B Y T

TRNG I HC DC H NI

NGUYN TH DUYN

NGHIN CU PHT HIN DEXAMETHASON

TRONG MT S CH PHM NG DC BNG

PHNG PHP SC K LP MNG

KHA LUN TT NGHIP DC S

H NI 2013

B Y T

TRNG I HC DC H NI

NGUYN TH DUYN

NGHIN CU PHT HIN DEXAMETHASON

TRONG MT S CH PHM NG DC BNG

PHNG PHP SC K LP MNG

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. PGS .TS. Nguyn Th Kiu Anh

Ni thc hin:

Phng phn tch , kim nghim v tng

ng sinh hc

H NI - 2013

LI CM N

Li u tin, em xin by t lng knh trng v bit n su sc n

PGS.TS. Nguyn Th Kiu Anh ngi trc tip hng dn v ch bo

tn tnh cho em trong sut qu trnh thc hin ti ny.

Em xin chn thnh cm n s gip nhit tnh ca cc thy c, anh

ch cng tc ti Phng Phn tch, kim nghim v tng ng sinh hc -

Vin cng ngh Dc phm quc gia trong qu trnh em lm thc nghim ti

phng.

Em cng xin gi li cm n n:

B mn Ha phn tch c cht.

B mn Dc c truyn.

to iu kin thun li cho em hon thnh kha lun ny.

V cui cng em xin by t lng yu thng, bit n ti gia nh v

bn b nhng ngi lun ng vin, gip em trong qu trnh hc tp v

hon thnh kha lun.

H Ni, ngy 20 thng 5 nm 2013

Sinh vin

Nguyn Th Duyn

MC LC

DANH MC CC BNG

DANH MC CC K HIU, CC CH VIT TT

DANH MC CC HNH V

T VN .................................................................................................. 1

CHNG I: TNG QUAN ........................................................................... 3

1.1. Vi nt v Glucocorticoid ..................................................................... 3

1.1.1. Tc dung v tc dng khng mong mun ca Glucocorticoid ......... 3

Tc dng ...................................................................................................... 3

Tc dng khng mong mun ...................................................................... 3

1.1.2. i cng v dexamethason v dexamethason acetat ...................... 4

1.1.2.1. Dexamethason ............................................................................. 4

1.1.2.2. Dexamethason acetat .................................................................. 6

1.2. Mt s nghin cu xc nh dexamethason v dexamethason

acetat .............................................................................................................. 7

1.3. Thc trng thuc ng dc v thc phm chc nng b trn

tn dc ......................................................................................................... 9

1.4. Phng php x l mu ..................................................................... 10

1.4.1. Phng php chit trc tip ............................................................ 11

1.4.2. Phng php chit lng lng .......................................................... 11

1.4.3. Phng php chit pha rn ............................................................. 11

1.5. Tng quan v sc k lp mng .......................................................... 12

1.5.1. Nguyn tc ...................................................................................... 12

1.5.2. Pha tnh ........................................................................................... 13

1.5.3. Pha ng .......................................................................................... 13

1.5.4. Dng c ........................................................................................... 14

1.5.5. ng dng ca SKLM ...................................................................... 15

1.5.6. u nhc im ca phng php sc k lp mng ....................... 17

CHNG II: NGUYN VT LIU, THIT B, NI DUNG V

PHNG PHP NGHIN CU ................................................................ 18

2.1. Nguyn vt liu, thit b ..................................................................... 18

2.1.1. My mc, thit b ............................................................................ 18

2.1.2. Ha cht, dung mi, cht chun ...................................................... 18

2.2. i tng, ni dung v phng php nghin cu ........................... 19

2.2.1. i tng nghin cu: .................................................................... 19

2.2.2. Ni dung nghin cu ....................................................................... 21

2.2.3. Phng php nghin cu. ............................................................... 21

CHNG III. THC NGHIM, KT QU V BN LUN ................ 23

3.1. Kho st v la chn iu kin phn tch. ........................................ 23

3.1.1. Chun b cc dung dch chun gc: ................................................ 23

3.1.2. X l mu ....................................................................................... 23

3.1.2.1. Mu rn ..................................................................................... 23

3.1.2.2. Mu lng ................................................................................... 24

3.1.3. Kho st, chn h dung mi sc k ................................................ 24

3.1.4. Kho st iu kin pht hin ........................................................... 27

3.2. nh gi phng php ....................................................................... 29

3.2.1. Tnh chn lc .................................................................................. 29

3.2.2. Gii hn pht hin ........................................................................... 30

3.2.3. lp li v tm li.................................................................... 33

3.3. p dng trn mt s mu th ............................................................ 36

3.4. Bn lun ............................................................................................... 42

KT LUN V XUT .......................................................................... 44

TI LIU THAM KHO

DANH MC CC K HIU, CC CH VIT TT

ACN

DAD

DM

DMKT

GC

HPLC

LC-MS

MeOH

Rf SK

SKLM

S/N

SPE

STT

TLC

TLTK

TPCN

tR UV

VIS

: Acetonitril

: Detector diod array

: Dung mi

: Dung mi khai trin

: Glucocorticoid

: Sc k lng hiu nng cao

: Sc k lng khi ph

: Methanol

: H s lu gi

: Sc k

: Sc k lp mng

: Signal/noise

: Solid phase extration

: S th t

: Sc k lp mng

: Ti liu tham kho

: Thc phm chc nng

: Thi gian lu

: T ngoi

: Kh kin

DANH MC CC BNG

Bng 1.1: Mt s nghin cu chit dexamethason v dexamethason acetat .... 7

Bng 1.2: Mt s nghin cu pht hin dexamethason v dexamethason acetat ... 8

Bng 1.3 : Mt s cht thng dng lm pha tnh cho SKLM ....................... 13

Bng 2.1: Cc mu thuc c phn tch ....................................................... 20

Bng 3.1. Kt qu xc nh LOD .................................................................... 33

Bng 3.3: Kt qu kim tra bng SKLM trn 5 ch phm ng dc ........... 38

Bng 3.4: Kt qu kim tra bng HPLC trn 5 ch phm ng dc ............. 41

DANH MC CC HNH V

Hnh 3.1. SK sc k vi h DM1 ................................................................. 25 Hnh 3.2. SK sc k vi h DM2 ................................................................. 25 Hnh 3.3. SK kho st DM trn nn mu rn ............................................... 27 Hnh 3.4. SK kho st DM trn nn mu lng ............................................. 27 Hnh 3.5. SK kho st tm iu kin pht hin (UV-254 nm) ..................... 28 Hnh 3.6. SK kho st tm iu kin pht hin vt (phun dung dch acid

sulfuric trong ethanol) ..................................................................... 28 Hnh 3.7. SK nh gi tnh chn lc (mu rn) ........................................... 29 Hnh 3.8. SK nh gi tnh chn lc (mu lng) ......................................... 29 Hnh 3.9. SK gii hn pht hin ca Dexamethason v Dexamethason

acetat ............................................................................................. 31 Hnh 3.10. SK analog xc nh LOD ........................................................... 32 Hnh 3.11. SK nh gi lp li ca phng php chit ........................... 34 Hnh 3.12. SK chp nh v analog ca dch chit mu t to R ................. 34 Hnh 3.13. SK chp nh v analog ca dung dch chun hn hp 0,5

mg/ml ............................................................................................ 35 Hnh 3.14. SK ca mu th .......................................................................... 37 Hnh 3.15: Sc k chun hn hp dexamethason v dexamethason

acetat ............................................................................................. 38 Hnh 3.16. Ph hp th ti pic cht chun ...................................................... 39 Hnh 3.17. SK mu thuc tr Vim mi ....................................................... 39 Hnh 3.18. Ph hp th ca mu thuc tr vim mi tng ng vi tR =

8,539 .............................................................................................. 39 Hnh 3.19.SK mu bt thuc ca c s ng nh Ton ............................ 40 Hnh 3.20. Ph hp th tng ng vi tR = 8,535 ca mu bt thuc ca c

s ng nh Ton ....................................................................... 40 Hnh 3.21. SK mu phong t nhc khp thy .............................................. 40 Hnh 3.22. SK mu phong t ct thng thy ................................................ 41 Hnh 3.23. Ph hp th tng ng vi tR = 8,546 ca mu phong t ct

thng thy ..................................................................................... 41

1

T VN

T th k trc, Y hc phng Ty, m ngi ta quen gi l Y hc hin

i, tr thnh h thng y hc chnh thng hu ht cc quc gia trn Th

gii, k c cc nc ng Nam . Th nhng, trong nhng nm gn y, cc

loi thuc c ngun gc t nhin ca Y hc truyn thng phng ng

(ng dc) ngy cng c nhiu ngi, c phng ng v phng Ty

ch , a chung v s dng ngy cng nhiu bi tnh an ton v hiu qu

ng tin cy, lu di ca n. Tuy nhin, nhm to ra nhng hiu qu tc thi

cho ngi bnh, gim au, n ngon, ng yn, tng cn, sinh lc di do...,

nhiu c s sn xut thuc y hc c truyn v c s cha bnh t nhn trn

tri php vo thuc ng dc, thc phm chc nng mt s loi tn dc.

