Nghien ciPU - Ky thuat
iTng dung cac tac nhan doi quang
hydroxyalkyi betacyclodextrin trong phan tich dong phan quang hoc proparanolol bang
phu'O'ng phap dien di mao quan
Le Thj Thu Cue', Nguyen Ngoc Vinh',
Tran Nguyen Quynh Trang^.Dang Van Tjnh^, Nguyen Dire Tuan^*
' Vien Kiem nghiem thuoc TP Ho Chi Minh
^ Khoa Duac, Dai hoc Y Duac TP Ho Chi Minh
Dat vln d§
Trong nhirng ndm g i n ddy, dien di mao quan Id phuang phdp hda ly duge edc nhd phdn tich lua chpn trong ITnh vue tdch ede dong phdn quang hpe vdi nhilu ly do: lugng m i u sir dung r i t it, dung mdi sii' dung trong qud trinh phdn tieh ehi Id dung d|eh dem do dd khdng anh hudng d i n mdi trudng, vide tdch cde dong phdn quang hoe ed t h i dat duge khi chi e i n thdm mdt lugng nhd tde nhdn ddi quang (chiral selector) vdo dung djeh dien ly n i n , Cde tde nhdn doi quang thudng duge s u dung Id cdc dang a, p, y-cyclodextrin, ede d i n c h i t cua cyclodextrin, protein, .,., Trong bdi bdo ndy, chiing tdi gidi thidu k i t qua phdn tdch hdn hgp racemic ciia propranolol b i n g phuang phdp didn di mao quan vdi mdt so tde nhdn doi quang Id p-cyclodextrin vd hai d i n c h i t hydroxyalkyi p-cyelodextrin: 2-0- (hydroxypropyl)-p-cyelodextrin (HP-p-CD) vd 2- 0-(2-hydroxybutyl)-p-cyelodextrin(HB-p-CD).
Nguyen lieu va phu'O'ng phap
C h i t ddi c h i l u , trang t h i l t bj, dung moi vd hda c h i t
Chat doi chieu
Propranolol hydroelorid - So Id:
QT118010903 - Vi$n Kiem nghidm thude TP Ho Chi Minh, hdm lugng 99,75%; dd i m 0,13%.
Trang thiet bj
Mdy di$n di mao quan Hewlett Paekard^°CE, d i u dd ddy diod quang, Cdt mao quan silica
nung chay, dudng kinh trong 50 \im, ehilu ddi tong cdng 64 cm, chilu ddi hieu qua 55,5 em.
Dung moi va hoa chat
2-0-(hydroxypropyl)-p-cyclodextrin (HP-p- CD) vd 2-0-(2-hydroxybutyl)-p-cycylodextrin (HB-p-CD) dd dugc tong hgp tai Vien Kiem nghiem thuoc TP Ho Chi Minh, beta- cyclodextrin (Merck) dat tieu chuan phdn tich.
Methanol dat tieu ehuan diing eho sde ky Idng (Merck), Tris (hydroxymethyl)-aminomethan (TRIS) vd acid phosphoric ddm ddc dat tieu chuan phdn tich (Merck),
Ddi tu'O'ng nghien cii'u
Vien ndn APO propranolol (propranolol hydroelorid 40 mg), so Id: HN1684, han diing:
10/2012, nhd san xuit: Apotex, Canada.
Phu'O'ng phap nghien cii'u
Trong d l tdi ndy, de tdch dong phdn propranolol trong hdn hgp racemic, ehiing tdi s u dung mpt sd tde nhdn doi quang nhu p-CD, HP-p-CD, HB-p-CD them vdo dung djeh didn ly n i n vd dua vdo mdt so tdi lieu tham khao d l dua ra d i l u kien dien di ban dau nhu sau: cdt mao quan 64 cm (55,5 cm) x 50 )im, 25°C, 20 kV, 50 mbar x 5 gidy, 214 nm, dung djch dien ly n i n TRIS 50 mM, pH 2,5 '^"^', Trong qua trinh thuc nghiem, ede y l u td anh hudng d i n qud trinh tdch dong phdn propranolol nhu ban c h i t vd nong dd tde nhdn doi quang, ban c h i t vd nong dd dung mdi hiru ca them vdo dung dich didn ly nen dd dugc khao sdt, Sau khi tim duge
Nghien CIFU - Ky thuat
-f.dieu kien tach dong phdn propranolol, se khao sdt tinh tuang thich he thong va tham djnh qui trinh phdn tich qua viec khao sat dp dung, dp lap lai, tinh tuyen tinh vd tinh dac hieu.
