Paracetamol (1)

5/11/2018 Paracetamol (1)

1/94

B A o cA o KET Q uA NGHI~N cU U f}~ TAl CAP B O

Ten de tal:NGHIEN cm r UNG DUNG PHUONG PHAp sA c KY LONGHI~U NANG CAO (HPLC) VA DO QUANG PHO UV-VIS DE

, '- . ,; if/'DINH TINH VA DINH LUONG CAC HOAT CHAT TRONG. MQT s6 THUOC CO'Ttr2 DEN 5 THANH PHAN

Chii nhlem de Uti: , ,T.S. THAI nUY THIN

Co quan chu trl:TRUONG DAI HOC nuoc HA NOI-

Co quan ph6i hop chinh:VIEN KlEM NGHIEM THUDe BO YTt. .

't


2/94

" " K K ,,', ,..."BAO CAO TONG KET DE TAl NGHIEN COOKHOA HOC CAP BO.

Co quan quan I y d~Uti : D O Y TtCO'quan chiI tra d~ tal: TRUONG I>.;.I HOC DUQC HA N(n

Ten d~tal:NGHIEN ccuUNG DUNGPHUONG PHAp sAc KY LONGA..... ., ~ NHI~U NANG CAO (HPLC) VADO QUANG PHO UV-VIS DE

DINH TiNH VA DINH LUONG cAc HOAT CHAT TRONG. MOT s6 THU6c COTtr2 DEN 5 THANH PHAN

ChiI nhiem de tal: Thai Duy ThinDanh sach nhUng nguOi thl}'c hi~n chinh:

1. PGS-TS Thai Phan Quynh Nhu2. NCS-DS Trdn vt Hung3. ThS Nguyin TUUngVy4. ThS vssnn5. ThS Nguyen Tudn Anh,6. DS CKI Hoang VeinDUe .

ThOi gian thuc hien de mi: Tit thang 1-2003 den thang 12 -2005.Tdng kinh phi dll(_1cduyet: 190.000,000d.T6ng kinh phi dll(_1ccAp: 190.000.000d.


3/94

MUCLUC.NQidung Trang

Phin A. TOM TAT cAc KET Q uA NOI B~T CUA DE TAL 11. Kt qua n6i b?t cua d~ ali . 12. Ap dung vao thirc ti~n xa h9i. 23. Danh gia tlnrc hien de tai d6i chiu voi de cuong nghien ciruda dtroc phe duyet. 2

4. Cac y kien de xuat. 3Phin B.N(nDUNGBAo cAo CHI TIET. 4

1.D~t vA n d~. 42. Tang quan d~ tai, 62.1. Tinh hinh nghien ciru trong va ngoai mroc. 62.2. T6ng quan v 8 cac plnrong phap phan tich. 63. DA i ttrC}'llgva phirong phap nghien cuu. 183.1. Thit k nghien ciru, 183.2. Chon mftu, ca mdu va d6i nrong nghien ciru. 183.3. Phuong phap nghien ciru. 193.4. Phuong phap xu I y s6li~u. 224. KSt qua nghien ciru. 23

4.1. Dinh tinh va dinh hrong paracetamol, bromhexin hydroc1orid,pseudoephedrin hydroclorid, clorpheniramin maleat va cafein bangphtrong phap HPLC. cs tri: PGS-TS Thai Phan Quynh Nhu. 234.2. Dinh hrong d6ng thai paracetamol (PA) va acid mefenamic

(AM) trong vien nen bao phim Pamesic bang phuong phap HPLC.en: tri: TS. Thai Duy Thin. 274.3. Nghien ciru dinh hrong ddng thai paracetamol (PA) va cafein

(CA) trong thu6c da thanh phfuJ bang HPLC. Chil tri: TS. Thai Duy Thin. 314.4. Nghien ciru dinh hrong d6ng thai paracetamol (PA) va ibuprofen

(IB) trong thuoc da thanh phan. Chi: tri: TS. Thai Duy Thin. 354.5. Dinh hrong d6ng thai paracetamol CPA) va quinin sulfat (QS)trong vien bao Antigrip F bang phuong phap HPLC.ci tri: TS. Thai Duy Thin 38


4/94

4.6. Dinh hrong dong thoi theophylin, ephedrin hydrochlorid vaphenobarbital trong thuoc h6n h


5/94

Phdn A . Tom tiit cae kit GUO n6i b(it cua oJ tai____________________ ..__ _ .. .7-.. ..__ ..__ ..__ _ .,.. ---- - - - - - --_ -.,. --_ .. - .. --- - - - - - - --> -PHAN A. , ..:; , ... ? X _ ? ~TOM TAT CAC KET QUANOI B~T CUABE TAl

1. K~t qui n6i b,t ella d@tiia. Dong gop mUi cila i I 2 ta ;.:. Chung toi da khai thac, irng dung hai phuong phap phan tich cong C1,lmanh, hiendai vito viec kiem nghiem cac thu6c c6 nhieu han mQt thanh phfut hoat chAt. d6 laphirong phap HPLC va phirong phap Quang ph6 tir ngoai phan tich da cAutir MCA,sredung thu~t toan th6ng ke trong viec danh gia ca c k~t qua pha n tich .

:. Xay dung duoc phuong phap, mo hinh qui trinh phan tich dinh tinh va dinh luongn6i chung d6i voi cac hoat chAt trong mQt cong tlnrc thuoc ph6i hop sir dung haiphirong phap tren .

:. (rng dung thanh ceng phirong phap, qui trinh phan tich da xay dung vao viec kiemnghiem mQt s6 cong thirc thu6c c6 til 2 toi 5 thanh phfut hoat chAt. Dang ti~p tucnghien ctru, danh gia tren mQt s6 cong thirc thuoc ph6i hep khac, d~c bi~t la cacthuoc c6 nhieu hon 5 thanh phfut boat chAt.

b. Kit qua C(l thi.:. Da xay dung diroc 10 qui trinh phan tich dinh tinh va dinh hrong cac hoat chAttrong 7 cong thirc thu6c ph6i hQ'Pph6 bi~n tren thi tnrong, nhieu hon mQt congthirc so voi du ki~n trong d cuong nghien ciru. Trong d6 c6 7 qui trinh sir dungplnrong phap HPLC va 3 qui trinh sir dung phirong phap quang ph6 tir ngoai pharrtich da cAuttl MCA .

:. Da khao sat tren 4 cong tlnrc thu6c c6 hai thanh phfut ph6i hQ'P,2 cong tlnrc thuocco ba thanh phfut ph6i hQ'Pv a 1con g tln rc thu oc c6 n am thanh phfut ph6i hQ'P

:. Cac nghien CUutren du da duQ'Ccac chuyen gia v ki~m nghiem kiem tra, thfundinh la co gia tri khoa hoc va y nghia tlurc tin, da diroc cong b6 tren cac tap chichuyen n ga nh nhu tap chi kiem n ghiem thuoc, ta p chi diroc hoc ... va tham gia baocao trong cac hQi nghi v diroc n6i rieng va hoa hoc n6i chung (xem phfut phu luc).

c. vi nghien CMU khoa hfJc12 cong trinh khoa hoc co lien quan da dircc cong b6.Tham gia 3 hQi nghi khoa hoc qu6c t~:

HQi n gh i diroc Dong d iro n g III 5/2003 0 Thai Lan .HQinghi hoa hoc A Au 0 Ha NQi 10/2003.HQi nghi direc Dong dirong IV 11120050Thanh ph6 He,Ch iMinh.

nhb trao d6i thong tin, ki~m clnrng va ban lu~ v phirong phap lien quan toi cacnQ i du n g c6 trong d e ti i cua chung toi.

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6/94

_?~4'!_:1~! ! . . ' ! ! _~4~ ~ ~ ~ _ M ~q _ ~ ! !_ 1 ) _ 4 ! _ k ( i L ~ ~ q _ ~ ~ _ ! ! ! ~ _d : Dong gop dao t(lo can b{j cho nganh.:. D~ tai da t~p hop va huy dQng duQ'cmQt sA hrong 1611can bQ tre cua tnrong D~hoc di roc Ha nQi~Vien kiem nghiem thu6c va mQt s6 Trung tam kiem nghiem diaphirong tham gia, gop phfut nang cao trlnh dQva kinh nghiem chuyen mon cho caccan bQ.

:. Gop phfut dao tao diroc 2 thac sy duoc hoc va mQt diroc sy chuyen khoa cdp 1.:. C6 12 sinh vien lam cong trinh t5t nghiep phuc vu cho nQi dung d~ tai, MQt s6 sinhvien sau khi t6t nghiep da phat huy t6t chuyen mon a noi cong tac (vien kiemnghiem thuoc, phong kiem nghiem xi nghiep, trung tam ki~m nghiem cac tinh,thanh) .

:. Htrong d a n nhieu sinh vien tham gia thuc nghiem khoa hoc phuc vu cho dS tAicok~t qua, trong d6 co 3 nh6m sinh vien tham gia bao cao tai hQi nghi khoa hoc tu6itre cua tnrong, 1 sinh vien tham gia bao cao hQinghi diroc dong duong,

2. Ap dung vao thjre tiin sin xuA t va dM sang xi h{)iK~t qua nghien CUu cua chung toi Iii co tinh kha thi va c6 y nghia thuc ti6n, diroc

vi~n kiem nghiem va m{>ts6 trung tam kiem nghiem img dung de kiem tra chdt hrongcac thuAc da thanh phfut dang hru hanh tren thi tnrong. MQt s6 chirong trinh dirocnghien cuu la de ap dung lam tieu chuan cho cac co so. MQt s5 chirong trinh dangdi roc cac co so tham khao d~ xay dung tieu chuan cho san phAro cua minh.3. Dauh gia thVC hifn di tai dai chi@u v6i di CVO'Dgughien ctiu di dtrqc phe

duyfta. tu d{j hodn thdnhDS tai hoan thanh dung ti~n dQvS thoi gian dang k Y .

b. ThVChifn cdc mflc tiiu nghien cu-uDa hoan thanh ddy dU theo muc tieu va cac nQi dung nghien ctru dllQ'ChQi dangkhoa hoc cua be>phe duyet, con thuc hi~n them diroc 1 cong tlnrc so voi dS cuong.Cac chi tieu khoa hoc dBra trong dS cuong duoc ton trong va thirc hien ddy duo

c. Kit qua l(lo ra so v6i dfl"kiln cua ban td cumrgCac k~t qua thu diroc hoan toan phil hop voi du kien.

d. Banh gia v;?csir dpng kinh phiDa sir dung kinh phi dung muc dich, hieu qua, chi tiBn dS tai theo dung qui dinh,

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7/94

_ r ~ 1 J _ 4 ~r . _ q _ , ! ! _ ~ 4~~ ~ _~ U I l ! ~ _ 1 } _ 4 ! _ ! ! _ t i _ ~~q_~~_!?!~ .4. Cae y kiin d~xuit.:. Chung t6i se tiep tuc nghien ci'ru d~ hoan chinh va rn a rQ ng hem tron g nhtrn g d tai

khac, t iSp we danh gia ca c phirong phap, qui trinh v a k~ t qua thu duoc; irng d1 J1 1 gnhieu h em t ro ng thuc t i ~n d~ co co sa d~ nghi dua rnQ t s6 chuyen luan thu6c dathanh phftn vao duoc d iS n V iet Nam trong I a n xud t b a n too.:. T iSp tuc nghien ciru, jrng dung h ai p hu emg phap dAxay dung cho n hiu cong thircthuoc da thanh phAn khac, d~c biet la plnrong phap qua ng ph6 illngoai p ha n tich dacA u illMCA. Ti~ p tuc rn a rQ ng kSt qua nghien C U u cho mot s6 thuoc co nhieuthanh phful hoa t chdt hem .

: . R A t rn on g ducc S 1 , l ' quan tam cua B Q Y t~ va cac co quan de d tai co t h e duoc apdung rQ n g ra i hem va rn a rQ ng cho nhiu s a n phAro khac .

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8/94

_ ! _ _ _1 ? . f i_ ~ _ ~ 4 ~ ! ! ~ .2. .... " x X. ? APHAN B. NQI DUNG BAO CAO cm TIET KET QUANGmENCUuDE TAIcApBQ

1. H~T vA N D E1.1. Tinh cAp thi~t cin nghien Ctlu cua d~UdTrong nheng nam gAn day, i : J mroc ta, thi wang thuoc dang phat trien nhanh ca v~s a n xudt v a kinh doanh. Cac thudc ngoai duqc cAp sd dang I c y hru hanh ngay cangnhieu, Theo th6ng ke cua C\lC quan Iy Duoc Vi~t Nam, hien da co khoang 6 nghin m~thang thu6c trong nuoc va khoang 4 nghin thu6c ngoai nh~p (iroc tinh co tren 800 hoatchAt) dang hru hanh tren thi tnrong Viet nam. Trong s6 do, cac thu6c da thanh phfm dB:va dang chiem m{>tt Y Ie cao.

D~ ph6i hop tac dung diroc Iy trong m{>tch pham thu6c va ti~n sir dung cho benhnhan, thu6c da thanh phfm thirong duqc ph6i hop cac hoat chAt trong cung m{>tnhomthuec nhir: cac khang sinh, cac vitamin, cac thu6c ha nhiet giam dau, thuoc s6t ret,thu6c chong lao V.v ... Cling co th~ kt hQP cac hoat chAt cua 2, 3 nhom thu6c nhirvitamin voi thu6c ha nhiet, khang sinh voi thu6c chong viem, thu6c nho mAt h6n hQP,thu6c dung ngoai hem hop v.v ...

Noi chung, tren the gioi, cac thu6c co nhieu thanh phfut dang diroc san xuAt va sirdung ngay cang nhieu. Nhu v@yviec nghien ciru phirong phap kiem nghiem cac thuocda thanh phfm 1 f t m{>tviec rAt cfm thiet d6i voi cac nha san xuAt, cac nha nghien ciru vkiem nghiem va cac co quan quan Iy chAt hrong thuoc,

Trong diroc di~n Viet Nam moi nhAt (D.D.V.N. III), hau nhu chua co cac chuyenIu~ ve thuec nhieu thanh phAn. Vi viY, vi~c nghien ctru d~ dira m{>tsf, tieu chuan chocac thu6c loai nay vao duoc di6n trong cac Ian xuftt ban toi lit rAt cfm thiet.

XuAt phat tir yeu cau thuc tS va cAp thiSt do, chung toi dii tiSn hanh nghien ciru d~tai:"Nghi2n CNU .mg dpng phrmng phap sdc kj long hi?u niing cao (HPLC) va doquang phD UV- Vis ttl lIinh tinh va djnh lurmg cac hopt chat trong m{jt sa thuac co

til'2din 5 thanh ph8n"

1.2. M\lC tieu nghien Ctlu.:. Xay dung qui trinh phan tich m{>t s6 thu6c da thanh phfut dB: chon (chua co tieu

chufut hoac co tieu chudn nhimg chua dAy du hoac co nhuoc di6m), dira trenphirong phap HPLC va phirong phap quang phd t lr ngoai phan tich da cAu tlr MeA.

:. Ap dung cac qui trinh tren d~ ki~m nghiem cac thuoc da thanh phAn co cong thtrcttrong 1 \ 1 ' , gop phdn nang cao chAt hrong thucc hru hanh tren thi wang.