Hu qu ca vic dng thuc ng y trn tn dc s rt trm trng v kh

lng trc c nhng mi nguy him, v ngi bnh c tin chc rng ang

ung thuc c ngun gc dc liu nn an ton v ung ko di.

Nhm glucocorticoid l nhm thuc chng d ng, chng vim mnh vi

nhiu tc dng ph, khong iu tr hp, cn c bc s k n v phi theo

di khi dng. Cc cht ny rt hay c trn mt cch kho lo v tinh vi vo

thuc ng dc c ch nh iu tr thp khp, hen ph qun, gouthay

thuc b do c tc dng nhanh, mnh v tc dng khng mong mun gy tch

nc, bo ph gi to.

c rt nhiu ch phm pht hin c cha corticoid nh: Tn ha truy

phong t thp thy, Hnh c khu phong t thp thy (trn betamethason)

[25], mu thuc bt nh xuyn ca Trung Quc c prednisolon acetat, Tng

ph on ca i Loan, Nam lin truy phong thu ct hong ca Trung Quc

c dexamethason acetat [10]. Dexamethason l mt glucocorticoid bn tng

hp, c tc dng (chng vim, chng d ng) mnh, ko di, khng gi mui,

2

nc. Do n c dng rng ri v cng d b trn tri php vo thuc

ng dc.

Nghin cu nh tnh, nh lng cc glucocorticoid trn tri php vo thuc

ng dc v ang thu ht c nhiu nhm nghin cu, c mt s

cng b v xc nh dexamethason, dexamethason acetat bng cc phng

php khc nhau nh: HPLC-MS, HPLC-DAD Tuy nhin cc phng php

ny yu cu my mc kh t tin. ng gp mt phng php phn tch

nhanh, n gin v kinh t hn trong xc nh cht cm dexamethason,

dexamethason acetat trong thuc ng dc, chng ti tin hnh thc hin

ti:

Nghin cu pht hin Dexamethason trong mt s ch phm ng dc

bng phng php sc k lp mng

Vi mc tiu c th sau y:

1. Xy dng c phng php pht hin dexamethason, dexamethason

acetat trong ch phm ng dc, thc phm chc nng bng sc k lp

mng

2. p dng phng php xy dng c pht hin dexamethason,

dexamethason acetat trong mt s ch phm ng dc, thc phm chc nng

(dng thuc nc v thuc rn) c nghi ng trn th trng.

3

CHNG I: TNG QUAN

1.1. Vi nt v Glucocorticoid

1.1.1. Tc dung v tc dng khng mong mun ca Glucocorticoid

Tc dng:

GC t nhin do vng b v thng thn sn xut ra gm 2 cht l

hydrocortison (cortisol) v cortison. GC tng hp gm nhiu cht khc

nhau. nng sinh l, cc cht ny cho cn bng ni mi, tng sc chng

ca c th vi stress v duy tr cc chc nng khc nhau ca c th. GC c

tc dng trn chuyn ha, trn cc c quan v tuyn [12]. Trong , tc dng

chng vim, chng d ng hay c p dng trong iu tr mt s bnh mn

tnh nh: thp khp, hen ph qun, vim xoang [12].

Tc dng khng mong mun: [5], [12].

- Tc dng trn s tng trng ca tr em: Mc liu t 5 mg/m2/ngy tr ln

gy chm ln tr em.

- Gy xp xng khi dng corticoid liu cao ko di.

- Lot d dy t trng t l khng cao (khong 1,8%) nhng nu gp thng

rt nng, thm ch c th gy thng d dy hoc t vong.

- Tc dng ph do dng ti ch: teo da, x cng b, vim da ng , mn

trng c hoc bi nhim nm, vi khun v virus, chm lin so, c thy tinh

th hoc tng nhn p, c bit khi dng lin tc v ko di.

- Hin tng c ch trc di i tuyn yn tuyn thng thn (HPA) [5].

Dng GC khng ng cch di ngy khi ngng thuc d gp hin tng suy

thng thn t ngt. Teo thng thn c th ko di nhiu nm sau khi iu

tr di ngy bng GC [12].

4

1.1.2. i cng v dexamethason v dexamethason acetat

1.1.2.1. Dexamethason

Cng thc cu to: [4], [7], [20], [21].

C22H29FO5 P.t.l: 392,5

Tn khoa hc: 9-fluoro-11,17,21-trihydroxy-16-methylpregna-1,4-dien

3,20-dion.

Tnh cht vt l: [4], [7], [20], [21]. Bt kt tinh trng hoc tinh th khng mu, khng mi. Chy 253 2550C

(km phn hy). Thc t khng tan trong nc, hi tan trong ethanol, kh tan

trong methylen clorid, tan trong aceton.

nh tnh: [4], [20]. - o ph hp th ca dn cht hydrazon: cho phn ng vi phenylhydrazin

trong mi trng acid cho dn cht phenihydrazon c mu vng (hp th cc

i 419nm).

- Ph hng ngoi ca ch phm phi ph hp vi ph hng ngoi ca

dexamethason chun.

- Dng phng php sc k lp mng: Dng bn mng Silicagel GF254 h

dung mi trin khai l butanol bo ha nc toluen ether (5: 10: 85).

- Phn ng mu: Oxy ha vi tc nhn H2SO4 c, c mu nht (thng

km hunh quang). Pha long vi nc, mu bin mt.

nh lng: [4], [20] - o quang ph hp th t ngoi kh kin (UV-VIS): Ha tan ch phm trong

5

ethanol; o khong 238,5nm, mu trng l ethanol. Ly E (1% 1cm) ca

dexamethason 238,5nm l 394 tnh kt qu.

- Phng php HPLC bng o din tch pc so vi cht chun.

Tc dng dc l: [6], [12] Dexamethason tc dng bng cch gn kt vo th th t bo, chuyn v vo

nhn t bo v tc ng n mt s gen c dch m. Dexamethason

c cc tc dng chnh ca glucocorticoid l chng vim, chng d ng v c

ch min dch, cn tc dng n cn bng in gii th rt t. V hot lc

chng vim, dexamethason mnh hn hydrocortison 30 ln, mnh hn

prednisolon 7 ln, thi gian tc dng ko di

Ch nh [6] - Liu php khng c hiu bng steroid, khi cn iu tr tch cc, nh iu tr

trng thi hen, bnh d ng nng, phn ng sau truyn mu, vim thanh qun

rt.

- Dng phi hp vi cc cch iu tr khc trong ph no, trong sc do nhiu

nguyn nhn khc nhau.

- Phng nga bng steroid trong phu thut khi d tr glucocorticoid c coi

l khng , tr suy v tuyn thng thn tin pht (bnh Addison).

- Dng dexamethason trc khi sinh trong chuyn d trc k hn (gia 24

v 34 tun) thc y qu trnh trng thnh thai (v d phi, mch mu

no).

- Liu php b tr bng dexamethason trong iu tr vim mng no ph cu.

- iu tr ti ch: Tim trong v quanh khp ngi bnh vim khp dng

thp, thoi ha xng khp, vim quanh khp, vim mm li cu. Cn c

dng ti ch trong mt s trng hp bnh l tai mi hng, nhn khoa, ngoi

da.

Liu dng : 0,5-10 mg/ngy [6], [10].

6

1.1.2.2. Dexamethason acetat

Cng thc cu to: [4], [7], [20], [21].

C24H31FO6 P.t.l: 434,5

Tn khoa hc: 9-fluoro-11,17-dihydroxy-16-methyl-3,20-dioxopregna-1,4-

dien-21-yl acetat

Tnh cht vt l: [4], [7], [20], [21]. Bt kt tinh trng hoc gn nh trng, a hnh. D tan trong ethanol 96% v

aceton, kh tan trong methylen clorid, thc t khng tan trong nc.

nh tnh:[4], [20]. - o ph hp th ca dn cht hydrazon: cho phn ng vi phenylhydrazin

trong mi trng acid cho dn cht phenihydrazon c mu vng (hp th cc

i 419 nm).

- Ph hng ngoi ca ch phm phi ph hp vi ph hng ngoi ca

dexamethason acetat chun (C). Nu cc ph thu c ca ch phm v ca

cht chun dng rn khc nhau, o thm ph dng lng: dng dung dch

bo ha (nng khong 3%) ca ch phm v ca cht chun trong

cloroform (TT) vi t bo o dy 0,2 mm.

- Phng php sc k lp mng

Bn mng: Silica gel GF254.

Dung mi khai trin: Nc - methanol - ether - methylen clorid (1,2 : 8 : 15 :

77).

7

Pht hin: Soi bc sng 254 v hin mu bng dung dch acid sulfuric

- Phn ng mu: Oxy ha vi tc nhn H2SO4 c, c mu nht (thng

km hunh quang). Pha long vi nc, mu bin mt trong ethanol.

- Khong 10 mg ch phm cho phn ng ca nhm acetyl.

