4. K^t lu^n
Da xay dyng dugc cac quy trinh xac djnh boat tinh ciia Sh^todomase tixing vien chua hdn hgp Streptokinase va Stt^ptodomase cd tuih d^c hieu trdn co chit ADN vi cho do tuyen tinh, do lap lai tdt, do dung phii hgp vdi phep thii dinh lugng. Qay trinh phu hgp vdi didu kien hi?n cd ttong phong thi nghiem vdi cic ttang thidt bi, hda chit thdng dung, ky tiiuit khdng phiic tap cd the thuc thi dugc d cic co sd kiem nghiem cap tmh, tiiinh pho va cac doanh nghi^. Tuy nhidn dd quy trinh chiing minh dugc tinh dac hieu mdt cic tdt nhit can phai thir vdi chuan don thanh phin chiia Streptodomase nhung vdi dieu kien hien nay chung tdi chua cd chuan don thinh phan Streptodomase nen chua tidn hinh thuc hien dugc mgt each day dii vi can hoin thien trong thdi gian tdi.
Tii lieu tham Idiao 1. Tieu chuin co sd HCTONASE Korean Dmg Co.Ltd.
2. Tieu chuin co sd Hawon - Strepase, Hawon pharmaceutical corporation.
3. TiSu chuin co sd Strase, Green Cross Corporati.
4. Deoxyribonuclease I,website: http://en.wikipedia.org/wiki/Deoxyribonuclease_I.
5. Ian C. Locke; Brian G. Carpenter. Functional characteristics of the streptococcal deoxynbonuclease "Sueptodomase", a protein with Dnase activity present in the medicament Varidase. Enzyme ADN Microbial Technology 35 (2004) 67-73.
SUMMARY
A simple and rapid method for analyzing of Streptodomase in combined pharmaceutical products containing 2 compounds of Streptokinase and Streptodomase have been developed and validated. The method is based on the digestion ofDNA nature to the lower viscid solution. And this change is determined by Ostwald viscometer. The activity of sample IS calculated with Streptodomase reference standard. This method showed linearity within the range of Streptodomase concentration of 5.0-16.0 lU/mi The experimental results proved that it is precise, accurate and suitable for the quality control of enzyme products containing Streptodomase combined of Streptokinase and Streptodomase.
XAY Dl/NG PHl/dNG PHAP C H I ^ T V A PHANTI'CH ATROPIN, S C O P O L A M I N
TRONG 2 LOAI THU6c HdTRd CAI NGHIEN
BANG PHl/ONG PHAP SAC KY LONG HlfU N A N G CAO (HPLC)
Dd THI BiCH THU4N, NGUYEN TUAN ANH, PH^M THJ GIANG Vien Kiem nghiem thuoc Trung uong
1. Dat van de
Nghien hiit li mgt t$ nan giy suy giim siic khde vi nhu Cedemex, Balok. Ngoai viec kidm tra chit lugng dao diic ciia con ngudi va la mgt van de biic xiic cua theo tieu chuan co sd, chiing tdi cdn kiem tra phat hi$n toin xa hgi. Vi vay vi?c phdng chong te nan ma hiy cic chit khdng dugc phep trong cac thudc fren nhu da vi dang la nhiem vy chung cua tit ci cic qu6c gia rotundm, naltrexol, clorpromazin, levomepromazin va tren the gidi. Ben canh viec quin 1;^ buon bin, sii dyng mgt sd chat an than gayngii thudng dung (phenobarbital, chat ma hiy, giio dye cho cgng ddng vd tic hai ciia ma diazepam, bromazepam, Zolpidem),
hiy, mdt h-ong nhung bien phap lam giam ganh nang De tiip tyc dinh gii chit lugng cac loai tiiudc cho gia dinh va xa hgi la giiip ngudi nghien ma ttiy cai hd ti-g cai nghien, nam 2009, ngoai vi$c kidm tra cic nghien bing tiiuoc cai va hd tra cai nghien, Ud ve vdi chit tren, chung tdi da tien hinh nghien ciiu xay dyng cugc song bmh tiiudng phuong phip chidt va phan tich afropin, scopolamin vi Hi?n nay, da cd mgt sd lo^ thudc hd trg cai nghien cic alcaloid cua li ca dgc dugc trong cac thudc hd frg cd ngudn gdc tii dugc li?u da dugc Bg Y td cip sd dieu tii cai nghien Cedemex va Balok [2].