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9/94

_ ! . . . _ _ l ? . ~ ~ _ ' ! 4 1 J _ ! ! ~ .:. Muc tieu nghien C U l l cu th~Trong pham vi 1 d~ ta i d.p bo chung toi chon mot s6 cong thirc thu6c c6 ill2 den 5

thanh phlin d~ nghien ciru (da diroc bOphe duyet), d6la cac thuoc han hQ'Psau:- Thu6c c6 2 thanh phfuI

+ Paracetarnol va cafein. ..., t 4 : _ 1A f M L C ~ M L+ Paracetarnol v a ibuprofen. {Ul,lL-t t-~~: [L~ t r ) C < U C W - [ ( -'\l 1L { )+ Paracetamol va quinin sulfat. (L ' ~ t \A ~ ~ f ' { _ c . . . A ) _

- Thuoc c6 3 thanh phdn+ Paracetarnol, clorphenirrarnin malea t va phenylpropanolarnin hydroclorid.+ Theophyllin; ephedrine hydroclorid v a Phenobarbital.

- Thuec c6 5 thanh phan+Paracetarnol; cafein; pseudoephedrin hydroclorid; brornhexin hydroc1orid vaclorphenirrarnin maleat.

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10/94

_ ~ _ _ ! _ 4 ~ g _ q ~ l ! _ , ! _ ! ~ ~ _ ~ ~ ~ ~ _2. TONGQUANTAILIBU2.1. Tinh hinh nghien eu-u trong va ngoai nwe

Trong nhtrng nam qua, a Vi~t Nam ta dd co nhi~u cong trinh nghien crru a cAp cos a va cAp bQ v~ ki~m nghiem cac thuoc da thanh phfin nhir:- Nghien ciru dinh lUQ11gd6ng thai cac vitamin tan trong mroc va vitamin tan

trong dAu trong thuec h6n hop multivitamin bang phuong phap HPLC [10].- Nghien cuu i rng dung quang ph6 UV-VIS , quang ph6 dao ham de din h hron g

cac thuec da thanh phfin [7; 8].Nghien ciru dinh hrong dang thoi cac hoat chAt trong mQt s6 thucc ha nhietgiam dau bang phirong phap HPLC [6;] ...

Tuy nhien, so voi tdc dQ phat tri~n cua thi tnrong thudc hien nay thi chua du dapimg, con rdt nhieu nQi dung cfin phai duqc nghien ciru d~ img dung trong thirc tin.

Tren the gioi, cac ch phfun thuoc da thanh phfin duqc nghien ciru rAt nhieu, nhirngphan 160 chi dirng a mire tieu chucln co s a .

Trong cac duoc di~n 160 va thong dung nhir BP, USP, EP, JP, Phap, Qudc t [19;23; 24; 25; 27; 28] ... chu yeu ghi cac dang thudc dan thanh phan,

Diroc dien Anh (BP1998 v a BP2000) co ghi mQt s 6 thuoc da thanh phk nhir cacchuyen lu~ thudc multivitamin, thuoc tiem lidocain va adrenalin ...

Rieng duoc di~n M y (USP 23, USP 24, USP 27 ... ) co sd thudc da thanh phfin nhieuhon va t~p trung vao mQt s6 nhom thuoc ph6 bien nhir: cac vitamin, nhom thudc hanhiet giam dau ...

Cac mroc trong khu V\lC Dong Nam A dd co nhtmg hQi nghi v~ cac phuong phapkiem nghiem ngoai diroc di~n. T6 clnrc Y te the gioi cling dd co nhtrng chirong trinhd~ t6 chirc hoc t~p va trao d6i gitra cac nuoc v~ vdn d~ nay.

2.2. TAng quan vi eac phuong phap phan tieh2.2.1. Phu01Ig ph tip siic kj long hifU nang cao (HPLC)a. K hai ni?m sac k Y long hi?u nang cao

La mQt phtrong phap tach hoa ly dua vao ai hrc khac nhau cua cac chAt khacnhau voi hai pha luon tiep xuc va khong dang tan voi nhau, mQt pha dQng va metpha tInh.

Pha dQng 1 (\chAt long chay qua cQt voi mQt t6c dQ nhdt dinh.Pha tinh chua trong cQt 1 (\mQt chAt rAn dd d irec ph an chia duo; dang ti~u phan

hoac mQt chAt long phu len mQt chAt mang rAn hay mQt chdt mang dd duoc biBn dbibfutg lien k B t hoa hoc voi cac nhom htru ca.Qua trinh site k Y x~y ra co th~ thea cac co ch: hftp phu, phan bd, trao dbi ion

hay ray phan ttl.-6-


11/94

_ ? _ . _ x ' _ 4 ~ g q ~ q _ ' ! _ ~ ~ ~ X ~ ~ 1 } _ .b. Cdc ilQiltamg ilgc trung c o o q u a trinh s A c 1 0 /.:. Thbi ,ian lltu v a thi tich lltuThai gian hru cua mQt chAt la thai gian tinh til Iuc tiem mdu vao cQt d~n khi chAt

do ra khoi cQtdat gia tri nAng dQcue dai va cho pic tren sAc k Y dA_N~u goi tRla thoi gian hru gift cua me)tchAt

thi : t'R = tR - t oTrong do: t'R hi.thoi gian hru thuc (thoi gian luu hieu chinh).

10la thai gian ch~t (thai gian khong hru gift).Trong cung mQt di~u kien sAc k Y e ta chon, thai gian hru cua m6i chAt la hang dinh

va cac chAt khac nhau thi thoi gian hru khac nhau t u y thuoc vao ban chat, cAutao vatinh chAtcua chAtdo. Vi v~y thai gian hru la d~i hreng dinh tinh cac chat,

Khi pha dQng chay qua cQt v m mQt t6c de)khong d6i thi thai gian hru co th~ thayth~ bang th~ tich hru. Th~ tich luu la th~ tlch pha dQng thu duoc sau cQttrong khoangthai gian nrong irng voi thoi gian hru,

_ _ _ _ _ _ _ _ _ _ _ ~ . 6 . W . _ _ _' ' ' . 1

- - - - - - - - ...- - - - - - - . - ..~ - - ; ~ ~ - - - - - -- - - - - - - - - ..--.~--~.~;I

lIOl;l ..... :i" - - - - - - - - ; ~ ~ - - - - - - - - - - 1 . I. . . . . - - - - - . . _ .. _ - _ .. . _ - - - _ . . . .- . ._ - _ . _ . _ - . . . . . . . . - - . . _ - - - - . . . . . . . . . . - . . . . . ~, ~ . : t . .Hinh 1. siic ky db cua hai chAtva cac thong s6 d@ctnrng

rIA .K hA b.f L Ie so p an 0Trong qua trinh sAc I c y luon co su phan b5 cua chAttan gitra pha dQng va pha tinh.

S\!,phan b6 nay dtroc d~c trung boi can bang phan b6 voi h~ s6 phan b6 duoc tinh theocong thirc sau:

Trong d6:K= Cs

CMCs, e M la nAng de)cua chAtphan tfch trong pha dQng va pha tinh 6thai di~m can bang.

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12/94

_ L r 4 ~ g _ p ' ! ' f ! _ , ! _ ~ ~ ~ ! . ~ ~ ! 1 . . _.:. Thira s a dung /U'(!IIg

Thira s3 dung IU Q 'Ilg lit d ~ i IUQ ' Ilg bi ~ u thi khll n ang phan bb cua chdt tan trong ha ipha cQng v a i s trc chua cua cQ t, n rc lit t Y s A g itra IUQ 'IlgchA t tan tron g pha tinh v a IUQ 'IlgchA t ta n trong pha dQng a thoi diem can bang

k'==Qs = =Kx VsQM VMTrong do Qs, QM li t hron g chA t tan co trong pha tin h v a pha dQng.

V s - V M li t the tich pha tinh v a pha dQng.C 6 the tlnh k' thea mQ t cong tln rc khac:

.:.Hi s A ehvn leeHai chdt chi diroc ta ch ra khi chung c6 cac gia tri k' khac nhau, h~ s b chon IQ c cho

bi~ t hieu qua tach cua h~ thbng sA c k Y .KB r, I RB -10 I'RBa==-==-= = = -KA k'A IRA -10 I'RAo day ta quy uoc chA t B bi hru gift m anh han chA t A, nhu v~ y a lu on 16 '1 1h a n 1, a

ca ng 1 6'1 1hi kha n ang t ach cua ha i chA t C Mg ro. Tln rong phan tich trong dieu kien atrong khoang 1 ,5 dn 2 . : . S a ilia IVthuv2t v a ehi2u eao ilia IVthuv2t

H ieu hrc cQ t tln ron g diroc bieu thi qua ha i thong s6: s e rn a Iy thuyet (N) hoa c chieucao dia ly thuyet (H ). C Qt sA c ky diroc coi n hu co N tA ng Iy thuyet, a m6i tA ng su phanb6 chA t tan v ito ha i pha la i d~ t den mQ t tran g tha i can bang moi. M6i tA n g dUQ 'C g illdin h n ln r mQ t la p pha tinh co chieu cao H . D fa ly thuyet dircc dinh nghia nhu lit mQ tkhu V \lC cua h~ thong phan tach m it trong do thit l~ p mQ t can bfutg nhi~ t dQng hocgina n6ng dQ trung bin h cua chA t t an trong pha tin h v a trong pha dQng.

86 dia ly thuyet N diroc t in h theo ca c cong thirc:N = =16X ( ! ! L ) 2 hoac 5,54X ( _ ! L _ ) 2W ~/2

W : C hieu r{>ngpic a da y pic.rong do~/2 : C hien rQng pic do (] m ra chieu cao pic.

C hieu cao cua dia Iy thuyet dugc t in h thea cong tln rc:H = ! : : . .N

Trong do L la chieu cao cua C Qt sA c ky,V6'i m Qt dit~ u kien sA c k Y nhA t din h, chieu cao dla 1 9 thuyet (H ) v a s6 dia 1 9 thuyet

(N) lit hang din h d6i v oi m6i chA t phan tfch.

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13/94

2. T6nq quan tai lieu. ..__..0'._ __ __~ _..__ _ __ __ __ __ _ L. : . B e Dhlin cidi (R~DQphan giai la d~i hrong bieu thi dQtach ella cac ehAtra khoi nhau trong mQt di8u

ki~n sitc I c y da cho. DQ phan giai ella hai pic canh nhau diroc tinh theo I trong 3 congthirc sau:

s, = 2 x V R B -tA)WA+WB1,18 x { t R B - tRA }W;'2(A) +WII2(B) ho~c

a-I k'--x Ba l+k' B- I Nx-4

.:. He sa dJi xfmg (VCho bit mire dQcan d6i cua pic sAcley, n6 duoc tinh theo cong thirc:

T=Wz2ATrong d6 Wx: la dQrQng day pic do a 1120chieu cao ella pic.

A : la khoang each til dirong vuong g6e ha til cue df;licua pic dn chandirong cong phia tnroc, 6 ta i 1120chien cao pic .

:. PhU'dng trinh Van-DeemterPhuong trinh Van-Deemter mo til anh huang ella tac dQ dong pha dQng va cac

thong s6 dQng hoc khac d~n hieu hrc cua CQtsAck YBH=A+-+Cxuu

H : Chi8u cao rna l y thuyet.u : Tac dQdong pha dQng.A, B, C : Cac h~ s6 thay d6i phu thuoc vao tirng CQtsAck Y .Trong d6: A: M6 U l a nh huang cua su khuech tan xoay (eddy diffusion).

B/u: M6 ta a nh huang cua su khuech tan doc cua cac phan t i T chAttan theo phirong dong chay cua pha dQng (longitadinaldiffussion).C: M6 ta a nh huang cua S1)' chuyen khai (mass transfer).

c. H~ th6ng HPLC: H~ thang may HPLC c6 cac bQph~ chinh sau:~ Binh chua dung moi (pha dQng).- Bam cao ap: dAypha dQng qua CQtsAc ley.- B(>phan tiem mdu: tiem vao cQtmQtthe tich mdu nhAt dinh,- CQt tach (pha tinh).- Detector.- May ghi tin hi~u hoac may vi tinh.

-9-


14/94

_ ? __ ! 4 ~ g _ ! J ! I _ l } _ , ! _ ! ? ! ~ _ ~ ~ ~ ~ - - - - - - - - - - - - - .

------- _-

Hlnh 3. Cac thanh phfut co ban cua mQtmay s&ck Y long hien dai

1_Khay chua binh dung moi (Solvent cabinet).2. BQdu6i khi chan khong (Vacuum degasser).3. B am cao ap (Pump).4. B{} tiem mdu 1 \ 1 ' d{}ng(Autosampler).5. BQdi~u nhiet cho cQt(Columm compartment).6. Detector.7. Module di~u khien (Control module).

Hlnh 2. h~ th6ng HPLC hoan chinh

d. Pha tinh.:. Khdi nimPha tinh trong HPLC leichAtnh6i cQt d lam nhiem vu tach mQt h6n hop chAt

phan tich, N61A nhirng ch~t rAn,x3p, kich tlnroc hat rAtnho, dirong kinh ca hatill 3 - 10um, dien tich b~ m~t hat thuong ill 50 ~500 m2jg .

:. Phiin loai- Can cu theo ban chAtchinh cua qua trinh sAc k Y trong cQttach, ngiroi ta chia n6thanh nhieu loai nhu hApphu, phan b6, trao d6i ion va ray phan t i r o Tirong irngvoi loai chAt nh6i nhu th~ nguoi ta co mQt loai sAc k Y rieng trong ky thu~tHPLC.

- Can cu theo trang thai rAn hay long cua pha tinh, ngtroi ta chia n6 thanh hailoai, n~u pha tinh hi chAtrfuI ta c6 sdc k Y long-ran (LSC), n~u pha tinh la chdtlong ta c6 sdc I c y long-long (LLC).

- Can cu theo de) phan C\lC cua pha tinh va pha dQng, c6 cac loai: sAc I c y phathuan (pha tinh phan C\lC con pha dQng it phan eire), sAcky pha dcio(pha tinh itphan cue con pha dQng thi phan cue), sdc k Y pha dao tao c~p ion va sAc I c y traod6i ion.

- Can cir theo c~u tnic x3p cua pha tinh la cac hat rAn,nguoi ta chia n6 thanh hailoai Ia xop toan phan ha t va xop b 8 m~tha t (xop chi la p v6 ngoa i).

- 10-


15/94

_ ? _ _ ! 4 ! ! g ! l _ q _ ' ! _ ! ? ! ~ _ ~ ~ f _ .

:. lJiiu kien d6i vu; mOt pha tlnh- Phai tro va h6n vtrng voi cac di6u kien cua moi tnrong sitc k Y .- C6 kha nang tach chon IQcmQt h6n hop chdt tan nhdt dinh trong di~u kien s~c

k Y nhdt dinh,- Tinh chAtb8 mat phai dn dinh (d~c biet l a ~c trung x5p cua no),- C a n hang dQng hoc cua su tach phai xay ra nhanh va I@plai t6t.- c o - hat phai wong d6i dAng nhAt.

e. Pha dong.:. Khai niemPha dc)ng la dung mol dung de rna giai chAt tan (chdt cAn phan tich) ra khoi cQttach d~ thirc hien mQt qua trinh sAc k Y _ Day la mot yu t6 rAt linh dQng va d dangthay d6i. N6 c6 th~ la mQt dung moi hoac h6n hop nhieu dung moi trQn Ifill voinhau thea nhtrng t Y I~ nh~t dinh. N6 cling c6 th~ Ia dung dich cac mu5i c6 chua cacchAtd~m, chAttao phirc ... N6i chung m6i loai sitc k Y se c6 cac he dung moi rita giairieng dS c6 duoc hi~u quit phan tach t5t nhAt.