Liu dng trung bnh: 0,5-10 mg/ngy [6], [10]. 1.2. Mt s nghin cu xc nh dexamethason v dexamethason acetat

Bng 1.1: Mt s nghin cu chit dexamethason v dexamethason acetat

TLTK Nn mu PP chit Dung mi/ct

[10] Thuc nc Chit pha rn Ct 250SPE (500mg C18

v HLB; 8ml

[11] Kem Trc tip Methanol

[9] Ch phm ng

dc (thuc bt)

Trc tip Aceton

[22] Vin nn Trc tip Cloroform

Thuc nc Pha long,

lng lng

Methanol-nc (1:1)

Ethyl acetat

[20] Bt Trc tip Ethanol

Thuc nc Pha long Methanol-nc

[16] Huyt tng

ngi

Chit lng lng Ethyl acetat

[17] Thc phm Chit pha rn C18 catridge

[19] Vin nn Chit trc tip Methanol

Mu Chit lng lng Aceton-Methanol (1:1)

8

Bng 1.2: Mt s nghin cu pht hin dexamethason v dexamethason acetat

STT Phng php iu kin phn tch TLTK 1 HPLC - Ct Inertsil C18 (250 x 4,6 mm; 5 m)

- Detector DAD: bc sng 240 nm - Pha ng: ACN-nc (35:65) - Tc dng: 1,6 ml/pht

[10]

2 TLC

- DMKT: H 1: Dicloromethan-ether-methanol-nc (77:15:8:1,2) H 2: Dicloromethan-methanol-nc (95:5:0,2) H 3: dicloromethan-methyl acetat-nc (100:50:50) (ly lp di) - V chm = 2 l - Pht hin: soi UV (254 nm), phun dung dch acid H2SO4 20% trong ethanol ri soi 366 nm, phun dung dch xanh tetrazolium kim.

[11]

3 HPLC

- Pha ng: ACN-nc (44-56) - Tc dng: 1,0 ml/pht - Detector (240 nm)

4 TLC - DMKT: Dicloromethan-ether-methanol-nc (77:15:8:1,2) - V chm = 5 l - Pht hin: soi UV (254 nm), phun dung dch acid H2SO4 20% trong ethanol.

[9]

5 HPLC - Ct C18 - Pha ng: ACN - H2O (28 : 72) - Pht hin bc sng 254 nm - Tc dng: 1,2 ml/pht - Cht chun ni: Triamcinolon acetonide

[16]

6 HPLC - Ct Metasil octadecyl silane, 5 m (25 cm x 4,6 mm) - Pha ng: MeOH-Nc (65:35) - Tc dng: 1 ml/pht - Detector UV (254 nm)

[15]

7 HPLC - Ct Diol vi ht c kch c 5 m - Pha ng: hexan propan-2-ol (90:10) -Detector DAD

[17]

8 HPLC - Ct C18 - Detector UV (239 nm)

[13]

9

1.3. Thc trng thuc ng dc v thc phm chc nng b trn tn

dc

Hin nay, tnh hnh thuc ng dc, thc phm chc nng b trn tri

php tn dc ang xy ra rt ph bin v c c quan qun l pht hin

nhiu cc vng min, t cc thnh ph ln n cc vng tnh thnh khc

trong ton quc. Cc ch phm b trn tn dc cng rt a dng v phong

ph, th on ca ngi sn xut lm gi ngy mt kho lo v tinh vi (trn

Tn dc vo v nang mm, lng trn c tnh theo liu dng ca

thuc,...). Trong nhm thuc ng dc b trn glucocorticoid hay gp

nht (v thuc c tc dng nhanh v mnh) v cng gy ra nhiu tc hi cho

ngi tiu dng [26], [27], [28].

Mt s thuc b pht hin nh:

- Nang mm Tng ph hon (Ceng Fui Yen) Malaysia sn xut,trn

Dexamethason, Cyproheptadin [24].

- Thuc Dn tc cu nhn vt, sn xut ti Campuchia, trn 4 loi Tn dc

Dexamethason, Diazepam, Paracetamol v Cyproheptadin [24].

- Khu phong t thp thy, v Truy phong t thp thy ca c s sn xut

thuc YHCT Hinh ha, TP H Ch Minh b trn betamethason [25].

-Thuc B t tm ca Phc Li ng- Nng c trn cyproheptadin v

dexamethason [23].

- Mt s ng dc v thc phm chc nng c ngun gc t dc liu trn

hot cht tng cng kh nng sinh l: Vin nang Supai 99 Tongkat Ali

plusdo Malaysia sn xut c cha Noracetildenafil; Vin nang Mnh Nam do

Cng ty TNHH Dc phm Ngc Khnh c cha Acetyldenafil; Vin nang

B Thn H Thnh do Cng ty TNHH H Thnh sn xut c cha

Sildenafil,...[24].

Vic trn thm tn dc vo ng dc khng ch l vn ca ring nc ta

10

m l vn ca nhiu nc khc: Trung Quc, Singapore, Malaysia, Nht

Bn, Campuchia,... Theo bo co ti hi ngh ha hp thuc ng dc khu

vc chu Thi Bnh Dng, nhiu mu thuc ng dc iu tr ho, hen,

thp khp b pht hin c trn prednisolon hoc dexamethason [10].

Vn thc phm chc nng hin nay:

- Sn xut: Mt s c s sn xut thuc cha bnh khng t GMP khng

c php sn xut dc phm chuyn sang sn xut TPCN do yu cu ca

sn xut ch phm ny khng kht khe nh thuc, ch cn cng b tiu chun

v chu trch nhim trc sn phm ca mnh, cc ch tiu cht lng ch yu

l tnh cht, kim loi c, nhim khun.

- Bn cnh cc c s sn xut thuc y hc c truyn nh l, gia truyn

ang dn sang sn xut kinh doanh TPCN. y l ngun gc dn n hng

lot TPCN c ngun gc YHCT nh: c hot thang k sinh, du gc...

Nhiu loi TPCN ng gi ging ht nh thuc cha bnh lm ngi dng

kh phn bit TPCN v thuc iu tr. y cng chnh l cc loi thc phm

chc nng c kh nng b trn tri php thuc tn dc nhm qung co,

chng minh tc dng tc thi ca sn phm thc y ngi mua, bn vi

gi cao.

- Trn tn dc tri php vo thuc ng dc v thc phm chc nng ngy

nhiu, do vic kim sot cht lng cng mang tnh cht thi s v cp

bch. c bit s bng n ca th trng TPCN trong my nm gn y

gy nhiu kh khn cho cc c quan qun l. V vy, vic xy dng cc

phng php pht hin cht cm trong ng dc v TPCN l rt cn thit.

1.4. Phng php x l mu [2]

Cc thuc ng dc thng l nhng thuc c thnh phn phc tp, do

phn tch cc thuc tn dc ln trong cc thuc ng dc th vic x

l mu, tch chit cc cht cn quan tm ra khi mu nh tnh v nh

11

lng l rt quan trng.

1.4.1. Phng php chit trc tip

Chit trc tip vi dung mi hu c l phng php chit ph bin nht

trong kim nghim thuc nhm ha tan dc cht. Ty vo tan ca dc

cht v tnh cht ca dung mi m ngi ta la chn cch ha tan nh ngm,

khuy, lc c hc, lc siu m.. Khi chit trc tip, cn c th thay i thi

gian chit, nhit ... tng hiu sut chit.

Phng php chit trc tip thng c p dng cho cc dng mu rn:

thuc bt, thuc vin, dc liu...hoc dng bn rn nh thuc kem, thuc m.

1.4.2. Phng php chit lng lng

L phng php chuyn cht phn tch ha tan trong mt dung mi sang

dung mi th hai khng ng tan vi dung mi th nht, ct thu hi dung mi

thu c cht phn tch. Ty vo bn cht cht phn tch v mc ch nghin

cu c th chit dng phn t hay chit cp ion, chit ni phc.

Cc yu t nh hng n qu trnh chit: tan ca cht phn tch

trong dung mi, nhit , s c mt ca cc cht ha tan khc trong dung dch.

ng dng: Chit lng lng c p dng kh ph bin trong kim

nghim cc thuc dng dung dch nh: thuc nc, siro, ru thuc...hay cc

mu dch sinh hc nh mu, nc tiu... Ngoi ra i vi mu phn tch dng

rn, ngi ta thng chuyn vo pha nc ri chit lng lng tch cc nhm

cht, tinh ch dch chit.

1.4.3. Phng php chit pha rn

L k thut x l mu da trn nguyn tc ca sc k lng nhm loi cc

cht c nh hng ti cht cn phn tch, lm giu cht trc khi phn tch

chng. Cc ti liu ting Anh gi l SPE (Solid phase extraction). K thut

ny c s dng t lu tinh ch dch chit trong chit xut lng lng.

Phng php ny lm gim ng k lng dung mi c s dng, chit c

12

chn lc cht cn quan tm, thi gian chit nhanh, hiu qu chit tt v lp

li cao.

Ngy nay SPE c p dng rng ri chit tch, tinh ch, lm giu

mu, c bit hiu qu cho cc mu dch sinh hc, phn tch vi lng, mi

trng...

1.5. Tng quan v sc k lp mng

1.5.1. Nguyn tc

Sc k lp mng (SKLM) (Thin Layer Chromatography) l mt k thut

phn tch dng tch cc cht trong hn hp.

Phng php SKLM bao gm pha tnh l mt lp mng cht hp ph c

ph trn mt mt phng tr, pha ng l cht lng gm mt hay nhiu dung

mi phi hp vi nhau, pha ng chuyn ng qua pha tnh di tc dng ca

lc mao dn [1].

