xác định một số corticosteroid trong thực phẩm chức năng bằng phương pháp...

B Y T

TRNG I HC DC H NI

NGUYN C THANH

XC NH MT S

CORTICOSTEROID

TRONG THC PHM CHC NNG

BNG PHNG PHP HPLC

KHA LUN TT NGHIP DC S

H NI - 2014

B Y T

TRNG I HC DC H NI

NGUYN C THANH

XC NH MT S

CORTICOSTEROID

TRONG THC PHM CHC NNG

BNG PHNG PHP HPLC

KHA LUN TT NGHIP DC S

Ngi hng dn: ThS. Bi nh Sn

Ni thc hin: Vin KNVSATTP Quc gia

H NI - 2014

LI CM N

Vi tm lng bit n su sc ti xin chn thnh cm n ThS. Bi nh Sn

giao ti v tn tnh hng dn trong sut qu trnh thc hin kha lun.

Ti xin chn thnh cm n cn b, cng nhn vin Vin kim nghim v sinh

an ton thc phm quc gia to mo iu kin thun li ti hon thnh ti.

Ti cng xin chn thnh cm n cc ging vin, nhn vin B mn Ha phn

tch - c cht, Trng i hc Dc H Ni gip ti trong sut qu trnh hc

tp ti trng.

Cui cng, ti xin chn thnh cm n gia nh, bn b quan tm ng vin

ti trong qu trnh hc tp v thc hin kha lun

H ni, ngy 7 thng 5 nm 2014

Nguyn c Thanh

MC LC

Trang

DANH MC CH VIT TT

DANH MC CC BNG

DANH MC CC HNH

T VN 1

CHNG 1. TNG QUAN 2

1.1. Gii thiu mt s steroid trong nghin cu 2

1.2. Vai tr ca nhm corticosteroid 4

1.3. Tnh hnh s dng corticosteroid 5

1.4. Cc phng php xc nh corticosteroid 6

1.4.1. Phng php ELISA 6

1.4.2. Phng php in di mao qun 6

1.4.3. Phng php sc k kh 6

1.4.4. Phng php sc k lng 7

1.5. C s l thuyt phng php sc k lng hiu nng cao 9

1.5.1. Nguyn tc chung ca sc k lng 9

1.5.2. Phn tch nh tnh v nh lng bng HPLC 11

1.5.3. Phng php chit pha rn 11

CHNG 2: I TNG V PHNG PHP NGHIN CU 14

2.1. Nguyn vt liu, thit b 14

2.1.1. Thit b 14

2.1.2. Dng c 14

2.1.3. Ha cht 14

2.2. Ni dung nghin cu 15

2.3. Phng php nghin cu 15

2.3.1. Phng php ly mu 15

2.3.2. Phng php phn tch 15

2.4. Thm nh phng php phn tch 16

2.4.1. Tnh c hiu v chn lc 16

2.4.2. Khong tuyn tnh v ng chun 17

2.4.3. Gii hn pht hin (LOD) v gii hn nh lng (LOQ) 18

2.4.4. lp li ( chnh xc) 19

2.4.5. ng 20

CHNG 3. THC NGHIM, KT QU V BN LUN 22

3.1. La chn phng php phn tch 22

3.2. Ti u iu kin xc nh cortiosteroid trn thit b HPLC 22

3.2.1. La chn ct tch 22

3.2.2. Kho st pha ng 24

3.2.3. Kho st gradient pha ng 26

3.2.4. Cc thng s ti u cho qu trnh tch sc k 28

3.3. Kho st qu trnh x l mu 29

3.3.1. La chn dung mi chit 29

3.3.2. Kho st quy trnh chit pha rn SPE 29

3.4. nh gi phng php phn tch 32

3.4.1. nh gi tnh chn lc ca bc sng phn tch 32

3.4.2. nh gi lp ca h thng 32

3.4.3. Lp ng chun 33

3.4.4. Gii hn pht hin, gii hn nh lng ca phng php 34

3.4.5. lp li v thu hi 36

3.5. Kt qu phn tch mt s mu thc 40

3.6. Bn lun 43

3.6.1. i tng nghin cu 43

3.6.2. Xy dng quy trnh phn tch 43

3.6.2. Kho st quy trnh x l mu 43

3.6.4. Thm nh phng php 44

3.6.5. Kt qu phn tch mu thc 44

KT LUN V KIN NGH 45

TI LIU THAM KHO

PH LC

DANH MC CH VIT TT

T vit tt Ting Anh hoc tn khoa hc Ting Vit

ACN Acetonitrile

AOAC Association of Official Analytical

Chemists

Hip hi cc nh ha hc

phn tch

HPLC High performance liquid

chromatography Sc k lng hiu nng cao

LOD Limit of detection Gii hn pht hin

LOQ Limit of quantification Gii hn nh lng

MeOH Methanol

MS Mass spectrometry Khi ph

ppb Parts per billion Phn t

ppm Parts per million Phn triu

RSD Relative standard deviation lch chun tng i

SPE Solid phase extraction Chit pha rn

UV Ultra violet T ngoi

Vis Visible Kh kin

v/v T l theo th tch

DANH MC CC BNG

Trang

Bng 1.1. Gii thiu mt s corticosteroid 2

Bng 2.1. Cc cht chun corticosteroid 14

Bng 2.2. Yu cu lp li ti cc nng khc nhau theo AOAC 20

Bng 2.3. thu hi chp nhn cc nng khc nhau theo AOAC 21

Bng 2.4. Quy nh v thu hi ca hi ng chu u 21

Bng 3.1. Bng kho st thnh phn pha ng 24

Bng 3.2. Hiu sut qua ct chit pha rn C18 vi dung mi ra gii MeOH 31

Bng 3.3. lp li dung dch chun hn hp 6 corticosteroid 33

Bng 3.4. Cc phng trnh hi quy tuyn tnh ca 6 corticosteroid 34

Bng 3.5. Gii hn pht hin v nh lng ca 6 corticosteroid 35

Bng 3.6. Nng cht phn tch cho vo nn mu cc mc khc nhau 36

Bng 3.7. lp li v thu hi ti nng thm chun thp 37

Bng 3.8. lp li v thu hi ti nng thm chun trung bnh 38

Bng 3.9. lp li v thu hi ti nng thm chun cao 39

Bng 3.10. Kt qu phn tch mu thc 42

DANH MC CC HNH

Trang

Hnh 1.1. Cu trc khung c bn ca steroid 2

Hnh 1.2. Sc k trong phn tch sc k 11

Hnh 1.3. Cc bc tin hnh ca qu trnh chit pha rn 12

Hnh 2.1. Lc quy trnh phn tch mu d kin 16

Hnh 2.2. ng chun trn nn mu thc 18

Hnh 2.3. Xc nh LOD bng cch tnh S/N 19

Hnh 3.1. Sc phn tch trn ct X-Brigde C18 23

Hnh 3.2. Sc phn tch trn ct HiQ SIL C18 23

Hnh 3.3. Sc phn tch vi pha ng H2O THF 24

Hnh 3.4. Sc phn tch vi pha ng H2O MeOH 25

Hnh 3.5. Sc phn tch vi pha ng H2O ACN 25

Hnh 3.6. Sc phn tch vi pha ng KH2PO4 ACN 25

Hnh 3.7. Sc phn tch theo chng trnh 1 26




Hnh 3.11. Sc phn tch iu kin ti u 29

Hnh 3.12. Sc phn tch dung mi v dung dch chun 6 corticosteroid 32

Hnh 3.13. Sc ti gii hn pht hin ca 6 cht phn tch 35

Hnh 3.14. Quy trnh phn tch mu thc 41

1

T VN

Theo nh ngha ca B Y t, thc phm chc nng l thc phm dng h

tr chc nng ca cc b phn trong c th ngi, c tc dng dinh dng, to cho

c th tnh trng thoi mi, tng sc khng v gim bt nguy c bnh tt. Mt s

nh sn xut v kinh doanh li dng lng tin ca ngi tiu dng nn thi

phng qu mc tc dng ca thc phm chc nng khin khng t ngi coi thc

phm chc nng nh l thn dc cha bch bnh.

Corticosteroid l nhm cht c gc steroid c tuyn thng thn ca c th

sn xut ra vi tc dng chng vim mnh, chng d ng v c ch min dch.

Corticosteroid c th s dng x l cc bin chng ca bnh ung th v iu tr

ung th, s dng trong cc trng hp vim khp nghim trng. Song, vic s dng

v iu tr bng corticosteroid cn c ch nh ca bc s, s dng sai mc ch v

liu lng gy ra nhng tc dng ph c th dn ti hu qu nghim trng.

Trong nhng nm va qua, ti Vit Nam cng nh trn th gii lin tip pht

hin cc vi phm trong vn v sinh an ton thc phm: Sa c cha melamine

tng m, sn phm h tr iu tr xng khp t tho dc nhng li cha

dexamethasone, piroxicam, furosemide

Do gip bo v quyn li ngi tiu dng cng nh h tr cc c quan

chc nng kim sot tt vn an ton v sinh thc phm nht l i vi thc phm

chc nng, chng ti tin hnh nghin cu Xc nh mt s corticosteroid trong

thc phm chc nng bng phng php HPLC vi cc mc tiu sau:

+ Xy dng quy trnh pht hin v nh lng mt s corticosteroid trong thc

phm chc nng.

+ ng dng quy trnh xy dng phn tch mt s sn phm thc phm

chc nng ang lu hnh trn th trng.