.:. Qua Irinh rira giiiiRihl giii ding dong: pha de)ngkhong thay dbi trong su6t qua trinh nra giai.Rira giai gradient: pha dQng lien tuc thay d6i (do t Y I~ cua cac thanh phAn tao nenpha dQng thay d6i) trong su5t qua trinh rna giiti. Luc nay, de)phan cue cua phadc)ngcling se hi bien d6i va thuong la tang hi~u quit tach .

:. Cae ylu t a ehinh can chu V Irong lila chen pha ilOng- B an chAtcua dung moi lira chon la m pha dQng.- Thanh phAn cac chAt tao ra pha dQng.- T6c de)dong pha dQng.- pH cua pha de)ng (d~c bi~t chu Y i J sAck Y trao d6i ion va sitc k Y cap ion) .

:. lJiu kien iliii vUi mot pha dong- Phai tro dAi voi pha tinh,- Hoa tan diroc chAtcAnphan tich,- B~n vtmg thea thei gian.- C6 de)tinh khiet cao.- Phai nhanh dat cac can bang trong qua trinh sAcky,- Phil hQ'Pvoi Ioai detetor dung d~ phat hien cac chAtphan tfch.- C6 tinh kinh t va khong qua hiem .

:. Pha dQng la mQt trong nhtrng yeu tA quyet dinh hieu suAt tach sAcky cua mQt hanhQ'Pm:u.N6 quyet dinh thai gian hru gift cac ch~t mau va hien qua su tach sic k Y oPha dQng c6 th~ anh huang dn:- DQchon IQccua he pha.- Thai gian hru gift ella chdt tan.

- 11 -


16/94

_ ? _ _ _ ! 4 ! ! g _ _ q ~ q _ , ! _ ! ? ! ~ _ ~ ~ f . ! 1 . .- H ieu 19c cua CQttach.- DQphan giai cac chAt trong mQtpha tinh.- Do r(lng va S\I can d6i cua pic sAck Y .

:. Tdt ca cac dung moi dung trong HPLC (k~ ca pha d(lng) d~u phai duoc dU6i khibang each dun nong, hilt chan khong, Hic sieu fun . .. Vi khong khi hoa tan trongdung rndi se 4 1 0 bot khi trong detector va gay nhiBu tin hieu.

f each ilanh gia pic.:. Dank gia dien tick pic: Dien tich pic cua mQt chAtnrong (mg voi tlmg IUQ11ghAtdo. D~ tinh dien tich pic, hien nay ngtroi ta thuong dung may tich phan di~n illgAnvol may vi tinh (sai s6 khoang 0,5%) hoac may tich phan co hoc (sai s6 khoang1,3%). Phuong phap nay co th~ dung cho cac pic khong bi troi duong n~n va ca picco dirong nen b] troi. Phuong phap nay chi d.n diem dau va diem cu6i cua pic dtrocnhan ra chinh xac va cho k~t qua tat d6i voi nang dQtrung binh, vira va cao .

:. Danh gia chiiu cao pic: Khi pic c6 dang khong dAithi chien cao pic (khoang eachgitra duong n~n va dinh pic) lit mQt dai hrong t Y l~ voi dien tich pic va n6 ding cothe dung d danh gia sdc k Y db. Phuong phap nay chi ap dung khi cac chi s6 k' lahang dinh, Voi pic co duong n~n bi nhiBu hoac pic hep thi vi~c xac dinh chien caopic se dBdang va chinh xac hemvi~c xac dinh dien tich pic.

g. each tinh kit q u a.:. Phlrung phap ngo(li chunn: La phirong phap dua tren co sa so sanh mu chufin vamiu thir trong cung di~u kien. K~t qua cua chAt chua bi~t dircc tinh toan so vcimiu chufin da bi~t tnroc nAng dQhoac suy ra tlr direng chuan .

:. Phuung phap n6i chunn: La plnrong phap cho them vao miu chuan va miu thirmot IUQ11ghAt khong d6i, rna trong cung di~u kien sAc k Y n6 co thai gian hru gdnvoi thoi gian hru cua chAt cAnphan tich trong miu thir. N6 direc tach hoan toan vaco nang dQgfut bang nllng dQcua chdt phan tich va co cAutnic hoa hoc nrong tu.

:. Phuung phtip them chunn: Chu yeu duoc sir dung trong k Y thu~t HPLC khi c6vftn de a nh huang cua cac chdt phu (vi du : ta diroc), Dung dich miu thir diroc themmQt lUQ11gxac dinh chit chuan, Cac pic thu diroc cua cll hai dung dich miu thir vamfiu thir them chAt chufut phai duoc do trong cung mQt dieu kien slic ky, K~t quaducc tinh toan dua vao str chenh l~ch nbng dQ (luong chAt chufuJ.them vao) va dQtang cua dien tich hoac chieu cao pic .

:. Phlrung phdp tinh theo phlin tram dien tich pic: Nang dQ cua mftu thir diroc tinhtoan dua tren dien tich pic tinh thea t Y 1~phftn tram dien tich pic chAt thir tren tAngdien tich toan bQpic co trong sAck Y db. Trong HPLC, phirong phap nay chi dungkhi co S \ 1 ' dap trng cua detector tren cac chAt la nhu nhau, n~u khong nhu nhau khido v oi m6i chAt can c6 h~ s 6 h i~u chinh .

- 12-


17/94

_ ? _ _ .4 !!g_!l!l_l}_'!_~~~L~~ '!: .. .. . _2.2.2. Phuong phdp quang phD tii"ngo{1iph8n tich i Ia c8u tii"(MeA)a. Cos o l Y thuyit.:. Nguyen tdc chung

Co so Iy thuyet cua phuong phap dua tren nguyen Iy "D(j hap th quang A coo moth6n h ( 1 J J trong dung d i c h tai m6t buue song a bat 1 0 / bfmg tang do hap thu quang cuam6i thanh phim tai buac song do".

Trong dung dich, dQ hAp thu quang va n6ng dO dung dich co m6i lien he voi nhautheo dinh lu~t Lambert - Beer:

A: d{>hAp thu quang ta i btroc song A . .E: dQ hAp thu rieng tai biroc song A . .C: n6ng dQ dung dich.d: b~dAy cf,c do.

ta co th~ viet gon lai nhu sau: A =K.C (voi K =E.d duqc goi la h~ sf, t Y 1~).Neu ta gia sir h6n hop cAn do co n thanh phan thi theo nguyen ly tren ta co:

Tai buoc song thit nhcft: A l =Kl.1CI + K1.2C2 +_ . + K1 .nCn (2)Khi de>hAp thu quang duqc do tai m buoc song, ta se thu duoc h~ g6rn m phirong

trinh, voi n A n sf, v~ n6ng dQ ( Cn ) va n. m A n sf, v~ h~ sf, t Y 1~ ( Km.n )

A =E.C.d (1)

Al =Kl.lCI + K1.2C2 + + Kl.nCnA2 =K2_ 1C1 + K2.2C2 + +K2.nCn (3)

AI KI.1 K L 2 . . K I.n CIA2 = K2.I K 2.2 ". K 2.n C2 (4)

~ a 1 1 1

A m K m . l K m . 2 .. K m . n e n[A] lit rna tr~n dO hAp thu quang, diroc do tren may.[K] lit rna tr~ he sf, t Y Ie, diroc xac dinh tnroc bdng each do ph6cua cac dung dich chuan h6n hop d hiet tnroc n6ng dQ.[C] la rna tr~ n6ng dQ cua cac thanh phAn trong h6n hop,

D~ co th~ giai duoc phirong trlnh (4) ta can phai chuyen rna tr~ ehu nh~t [Klm.nthanh rna trin vuong bang each nhan voi rna tr~ chuyen vi cua K.

Gia sir m > n, khi do ta co th~ viet phirong trinh (4) dum dang sau:

Trong do:

A m.1 =[Klm.n c., (5 )Nhan ca hai ve cua phirong trinh (5) voi rna tr~ chuyen vi cua K lit [K']m.n ta diroc

- 13-


18/94

2. Tone quan fai li~u______.__0._._ _ .,. ---- ----_.,.-or - .. - - - - - - - - - - - - - - - -- - - - _

---+ [Z]n.l = [T ]m .n . C n. tTrong thirc te cua bai toan, C lit nang dQ chua hiet cdn tim cua rndu tlnr, v~y ta c6

th~ viet phirong trlnh tren duoi dang nhir sau- -1Cn.t= [T] m.n . [ Z ]n .l (6)

Nhir v~y, d~ xac dinh nbng de>chua hiet cua rndu tlnr, diSu quan trong la ta cdn phaigiai ducc phirong trinh (6) dua tren viec xac dinh rna tr~ h~ s6 hAp thu [K]m.n thongqua do de>hAp thu A m.t :. Gitii he Cremer bdng ohufTIIg ohao Gauss

Me>t h~ phuong trinh dai s6 tuyen tinh chi tim diroc nghiem, khi s6 plurong trinhcua h~ 100 han hoac bAng s6 A n (nghia lit ta phai c6 m ~ n). Trong wang hop t6i thieuvoi m = n, khi d6 ta se c6 h~ n phuong trinh n A n va rna tr~ [K] lit rnQt rna tr~ vuong(kich cO'n x n), day chinh l a he Cremer quen thuoc trong dai sf, tuyen tinh. D~ giai loaih e nay ta dung phirong phap Gauss.

Xet h~ phuong trlnh Cremer sau:al.1x] + aI.2x2 + + al.nXn= b,a2.lx] + a2.2x2+ + a2.nxn=b2

co rna tr~ h e sf,A=

au a u . . . a 1 . 1 1a 2 . l a2.2 a2.n (7)

Phuong phap Gauss la phuong phap giai bang each dung cac bin d6i sa cAp d~dua he da cho vS he tam giac co dang:

a1.1xI+ a1.2x2+ + al.nXn=bIa2.2x2+ + a2.nXn=b2

co rna tr~ h~ sf,

A=au ~.2 a 1 . no a 2.2 a2.no

(8)o a n .1I

Voi gia thiet ~.m ;f 0, khi do viec giai he tam giac tren rAt don gian.Tir phuongtrinh cu6i ta se tim duoc xn, th len cac phirong trinh tip tren tim ducc Xn-l, Xn.2 ... vaXl a plnrong trinh dAu tien .:. Phlin dch chuan. danh gia chat lup?y! cda phlin tich chuan bdng cach ap dung

thull binh phufTllg tAi thiiu va cdc thOng k2 tUfTllg quanThuat binh phtrong tf,i thieu (least square method) se lam cho dQ dung trong viec

phan tich chuan cao hon vi n6 la m giam thieu S\l' sai khac gitra n6ng dQ tlnrc (enteredcon cen tra tion ) v a nAng de>tin h toa n duqc (ca lcula ted con cen tra tion ).

- 14-


19/94

_ ? __ ! . _ 4 ~ g _ q ~ f ! _ , ! _ ! ~ ~ _ ~ ~ ~ l } _ .Gilt thh~ts6 hreng cac h6n hop chuan duoc sir dung Ia p. Theo dang rna tr~n cua h~

(4) 6' tren,Chung ta d bit muon xac dinh duoc rna tr~ he sA [K] cua h6n hop gAmn chAtthi

ta phai sir dung t6i thieu n dung djch han hop chuan va do tai tbi thieu la n biroc song(rn = n = p) de co n2 phuong trinh xac dinh rn9t nghiem duy nhAt cua h~ gAm n2 phclntir cua K,Vi du: muon xac dinh cac h~ s6 K cua h6n hQP gAm 2 chAt, ta phai sir dung tbi

thieu 2 dung dich h6n hop chudn va do rn~t d9 quang ta i t6i thieu 2 buoc song khi dota se co 2 x 2 =4 phuong trinh xac dinh cac gia tri cua h~ s6 t Y Ie Km.n,cu the nhtr sau:

T ai b iro c song Idung djch han hop chuan 1: Al =Kl.lC1 +K1.2C2dung dich han hop chuan 2: A2 = Kl.1C3+KI.2C4

Tai buoc song 2dung dich h6n hop chuan 1: A3=K2.IC1 +K2.2C2dung dich h6n hop chudn 2: ~ =K2.lC3+ K2.2C4

Tren tlnrc t, ta co t h e giam sai s6 trong phan tich chuftn bang each ap dung nguyenly binh phirong tbi thieu "su dung sa /U(Y11gcac dung dich h6n hap chudn nhiJu han s6cdu tU (p>n}", khi do he tren se tro thanh ~~heren xac dinh- overdetermined system".V~ nguyen lAc,de giai loai h~ nay ta dua v~ viec giai h~ Cremer, khi do m6i In chungta chi c a n dung gia tri phan tich cua n dung dich chuan trong s6 p dung dich sir dung (p> n), v~y chung ta g~p bai toan t6 hop ch?p n cua p phn t i r o Theo cong thirc tinh t6hop ta co: e n = p!

P nL(p-n)!, e n e nKet qua la ta se co p tnrong hop, nghia la ta se co nrong ung p h~ Cremer va(9)

thu duoc e; nghiem, m6i nghiem gAm n2 gia tri nrong img voi n2 phcln tir cua he sf>t Y I e K .