dang ky sin xuit luu hinh. Tii nam 2006 d ^ nay, Yietx Trong bai bao nay, chiing tdi xm gidi thieu ngi Kidm nghi?m thudc Trung uong hen hac liy miu kidm dung xay dyng phuong phap chiet xuat vi phan tich tra, danh gia chat lugng cac loai thuoc hd trg cai nghien atropin va scopolamin bing phuong phip sic ky long
T9P chi Kl^M NGHIEM THUOC - So 4.2011; T^p 9.(34) 15
hieu nang cao trong 2 loai thudc hd trg cai nghien da ndi tren.
2. Thuc nghiem
2.1. Thiit bi, dt/ng cut, hod chat, chdt chudn 2.1.1. Thietbi, dungcu
Da dugc hieu chuin theo ISO/IEC 17025 v i GLR - May sac ky long hieu nang cao Shimadzu UFLC vdi detector DAD.
- Miy ly tam va cac dung cu thuy tinh cin thidt.
2.1.2. Hod chdt, chdtchudn - Acetonitnl loai dimg cho sic ky Idng;
- Kali dihydrophosphat PA;
- Triethylamin PA, acid sulftuic PA;
- Chat chuan phdng thi nghiem Scopolamin HBr;
- Chuan Atropin sulfat ciiaAsean, sd Id M.I85015.
2.2. Doi tirang vd phuang phap nghien cuu 2.2. J. Doi tuong nghien cuu
2.2.1.1. Vien nang Cedemex: Hai Id thudc hd trg cai nghien ma tuy Cedemex do cdng ty TNHH Dugc pham Que Lam san xuat (Bang 1).
Bang 1. Cac mdu thuoc vien nang Cedemex lay de kiem tra chdt luong
Lo san xuat
01
02 Han dung
03/2010
12/2010 So ddng ky
QLDB-47-07
QLDB-47-07
N&i lay mdu
Tning tam chiia benh giao dye lao dong xa h5i
tinh Hoa Binh Vi?n nghien cmi dieu tn cac benh hiem ngheo
- km 10, ducmg 5, Khu CN Phil Thuy - Gia Lam,
Ha Noi 2.2.1.2. Hoan Balok: Hai Id thudc hd trg cai nghien ma tuy Balok do Benh vien da khoa Bao long - Cd Ddng - Son Tay - Ha ndi san xuat (Bang 2).
Bang 2. Cac mdu thuoc hoan Balok lay de kiem tra chdt luong
Lo sdn xuai
010708
021108 Han ddng
07/2010
11/2010 So ddng /^
Khong CO
Khdng CO
Noi lay mdu Benh vien da khoa Bao long - C6 Dong - Son Tay
- Ha npi.
Cong ty CP Bao Long- Trung tam k^ thua ung dyng YHCT - 54 Chiia Lang - Ba Binh - Ha NQI .
2.2.2. Phuong phdp nghien cuu 2.2.2.1. Didu kien sic ky
- May sac ky long hieu ndng cao. May Shimadzii
- UFLC; [ - Cot: Cgt phan tich pha dao C18 (250 x 4,6 mm, 5
Mm);
- Detector: UV 216 nm; : - Pha dong: La hdn hgp 2 dung moi A va B.
- Dung mdi A: Acetonitril
- Dung mdi B: Pha hdn hop ^jmg dich kali dihydrophospahat 0,02M - tiiethylamin (100:0,3), diSu chinh pH ddn 3 bing acid phosphoric dac. . . .