2

CHNG 1. TNG QUAN

1.1. Gii thiu mt s corticosteroid trong nghin cu

Corticosteroid l nhm cht trong cng thc c khung steroid v c chung cu

to l ol - 21 - dion - 3,20 pregna - 4 - en v rt t ngoi l.

Hnh 1.1. Cu trc khung c bn ca steroid

Di y l mt s corticosteroid c nghin cu trong ti ny:

Bng 1.1 Gii thiu mt s corticosteroid

TT Tn

Cng thc

phn t

Khi

lng

phn t

(g/mol)

Tnh cht vt l Cng thc cu to

1

Cortison acetat

21-(acethyloxy)-

17-

hydroxypregna-

4-ene-3,11,20-

trione

C23H30O6 402,49

Tinh th mu

trng, nhit

nng chy 220-

2400C.

2

Hydrocortison

acetat

(11)-11,17,21-

trihydroxypregn

a-4-ene-3,20-

dione 4-amino

C23H32O6 404,2

Tinh th mu

trng tan tt

trong dung mi

hu c.

3

TT Tn

Cng thc

phn t

Khi

lng

phn t

(g/mol)


3

Prednison

17,21-

dihydroxypregn

a-1,4-diene-

3,11,20-trione

C21H26O5 358,428

Dng tinh th

hoc bt mn

mu trng,

khng mi v

c v hi ng.

4

Prednisolon

(11)-11,17,21-

trihydroxypregn

a-1,4-diene-

3,20-dione

C21H28O5

360,444

Dng bt kt

tinh mu trng,

khng mi. t

tan trong nc,

tan trong dung

mi hu c.

5

Methyl

prednisolon

(6, 11)-

11,17,21-

trihydroxy-6-

methylpregna-

1,4-diene-3,20-

dione

C22H30O5 374,471

Dng bt kt

tinh mu trng,

im nng

chy t 262

264oC.

4

TT Tn

Cng thc

phn t

Khi

lng

phn t

(g/mol)


6

Betamethason

(8S,9R,10S,11S,

13S,14S,16S,17

R)-9-fluoro-

11,17-dihyroxy-

17-(2-

hydroxyacethyl)

-10,13,16-

trimethyl-

6,7,8,9,10,11,12,

13,14,15,16,17-

dodecahydro-

3H-

cyclopenta[]ph

enaanthren-3-

one

C22H29FO5 392,461

Dng tinh th,

bt mu trng,

khng mi.

Nhit nng

chy 231-

234oC.

1.2. Vai tr ca nhm corticosteroid [1], [2]

Da vo tc dng dc l c bn, cc corticosteroid c phn thnh hai dng:

mineralocorticoid v glucocorticoid. Cc mineralocorticoid lm thay i cn bng

nc - in gii bng cch gip ti hp thu natri, bi tit hydro v kali ng thn

xa, gy ph v cao huyt p. Glucocorticoid cng c mt s tc dng ca

mineralocorticoid ng thi tham gia vo mt s chu trnh chuyn ha khc gm:

ti to ng, phn b li m, chuyn ha protein v cn bng calci.

5

Khng vim v c ch min dch: Glucocorticoid c tc dng chng li cc

biu hin ca qu trnh vim, do lm gim tc dng hoc c ch cc cht trung gian

gy vim nh cytokin, ecosanoid, gim gii phng histamin.

Tc dng chuyn ha: Glucocorticoid lm tng ng huyt lc i cung

cp glucose cho cc m ph thuc glucose nh no v tim.

Tc dng ln cht in gii v cn bng nc: Mineralocorticoid liu cao gy

gi natri v nc, ng thi gy o thi kali, iu c th dn n ph v nhc

c. Cc dn xut tng hp t gy tc dng ny. Ngn hp thu calci rut v tng

o thi qua thn nn gim d tr calci trong c th. S o thi Ca2+ qua thn lm

tng calci niu. C th tr calci niu do mineralocorticoid bng li tiu.

1.3. Tnh hnh s dng Corticosteroid

Corticosteroid l nhm thuc c s dng rng ri nht trn th gii trong

nhiu nm qua. Corticosteroid thng c coi l con dao hai li m c hai li

u ht sc sc bn.

Nm 1949, Hench, Richstein v Kendall dng cortison iu tr cho mt

ph n 29 tui gn nh tn ph v chng thp khp. Thnh cng ny m ra cho

nhng ngi bnh thp khp mt nim hy vng ln, v cho n thi im vic

gip cho ngi bnh gim bt au n v em h tr v vi cng vic thng nht

hu nh l iu bt kh khng. Nhng t ph trong iu tr vim khp em li

cho kp ca Hench gii Nobel y hc danh gi vo nm sau , nm 1950.

Corticosteroid tr thnh mt thn dc, cc labo tm cch sn xut cc dng

thuc c th tim, c tc dng nhanh hn, hoc thay i cu trc ha hc ko

di thi gian tc dng ca thuc; cc nh lm sng hc th tm cc ng dng iu tr

trong nhiu loi bnh khc nhau t thp khp, cho n hen suyn, d ng, ph, m

ay, sc thuc, cc bnh l ti cc c quan khc nhau nh thn, da, mch mu,... c

cn nguyn min dch. Trong nhiu trng hp, corticosteroid cho thy nhng kh

nng a dng ca n v ha hn cho vic gii quyt mt s bnh l mn tnh.

Tuy nhin, vic s dng t v i tr cng lm cho ngi ta pht hin ra

nhng tc dng ph ca thuc sm hn. Hng lot cc bin chng nguy him do

6

qu liu corticoid nh suy tuyn thng thn, xut huyt tiu ha, long xng, xp

t sng, g vo ct sng, tng ng mu, tng huyt p, teo c, ri lon tm thn,

trm cm, c thy tinh th, suy gim chc nng min dch gy nhim trng c hi,

chm pht trin th cht tr em lm cho ngnh y phi a ra nhng cnh bo

khn cp cho vic lm dng corticosteroid.

Hin nay Vit Nam, tnh trng lm dng cc loi thuc corticosteroid ang

din ra kh ph bin cc c s khm cha bnh t nhn, thuc nhiu tnh thnh

trong c nc. Rt nhiu ngi bnh ang phi hng ngy gnh chu nhng hu qu

khn lng do vic lm dng cc thuc ny gy ra.

1.4. Cc phng php xc nh corticosteroid.

1.4.1. Phng php ELISA (enzyme-linked immunosorbent assay) [11]

M.Ebrahimi [11] phn tch 17, 20-dihytroxy-4-pregenen-3-one s dng

enzyme acethylcholinesterase lm cht pht hin. Phng php da trn nguyn l

s lin kt c hiu gia khng nguyn v khng th to thnh cng hp khng

nguyn - khng th. Cng hp khng nguyn - khng th ny s tip hp vi khng

th th 2 c nh du bng enzym acethylcholinesterase. Phc hp ny s c

to mu vi thuc th Ellman v o quang bc sng 405nm. Phng php cho

kt qu nhanh chng, chnh xc v nhy cao.

1.4.2. Phng php in di mao qun [9]

Phng php phn tch cortisol t do trong nc tiu c Lokinendi V.Rao v

cng s pht trin nm 1999 [9]. Mu c lm giu bng k thut chit pha rn s

dng ct SPE C18 bin i trn nn cht tr ca polytetrafluoroethylane. Mu nc

tiu c a nhanh qua ct c hot ha trc vi 250l methanol v 1ml

nc ct. Sau , ct c ra 2 ln, mi ln 1ml hn hp aceton : nc t l 1 : 9

(v/v). Cui cng, ra gii vi 80l ACN v phn tch trn h thng in di mao

qun. S dng h m 75mmol/l SDS, 20% ACN (v/v) v 20mmol/l MES (pH 6,0).

ng mao qun ng knh trong 50m x 375m x 37cm. Mu c bm vo h

thng di p sut 5psi trong 20s v nhit ct c duy tr 16 0,10C, detector

7

UV bc sng 254 nm v th 10kV. Phng php cho thu hi 89 - 94%, gii hn

pht hin l 10g/l.

1.4.3. Phng php sc k kh [14]

Theo ti liu hng dn ca hng Thermo Fisher Scientific [14], phng php

GC-MS/MS c th phn tch c cc steroid. Thng s thit b gm: th tch bm

mu l 1l, s dng ct Rtx-CLP 0,32mm x 20m x 0,5micron v tc kh l

1ml/pht. Chng trnh gradient nhit ci t 600C trong 1 pht sau tng ln

2200C tc 400C/pht, tng tip ln 2600C tc 10o/pht v gi 1 pht nhit

2600C, tip tng ln 3000C vi tc 400C/pht v gi 10 pht nhit

ny. Hng kho st thy phng php c nhy cao, khong tuyn tnh t 4pg n

4ng. Gii hn pht hin trong mu nc tiu thm chun l 50ng/ml. Cc steroid c

n 70% c mnh con tng ng nhau khi qut ph.

Cc steroid l cc hp cht c khi lng phn t tng i cao, do mun s

dng phn tch bng phng php sc k kh, yu cu phi c dn xut trc vi

cc thuc th to hp cht d bay hi ri mi tin hnh phn tch. Do , ha

cht cng nh thao tc thc hin phc tp, qu trnh dn xut c th khng hon

ton gy ra sai s phn tch.

1.4.4. Phng php sc k lng

Sc k lng l phng php tch c s dng ph bin nht trong lnh vc

phn tch hin nay v phm vi ng dng rt rng ri.