Vi d\1: vfut voi tnrong hop han hop co 2 chAt 6' tren, d~ phan tich chuan chung ta seT ~ , , c:dung 5 ChUMhon hop. Neu gia thiet chi do tai hai btroc song thi se co 5 = 10

tnrong hop va se co 10 gift tri rna tr~ chuan hoa K, co nghia la ta dd phai giai 10 hephuong trinh, mai he c o 4 phuong trinh d~ xac dinh 4 A n s6 la cac h~ s6 t Y Ie K cua 2chAt tai hai buoc song.Til cac gift tri K nay, tinh duQ'c rna tr~ trung binh [K]tbva se xac dinh duoc nongdQcua m6i t h r u m phn trong dung dich chuan h6n hop theo cong thirc:

[C ] = [K]tb-1.[A] (10)- 15 -


20/94

_ 7 . . _ _ ! . _ 4 ! ! g _ ! l ! ,: l ! _ _ ,! _ ! ~ ~ _ ~ ~ ~ 1 } _ ._ _ . . _ . _ . _So sanh cac gia tri C nay voi gia tri nAng de. rna ta pha ban dAucho m6i thanh phAn

trong h6n hop ta se tinh duqc S\l' sai khac gitra nAng de. tinh diroc a tren va nAng dQthirc cua cac dung dich chuAn h6n hop, Sau do, ta co th~ dung cac thong ke nrong quand~ danh gia sai sa cling nhu chAthrong dtrong chuan .:. Phlin tich mflu Ihir va ap dung Ihu{il loan hinh phu01Ig ta ; Ih;2u Irong phlin tich

mduthirSau khi phan tich chuan d~ xac dinh rna tr~ h~ sa t Y l~ [Kltb va chAt hrong cua no

dira vao cac thong ke da: neu, rna tr~ [Kltb duoc sir dung d~ tinh true ti~p nAng de.chua bi~t cua miu thir. Theo nguyen Iy binh phuong tai thieu, khi tang sa buoc songpharr tich se giam duqc sai sa cua phep do (giam gia tri t&ng binh phtrong cua su saikhac gitra n6ng dQtrung binh va n6ng di) cua nrng tnrong hop t&hop biroc song), vasai sa se cang nho khi sa bircc song pharr tich cang tang. Do do, d~ giam sai sa eftaphep do, chung ta se dung sa hrong cac buoc song phan tich Ian hon sa cAu tir trongh6n hop (m > n) bang each sir dung them nhieu thong tin hon cua ph&.Nhu v~y, d~ tinh n6ng dQchua bi~t cua miu thir, m6i IAnta chi cAnsir dung gia tri

di) hAp thu quang do tai n buoc song trong sa m biroc s6ng pharr tich, ta lai g~p baitoan t6 hop ch~p n cua m phfut t i r o Vfut a vi du tren, trong tnrong hop dung 6 bmrc

-" C2song pharr tich cho hon hop gem c6 hai chat, chung ta se giai duoc 6 = 15 gia tricho n6ng dQcua m6i thanh phAnva k~t qua se la gia tri trung binh cua IS gia tri rna tatinh duoc,b. Ph6n mJm cho phdn tich da ctiu tu MeATh\I'Cte, co sa Iy thuyet cua phuong phap pharr tich da cAutil (MCA) cling nhu cac

thuat toan d~ giai quyet no da:eo ti c lau, va da:c6 t h S thuc hien diroc ngay ti t khi comay quang ph&UV-Vis. Tuy nhien, do han ch~ v~ di) ehinh xac cua cac thi~t bi phantich, cilng nhu han ch~ v~ thi~t bi xir Iy sa li~u dn d~n ket qua phan tich khong chinhxac hoac co t h e do duqc chinh xac nhung lai phai kha cong phu trong viec tinh toank~t qua.Nam 1989-1990: Hang Beckman gioi thieu phAn mem pharr tich quang ph6 MCA

tren cac may quang ph6 Beckman DU thong thuong, D~ phan tich MCA tren the h~may nay, tnroc h~t chung ta phai lua chon mi)t sa buoc song pharr tich thich hop daivoi m6i thanh phArr. sau do tien hanh phan tich cac miu chufut va miu tlnr, cuai cungla xir Iy k~t qua dua vao phArrmem tren may tinh di~n nr,

C a c th~ he may quang ph6 hien d~i sau nay nhu: DU 650i, DU 7000 cua hangBeekman hay HP8452, HP8453 cua hang HP e6 su cai tien ro ret ca v~ phAn cirng vaphArrmem, VS phan cirng, cac may diroc trang bi bi) vi xir Iy tac di) cao, cAu hinhquang hoc cai ti~n. detector chu6i diot dUQ'cnbi voi bQ truyen va bien dc3idft li~u tacdi) eao, cho phep phan tich ph6 nhanh hon, tang di) nhay va di) l~p lai cua biroc song.

- 16-


21/94

_ ~ _ _r 4 ! ! g _ q ~ q _ , ! _ ! ~ ~ _ ~ ~ f . ' ! ' : . .d6ng thoi thu diroc toan be. dit li~u ph6 trong khoang biroc song da chon dung chophan tich. Su cai ti~n ve giao di~n phfut mem, dung thuat toan chuyen h6a chu6iFourier trong x u l y du li~u ph6~ cling nhir Ia ap dung cac thong ke toan hoc da lam choviec tinh tom k~t qua t r a nen dan gian han voi sai s6 t6i thieu.Hien nay a nhieu co s a kiem nghiem chua dugc trang b] cac th~ h~ may quang phA

hien dai nhu tren, vi v~y chung toi da thi~t l~p mQt phdn mem x u ly k~t qua cho phepap dung ky thuat MCA de phan tich duoc mQt s6 chcpham c6 ill2 d~n 3 thanh phfuJ.hApthu quang tren may quang ph6 thong thuong. Chuang trinh x u I y nay rna thuat giaicua n6 dua tren viec giai quyet hai bai toan chu y~u~do la: plnrong phap Gauss de khirh~ plurong trinh Cramer; t6 hQP de x u l y cac kctqua thu duQ'c til nhieu h~ Cramer (h~tren xac djnh-overdetermined) cung voi viec loai be cac kctqua qua sai l~ch theonguyen ly cua thuat binh phuong t6i thieu (least square method).

Hinh 4. Cira s6 chuang trinh phan tichda cAutilMCA.

Hinh 5. Thictl~p cac thong s6 chung

- 17-


22/94

} _ ._ P _ ~ U _ l [_ ( ! ! l_ g _ _ v _ ~ l!!!'!l!!!g p _ ~ _ q p __,!g!!~~!!: _ 0 ! _ - - - - _ _ - - _ .3. DOl TUONG vA PHU ONG PHA !> N GHIE N CUU3.1. Thi~t k~ nghien ctruf)~ tili (hrgc thi~t k~ theo mo hinh nghien cnu thvc nghi~m t~li pheng thi

nghiem,NQi dung nghien ciru:.:. Thir nghiem tim kiem ky thuat hoac giai phap d~ loai trir anh huang cua cacthanh phfut khac (k~ cft ta duoc), khai thac cac irng dung cua phuong phap s~cky long hieu nang cao (HPLC) va phirong phap quang phi, UV-Vis hien dai vanviec phan tich cac thuoc da chon .

:. Nghien ciru xay dung cac phirong phap dinh tinh va dinh hrong d6ng thai nhieuhoat chAttren cac thi~t bi HPLC va quang ph6 UV-Vis hien co.

:. Nghien ciru phirong phap xu I y mdu, khao sat tinh tuyen tinh, chirng minh bingthuc nghiem tinh dung, tinh chinh xac, ...

:. Danh gia ich lei va tinh kha thi cua phirong phap .:. U ng dung cac phirong phap da xiiy dung vao viec kiem nghiem mQt s6 thuocdang hru hanh tren thi tnrong.

D\1kien xay dung phuong phap dinh tinh va dinh luong mQt s6 thuoc co chua ti r 2d~n 5 thanh phdn, cu th~ nhu sau:

- Thuoc co 2 thanh phan:+ Paracetamol va cafein.+ Paracetamol va ibuprofen.+ Paracetamol va quinin sulfat.+ Paracetamol va acid mefenamic.Thu6c co 3 thanh phan:+ Paracetamol, clorphenirramin maleat va phenylpropanolamin hydroclorid.+ Theophyllin, ephedrin hydroclorid va phenobarbital.Thu6c co 5 thanh phdn:+ Paracetamol, cafein, pseudoephedrin hydroclorid, bromhexin hydroclorid va

clorphenirramin maleat.Mfii chuong trinh cho mQt cong tlnre thn8c se IimQt ti~n d~ tili.

3.2. Chon min, crr min v a dBi ttrQ'Dgnghien ctruChpn mau v a cOmau

Chon cac m~u thudc din hinh, co t ir hai thanh phan tra.!l9l cho cac tiu d~ tai.M6i tieu d~ tai chon t6i thiu la 2 mdu thuoc d~ nghien ciru voi s6 hrong dli dlam cac khao sat thuc nghiem,

lJtJi t lf(1 llg n g h ie n c a u C ac mau thuoc ap dun g tuy theo thi tn ron g cun g c~ p.- 18 -


23/94

_ ~ _ _ ! _ ~ U ' ! _ f { ! ! g _ _ ' : ~ l ! ! ? ' ! _ ' ! ! } g p _ ~ _ 4 P _ _ 1 J g ! ? ! ~ ! ! E ~ .3.3. Phuong phap nghien em.3.3.1. PhU'01lgphap siic kj long hi?u nang cao (HPLC)

a. Chi lieu nghien cuuCac chi tieu nghien ciru duoc mo ta 6 sa db sau.

b. Phuong phdp xac dinh cac chi lieu nghien cuu.:. L U'a chon di2u kien sac k V- Thit~t bi HPLC

V6i may HPLC ta quan tarn nhat toi detector, co th g~p mQt s6 loai detectorsau: detector hdp thu tir ngoai - kha kien (UV- Vis), detector hftp thu tir ngoai (UV),detector huynh quang (FLD: fluorescence detector), detector chiet suat vi sai,detector ion hoa ngon hra, detector hong ngoai (IR) ...

Ph6 bien nhAt lit detector UV- Vis (l90nm - 900nm) nhir: detector mang diode(DAD: diode array detector), detector do a nhieu bUGC song (MWD: multiwavelength detector), detector do a buoc song thay dbi (VWD: variablewavelength detector) ...

Trong do detector mang diode hi loai detector hi~n d~i co dt) nhay rAt cao, d~cbiet li t co kha nang quet ph6 va do nhieu buoc song cung mt)t luc, do do d6i voimot h6n hQP gbm nhieu thanh phfut ta co th chon cho m6i thanh phan mt)t bu6csong ph at hien thich hQP nhftt.- Loai ct)t dung phan tich (pha tinh): cQt cua hang nao, kich thtroc cQt ra sao, cac

thong s6 v Ioai pha tinh, cO"hat, < I Q xfip hat ...- Pha dt)ng:+ Thanh phan va t Y l~ cac thanh phfut cua pha dt)ng, pH ." n~u hi rna giai ding

dong (isocratic).+ Thanh phan va chuang trinh dung moi n~u lei r n a giai gradient.

- 19 -


24/94

_Lp~~~ l ! _ f ! ! ! g _ _ ~ ~ p ! ! l ! ' ! ! } g _ p ~ _ q p _ _ , ! g ! !~ ? ! !E~ . .Luu y: Pha d(>ng la m(>t y~u t6 rAt linh d(>ng, ta co t h d dang thay d6i dS

thu dtrcc k~t qua tach t6t nhAt bang each thay d6i tY l~ cac thanh phan, thay d6ipH ...Bmrc song phil! hien: phai thich hop voi m6i thanh phftn.

- T6c de>dong pha de>ng:khao sat d~ chon t6c d(>phil hop nhat.Th~ tich tiem: thtrong la 20Jll, 50Jll.

- Nhi~t dQ c(>tphan tich .:. Khao Slit do turin tinh CUD cae thimh phOn can phlin tich

Ta bi~t rang, nong de>cua m6i chftt phan tich deu co m6i quan h~ tuyen tinhvoi dien tich hay chieu cao pic sitc k Y (trong mQt khoang n6ng dQ nhdt dinh naodo). Nghia la trong khoang n6ng d(> do thi dien tich (S) hay chieu cao (h) picphu thuoc vao nbng d(>(C) theo mt)t ham b?c nhdt.

S=a.C + b hoach = a.C + b (a, b la h~s6).

Chung ta can phai xac dinh khoang tuyen tinh nay bang thirc nghiem tir dolam co sa d~ pha cac dung djch phan tich co n6ng dt) nam trong khoang timdiroc.

Ti~n hanh pha mt)t dung dich g6c, sau do pha loang dung dich g6c nay d~thu duoc m(>t day dung dich chuan bi~t truce n6ng dt). Bern cac dung dichchuan nay chay s~c k Y d~ thu duoc gia tri dien tich hay chien cao pic, l?pphirong trinh h6i qui, daub gia dQ tuyen tinh thong qua h~ sf>nrong quan r .

:. Chu8n hi cae dung dich phlin tichTrong phtrong phap HPLC, thong thtrong ta tinh k~t qua bang each so sanh

mau chuan va mftu thir trong cung dieu kien, Do do, ta din chuAn hi cac dungdich chuan va cac dung dich thir cho qua trinh sitc ky .

:. Khao sat tinh thich h{!JJclio he thAngYeu cftu a buoc nay la phai kiem tra xem cac di~u kien site ky ta da lua chon

co thich hop khong? Ta thtr chay me>tmftu phan tich vai dieu kien sitc ky dachon, neu cac pic tach khoi nhau hoan toan, pic nho, gon la duoc .

:. Khao sat do chinh xtic CUD phU01lg phapBe> chfnh xac tlnrong duoc xac dinh thong qua d(> l~p lai cua phirong phap

(each thong thirong nhdt Hi xac dinh d(>lech chuan nrong dbi cua k~t qua phantich RSD - theo cac duoc di~n hoac sai s6 nrong d6i).

Tien hanh khao sat d(> l~p lai bfing each dinh hrong rieng biet tren nhi~umau gi6ng nhau ella chat ca n phan tich r6i xu Iy ket qua bang phirong phapthong ke,

- 20-


25/94

} . _ _} _ ~ U l ! ' _ f ! ! 1 : g _ _ ~ ~ p ! ! ' ! _ ( ! ! ! Z p _ ~ ! P _ _ , ! g ~ ! ? ~ _t ; _ ~ _ _ _- 0.:. Khao sal do dung cua phuO'ng phap

De>dung duoc xac dinh thong qua kha nang thu hbi cua k~t qua phan tichkhi dung phirong phap them chuan,

Them vao mdu thir met hrong ehinh xac ehdt chuan da bi~t tnroc ham hrongsao eho dung dich eu6i cling co nbng d{>nk trong khoang tuyen tinh da khaosat, xac dinh phan tram tim lai,

3.3.2. Phuong phap quang phD phon tick da cdu tIC(MCA)a. Chi lieu nghien ctruCac chi tieu nghien ciru duoc mo til a sa db sau.b. Phuong phdp xac dinh cac chi lieu nghien cUu

.:. Khao sat Dhb hap thu UV v a Iua chon cac huuc song. hoac gil'; song phontlch

.:. Xac dinh khoang nang do phon tlch

.:. Chuan hi day dung dich chunn hDn h(!fJ

.:. Do dOhap thu quang cua cac dung dich chuan han hem

.:. Xoy dU'ng. danh gia. tai uu duirng chunn hdng phOn m2m tTen may tlnh

.:. Phon Ilch mau thu

.:. Danh gia do dung v a do chinh xac cua phuO'ng phapPhdn nay tuong 1 \ 1 nhu d6i voi phirong phap HPLC.

[Kh aO sa t phA hAp t 1 1 \ 1 UV va IvachQn elk btr6c song, ]hoac giai song phan tich. 0

J

CO ]

)

- 2 1 -


26/94

_L_f!~U'!_(!:!g_ l ! ~ l! ! !~ ,!?,!gp~_qp:_ng~~~ !!_ ,!_~ . _3.4. Phuong phap xir I y s6 Ii~u- Danh gia phirong phap phan tich va cac k~t qua phan tich: sir dung phuong phapthong ke, str dung cong cu h6 tro lit chuong trinh Microsoft Excel.Str dung phuong phap h6i qui tuyen tinh, thuat binh phirong t6i thieu d~ danh giachuan va thir,

Cac 49c trung th8ng k2 r e xu Ij kit qua phiin tich:+ Gia tri Trung binh:

1 nx : ; : ; : - "'x~ in i-I

+ DQ lech chuan (standard deviation): n-1

+ Sai s6 chuan:+ DQ lech chuan tuong d6i (relative standard devitation): RSD (%) = ~ x 100x

- 22-


27/94

. 1 : . ' . E ! . q ' ! _ q . ! ! & ~ ! ? _ ' } .~ 0 . : t . . _ _ . . ' . . . . . . . . . _4. KET QUA NGHI"EN CUu4.1. Djnh tinh v a djnh hr4]llg paracetamol, bromhexin hydroclorid,

pseudoephedrin hydroclorid, clorpheniramin maleat v a cafein bing phuongphap HPLC

Vien nang ha nhiet, giam dau, chua ho "Cotussin" do cong ty diroc pham TienGiang nghien diu san xudt c6 cong thirc nhu sau:

- Paracetamol (PA) 280 mg- Cafein (CA) 30 mg- Pseudoephedrin hydroc1orid (PE) 10 mg- Bromhexin hydroc1orid (BH)- Clorpheniramin maleat (CM)

84

mgmg

Trong cac U t i li eu tham khao, chua thdy san pham nao c6 cong thirc nhu tren,Vi v~y de giup cac co s6 xay dung tieu chuan cho san pham cua minh, chung toi

dl1nghien C U u phirong phap dinh hrong 5 hoat chdt tren trong vien h6n hop bangphuong phap HPLC voi di~u kien sitc ky long pha thuan, detector UV va chuang trinhsAcky d~ng dong.

