NGHIEN CJJVKHOA HOC
DINH LL/ONG BETA CAROTEN TRONG V I £ N NANG M^M VINPHALAF B A N G PHl/GTNG PHAP HPLC
LE THI THIEN Hl/ONG. PHAN THJ THUY CHI Vi$n Kiim nghidm thuoc Trung u&ng
1. Dat van de
Beta caroten la tien chat ciia vitamin A nhung no khdng chi cd vai tro nhu nhung gi ma vitamin A cd ma con sd hiru nhimg tac dung sinh hpc rdng rai khac. Beta caroten giiip ca thd phdng tranh duac tinh trang thidu hut vitamin A, ngan chan mu Ida, lam lanh manh hda he mien dich... Beta caroten con lam het sach nhirng nguyen tir dxy tu do dang du thira dien tir trong da, bao ve da khdi tia cue tim.... Ben canh dd, beta caroten con sd hiiu mdt khd nang chdng dxy hda uu viet vi nd cd tac dung khir hdt gdc tu do du thira trong ca the, bao ve mang te bao, cham lai lao hda, ngan ngira ung thu...
C6 rat nhidu che pham phdi hop beta caroten, vitamin C, vitamin E ngoai ra cdn cd them selen;
glutathion hoac cac vitamin khac nhu vitamin Bp Dupc didn My cd cac dang che phdm thudc cd chiia beta caroten va dinh lupng hoat chat nay bang phuang phap do quang sau khi tien hanh qud trinh chidt tdch phiic tap. Dd gdp phan kiem tra chit luang thudc chiing tdi da trien Idiai va tham dinh phuang phap dinh luong beta caroten trong vien nang mdm Vinphalaf cd cdng thiic bao chd nhu sau:
- Hon dich 30% beta caroten - Vitamin C {Acid ascorbic) - Vitamin E (DL - a - Tocopherol) - Vitamin B, (Thiamin hydroclorid) - Vitamm B^ (Pyndoxin hydroclorid) - Ta duoc: {Dau dau nanh, ddu co, sdp ong trdng, lecithin, gelatin, glycerin, nipagin, nipasol, ethyl vanillin, titan dioxyd, phdm mau chocolate, nuac tinh khiet).
50,0 mg 500,0 mg 400 IU 5,0 mg 5,0 mg
Vira dli 1 vien
2. Thvc nghi§m va ket qua
2.1. Thiet bi, hod chat, chdt chudn va ddi tucmg nghien ciru
2.1.1. Thiet bi, dung cu: DS duac hieu chudn theo ISO/IEC 17025.
- May sac ky Idng hi?u nang^ cao Agilent Technologies vdi detector DAD.
- Cdt LiChrospher® 100 RP-18 (250 x 4 mm;
5 pm).
- Can phdn tich Mettler Toledo AB 204 dd chinh xac 0,1 mg.
- Bd Ipc diing cho sdc ky vdi mang Ipc 0,45 \im.
- Cac dung cu thuy tinh chinh xac: binh dinh miic, pipet.
21.2. Chat chudn: Beta caroten RS Ngudn gdc: PHIL Inter pharma.
SdId:HY20110308C.
Ham luong: 33,53% (C^gH^g) tinh theo nguyen trang.
2.1.3. Hoa chdt thuoc thic - Acetonitril (Merck, HPLC) - Ethanol (Merck, HPLC) - Cloroform (Merck, PA) - NudcRO
2.1.4. Doi tuang nghien ciru: Vien nang mem VINPHALAF (Ld: 11004) do Cdng ty Cd phin Dupc pham VTnh phiic san xuit.
2.2. Phuang phdp nghien cuu 2.2.1. Diiu kien HPLC
Pha ddng : Acetonifril - Ethanol (1:1).
- C p t : LiChrospher® 100 RP-18 (250 X 4 mm; 5 (im).
- Detector DAD : 450 nm.
Tdc dp ddng 1 ml / phiit.
Thd tich tiem 20 \i\.