Cc cht phn tch di chuyn vi tc khc nhau ty thuc theo bn

cht ca chng, c tch ring v cc v tr khc nhau trn bn mng. S

tch cc cht khc nhau da trn tnh cht phn cc ca cc thnh phn trong

dung dch phn tch. C ch tch c th l phn b, hp ph, trao i ion, sng

lc phn thoc phi hp nhiu c ch [1].

i lng c trng cho mc di chuyn ca cc cht phn tch l h s lu

gi Rf. Tr s ca n c tnh bng t l gia khong cch di chuyn ca cc

cht phn tch v khong cch dch chuyn ca pha ng [1]:

Rf = dR / dM

Trong :

dR : Khong cch t im xut pht t tm n vt phn tch (cm),

dM : Khong cch t im xut pht n mc dung mi pha ng (cm)

Rf : C gi tr dao ng gia 0 v 1.

13

1.5.2. Pha tnh

Pha tnh ca SKLM l cc ht c kch thc 10 30 m c ri u v

kt dnh thnh mt lp mng ng nht dy khong 250 m trn gi thy

tinh, nhm hoc nha. Ngi ta thng a thm cht pht hunh quang

khng tan vo pha tnh pht hin cht phn tch. Mt s cht thng dng

lm pha tnh cho SKLM c trnh by trong bng sau:

Bng 1.3 : Mt s cht thng dng lm pha tnh cho SKLM

Pha tnh C ch sc k ng dng phn tch

Silica Hp th Acid amin, hydrocarbon, alkaloid,

vitamin

Dn cht siloxan Phn b Cc cht t phn cc

Cellulose Phn b Acid amin, carbohydrat, nucleotid

Alumina Hp th Hydrocarbon, alkaloid, cht mu thc

phm, lipid

Ct bin Phn b ng, acid bo

Cellulose trao i

ion

Trao i ion Acid nucleic, nucleotid, ion kim loi,

halogenid

Gel sephadex Loi c Polymer, protein, phc kim loi

Trong nhng cht lm pha tnh k trn, ph bin nht l Silica (SiO2) v

Alumina (Al2O3) [1],[8].

1.5.3. Pha ng

Pha ng thay i ty thuc vo c ch sc k, c th s dng mt hoc

hn hp nhiu dung mi vi t l thch hp. Trong SKLM vic chn dung

mi c ngha rt quan trng. Ty thuc vo tnh cht phn cc v i lc vi

pha tnh, ta la chn dung mi ph hp lm pha ng khai trin. Mun tch

cc cht khng hoc t phn cc phi dng cht hp ph hot nng cao v

14

dung mi khai trin yu (t phn cc) v ngc li [1].

Tuy vy la chn ti u ha sc k thng da ch yu vo kinh nghim. Sau

y l mt s gi chung nht cho pha ng SKLM [1]:

- Dung mi cn c tinh khit cao.

- Cn iu chnh sc ra gii ca pha ng tr s Rf nm trong khong 0,2

0,8 t phn gii cc tr.

- Cht phn tch dng ion hay phn cc c ra gii tt bng dung mi phn

cc nh hn hp n-BuOH : H2O. Thm mt t acid acetic hoc amoniac vo

nc s lm tng tan ca base hoc acid tng ng.

- Khi dng silicagel hoc cc cht hp ph phn cc khc, phn cc ca

pha ng s quyt nh tc di chuyn ca cht phn tch v tr s Rf ca

chng. Nu thm mt t dung mi t phn cc nh ether ethylic vo dung mi

khng phn cc nh methylbenzen th s lm tng ng k tr s Rf.

1.5.4. Dng c

Dng c chm sc k

C nhiu dng c chm sc k nh: mao qun thy tinh, pipet nhn,

micropipet vi nhiu c khc nhau, my chm sc k vi bm tim mu

Hin nay c rt nhiu loi my chm sc k vi phn mm iu khin cho

php thay i c th tch chm, s lng mu chm trn mt bn mng, v

tr chm mt cch chnh xc. Ngi dng c th chm trn hoc vch theo

yu cu.[14]

Cc loi my thng dng l Nanomat (CAMAG, Muttenz, Switzerland),

Linomat 4, 5 (CAMAG, Muttenz, Switzerland), TLS100 (Baron, Reuchenau,

Germany); AS30, TLC Spotter PS 01 (Desaga, Heidelberg, Germany),

Automatic TLC Sampler 3, 4 (ATS 3; ATS 4 CAMAG). Trong , hay s dng

nht l my chm sc k Linomax 5.0 kt hp vi mt my tnh chy phn mm

winCATS. Cng c th s dng ring cc thit b ty theo nhu cu [14]

15

Bnh chy sc k

Bnh chy sc k c th l hnh hp hoc hnh tr ch nht bng thy

tinh trong sut. Thng dng loi bnh c in y phng hoc bnh y hai

ngn (twin trough chambers TTC) vi cc kch thc khc nhau, bnh hai

ngn dng tit kim dung mi hn. Bnh c np y bng thy tinh hoc thp

khng g m bo to bung khai trin kn. Ngoi ra cn c th s dng

cc cc thy tinh hnh tr c np y kn trin khai bn mng vi kch

thc nh [1], [14], [18].

Thit b pht hin sc k [1], [14], [18].

- Bnh phun thuc th hin mu: TLC Sprayer, Reagent Spray, TLC spray

cabinet (CAMAG), Sprayer SG1, Autosprayer ChromaJet DS 20 (DESAGA).

- n t ngoi pht cc bc x c bc sng 254nm v 366nm nh UV Lamp

(CAMAG).

- Densitometer: Thit b o cng tia phn x t b mt bn mng khi soi

di n UV-VIS.

1.5.5. ng dng ca SKLM

SKLM l mt k thut c s dng trong nhiu lnh vc ha hc, dc

phm, ha sinh, lm sng vo 3 mc ch sau:

nh tnh

Thng da vo tr s Rf ca mu th v mu chun chy sc k trong

cng iu kin. Mi cht trong mt h sc k c mt gi tr Rf c trng. Gi

tr Rf ph thuc nhiu yu t nh cht hp ph (loi cht hp ph, hot tnh,

b dy cht hp ph), pha ng (thnh phn, tinh khit, tc di chuyn

ca pha ng...), bo ha dung mi trong bnh chy sc k, chiu di khai

trin, nhit , lng mu, cch xc nh tm, chnh xc ca php o

di vic nh tnh c chnh xc, khng c thay i iu kin thc

nghim v nn tin hnh mt lot php o xc nh gi tr trung bnh.

16

i khi do sc k lin tc khng xc nh c tuyn dung mi pha ng,

ngi ta dng h s lu gi tng i Rr c trng cho cht phn tch [1]:

Rr = dR,x / dR,c

Trong : dR,x : l ng i ca cht phn tch (cm)

dR,c : l ng i ca cht chun (cm)

(gi tr Rr cng gn 1 th cht phn tch v cht chun cng ng nht)

Ngoi tr s Rf, ngi ta cn da vo mu sc vt hoc qut ph vt nh

tnh cc cht.

Th tinh khit

y l ng dng rt ph bin ca SKLM kim tra s tinh khit ca

cht chit xut t dc liu, l bin php n gin kim tra cc tp cht i

km vi dc cht khi iu ch [1]. Khi khai trin dung dch mu vi cc h

dung mi khc nhau khng pht hin vt l ngoi vt chnh ta c th kt lun

cht chit xut tinh khit. Hin nay ng dng th tinh khit khi chit phn

on v thng dng nht xc nh cht tinh khit khi chy sc k ct.

nh lng v bn nh lng [1]

Bng cc bin php chnh xc ha lng mu a ln bn mng c th

p dng SKLM nh lng. Ty theo phng tin s dng m chnh

xc ca php nh lng cp khc nhau.

C hai cch nh lng cc cht trong vt sc k:

nh lng sau khi tch cc cht ra khi bn mng. Tch chit cht phn tch trong vt sc k bng dung mi thch hp. Sau khi

lm sch dch chit, nh lng cht phn tch bng mt k thut thch hp

(ph hp th, hunh quang, cc ph). Phng php ny hin nay t dng v

c nhiu tr ngi, li mt thi gian.

nh lng trc tip trn bn mng: o din tch hay cng mu ca vt sc k. Hin nay dng 2 k thut:

17

- Densitometer: chiu chm tia vo vt sc k v o cng hp th hoc

hunh quang.

- X l nh vi camera k thut s:

Qut bn mng vi h thng phn tch hnh nh, nht l camera k thut s c

phn gii cao thu nhn hnh nh ca vt sc k. X l d liu nh bng

my tnh.

1.5.6. u nhc im ca phng php sc k lp mng

u im [1]

- Chi ph thit b n gin, khng i hi nhiu thao tc qu kh nh nhiu

phng php khc m vn cho mt chnh xc c th chp nhn c.

- Pht hin c tt c cc cht k c cc cht khng di chuyn theo pha ng

(nm im xut pht).

- Thc hin tch d dng cc mu c nhiu thnh phn: C th thc hin sc

k ng thi 10 20 mu hoc hn, so snh trc tip mu th vi mu chun.