Chuong trinh gradient dung moi:
Thdigian (phut)
0-22 22-23 23-33 33-34 34-50
Dung moi A (%, tt/tt)
12 12-*50
50 5 0 ^ 1 2
12
Dung moi B (%, tt/tt)
8S 88-^50
50 50-.88
88
KiSurmiglSl
Dingdong Gradient >'^
Dang ding ; Gradient '•' Ding dong - Toe dQ ddng: 1,0 ml/phut.
- The tich tiem: 10 |il
2.2.2.2. Chuan hi cac dimg djch thu va dung dich chuan
Dung dich thu: Lay mdt lugng bgt thudc tuong duong 5 vien ddi vdi mau cedemex hoac 3 vien voi mlu balok cho vio binh ndn 250 ml, them chinh xac 50,0 ml dung dich acid sulfiiric 1 % (TT), lie deu hong 5 phut, cin binh. Dun tren each thiiy sdi trong 1 gid, de ngudi, cin lai binh va bd sung lugng dimg dich acid sulfiiric 1% hao hyt. Ly tam, lgc, bd 5 ml dich lgc dau.
Hiit chinh xac 25,0 ml dich lgc sau cho vao binh g ^ kiem hda dich lgc bang dung dich amoniac 6N den pH = 9-10, de yen khoang 30 phut, chiet 3 Ian, moi Ian 40 ml cloroform (TT). Gdp cac dich chiet clorofonn, cd tren each thiiy den gan can rdi liy ra de bay hoi hi nhien den c^n. Hoa cin trong 10,0 ml dung djch sulfuric 1%, lgc qua mang lgc 0,45 |im lam dung djch thir [1].
Dung dich chuan:
- Dung dich atropin sulfat trong acid suliuric 1% co ndng do khoing 150 |ig/ml.
- Dung dich scopolamin hydrobromid trong acid sulfuric 1% cd ndng do khoang 100 ^g/ml.
T?p chi KIEM NGHIEM THUOC - So 4.2011; Tap 9.(34)
2.2.2.3. Dinh gia kdt qui
- Dinh thih: So sadi thdi gian luu, phd UV-VIS cua cac pic tuong ftng trong sic 1^ dd ciia dung dich thii va vdi dung dich ^lulli atropin, scopolamin.
3. K^t qui \ii ban lu^n
3.1. Ket qua phan tich atropin vd scopolamin trong 1^ mau nghiin cuu
Tien hinh chidt cic dung djch phin tich theo quy trinh gbi d myc 2,2.2.2 va phan tich theo didu kidn sic ky da neu d phan 2.2.2.1 thu dugc cac ket qua sau:
* Mdu Cedemex: Sac ky dd cac dung dich thir d hai Id 01 v i 02 khdng cd pic cd cung thdi gian luu vdi pic cua atropin vi scopolamin trong sic ky dd cua dung dich chuan (Hmh 1).
* Mau Balok: Sic ky dd cac dung djch thir d hai Id 010708 v i 021108 khdng cd pic cd cimg thdi gian luu vdi pic cua atropin va scopolamin trong sic ky dd cua dung dich chuan (Hinh 1).
* Sac ky do ciia dung dich thii tren ca hai miu Cedemex v i Balok cd them chuan deu cd xuat hien pic cd ciing thdi gian luu vdi pic ciia atropin vi scopolamin trong sac ky dd ciia dung dich chuan.
:Jt - *'—»- " '"^ \
jQV
^-IJi—«AA-jv4ij\A_-r-« IL
Hinh I. Sdc Ic^ do HPLC phat hien Atropin va Scopolamin trong mdu Cedemex vdBaloli A, B: Mdu Cedemex 16 01, 02 QD: MduBalolilo 010708 vd 021108 E: Mdu Cedemex 16 02 them 2 chuan F: Mdu Baloli 16 010708 them 2 chudn G: Chudn Scopolamin (1,^:10,8phut) H: Chuan Atropin (1^^20,3 phiil)
Tfp c U K d M NGHIEM THu6c - So 4.2011; T$p 9.(34)
3.2. Khao sdt vd danh gid phuang phap 3.2.1. Khao sdt khoang tuyen tinh cuaphuangphdp
Pha hai day dung dich chuan atropin sulfat va scopolamin hydrobromid trong acid sulfuric 1% cd ndng do hi 75 |ig/ml ddn 750 |ig/ml ddi vdi atropin va 50 |ig/ml den 500 fig/ml vdi scopolamin, tidn hanh sac ky theo dieu kien da md ta d muc 2.2 2.1, ket qua dugc the hien d Bang 3,4 va Hinh 2, 3.
