j j g h i e n CLFU - Ky thuat
^hien ciru dinh lucrng cac hoat chat
r
trong sinh khoi thong do
> r
bang sac ky long hieu nang cao
Vu Binh Dirong^, Nguyen Van Long^, Pham Thi Thanh Ha', Le Thi Thien*, Dao Van Don^
Trudng dgi hoc Dugc Hd Ngi; ^ Hge Vien Qudn Y
D a t v a n d e
Thong do Viet Nam {Taxus wallichiana) Id dugc lieu quy phan b i chii y i u tai eae huyen Dire Trgng, Dan Duang, Lae Duang vd thdnh p h i Od Lat, tinh Ldm D i n g . Theo ede tdi lieu nghien ciru cua cac nhd khoa hge, trong thong do Viet Nam co chua cac boat chat co tae dung ipc c h i t i bdo ung thu nhu: paelitaxel (taxol), cephalomannin hoac cae ehit cd the ban t i n g hgp ra cae thuic d i i u tri ung thu nhu baeeatin III, 10 deacetyl baeeatin III, brevifoliol...
Hien nay, tai Trung tam Nghien euu ung dung Sinh - Y- Duge (Hpc vien Quan Y) da budc d i u thdnh cdng trong viec nu6i c i y tac sinh khii t i bdp thong dd trong phong thf nghiem, dang tang d i n qui mo san xuat cdng nghiep .
De gop phin ddnh gid c h i t lugng eua sinh khii th6ng do san x u i t tai Hge vien Quan y, chiing tdi tiin hdnh d i tai ndy vdi mue tieu xay dung qui trinh djnh lugng eae boat c h i t chinh (paelitaxel vd baeeatin III) trong sinh k h i i thong do va ung dung qui trinh da xdy dung trong danh gia ehit lugng sinh k h i i thdng dd.
N g u y e n lieu v a t h i e t bj
Sinh khii th6ng do (Hge vien Qudn y eung cip, L6 SX 050909; 060909; 070909).
He thing HPLC Alliance Waters 2695D, autosampler, buing gia nhiet cot. Detector PDA, Phin m i m xu' ly s i lieu Empovi/er® 2, cpt Fortis Phenyl (250 x 4,6 mm, 5 pm) va Cpt Luna L43 (250 x 4,6 mm; 5 pm); Can phan tieh Sartorius (dp chfnh xdc 0,1 mg), mdy ly tam lanh UNIVERSAL 320 (Hettich - Due), may khuiy tu gia nhiet RCT Basic, IKA® USA, mdy l i c MSI Minishaker, IKA® USA, tu lanh am sau DF8514 (llshinlab - Han Q u i c j , nhiet dp am t i i da - 80°C, micropipet_1-10iil, lO-IOOiil, lOO-IOOOiil, bp phan lpc: phiu Ipc Buchner, bp phan Ipc m i u , mang Ipe 0,2Mm; 0,45Mm.
TAP CHI DUQC HOC - 9/2010 (SO 413 NAM 50)
Cdc ehit ehuin paelitaxel va baeeatin III (Sigma - My).
ThLPC n g h i e m v a k § t q u a Phipcng phap xd" ly m l u
Sau khi tham khao m6t s i tai lieu ' va lua chpn cae d i i u kien hien eo, chung toi t i i n hdnh khao sat phuang phdp xu' ly m i u vdi cdc d i i u kien sau:
- Chiit long - Idng vdi h i n hgp dung mdi methanol - dieloromethan (3:1, tt/tt), ehiit nhiiu l i n , r i i phan tich ngay b i n g HPLC.
- Chiit long - r i n vdi dung m6i ehiit methanol, chiit nhiiu l i n , sau do tinh c h i b i n g qua cot chiit pha r i n trudc khi chay HPLC
K i t qua s i c ky d i cua 2 phuang phdp XLP ly m i u t h i hien d hinh l a va l b eho thiy: phuang phap ehiit long - r i n eo qua ept ehiit pha r i n thi pie boat c h i t paelitaxel va baeeatin III tdeh ro rang ra khoi hoat chit, d i n g thdi kha nang chiit boat c h i t t i t han phuang phap ehiit long - long.
Vi vay, ehung toi chpn phuang phap xu- ly mdu nay eho nghien euu t i i p theo.