1.4.4.1. Phng php sc k lng khi ph LC-MS [7], [13], [15]

Robin Difrancesco v cc cng s [13] s dng phng php sc k lng

khi ph xc nh mt s steroid trong huyt thanh ngi. Mu c thm

chun, ly tm loi b cn, dch trong c chit qua ct chit pha rn Oasis HLB

(1ml, 30mg). Dch ra gii c lm kh v ha cn vi 1ml pha ng ri em

phn tch LC-MS/MS. Gii hn pht hin 3,6 - 7,2ppb i vi cc steroid, khong

tuyn tnh t 3,6ppb - 50ppm, thu hi nm trong khong cho php ca cp hm

lng phn tch, s lp li trong ngy c lch chun tng i RSD 0,76%,

trong cc ngy khc nhau c RSD 9,57%.

8

nh lng hm lng prednison, prednisolon, dexamethasone v cortisol

trong dch huyt thanh, tc gi Valerie A.Frerichs, Kathleen M. Tornatore [15] cng

dng phng php LC-MS. Cc cht c chun b trong hn hp dung mi gm

50% methanol v 50% dung dch m acetat 5mM, pH 3,5 nng 1ppm. Mu

c thu thp, thm chun sau c bo qun -700C trong 3 thng. Khi phn

tch, gi ng mu v tin hnh x l chit tch cc steroid. Gii hn pht hin ca

phng php t 0,2 - 0,58ppb v gii hn nh lng ca phng php t 5,4 -

10,7ppb tng ng vi tng hp cht.

Adam Tolgyesi v cng s [7] nm 2011 phn tch cc corticosteroid trong

m ln s dng sc k khi ph. Cc tc gi tin hnh nghin cu i vi 5 cht

thuc nhm corticosteroid bao gm methyl prednisolon, methyl prednison,

prednisolon, dexamethasone, flumethasone. Phng php c thu hi t 81-

100%, gii hn pht hin trong khong 0,1 - 0,3g/kg, thi gian phn tch ch

7,5pht. lch chun tng i trong phng th nghim v ti lp gia cc

ngy phn tch cng cho kt qu trong gii hn cho php.

1.4.4.2. Phng php sc k lng vi detector hunh quang [16]

Nhiu tc gi nghin cu s dng detector hunh quang tng nhy v

chn lc. Bn thn cc steroid khng c kh nng pht hunh quang, v vy khi

s dng phng php ny cc steroid c to dn xut c kh nng pht hunh

quang.

Wei Gao v cng s [16] xc nh cortisol trong tc bng cch cho cortisol to

dn xut vi axit sulfuric trong methanol (70:30 theo th tch) trong 2 pht nhit

700C. Dn xut sau c cho qua ct SPE C18 lm sch v lm giu mu.

Phn tch trn h thng sc k vi cc iu kin: Ct ODS C18 (250cm x 4,6mm x

5m), pha ng gm nc v methanol, detector hunh quang vi bc sng kch

thch l 360nm v pht x l 480nm. Gii hn pht hin ca phng php l

1pg/mg. Hiu sut thu hi qua ct SPE l 81,4 3,3% i vi ni chun, 100,3

5,2% i vi cortisol nng 20ng/ml. lch chun tng i gia cc ngy l

7,3%.

9

1.4.4.3. Phng php sc k lng vi detector UV-Vis [8], [12]

phn tch tn d dexamethasone v prednisolon trong sa b, Eszter Desi v

cng s [8] s dng phng php chit pha rn x l mu v phn tch trn

sc k lng vi detector UV. Phng php s dng ct C18, pha ng gm ACN v

nc ct vi chng trnh gradient, detector 240nm, th tch bm mu 100l.

Phng php phn tch c th ng dng phn tch dexamethason v prednisolon

trong sa t yu cu cho php ca Chu u vi gii hn pht hin ca

dexamethason v prednisolon ln lt l 0,075g/kg v 0,05 g/kg.

Qiang Fu v cng s [12] phn tch 26 hp cht lin quan vi betamethason

bng sc k lng hiu nng cao pha o. Betamethason v cc hp cht lin quan

c chun b trong hn hp dung mi axit acetic v ACN t l 0,1:100 (v/v).

Phng php s dng ct C18 ACE3 (15cm x 4,6mm x 3m), pha ng gm 2 knh:

knh A l 0,1% methanesulfonic acid, knh B l hn hp tert-butanol v 1,4

dioxane t l 7:93 (v/v) vi chng trnh gradient pha ng, tc dng 1ml/pht,

th tch bm mu l 50l, detector UV 254nm. Tng thi gian phn tch l 68 pht.

Phng php tch c cc chun r rng, peak cn i, gii hn nh lng v

gii hn pht hin thu c ca betamethason l 0,05mg/kg v 0,02mg/kg, vi cc

hp cht khc l 0,01mg/kg v 0,02mg/kg.

1.5. C s l thuyt phng php sc k lng hiu nng cao

1.5.1. Nguyn tc chung ca sc k lng [3], [4], [5]

Sc k lng l qu trnh tch xy ra trn ct tch vi pha tnh l cht rn hoc

cht lng v pha ng l cht lng (sc k lng - rn, lng-lng). Mu phn tch

c chuyn ln ct tch di dng dung dch. Khi tin hnh chy sc k, cc cht

phn tch c phn b lin tc gia pha ng v pha tnh. Trong hn hp cc cht

phn tch, do cu trc phn t v tnh cht l ho ca cc cht khc nhau, nn kh

nng tng tc ca chng vi pha tnh v pha ng khc nhau, chng di chuyn vi

tc khc nhau v tch ra khi nhau.

1.5.1.1. Pha tnh trong sc k lng

10

Tu theo bn cht ca pha tnh, trong phng php sc k lng pha lin kt

thng chia lm 2 loi: sc k pha thng (NP-HPLC) v sc k pha o (RP-

HPLC).

+ Sc k pha thng: pha tnh c b mt l cc cht phn cc ( l cc silica

trn hoc cc silica c gn cc nhm ankyl c t cacbon mang cc nhm chc

phn cc: -NH2, -CN)

+ Sc k pha o: pha tnh thng l cc silica c ankyl ho, khng phn

cc, loi thng dng nht l C18H37

u im ca sc k pha o l tch v phn tch cc cht c phn cc rt a

dng: t rt phn cc, t phn cc ti khng phn cc. Hn na, trong rt nhiu

trng hp th thnh phn chnh ca pha ng li l nc nn rt kinh t.

1.5.1.2. Pha ng trong sc k lng

Pha ng trong sc k lng l thnh phn c cho i qua ct lin tc phn

tch cc hp cht trong mt hn hp, c nhng yu cu sau:

+ Pha ng phi tr vi pha tnh

+ Bn vng, n nh v khng b phn hu trong qu trnh chy sc k

+ Ho tan c mu

+ Phi c tinh khit cao, c nht thp v ph hp vi detector

C th chia pha ng lm hai loi:

+ Pha ng c phn cc cao: c thnh phn ch yu l nc, tuy nhin

phn tch cc cht hu c, cn thm cc dung mi gim phn cc. Pha ng

loi ny c dng trong sc k pha ngc.

+ Pha ng c phn cc thp: l cc dung mi t phn cc nh cyclopentan,

n-pentan, n-heptan, n-hexan, 2-chloropropan, cacbondisulfua (CS2), CCl4, toluene

tch mt nhm cht thng thng pha ng mt thnh phn i khi khng

p ng c kh nng ra gii, ngi ta thng phi hp 2 hay 3 dung mi c

c dung mi c phn cc t thp n cao ph hp vi php phn tch. S thay

i thnh phn pha ng i khi din ra theo thi gian, trng hp ny ngi ta gi

l gradient pha ng.

11

1.5.1.3. Detector trong sc k lng

Detector l b phn quan trng quyt nh nhy ca phng php. Tu thuc

bn cht l ho ca cht phn tch m la chn detector cho ph hp

+ Detector quang ph hp th phn t UV-Vis: p dng cho cc cht c kh

nng hp th nh sng trong vng t ngoi (UV) hoc vng kh kin (Vis).

+ Detector hunh quang: s dng pht hin cc cht c kh nng pht hunh

quang. i vi nhng cht khng c kh nng nh vy, cn phi dn xut ho cht

phn tch, gn n vi cht c kh nng pht hunh quang hoc cht phn tch phn

ng vi thuc th to thnh sn phm c kh nng pht hunh quang.

+ Detector dn: ph hp vi cc cht c hot tnh in ho: Cc ion, cc hp

cht c tnh dn in

Hin nay, cc detector hin i ngy cng c pht trin nh: detector PDA,

MS, cho nhy v chn lc tt hn.

1.5.2. Phn tch nh tnh v nh lng bng HPLC [3], [4]

Phn tch nh tnh: i lng c trng cho s tch sc k l thi gian lu ca

cc cht. Da vo thi gian lu ca cht phn tch trong chun v trong dung dch

phn tch nh tnh tng cht trong hn hp.

Hnh 1.2. Sc k trong phn tch sc k

nh lng cht phn tch, dng phng php ng chun, thm chun

hoc so snh.

12

1.5.3. Phng php chit pha rn [5]

Chit pha rn (SPE) l mt phng php chun b mu vi mc ch lm

giu v lm sch mu phn tch t dung dch bng cch hp ph ln ct chit pha

rn. Sau cht phn tch c ra gii bng mt lng nh dung mi thch hp.

Cc cht nh hng c loi b.

