• Nghien ciru - Ky thuat
4. Durgasrinivas D., Venkateswararao P., Ramanjeyulu KV., Vinay PoUian, Ravali K.. Suresh B.
(2012), "Method development and validation for Vne assay of zoledronic add in phannaceutical dosage form using high performance liquid chromatography technique", Intemational Joumal of Biological &
Pharmaceutical Research, Vol.3(7), pp. 911-917.
5. Mastanamma, G. Suresh, D. Sindu Priya and J. V. L. N. Seshagiri Rao (2012), "A validated RP- HPLC method for the estimation of zoledmnic acid", Intemational Joumal of Pharmaceutical Sciences and Research. Vol. 3(3). pp. 826-829.
Ngliien cmi thiet lap cliat doi ctiieu hispanon va leoheteronin B tir fch mlu
{Leonurus heterophyllus)
Ha Dieu Ly', Huynh Thi Mai TVan^
Khoa Thiet lap chdt chudn-Chdt dot chieu Vien Kiem nghiem Thuoc Thdnh pho Ho Chi Minh ' E-mail: [email protected]
S u m m a r y
Hispanon and leoheteronin B were isolated from Leonurus heterophyllus and further established as reference substances under the guide of Asean-USP and ISO 34. By the developed method, which was validated to show specific, accurate and precise, the contents of hispanon and leoheteronin B were 96.68 % and 100.00 %, respectively. That meant they promised to serve as references in quality control of medicinal plant materials and herbal products.
Keywords: Hispanon, leoheteronin B, Leonurus heterophyllus.
D a t v i n d e
Trong vdi thdp ky gan ddy. cdc nude trdn the gidi dang d l y mgnh vigc nghidn edu, bdo ehd va sdn xudt cdc chd p h i m cd ngudn gde t y nhidn de hd trg, phdng ngda vd dieu trj bgnh. Hign nay cd hdng nghin logi chd phdm t u duge ligu d u g c sdn x u l t vd luu hdnh '^'.
Ddng hdnh vdi s y phat tridn do. thyc trgng e h l t lugng d u g c Hdu va thudc ddng y, thudc t u duge lidu trdn thj trudng nude ta hidn nay cdn mdt sd bdt cdp: d u g e lidu nhap khdng rd ngudn gde, khdng rd tidu e h u l n , khdng cd phidu kidm nghidm chdt lugng...Dd kidm tra d u g c lidu cdc CO s d sdn x u l t vd co quan kiem nghigm phdi mua e h l t e h u l n ddnh d i u t u nude ngodi vdi gid r l t d i t vd nhidu khi khdng cd san trdn thj topdng. Do dd, vide thidt ldp chat chudn ddnh d i u t d d u g c lidu ngdy cdng trd nen can thidt ddi vdi cdng tdc bdo ddm e h l t lugng thudc t u giai dogn lya chgn nguydn lidu ddu vdo, ddn vide xdy
dyng tidu ehuln eo s d cung nhu cdng tdc gidm sdt chat lugng thudc d u g c ligu luu hdnh cua c o quan qudn ly, ddng thdi tidt kigm ngogi tg nhgp ehuln t u nude ngodi.
Trong bai bdo trude'^', chung tdi da chiet xudt phdn Igp, xac djnh c l u true vd xdy dyng bg d u lieu chuan cCia c h i t phdn lap hispanon vd leoheteronin B t u ich m l u . Trong bdi bdo nay chung tdi tiep tye t h i m djnh quy trinh danh gid vd xae djnh do tinh khidt cua hispanon va leoheteronin B dd lam e h l t ddi chidu, phgc vu eho cdng tde kidm tra e h l t lugng ich mau cac san p h i m ed ehua ich mau luu hdnh trdn thj trudng.
D o i tu'p'ng v a p h u ' o n g p h d p n g h i e n CU'U
Mau nghien c u u
Hispanon vd ieohereteronin B ehidt xult tu ich m l u eua Khoa Thidt ldp Chit chuln-ChIt ddi chidu, Vidn Kidm nghi§m thudc TR Hd Chi Minh, ndm 2012.