2.2.2 Phuang phdp xir ly mdu - Chudn bi dung dich chudn:
Cdn chinh xdc mpt lupTig chat chuin tuang duong vdi khoang 15 mg Beta caroten (C^^^ vao binh dinh miic 100 ml, them 70 ml cloroform (TT) Idcl^
dd hoa tan. Bd sung cloroform ddn vira dii, Idc dSu.
Hiit chinh xac 2,0 ml dung dich nay, pha loang bang Tap chi KIEM NGHI$M THU6C - So 3.2012; T|p 10.(37)
pha dpng ddn viia dii 50 ml, lie ddu. Lpc qua mdng Ipc 0,45 pm vd tidn hdnh do HPLC.
- Chudn bi dung dich thir:
Can chinh xdc mOt lu^ng hon hpp thudc chto trong nang tuong duong vdi khodng 15 mg Beta caroten (C^t^jg) vao binh djnh miic 100 ml, them 70 ml cloroform (TT), Idc ky 15 phfit. Bd sung cloroform d ^ vira d i , lie ddu, Ipc qua gidy lpc. Hut chinh xac 2,0 ml dich lpc, pha loang bing pha ddng ddn viia du 50 ml, lie deu. Lpc qua mang lpc 0,45 pm vd tidn hdnh do HPLC.
3.K4tquiivdbdnlu$n 3.1. Chgn ^u ld$n sdc k^
Chung tdi dd khdo sdt vd lua chpn dupc didu kien sic k^ nhu da ndu d phdn 2.2.1, sic k^ dd thu dupc ciia dung djch chuan vd thir thd hi^n d Hinh 2 vd 3.
Tren sdc k^ dd chiing tdi thiy pic ciia beta caroten ddi xiing, gpn. Nhu vdy vi^c I\ra chpn cdc didu kien sdc ky^ Id phd hpp.
fr iK\Kwm.n*\^¥mm^Katmmm^i\»l^mKTi
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Hinh 1. SKD dinh lupng beta caroten cUa mdu trdng
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1 a d Hinh 3. SKD dinh luang beta caroten cUa mdu thir
3.2. Thdm ^nh phuang phdp 3.2.1. Tinh dgc hieu
Dp dac hifu cua phuong phap dugc ddnh gid thdng qua kdt qua phdn tich thu dupc trdn nen miu tring khdng chiia beta caroten, dung dich chudn beta caroten va dung djch thu chuin bj tir vien nang mdm VINPHALAF.
- Nin mdu trdng khong chua beta caroten: Cdn khodng 1,4 g miu gid dupc (bao gdm cdc ho^t chat khac va hdn hpp cdc ta duac theo cdng thiic bdo ch^, xu 1^ theo quy trinh xic 1^ miu thii (muc 2.2.2).
- Chudn bi dung dich chudn va dung dich thir:
Chuan bi nhu muc 2.2.2.
Kit qud khdo sdt tinh dgc hieu:
- Thdi gian luu cua pic beta caroten trdn sdc ky do cua dung dich thu tuang iing vdi dung dich chudn.
- Phd UV cua pic beta caroten trdn sic k^ dd ciia dung dich thir va dung dich chudn gidng nhau.
- Mdu gia dupc khdng cd pic tai thdi gian luu tuang umg vdi pic beta caroten.
Nhu vay phuang phap phan tich ddp iing dupc ydu ciu vd dd dac hidu vdi beta caroten, khdng bj dnh hudng bdi cac thanh phan ta dupc cd mat trong cdng thuc bao chd ciing nhu cdc dnh hudng khde trong qua trinh chuan bi mau.
3.2.2. Do thich hgp cua hi thing
Tidm 6 lan dung djch chuan. Ghi lai cac sdc ky do, xac djnh cdc thdng sd thdi gian luu, di$n tich pic, h^
sd ddi xiing, sd dia ly thuydt ciia pic beta caroten.