Liy 200 mg b6t mjn sinh khii th6ng dd chiit sieu am bing dung mdi methanol (12 ml x 3 lin), ly tam 3000 vong/phiit l i y phin djch trong phfa tren, b i c hai methanol tdi khi con 10 ml, bo sung them 10 ml natri clorid 5% vd chiit bing dung mdi dieloromethan (10ml x 4 lin). Djch ehiit dieloromethan dugc Ipc qua 3g natri sulfat khan, djch Igc dugc b i c hai tdi cdn. Hoa tan e i n vdi 2 ml dielcromethan roi chuyen vao cpt silica gel Ig.
Rira cpt vdi dieloromethan (2x2ml) sau d6 vdi aceton - dieloromethan (4:96, 2x5ml), bo djch ru-a.
Rua giai vdi aceton - dieloromethan (50:50, 2x5ml) gop nude rua, b i c hai tdi e i n (N2, 35°C), boa tan c i n trong 1ml methanol trudc khi dua vao phan tich HPLC.
31
N g h i e n CLFU - Ky t h u a t
in 111 • REcftad
QOB I {, I
aji|.i^^/ii^U..4^_^jJ!j
1 Qt25i
i
ooisl
QOIOI I QOSJ 1
F^itad
ll|j^-J---.U-- ^j^h-
5 0 3 WOD 15ID 2DCD S C O 2DCD 35C0 « C D 4 5 0 3 SQCO S C D eQCC 2100 aacD
- A - ^ J , .
Hinh 1: Sic ky dd miu sinh khdi thdng do xir ly theo phwang phip chiit long-long (a) vi long - ran (b) Khao sat dieu kien sac ky
Chung tdi da tiin hdnh khao sat diiu kien s i c ky: Cpt C18, C6t Fortis phenyl (Phenyl)'^' vd Luna
\2 51
L43 (Pentafluorophenyl) ' , pha dpng Id acetonitril - nude - methanol vdi nhiiu chuang trinh gradient
Bang 1: Chwang trinh pha ddng
khac nhau. K i t qua chiing t6i tim duge diiu kien sic ky nhu sau: Cot Luna L43 (250x4,6mm; 5pm), nhiet do cot 25°C; detector PDA 228 nm; t h i tich m i u bam 10 pi, nhiet d6 autosampler 4°C; chuang trinh pha dgng duge trinh bay d bang 1.
Thai gian 0 40 50 60
(phut) Toe do (ml/phut) 1
1 1 1
Acetonitril (%) 25 65 25 25
Nwac (%) 75 35 75 75 Tinh dac hieu va tuxyng thich cua phuong phap
T i i n hdnh phan tich m i u tring, m i u h i n hap 2 chuin paelitaxel va baeeatin III eo ning d6 20 pg/ml va m i u thir sinh khii th6ng do. K i t qua d hinh 2 vd bang 2 cho thiy trong m i u tring
khong eo pic tai eae thdi diem x u i t hien ede pic cua paelitaxel vd baeeatin III; cdc pic can d i i , tach nhau ro rdng; thdi gian luu vd dien tieh pie cua paelitaxel vd baeeatin III ehuin co d6 lap lai cao(n = 5, RSD < 1%).
A J ,
Qcm'
500 o n 1503 2300 2500 3303 3E0D -OCD « 0 D 3303 S O ] 6303
Miles
K--it-
• E a ) « D z m 2 0 O 2 i a ) H i i a D 3 i ! ) 3 e a ) 3 i ( i i Mite
Hinh 2: Sac ky dd hdn hgp chuin (a) vi mau thw (b) Bang 2: Do lap lai cua he thing
n = 5
Jf ±SD RSD (%)
Paelitaxel TR (phut)
31,11 ±0,08 0,25
S (pV*giay) 202941 ± 753,32
0,37
Baeeatin III TR (phut)
18,63 + 0,06 0,31
S (pVgiay) 128015 ±1182,64
0,92
32 TAP CHI DUOC HOC - 9/2010 (SO 413 NAM 50)
• N g h i e n
CLFU- K y t h u a t
Khoang tuyin tinh '>
Pha cac hin hap ehuan co ning dp 5; 10; 20;
50; 80; lOOpg/ml trong methanol. Kit qua d bang 3 vd hinh 3 cho thiy trong khoang ning dp khao sat, dien tich peak va ning d6 paelitaxel vd baeeatin III CO su tuang quan tuyin tinh vdi R^ ~ 1.