Ti^P C H i D i r p C H p c . 1/2014 (S6 453 N A M 54)
• Nghien CIFU - Ky thuat
T h i e t b j , d u n g c u , hda c h a t
May s i c ky long cao dp Shimadzu LC-20A vdi detector PDA, methanol (HPLC), acid phosphoric (PA). Thiet bj va dung cu da d u g e hieu chuan theo ISO/IEC 17025 vd GLP.
Phirong phap n g h i d n c u u
- Dira vio bg du- lieu chuan da xay dung ^', tidn hdnh t h i m dinh quy trinh phan ti'ch theo eae chi tidu: tinh dae higu, tinh tuydn tinh, do chinh xae, do dung i^^
• Dieu kidn s i c ky n h u sau: Cdt Phenomenex Luna C-8 (150 x 4,6 mm, 5[im), detector PDA b u d e sdng 254 nm, tde do ddng 1,0 ml / phut, thd tieh tiem 20 ^1.
• Pha ddng: MeOH - H3PO,0.1 % (pH 3,0) theo c h u o n g trinh dung mdi.
Thai gian (phOt) 0-20 20-35 35-50 50-60
MeOH 6 3 ^ 7 0 70 ^ 100 100 -> 63
63 HfO, 3 7 - ^ 3 0
3 0 - > 0 0 ^ 3 7 37 Dung dich 1: chuan bj mau hispanon cd nong do khoang 0,025 mg/ml methanol.
Dung dich 2: chuan bj mdu leoheteronin B ed ndng do khoang 0,025 mg/ml methanol.
Bdng 1: Kit qua tinh tuung thich he thing sic ky
- Thiit lap chat doi chieif*^
Banh gia do ddng nhit cua qua trinh ddng lg '^'^
L l y m l u ngdu nhidn theo v N +1 tnang dd N la tdng sd Ig. Tidn hanh xae djnh chi tieu tinh khidt sac ky t d n g lo theo p h u o n g phdp da xdy d y n g . Ddnh gia theo ky thudt thdng ke phan tich ANOVA-one way so sanh 2 gia trj F^, va F,^
Banh gia chit chuin thong qua lien phong thi nghidm i^'^'
Hai phdng thi nghidm d u g c lya chgn de gir\
m l u phai dat tidu ehuan GLP hoae ISO 17025.
Moi phdng thi nghigm se nhan dug'c sdu lg m l u ngdu nhien kdm theo quy trinh phdn tieh vd cac tai lidu lidn quan.
Phuang phap xu ly s6 lieu - Xac dmh gii tri in dinh '^'
T u kdt qua cCia 2 PTN, xac djnh gia trj an djnh theo I S 0 1 5 3 2 8 v a 17043.
K e t q u a v a b a n l u a n T h a m d i n h q u y t r i n h p h a n t i c h Tfnh tuvng thich he thong
D g lech chuan (RSD) cua dien tich pic £ 2,0
%; dd Idch ehuan (RSD) eua thdi gian luu £ 1,0
%; hg sd doi x u n g T= 1,097 (0,8 £ T < 1,2): cac thdng sd khao sat dat yeu c l u tinh tuong thich he thdng.
STT TB (n=6)
RSD(%)
Leoheteronin B yphut) Dien tich pic
11,736 1282617 0,16 0,02
He so d6i XLpng 1,085
y p h u t ) 31,130 0,01
Hispanon
Dien tich pic H# so doi xCpng 1284413 1,097
0,21 Tinh dac hieu
Lan lugt tidm m l u t r l n g la pha ddng, dung djch 1, dung djch 2 vao hg thdng sac ky
Hinh 1 : Sdc ky do miu trdng Hinh 2: Sdc k'jf do vd d0 tinh khiit ciia leoheteronin B vd hispanon
TAP CHi DirOC HOC - 1/2014 (S6 453 N A M S ^ S .
• Nghien CIPU - Ky thuat
Nhgn xdt: Sac ky dd m l u t r l n g Widng xuat hidn pic d n khdo sdt; Pie chinh khdng x u l t hign t^p khi s u dung chuc nang kiem tra dd tinh khiet pie. Phuong phdp phan tich cd tinh dac hidu vdi leoheteronin B vd hispanon.
Dg lap lai cua phuvng phap
C h u i n bi dung djch hispanon va leoheteronin B ed cac ndng do tuong ung 80 %, 100%, 120%
ndng do do. Moi ndng d^ thye hidn 3 m l u .