Ct SPE c nhi bng nhng ht nh, xp c cc nhm chc khc nhau. Cht

lng qua ct di tc dng ca p sut hoc chn khng.

Cc bc tin hnh trong qu trnh chit pha rn:

Hnh 1.3. Cc bc tin hnh ca qu trnh chit pha rn

Bc 1: Hot ho cht hp ph pha rn

+ Lm t vt liu nhi, chuyn cc nhm chc ca cht hp ph v dng hot

ng

+ Loi khng kh trong cc khong trng trong lp cht hp ph

+ Khng c cht hp ph b kh

Bc 2: Mu v cht phn tch c chy qua ct

+ Cht phn tch c lm giu trn cht hp ph

13

+ Mt vi thnh phn nh hng khc cng c th b gi li

+ Cc thnh phn khng b hp ph b loi ra lm sch mu phn tch

Bc 3: Loi b cc cht gy nh hng ra khi ct

+ Gi li cht phn tch

+ Loi b cc cht gy nh hng, tp bn

Bc 4: Gii hp cht phn tch bng dung mi thch hp

+ Ph v tng tc gia cht phn tch v cht hp ph vi mc ch tch c

cht phn tch vo dung dch ra gii

+ Dung mi c chn ph hp t hiu qu cao ng thi cng t cht gy

nh hng ti php phn tch cng tt.

Hin nay, phng php chit pha rn ngy cng c s dng rng ri v ph

bin do nhng u im vt tri sau:

+ Hiu sut thu hi cao

+ Kh nng lm giu, lm sch cht phn tch ln

+ Gim lng dung mi s dng

+ C kh nng kt hp cc phng php phn tch

+ An ton, n gin, d s dng, c th tin hnh hng lot

Vi i tng nghin cu l thc phm chc nng, thnh phn rt a dng. Vic

lm sch dung dch phn tch trc khi a vo phn tch trn h thng HPLC l rt

quan trng. Khi dung dch phn tch cng sch, s nh hng nn i vi cht phn

tch cng t, ng thi tui th ca ct sc k cng c ko di.

14

CHNG 2. I TNG V PHNG PHP NGHIN CU

2.1. Nguyn vt liu, thit b

2.1.1. Thit b

H thng sc k lng hiu nng cao ca Shimadzu, My lc vortex VELP, My

li tm lnh Hermle, Cn phn tch (0,1mg v 0,01mg), Cn k thut (0,01g), My ct

quay chn khng Eyela.

2.1.2. Dng c

ng ly tm 50ml, ng ong, Cc loi pipet, Mng lc mu 0,2m, Bnh nh

mc, ng nghim thy tinh c nt xoy, B chit pha rn SPE...

2.1.3. Ha cht

+ Cc corticosteroid chun

Bng 2.1. Cc cht chun corticosteroid

STT Tn chun Xut x tinh khit

1 Cortison acetat Vin kim nghim thuc TW 99,7%

2 Hydrocortison acetat Vin kim nghim thuc TW 99,8%

3 Prednison Vin kim nghim thuc TW 99,8%

4 Prednisolon Vin kim nghim thuc TW 99,7%

5 Methyl prednisolon Vin kim nghim thuc TW 99,9%

6 Betamethason Vin kim nghim thuc TW 100,1%

- Dung dch chun gc 1000ppm: cn chnh xc khong 0,1000g trn cn phn

tch 10-4g tng cht chun corticoteroid ha tan v nh mc 100,00ml bng

methanol. Bo qun trong t lnh 40C, s dng trong 6 thng.

- Dung dch chun trung gian 100ppm: ly chnh xc 1,00ml dung dch chun

gc 1000ppm vo bnh nh mc 10,00ml v nh mc ti vch bng methanol.

- Dung dch chun hn hp trung gian 10ppm: ht ln lt 1,00ml mi chun

n 100ppm vo bnh nh mc 10,00ml, nh mc ti vch bng methanol.

- Cc dung dch chun lm vic nng 1, 2, 5ppm c pha trong methanol.

15

+ Cc loi ha cht, dung mi khc: Methanol (Merck 99,9%), Acetonitril

(Merck 99,9%), n-hexan (Merck 99,9%), Kalidihydrophotphat (Merck), Nc ct

dng cho sc k lng.

+ Chun b pha ng: Knh A l dung dch KH2PO4 20mM trong nc, knh B

l ACN.

2.2. Ni dung nghin cu

+ i tng nghin cu: Thc phm chc nng

+ Ni dung nghin cu ca ti ny l:

- Xy dng quy trnh phn tch mt s cht thuc nhm corticosteroid bng

k thut sc k lng vi detector UV

- Ly mu v p dng phng php nh gi mt s mu thc t.

2.3. Phng php nghin cu

2.3.1. Phng php ly mu

+ i tng mu: Thc phm chc nng c tc dng h tr trong vic gim

cn, lm p, tng cng sinh lc, h tr trong iu tr v phng vim xng khp.

+ Phng php ly mu: ngu nhin

+ a im: Thnh ph H ni

+ n v mu ly: hai n v mu (hp, gi, l)/mu

+ Bo qun v lu tr mu: iu kin thng.

2.3.2. Phng php phn tch

+ Nguyn tc: Cc corticosteroid c tch ra khi nn mu bng methanol, c

ui dung mi methanol. Ha tan phn cn trong nc, lm sch qua ct chit pha

rn C18, dch sau khi qua ct SPE c tch v nh lng bng k thut sc k

lng hiu nng cao s dng detector UV.

+ Quy trnh phn tch d kin nh sau:

16

Hnh 2.1. Lc quy trnh phn tch mu d kin

+ Tm tt quy trnh:

- Cn mu vo ng ly tm, thm dung mi chit corticosteroid

- o trn u mu bng my lc Vortex

- Rung siu m tng hiu sut ha tan cht phn tch vo dung mi chit

- Ly tm vi tc cao, phn tch phn dung dch v lng cht phn b

- C quay gn cn, ha cn, cho dch qua ct SPE lm sch v lm giu cht

phn tch

- Bm dch ra gii vo h thng HPLC nh tnh v nh lng cht phn

tch c trong mu.

2.4. Thm nh phng php phn tch [6]

2.4.1. Tnh c hiu v chn lc

+ Tnh c hiu: l kh nng pht hin c cht phn tch khi c mt cc tp

cht khc. Tnh c hiu thng lin quan n vic ch xc nh mt cht phn tch.

+ Tnh chn lc: l khi nim rng hn tnh c hiu, lin quan n phn tch

nhiu cht trong cng mt quy trnh. Tnh chn lc c th bao trm c tnh c hiu

Cn mu

Chit, ly dch

Lc

Dch ra gii

HPLC

C quay gn kh

Ha cn, qua ct SPE C18

17

do cc phng php phn tch thng c nhiu cht cng xut hin, nn khi nim

tnh chn lc thng mang tnh khi qut hn.

+ Cch thc hin: xc nh tnh c hiu/chn lc ca phng php, b tr

th nghim nh sau:

- Phn tch mu trng, lp li ti thiu 6 ln vi tng loi nn mu. Mu trng

phi khng c cho tn hiu cht phn tch.

- Phn tch mu th hoc mu thm chun, lp li ti thiu 6 ln. Mu thm

chun hoc mu th phi c tn hiu cht phn tch.

2.4.2. Khong tuyn tnh v ng chun

+ Khong tuyn tnh ca mt phng php phn tch l khong nng tn

hiu o v nng cht phn tch c quan h tuyn tnh vi nhau.

+ Cch xc nh: cn khong 6 - 10 dung dch chun c nng khc nhau,

thc hin o tn hiu ca cc dung dch v kho st s ph thuc ca tn hiu vo

nng .

+ Xy dng ng chun: C nhiu loi ng chun khc nhau ty thuc

vo cc phng php v k thut khc nhau, c mt s loi ng chun ch yu

sau:

ng chun vi chun tinh khit:

Chun b dy dung dch chun, o tn hiu y tng ng vi nng x. Nu s

ph thuc l tuyn tnh, ta c ng chun l phng trnh ng thng: y = ax + b.

ng chun trn nn mu trng.

Phn tch cc mu trng thm chun vi nng khc nhau, trong khong tuyn

tnh c lng trn. V th ph thuc gia tn hiu o v nng . ng

chun xy dng trn nn mu trng thng cho tin cy cao hn so vi xy dng

trn chun tinh khit v c th loi tr mt phn nh hng ca nn mu. Tuy nhin

thc t kh tm c nn mu trng ph hp. Do , thng s dng phng php

lp ng chun trn nn mu thc.

18

ng chun trn mu thc:

Phn tch mu thc c cho thm cc nng chun khc nhau tng t nh vi

mu trng. V th tn hiu o ph thuc vo nng chun thm, dng ng

chun trn nn mu thc thng c dng nh sau:

Hnh 2.2. ng chun trn nn mu thc

Khi s dng ng chun trn nn mu thc c th loi tr c cc

nh hng ca nn mu n kt qu phn tch.

2.4.3. Gii hn pht hin (LOD) v gii hn nh lng (LOQ)

+ Gii hn pht hin (LOD): l nng thp nht ca cht phn tch m

phng php c th nh tnh c trong iu kin th nghim c th. LOD c th

c xc nh l nng ca cht phn tch m ti tn hiu cht phn tch gp 3

ln tn hiu nn.

