Nghien CLFU - Ky thuat
Nghien cmi ling dung ky thuat
dien di mao quan vung trong phan tich khang sinh nhom cephalosporin
U-
Dat van de
Hien nay cephalosporin duge coi la mgt nhdm khang sinh quan trgng va thudng dung nhit.
NhO'ng nghien cuu hda hgc khang sinh cephalosporin cho thiy ehung la d i n c h i t aeyl hoa eua A7AC mang c i u true A^-cephem. Khung ciia cephalosporin (A^-cephem) gdm vdng f>- lactam (azetidin) k i t hgp vdi vdng dihydromethathiazin, trong dd A phai la A^ mdi dii lien hgp dien tir vdi vdng p-laetam d i hgp c h i t cd tac dung sinh hge. Dien tir TT d A^cung vdi cac eromophor (nhan tham) lam eho phd h i p thu UV ciia tu'ng cephalosporin cd cue dai xac djnh.
Cae phuang phap djnh lugng khang sinh nhdm P-laetam ehinh thire duge quy djnh trong duge diin eae nude nhu Anh. My, Viet Nam gdm 5 ky thuat Do iod. do t h i . do quang. HPLC va phuang phap vi sinh vat Ben canh dd da ed nhiiu phuang phap khae dugc sir dung trong cae tieu ehuin ca sd hoac dugc edng bd tren cae sach chuyen khao va tap chi. Cae phuang phap nay duge ggi ehung dudi ten cac phuang phap ngoai dugc diin. bao gdm 4 nhdm: Quang phd (chu y i u quang phd UV-VIS va huynh quang); HPLC; Dien hda (phep do von - ampe) va Dien di mao quan.
Dien di mao quan la mgt u'ng dung mang tinh ky thuat cao. cd the diing de phan tich eae c h i t ion hda va ca nhO'ng c h i t khdng ion hda, nguyen ly dan gian. ddng thdi sir dung dung mdi re tiin it ddc hai (cac dung dich dem). Cho d i n nay, dien di mao quan v i n la mdt ky thuat mdi dang phat triin nhanh nen nhirng u'ng dung eua nd ludn duge md rdng tir nam nay qua nam khae.
Mdt sd nghien ciru v i phan tich va nghien ciru v i su dn dinh nhdm khang sinh cephalosporin trong
Nguyen Van Yen, Nguyen Thi Thanh Phirong 'SaYrSmNoi
e h i phim va trong djeh sinh hgc (huyit tuang, nude tiiu, mat) bing mode ClE '^•^•^' va MEKC '"*'*' da dugc cdng bd. LTu d i i m ndi bat cua ky thuat CE trong nghien ciru nhdm khang sinh p-lactam la kha nang tach dugc hdn hgp nhiiu thanh phan, cd t h i tach hdn hgp 1 4 - 2 0 chit ed c i u triic hda hgc tuang ty nhau '^•^'.
Ky thuat Dien di mao quan (CE) da dugc dua vao phin phuang phap ehung eua DDVN IV. nhung cac chuyen luan sir dung ky thuat CE chua cd nhiiu. De phat triin irng dung ky thuat nay d Viet Nam, trong chuyen luan nay ehung tdi nghien eu'U xay dung chuang trinh dien di mao quan vung CZE phan tieh hdn hgp 5 khang sinh nhdm cephalosporin sir dung nhiiu nhit a Viet Nam, mac du hdn hgp khang sinh nay khdng duge su' dung trong thuc t i diiu tri nhung nghien cu=u nay n h i m chu'ng td kha nang irng dung cua ky thuat CE ddi vdi viec phan tich cac khang sinh nhdm cephalosporin ndi ehung.
De minh hga nghien ciru nay, ehung tdi chon khang sinh eefixim (mdt khang sinh ed t/ le cao cae Id thude kem c h i t lugng, chi phi cho phan tieh b i n g HPLC d i t nhit) trong xay dyng quy trinh djnh lugng eefixim trong e h i p h i m .
Phu'O'ng p h a p , thiPC n g h i e m Hda chit va thiit bj
Thiet bj
- May dien di mao quan Agilent 3D CE, May HPLC Shimadzu UV SCL-10AVP.
- Cdt phan tieh: Cdt Agilent HPCE 50nm.
l=56em va 75LLm, l=56em
Chat chuin, hoa chat dung moi
- C h i t ehuin Qudc gia: cephadroxin, cephalexin, cephaelor, cefotaxim, eefixim.