A. Th.,c nghi~mA.1. May moe, dung miJi, hoa chata. May moe dung C{l

- May site k y long hieu nang cao HP 1100 voi detector UV - VIS.- Ce>tLichrosorb Si 60 (250 x 4 mm; 5 urn).- May lite sieu am.- Can phan tich voi de>chinh xac 0,1 mg.

b. Hod chdt dung moi- Methanol tinh khiet site k y o- Dung dich d~m Amoni nitrat pH 9,5 (884 ml mroc cho them 94 rnl Amoniac

d~m d~e (25 - 28%) va 21,5 ml acid nitric (69 - 71%), khuay d~u, d~ nguoi,f)i~u chinh pH dn 9,5 bang acid nitric hoac Amoniac. LQc qua mang loc 0,45urn.

- Dung moi A: h6n hop Methanol: Nuoc (1 : 1).

A.2. Phu01Jg phapa. Dieu kien sac l e Y

- M ay sac ky long hieu nang cao HP 1 100 - Hewle tt Packa rd .- 23 -


28/94

4. Kit aUG nehien cUu_ __ __ . .. .~ . . ~ : :: :l . . . . . . . , ._. _0': _ .._...... .. .. .._.. .... ._ ..... ..__ .._T- CQt Lichrosorb Si 60 (250 X 4mm; 5 urn),- Detector UV - VIS: 257 nm do PE va CM; PA; 298 nm do CA va BH.- Pha dong: Methanol- Dung dich dem Amoninitrat pH 9,5 (45 : 6)- T6c dQ dong:

1,5 ml/phut do PE va CM.1,0 ml/phut do CA va BH.1,0 ml/phut do PA.

- Th tich tiem: 20 ul,

b. Cdc dung dich H em s d c k Y.:. Dung dich thir

- Dung dich thu dS dinh luong PA: Can chinh xac mQt hrong bQt vien tucmg irngvoi 30mg PAvao binh 50ml, them 30ml pha dQng, u ie khoang 10 phut, thempha dQng d~n vach, l~c d~u, IQc qua gifiy IQc thuong. Hut 2ml dung dich trencho vao binh dinh mire 20ml, them pha dQng toi vach, l~c d~u, sau do IQc quamang IQc 0,45Jlm.

- Dung dich thir dS dinh hrong CA, PE, BH va CM: Tron d~u va nghien minhrong bQt vien lfiy tir 20 nang. Can chinh xac mQt hrong bQt vien nrong irng voi2 vien, them 30 ml pha dQng, l~c khoang 10 phut. Dung 30 mi mroc d chuyenh6n hop nay vao binh gan. Them 10ml NaOH IN, l~c ky rbi chiet bangCloroform 3 IAn x 30ml. GQP dich chiet, b6c hoi tren each thuy d~n h~tCloroform. Hoa tan c~n trong 20mI dung dich HCI 0, IN rbi them methanol viradu 200mI, l~c d~u. LQc qua gifiy IQc thuong, sau do IQc qua mang loc 0,45 urn .

:. Dung dich chufut- Dung dich chuan d dinh hrong PA: Pha chfit chuan PA trong dung moi pha

dong d thu ducc dung dich PA co nong dQ chinh xac khoang 30J..lglml.- Dung dich chuan d dinh hrong CA, PE, CM va BH:

+ Can chinh xac khoang 40mg CM, hoa tan trong pha dQng vita du 25ml(dung dich CM)

+ Can chinh xac khoang 40mg BH, hoa tan trong pha dQng vira du 25ml(dung dich BH)

+ Can chinh xac khoang 560mg PA, 60mg CA, 20mg PE, cho vao mot c6c como, them chinh xac 5mI dung dich CM, 10mI dung dich BH, 15mI phadQng, khuay cho tan h~t rbi xir Iy ti~p nhu mau thir, tir doan " dung 30mimroc d chuyen h~t h6n hop nay vao binh gan ... "

Tinh k~t qua: Dtra vao dien tich hoac chieu cao cua pic chuan va t lnr tirong (mg.

- 24-


29/94

4. Kh q _ u a nghien cuu_ _ _ _ _ _ _ _ _ _ __ . __ _ . . . _ . . LB. K~t qua v a bifn lu,n

V6i chirong trinh s~c ky da:neu, s~c ky d6 da:tach cac chat CA va BH; PE va CMnhu hinh 1.1 va hinh 1.2.

80-~


30/94

~~ .~~4! .q ' !_q~g_~ !~ '}~~ .. ~ ~~~~~ ~ " .~ ~ "~" ~.~ ' .ducc b6c hoi tren each thuy d~n kho, hoa tan cin trong 5ml dung dich HCI O,lN themMethanol vim du 20ml, rbi do HPLC theo chU011g trinh tren. K~t qua thir tren 3 binhd~u khong xUdt hien pic tren s~c ky db. Nhir v~y, hoat chdt dll dircc chi~t h~t 6 3 IAndAu. Kha nang chi~t duoc dam bao,B.3. Dp I~p l(1i, sai s a v a dp dungCac thir nghiem nay ducc thirc hien tren mfiu vien Cotussin cua cong ty DirocphAm Ti~n Giang, SKS: 071199. K~t qua diroc gioi thieu trong bang 1.2.

Bang 1.2. K~t qua khao sat dQ l~p lai va dQ dung tren mftu COTUSSINs A li~u t h A n g ke Thanh phanPA CA CM PE B H

GiA tri trung binh X ( mg I vien ) 278,4 29,05 4,38 9,21 7,26D C ) l~ch chuan S 0,63 0,38 0,08 0,17 0,15DQl~ch chuan nrong d6i 0,23 1,31 1,83 1,85 2,07Ty l~ tim l{lidiroc ( %) ( n =3, hrong chuan 99,0 99,1 98,5 99,0 98,4them vito 20% m6i chat)

K~t qua khao sat cho thy dQ l~ch chuan nrong d6i tuy thea nrng tnrong hop tir0,23 - 2,07% va t Y l~ tim lai diroc hoat chdt tir 98,4 99,1%.


31/94

4. Kit nUG nehien cuu.. ~.. ::t _.." _.._0_ .... _.. .... .... ..._ .... .. N . _ . _ . . . . . . _ . . . - - . . - _4.2. Binh hrong dAng thm Paracetamol (PA) va Acid mefenamic (AM) trong vien

nen bao phim Pamesic bing phmmg phap HPLC

Vien bao Pamesic do cong ty v~t tir Yt~ Ti~n Giang nghien ciru san xuit co thanhphan nhir sau:

Paracetamol (PA) 500mg.Acid mefenamic (AM) 250mg.

Trong cac diroc dien hien hanh [19; 23; 27; 29 ...] chua co chuyen luan v~ haithanh phan nay. Hai chit tren co de) tan nhir nhau (d~u d6 tan trong kiem) nen rftt khochiet tach. Vi v~y chung toi da:nghien CUu phuong phap dinh tinh va dinh IUQ11g6ngthai eel2 thanh phan tren trong h6n hop thuoc voi muc dich cling co s a xay dung tieuchuan cho san pham maioA. Th\l'c nghifmA.I. Thi~thi, hod chtit

- May sAcky long hieu nang cao HP 1100 voi detector UV -Vis.- C9t Lichrosorb RP18 (250x4 mm; 1011m).- Be)IQcdung moi va IQCmftu voi duong kinh 16mang IQc0,45 11m.- Chftt chuan paracetamol va acid mefenamic do Vi~n Kim nghiem - BQY t

cung cap.- Acetonitril, tetrahydrofuran loai dung cho HPLC.- Acid phosphoric tinh khit phan tich,

A.2. Phuong ph tip nghien caua. fJiJu kien sac I c Y

- CQtLichrosorb RPI8 (250x4 mm; 10Ilm).- Detector UV d~t a buoc song 279 nm.- Pha dong: Acetonitril - mroc - tetrahydrofuran (3 : 8 :9) dieu chinh dn pH

2,2 bang acid phosphoric.- T6c d9 dong 1ml/phut,- Th tich tiem 20111 .

h. Chu{m bi dung dich- Dung dich chuan: Can chinh xac khoang 0,1 g chdt chuan PA va khoang 0,05

g chit chuan AM, hoa tan bang 5 ml THF, sau do pha loang ba.ng dung moipha dong d diroc dung dich chuan co n6ng dQ cua PA khoang 80 ug/ml vacua AM khoang 40 ug/rnl,

- Dung dich thir: Can 20 vien, tinh kh6i IUQ11grung binh vien, nghien thanh be)tmin va tron deu. Can chinh xac met luon g bO t vien nrong (rug voi kh6i I U Q ' l 1 g

- 27-


32/94

4. Kit aua nehien cuu__ __ __ __ _ :::l 0 ~ ............__--....r _ . - - -_ . - -- - - - _ . - -- - . _ . - _ . -_ . - _ . - - - - _ .cua khoang 50 mg AM, hoa tan bang 5 ml THF, pha loang bang dung moi phadng d~ duoc dung dich thir co nong de>cua PA khoang 80 ug/ml va cua AMkhoang 40 ug/ml lac du. LQc qua mang IQc 0,451J.ffi.

B. K~t quaB.1. Khao sat tinh thich h{1JJcua h? thang sAc kj

V6i diu kien s~c ky du chon nhtr tren, s~c ky db thu diroc a hinh 2.1 cho thayPA va AM diroc phan tach hoan toano

mAU N16014012010080604020o~----~~--------~--~--~

Hinh 2.1. S~c k Y db thu duoc cua dung dich hem hop PA va AMB.l. Khiio sat khoang tuy2n tfnh

Chung t6i ti~n hanh khao sat de>tuyen tinh gitra neng dQ cac chat d.n dinh hrongvoi dien tich pic tuong irng tren cac chftt chuan PA va AM. Chuan bi me)t day dungdich h6n hop chuan trong pha de)ng c o nong de) cua PA bi~n Thien trong khoang 20 -200 ug/rnl va cua AM bien thien trong khoang 10 - 100 ug/ml, k~t qua khao sat de)tuyen tinh diroc trinh bay a bang sau:

Bang 2.1. Ket qua khao sat de>tuyen tinhSTT PA AMNang d


33/94

4. Kit q ua ng _h ien c uu___ .~ . __ _ ~ T ~ _ r L L+~ -+_ - - - - - - - - - - - -+ ~+- - - -- - --- .... - - --- --. ----.

Paracetamol

45004000 50003500 400030002500 300020001500 20001000 1000500

0 00 50 100 150 200 250 0

Acid mephenamic

50 100 150 I

Hinh 2.2. D b thi bieu di~n S\l phu thuoc tuyen Hinh 2.3. D b thi biu di~n su phu thuQC tuyentinh nA n g dQ v a dien tich pie wang irng cua P A tinh nA n g dQ v a dien t ich pie n rong irn g cua AM

Nh,n xet: Trong khoang nbng de>da khao sat co SI! nrong quan tuyen tinh gifradien tich pic va n6ng dQ voi h~ s6 nrong quan r~t gful 1 (r = 0,9996 - 0,9999). Chungt6i chon n6ng dQ dung dich thir va chuan d~ dinh IUQ' I lg nim trong khoang tuyen tinhda khao sat (P A khoang 80 ug/ml va AM khoang 4 0 ug/ml).B.3. Khao sat 49 /(ip /{li cua phuang phdp

DQ l~p lai cua phuong phap diroc ti~n hanh tren hai miu thir vien nen bao phimPamesie. K ~ t qua khao sat de>l~p h i l i diroc trinh ba y (] ba ng sau:

B a n g 2.2a. K ~ t qua khao sat dQ l~p lai tren miu vien nen bao phim PamesieMJu 1SKS: 011102PA AMSIT Ham hrong (mg/vien) So lieu thong ke Ham hrong (mg/vien) So lieu thong ke

1 511,79 247,542 508,46 - 247,75 -3 506,95 X = 506,08 248,50 X =248,554 504,15

S =4,21250,78

S = 1,22RSD%=0,83% RSD%=O,49%5 499,32 247,75

6 505,83 248,97

- 29-


34/94

: t _ _li~~~~q_! . ~ ~ _ i _ ~ ' ! .< : ~ - - - - - - - - - - - - - - - - - - - - - - - - - - _ . - _ . - - - - - - - - - - - - - - - - - - - - - - - . - - - - - - - - - - - - - - - - - - - - - - - - . . . . - - - - - - - - - - - - - - - - -Bang 2.2h. K et qua khao s a t dQl~p lai tren miu vien nen bao phim Pames ic

Mau 2 SKS.- 021202PA AMSIT H a m luong (mg/vien) So lieu thong ke H a m hrong (mg/vien) So li~u thong ke

1 498,21 255,022 495,07 - 252,31 -3 503,25 X = 495,61 250,63 X = 252,884 488,13 S =6,75 252,55 S = 1,465 487,30 RSD%= 1,36% 253,68 RSD%=0,58%6 501,71 253,08

Nb,n x tt : K et qua tren cho thA y phirong phap co dQ l~ p laicao v6i dQ l~ ch ch ua ntuong d6i khoang tir 0,49 - 1,36%_B.4. Khdo sat 4 9 dung

K hao sa t dQ dung bang phuong phap them , them vao m iu thir da : hiet wac hamhron g m 9t luong chinh x ac chA t chudn tuong img v6i khoang 20% hrong co trong m iuthir, K et qua khao s a t de>dung ducc trin h ba y i J bang s au :

Bang 2.3. K et qua khao s a t dQdungPA AMSIT Luong them Luong tim thay Luqngthem Luong tim thay

vito (mg) (mg) % tim la i vito (mg) (mg) %timl~1 20,32 20,09 98,9 9,99 9,76 97,72 20,32 19,97 98,3 9,99 9,77 97,83 20,32 20,25 99,7 9,99 9,88 98,9TB 98,9% 98,2%

Nhin xet: PhU 01 lg phap co de>dung cao vei kha n ang tim lai cua PA la 98,9% v acua AM la 98,2%.

- 30-


35/94

4. Kit q ua nehien cUu_________.. 0 .. ..__ ____ .. __ ____ _ .4.3. Binb hrqng dAng tbm paracetamol (PA) v a cafein (CA) trong thu6c da thanh

phAn bing phmrng phap HPLCThu6c da thrum phdn bao g6m paracetamol va cafein la mot dang ph6i hop thong

dung, vi du vien Pasepan cua CTDF Ha Tay hay vien Sedapa ella XNDFTW 2./ ;D e dinh tinh va dinh hrong cac hoat chat trong h6n hop thuoc tren thirong apdung theo tieu chan cc sa (chiet tach r6i do quang) [2,16]. Phuong phap nay co nhiroc

di~m la phai qua biroc chiet tach kha phirc tap, d e m~c sai s6, t6n nhieu thai gian, dungmoi va hoa chat,

Chung t6i dff nghien ciru mQt chuang trinh HPLC dan gian va tach t6t hanchuang trinh dff de c~p trong cac tai lieu tham khao [29] de dinh tinh va dinh hrongd6ng thai 2 thanh phdn tren,A. Thuc nghi~mA.I. Hoti chiit, thiit b i

- May s~c k Y long hieu nang cao HP 1100 voi detector UV.- CQtLichrosorb RPI8 (250x4 mm; lOf-Lm).- Be>IQcdung moi va IQcmftu voi dtrong kinh 1 6 mang IQc0,45 urn.- ChAtchuan paracetamol va cafein do Vi~n Kiem nghiem cung cftp.- Methanol loai dung cho HPLC.- Ethanol tuyet d6i (PA), acid phosphoric (PA).- Dung moi A: Ethanol- mroc (1:1).