Cch xc nh LOD: Phn tch mu nng thp cn c th xut hin tn hiu

ca cht phn tch. S ln phn tch lp li 4 ln. Xc nh t l tn hiu chia cho

nhiu (S/N), trong S l chiu cao tn hiu ca cht phn tch, N l nhiu ng

nn.

LOD c chp nhn ti nng m S/N = 3

19

Hnh 2.3. Xc nh LOD bng cch tnh S/N

+ Gii hn nh lng (LOQ): l nng ti thiu ca mt cht c trong mu

th m ti ta c th nh lng c bng phng php phn tch v cho kt qu

c chnh xc mong mun.

Cch xc nh LOQ: C th xc nh da vo t l tn hiu cht phn tch v

nhiu ng nn. LOQ c th c xc nh l nng ca cht phn tch m ti

t l S/N = 10. Do , c th tnh LOQ t LOD theo cng thc: LOQ = 3

10LOD.

2.4.4. lp li ( chnh xc)

+ lp li ( chm) th hin s gn nhau ca cc kt qu o so vi gi tr

trung bnh, l mc thng nht ca cc kt qu th ring bit khi quy trnh phn

tch c p dng lp li trn cng mt mu. lp li c th hin bng lch

tng i RSD.

+ Cch xc nh: Tin hnh th nghim lp li n ln (thng thc hin 6 ln).

Tnh lch chun tng i RSD ca hm lng cht phn tch. Cc cng thc

tnh ton lin quan nh sau:

i lng o ngha

Gi tr trung bnh: n

ix

xn

i

1

x l gi tr trung bnh ca tp hp cc

gi tr xi.

lch chun: SD1

)( 2

n

xxi SD ni ln mc dao ng ca cc kt

qu o xi xung quanh gi tr trung bnh.

lch tng i: %100x

SDRSD

Biu th s bin ng, phn tn ca cc

kt qu th nghim

20

+ nh gi lp li hay chm ca phng php: yu cu RSD khng c

cao hn gi tr cho php. Di y l tiu chun thng c s dng:

Bng 2.2. Yu cu lp li ti cc nng khc nhau theo AOAC

TT Hm lng % n v RSD (%)

1 100 100% 1,3

2 10 10% 1,8

3 1 1% 2,7

4 0,1 0,1% 3,7

5 0,01 100ppm 5,3

6 0,001 10ppm 7,3

7 0,0001 1 ppm 11

8 0,00001 100ppb 15

9 0,000001 10ppb 21

10 0,0000001 1ppb 30

2.4.5. ng

+ ng ca phng php: ch mc gn nhau gia gi tr trung bnh ca

kt qu th nghim v gi tr thc hoc gi tr c chp nhn l ng ().

+ C nhiu cch tnh ng nh: dng so snh vi phng php chun, tnh

thu hi, s dng vt liu chun... trong nghin cu ny chng ti xc nh

ng thng qua vic xc nh thu hi.

+ Tnh thu hi: thu hi c xc nh da trn k thut thm chun.

thu hi H c tnh nh sau:

%100)(

c

mcm

C

CCH

Trong : Cm+c: Nng ca mu thm chun

Cm: Nng ca mu

Cc: Nng ca chun thm vo mu

+ nh gi thu hi:

Sau khi tnh thu hi, so snh kt qu tnh c vi cc gi tr cho php.

21

Bng 2.3. thu hi chp nhn cc nng khc nhau theo AOAC

TT Hm lng (%) n v thu hi (%)

1 100 100% 98-102

2 10 10% 98-102

3 1 1% 97-103

4 0,1 0,1% 95-105

5 0,01 100ppm 90-107

6 0,001 10ppm 80-110

7 0,0001 1 ppm 80-110

8 0,00001 100ppb 80-110

9 0,000001 10ppb 60-115

10 0,0000001 1ppb 40-120

Bng 2.4. Quy nh v thu hi ca hi ng chu u

TT Hm lng cht n v thu hi (%)

1 1 g/kg 1ppb 50-120

2 Trn 1 g/kg n di 10 g/kg Trn 1 n di 10ppb 70-110

3 10g/kg 10ppb 80-110

22

Chng 3. THC NGHIM, KT QU V BN LUN

3.1. La chn phng php phn tch

Cc steroid trong phn t c ni i lin hp nn c kh nng hp th UV do

c th s dng detector hp th phn t phn tch. Cht phn tch sau khi c

tch khi ct sc k, c dn vo flowcell ca u o v c chiu bi mt chm

tia t ngoi. S hp th tia bc x bi cc cht tan tun theo nh lut Lambert-

Beer.

Tham kho mt s ti liu [2], [5], [8], [12] chng ti tin hnh xc nh cc

corticosteroid trn h thng sc k lng HPLC-UV vi cc iu kin s b nh sau:

+ Ct pha o C18

+ Detector: UV 254nm

+ Tc dng: 1mL/pht

+ Nhit ct: 35oC

+ Th tch bm mu: 20L

3.2. Ti u iu kin xc nh corticosteroid trn thit b HPLC

3.2.1. La chn ct tch

Ct tch c vai tr quan trng trong vic tch cc cht khi nhau. Cc cht

thuc nhm corticosteroid hu nh khng phn cc [2] nn chng ti kho st la

chn phn tch hn hp cht trn mt s ct pha o C18 :

+ Ct Xbridge C18 (150mm 2,1mm 3,5m) v tin ct.

+ Ct HiQ SIL C18 (250mm 4,6mm 5m) v tin ct.

C nh cc iu kin phn tch khc:

+ Pha ng: knh A: dung dch KH2PO4 20mM trong nc; knh B: ACN

+ Detector UV ti bc sng 254nm

+ Tc dng 1ml/pht

+ Th tch bm mu 20L

+ Nhit ct 350C

Kh nng tch 6 corticosteroid trong hn hp c th hin trong sc di

y.

23

Hnh 3.1. Sc phn tch trn ct X-Brigde C18

Hnh 3.2. Sc phn tch trn ct HiQ SIL C18

Vi ct Xbridge, v ng knh trong ca ct nh, th tch ct nh nn cc peak

ra sm v khng tch c khi nhau, thi gian lu cht phn tch ngn rt d ln

vi tp. Hn na, ct ht nh th p sut ct cao, nu hot ng lin tc p sut

cao nh vy bm d xy ra hin tng p sut khng n nh, tc dng khng

chnh xc.

24

Vi ct HiQ SIL C18 cc peak tch c khi nhau, peak tng i nhn v

cn xng. Do vy chng ti la chn ct HiQ SIL C18 (250mm x 4,6mm x 5m)

tin hnh phn tch tip theo.

3.2.2. Kho st pha ng

Pha ng l yu t quyt nh n hiu qu tch sc k. Nhn chung pha ng

c th nh hng n chn lc ca h pha, thi gian lu gi, hiu lc ca ct

tch, phn gii, rng ca peak sc k. kho st pha ng s dng, chng

ti c nh cc iu kin phn tch v thay i cc pha ng khc nhau.

Bng 3.1. Bng kho st thnh phn pha ng

STT H pha ng Kt qu

1 Knh A: H2O - Knh B:

tetrahydrofuran (THF) 50: 50 (v/v)

Tn hiu cht phn tch rt thp, tn

hiu nhiu nn cao

2 Knh A: H2O - Knh B: MeOH

50: 50 (v/v)

p sut ct cao, khng tch c cc

peak

3 Knh A: H2O - Knh B: ACN Cc peak tch tng i, thi gian

phn tch di

4 Knh A: Dung dch KH2PO4

20mM - Knh B: CAN

Cc peak tch tt, p sut ct n nh,

peak cn i

Hnh 3.3. Sc phn tch vi pha ng H2O THF 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 min

0

5

10

15

20

25

30

35

40

45

mVDetector A Ch1:254nm

/12.

999

/14.

147

/18.

232

/23.

808

25

Hnh 3.4. Sc phn tch vi pha ng H2O MeOH

0.0 5.0 10.0 15.0 20.0 25.0 30.0 min

-50

-25

0

25

50

75

100

125

150

175


/8.6

81

/10.

124

/11.

617

/15.

721

/18.

074

/28.

501

/30.

229

Hnh 3.5. Sc phn tch vi pha ng H2O ACN

Hnh 3.6. Sc phn tch vi pha ng KH2PO4 ACN

26

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 min

-5.0

-2.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

20.0


/16.

263

/16.

901

/18.

832

/23.

038

Khi s dng pha ng l nc v dung mi THF, tn hiu cht phn tch cng

mt nng so vi khi s dng h pha ng khc thp hn nhiu. Nu s dng h

pha ng ny, nhy ca phng php khng cao. ng thi, p sut ct khi s

dng h pha ng ny tng i cao, sau mt thi gian phn tch, thi gian lu ca

cht phn tch khng n nh, do vy khng th phn tch hng lot.

Vi h pha ng gm nc v methanol, p sut ct cao v kh nng tch km.

Vi pha ng knh A l nc, knh B l ACN, hn hp c tch ra r rng

nhng thi gian lu ko di, tn hiu ng nn cao v khng phng.

Khi s dng h pha ng gm knh A l dung dch KH2PO4 20mM v knh B

l ACN, cc peak sc k tch nhau r rng, thi gian lu ca cc cht c ra gii

ra hp l. Do vy, chng ti quyt nh la chn pha ng ny.

3.2.3. Kho st gradient pha ng

Cc hp cht corticosteroid c cu trc gn ging nhau. Do vy nhm mc ch

gim thi gian lu, tng hiu qu tch sc k, chng ti c nh cc iu kin khc

v tin hnh kho st pha ng c s bin i nng theo thi gian nhm ti u

ha kh nng tch 6 cht.