- Cac hda c h i t dat loai P.A (Merck).
44
TAP CHI DU^OC HOC - 12/2010 (SO 416 NAM 50)
Nghien CLFU - Ky thuat
Doi tu'O'ng nghien CIFU
- Vien nang FIXMA (Cefixim lOOmg). SKS:
FM02- HD: 11/12/2009
- Nai san xuit: Clesstra Healthcare PVT., Ltd. - India
K § t q u a v a b a n l u a n
Xay dyng chu'cng trinh CE phan tich hdn hgp 5 khang sinh nhdm cephalosporin
Cac dieu kien phan tich Chuin bi cac dung djch dem TT
1 2 3 4
Dung djch dem Citrat20mM, Borat 15mM
Citrat 20mM Borat 15mM Na2HPO4l0mM: MeOH 8 : 2
Ki hieu A l A2 A3 A4 Chuan bi dung dich phan tich hdn hap cephalosporin:
- Pha rieng biet cae dung djch me ndng do Img/ml cephalexin (CFL-M); eephadroxil (CFD- M); eefixim (CFX-M); cefotaxim (CFT-M);
cefaclor (CFC-M) bing each can chinh xac khoang 50mg hoat chit, them 5ml methanol (MeOH), them nude vu'a du 50ml.
- Hilt chinh xae 1ml dung djeh CFL- M, 1ml dung djch CFD- M, 1ml dung dich CFX- M, 1ml dung djeh CFC- M va 1ml dung djeh CFT- M cho vao binh djnh mirc 50ml. Them dung dich dem
v.d 50 ml. Lge qua mang Ige 0,2|j,m. Thu duge cac dung djeh:
+ Vdi dem A l thu dugc hdn hgp dung djeh k y h i e u S H A I .
+ Vdi eae dem A2; A3; A4; A5 thu 6\jegc cac dung djeh tuang u'ng SHA2; SHA3; SHA4; SHA5.
- De xae djnh pic eua mdt c h i t trong mdt chuang trinh dien di, pha cac dung dich ddi chiiu ed eung ndng do trong cac dung djch dem tuang irng.
- De xac djnh tinh chgn Igc, them tirng chuin vao dung djeh hdn hgp.
Diiu kien dien di:
+ Mode: Dien di mao quan vung CZE.
+ Cdt Agilent HPCE 50|j.m, I = 56em.
+ Dien t h i 2 d i u cdt mao quan: 25 KV +Xir ly mao quan: 1,00 phiit vdi nude; 2,00 phut vdi NaOH 0,1N; 2,00 phut vdi nude; 5,00 phiit vdi dung djeh dem khao s a t
+ Thdi gian tiem m l u : 5s.
Ket qua phan tfch
Khao sat, lira chon dung djch dem
T i i n hanh phan tieh cac dung djch hdn hgp cephalosporin vdi eae dung djch dem A l , A2, A3, A4, song song vdi phan tich cac dung djeh ddi chiiu tuang u'ng trong eung d i i u kien bing chuang trinh dien di tren, k i t qua dugc tdm t i t d bang sau:
Bang 1: Khao sat anh hwang cua cac he dem din kha nang tach cac cephalosporin
Dd
SHA1 SHA2 SHA3 SHA4
Dien the 25 KV 25 KV 25 KV 25 KV
Dieu kien phan tich
A nm Dem 270 A l 270 A2 270 A3 283 A4
CFC
5,7S
Th&i gian di chuyen cac chit
> CFL CFC CFT Co 4 dinh pic khong tach rdi
Co 4 dinh pic khong tach rdi Co 4 dinh pic khong tach rdi
6,33 6,95 7,52 CFX
9,76 10,37
7,86 17,53
(MD C.S^TW.Ii BlI-.!! (CkWIOVSCr » 0*0 c.sg^ro.ii p.»(t(((CHW>s\icr mi.ci)
s 5 .
» i
ML
Hinh 1 : Dien di dd nen dem A4 Hinh 2: Dien di dd cua hdn hgp 5 khang sinh cephalosporin trong dem A4
TAP CHI DUOC HOC - 12/2010 (SO 416 NAM 50)
45N g h i e n CLFU - Ky t h u a t
Nhan xet:
+ Chuang trinh dien di tren vdi dem. A4 cd 5 pic tach rdi nhau va ed thdi gian di chuyin tuang u'ng vdi pie thu dugc tren dien di dd eua m i u ddi chiiu tuang irng trong eung d i i u kien.