Hinh 2. SKD dinh luang beta caroten cua mdu chudn
Tfp chf K I £ M N G H I $ M T H U O C - S6 3.2012; T$p 10.(37)
Bang 1. Ket qua khao sdt do thich hap cua he thong sac ky
Bing 2. Kit qua khao sat tinh tuyin tinh
Su
1 2 3 4 5 6 T.binh RSD (%)
Thdi gian liru (min.) 4,393 4,378 4,378 4,375 4,369 4,366 4,377 0,216 < 1,0%
Difn tich pic (mAU.s)
762,31 763,21 762,84 762,34 762,97 763,13 762,80 0,051 < 2,0%
doi xirng 0,85 0,85 0,85 0,84 0,84 0,84
< 2 S6 ^a ly
Ihuyit 1397 1422 1387 1419 1381 1380
>1000 6 9 thich hop cua h | th^ng dat yeu cau.
Stt
1 2 3 4 5
%S0 v&i
^tth lu^mg
50 80 100 120 150
Lirffng can chh
chuan (mg) 22,6 36,1 45,1 58,5 67,7
Binh
^ h miic (ml)' 100 100 100 100 100
Bppha loang 2->50 2->50 2->50 2->50 2->50
NSiig0 beta caroten
(pgAnl) 3,031 4,842 6,049 7,846 9,080
Diii," tich pic (mAVj) :
380,7^ 612,24 763,19!
996,24 1127,92 H$ s£ tirtfng quan: 0,9996
Hi s6 gic: 124,54 "
H? s6 chin (intercept): +7,7304
_ 1000
1 BOO
1
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^L ^^ IHIHI^^H
^^^Bt ^^^^^^1
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ODD 2.0CO 4.CHn 6 D W 3 0 0 0 lO.OW NAng S ^ bMaciroMn (mcg/ml)
3.2.3. Do tuyen tinh
Khao sat sir tuong quan giua y (dien tich pic) va x (n6ng do); bang phuong phap binh phuong cue tieu, ket qua cho thiy he s6 r^ = 0,9992 (-» r = 0,9996) chihig t6 CO su tuong quan tuy^n tinh giiia ndng dp va
dien tich pic (Bang 2, Hinh 4). . . - , , Hinh 4. Do tm bieu dien moi tuang quan tttyen ttnh giira
nong do va di$n tich pic 3.2.4. Do diing
- Chudn bi mdu chudn: Can chinh xac 45,1 mg chit chuan beta caroten va chuan bi nhu muc 2.2.2.
- Chudn bi cdc mdu thir: Them chinh xac 1 lugng chuan beta caroten tuong ung voi 80,100 va 130% Mm luong ghi tren nhan vao cac mau gia dugc. Moi miic nong dp lam 03 man. Tien hanh theo qui trinh phSn tich.
Bdng 3. Ket qud khdo sdt do dung Sn
1.
2.
3,
4 5.
6.
7.
8 9
Ltrfng litem vao so v&i ntian 80%
80%
80%
100%
100%
100%
130%
130%
130%
Lir^g cin mau gii dirffcig) 1,4112 1,4116 1,4113
1,4118 1,4110 1,4108
1,4115
!,4116 1,4116
Lirang can chat chuin beta caroten litem vao
mau (mg) 36,6 36,2 37,2
48,3 49,6 47,8
57,6 57,7 58,4
Lir(mg beta caroten lhi^ti(C^H^ them
vao miu (mg) 12,27 12,14 12,47
16,19 16,63 16,03
19,31 19,35 19,58
Di^n tich pic beta caroten (mAV.Sj
(tli,il 616,50 627,43
815,88 841,62 804,11
973,78 969,55 993,28
Lu^tg beta caroten (C^Hj^
tim lfi (mg) 12,40 12,22 12,44 TB 16,17 16,68 15,94 TB 19,30 19.22 19,69 TB
%ThullSi
101,06 100,66 99,76 100,«;
RSD-0,6!%
99,88 100,30 99,50 99,89;
RSD = 0.40%
99,95 99,33 100,56 99,95;
RSD = 0,66%
% Thu h6i trung binh = 100,11% ; RSD = 0,54%
Tap chi KIEM NGHIfM THUOC - So 3.2012; Tip 10.(37)
3.2.5. Khodngxdc dfnh: Tii kdt qud dO diing suy ra khoang xac dinh tii 4,86 den 7,83 pg/ml.
3.2.6. DQ lap lgi
- Chudn bi dung dich chudn: Cdn chinh xdc 45,1 mg chat chuan Beta caroten vd chuan bj nhu myc 2.2.2.