Chng trnh 1: C nh t l ACN l 50%

Hnh 3.7. Sc phn tch theo chng trnh 1

Cc hp cht trong hn hp khng tch ra khi nhau, thi gian lu ko di. Hn

na, chn peak dong, peak t lm gim nhy ca tn hiu phn tch.

27

Chng trnh 2:

Thi gian (pht) 0,01 3,00 10,00 13,00 13,01 18,00

% ACN 50 60 100 100 50 50


Vi chng trnh 2, cc cht tch ra c khi nhau mt phn, tuy nhin

hiu lc tch cha tt. c bit, prednison v prednisolon b chp hn vi nhau,

khng tch c. Thi gian lu ca cc cht tng i ngn, do cn gim nng

ACN giai on u.

Chng trnh 3:

Thi gian (pht) 0,01 3,00 8,00 13,00 13,01 18,00

% ACN 40 60 90 90 40 40


28

Thi gian lu ca cc cht c ko di ra mt cht, nhng cc peak vn

cha tch c khi nhau hon ton. c bit, hydrocortison acetat v cortison

acetat b chp vo vi nhau.

Chng ti quyt nh ko di thi gian tng nng ACN trong khong u

v tin hnh tch sc k theo chng trnh 4 di y:

Thi gian (pht) 0,01 6,00 10,01 13,00 13,01 18,00

% ACN 40 60 90 90 40 40


Vi chng trnh ny, cc cht tch ra c khi nhau, thi gian lu hp l,

cc cht c ra gii ra trong khong thi gian t 5 - 14 pht. Tng thi gian phn

tch l 18 pht loi b ht cc tp khc cn li v n nh pha ng v iu

kin ban u.

3.2.4. Cc thng s ti u cho qu trnh tch sc k

Sau khi tin hnh kho st cc iu kin cho qu trnh phn tch trn h thng

HPLC, chng ti quyt nh la chn cc thng s nh sau:

+ Ct HiQ SIL C18 (250mm x 4,6mm x 5m) v tin ct.

+ Pha ng gm 2 knh: knh A l dung dch KH2PO4 20mM v knh B l

ACN, theo chng trnh gradient:

Thi gian (pht) 0,01 6,00 10,01 13,00 13,01 18,00

% ACN 40 60 90 90 40 40

29

+ Detector UV bc sng 254nm

+ Tc dng: 1ml/pht

+ Th tch bm mu: 20l

+ Nhit ct: 35oC

Hnh 3.11. Sc phn tch iu kin ti u

3.3. Kho st qu trnh x l mu

Quy trnh x l mu d kin c trnh by lc 2.1. Vi quy trnh d

kin ny, chng ti tin hnh la chn dung mi chit corticosteroid t thc phm

chc nng v kho st quy trnh lm sch qua ct chit pha rn SPE.

3.3.1. La chn dung mi chit

Theo cc ti liu tham kho [5], [8], [13], chng ti quyt nh la chn dung

mi chit l methanol v hiu qu chit tt, ph bin v gi thnh ph hp.

3.3.2. Kho st quy trnh chit pha rn SPE

Cc sn phm thc phm chc nng thng c nn mu rt phc tp. Nu khng

c lm sch, khi phn tch trn h thng sc k d gy bn ct, sau mt thi gian

lm tng p sut ct, nh hng n hiu lc tch v lm gim tui th ct. ng

thi, tp nhiu cng lm gim tnh chn lc i vi cc cht phn tch. Do cn

phi lm sch mu trc khi bm vo h thng HPLC.

Bn cht ca cc hp cht corticosteroid l t phn cc, do chng ti la chn

ct chit pha rn C18 cho mc ch ny. ng thi khi a mu qua ct chit pha

30

rn cn c kh nng lm giu mu, to iu kin thun li cho phn tch lng vt

v siu vt.

3.3.2.1. Kho st dung mi np mu qua ct SPE

Pha chun hn hp trong hai dung mi l nc v methanol. Sau cho ton b

dung dch chun hn hp qua ct SPE, tc 1ml/pht, hng dch chy qua ct

trong qu trnh np mu, ra tp, ra gii. Sau , cc dch ny c phn tch trn

h thng sc k. Kt qu thu c:

+ Vi hn hp chun c pha trong dung mi methanol: dch qua ct thu c

cht phn tch, dch ra tp khng pht hin cht phn tch, dch ra gii ch thu

c tn hiu rt thp ca cht phn tch c nng gii hn pht hin, c cht

khng c tn hiu trong sc . Nh vy, vi dung mi methanol kh nng lu gi

cht phn tch trn ct SPE rt km.

+ Vi hn hp chun c pha trong nc: dch qua ct v ra tp khng thu

c tn hiu cht phn tch, ch thu c tn hiu ca cht phn tch trong dch sau

qu trnh ra gii. Nh vy, trong trng hp ny kh nng lu gi ca ct SPE l

rt tt.

Do , trong nghin cu ny cht phn tch c ha tan trong nc trc khi

tin hnh lm sch (lm giu) trn ct chit pha rn.

3.3.2.2. Kho st dung mi ra gii

Cho 20ml hn hp chun pha trong nc qua ct SPE C18, tin hnh ra gii

bng 3 dung mi:

+ Ethylacetat,

+ Hn hp methanol : ethylacete t l 50 : 50 (v/v)

+ Methanol

Dch ra gii sau c bm vo h thng sc k, phn tch ng thi chun

v dch ra gii qua ct SPE. Tnh hiu sut thu hi qua ct.

+ Vi dung mi ra gii l ethyl acetat: tn hiu cht phn tch thu c thp.

c bit, khng c tn hiu ca hydrocortison acetat. Hiu sut qua ct t 20-35%

ty tng cht. C th kt lun kh nng ra gii ca ethyl acetat km.

31

+ Vi dung mi ra gii l hn hp methanol - ethyl acetat: h s thu hi qua

ct khi s dng h dung mi ny t 68 - 84% ty tng cht. Nh vy, h dung mi

c th ra gii c hn hp cht phn tch nhng hiu sut cha cao.

+ Vi dung mi ra gii l methanol: kt qu thu c nh sau:

Bng 3.2. Hiu sut qua ct chit pha rn C18 vi dung mi ra gii MeOH

Dung dch chun Ln 1 Ln 2 Ln 3 TB H (%)

Prednisolon

3,30ppm 2,74 2,88 2,72 2,78 84,3

Prednison

3,49ppm 3,31 3,36 3,36 3,34 95,8

Methyl prednisolon

3,34ppm 3,19 3,14 3,17 3,17 94,8

Betamethason

3,34ppm 3,30 3,32 3,31 3,31 99,1

Hydrocortison acetat

3,18ppm 2,88 2,97 2,99 2,95 92,7

Cortison acetat

3,94ppm 3,70 3,68 3,70 3,69 93,8

Nh vy, khi s dng dung mi ra gii methanol hiu sut thu hi tt, t t

84,3% - 99,1%. Do , chng ti quyt nh la chn methanol lm dung mi ra

gii.

Sau khi la chn c dung mi ra gii, chng ti tin hnh kho st th

tch dung mi ra gii. Tin hnh qua ct, ra gii vi 5ml methanol, hng dch ra

gii. Sau , tip tc thm 2ml dung mi methanol qua ct, hng dch sau ct. em

dch thu c bm vo h thng sc k, kt qu vi 5ml dch u hiu sut t

khong 90%, vi 2ml dch sau, khng c tn hiu ca cht phn tch. Vi kt qu

ny, cho thy 5ml dung mi ra gii cht phn tch.

Tm li, quy trnh qua ct SPE l:

+ Hot ha ct: 5ml n-hexan, 5ml methanol, 5ml nc

32

+ Np mu: Cho ton b dung dch cht phn tch pha trong nc qua ct, tc

1ml/pht

+ Ra tp: 5ml nc, 5ml n-hexan

+ Ra gii: 5ml methanol

3.4. nh gi phng php phn tch

3.4.1. nh gi tnh chn lc ca bc sng phn tch

Bm hn hp chun 6 corticosteroid vo h sc k vi cc iu kin ti u,

pht hin bng detector UV 254nm, sc thu c tn hiu ca 6 peak tng i

cn xng, vi thi gian lu t 5 - 15pht. Trong khi , khi bm dung mi

methanol, khng thu c tn hiu peak trong khong thi gian 5 - 15 pht. Nh

vy, ti bc sng 254nm c th m bo tnh chn lc cho phn tch cc

corticosteroid trong ti ny.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

-250

0

250

500

750

1000

uV

Hnh 3.12. Sc phn tch dung mi v dung dch chun 6 corticosteroid

3.4.2. nh gi lp ca h thng

Bm hn hp dung dch 6 cht chun ti nng 3 - 5ppm tng ng tng cht

vo h thng HPLC, tim lp 6 ln v tnh lp li ca thi gian lu (tR) v din

tch peak (S) i vi tng cht.