Chiiu cao pie ciia mgt hoat c h i t lang len khi them mdt Iugng chuin tuang ung. Vdi k i t qua thye nghiem tren, chuang trinh phan tieh dam bao tinh dac hieu. Sir dung dem A4 trong cae nghien eiru tiip theo.
+ Khi e i n xay dyng quy trinh djnh lugng Bang 2: Phwang trinh hdi quy va he sd twang
theo ky thuat ndi ehuan, ed the dya vao' dien di dd (hinh 2) de lya chgn c h i t ndi c h u i n phu hgp eho djnh lugng mdt khang sinh.
+ Thanh p h i n va pH eua dung dich dem la mdt y i u td quyit djnh hieu lye tach ciia qua trinh dien di.
Khao sat khoang tuyin tinh:
K i t qua khao s^t cho t h i y sy tuang quan tuyin tinh giCra ndng do va dien tich pie eua mil chit trong khoang ndng' do khao sat dugc the hien trong bang 2.
quan giira ndng do va dien tich pic TT
1 2 3 4 5
Hoat chit Cephalexin Cephadroxil
Cefaclor Cefotaxim
Cefixim
Khoang tuyen tinh (jig/ml) 5 + 50
1 0 * 5 0 5 + 50 3 + 50 3 + 50
Phwang trinh hoi quy y = ax + b y = 1,588x+ 13,07 y = 0,756x+14,70 y = 1,089 x + 12,71 y = 2,699x+ 19,04 y = 2,334x+ 17,34
R' 0,9982 0,9991 0,9983 0,9997 0,9988 Khao sat do lap lai: T i i n hanh tren cac dung
djeh ddi chiiu ed ndng do mdi hoat c h i t 20
^ig/ml. Dugc do lap lai 5 lan khae nhau. K i t qua dugc trinh Bang 3: Khao sat do lap lai
a eae thdi diim bay d bang 3.
Lan khao sat
1 2 3 4 5 TB RSD%
Cephalexin tM
6,431 6,438 6,407 6,329 6,424 6,406 0,69
Spic 45,47 43,82 44,94 44,61 45,36 44,84 1,484
Cephadroxil tM
5,507 5,702 5,645 5,714 5,797 5,67 1,895
Spic 30,27 29,64 28,89 30,13 30,27 29,93 2,330
Cefaclor tM
6,826 6,915 6,955 7,001 6,986 6,937 1,007
Spic
35,76 33,93 35,07 34,73 35,58 34,81 1,725
Cefotaxim
tM 7,676 7,523 7,508 7,583 7,611 7,580 0,900
Spic
74,74 72,71 72,14 75,28 74,77 73,93 1,898
Cefixim
tM 17,534 17,587 17,503 17,372 17,416 17,428 0,500
Spic
66,16 64,38 64,82 65,58 65,32 65,25 1,052
Nhan xet: K i t qua dien di 5 l i n lap lai trong d i i u kien da chgn eho do lap lai dat yeu c i u vdi sai sd tuang ddi eiia thdi gian luu va sai sd tuang ddi eua dien tieh pic < 3%.
LPng dung trong phan tich dmh lu'O'ng khang sinh cefixim trong c h i phim
T i i n hanh djnh lugng hoat c h i t cefixim trong c h i p h i m vien nang FIXMA (Cefixim 100mg) bing chuang trinh dien di da chgn nhu tren.
Ddng thdi tiin hanh dinh lugng khang sinh tren bing phuang phap HPLC theo BP 2003 de so sanh k i t qua. Quy trinh djnh lugng eeffixim trong c h i p h i m dugc tdm t i t nhu sau:
Can xac dinh KLTB vien. Can lugng ehi pham tuang u'ng vdi 50 mg hoat c h i t Them 3 ml MeOH, them nude v.d 50 ml (Ddi). Hut ehinh xac 1,0 ml Dd 1, them dung djeh dem A4 v.d 50 ml, thu duge dung djeh thu'.
Can ehinh xac khoang 50 mg c h i t ddi chiiu.
Them 3 ml MeOH, them nude v.d 50 ml.(DdC).
Hut chinh xac 1,0 ml Dd C, thgm dung djc dem A4 v.d 50 ml, thu 6wgc dung djch ddi chiiu.
T i i n hanh triin khai djnh lugng eefixim bing chuang trinh dien di nhu da nghien eu'U.