- SKLM cung cp thm thng tin bn nh lng v cc thnh phn chnh ca

thuc v cc tin cht lm thuc, do n cho php nh gi cht lng

thuc.

Nhc im [1]

- Trong mt s trng hp lp li ca tr s Rf thp do thnh phn pha

ng thay i trong qu trnh trin khai sc k.

- Tng gin rng pic sc k do khuch tn v tc dng ca pha ng thp.

18

CHNG II: NGUYN VT LIU, THIT B, NI DUNG

V PHNG PHP NGHIN CU

2.1. Nguyn vt liu, thit b

2.1.1. My mc, thit b

Bn mng silicagel GF254 ca Merck (c). Cn phn tch Mettler Toledo (Thy S) (d=0,1 mg) Cn phn tch Mettler Toledo (Thy S) (d=0,01 mg) My chm sc k CAMAG Nanomat 4 ; my chm sc k Linomat 5.0;

bnh trin khai sc k CAMAG; bnh phun sc k CAMAG; bung soi

bn mng VILBER LOURMAT, h thng CAMAG REPROSTA 3 vi

s h tr ca phn mm winCATS.

H thng my HPLC Agilent Technologies 1260 Infinitive ca M v phn mm Aligent Chemstation version B

My siu m Bandelin-Sonorex super RK 106 (c). Bp un cch thy T sy Shellab (c) T sy chn khng Pipet t ng 10-100l, 100-1000l. Bnh nh mc, pipet thy tinh, ng ong v cc dng c phn tch cn

thit khc.

2.1.2. Ha cht, dung mi, cht chun

Ha cht dung mi :

- Methanol , aceton tinh khit ha hc ca Trung Quc

- Dicloromethan, diethyl ether, butanol, toluen, ethyl acetat tinh khit phn

tch ca Merck, c

19

- Nc tinh khit dng cho sc k

- Acid sulfulric, ethanol Trung Quc

Cht chun:

- Dexamethason cht chun i chiu, SKS: 0197025, hm lng 99,61%

(khan), m 0,13 do Vin kim nghim Trung ng cung cp

- Dexamethason acetat cht chun i chiu, SKS 0200014,hm lng 99,6%

do Vin kim nghim Trung ng cung cp

2.2. i tng, ni dung v phng php nghin cu

2.2.1. i tng nghin cu:

Qua tnh hnh thuc ng dc v thc phm chc nng b trn corticoid v

cc dng bo ch ca chng trn th trng, chng ti chn cc ch phm c

cng dng cha thp khp, cha Gout, hoc cc thuc b dng lm i

tng nghin cu.

Mu trng (nn mu): Ch phm s b kho st khng c cht cn phn tch.

Dng rn: (mu R) Vin Gout Tm Bnh c cng thc:

Cao l si rng

Cao Hy thim

T gii

Ct kh c

M tin ch

ng quy

25 mg

25 mg

60 mg

60 mg

10 mg

60 mg

c hot

Th phc linh

Ngu tt

trng

T dc va 1 vin

60 mg

65 mg

40 mg

65 mg

500 mg

20

Dng lng: (mu L) Siro HTin YBA c cng thc:

Bch trut Hong k ng sm Phc thn Mc hng Trch cam tho

12 g 12 g 06 g 12 g 06 g 04 g

ng quy Vin tr Toan to nhn Acid benzoic ng knh Nc ung va

04 g 04 g 12 g 0,125 g 100 g 125 ml

Mu th: Cc ch phm ng dc c cng dng cha khp, cha Gout thu thp c trn th trng hoc mu thuc do cc c nhn, n v nghi

ng gi ti Vin kim nghim thuc Trung ng

Bng 2.1: Cc mu thuc c phn tch

TT mu

Tn mu Thnh phn Ni sn xut Ngun gc

1 Thuc tr Vim mi

Thc a, ngu tt, i hi..

Thuc gia truyn ng y Thi Nam (Ch Mi, An Giang)

Mu do ch Trnh Th Quy (Vin KN) cung cp

2 Bt thuc ca c s ng nh Ton

Khng ghi trn nhn

Khng r Mu do ch Trnh Th Quy (Vin KN) cung cp

3 Phong t nhc khp thy

c hot, thanh to, trng, ngu tt, ng quy

C s sn xut v kinh doanh thuc YHCT c Th Sanh

Mu do ch Trnh Th Quy (Vin KN) cung cp

4 Vin Gout Tm Bnh

Cao l si rng, ng quy, c hot, trng..

Cng ty TNHH Dc Phm Tm Bnh

Mua ti H Ni

5 Phong t ct thng thy.SK: V1338-H12-10 S l SX:01-12-10

c hot, tang k sinh, trng, ngu tt

C s sn xut thuc thnh phm YHCT An Tin

Mu lu ti Vin kim nghim

21

2.2.2. Ni dung nghin cu

X l mu bng k thut chit rn lng, lng lng

Kho st, chn h dung mi sc k ph hp pht hin v tch r rng

ring bit c 2 cht phn tch ra khi nn mu

Kho st chn iu kin pht hin, nhn bit cc cht trn bn mng

nh gi tnh chn lc, lp li, tm li ca phng php

Xc nh gii hn pht hin cht phn tch

p dng phng php phn tch xy dng xy dng kim tra mt

s mu thuc ng dc v thc phm chc nng

nh gi kt qu phn tch mu th

2.2.3. Phng php nghin cu.

X l mu:

Da trn nguyn tc ca cc k thut x l mu dc liu, tnh cht l ha

ca cht phn tch, qua tham kho ti liu [9], [10], v iu kin thc t,

tin hnh x l mu theo phng php sau:

- Dng rn: s dng phng php chit trc tip bng dung mi aceton.

- Dng lng:s dng phng php chit lng lng bng dung mi ethyl

acetat.

S dng phng php SKLM xy dng iu kin pht hin cht phn

tch trong mu t to c gy nhim cht cn phn tch.

Mu t to: Mu trng c cho thm dexamethason v dexamethason acetat

chun.

- Mu t to dng rn:

Liu dng ca mu trng R l: 3 vin/ln

Liu dng ti thiu ca dexamethason, dexamethason acetat l: 0,5 mg

Do t l trn to mu t to (mu trng + chun) kho st l 0,5 mg

dexamethason v 0,5 mg dexamethason acetat trong 3 vin mu trng R

22

- Mu t to dng lng: T l trn to mu t to (mu trng + chun)

kho st l 0,5 mg dexamethason v 0,5 mg dexamethason acetat trong 10

ml mu trng L

p dng phng php phn tch xy dng xy dng kim tra mt

s mu thuc ng dc v thc phm chc nng. Quan st bn mng

di nh sng t ngoi 254 nm , i chiu Rf ca cc vt ca mu th vi

Rf ca cht chun

So snh nh gi kt qu phn tch mu s dng SKLM vi iu kin

kho st v kt qu phn tch mu bng HPLC vi iu kin phn tch theo

ti liu [10]:

- Ct Inertsil C18 (250 x 4,6 mm; 5m)

- Detector DAD: bc sng 240 nm

- Pha ng: ACN-nc (35:65)

- Tc dng: 1,6 ml/pht

- Qut ph t 200-400 nm

Sau khi tin hnh thc nghim,thu thp d liu, x l v nh gi kt qu

dng phn mm Excel 2007

23

CHNG III. THC NGHIM, KT QU V BN LUN

3.1. Kho st v la chn iu kin phn tch.

3.1.1. Chun b cc dung dch chun gc:

Dung dch chun dexamethason 1 mg/ml trong methanol: Cn chnh xc khong 5 mg dexamethason vo bnh nh mc 5 ml, thm 3 ml

methanol, lc cho tan hon ton. Thm methanol va n vch. Lc

u.

Dung dch chun dexamethason acetat 1 mg/ml trong methanol: Cn chnh xc khong 5 mg dexamethason acetat vo bnh nh mc 5 ml,

thm 3 ml methanol, lc cho tan hon ton. Thm methanol va n

vch. Lc u.

Dung dch chun hn hp: Cn chnh xc khong 10 mg tng cht chun vo bnh nh mc 10 ml, thm 5 ml methanol, lc cho tan hon

ton. Thm methanol va n vch. Lc u.

3.1.2. X l mu

3.1.2.1. Mu rn

Tin hnh chit mu bng phng php chit rn lng, s dng dung

mi l aceton

Quy trnh chit mu rn nh sau:

+ Mu trng R: Ly lng bt thuc tng ng khi lng ca 3 vin mu

trng R vo bnh nn c nt mi, thm 15 ml aceton lc k v siu m 10 pht,

lc. Chit tip 2 ln, mi ln 5 ml aceton. Dch chit c bay hi dung mi

trn cch thy ti cn. Ha tan cn trong 1 ml methanol.

+ Mu t to R: Ly lng bt thuc tng ng khi lng ca 3 vin mu

R vo bnh nn c nt mi, thm vo 1 ml dung dch chun hn hp nng

0,5 mg/ml ri dng a thy tinh khuy, trn k to thnh mu c 2 cht

24

cn nghin cu, tip tc tin hnh quy trnh x l mu nh i vi mu trng

R.