33

Bng 3.3. lp li dung dch chun hn hp 6 corticosteroid

Chun Ln 1 Ln 2 Ln 3 Ln 4 Ln 5 Ln 6 TB RSD

(%)

Prednisolon

3,30ppm

S 109230 110166 110106 110268 110160 109456 109898 0,40

tR 5,968 5,989 5,975 5,969 5,994 5,943 5,973 0,30

S 178856 179126 179503 179099 178879 178021 178914 0,28

Prednison

3,49ppm tR 6,424 6,479 6,468 6,476 6,449 6,459 6,459 0,32

Methyl

prednisolon

3,34ppm

S 170118 170100 170193 170637 170426 170279 170292 0,12

tR 8,086 8,099 8,077 8,114 8,065 8,075 8,086 0,22

Betamethason

3,34ppm

S 141824 141915 141552 141690 141317 141598 141649 0,15

tR 8,997 9,036 9,013 9,051 9,025 9,037 9,026 0,21

Hydro cortison

acetat

23,18ppm

S 136572 136655 137299 136774 137471 137289 137010 0,28

tR 12,576 12,598 12,573 12,615 12,596 12,612 12,595 0,14

Cortiosone

acetat

3,94ppm

S 146152 145596 144686 145028 145229 145286 145330 0,35

tR 13,916 13,965 13,909 13,954 13,932 13,948 13,940 0,16

Qua kt qu trn cho thy lp li ca thit b tt, lch chun tng i ca

din tch peak t 0,12 - 0,40% v ca thi gian lu t 0,14 - 0,32%. Nh vy, h

thng n nh tt cho phn tch 6 hp cht corticosteroid.

3.4.3. Lp ng chun

T dung dch chun gc, pha cc dung dch chun trung gian v hn hp cc

chun, bm vo h thng sc k. Thit lp phng trnh tng quan gia nng

cht phn tch v din tch peak ca tng cht theo phng trnh hi quy tuyn tnh.

34

Bng 3.4. Cc phng trnh hi quy tuyn tnh ca 6 corticosteroid

TT Cht chun Khong nng

Phng trnh hi qui H s R2

1 Prednisolon 0,33 - 120ppm y = 30081x + 15006 0,999

2 Predisone 0,35 - 140ppm y = 45537x + 31200 0,999

3 Methyl

prednisolon 0,34 - 136ppm y = 47321x + 15728 0,999

4 Betamethason 0,33 - 134ppm y = 35662x + 47543 0,996

5 Hydro cortison

acetat 0,32 - 127ppm y = 35068x + 51852 0,994

6 Cortison acetat 0,39 - 158ppm y = 32417x + 33130 0,998

Nh vy, ng chun ca cc corticosteroid u c h s tng quan tt R2 >

0,99. Khong tuyn tnh ca cc cht tng i rng t khong 0,3ppm n trn

120ppm ty tng cht, v vy rt thun li cho vic xc nh corticosteroid trong

cc i tng khc nhau.

3.4.4. Gii hn pht hin, gii hn nh lng ca phng php.

Tin hnh xc nh LOD: da vo t s S/N = 3 tm gii hn pht hin ca

cht phn tch, gii hn nh lng c tnh t gii hn pht hin:

LOQ = 3

10LOD

Bm vo h thng sc k hn hp 6 chun v thu c sc k . Pha long

hn hp chun n khi trn sc thu c tn hiu cht phn tch so vi nhiu nn

c t l bng 3 v chiu cao.

35

Bng 3.5. Gii hn pht hin v nh lng ca 6 corticosteroid

Cht phn tch

LOD trong

dch bm

sc k

(ppb)

LOD ca

phng

php

(2g/5ml)

(ppb)

LOQ trong

dch bm

sc k (ppm)

LOQ ca

phng

php

(2g/5ml)

(ppm)

Prednisolon 66 165 0,218 0,54

Predisone 70 175 0,231 0,58

Methyl prednisolon 68 170 0,224 0,56

Betamethason 66 165 0,218 0,54

Hydrocortison acetat 64 160 0,211 0,53

Cortison acetat 78 195 0,257 0,64

Hnh 3.13. Sc ti gii hn pht hin ca 6 cht phn tch

Nh vy, gii hn pht hin ca cc cht trong khong 66 - 78ppb v gii hn

inh lng trong khong 0,211 - 0,257ppm. Vi kt qu gii hn pht hin v gii

hn nh lng ny, phng php nhy pht hin cc cht phn tch trong

i tng nghin cu l cc loi thc phm chc nng.

36

3.4.5. lp li v thu hi

y l hai yu t quan trng nh gi hiu qu ca phng php phn tch.

Chng ti tin hnh thm chun trn nn mu thc tin hnh xc nh ng thi

hai yu t ny. S dng mu thc phm chc nng khng pht hin cht phn tch,

thm chun ti 3 mc nng (thp, trung bnh v cao) i vi mi cht. Mi mc

chng ti tin hnh lm lp li 6 ln.

Tin hnh x l mu theo quy trnh kho st, thm chun vo mu sau khi

cn, tin hnh phn tch theo cc thng s ti u la chn.

Bng 3.6. Nng cht phn tch cho vo nn mu cc mc khc nhau

STT Cht phn tch Mc thp

(ppm)

Mc trung

bnh (ppm)

Mc cao

(ppm)

1 Prednisolon 2,64 6,60 66,00

2 Predisone 2,79 6,98 69,80

3 Methyl prednisolone 2,72 6,80 68,00

4 Betamethason 2,67 6,68 66,80

5 Hydrocortison acetat 2,54 6,36 63,60

6 Cortison acetat 3,15 7,88 78,80

37

Bng 3.7. lp li v thu hi ti nng thm chun thp

Chun Ln 1 Ln 2 Ln 3 Ln 4 Ln 5 Ln 6 TB SD RSD

(%)

HTB

(%)

Prednisolon Cthm 2,64 ppm

Cthu li 2,41 2,39 2,38 2,78 2,29 2,58 2,47 0,18 7,21 93,6

Prednison Cthm 2,79ppm

Cthu li 2,83 3,05 2,77 2,71 2,77 2,46 2,76 0,19 6,89 99,1

Methyl

prednisolon

Cthm 2,72ppm

Cthu li 2,75 2,44 2,44 2,84 2,50 2,72 2,62 0,18 6,72 96,1

Betamethason Cthm 2,67ppm

Cthu li 2,92 2,88 2,75 2,83 2,76 2,57 2,78 0,12 4,47 104,3

Hydro cortison

acetat

Cthm 2,54 ppm

Cthu li 2,47 2,38 2,51 2,52 2,37 2,43 2,45 0,06 2,62 96,3

Cortison

acetat

Cthm 3,15ppm

Cthu li 3,01 3,19 3,21 3,05 3,37 2,91 3,12 0,16 5,29 99,1

38

Bng 3.8. lp li v thu hi ti nng thm chun trung bnh

Chun Ln 1 Ln 2 Ln 3 Ln 4 Ln 5 Ln6 TB SD RSD

(%)

HTB

(%)


Cthu li 5,53 5,81 5,53 5,80 6,08 6,44 5,87 0,34 5,95 89,4


Cthu li 7,28 7,26 7,12 6,85 6,56 6,76 6,97 0,29 4,20 99,9

Methyl

prednisolon

Cthm 6,80ppm

Cthu li 5,80 6,69 6,53 6,62 6,66 6,63 6,49 0,34 5,27 95,4


Cthu li 7,36 7,12 6,66 6,41 6,43 7,09 6,84 0,40 5,83 102,5

Hydro cortison

acetat

Cthm 6,36ppm

Cthu li 5,57 5,42 5,46 5,95 6,14 5,60 5,69 0,29 5,08 89,5

Cortison acetat Cthm 7,88ppm

Cthu li 7,97 8,19 8,10 8,10 7,88 7,48 7,95 0,26 3,22 100,9

39

Bng 3.9. lp li v thu hi ti nng thm chun cao

Chun Ln 1 Ln 2 Ln 3 Ln 4 Ln 5 Ln6 TB SD RSD

(%)

HTB

(%)


Cthu li 62,50 60,10 58,71 60,34 56,18 62,51 60,06 2,40 4,00 91,0


Cthu li 66,66 59,94 62,16 63,86 64,12 66,77 63,92 2,63 4,12 91,6

Methyl

prednisolon

Cthm 68,00ppm

Cthu li 72,59 68,06 69,21 63,01 71,05 70,70 69,10 3,37 4,87 101,6


Cthu li 72,77 69,86 67,11 71,95 70,19 63,42 69,22 3,45 4,98 103,6

Hydrocortison

acetat

Cthm 63,60ppm

Cthu li 58,31 57,09 52,07 56,00 53,33 59,23 56,00 2,81 5,02 88,0

Cortison acetat Cthm 78,80ppm

Cthu li 71,95 68,48 72,87 70,95 74,08 74,83 72,19 2,29 3,18 91,6

40

Nh vy:

+ mc thm chun thp, thu hi t t 93,6 - 104,3%, lch chun tng

i trong khong 2,62 7,21%

+ mc thm chun trung bnh, thu hi t t 89,4 - 102,5%, lch chun

tng i trong khong 3,22 5,95%

+ mc thm chun nng cao, thu hi t t 88,0 -103,6%, lch chun

tng i trong khong 3,18 5,02%

So snh vi yu cu ca AOAC [10], hiu sut thu hi v lch chun u nm

trong khong cho php. Nh vy, phng php c chm v ng t yu cu.