- Dung dich ihu: chuan bj nhu m\ic 2.2.2.
Bdng 4. Kit qud khdo sdt dg lap lgi ' Di?n tich pic tmng binh miu chuin: 762,80
- Khoi lirang vien tnmg binh: 1,4498 g SII
1 2 3 4 5 6
Lu^nm can mSu thA(g) 1,3139 1,3252 1,3126 1,2862 1.3558 1,3189
Difn lichpic beta caroten (mAU.s) 780,90 782,80 776,75 761,40 801,65 783,19 IVnng binh
Kit qud Oinh Itrpng (%) 113,88 113,18 113,39 113,43 113,29 113,78 113,49 RSD (%) = 0,24% < 2,0%
3.2.7. DQ chinh xdc trung gian
stt
1 2 3 4 5 6
Bdng S. Ket qud khdo sdt dg chinh xac trung giar KNV:1
Thilt bi phSn tich: Agilent technologies 1200 LiT^ng can chat chuan: 4S,1 mg Di^n tich pic trung bhih mau chuan: 762,80 Khoi lu^mg vi€n tnmg buih: 1,4498 g (Liyicgtqu4tird$15pl^i)
Lufmg cin mau Ihir (g) 1,3139 1,3252 1,3126 1,2862 1,3558 1,3189
Dl^n Itch pic 780,90 782,80 776,75 761,40 801,65 783,19
% Him lir0tg 113,88 113,18 113,39 113,43 113,29 113,78
•nung Unh -113,49%; RSD - 0,24%
KNV: 2
Thiet bi phan tich: Agilent technologies 1200 Luong can chat chuan: 45,7 mg D i ^ tich pic trung bmh miu chuan: 773,10 Khoi luong vien tnmg binh: 1,4492 g
Lirpng cin muu Ihir (g) 1,3136 1,3755
Difn lich pic 777,79 814,42 1,3604 808,79 1,3822 817,64 1,3652 803,17 1,3841 1 816,14
Tmng binh = 113,24%; RSD =
% Him lirpng 113,38 113,38 113,85 113,28 112,66 112,91 0 3 7 % K^t quii djDh l i i ^ g tmng binh = 113,37% ; RSD = 0,32% < 2,0% (n=12)
D$ chinh xic trung gian tl^t y£u c^u 4 . K e t l D | i i
Phucmg ph4p sic Ic^ 16ng pha dao da dugc thik lap dl dinh lugng beta caroten trong che pham dang thuoc nang mem. Phuong phap da xay dung su dvmg cgt LiChrospher® 100 RP-18 (250 x 4 mm; 5 ^m) trong qui trinh phta tich. Pha d$ng 14 h6n hgp Acetonittilvi Ethanol (1:1), t6c dddong 1 ml/phiit, thS tich tiSm 20 pi;
detector DAD a but]^: song 450 nm. Phuong phap dugc thim djnh vh tiiih dac hieu, dp thich hgp ciia hp th6ng, do tuyen tinh, khoing xic dinh, dp ddng, dp IJp lai, dO chinh xic tmng gian. Ket qua thim djnh cho thiy phuang phip dugc xiy dvmg hoin toin phu hgp cho vipc dinh lugng beta caroten trong chj phim viSn nang mjm VINPHALAF v i c6 th6 ap dvmg dinh lugng hojt chit niy trong cac ch6 phim vi6n nang m t o co thinh phia tuong tvr.
Tii lipu tham khao 1. Dugc dien Vi?t Nam, Lan xuat ban thir tu, 2009.
2. BP.2010.
T»p cbi KitM NGHIfM THU6C - S6 3.2012; T|p 10.(37)
3. USP. 34,
4. Sandie Lindsay, High Performance Liquid Chromatography, 2nd edition, 1994.
SUMMARY
A reversed-phase liquid chromatography method was developed for assay of beta carotene in soft capsule. The method was carried by using a LiChrospher 100 RP-J8 (250 x 4 mm; 5 /im) column and a mixture of acetonitrile and ethanol (1:1) as mobile phase. The flow rate of mobile phase is maintained at 1.0 ml per minute, injection volume: 20 fil, analyle was detected by a DAD detector set at 450 nm. The method was validated in specificity, linear range; accuracy, precision.