+ Cc mu th: ly lng bt thuc tng ng vi liu dng trong mt ln,

chit theo quy trnh trn.

3.1.2.2. Mu lng

Chit lng lng bng ethyl acetat

Quy trnh chit mu lng:

+ Mu trng L:

- Ly 10 ml mu trng L. Lc k 3 ln vi dung mi chit, mi ln 10 ml.

yn cho tch lp. Gn ly lp dung mi hu c.

- Lc qua giy lc vo mt cc c m.

- Bc hi cch thy n cn.

- Ha tan cn trong 1 ml methanol.

+ Mu t to L: Ly 10 ml mu trng L, thm vo 1 ml dung dch chun

hn hp nng 0,5 mg/ml ri lc k to thnh mu c 2 cht cn nghin cu,

tip tc tin hnh quy trnh x l mu nh trn

+ Mu th: ly 10 ml mu th tin hnh chit nh i vi mu trng L

3.1.3. Kho st, chn h dung mi sc k

Kho st vi cc cht chun (cha c nn mu) Chng ti chn kho st cc h dung mi pha ng sau la chn dung mi

c kh nng tch c r rng ring bit hai cht phn tch.

H DM1: Butanol bo ha nc toluen diethyl ether (5 : 10 : 85)

H DM2: Nc methanol diethyl ether dicloromethan (1,2 : 8 : 15 : 77)

+ Tin hnh:

- Hot ha bn mng silica gel GF254 110oC trong 30 pht.

- Cho dung mi vo bnh trin khai, bo ha hi dung mi bng cch lt giy

lc xung quanh thnh trong bnh giy lc thm u dung mi. y kn bnh

25

v yn 1 gi. Lng dung mi cn ly sao cho sau khi thm u giy lc

cn li mt lp dy 5-10 mm

- Chun b mu: t cc dung dch chun gc chun b mc 3.1.1, pha cc

dung dch chun lm vic c nng 0,5 mg/ml

- Chm cc dung dch chun dexamethason, dung dch chun hn hp, dung

dch chun dexamethason acetat nng 0,5 mg/ml trong methanol trn cng

mt bn mng (chm vch bng my chm sc k Linomat 5.0)

- Th tch chm: 5 l

- Lm kh vt v a bn mng vo bnh trin khai bo ha dung mi

- Trin khai sc k cho n khi dung mi chy c khong 8 cm th ly bn

mng ra v kh.

- Pht hin vt: n t ngoi bc sng 254 nm.

Sau khi tin hnh sc k, kt qu c th hin hnh 3.1 v 3.2.

1 2 3

Dexamethason acetat

Dexamethason

1 2 3

Hnh 3.1. SK sc k vi h DM1

Hnh 3.2. SK sc k vi h DM2

26

1: dung dch chun dexamethason acetat 0,5 mg/ml

2: dung dch chun hn hp 0,5 mg/ml

3: dung dch chun dexamethason 0,5 mg/ml

H DM1: Trn SK, hai cht phn tch tch r rng ring bit, tuy nhin h

s Rf thp (Rf ca dexamethason l 0,21, Rf ca dexamethason acetat l 0,44),

thi gian trin khai sc k di

H DM2: Trn SK, hai cht phn tch tch r rng ring bit, tr s Rf ph

hp (Rf ca dexamethason l 0,57, Rf ca dexamethason acetat l 0,74).

Nhn xt: C hai h dung mi u c kh nng tch tt hai cht phn tch.

Tuy nhin, thi gian phn tch ca h DM2 nhanh hn v tr s Rf ph hp

hn. Do vy, chng ti la chn DM2 tin hnh kho st trn nn mu ch

phm ng dc

Kho st vi mu t to. Tin hnh chit mu trng, mu t to theo quy trnh mc 3.1.2

Chm ln bn mng 5 l dch chit mu t to (trng + chun), dung dch

chun hn hp 0,5 mg/ml trong methanol.

Sau khi trin khai sc k, kh bn mng v pht hin vt bng soi di n

t ngoi bc sng 254 nm

Kt qu s dng h DM2 th hin hnh 3.3 v hnh 3.4.

27

1 2

Dexamethason acetat Dexamethason

1 2

Hnh 3.3. SK kho st DM trn nn mu rn

1: dch chit mu t to R 2: dung dch chun hn hp 0,5 mg/ml

Hnh 3.4. SK kho st DM trn nn mu lng

1: dung dch chun hn hp 0,5 mg/ml 2: dch chit mu t to L

Nhn xt: Kt qu kho st cho thy trn SK, dch chit mu t to cng

xut hin hai vt c Rf tng ng vi hai vt ca dung dch chun hn hp,

hai vt ny tch ra ring bit r rng v tch ra khi cc vt ca nn mu.

Vy c th chn h DM2 lm dung mi trin khai SKLM pht hin nhanh

dexamethason v dexamethason acetat trn tri php trong cc ch phm

ng dc.

3.1.4. Kho st iu kin pht hin

Chm dung dch chun hn hp c nng gim dn

Cch 1: soi n t ngoi c bc sng 254 nm

Cch 2: Phun dung dch acid sulfuric 20% trong ethanol, sy 120oC trong

28

10 pht ri soi n t ngoi bc sng 366 nm. Cc cht phn tch s cho

mu v hunh quang xanh nht.

Hnh 3.5. SK kho st tm iu

kin pht hin (UV-254 nm)

Hnh 3.6. SK kho st tm iu kin

pht hin vt (phun dung dch acid sulfuric trong ethanol)

Nhn xt: Pht hin bng cch phun dung dch acid sulfuric 20% trong

ethanol ri soi n t ngoi bc sng 366 nm c nhy cao hn. Nhng

do iu kin thc t, chng ti ch s dng cch pht hin vt bng n t

ngoi bc sng 254 nm.

Vy iu kin sc k pht hin dexamethason v dexamethason acetat

c la chn l:

- Bn mng silica gel GF254

- Dung mi khai trin: Nc methanol diethyl ether dicloromethan (1,2 :

8 : 15 : 77)

- Th tch chm: 5 l

- Pht hin vt: nh sng t ngoi 254 nm hoc phun dung dch acid sulfuric

20% trong ethanol, sy 120oC trong 10 pht ri soi n t ngoi bc

sng 366 nm.

29

3.2. nh gi phng php

3.2.1. Tnh chn lc

Chun b mu:

- Dung dch chun hn hp nng 0,5 mg/ml: pha long gp i dung dch

chun hn hp nng 1 mg/ml.

- Dung dch mu trng, mu trng thm chun (mu t to) chun b theo quy

trnh mc 3.1.2

Tin hnh sc k cc dung dch mu chun, mu trng, mu trng thm chun.

Kt qu c th hin hnh 3.7 v hnh 3.8

1 2 3

Dexamethason acetat Dexamethason

1 2 3

Hnh 3.7. SK nh gi tnh chn lc (mu rn)

1: dch chit mu trng R 2: dch chit mu t to R 3: chun hn hp 0,5 mg/ml

Hnh 3.8. SK nh gi tnh chn lc (mu lng)

1: chun hn hp 0,5 mg/ml 2: dch chit mu t to L 3: dch chit mu trng L

30

Nhn xt:

- Trn SK ca mu trng thm chun c xut hin hai vt c Rf v mu sc

tng ng hai vt thu c trn SK ca dung dch chun hn hp

- Mu trng khng cho vt tng ng v v tr vi vt dexamethason v

dexamethason acetat chun

Kt lun: Phng php c tnh chn lc cao

3.2.2. Gii hn pht hin

Gii hn pht hin chnh l nng cui cng m mt thng quan st c

hoc l nng m ti c t l S/N bng 3 (tn hiu p ng ca pic gp

khong 3 ln ng nn nhiu sau khi x l hnh nh bng phn mm

Videoscan)

T dung dch chun hn hp gc 1 mg/ml (S1), tin hnh pha long c cc

dung dch chun hn hp c nng gim dn (S2-S5) nh sau:

S1 S2 S3 S4 S5 Nng

1 mg/ml

0,5 mg/ml

0,25 mg/ml

0,125 mg/ml

0,05 mg/ml

Th tch S1(l)

1000 500 250 125 50

Thm MeOH va

1000 1000 1000 1000 1000

Cho vo cc mu trng R ln lt 1ml cc dung dch chun hn hp chun

b trn ri tin hnh chit nh mc 3.1.2.1. Sau , tin hnh sc k vi

iu kin phn tch xy dng c.

+ Quan st bn mng di n t ngoi bc sng 254 nm. Xc nh nng

thp nht cn c th quan st c vt ca 2 cht phn tch trn bn mng.

Kt qu th hin hnh 3.9.

31

1 2 3 4 5 6 7 8

Hnh 3.9. SK gii hn pht hin ca Dexamethason

v Dexamethason acetat

Dung dch chun hn hp nng 0,5 mg/ml: 1, 2

Dung dch chun hn hp nng 0,25 mg/ml: 3,4

Dung dch chun hn hp nng 0,125 mg/ml: 5,6

Dung dch chun hn hp nng 0,05 mg/ml: 7,8

Gii hn pht hin (LOD) ca dexamethason v dexamethason acetat khi pht

hin bng n UV-254 nm ng vi nng mu th 0,25 mg/ml, vi th tch

chm 5 l l: 5 l x 0,25 mg/ml = 1,25 g

+ X l hnh nh bng phn mm Videoscan thu c SK analog.