Bdng 2: Kit qua khao sdt do lap lai cua phuang phap
Hispanon Leoheteronin B
Mire nong dg
chuin do Nong dp (pg/ml) Dien tich pic RSD Ndng dp (pg/ml) Didn tich pic RSD 20,55
20,55 20,55 25,69 25,69 25,69 30,83 30,83
1026595 1026919 1027232 1284592 1283632 1284591 1556707 1556127
TB (%)
Nhdn xdt: RSD £ 2,0 % dat ydu cau dd ehinh xdc.
Khoing tuyen tinh
C h u i n bj dung djeh hispanon va leoheteronin B ed cdc ndng dg tuong ung 25 %, 80 %, 80%, 100%, 130% ndng dg do. Tien hdnh s l e ky vdi dieu kign da chgn.
Bdng 3: Kit qua khio sdt khoing tuyin tinh
STT
1 2 3 4 5
Hispanon Ndng d^ (pg/ml)
6,42 16,06 20,55 25.69 32,12
Di^n tich 315912 801081 1026595 1297256 1624655
Leoheteronin B Nong dd (pg/ml)
6,33 15,81 20,24 25,30 31,63
Dien tich 312302 791576 1021522 1284541 1600159
S t r 6 n a t u y t n t i n h cOa hispanon
IBOOOOQ .S09TB.-1S017
14O0OO0 ' " • y^
^ eooooo j^
zooooo 0 •
D 10 20 « N&ngdf (to
E)(M>ng tuyjn Unh c d i l t a h « t * r o n l n B
: tgoon
y = 5103ex-11707 j)^
R' = 1 y ^
.:.. " "
Hinh 3: BuCmg biiu diin mii tuang quan di$n tich dinh theo ning di
T A P C H i D U p c HQC - 1/2014 (S6 453 N A M 54)
• Nghien ctpu - Ky thuat
Khao sdt s y t u o n g quan giua y (dien tich dinh) va X (ndng dd), bdng p h u o n g phdp tdng binh p h u o n g c y c tidu, kdt qua cho t h i y he sd t u o n g quan r > 0,999, suy ra cd s y t u o n g quan tuydn tfnh chgt chd giua ndng do vd didn tich dinh.
Bg dungphuxmg phip
T u kdt qua cOa do ehinh xde d 3 m u c ndng do 80, 1 0 0 , 1 2 0 % so vdi ndng do do, tinh ndng do tim lai d y a t d d u d n g hdi quy, suy ra ti Id hdi phue.
Bang 4: Kit qua khao sat do dung cua phuang phap
Hispanon Leoheteronin B
% do chinh xac trinh hdi quy phuc % pic (pg/ml) (pg/ml)
100%
120%
1026595 1026919 1027232 1284592 1283632 1284591 1556707 1556127
20,55 20,55 20,55 25,69 25,69 25,69 30,83 30,83
20,13 20,14 20,15 25,19 25,18 25,19 30,53 30,52
(pg/ml)
1021893 1019616 1023058 1282317 1281613 1283699 1540122 1541679
20,24 20,24 20,24 25,33 25,33 25,33 30,36 30,36
20,02 19,98 20,05 25,12 25,11 25,15 30,18 30,21
7y/e Mire Ning do Ning do tinh Nina dido " " " a ' ' ' " " ' '
nong dp Dien tich do do tif do tirphmyng Tfldhil Dien tich ,,•, M ^i,,.), tirphuvng ,,.
" ' . . . . ' " ^ " ^ ^ trinhhoiquy . . .
xac (pg/ml) ,„„,J,' phuc%
TB (%) 98,40
RSD= 0,553
99,23 RSD= 0,234 Yeu cau: 98,0 -102,0 %. Vay do Sung cua phu'ong phap 98,03 % - 99,48 % la ctiap nh?n duac.