Bang 3: Sw phu cua dien tich pic vao ndng dd cac chuin
~^~^~-Diiiiidch pic (pV*
Nong do (pg/rfil)~-—^__^
5 10 20 50 80 100 Y = aX + b
s) Paelitaxel
93395 203336 403131 1020613 1618654 2068974 Y=20610X-
8919,2
Baeeatin III
59921 128307 264345 645826 1027586 1337309 Y=13229X-
7051,4 0,9998 0,9991
I
•S • Paelitaxel
I » Baeeatin III:
50 100 N o n g d o ((Ag/iTil)
Hinh 3: Dd thi tuyen tinh cua paelitaxel vi baeeatin III
Do dung
T i i n hdnh theo phuang phdp them ehuin.
Mdu thLP sinh khii th6ng do duge them ehinh xdc mpt lugng ehuin paelitaxel (lOOpg) vd Baeeatin III (80pg) r i i XLP ly mau theo quy trinh da xae djnh, m i u d i i chipng duge them methanol, t i i n hanh djnh lugng xdc djnh ty le tim thiy paelitaxel vd baeeatin III. K i t qua thu duge d bang 4 eho thiy phuang phdp eo dp diing cao vdi ty le tim thiy g i n 100%.
Bang 4: Ty le (%,) tim thiy paelitaxel va baeeatin III
Mau
% tim thay Paelitaxel Baeeatin III
X ± SD (n = 5) 94,55 11,78 93,37 ±1,67 Do dn djnh
Tiin hdnh chiit mpt m i u sinh khii th6ng do theo quy trinh XLP ly miu da xdc djnh. Song song TAP CHi DUOC HOC - 9/2010 (SO 413 NAIM 50)
chuin bj mpt m i u ehuin h i n hgp ciia paelitaxel va baeeatin III, tiin hdnh s i c ky ngay va tiip tue eu 2 gid mpt lin trong vong 6 gid. K i t qua thue nghiem eho thiy trong 6 gid phan tieh lugng paelitaxel va baeeatin III giam nhieu nhit eon lin lugt 99,14% va 99,35% trong chuan, 99,02% va 98,94% trong m i u thii' so vdi lugng ban d i u . Vay m i u ehuan va thu d i u on djnh trong thdi gian phan tieh.
Djnh lu'gng paelitaxel va baeeatin III trong sinh khdi thdng dd
Djnh luang 3 m i u sinh khii thdng dd lay tu mdi trudng B5 + NAA d 3 16 khde nhau: 050909;
060909; 070909).. Tiin hanh xii' ly m i u theo quy trinh da xae djnh vd s i c ky theo cdc thdng s i da khao sdt dugc. Kit qua hdm lugng paelitaxel va baeeatin III trong sinh khii thdng dd Viet Nam la:
0,0137 ± 0,00030% va 0, 0081 ± 0,00015%.
K§t luan
Da xay dung phuang phap djnh lugng ding thdi Paellitaxel vd Baeeatin III trong sinh khii thong do Viet Nam bing HPLC vdi diiu kien s i c ky la ept Luna L43 (Pentafluoro phenyl); pha dpng acetonitril va nude ehay theo chuang trinh gradient; detector PDA 228nm. Phuang phap ndy co dp dac hieu, dp tuyin tinh, dp diing cao, co the djnh lugng paelitaxel vd Baeeatin III d ning d6 5 pg/ml. Kit qua djnh luang paelitaxel va baeeatin III trong m i u sinh khii thong do theo phuang phdp da xay dung tuang irng Id: 0,0137% va 0,0081%.
Summary
An HPLC method for simultaneous determination of Paelitaxel and Baeeatin III in Taxus wallichiana cellmass was developed with a mobile gradient phase of acetonitrile and water and PDA detection at 228nm. The method was validated. The calibration curves of both Paelitaxel and Baeeatin III had a satisfactory correlation coefficient (close to 1.000). The recovery rates of extraction were about lOOVo for both Paelitaxel and Baeeatin III. Standard and test samples were stable during the analysis. Quantitative analysis of them in Taxus wallichiana cellmass gave:
Paelitaxel - 0.0137%o and Baeeatin III - 0.0081 %.