Kt qu c trnh by hnh 3.10 v bng 3.1.

32

(a)

(b)

(c)

Hnh 3.10. SK analog xc nh LOD

(a). Dung dch chun hn hp nng 0,5 mg/ml

(b). Dung dch chun hn hp nng 0,25 mg/ml

(c). Dung dch chun hn hp nng 0,125 mg/ml

33

Bng 3.1. Kt qu xc nh LOD

Dexamethason Dexamethason acetat Nng

Chiu cao pic

Chiu cao nhiu

S/N Nng

Chiu cao pic

Chiu cao nhiu

S/N

0,5 mg/ml

2601,4 223,1 11,66 0,5 mg/ml

1756,3 223,1 7,87

0,25 mg/ml

1658,7 251,4 6,6 0,25 mg/ml

1109,9 251,4 4,415

0,125 mg/ml

884,2 209,7 4,22 0,125 mg/ml

620,2 209,7 2,96

Vy gii hn pht hin (LOD) ca dexamethason v dexamethason acetat khi

x l hnh nh SK bng phn mm Videoscan l: 5 l x 0,125 mg/ml =

0,625 g/vt

Nhn xt : C th s dng SKLM kho st s b v pht hin nhanh cht

dexamethason v dexamethason acetat c th trn tri php trong thuc ng

dc vi nhy l 1,25 g/vt khi quan st bng mt thng di n t

ngoi bc sng 254 nm v 0,625 g/vt khi x l hnh nh bng phn mm

winCATS.

3.2.3. lp li v tm li ca phng php

lp li c xc nh da vo kt qu phn tch 6 ln song song trn

mu t to R. Kt qu nh gi da vo RSD gia cc ln lm. Yu cu RSD

ca Rf phi nh hn 2%, RSD ca Spic nh hn 10%.

tm li: T p ng phn tch ca mu chun v cc mu th, tnh

tm li theo cng thc sau:

H = Sth / Schun x 100

- Chun b mu:

Mu t to R: chit nh mc 3.1.2.1, tin hnh trn 6 mu nh nhau.

Mu chun: dung dch chun hn hp nng 0,5 mg/ml trong methanol

34

- Chm ln lt 6 dch chit mu t to R v mu chun ln bn mng.

- Trin khai SKLM vi iu kin kho st.

Kt qu th hin hnh 3.11.

1 2 3 4 5 6 7

Hnh 3.11. SK nh gi lp li ca phng php chit

Dch chit mu t to R: 1, 2, 3, 4, 5, 6

Dung dch chun hn hp 0,5 mg/ml

- X l kt qu bng phn mm Videoscan

Kt qu c th hin hnh 3.12

Hnh 3.12. SK chp nh v analog ca dch chit mu t to R

35

Hnh 3.13. SK chp nh v analog ca dung dch chun hn hp

0,5 mg/ml

Bng 3.2: Kt qu xc nh lp li v tm li:

Mu Dexamethason Dexamethason acetat Rf A (din

tch) % tm li Rf A (din

tch) % tm li

1 0,483 92114,6 94,74 0,753 79484,2 74,30 2 0,494 97563,9 100,34 0,757 68904,4 64,41 3 0,490 88402,9 90,92 0,763 68768,7 64,28 4 0,488 101062,9 103,94 0,770 71058,6 66,43 5 0,475 107389,5 110,45 0,769 75814,7 70,87 6 0,489 102535,0 105,45 0,778 76047,8 71,09 GTTB 0,486 98178,13 100,97 0,765 73346,4 68,564 RSD% 1,37 7,13 7,13 1,20 6,00 6,00 Chun 0,5 mg/ml

0,488 97231,9 0,777 106974,9

Nhn xt

+ Bng quan st: nhn thy 6 ln chm lp li ca 6 ln chit c Rf tng i

ngang bng nhau, cng vt tng t nhau.

+ Bng tnh ton da vo kt qu phn tch SK bng phn mm Videoscan

bng thy RSD ca Rf ca dexamethason l 1,37%, ca dexamethason acetat

36

l 1,2%; RSD ca Spic ca dexamethason l 7,13%, ca dexamethason acetat

l 6,00%. Cc gi tr RSD ny nm trong khong p ng yu cu v lp

li.

+ Hiu sut chit dexamethason l 100,97% , ca dexamethason acetat l

68,564 %. Hiu sut chit ny kh cao nn c th s dng quy trnh chit ny

phn tch mu thuc ng dc.

Kt lun: Quy trnh phn tch c lp li ph hp tin hnh kim tra cc

mu th.

3.3. p dng trn mt s mu th

Thu thp mt s ch phm ng dc c cng dng cha khp, thuc b,..

trn th trng hoc mu thuc do cc c nhn, n v nghi ng gi ti Vin

kim nghim thuc Trung ng

- Tin hnh phn tch cc mu th trn theo quy trnh xy dng. Sau khi

trin khai sc k, sy kh bn mng v quan st di nh sng t ngoi 254

nm. Kt qu th hin hnh 3.14 v bng 3.3.

37

1 2 3 4 5 6 7

Hnh 3.14. SK ca mu th

1: Thuc tr vim mi

2 : Bt thuc ca c s ng nh Ton

3: Phong t nhc khp thy

4: Vin Gout Tm Bnh

5: Chun hn hp nng 1 mg/ml

6: Chun hn hp nng 0,5 mg/ml

7: Phong t ct thng thy

38

Bng 3.3: Kt qu kim tra bng SKLM trn 5 ch phm ng dc

STT Tn mu Dexamethason Dexamethason

acetat

1 Thuc tr Vim mi + -

2 Bt thuc ca c s ng nh Ton + -

3 Phong t nhc khp thy - -

4 Vin Gout Tm Bnh - -

5 Phong t ct thng thy + -

- Tin hnh phn tch cc mu th bng phng php HPLC theo iu kin

sc k xy dng trong ti liu tham kho [10]

iu kin sc k:

- Ct Inertsil C18 (250 x 4,6 mm; 5 m)

- Detector DAD: bc sng 240 nm

- Pha ng: ACN-nc (35:65)

- Tc dng: 1,6 ml/pht.

- Qut ph t 200-400 nm

Song song tin hnh phn tch mu chun hn hp.

Kt qu phn tch th hin cc hnh v bng sau:

Hnh 3.15: Sc k chun hn hp dexamethason

v dexamethason acetat

39

Dexamethason Dexamethason acetat

Hnh 3.16. Ph hp th ti pic cht chun

Hnh 3.17. SK mu thuc tr Vim mi

Hnh 3.18. Ph hp th ca mu thuc tr vim mi tng ng

vi tR = 8,539

40

Hnh 3.19.SK mu bt thuc ca c s ng nh Ton

Hnh 3.20. Ph hp th tng ng vi tR = 8,535 ca mu bt thuc ca c

s ng nh Ton

Hnh 3.21. SK mu phong t nhc khp thy

41

Hnh 3.22. SK mu phong t ct thng thy

Hnh 3.23. Ph hp th tng ng vi tR = 8,546 ca mu phong t ct

thng thy

Bng 3.4: Kt qu kim tra bng HPLC trn 5 ch phm ng dc

STT Tn mu Dexamethason Dexamethason acetat

1 Thuc tr Vim mi + -

2 Bt thuc ca c s ng

nh Ton

+ -

3 Phong t nhc khp thy - -

4 Vin Gout Tm Bnh - -

5 Phong t ct thng thy + -

42

Nhn xt: Cc mu th dng tnh trong phng php SKLM cng dng tnh

trong phng php HPLC. l cc mu:

- Thuc tr vim mi ca c s thuc gia truyn ng y Thi Nam

- Bt thuc ca c s ng nh Ton

- Phong t ct thng thy ca c s An Tin

Cc mu th m tnh trong phng php SKLM cng m tnh trong phng

php HPLC

V th c dng phng php SKLM sng lc trc khi phn tch bng

HPLC

3.4. Bn lun

Chun b mu th: i tng kho st l thuc ng dc v thc phm chc nng thuc

nhm c tc dng cha thp khp, nhc xng, thuc lm tng cn, kch

thch n ngon. V th c th thu thp mt s mu ch phm ng dc trn th

trng, kho st s b khng nh trong khng c cht cn phn tch v

s dng n lm nn mu kho st xy dng phng php. Hoc c th xy

dng cng thc mu t to c thnh phn tng t nh mt s ch phm

t dc liu.

gy c tc dng (gim nhanh cc triu chng au nhc, vim..,

lm tng cn, kch thch n ngon), mc liu ti thiu ca dexamethason v

dexamethason acetat l khong 0,5 mg/ln, do vy liu m ngi ta trn vo

trong thuc ng dc cng phi tng ng nh vy. Do vy, chng ti

xy dng cng thc mu t to kho st phng php vi lng 2 cht

phn tch trn vo l 0,5 mg mi cht trong lng mu trng tng ng vi

mt liu dng ca mu trng . i vi mu t to dng rn, chng ti trn

theo t l 0,5 mg mi cht phn tch trong lng bt thuc c trong 3 vin

Gout Tm Bnh (v liu ca ch phm ny l 3 vin/ln). i vi mu t to

43

dng lng, chng ti trn theo t l 0,5 mg mi cht phn tch trong 10 ml

siro HTin YBA.