Bang 3. Ket qua khao sdt dp tuyen tinh cua atropin
Dung djch
Nong d9 (Mg/ml)
Dien tich pk 1
76,5
592385 2
153
1182119 3
191,25
1485565 4
382,5
2815979 5
765
5852610 PluroTig trinh hoi qui: y = 7605x + 424,43
He so tiroTig quan: r = 0,9994
? 4. Ket qua khao sdt khoang tuyen tinh ciia scopolamin
Dung dich
Nong do (fig/ml)
Dien tich pic
!
49,75
328750 2
99,5
651082 3
124,375
877575 4
248,75
1770159 5
497,5
3482373 Phuwng trinh hdi qui: y = 7065,5x - 19188
He so tiroTig quan: r = 0,9996
Oi^Cmg h
1
* : < , . . V * n . n .
= 7605x^424- R' = 0 9 9 9 4
5. / ^
° 200
] " ' ^
° N6ng d * { n eg/ml)
Du^ng h^i qui tuyen tinh cua Scopolamin
.g
«
c a
.
y = 7065 5x - 1918B J R^ = 09996 ^ ^
^y^
y^
^y^
y^
y^
N&ng d$ (mcg/ml)
%
f^
• 'Hinh 3. Ket qua khao sdt khoang tuyen tinh cua Scopolamin
Nhgn xet: Ket qua khao sat cho thay vdi khoang ndng do cua atropin tir 75 |ig/ml den 750 pg/ml va scopolamin tir 50 ng/ml den 500 |ig/ml cd su tuong quan tuyen tinh giua nong do va dien tich pic. Su phu thudc niy khi chat che the hien qua dudng thing hoi qui vi he sd tuong quan r « 1. Khoang ndng dp khao sat rgng, phii hgp vdi ndng do boat chat can djnh lugng trong mau dugc lieu thudng cd su bien ddi nhieu ve ham lugng boat chit, ndng do can dudi cua khoang tuyen tinh li kha thip cho phep ta dinh lugng dugc miu CO ham lugng hoat chit nho.
3.2.2. Khao sdt do lap lai vd do dung cuaphuangphdp Do diing ciia phuong phap dugc xac dinh bang phuong phap them. Them vao mau thii mgt lugng chat chuin atropin sulfat va scopolamin hydrobromid sao cho ndng do cua atropin va scopolamin nam trong khoang tuyen tinh da khao sit. Sau dd, tien hinh chiet nhu da md ta d muc 2.2.2.2, tidn hinh chay sac ky theo dieu kien da lua chgn, lap lai thuc nghiem 5 lin. Dira vao lugng cac chat chuin da them vao vi cin cii vao dien tich pic thu dugc tren sac ky dd tinh lugng cac chat thu hdi lai.
Do cac mau nghien ciiu Cedemex va Balok khong phat hien thay atropin v i scopolamin khi thir nghiem, do do do lap lai cua phuong phap dugc chung toi tinh toan dya tren ket qua cua do lap lai cua cac miu thii xac dinh do dimg.
Ket qua thu dugc ghi d Bang 5.