Tai lieu tham khao
1. Vu Binh Duang, Nguy§n Van Long, Hoang Van Luong, Le Bach Quang (2008), Sinh khii t§ bao thuc vat, hudng mdi trong san xuit nguyen lieu lam thu6c, Tap chi Thong tin YDwge, (12), tr. 6-9.
2. Dwge dien My 24, 29 (USP 24, 28), CDrom 33
Nghien CLFU - Ky thuat
3. S. Bala, G.C. Uniyal, S.K. Chattopadhyay, V.
Tripathi, K.V. Sashidhara, M. Kulshrestha, R.P.
Sharma, S.F. Jain, A.K. Kukreja, S. Kumar (1999), Analysis of taxol and major taxoids in Himalayan yew, Taxus wallichiana, Joumal of Chromatography A (858), pp. 239-244.
4. S. Li, Y. Fu, Y. Zu, R.Sun, Y.Wang, L. Zang, H.
Luo, C. Gu, T.Efferth (2009), Determination of paelitaxel and other six taxoids in Taxus species by high-performance liquid chromatography- tandem mass spectrometry, Joumal of Pharmaceutical and Biomedical Analysis (49), pp. 81-89.
5. Phenomenex, Chromatography Product Guide 09/10, pp. 152-153.
Ket qua hmc dau ve thay doi kha nang thye hanh tir mot mo hinh can thiep htfOTig toi muc tieu su* dung khang sinh
r r
hop ly cho can bo y te tuyen xa tai huyen My Loc, tinh Nam Dinh
Ngo Huy Hoang\ Hoang Thi Kim Huyen^, Joy Notter^
'Trudng dgi hoc Dieu dudng Nam Dinh
^Trudng dgi hge Dugc Hd Ndi
Dat van de
Trong he thing y t i nude ta, tram y t i la nai d i u tien thue hien ede djch vu eham soc sue khoe eho nhan dan. Vdi chfnh saeh bao hiem y t i hien nay va mue tieu t i i n tdi bao hiem y t i toan dan, cong tdc khdm chira benh Id boat dpng ehii y i u hang ngay eiia can bp y t i (CBYT) xa. Trinh dp khdm benh vd ke dan eua CBYT d nhiiu nai con ban e h i , gay anh hudng khdng nhd kh6ng chi d i n viee SLP dung khdng sinh khdng dung ma eon la mpt treng nhu'ng nguyen nhan chu y i u gdy nen hien tugng vi khuin khdng thuic vd nhirng he luy anh hudng d i n sire khoe cpng d i n g cijng nhu Idm tang chi phi eham soc y te '''I
Muc tieu eua nghien euu nay nhim dinh gii hieu qua cua viec ip dung mdt md hinh tap huin nhim nang cao kha nang thwe hinh cho can bo y ti tuyin xa di han chi viec sir dung khang sinh ehwa hgp ly, gop p h i n nang cao chit lugng kham chCra benh d tuyin xa, tu do CO duge nhijng d i x u i t eho viee xdy dung vd 34
Birmingham City University hoan thien mpt chuang trinh tap h u i n co the dp dung chung eho y t i tuyin xa.
D o i tu'O'ng v a Phu'O'ng p h a p n g h i e n CLPU
Ddi tu'gng nghien cu'u
Bai viit ndy Id bude t i i p theo ciia ciing met nghien ciPU can thiep sau khi da tap huin ndng eao trinh dp vd nhan thiPC tren ciing d i i tugng vd phuang phdp nghien ciPU.
D i i tugng nghien cuu Id cae CBYT dang edng tdc tai cdc tram y te xa thuoe huyen My Loe, tinh Nam Djnh vdi cdc tieu chuan lua chpn va loai trip sau:
- Tieu chuan lwa chgn:
T i t ea cdc CBYT dang eong tdc tai ede tram y t i xa eiia huyen My Lpe tinh Nam Djnh tai thdi diim triin khai chuang trinh tap h u i n (thdng 3 nam 2010).
- Tieu chuan loai trir:
NhO'ng ngudi tip c h i i tham gia hoae v i n g mat dot x u i t tai thdi diem tap huin.
TAP CHI DUOC HOC - 9/2010 (SO 413 NAM 50)