Sau khi triin khai, phan tich phd dien di va tinh toan k i t qua.
46 TAP CHl DUOC HOC - 12/2010 (SO 416 NAM 50)
N g h i e n CLFU - K y t h u a t
Hinh 3: Dien di dd dung dich ddi chieu So sanh k i t qua djnh lugng dugc vdi phuang phap HPLC theo BP 2003, xii' ly thdng ke, sii' dung test t 6e so sanh 2 GTTB, test F de so sanh phuang sai cua 2 phuang phap, k i t qua duge trinh bay d bang 4:
Bang 4: Phan tich thdng ke kit qua dinh Iwgng cefixim trong chi phim
Vien nang FIXMA (Cefixim 100mg) Nai san xuit: Clesstra Healthcare
SKS: FM02 HD: 11/12/2009 Ket qua
HLTB (%) RSD (%) Co mau
S' TestF
Testt
HPLC 99,99 0,94
5 0,8771
F(0,05;5) =
" to(0,05;5) =
6,39 2,304
CZE 98,77 0,81
5 0,6343 F = 1,38 t =2,204 Nhan xet: Chuang trinh dien di mao quan trdn djnh lugng cefixim trong e h i p h i m hoan toan tuang thieh. K i t qua cho thiy do chinh xae ciia 2 phuang phap HPLC va CZE la nhu nhau.
Ham lugng eefixim trong e h i p h i m la 99,38%;
Phuang phap CZE ed ehi phi it han nhiiu so vdi phuang phap HPLC.
Ket luan
Chuang trinh dien di mao quan viing de phan tieh eae khang sinh nhdm cephalosporin tren da duge thim djnh theo cac chi tieu cua duge dien.
Nghien ciru nay da chirng minh duge ky thuat CZE phii hgp vdi viee phan tieh cac cephalosporin va cd mdt sd uu diim han so vdi HPLC nhu: viec ehuin bj nhanh, dung mdi re tiin, it doe hai. Viee phan tich hdn hgp 5 cephalosporin eho thiy kha nang tach phu thugc vao cac dung djch dem va pH eua dung djeh dem. -Cac dem A l , A2, A3 khdng tach rdi duge cae cephalosporin nhdm 2 trong cung mdt hdn hgp nhung tu'ng chit ed t h i tach duge. Ap dung eae k i t qua tren, viee phan tich eae cephalosporin khac (khdng 6wgc neu ra trong nghien ciru nay) cung cd the dugc thye hien tuang ty.
Hinh 4: Dien dl dd dung dich thw
Summary
An capillary zone electrophoresis method determines mixture of 5 cephalosporins:
The electrophoresis conditions are as follows:
+ Buffer solution: Na2HP04 WmM: MeOH (8:2) buffer
+ Agilent HPCE column: 50pm, I = 56cm.
+ The applied voltage was: 25 KV + Detector DAD: 270 nm.
+ Preconditioning: 1,00 minute with water;
2,00 minutes with 0,1 N sodium hydroxide solution; 2,00 minutes with water; 5,00 minutes with buffer solution.
+ Inject time: 5s.
In the present work, the fine (5) most frequently used cephalosporins were analysed by means of CZE. In the case of the separation conditions optimised for 5 cephalosporins, the precision was less than 3%, and the method was linear within the range of cephalosporins concentration of 3 ^ 50 pg/ml with R^>0,998 .
A mixture of cephalosporins Is generally not used In medicine. The reason to study a great number of cephalosporins was to prove the applicability of CE for the analysis of general cephalosporins.
Tai lieu tham khao
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Okamoto, J. C/iromatogr (1992), 590 p.326.
3. Y. Mrestani, R. Neubert, J. Schiew/e, A. HartI, J.
Chromatogr B 690, p.321.
4. H. Nishi, N. Tsumagari, T. Kalimoto, S. Terabe, J. Chromatogr (1989), 477, p 259.
5. Y-M. Li, Y. Zhu, D. Vanderghinste, A. Scepdael, E.
Roets, J. Hoogmartens,(1999) Electrophoresis 20, p.127.
6. Attila Caspar, Melinda Andrasi, Szilvia Kardos, J. Chromatogr (2002) B 775 p. 239 - 246
7. BP 2005, p 914.
8. Hows M.E.P., Perett D., Kay J., (1997) J.
Chromatogr. 590, p 364 - 368.