Chuan bi mau
Dung dich miu ddi chiiu: can chinh xdc khoang 50 mg propranolol hydroelorid, cho vdo binh djnh mirc 100 mii hda tan trong 20 ml methanol, sau dd them nud'c cat khu" khodng vira du the tich, Idc diu. Lay chinh xdc 5 ml dung djch tren, cho vdo binh djnh mire 50 ml, them nud'c cit khu" khodng vira du the tich, lie ddu. Lpc qua mdng Ipc millipore 0,2 pm.
Dung dich miu thir: cdn 20 vien, xdc djnh khdi lugng trung binh eua mpt vien, nghiin thdnh bpt min. Can mpt lugng bpt vien tuang Crng 50 mg propranolol hydroelorid, cho vdo
binh djnh mirc 100 ml, them 20 ml methanol, lie ky, sau dd them khoang 70 ml nud'c cat khu- khoang, sieu dm khoang 15 phiit, de ngudi, them nud'c cat khu khoang vira du the tich, lie diu. Lpc qua giay Ipc (loai bo 20 ml djch lpc dau). Lay chinh xac 5 ml djch Ipc, cho vdo binh djnh mirc 50 ml, them nud'c c i t khu" khodng vira du the tich, Idc deu. Lpc qua mdng lpc millipore 0,2 pm.
Dung dich miu tring: dugc chuan bj tuang tu mdu thCr nhung khdng cd chat khao sdt
Kdt qua va ban luan
Khao sat dilu kien phan tich
Anti hird/ng tac nhan doi quang p-CD, HP- p-CD, HB-p-CD
Hinh 1: Anh htrdng tic nhin ddi quang trin do phin giai ddng phin propranolol. Dieu kien dien di:
cdt mao quin 64 cm (55,5 cm) x 50 fum, 2^C, 20 kV, 50 mbar x 5 giiy, 214 nm, dung djch diin ly nin TRIS 50 mM cd gia tri pH 2,5; (a) 20 mM p-CD, (b) 20 mM HP p-CD, (c) 20 mM HB p-CD.
Nhin xit: vdi tde nhan p-CD kha ndng tdch dong phdn propranolol rit kem. Su thay doi hda hpc cua nhdm 2-hydroxy eua p-CD vd'i nhdm hydroxyalkyi 1dm eho khoang sa nude cua p- CD linh ddng han. Do dd, tinh chpn Ipc ddi vdi dong phdn propranolol cua tde nhdn HP p-CD va HB p-CD cao han p-CD, the hien mdt su cai thidn rd r^t v l dp phan giai cua hai dong phdn (dp phdn giai lin lugt Id 0,94 vd 1,21 vd'i tde nhdn doi quang HP p-CD vd HB p-CD, Nhu vdy hai tde nhdn doi quang HP p-CD vd HB p-CD dugc lua chpn cho nhirng khao sdt tilp theo nhdm muc dich tdch hodn todn hai dong phdn propranolol (yeu cau ve dd phdn giai > 1,5).
Anh huKmg dung moi hOu cc va nong do tac nhan doi quang
Cde ket qua khao sdt anh huang cua mdt so dung mdi hiru eg nhu methanol, ethanol, n- propanol vd aceton dugc them vdo dung djch di$n ly nen cho thay methanol Id dung mdi hiru eg thich hgp nhat de cai thien dd phdn giai. Khi them 20% methanol vdo dung djch di$n ly nin, dp phdn cue cua dung djch se bj giam di, Idm tdng tinh chpn Ipc cOa cdc tde nhdn d6i quang vdi cae dong phdn.
Anh huxmg nong d^ tac nhSn d6i quang Khao sdt anh huang nong dd HP p-CD vd HB P-CD d cdc gid tri 10 mM, 20 mM vd 30 mM.
22 TAP CHI DUQC HQC - 12/2009 (SO 404 NAM 49)
Nghien CCPU - Ky thuat
Rs=l,08 Rs=1.39 Kv 1.".!
L
Hinh 2: Anh hirdng ndng do HP p-CD trin do phan giii ddng phan propranolol. Dieu liien dien di: cdt mao quin 64 cm (55,5 em) x 50 pm, 2^C, 20 kV, 50 mbar x 5 giay, 214 nm, dung djeh dien ly nin TRIS-phosphat 50 mM gii tri pH 2,5 vi 20% methanol; (a) lOmM HP p-CD, (b) 20 mM HP p-CD, (c) 30 mM HP p-CD.