Hinh 2. Ket qua khao sdt khoang luyen tinh ciia Atropin
Tap chi KIEM NGHIEM T H U O C - So 4.2011; T | p 9.(34)
Bang 5. Ket qua khao sdt do Igp lai vd do HoiftchSt
Atropin STT
1 2 3 4 5 S6 li^u thong kS
1 2 3 4 5 So USu thing kg
Ltrtfng them vdo (fig) 3060 3060 3060 3060 3060
dung cita phuan MiSu Cedemex
Lugng tim thay (fg) 2699,7 2800,4 2729,8 2705,7 2606,7
gphdp tren mdu Cedemex vd Balok
%
dml^i 88,23 91,52 89,21 88,42 85,19 x = 88,51%; S =2,27;
RSD ^ 2,56%
1990 1990 1990 1990 1990
1,73019 1,72783 1,73982 1,72560 1,75646
86,94 86,83 87,43 86,71 88,26 x = 87,24%; S = 0,64;
RSD = 0,73%
Mdu Balok Lir^g them
vdo (pg) 2130 2130 2130 2130 2130
Lmmg lim th^
<f^g>
2140,5 2179,4 2180,0 2175,3 2147,4
%
tim l^i 100,49 102,32 102,35 102,13 100,82 K= 101,62%; S = 0,89;
RSD = 0,88%
1485 1485 1485 1485 1485
1300,5 1310,4 1304,2 1325,9 1311,5
87,58 88,24 87,82 89,29 88,32 lt = 88,25%; S = 0,66;
RSD = 0,74%
Nhgn xet: Cic ket qua thyc nghiem cho thay - Khi nang tim lai trung binh vdi atropin li 88,51% d mau Cedemex va 101,62% d mau Balok;
vdi scopolamin, cac ty 1^ nay lin lugt li 87,24% vi 88,25%. Ket qui nay cho thay phuong phap da xay dyng chip nhan dugc ddi vdi muc dich xic dinh cic thinh phan cd trong mlu che pham ddng dugc (loai miu cd nen miu phuc tap).
- D§ l§ch chuin tuong ddi tren ci hai ho^t chit d hai mlu la kha thip (Afropin: 2,56% vi 0,88%;
Scopolamin: 0,73% vi 0,74%). Nhu vay phuong phip cd dg Lap lai tdt.
Vdi ket qui nay cho phep ta ip dung quy trinh di xic dinh atropin va scopolamin trong cic chd phim Cedemex vi Balok.
3 2.3.. Xdc dinh giai hgn phdt hien vd giai hgn dinh lucmg
* Gidi h^n phit bi^n (LOD):
Pha loang din cac dung dich chuin Afropin, Scopolamin bing dung dich thu cho tdi khi thu dugc ndng dQ tdi thieu mi tai dd cd the dgc dugc 6ap lii^, so bg xic dinh gidi ban phit hien. Tien hanh phan tich 7 mlu da pha loang den ndng dO tdi thiiu nhu tren.
Cy the tien hanh ohu sau: Chuin bi 7 dung dich thir tren miu khdng phat hi?n thiy atropin, scopolamin;
pha loang dung dich chuin atropin (ndng dg 153 p.g/ml) va dung dich chuin scopolamin (ndng dO 99,5 |ag/ml) gip 20 lin tren ndn cac dung dich thii
T?p chi KlfeM NGHI$M THU6c - S6 4.2011; T§p 9.(34)
da chuan hi (vdi ndng do niy xic djnh dugc cic pic atropin va scopolamin). Xac dinh gii tri SD ciia dien tich pic atropm va scopolamin, tinh ndng dg gidi ban phat hien theo cdng thiic [3]:
t^ X SD X 20 LOD (jig/vien) =
M x n Trong dd :
t^ l i gia tri tra bing Student vdi do tin cay 98%, b^ctydo(n-l).
SD la dg lech chuan giua cac mau thir.
M la he sd gdc ciia phuong trinh hdi qui tuyen tinh.
20: he sd pha loang ciia dung dich thii.
n: sd vidn de chuan bi dung dich thii (n=5 vdi mau Cedemex, n=3 vdi mau Balok).
Ket qua ghi d Bang 6.
Bang 6. Kit qud khao sdt nong dg gioi hgn phdt hien ciia atropin vd scopolamin voi nen mdu vien Cedemex
vd Balok.