Validation results proved that the developed method was suitable for assay for beta carotene in soft capsule and used to determme beta carotene in Vinphalaf soft capsule.
DINH Ll/OTNG CAC DANG CLORPHENIRAMIN
DANG DI/OC sis DUNG TRONG CHE PHAM THUOC B A N G P H I / O N G PHAP HPLC TREN C 6 T sAc Kt CHON LOC D 6 | QUANG
LE DINH CHI Tru&ng Bqi hoc Duac Hd Noi 1. Bat vSn de
Clorpheniramin la mot hoat chit co hoat tinh khang Histamin hien dang duac su dung ddng then duoi dang hon hop racemic cua hai dong phan doi quang (dexclorpheniramin va levoclorpheniramin) va dang dexclorpheniramin tinh khiet. Tren thi truong Viet Nam, hai dang hoat chat nay dang duoc sii dung dpc lap hoac phoi hgp voi nhi8u hoat chat khac, song trong Dugc di^n Viet Nam vin chua co chuyen luan nao dua ra phuang phap chinh thong cho phep dinh lugng rieng mpt each dac hieu dexclorpheniramin trong che pham thuoc. Mpt phuang phap nhu vay phai cho phep tach rieng biet hai dong phan doi quang cua clorpheniramin, nho do dexclorpheniramin se dugc dinh lugng ma hoan toan khong bi anh huong boi sir co mat cua levoclorpheniramin. Nhiing nghien ciiu dS dugc cong b6 chung toi tim duac [2],[3] moi chi danh gid d6n kha nang tach hai dong phan d6i quang ciia clorpheniramin ma chua danh gia miic dp phu hgp khi ap dung tren ch8 pham, nhat la che pham da thanh phan, va dp dac hi8u khi phan tich dong phan doi quang ciia clorpheniramin trong dieu kien co mat cac hoat chat khac. Yeu cSu v6 dp dac hi6u da dugc chiing toi giai quyet trong mgt nghien Cliu da cong bo ve dinh tinh phan biet clorpheniramin racemic va dexclorpheniramin [I]. Trong nghien cuu nay, chiing toi thuc hien xay dung mgt quy trinh phan tich bang HPLC cho phep dinh lugng ca hai dang clorpheniramin hien dang dugc sii dung trong ch6 pham thuoc: dang clorpheniramm racemic va dang dexclorpheniramin.
LE THI HL/ONG HOA Vi^n Kiem nghiem thuoc Trung ucmg
2. Thirc nghiem
2.1. Thiet bi, diing cii, hda chdt vd chdt chudn 2.1.1. Hoa chdt, chdt chudn
- Acetonitril, isopropanol loai dung cho HPLC;
- Triethylamin, acid acetic bang loai tinh khiSt phan tich;
- Chat chuan clorpheniramin maleat ciia Vi?n Kiam nghiem thu6c Tmng uong (SKS: 0198032;
ham iugng 100,0 % tinh theo che pham khan; dp am 0,10%);
- Chat chuSn dexclorpheniramin maleat cua USP (Lot H0D199; ham lugng 99,9 % tinh theo nguyen trang).
2.1.2. Thietbi, dungcy
- May sac ky long hi6u nang cao Merck - Hitachi dugc trang bi detector UV-VIS;
- Can phan tich Sartorius ED224S co do chinh xac tai 0,1 mg;
- May do pH Metrohm 691;
- Cpt sac ky chpn Igc doi quang Cyclobond I 2000 (250 X 4,6 mm, 5 ^im) co chiia pha tinh la P-cyclodextrin;
- Cac thiet bi thi nghiem c£in thi6t khac (dung cy thiiy tinh chinh xac c4p A, may iSc sieu am, bo IQC dung moi...).
Cac thiet bi phan tich sii dung trong qua trinh nghien cuu deu dugc hi?u chuSn va sii dung theo diing cac quy dinh cua ISO/IEC 17025 va GLP
10 T?p chi KIEM NGHlfM THU6c - So 3.2012; T?p 10.(37)