A.2. Phuong phtip nghien cuua. DiJu kien sac I c y

- CQt Lichrosorb RP18 (250x4 mm; 10f-Lm).- Detector UV di.\t6 buoc song 273 nm.

Pha dong: Dung dich acid phosphoric 0,75% trong h6n hop dung moimethanol - mroc (35:65).

- T6c dQdong 1ml/phut,- The tich tiem 20f.11.

h. Chudn hi dung dich- Dung dich chuan: Chuan bi mQt dung dich h6n hop chuan trong dung m6i Aco nong dQcua PA khoang 600 ug/rnl va cua CA khoang 20 ug/ml.

- Dung dich thir: Can 20 vien, tinh kh6i hrong trung binh vien, nghien thanh bQtmin va trQn deu. Can chinh xac mot hrong bQt vien urong img voi kh6i hrongcua khoang 60 mg PA, hoa tan va them dung moi A d~n vira du 100 ml, IQc ?--ua giAy IQcthirongfbo khoang 20 ml dich IQcdu). Sau do IQcqua mang IQc0,45f.1m.

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4. Kit qua nehien cUu._.. .. ....0 .... ...... .. .. - - - -__....-- - - - -_..-- --- -- ....._....-- -- -.......- -- -- --- -- -- -- -- _..-- --- -- .. 150 ~ 750 10 ~ 50tuyen tinh dff khao satPhuong trinh hoi quy Y = 15,2x + 154,9 Y =46,4x + 29,3H~ so tuong quan r = 0,9995 r= 0,9996

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4. Kit nUG nehien cuu_ _ _ ::t O~ .+ T __ - - - T _ - - -~ _ - - _ . _ - - - - - - - - - - - - - - - . - - - - - - - - - - . - - . - - -

Paracetamol1 5 0 0 0 2 5 0 0

2 0 0 010000 1 5 0 05 0 0 0 1 0005 0 0

0 00 2 0 0 40 0 60 0 800 0

Cafein

20 40 60

Hinh 3.2. D b thj bi~ u di~ n S,! phu thuQc tuyen Hinh 3.3. D b thj bi~ u di~ n su phu thuoc tuyentinh nbng dQva dien tich pie tirong ling cua PA tinh nbng dQva di~n tlch pie tuong ling cua CAB.2. Kit qua khao sat i/{j 19P19;

DQ l~p lai diroc khao sat tren hai miu vien nen SEDAPA do XNDPTW II sanxudt c6 thanh phfut:

Paracetamol:Cafein:Ta duoc:

Mltu 1 SKS : 350900Mltu 2 SKS : 361000

300 mg.10mg.vira du 1 vien

Bing 3.2. K~t qua khao sat de>l~p lai tren miu SEDAPA. MJu 1 SKS: 350900PA CASIT H a m hrong (mg/vien) So li~u thong ke H a m luong (mg/vien) So li~u thong ke1 291,3 9,762 288,1 - 9,52 -X = 288,4 X - 9,643 288,6 (mg/vien) 9,66 (mg/vien)4 287,9 S = 1,52 9,57 S = 0,095 287,7 RSD%=0,53% 9,63 RSD% = 0,93%6 286,9 9,71

Bing 3.3. K~t qua khao sat dQl~p lai tren mu SEDAPA, Mdu 2 SKS: 361000PA CASIT Ham hrong (mg/vien) So li~u thong ke H a m hrong (mg/vien) So li~u thong ke

1 301,81 9,712 301,36 - 9,83 -X = 302,62 X - 9,833 303,30 (mg/vien) 9,82 (mg/vien)4 305,40 S = 1,54 9,86 S = 0,095 302,37 RSD%= 0,51% 9,97 RSD% = 0,92%6 301,44 9,75

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4. Kit qua nehien C U l l_________ _ 0.: .. .. ~__..__ ...... .. ...... .. - - - - - - - - - .._....- - - _..- _...__.."- _.....< . . - - _ .K~t qua khao sat dQ l~p lai a tren eho thdy sai s6 tirong d6i til 0,54 den 1,07%,

voi sai s6 nay eho phep ap dung duoc phirong phap tren.

B.3. Kit qua khao sat dp dungKhao sat dQ dung tren hai ma.u SEDAPA a tren bing phuong phap them chuan,

kt qua diroc trinh bay a bang 3.4 va 3.5.Bang 3.4. Ket qua khao sat dQdung tren ma.u SEDAPA, Mau J SKS : 350900PA CA

STT Luong them Luong tim Lugng them Luong tim % tim laiVaG (mg) thdy (mg) %timl~ vao (mg) thay (mg)

1 20 20,2 101,0 0,30 0,305 101,72 20 19,6 98,1 0,30 0,293 97,83 30 30,1 100,3 0,60 0,600 100,04 30 29,4 97,9 0,60 0,582 97,05 40 40,1 100,2 0,90 0,896 99,56 40 39,1 97,7 0,90 0,884 98,2TB 99,2 % 98,9%

Bang 3.5. K~t qua khao sat dQdung tren mdu SEDAP A, MJu 2 SKS : 361000PA CA

SIT Lugng Luong tim Luong them Luong timthem VaG % tim lai % tim laithdy (mg) vao (mg) thdy (mg)(mg)I 20 20,1 100,4 0,30 0,300 100,12 20 20,3 101,3 0,30 0,298 99,43 30 29,6 98,5 0,60 0,592 98,74 30 29,7 99,0 0,60 0,595 99,15 40 39,7 99,3 0,90 0,886 98,56 40 39,5 98,8 0,90 0,898 99,8TB 99,5 % 99,2 %

Kt qua tren cho thdY phftn tram tim lai cua PA va CA tren hai mfiu dat tir 98,9d~n 99,5 %, phirong phap c o dQdung t6t.

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4. Kit aua nehien elm___ .. .A. __o~ .,,____ _..,_. _ __ __ __ ____ __ -__ -- -- -- - .4.4. Nghien Ctlu dinh hrQ'llg dAng thM paracetamol (PA) v a ibuprofen (IB) trong

thu6c da thanh phin bAng phmrng phap HPLCMQt trong nhtrng dang thong dung tren thi tnrong hien nay la h6n hop

paracetamol va ibuprofen (vi du: vien nen Alaxan cua xi nghiep United Pharma Vi~tnam [II], vien nen Pi-Antalvic [5] cua cong ty Diroc v~t nr Y t~ Tra Vinh, vien nenMK protamol cua cong ty cb phan Duoc pham M e Kong ...). Diroc diSn Vi~t Nam clingnhu cac ducc diSn khac hien nay dSu chua co chuyen lu~n cho dang bao ch nay. Muctieu cua chung toi la nghien ciru mQt s6 phirong phap dinh hrong paracetamol vaibuprofen co trong thanh phk mot dang thuoc voi muc dich lam phong phu them vadanh gia cac plnrong phap eho cac C < Y s a hra chon ap dung. D6ng thai mong duoc gopph~ xay dung cac tieu chuan chdt IUQl1gella nganh cling nhu cua QU6c gia cho cacdang thuoc co thanh phan nrong nr.A. Th\lc nghi~mA.1. Thi2t hi, hoa chat

- May sAc k Y long hieu nang cao MERCK-HITACHI 655A-12 voi detector UV-Vis 655A-22.

- CQtLichrosorb RP 18 (250x4 mm; I urn),- BQ IQcdung moi va IQcm~u voi dirong kinh 16mang IQC0,45 urn,- Ch~t chuan paracetamol va ibuprofen do vi~n kiem nghiem cung c~p.- Diethyl ether, ethanol vc'', natri sulfat khan, dung dich NaOH O,IN va O ,O lN .

A.2. Phllung phap nghiin cuua. DiJu kien s d c k Y

- CQtLichrosorb RP 18 (250x4 mm; 1 0 J lm ) .- Detector UV d~t a biroc song 224 nm.- Pha dong: Acetonitril- acid phosphoric 0,1% (60:40).- T6c dQdong 1 ml/phut,- Th~ tich tiem 20Jll.

b. Chudn bi dung dich- Dung dich chuan : Chuan bi mQt dung dich h6n hop ehuAn trong pha dQng co

nong dQella PA khoang 65 ug/ml va cua IB khoang 40 ug/ml,- Dung dich thir: Can 20 vien, tinh kh6i IUQl1grung binh vien, nghien thanh bQt

min va trQn d~u. Can chinh xac mot hrong bQt vien nrong irng voi kh6i hrongcua khoang 32,5 mg PA, hoa tan va them pha dQng d~n vira du 50 ml, IQc (bokhoang 20 ml dich 19 C dfiu). Hut chfnh xac 2,0 ml dich IQctren cho vao binhdinh mire 20,0 mI, them pha dong d~n vita du, I~c d~u. LQc qua mang loc0 ,45J lm.

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_( l! i !_ q~4_ lJ .g~ j~ '! _c : ~ - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -B. K~t qua

Voi di~ u kien s~ c ky da chon nhu tren, cluing t6i thu diroc s~ c ky db cho thely PAva IB duoc phan tach hoan toano

.rHinh 4.1. S~ c k Y db thu diroc cua dung dich h6n hop PA va IB

B.l. Khao sat khoang tuyin tinhChung t6i ti~ n hanh khao sat dQ tuyen tinh gitra nA ng dQ cac c he lt cAn dinh hrong

voi dien tich pic nrong irng tren cac chelt chuan PA v a IB_ Chuan bi mot day dung dichhem hQ 'P chuan trong pha dng co nong dQ cua PA bi~n thien t rong khoang 26 - 130ug/ml va cua IB bien thien trong khoang 16 - 80 ug/ml, k~t qua khao sat dQ tuyen tinhduoc trin h bay a bang sau:

Bang 4.1. K~t qua khao sa t dQ tuyen t inhPA IBSTT Nbng dQ(ug/ml) Dien tich pic N6ng dQ(ug/ml) D ien tich pic

1 26 1970 16 9582 52 3628 32 15213 78 5475 48 21484 104 7168 64 27115 130 8922 80 3356Khoang nong dQtuyen tinh dll khao 26 ~ 130 16 ~ 80satPhuong trinh hoi quy Y = 67,lx + 199,4 Y = 37,4x + 343,0H~so tuong quan R = 0,9999 R = 0,9997

- - ...-------- - --_._._-- --.~---Paracetamol

10000 l + J . " ~ > " , C ) " : : : i [ : ' " ' , L > - " ' ~ ~ ~ ' _ ; " ; : ; " F iC ' : " % 8000600040002000o

o 50 100 150Hinh 4.2. D 6 thi bieu din sir phu thuoc tuyentinh n6ng dQva dien tich pic n ron g im g ella PA

Ibuprofen4000300020001000

o6 020 40 80 1 00

Hinh 4.3. Db thi bieu dien SlJ phu thuoc tuyentinh n6ng dQva di~n tich pic nrong irng cua IS

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_ ~ _ _ ! : 4 ! _ q l f _ 4 _ ~g~!~,!~~- ---- ----------------------------------------------------------------'---------------------.Nb,n xet: Trong khoang nA ng de) da khao sa t co S 1 , 1 ' n ron g quan tuy~ n tinh gitra

d i~n tich pic v a nA ng de) voi h~ s6 nrong quan rA t gA n 1 (r = 0,9997 - 0,9999). Chungtoi chon n an g de) d un g dich thu v a chuan d~ dinh hrong n ~m tron g khoang tuyen t inhda khao s a t (PA khoang 65 ug/ml va IB khoang 40 ug/ml).B.2. Khdo sat a p lpp l{li ella phuang phap

D


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4. Kt qua nehien cUu___..,,_.___ _ .._ o~ .......... ................ .... - - - - -- - - - - - - --_ . _ ..- -- - - - -_ ..........- - - - - - - - - - _ --- - - - - - - - - - - _ .4.5. Djnh hrqng dang thei paracetamol (pA) va quinin sulfat (QS) trong vien bao

phim Antigrip F bAng phuong phap HPLCTren thi tnrong hien co mQt s6 che pham nhu vien AntigripF va Tiphagrip cua

CTD-VTYT Ti~n Giang (Tiphaco), vien Anogin quinin cua CTD- VTYT Tra Vinhph6i hop paracetamol va quinin sulfat dS di~u tri cac clnrng dun s6t, nhirc dau, dauminh may. Vien nen Antigrip F co cong thirc nhu sau:

Quinin suifat 30 mgParacetamol 150 mgTa diroc vd 1 vienD e dinh hrong cac thanh phan nay, h.uh~t cac diroc dien [1; 15; 19; 27; 29J chi

co phirong phap d6i voi ch~ pham don thanh phfut, con voi cac ch~ phAm da thanhphan thi tieu chuan cua cac nha san xuAtdira ra rdt khac nhau: Phuong phap quang ph6hApthu tir ngoai-kha kien, phirong phap chuan de>moi tnrong khan, phuong phap acid-base. Cac phirong phap nay thtrong phai chiet tach phirc tap, t6n nhieu thai gian [3].

Vi v~y chung t6i tin hanh nghien ciru dinh tinh va dinh hrong dAng thaiparacetamol va quinin sulfat trong vien nen Antigrip F bang phuong phap HPLC voidi~u kien sAck y pha dao tao cap ion.A. Th,!c nghifmA.I. Thit hi, hod chat

- May sAcky long hieu nang cao HP 1100.- Cot Lichrosorb RP 18 (250x4 mm; 10J.lm).- Be>loc dung moi va loc mdu voi dtrong kinh 1 6 mang lQC 0,45 urn.- ChAtchuan paracetamol va quinin sulfat do Vien Kiem nghiem cung cAp.- Methanol loai tinh khiet s~c ky,- Acid acetic 4%, diethylamin, natriheptansulfonat.- Dung moi: Ethanol - mrcc (1: 1)

A.2. PhU'lIng ph tip nghien cua. DiJu kien s d c k Y

- CQtLichrosorb RP 18 (250 x 4 mm; Sum).- Detector UV: 235nm.- Pha dQng: Ll g natriheptansulfonat trong 1 lit h6n hop methanol-acid acetic4%-diethylamin ti I e (650: 350: 1).- T6c dQdong: Irnl/phut.

- ThS tich tiem: 20f.!1.- Nhiet dQphong.