Thiet lap chat do! chieu
Danh gia do dong nhat trong qua trinh dong Ig Bang 5: K4t qua xac dinh do ddng nhM trong qua trinh dong lo
STT 1 2 3 4 5
Dd tinh khiSt leoheteronin B Loso
1 2 7 8 9
Lin 1 (%) 100,00 100,00 100,00 100,00 100,00
Lin 2 (%)
100,00 100,00 100,00 100,00 100,00STT 1 2 3 4 5 6
Dp tinh khlet hispanon Lg so
2 3 5 9 10 13
Lan 1 (%) 96,71 96,63 96,58 96,6 96,23 96,31
Lin 2 (%) 96,25 96,31 96,78 96,43 96,27 96,54 'Xac djnh dd tinh khiit dua vao % didn tich dinh trong sic k^ do theo dieu kidn da thim djnh
Vgy cac lg leoheteronin B trong qua trinh ddng Id ddng nhdt.
Phan tich p h u o n g sai mdt yeu td theo Anova cua hispanon eho ket qua
TAP CHi DirpC HQC - 1/2014 (S6 453 NAMSfly
• Nghien ciru - Ky thuat
F^ = 0,154 < F^=4,6. Vdy cac Ig trong qud trinh ddng eung ddng nhat Xac djnh gii trf in djnh- Banh gia chit chuan thdng qua lien phong thi nghigm
Bdng 6: Kit qui xdc djnh dd tinh khiit lien phdng thi nghidm
STT 1 2 3 4 5 6 TB
Leoheteronin B PTN1
100,00 100,00 100,00 100,00 100,00 100,00 100,00
PTN2 100,00 100,00 100,00 100,00 100,00 100,00 100,00
Hispanon PTN1
96.75 96,60 96,75 96,31 96,58 96,78 96,63
PTN2 96.63 96,99 96,59 96.53 96,23 96,28 96,54 PTN1: Khoa V$t /j? do ludng, Vidn Kiim nghi$m thuic TR Ho Chi Minh
PTN2: Khoa Kiim nghidm My pham, Vi$n Kiim nghi$m thuic TP. Ho ChiMinh
Nhdn xdt: Gid trj In djnh eua leoheteronin B cCia 2 phdng thi nghidm Id 100.00 % Bdng 7: Kit qui xdc djnh gid tq in dinh ciia hispanon
S6 tin Ifp
x,-s x,*s
TB Bt lich s
X-m6il S-m6il
96,64 0,012 96,68 0,125
96,54 96,90 96,68 0,011 36,68 0,120
»%
96,50 96,85 96,68 0,011 96,66 0,120
''„
96,50 96,85 96,68 0,011 96,68 0,120
Nhin xdt sau ba lln thay ddi. S' = 0.120 khdng thay ddi, khi dd gid trj X* duge chgn Id 96,68 %. Td kdt qud trdn tinh z-seore eua 12 gid tri ddu ed |z| < 2,0.
Vdy gid trj I n djnh cda hispanon: 96,68 % Dd khdng ddm bdo do
1.25 1,25
(J= xS- xO.OII =0,004 < 0 , 3 x S = 0,3xO,120 = 0,036 Vp yll2
DO khdng ddm bdo do md rdng U^pxk = 0.036X 2 = 0,072 ( hd sd phd k = 2 d o.^^)
Kdt Iu9n
Vdi chit ehidt cd l$p dKsgc td (eh mlu. ehdng tdi dd xdy dyng quy trinh ddnh gid vd thim (^nh quy trinh theo hudng din cua ICH vd thidt ldp dugc 2 ehlt ddi chidu Id hispanon vd leoheteronin B theo hudng din cCia Asian-USP vd ISO guide 34. Chit ddi chidu thidt ldp duge cd dd tinh khidt
2 95 %, ddp ung mge dich su dyng cho chat ddi chidu ehidt tu duvc lidu. phyc vy eho edng tac kidm tra chit lugng dug^e lidu va thudc tu dugc tidu fch mlu.
Tai li$u t h a m khao
1.BdY ti, Di dn phdt triin vd quy ho^ch dugc li$u (2011).2. Huynh Thj Mai Trang, V5 Thj Kim Huang, Ha Di$u Ly, (2012), Chidt xudt tinh chd Hispanon va Leoheteroin B td ich mlu {Leonums hetemphytlus) dd Idm chdt ddi chidu, T^p chi Dugc hqc. So 440. trang 20-24.
3. ICH guideline Q2A.
4. Asean document guidelines (2007). Guideline for the establishment, handling, storage and use of Asean reference sustances.
5. ISO guide 34:2009, General requirements for the competence of reference material producers.
6. ISO 13528 vd ISO 17043.