HocU chat
Atropin Scopolamin
Kh qud thong ki Mdu Cedemex
SD 21895,3
4931,9 LOD (Hg/vien)
36,2 8,8
Mau Balok
SD 6276,9 3079,7
LOD (ng/viSn)
17,3 9,1 Ghi chd: Gii tri t^ = 3,143 (vdi n = 7, P = 0,98)
* Gidi ban dinh lugng (LOQ):
Gidi han dinh lugng li lugng thap nhat cua chat can tim cd Uong mau thir cd the phit hien dugc ve mat dinh lugng ghi d Bang 7, vdi do diing va do chinh xac thich hgp. Thudng trong phin tich kiem ngbidm gidi ban dinh lugng dugc tinh bing 3,3 lin gioi han phat Men.
Bang 7. Ket qud gioi hgn dinh luong cua atropin vd scopolamin vdi nen mau vien Cedemex vd Balok.
Hoqt chat
Atropin Scopolamin
LOD (ftg/viin) M&u Cedemex
119,5 29
LOD (pg/vien) MSu Balok
57,1 30,1
4. K^t luan
- Da xay dyng phuong phip dinh tinh, dinh lugng scopolamin va atropin trong 2 loai thudc hd trg cai nghien cd nguon gdc tir dugc li§u la vien nang Cedemex va hoin cimg Balok. Phuong phip cd dd tuyen tinh, dd chinh xac v i lap lai tdt, cd gidi ban phit hien thip, phu hgp de phin tich phat hien atropin va scopolamin tren cac mau cd nen mlu phiic tap nhu Cedemex vi Balok.
- Da i p dung phuong phap xay dung de phin tich scopolamin v i atropin trong 04 Id thuoc thugc 2 loai chi pham trdn, kdt qui khdng phat hifn thay Scopolamin vi atropin trong c i 2 loai thudc da v i dang dugc sii dung tai cic CO sd cai nghien ma hiy.
T i i li|u tham khao
1. Bii giang Dugc lieu Tap 2- Bg mdn Dugc lieu-Trudng dai hgc Dugc H i Ngi, Nam 1998
2. Bio cao "Danh gia chat lugng cua mdt so thudc hd trg cai nghiem dang dugc sii dyng tai mgt sd co sd dieu tri " nam 2009. Khoa Ddng dugc Vien Kiem nghiem thudc TW.
3. Daniel C. Harris. Quantitative Chemical Analysis Sixth edition, W.H. Freeman and Company 2002.
SUMMARY
TheRP- HPLC method with DAD detector/or determination of Atropine and Scopolamine in the detoxication Drugs (Balok and Cedemex) is established.
Chromatographic condition consisted of - Column: Inersil ODS-3 (250x 4.6 mm. 5 \un):
- Injection volume: 10 ill, flow rate: 1 ml/min -Detector: UV216nm
- Mobile phase: Mixture of Acetonitrile and 0.02M Phosphate buffer pH 3 with Gradient mode.
Experimental results shown that the method was accurate, precise and the limit of detection was low (36.2 \xg/capsule and 17.3 \i.g/pill for atropine; 8.8 \ig/capsule and 9.1 ^xg /pillfor scopolamine in Cedemex Capsules and Balok Pills, respectively).
XAC DINH HAM Ll/ONG CHl TRONG M O T SO MAU MY PHAM BANG
PHl/ONG PHAP Q U A N G P H 6 HAP THU NGUYEN TONGUY£N VAN HA, Li THIHUONG HOA, TRINH VAN LAV Vi^n Kiem nghiem thudc Trung u&ng
I. Dat van Hk
Chi (Pb) li nguyen td thudc nhdm cic kim loai nang cd dgc tinh cao ddi vdi ngudi v i ddng v^t khi bi nhilm.
Qua. trinh nhiem dgc chi cd the do tiep xuc qua da, hit did ho^c qua dudng an, udng. Do do, chi cd ti-ong
danh sich cac chit bi cim sii dyng trong my pham cua ASEAN ciing nhu ciia chau Au [1]. Tuy nhien, viec xac dinh ham lugng chi trong my phim g|^ phii nhung khd khan do nen mlu phiic tap v i gidi ban clu hx)ng miu thip. Trong Thdng bio Kidm nghi?m sd 4
T?p chi H E M NGHI$M THUOC - So 4.2011; TSp 9.(34)