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4.Kit aua nehien cUu. _ . Or A .._.. Q~...........__.,. ... ... ... ..._... ... ... ... ..._... ... --- -_...- _...--...- ---_...-----_......_...b. Chudn bi dung dich

- Dung dich chuan: Chuan bi mot dung dich chuan cua h6n hop hai thanh phantren trong dung moi ethanol - mroc (1: 1) co n6ng d


44/94

4. Kit aUG nehien cUu. ::t O~ .. ..~.. - - - ~~T ~ - - _ . - + _ .. - - ~ .

Paracetamol15000 2500200010000 1500

5000 10005000 0

0 50 100 150 200 0

Quinin sulfat

10 20 30 40

Hinh 5.2. Db thi bieu di~n su phu thUQC tuyen Hlnh 5.3. Db th i bieu di~n S\lphu thuQC tuy~ntinh nbng dQ va dien tich pic nrong img cua P A tinh nbng dQ va dien tich pic nrong img ella QS

Nh,n xet: Cac k~t qua tren cho thdy, trong khoang ndng dQ khao sat, c6 sunrong quan tuyen tinh gifra gia tr i dien tich pic va ndng dQ (r = 0,9999). Nong dQ dungdich chuan va dung dich thu cua chung toi nk trong khoang tuyen tinh dii khao sat.B.2. Kit qua khaoStit dp /(ip /(li

DQ l~p lai cua phuong phap diroc tien hanh tren mdu thtr la vien nen bao phimAntigrip F, k~t qua khao sat dQ l~p lai duoc trinh bay a bang sau:

Bang 5.2. K~t qua khao sat dQl~p laiSIT PA QS

Ham luqng (mglvien) So lieu thon_g_e Him lugng (Il!glvien) So li~u thong k e1 153,12 28,362 149,60 - 28,17 -3 150,06 X = 150,60 28,10 X -28,664 148,79 S = 1,66 28,88 S = 0,525 149,89 RSD% = 1,10% 29,01 RSD% = 1,81%6 152,15 29,45Nb,n xet: Phuong phap c o dQ l~p lai eao voi dQ lech chuan nrong d6i 1,10 - 1,81.B.3. Kit qua khaosalll(j dung

Khao sat dQ dung bAng phuong phap them, k~t qua diroc trinh bay 6 bang sau:Bang 5.3. K~t qua khao sat dQ dung

PA QSSIT Luqng them Luong tim % tim 1~ Luqng them Luong tim % tim 1~V 3 . 0 (mg) tMy (mg) vao (mg) tM_y(mg)1 7,5 7,43 99,0 1,5 1,47 98,02 7,5 7,38 98,4 1,5 1,48 98,63 7,5 7,48 99,7 1,5 1,52 101,3IB 99,0 99,3

Nb,n xet : Phuong phap co de>dung cao voi kha nang tim lai cua PA la 99,0% v aella QS Icl99 ,3% .

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_ ~ _ _ f ~ ! _ q ' ! _ q _ ! . l . g ~ j ~ r : _ ~ - - - - - - - - - - - - - - - - - .4.6. Binh hrQ'llg dang thot theophylin, epbedrin bydroclorid v a phenobarbital

trong thuAc han hQ'Ptr] hen suy~n bing phuong phap HPLC

Tren thi tnrong co nhi~u loai thuoc chua hen co 3 thanh phan theophylin,ephedrin hydroclorid, phenobarbital nhir Etadasmyl, Tiphasmyl (C6ng ty DuQ'C va v~ttu y te Ti~n Giang); Asmin (Xi nghiep Duoc pham trung irong 25); Dasmin (Xi nghiepDiroc pham trung trong 2); Asmalin (Xi nghiep Diroc pham trung irong 5); Antasma(C6ng ty DuQ'C va v~t illy t~ Ben Tre); Tedralas (Labomed - Phap), Trong cac Dirocdi~n hien co a Viet Nam va met s6 tai li~u tham khao khac chi co chuyen luan kiemnghiem cac dang bao ch~ don thanh phdn cua cac hoat chat tren va phirong phap dinhhrong co th~ la: do m~t dQ quang, dinh IUQ1lg trong m6i tnrong khan, plurong phapchuan dO.acid - base, phtrong phap so mAu. Nhir v~y muon dinh hrong cac thanh phantrong h6n hop thi phai thirc hien vi~c chiet tach plnrc tap, Hon nita, S l , C chenh lech v~hrong gitra cac thanh phn rat Ian trong do hrong Phenobarbital rat nho him cho ktqua kho chinh xac phu thuoc nhieu vao nguoi thtrc hien.

)~ khAc phuc nhiroc di~m nay, chung t6i da tin hanh nghien ciru phuong phapdinh hrong d6ng thai Theophylin, Ephedrin hydroclorid, Phenobarbital trong thuoch6n hop bang phuong phap sic ky long hieu nang cao.

A. Thtre ngbi~mA.I. Thiit hi, hoa chat

- May sic k Y long hieu nang cao MERCK-HITACHI 655A-12 voi detector UV-Vis 655A-22.

- CQt Lichrosorb RPl8 (250x4 mm; IOum).- Be) IQc dung m6i v a IQCmdu v oi d iro ng kinh 16 rnang IQc 0,45 urn.- Cac chat chuan theophylin, ephedrin hydrochlorid va phenobarbital do Vien

K i em n ghiem cung cAp.- Methanol loai dung cho HPLC.- Natri heptansulfonat tinh khit sAc ky.- Dung dich d~m phosphat pH 3,5: hoa tan 12 g NaH2P04 trong 1000 ml rnroc

c f U , di~u chinh v~ pH 3.5 bang acid phosphoric. LQc qua mang IQc 0,45 urn.

A.2. Phuong phap nghien cu-ua. ou kien sac kf- Ce)t Lichrosorb RP18 (250x4 mm; 10llm).

- Detector UV dij.t a biroc s6ng 240 nm.- 41 -


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__ q l } _ l ! _ ~ g _ ~ ! ~ l } _~0f .- Pha dQng: Hoa tan 1,0 g natri heptansulfonat trong 1 lit h6n hop cua methanol -

d~m phosphat pH 3,5 (38 : 62).- T6e dQ dong 1 ml/phut.- Th~ tfeh tiem 20J.!1.

b. Chudn bi cac dung dich tiem- Dung dich chuan: Hoa tan cac chAt chuan theophylin, ephedrin hydrochlorid vaphenobarbital trong h6n hop dung moi ethanol - mroc (70 : 30) d~ duoc dungdich h6n hop chuan co nbng dQ cua theophylin khoang 1,6 mg/ml; cuaephedrin.HCI khoang 0,2 mg/ml va cua phenobarbital khoang 0,1 mg/ml.

- Dung dich thir: can 20 vien (d6i voi vien bao dirong phai rica h~t lap vo bao cuavien, sAy a 50C trong 1 gio rbi moi dem can), tinh kh6i hrong trung binh cuavien, nghien min rbi trQn d~u. Can chinh xac mQt hrong bQt vien nrong irng voikh6i hrong cua khoang 80 mg theophylin, cho van binh dinh mire 50 ml, them15 ml mroc cAt, lAc ky, them ethanol tuyet d6i d~n vira du th~ tich, lAc d~u, IQcqua gidy IQc thuong, rbi IQc qua mang lee 0,45J.!m.

B. Kit quaV6i chuong trinh sdc ky nhu tren, tren sic ky db thu diroc cac pic tach khoi nhau

hoantoan

Hinh 6.1. SAc ky db cua h6n hop 3 thanh phan (tr (theophylin) 4,36 phut;ephedrin.HCl 7,44 phut va cua phenobarbital 12,05 phut)B.1. Khao Slit t I P tuyn tinh

Chuan bi m


47/94

4. Kit aua nehien cuu________.x 0": .._ .._ .,._ .._- - - - - -- __ -- -- -- -- .

6 05040302010o ,L_.__0~2_._~~~4 0:~

Bang 6.2. K et qua khao sa t dQ tuyen tinh dbi voi H1nh 6.3. D 6 thi bi~ u di~ n su phu thuoc tuyenephedrin tinh n6ng dQ va dien tich pic cua ephedrin

STT N on g dQ (m g/m l) Dien tich picI 0,4 957,22 0,8 1840,33 1 ,2 2851,14 1 ,6 3736,05 2,0 4 685,2Khoa ng nong dQ tuyen tinh: 0,4 - 2,0 (mg/m l)Phuong trinh h&i quy : y =2337,9 x + 8,5H~ s6 n rong quan : r = 0,9998, , ,Bang 6.1. Ket qua khao sat dQ tuyen t inh doi voi

theophylin

STT N&n g dQ (m g/m l) D ien tich pic1 0,1 1 0,32 0,2 20,13 0,3 32,14 0,4 4 2,55 0,5 55,0

K hoang n fmg dQ tuyen tinh : 0 ,1 - 0 ,5 (mg/m l)Phuong trin h h6i quy : y = 111,8 x -1,54H~ s6 tirong quan : r = 0,9993

STT Nong dQ (mg/ml) D ien tich pic1 0,05 52,52 0,10 105,03 0,15 160,54 0,20 208,55 0,25 258,3K hoang nong dQ tuyen tinh: 0,05 - 0,25(mg/m l)

Phuong trin h h6i quy : y = 1030,2 x + 2,43H~ s A nrong quan : r = 0,9997Bang 6.3. K t qua khao sat dQ tuyen tinh d6i voipheno ba rb it a l

Theophylin50004000300020001000o

o 1.5 2 2.5.5Hinh 6.2. D 6 thi bi~ u di8n su phu thuoc tuyentin h n6ng dQ va dien tich pic cua theophylin

Ephedrin.HCI

r------------ - -\ 300 Phenobarbital

25020015010050o

0.1 0.2 0.3

Hinh 6.4. D 6 thi bi~ u di8n su phu thuec tuyentfnh n6ng dQ va dien t ich pic cua phen ob arbita l

B.2. Khiio sat iI(j J (ip J {liChung toi ti~n hanh khao sat de;,lijp lai tren hai mdu vien nen va vien bao dirong:Mfru 1: Vien bao duong Etadasrnyl, SKS : 011099

Mfru 2: Vien nen Typhasmyl , SKS : 010600

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_~ -_ _! i ! _ q l } _ q _ ! ' _ g _ ~ ! ~ f } _ ~ 0 ! - - - - - - - . - - - - - . . . - - - - . , .

Chuan bi cac dung dich h6n hop chuan va thir co n6ng dQtuong ung nhu sau:Theophylin: 1,6 mg/mlEphedrin.HCI: 0,2 mg/mlPhenobarbital: 0,1 mg/ml

K ch quit khao sat dQ l~p lai duoc trinh bay (]bang 6.4 v a 6.5 diroi day

Bang 6.4. K~t qua khao sat dQl~p lai tren mftu EtadasmylSIT Ham hrong hoat chat trong mot vien (mg)Theophylin Ephedrin.HCI Phenobarbital1 41,18 7,85 2,552 41,49 7,91 2,573 41,68 7,79 2,504 41,41 7,83 2,475 41,02 8,12 2,486 40,82 7,90 2,47Tinh thong keX 41,20 7,90 2,51S 0,32 0,12 0,04RSD(%) 0,78 1,52 1,59

Ba ng 6.5. Ket qua khao sat dQ l~p lai tren m iiu TyphasmylSTT Ham hrong heat chat trong mQt vien (mg)Theophylin Ephedrin.HCl Phenobarbital1 80,02 10,67 4,952 80,78 10,72 4,903 81,04 10,40 4,914 80,46 10,71 5,005 81,10 10,59 4,886 80,31 10,52 4,90Tinh thong keX 80,6 10,6 4,92S 0,43 0,16 0,04RSD (%) 0,53 1,51 0,81

Nh,n xet: Phuong phap co dQI~p lai t6t.


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_ ~ . _ _ f : ~ ! _ q _ 1 ! _ q _ ! l _ g _ ~ j ~ 1 } _~ ._. ---- .B.3. Khiio sat i l P dung

Khao sat dQ dung bfutg phirong phap them, them vito miu thir mQt luong chinhxac chdt chuan bang khoang 20 - 30 % hrong co trong miu thir, ti~n hanh nrong nrphep thir dl) l~p lai. K~t qua diroc trinh bay 6 bang 6.6 va 6.7 dum day:

Bang 6.6. K~t qua khao sat dl) dung tren miu vien bao dirong EtadasmylTheophylin Ephedrin.HCl PhenobarbitalThem Tim % tim Them Tim % tim Them TIm thay %vao thAy vao thfty vao tim(mg) (m_g) ll;li (mg) (mg) Il;li (mg) (mg) lai20,0 19,8 99,0 3,0 2,95 98,3 1,0 0,98 98,020,0 19,5 98,0 3,0 2,99 99,7 1,0 0,97 97,020,0 20,1 101,5 3,0 2,93 97,7 1,0 0,99 99,0Trung blnh 99,1 98,6 98,0

Bang 6.7. K~t qua khao sat dl) dung tren mlm vien bao dirong TyphasmylTheophylin Ephedrin.HCI PhenobarbitalThem Tim % tlm Them Tim % tim Them Tim thay %vao thfty vao thAy v ao tim(rng) (mg) I~ (mg) (mg) Il;li (mg) (mg) l;,1i40,0 39,6 99,0 5,0 5,10 102,0 2,0 1,99 99,540,0 38,9 97,3 5,0 4,85 97,0 2,0 1,98 99,040,0 39,8 99,5 5,0 4,99 98,0 2,0 1,94 97,0Trung binh 98,6 99,0 98,5

Nh,n xet: Phuong phap co dQdung cao voi ca ba thanh phan.

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_~ .__!_ ' ! _ q _ t ! . g _ ~ j ~ t } _~0! . ----.._.. .....-_..-_.......4.7. Djnh hrqng dAng thM paracetamol, phenylpropanolamin hydroclorid v a

clorpheniramin maleat bing phuong phap HPLCTren thi tnrong hien nay co nhieu dang thuoc co k~t hQ'Pcac thanh phAn nhu tren,

trong do vien nen Decolgen do cong ty United Pharma san xuat co cong thirc:Paracetamol (PA) 500 mgPhenylpropanolamin.HCL (PH) : 25 mgClorpheniramin maleat (CM) 2 mg

Voi nhiing hoat chAt tren, trong cac duoc di~n thong dung hien hanh [I, 19; 27;29] chu yu chi co dang thuoc dan thanh phan. Trong diroc di~n My [29] va tieu chuanco sa [4; 12], co sft dung phirong phap HPLC d~ dinh hrong cac hoat chiit trong h6nhQ'P tren, nhirng m6i chAt phan tich duoc su dung mQt chuang trinh sitc k Y khac nhau.

Nham tim met phuong phap chung, co th~ dinh tinh va dinh hrong nhanh 3 thanhphan trong h6n hQ'P thuoc, chung toi nghien ciru xay dung mQt chuang trinh sitc ky coth~ phan tich d6ng thai cac hoat chat tren.A. Th\l'c nghi~mA.I. Hod chat, nguyen li?u

- Vien nen Decolgen co cong thirc nhu tren.Mdu 1 co s6 kiern soat: 402221Mau 2 co s6 kiem soat: 404971

- ChAt chuan cua Vi~n Kim nghiem: chuan paracetamol (ham hrong 100%),chuan phenylpropanolamin hydroclorid (ham hrong 99,66%), chuanclorphenirarnin maleat (ham hrong 99,86%).

- Trimethylamin loai dung cho HPLC.- Acetonitril loai dung cho HPLC.- Acid phosphoric loai tinh khiet phan tich.- Niroc cAt.

