanchtchgia cng dng o c vnngcefoperazontrongkhong0,010,07ppm(R2=0,999,n=6) (hnh3.5, 3.7).Ccph cann mvn nng khng xut hin pckhc trong khong th xut hin pc cefoperazon ( 0,3 1,2V) chng t phng php ny c tnh chn lc. Ngoi ra, s c mt ca sulbactam khng lm nh hngnchnhxcvkhongtuyntnhcaphpnhlng cefoperazon (hnh 3.6, 3.7) v (bng 3.4). Ktqukimnhttheocpvisliuthuct 6chphmkhcnhau chothyskhcbitvngcaphpnhlngbngccphsng vung v HPLC c quy nh trong Dc in M khng ngha thng k (p > 0,05). Hm lng thc t ca cefoperazon trong cc ch phm c kho st t khong 99 102% so vi hm lng ghi trn nhn (bng 3.7) (t yu cu theo quy nh ca Dc in M: Cefoperazon trong thuc bt pha tim phi t 90 120% so vi hm lng ghi trn nhn) [28]. 42 KT LUN V XUT Nh vy trong lun vn ny, cc vn sau c gii quyt: 1.Lachnciukintiunhlngcefoperazonbngphng php cc ph sng vung vi in cc git thy ngn treo. 2.Xydngcphpnhlngcefoperazontrongccchphm,vi khong nng tuyn tnh 0,01 0,07 ppm, tha mn cc yu cu v lp (RSD