R s = l . 5 6
R s = l . 8 2
L
Rs=l,45
Hinh 3: Anh hwdng ndng dd HB p-CD trin do phin giii ddng phin propranolol. Dieu Ifien dien di: cdt mao quin 64 cm (55,5 cm) x 50 /jm, 2^C, 20 kV, 50 mbar x 5 giiy, 214 nm, dung dich diin ly nin TRIS-phosphat 50 mM gii tri pH 2,5 vi 20% methanol; (a) WmM HB p-CD, (b) 20 mM HB p-CD, (c) 30 mM HB p-CD.
Nhin xit:
Khi nong dp HP p-CD tdng thi dp phdn giai vd thd'i gian di ehuyen tang, Cy ndng dp HP p- CD 30 mM, hai pic dong phdn tdch hodn todn (dp phdn giai > 1,5). Neu t i l p tuc tdng ndng dp tde nhdn ddi quang, dp phdn giai chT tang nhe nhung se kdo ddi thd'i gian phdn tieh.
Khi nong dp HB p-CD tdng thi thdi gian di chuyen tdng vd ed s u thay doi ve dp phdn giai.
O nong dp HB p-CD 20 mM, hai pie dong phdn tdch hodn todn (dp phdn giai > 1,5). N l u t i l p tue tdng nong dd tde nhdn doi quang, do phdn giai giam vd thdi gian phdn tich kdo ddi (tren 50 phiit).
Qua khao sat tren, chpn nong dd HP p-CD 30 mM vd HB p-CD 20 mM Id thich hgp v l dd phdn giai vd thd'i gian phdn tieh.
Nhu vdy dieu kien dien di thich hgp de tdch dong phdn propranolol Id:
+ B i l u kien dien di 1: opt mao quan 64 cm (55,5 cm) X 50 nm, 25°C, 20 kV, 50 mbar x 5gidy, 214 nm, dung djch dien ly n i n TRIS 50 mM pH 2,5 cd 20% methanol, 20 mM HB p-CD,
+ Dieu kien dien di 2: cdt mao quan 64 cm (55,5 cm) X 50 nm, 25°C, 20 kV, 50 mbar x 5gidy, 214 nm, dung djch dien ly nen TRIS 50 mM pH 2,5 ed 20% methanol, 30 mM HP p-CD
Nghien CLPU - Ky thuat m
mAU
8 I
6 ] 4 j 2 : 0
10 15 20 25 30
1
o
anol opr
Q. '
:
••
.
35 o
-CO CO
lo rano rop
, a.
40
min
mAl
8 6 4 2 0
10 15 20 25 30
0 ' 2
1 i
2 'iii 2
a. °- 1 I
35 40
min
Hinh 4a: Diin di dd dung dich mau ddi chiiu (hdn hop racemic cua propranolol) irng vdi diiu kien
diin di 1
Hinh 4b: Dien di dd dung dich miu thir (hdn hop racemic cua propranolol) irng vdi d i l u kien diin
di1
mAU
8 6 4 : 2 : 0
10 15
CM <J0 CO t
^ i
ra ra a. o o.
o
20 25 30 35 40
min
mAU
8 6 4 2 0
10 15 20 25 30
in
35 40
min
Hinh 5a: Dien di dd dung dieh miu ddi chiiu (hdn hap racemic cua propranolol) irng vdi diiu kiin
diin di 2
Hinh 5b: Diin di dd dung dich miu thir (hdn h(?p racemic ciia propranolol) irng vd'i diiu kiin diin
di2 Khao sat tinh tu'O'ng thich he thdng
Bang 1: Kit qui khio sit tinh twang thich hi thdng ciia phwang phip (n-6)
HP p-CD HBfi-CD
Mau Dong phan propranolol RSD DQ RSD
Thai gian di chuyen ConrArea ^ ... Thai gian di chuyen CorrArea giai
D(>
phin giii
•Chuan Pic dong phan 1 Pic a6ng phan 2 Pic(36ng phan 1
0,86%
0,87%
0,24%
0,24%
0,48%
0 , 2 1 %
1,73 0,55% 0,72%
0,55% 0,31%
1,83
ThCc
Pic(36ng phan 2 0,24% 0,17% 1,76
0,73% 0,39%
0,74% 0,32%
1,82
24 TAP CHi DUQC HQC - 12/2009 (S6 404 NAM 49)
Nghien CLPU - Ky thuat
K i t qua thong ke cho thay, sau 6 lan dien di thdng so CorrArea vd thd'i gian di chuyen cd RSD < 2%; gid trj dp phdn giai Id-n han 1,5.