A.2. Thi2t hi, d(lng C(I- May sitc ky long hieu nang cao: DIONEX-Detector UV diode array.- CQt Nuc1eosil C18 (250mm x 4mm, 5J,!m).- BQ loc dung moi va bQ IQc mau voi dirong kinh 16mang IQc 0,45 urn.- M ay lAc s ieu am-SONOREX.- May do PH Meter 3305-Jenway.- Can phan tich Mettler AB 204 dQ chinh xac 0,1 mg.- Dung cu thuy tinh: binh dinh mire 1000,Oml; 100,Oml; 50,Oml; 25,Oml; 20,Oml;

lO ,Oml; 6ng dong , pipet chin h x ac, pipet thuon g, c6c co mo ...- 46-


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_ ~ . _ ! f 4 ! _ q _ l _ I _ q _ ~ g ~ ! ~ ' Y _-:~ --- ------------------------- ---------------------------------------------------_ .B. K~t quaB.1. Khao st i t Ivachon i l i2u kirn sdc kf

Khao sat ph6 til ngoai cua cac ehftt phan tich:

O .B

5 0.0'!gc~., 0.4~~.e'" 0.20200 300

Hinh 7.1. Ph6 hftp thu UV-VIS cua PA trong dung moi pha dQng2 .5

2:: ;5 - 1. 5cc:;""3.

0.50:' NLD0

Hinh 7.2. PhA hApthu UV-VIS cua PH trong dung moi pha dQng

1 . 7 5

1. 551 125~0,, 0.75.0.~

0.5025

Hinh 7.3. PhA hApthu UV-VIS cua CM trong dung moi pha dQng

Sau qua trinh thtrc nghiem, chung toi da Iva chon ducc cac diu kien s~e k Y d~dinh hrong 3 thanh phfut PA, PH va CM nhu sau:

May sie ky long hieu nang eao DIONEX-Detector UV diode array cua bQmonHoa diroc, tnrong Dai hoc diroc Ha noi.

- CQtNucleosil C18 (250 mm x 4 mm; 5 um),- 47-


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4. Kit nUG nehien Clfu_________ _ : :: t. 0 ---- __------------------------------------------..,_op + - _ .. -- - - - - - - _- Detector UV (] 216nm d~ do PH v a CM, (] 244nm d~ do PA.- Pha dQng: Hoa tan 2,5 g kali dihydrophosphat trong 1 lit h6n hop cua nirrrc -

acetonitril - triethylamin (968:30:2), dieu chinh den pH 2,7 b~g acidphosphoric.

- T6c dQdong 1,2 ml/phut.- Th~ tich tiem mdu: 20 ul,- Nhiet d


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_ ' ! _ . l ~ / ! _q , !C ! _ ! ! g _ J J ! ~ , !_ ~0f..._..___.._.__-_.'..---_.----_.-_-_.--_._.-"_._.-...-_-'.... -_._.-.,._.-.._._._.'---..----.B.3. Khao sat dp chinh xdc ciia phuang ph6p

.:. Dung dich thu d~c (dung d~ do PH va CM): Can 20 vien, xac dinh kh6i luongtrung binh vien, nghien thanh bQt min . C a n chinh xac kh6i luong bQt vientuong irng voi kh6i hrong cua khoang 2/5 vien cho vao binh dinh mire 100,0ml, them mroc, lAcsieu am d~ hoa tan khoang 10 phut, them mroc d~n vach, lAcd~u. LQequa mang Ioc 0,45 J.Lm.Duoc dung dich thfr d~c.

:. Dung dich thir loang (dung d8 do PA): Hut chinh xac 1,0 ml dung dich thtr d~ceho vao binh dinh mire 50,0 ml, them mroc d~n vach.fac d~u.

K~t qua khao sat dQchinh xac diroc trinh bay 6 bang 7.5, 7.6, 7.7.Bang 7.5. N6ng dQva dien tich pie cua dung dich chuin

CMtchufut LUQ'Ilgcan (g) N on g dQ (ug/m l). Di~ntich ~ePA 0,2012 40,24 43,19PH 0,5038 100,76 17,02eM 0,0448 8,96 4,96

Bang 7.6. Luong can bQtvien va di~n tieh pie cua dung dich thirM =658,10mg Dien tich picSIT Luong can bQtvien (g) PA PH CM

1 0,2636 44,76 17,13 4,602 0,2632 44,56 17,22 4,623 0,2636 44,89 17,23 4,644 0,2628 44,69 17,17 4,635 0,2668 44,91 17,40 4,706 0,2657 44,85 17,62 4,54

Bang 7.7. K~t qua khao sat dQchinh xac ella phirong phapPA PH CM

IT Ham hrong s A li~u Ham hron g s A lieu H am hron g s A lieu(rng/vien) th8ng ke (mg/vien) themgke (rng/vien) th6ng ke

1 520,57 25,41 2,07- - -2 519,03 X=519,43 25,49 X =25,51 2,08 X = 2,083 522,08 S"" 2,34 25.47 S = 0,164 2,09 S = 0,0234 521,34 RSD%"'" 25,45 RSD%= 2,09 RSD%=5 516,05 0,45% 25.41 0,64% 2,09 1,110;06 517,50 25,84 2,03

K~t qua trinh bay 6 bang 7.7 eho thdy dQl~eh chuan nrong d6i cua PA la 0,45%,ella PH lit 0,64%, ella eM IA 1,11% .


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4.Kit aua nehien C U l l.."_ ::l.__ o _ , . _ _ _ . . _ _ _ _ _ _ _ . . _ _ _ . . _ _ . . . . . . . . - - _ . . - - - - - - _ - - - - _ . . - - - - - .B.4. Khiio sat t IP dung ciia phuong phtip

DQ dung cua phuong phap duoc xac dinh bang phirong phap them chuan. K~tqua khao sat dQdung diroc trinh bay a bang 7.8:

Bang 7.8. K~t qua khao sat dQdungHo~t Luong bot Luong co s a n Luong Luqng Ty l~ thuSTT trong mdu thir chudn them chuan timchAt vien (g) (mg) vito (m_) th~ (m_&) h6i (%)1 0,0129 10,18 9,84 9,95 101,12PA 2 0,0131 10,34 9,84 9,75 99,093 0,0137 10,81 9,84 10,01 101,73Trung binh 100,65

1 0,0129 0,50 23,52 23,13 98,34PH 2 0,0131 0,51 23,52 23,38 99,40

3 0,0137 0,53 23,52 23,84 101,36Trung binh 99,701 0,0129 0,040 2,30 2,260 98,26

eM 2 0,0131 0,041 2,30 2,275 98,913 0,01327 0,043 2,30 2,338 101,65Trung binh 99,61

K~t qua & bang 7.8 eho thAy kha nang tim lai til 99,61% d~n 100,65%, co nghiala plnrong phap co dQdung cao, ap dung d~ dinh I U Q ' l 1 g eho k~t qua t6t.

B.S. Ung d(lng di dinh tinh va dinh /U'p7Igd6ng thm hnn h(YpPA, PH va eM trongvien nen DecolgenChung toi ti~n hanh dinh tinh va dinh hrong dbng thai PA, PH va Clvl tren mdu 2

vien nen Decolgen. K~t qua dinh hrong duoc trinh bay a bang 7.9.Cac s~c ky db thu diroc cua dung dich mdu t lnr va mdu chuan diroc trinh bay &

cac hinh 7.6, 7.7 va 7.8.

- ~ ~ = = ~ ~ - - - - - - - - ~ - - - - - - - - - - - - - - ~ ~. . . . .. . . .-

Hinh 7.6. Sdc k y d b cua dung dich mdu chuan- 50-


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4. Kit aua nehien Czlu_________:;:{ 0. -------------------------------- ------------------------_...-~.'13.

W","'"WVL.:21t

-t c . . . 14.0 11.:)t . . . n ' _ o 11.QHinh 7.7. SA c k Y db cua dung dich mdu thu do PH va CM (y burro s6ng 216nm

IRDeO

Hinh 7.S. SA c l e Y db cua dung dich mdu thir do PA a btroc song 244nmBang 7.9. K t qua dinh hrong

Ho~tchat PA PH eMHam hrong Ham Ham hrong Ham Ham hrong Hamhrong so hrong so hrong soSTT vien " nhan vien v6i n h a n vien voi n h a n(mg/vien) Vat (mg/vien) (mg/vien)(%) (%) (%)

1 512,88 102,58 25,30 101,20 2,06 103,02 512,19 102,44 25,36 101,44 2,03 101,53 512,10 102,42 25,42 101,68 2,08 104,04 509,83 101,97 25,50 102,0 2,09 104,505 518,09 103,62 25,17 100,68 2,04 102,06 514,11 102,82 25,36 101,48 2,06 103,0TB 513,2 102,64 25,35 101,41 2,06 103,0

Tir cac hinh 7.6, 7.7 va 7.8 cho thA y gia tri tha i gian hru cua cac chAt a mdu thirso voi mdu chuan la nrong duong nhau.K t qua a bang 7.9 cho thA y ch~ pham dem dinh hrong dat tieu chuan v~ hamI U Q 1 1 g cua PA, PH va CM.

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_ ( _ ~ ~ ! _ q _ ~ q _ ~ g _ ~ ! ! _ ' ! _ t : ~ - _4.8. Binb hrqng dang tbOi paracetamol va ibuprofen trong vien nen bing

phmmg phap toan pbaNgoai cac phirong phap da noi a phfm tren (phirong phap quang ph6 UV-VIS

thong thuong sau khi chiet tach, phirong phap HPLC), chung tbi da nghien ciru thanhcong phuong phap phan tich toan phd d~ dinh hrong d6ng thai paracetamol vaibuprofen.

A . ThVC ngbi~mA.1. Thiit bi, dflng cfl

- M ay quang phd UV-VIS Beckman DU 650i.- can phan tich co dQ ehinh xac 0,1mg.- Cac dung cu thuy tinh chfnh xac: Binh dinh rmrc, pipet.

A.2. Hod chat, nguyen vpt /i~u- Chdt chuan: paracetamol, ibuprofen dat tieu chuan BP 1993.- Mftu tlnr: Vien nen Dibulaxan, SKS: 020601.- Dung dich d~m phosphat pH 7 pha thea DDVN III.

B. Khio sat va xiy dl}'llg phuong phapB.1. Pha cdc dung dich chu8n g6c paracetamol va ibuprofen.:. Dung dich chun g6e paracetamol (PG): Ca n chinh xac khoang 80 mg paracetamol,hoa tan trong binh dinh mire bang d~m pH 7 vira dil 100ml, 1rQnd~u. Ldy 10 mldung dich pha loang bing dem pH 7 vira du 100ml, 1rQnd~u, diroc dung dich chuang6c paraeetamol co n6ng dQ80 meg/ml.

.:. Dung dich chuan g6e ibuprofen (IG): Can chinh xac khoang 50 mg ibuprofen, hoatan trong binh dinh mire bang dem pH 7 vira dli 100ml, tron d~u. L~y 10 ml dungdich nay pha loang bang d~m pH 7 vira dli 100ml, trQn d~u. diroc dung dich chuang6c ibuprofen co nbng dQ50 mcg/m!.

B.2. Xtiy dvng phuang phapa. Xac dinh giai song phdn tich

Ghi phA h~p thu quang cua m6i dung dich thanh phfut rieng re ill 200nm dn300nm, ket qua ph6 ghi diroc nhu & hinh 8.1.

Tren hinh 8.1 eho th~y ibuprofen (1) h~p thu cue dai tai 222nm, paraeetamol (2)hdp thu eire dai tai 243nm va dQhdp thu cua dung dich h6n hQ'P(3) i . : 1 biroc song diroi

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4. Kit nUG nehien cuu_ _ _ _ _ _ _ _ _ : : : : t 0 .,... .,.......,.--_.....--_.._.......----_....._-----_..----_.....--_..-..........._.215nm la luang d6i Ia n (> 1,0). Do do chung toi chon khoang buoc song phan tich ehohai ch~t til 215nm d~n 300nm.

VavelBngtb (n.)

SIIo:Iothin9; NoneL m m ~ ~ - - - - - - - - - - - - - - - - - - - - ~ - - ~Functions; Sc::.an

Hinh 8.1. PhAUV-VIS ella ibuprofen (1) n6ng dQ7,576 ug/ml paraeetamol (2) n6ngdQ 10,543 ug/ml va h6n hop ella cluing (3) trong d~m pH 7

b. Xae dinh khoang n6ng d9 co luang quan tuyen tinh VGid9 hJp thu cua m6i chdt taicac buac song qrc daiChung toi da pha day gbm 5 dung dich chuan co nang dQ bien thien til 5 ug/ml

dn 11 ug/ml cho ibuprofen, til 8 ug/ml d~n 15 ug/ml cho paraeetamol. Ti~n hanh dom?t dQquang cac dung dich ibuprofen tai 222nm va paracetamol tai 243 nm.

Trong khoang n6ng dQ d khao sat cho th~y co su luang quan tuyen tinh gitran6ng dQva d{)hAp thu quang ella m6i ch~t tai biroc song khao sat. K~t qua duoc trinhbay (y bang 8.1.

Bang 8.1. Mbi nrong quan gilra n6ng dQva m~t dQquang.Ibuprofen I uz/ml i 4,843 i 6,457 1, 8,071 i 9,686 I 11,300~ ~ _ , , ~ , _ , _ _ ,_ " ' c n . n . _ . _ . ~ _ ~ _ '_ " " " '_ n . n . . ' . n .n ._ . _ _ ~_ _ . n _ ._ . _ . _ . . . ._ ._ ~ nf ._ ._ . .._ _ ._ _ . _ ._ .__ n . n ._ ._ _ . .. .. . _~ c n .n . _ ._ . _. ~ . ' . . n .' .. _ . _ . ~ . _ + _ _ n . n . ' . . _ . , . ' _ ~ n . n . n . n . . U . L rD222 ! 0,22317 ! 0,29743 ! 0,37445 i 0,45714 i 0,52569".~__ . .__ ~ _ _ ._"._L~_ ._ ._ ._ .. . ._ . __ _~_~_ . __ ".l ___._.". ._._ ...".__ ~ __.__ "~ ._ ".." " .Duong hoi quy D =O,047374.C - 0,00679 voi r =0,9994Paracetamol uz/ml I 5,168 i 7,752 I 10,336 I 12,920 i 15,504. . .J : " . . J . 7 . . _ . . ~ . " _ _ ._ ._ . ~ " ~ , _ . . . _ " "----!_ .. ._ ._ ._ . ._ _ _ _._ ._ ."."~_._ ._ ."._." + _ . . _ . " . " _ _ . _ " . . " .. _ _ . _ . _. .-l- __ ".. "___._ ".D243 J 0,3348 i 0,5066 ! 0,6763 ! 0,8498 I 1,0186._ _ ._ .u ._ . ~ _ ._ . _ . _ . u _ ~ _ _ _ ._ ._ . u _ .u _ _.l__ ..,._._........,_,.,._._. __ ~ __ . .....L ~ _. _._ .u .~ -+ -n _. _. _ . . __ _ ~_ . . ~ __ n._ ., . .' . _~ _._ n_ n. _ .. _. ,. _. ~ . ,_ ._ _ . U .U _ L ~ _ . _ _ u _ . _ _ , . . u . ,. . , . _ _ _ . , . _ .Duong hoi quy D =0,06621.C - 0,0019 V0 1 r = 0,9999

Til k~t qua khao sat m6i tirong quan gitra dQ hApthu quang va n6ng de)cua m6ithanh phan, t:5' I e phbi hQ'Pgitra hai eh~t trong cong thirc ella vien nen Dibulaxan,chung toi chon khoang nong d~ phan tich cho paracetamol tir 8 ug/ml d~n 12 ug/ml vaibuprofen til 5 ug/ml dn 8 ug/rnl.c. Pha day dung dich chudn h6n h(IJJ

Dimg pipet chinh xac phbi hQ'Ptil dung dich chuan g6c PO va 10, sir dung d~mpH 7 lam dung mei pha loa

Paracetamol (1)

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