Phng php phn tch Qua phn tch, chng ti nhn thy kt qu th bng c hai phng php

SKLM v HPLC l tng ng. Nhng mu cho kt qu m tnh vi phng

php SKLM cng cho kt qu m tnh vi HPLC.

Do vy c th s dng phng php SKLM thm d s b khi phn tch

cc cht trc khi s dng HPLC phn tch chng. Phng php SKLM l

phng php n gin, d thc hin, c th phn tch nhiu mu cng mt lc

v trong cng iu kin th nghim vi cht chun.

Phng php SKLM khng tht c hiu, gii hn pht hin cao. Tuy

nhin, trong trng hp ny, chng ti nhn thy vt ca hai cht phn tch

tch nhau r rng ring bit v tch ra khi cc vt ca nn mu.

V vy c th s dng phng php ny pht hin nhanh s c mt ca

dexamethason v dexamethason acetat trn tri php trong cc ch phm

ng dc.

44

KT LUN V XUT Sau khi hon thnh kha lun Nghin cu pht hin Dexamethason trong

mt s ch phm ng dc bng phng php sc k lp mng, chng

ti thu c cc kt qu sau:

1. Kho st, la chn iu kin phn tch ph hp pht hin dexamethason

v dexamethason acetat trn tri php trong ch phm ng dc. C th l:

+ X l mu: i vi dng rn, s dng phng php chit trc tip bng

dung mi aceton. i vi dng lng, s dng phng php chit lng-lng

bng dung mi ethyl acetat.

+ iu kin sc k:

- Bn mng silica gel GF254

- Dung mi khai trin: Nc methanol diethyl ether dicloromethan (1,2 :

8 : 15 : 77)

- Th tch chm: 5 l

- Pht hin vt: soi di nh sng t ngoi 254 nm hoc phun dung dch acid

sulfuric 20% trong ethanol, sy 120oC trong 10 pht ri soi n t ngoi

bc sng 366 nm.

2. nh gi phng php phn tch vi cc ch tiu: tnh chn lc, gii hn

pht hin, lp li v tm li ca phng php.

3. ng dng phng php xy dng xc nh s c mt ca Dexamethason

v dexamethason acetat ca 5 mu ch phm ng dc thu thp c. Kt

qu cho thy trong 5 mu c kim tra th c 3 mu c dexamethason. l

cc mu sau:

- Thuc tr vim mi ca c s thuc gia truyn ng y Thi Nam

- Bt thuc ca c s ng nh Ton

- Phong t ct thng thy ca c s An Tin

45

XUT 1. Tip tc nghin cu, hon thin quy trnh c th p dng phng

php phn tch ny cc phng th nghim

2. Tip tc kho st pht hin cc glucocorticoid khc b trn tri php

trong cc ch phm ng dc nhm m bo an ton cho ngi s

dng. Ngoi ra, tin hnh thm trn mt s thc phm chc nng do

vn thc phm chc nng ang b trn tri php tn dc ang l

mt vn mang tnh thi s.

3. Nghin cu phng php pht hin cc nhm khc trn tri php

vo ch phm ng dc nh: nhm thuc khng histamin, nhm thuc

h nhit gim au, nhm thuc tng cng sinh lc

TI LIU THAM KHO

TI LIU TING VIT

1. Trn T An (2006), Trng i hc Dc H Ni, Ha phn tch, tp

II, tr 214-222.

2. Nguyn Th Kiu Anh (2011), Cc phng php x l mu, Ti liu

ging dy sau i hc, Trng i hc Dc H Ni.

3. Kurt Randerath, Nguyn Hu By (dch) (1974), Sc k lp mng,

NXB Y hc, tr. 6-7, 22-45, 70-126.

4. B Y t (2009), Dc in Vit Nam IV, Nh xut bn Y hc.

5. B Y t (2007), Dc lm sng, Nh xut bn Y hc, tr 210-212

6. B Y t (2002), Dc Th Quc Gia Vit Nam, Nh xut bn Y hc

7. B Y t (2007), Ha dc tp II, Nh xut bn Y hc , tr 57

8. Nguyn Vn n (1985), Phng php nghin cu ha hc cy thuc,

NXB Y hc, tr 42-57.

9. Hong Th Hoa (2007) Nghin cu pht hin dexamethason c th trn

tri php trong thuc ng dc bng phng php sc k, kha lun

tt nghip dc s kha 2003 2007, trng i hc Dc H Ni.

10. Trnh Th Quy (2012), Nghin cu pht hin mt s glucocorticoid

trn tri php trong ch phm ng dc bng sc k lng hiu nng

cao, lun vn thc s dc hc, trng i hc Dc H Ni.

11. Hong Thanh Tm (2008), Xy dng phng php pht hin mt s

cht thuc nhm glucocorticoid trn tri php trong m phm, lun vn

thc s dc hc, trng i hc Dc H Ni.

12. Mai Tt T, V Th Trm (2007), Dc l hc tp II, Nh xut bn Y

hc, tr 290-294.

TI LIU TING ANH

13. Chen Q, Zielinski D, Chen J, Koski A, Werst D, Nowak S (2008),

A validated, stability-indicating HPLC method for the

determination of dexamethasone related substances on

dexamethasone-coated drug-eluting stents, J Pharm Biomed Anal,

48(3), pp 732.

14. Elke Hahn Deinstrop (1999), Applied Thin Layer Chromatography,

Wiley VCH, Germany, tr 53, 57, 61-63, 87-92, 99-112.

15. Garcia CV, Breier AR, Steppe M, Schapoval EE, Oppe TP (2003),

Determination of dexamethasone acetate in cream by HPLC, J

Pharm Biomed Anal,31(3), 597-600.

16. Kumar V, Mostafa S, Kayo MW, Goldberg EP (2006) HPLC

determination of dexamethasone in human plasma and its application to

an in vitro release study from endovascular stents, Pharmazie,61(11)

pp 908.

17. Huetos O, Ramos M, Martin de Pozuelo M, San Andres M, Reuvers

TB (1999), Determination of dexamethasone in feed by TLC and

HPLC, Analyst, 124(11), pp 1583.

18. Joseph Sherma (2003), Handbook of Thin Layer Chromatography,

Newyork, chapter 4 (99-135), chapter 18 (535-565).

19. M Amin (1988), Quantitative thin-layer chromatographic

determination of dexamethasone and dexamethasone sodium

hydrogen sulfate in blood and in pharmaceutical preparations ,

Analytical and Bioanalytical Chemistry, 329(7), pp 778-780.

20. The British Pharmacopoeia 2010.

21. The European Pharmacopoeia 6th Edition

22. The Unitied State Pharmacopoeia 2007.

TRANG WEB

23. Nguyn ng-Nam Phng (2012), Thuc gia truyn kch n b pht

hin trn ng dc, vnexpress.net. http://giadinh.vnexpress.net/tin-

tuc/song-khoe/tre-em/thuoc-gia-truyen-kich-tre-an-bi-phat-hien-tron-

doc-duoc-2279530.html. Ngy truy cp 04/05/2013

24. Nguyn ng Lm (2012), Cht lng dc liu, ng dc trn th

trng hin nay, nhng thch thc, caythuocquy.info.vn.

http://caythuocquy.info.vn . Ngy truy cp 11/05/2013

25. Vng Linh (2010), Him ha khi v tnh dng thuc b trn corticoid,

vnexpress.net.http://giadinh.vnexpress.net/tin-tuc/to-am/hiem-hoa-khi-

vo-tinh-dung-thuoc-bi-tron-corticoid-2272316.html. Ngy truy cp

03/05/2013

26. L Quc Thnh (2009), Cnh gic vi thuc ng y trn tn dc,

suckhoedoisong.vn.http://suckhoedoisong.vn/20090323095045330p44c

60/canh-giac-voi-thuoc-dong-y-tron-tan-duoc.htm. Ngy truy cp

02/05/2013

27. Tng hp (2012), nh ch lu hnh thuc ng y c cha tn dc

paracetamol v dexamethason, songmoi.vn. http://songmoi.vn/doi-

song-suc-khoe/dinh-chi-luu-hanh-thuoc-dong-y-co-chua-tan-duoc-

paracetamol-va-dexamethason. Ngy truy cp 04/05/2013

28. V Tun-Quang Duy (2012), Trn tn dc vo ng dc: La v

hi ngi bnh, dantri.com.vn. http://dantri.com.vn/suc-khoe/tron-tan-

duoc-vao-dong-duoc-lua-va-hai-nguoi-benh-563974.htm. Ngy truy

cp 04/05/2013

PH LC

Hnh nh cc mu th:

Mu 1: Thuc gia truyn ng Y Thi Nam

Mu 2: Bt thuc ca c s ng nh Ton

Mu 3: Phong t nhc khp thy

Mu 4: Vin Gout Tm Bnh

Mu 5: Phong t ct thng thy

/B Y TTRNG I HC DC H NITRNG I HC DC H NIH NI - 2013