Vdy phuang phdp dat tinh tuang thich he thong.
Tham djnh phu'ang phap Tinh dac hieu
T i l n hdnh dien di mdu trdng, mau doi c h i l u vd mdu thir theo d i l u kien dien di 1 vd 2. K i t qua cho t h i y m i u t r i n g khdng cd pic trimg vdi
pic c h i t phdn tich. Dien di dd mdu thir giong didn di do mdu doi chieu. Khi thdm chat doi c h i l u propranolol vdo m i u thu- thi dien tieh pic ciia c h i t them vdo tdng. Pho tCr ngoai tai thd'i gian luu cua cdc pic trong mdu thCr giong pho tir ngoai eua mdu doi c h i l u . Dd tinh khiet cua cac pic Id-n han 99%. Hai pic cua hai dang dong phdn cd pho UV-Vis giong nhau. Vdy phuang phdp cd tinh dae hieu.
Do dung, do lap lai va khoang tuyen tinh
Bang 2 !: Kit qui khao sit do dung, do lip lai vi khoing tuyin tinh
Pic dong phan 1 Pic dong phan 2
HP p-CD
H B p - C D
Phuang trinh hoi qui
Khoing tuyen tinh (/jg/ml) Do l$p lai (n=6)
Do dung (n=9) Phuong trinh hoi qui
Khoang tuyen tinh (fig/ml) DQ lip lai (n=6)
DQ dung (n=9)
y = 0.0024X + 0,0018 y = 0,0024x + 0.0015 R = 0,9994 R = 0,9995
1- 200 (h6n hcrp racemic) RSD = 0,22% RSD = 0,17%
RTB = 98,36% RTB = 99,59%
y = 0,0024x - 0,0003 y = 0.0024x - 0,0007 R = 0,9999 R = 0,9999
1- 200 (h6n hpp racemic) RSD = 0,40% RSD = 0.32%
RTB = 98,83% RTB = 99.64%
f^re: ty li hdi phuc trung binh.
K i t qua thong ke cho t h i y ty le hoi phuc ndm trong gid-i han cho phdp cua ty le hoi phuc ly t h u y l t (98% - 102%) vd RSD < 3% ddi vai dp ldp lai. Qui trinh phdn tieh cd khoang t u y l n tinh rpng vdi hd so tuang quan cao.
Kdt luan
Bdng phuang phdp didn di mao quan, chimg tdi dd khao sdt mdt so dieu kien anh huang d i n qud trinh tdch hai dang dong phdn trong hon hgp racemic cua propranolol vdi viee irng dung p-CD vd hydroxyalkyi p-CD (HP p- CD, HB p-CD) Idm tac nhdn doi quang. Da xdy dung quy trinh xdc djnh hai dang dong phdn quang hpc cua propranolol vdi hai tde nhdn ddi quang HP p-CD vd HB p-CD . Quy trinh ndy cd tinh ddc hidu, cho k i t qua chinh xde vd cd tinh ldp lai.
Summary
Two capillary electrophoretic methods for separation of the enantiomers of propranolol were established and validated. The two enantiomers were separated by a fused-siliea
capillary with TRIS running buffer (50 mmol L'\
pH 2.5) containing either 30 mmol L'^
hydroxypropyl P-cyclodextrin (HP P-CD) or 20 mmol L"' hydroxybutyl P-cyclodextrin (HB p- CD). The effects of solvent and chiral selective concentration on the separation were investigated. The quantitation range for both enantiomers at 214 nm was 1 - 200 pg mL'\
tntra-day relative standard deviation (RSD;
n = 6) was < 3%. The recovery range was 98%
- 102%. The methods were applicable for quantitative determination of the propranolol enantiomers in pharmaceutical preparations and chiral impurities in raw propranolol materials.
Tai lieu tham khao
1. Hiroyukl NIshi, Journal of chromatography A, vol. 735, issue 1 - 2 (1996), 57- 76.
2. Salvatore Fanali, Journal of Chromatography y4. vol. 735(1996). 7 7 - 1 2 1 .
3. Yunhe Wei, Jian Li, Chenfu Zhu, Aiyou Hao, and Minggang Zhao, Analytical Sciences (2005), vol.
21.959-962