3.5. Kt qu phn tch mt s mu thc

Sau khi kho st cc iu kin phn tch v nh gi thm nh phng php,

chng ti cho rng c th s dng phng php ny phn tch 6 corticosteroid trong

thc phm chc nng. Do vy, chng ti p dng phn tch mt s mu thc t ang

c lu hnh trn a bn H Ni theo quy trnh sau:

41

QUY TRNH X L MU THC

Hnh 3.14. Quy trnh phn tch mu thc

Cn 2-5g mu vo ng ly tm 50ml

+ 30ml methanol

+ Hot ha: 5 ml n-hexan, 5ml MeOH,

5ml nc

+ Np mu: tc 1ml/pht

+ Ra tp: 5ml nc, 5ml n-hexan

+ Ra gii: 5ml MeOH

Lc Vortex, rung siu m 30pht

Ly tm, lc dch vo bnh c quay

HPLC

C quay cn khong 5 ml

Ha cn vi khong 20ml nc, cho qua ct SPE C18

42

Bng 3.10. Kt qu phn tch mu thc

TT Tn mu Prednisolon Prednison Methyl

prednisolon Betamethason

Hydrocortison

acetat

Cortison

acetae

1 H tr tng cng sinh lc 1 (-) (-) (-) (-) (-) (-)



4 Tc dng h tr mc tc (-) (-) (-) (-) (-) (-)

5 B thn 1 (-) (-) (-) (-) (-) (-)

6 B thn 2 (-) (-) (-) (-) (-) (-)

7 H tr iu tr xng khp 1 (-) (-) (-) (-) (-) (-)

8 H tr iu tr xng khp 2 (-) 0,62 ppm (-) (-) (-) (-)

9 Phng nga bnh gout (-) (-) (-) (-) (-) (-)

Ghi ch: (-) l khng pht hin cht phn tch trong mu

Trong 9 mu phn tch, hu ht khng pht hin cc corticosteroid, ring mu h tr iu tr xng khp 2 pht

hin thy nng prednison mc 0,62ppm trong mu. M thnh phn ny khng c cng b trn nhn ca sn phm.

43

3.6. Bn lun

3.6.1. i tng nghin cu

Hin tng thm cc cht c vo thc phm ni chung v thc phm chc

nng ni ring ngy cng tr thnh hin tng ph bin, gy nhiu bc xc cho

ngi s dng. V vy, chng ti la chn ti ny nhm xy dng phng php

xc nh corticosteroid trong thc phm chc nng, t gip cc c quan chc

nng kim sot tt vn an ton v sinh thc phm nht l i vi thc phm chc

nng, v bo v quyn li ca ngi s dng.

Tuy nhin trong ti ny chng ti cha c iu kin ly mu i din m mi

thc hin ly mu ngu nhin trn a bn H ni vi s lng mu cn nh.

3.6.2. Xy dng quy trnh phn tch

Phng php sc k lng rt ph hp cho vic xc nh steroid nn n c s

dng rng ri. Cc tc gi nghin cu bng phng php sc k lng trn nhiu loi

detector khc nhau. Trong detector UV l loi detector thng dng nht v c

s dng sm nht. V cc steroid trong phn t c nhiu ni i lin hp nn c kh

nng hp th ti cc bc sng ngn, nn s dng detector hp th phn t phn

tch l ph hp. Chng ti quyt nh la chn phng php HPLC-UV xc nh

cc corticoid, phng php ny s c kh nng ng dng rng ri trong cc phng

kim nghim do tnh ph bin ca thit b. Sau khi kho st, chng ti tm c

c iu kin ti u cho qu trnh xc nh trn h thng HPLC, s dng ct tch

ph bin, dung mi pha ng thng dng, gi thnh thp.

3.6.3. Kho st quy trnh x l mu

Qua tham kho ti liu, chng ti la chn methanol l dung mi chit

corticoid t nn mu thc phm chc nng. Tuy nhin, cc sn phm thc phm

chc nng, thnh phn a dng t ngun nguyn liu sn xut ban u, do nn

mu phn tch rt phc tp. Nu khng c lm sch, khi phn tch trn h thng

sc k d gy bn ct, sau mt thi gian phn tch, p sut ct tng, hnh dng pic

xu, tui th ct ngn. ng thi, tp nhiu cng lm gim tnh chn lc i vi

cc cht phn tch. Do , cn c bc lm sch qua SPE. ng thi, khi mu c

44

x l vi k thut chit pha rn, quy trnh ny s lm giu mu ln mt h s thch

hp, thun li cho phn tch lng vt.

Qua thc nghim, chng ti chn c cc iu kin cho qu trnh chit pha

rn vi hiu sut thu hi khong 90%.

3.6.4. Thm nh phng php

Sau khi xy dng quy trnh phn tch, chng ti tin hnh thm nh phng

php theo AOAC. Cc tiu ch thm nh u t theo yu cu ca AOAC, khong

tuyn tnh ca phng php rng, cho php nh lng corticoid trong cc i

tng a dng.

3.6.5. Kt qu phn tch mu thc

Do iu kin hn ch v thi gian nn chng ti mi phn tch c 9 mu thc

t v pht hin 1 mu c s xut hin ca corticoid. Tuy nhin, hm lng thu c

thp nn c th l dng tnh gi. Hn na, cch ly mu khng i din nn cha

th kt lun chnh xc v mu thc phm chc nng ny.

45

KT LUN V KIN NGH

Sau khi tin hnh nghin cu ti xc nh hm lng mt s

corticosteroid trong thc phm chc nng bng phng php sc k lng hiu nng

cao vi detector UV, chng ti thu c cc kt qu sau:

1. ti u ha c cc iu kin chy sc k cho xc nh 6 corticosteroid

bao gm:

+ Kho st c cc iu kin cho phn tch sc k: ct sc k HiQ SIL C18

(250mm 4,6mm 5m); pha ng gm: knh A l dung dch KH2PO4 20mM

trong nc, knh B l ACN; chng trnh ra gii gradient, detector UV bc sng

254nm.

+ Xy dng c quy trnh lm sch mu bng ct chit pha rn SPE C18: Mu

c chit trong methanol, c gn cn ri ha cn trong nc trc khi qua ct,

dung mi ra gii l methanol vi th tch l 5ml.

2. Xy dng ng chun t 0,3ppm n trn 120ppm, vi 6 cht u c h s

tng quan R2 > 0,99, phng php c gii hn pht hin cc corticosteroid t 66

78ppb, gii hn nh lng t 0,211 - 0,257ppm, lch chun tng i t 2,62

7,21% v thu hi t t 88,0 104,3%.

3. phn tch 9 mu thc phm chc nng lu hnh trn a bn H Ni, v

pht hin 1 mu c cha prednison.

T cc kt qu thu c, chng ti nhn thy phng php ny c th p dng

phn tch 6 corticosteroid vi tin cy cao. Trong thi gian ti, chng ti s tip

tc nghin cu phng php phn tch ng thi nhiu cht thuc nhm steroid

hn. V m rng i tng phn tch trn cc i tng khc nhau khng ch trong

phm vi thc phm chc nng.

TI LIU THAM KHO

Ting Vit

1. B Y t (2007), Dc l hc tp 2, Nh xut bn Y hc, H ni.

2. B Y t (2007), Ha dc tp 2, Nh xut bn Y hc, H ni.

3. Trn T Hiu, T Vng Nghi, Nguyn Vn Ri, Nguyn Xun Trung (2007), Ha

hc phn tch phn 2, Nh xut bn Khoa hc v k thut, H ni.

4. Cao Minh Quang (2006), Sc k lng hiu nng cao, Nh xut bn Y hc, Thnh

ph H Ch Minh.

5. Nguyn Vn Ri (2009), Cc phng php tch, khoa Ha hc trng i hc

Khoa hc T nhin, i hc Quc gia H ni.

6. Vin Kim nghim An ton V sinh thc phm quc gia (2010), Thm nh

phng php trong phn tch ha hc v vi sinh, Nh xut bn Khoa hc v k

thut, H ni.

Ting Anh

7. Adam Tolgyesi, Virender K.Sharma, Jeno Fekete (2011), Development and

validation of a method for determination of corticosteroids in pig fat using liquid

chromatography - tandem mass spectrometry, Journal of Chromatography B, 879,

403 - 410.

8. Eszter Desi, Agnes Kovacs, Zoltan Palotai, Aniko Kende (2008), Analysis of

dexamethasone and prednisolone residues in bovine milk using matrix solid phase

dispersion-liquid chromatography with ultraviolet detection, Microchemical

Journal, 89, 77-81.

9. Lokinendi V.Rao, John R.petersen, Michael G.Bisell, Anthony O.Okorodudu,

Amin A. Mohammad (1999), Development of a urinary free cortisol assay using

solid-phase extraction-capillary electrophoresis, Journal of Chromatography B,

730, 123-128.

10. Magali Hay, Piere Mormede (1997), Improved determination of urinary

cortisol and cortisone, or corticosterone and 11-dehydrocorticosterone by high-

performance liquid chromatography with ultraviolet absorbance detection, Journal

of Chromatography B, 702, 33-39.

11. M.Ebrahimi A rapid ELISA method for 17, 20-dihydroxy-4-pregenen-3-one

(17,20P) hormone using Acethylcholinesterase as tracer, Original artical.

12. Qiang Fu, Minshan Shou, Dwight Chien, Robert Markovich, Abu M.Rustum

(2010), Development and validation of a stability- indicating RP-HPLC method

for assay of betamethasone and estimation of its relates compounds , Journal of

Pharmaceutical and Biomedical analysis, 51, 617-625.

13. Robin Difrancesco, Valerie Frerichs, Julie Donnelly, Colleen Hagler, Jill

Hochreiter, Kathleen M. Tornatoer (2007), Simultaneous determination of

cortisol, dexamethasone, methylprednisolone, prednisone, prenisolone,

mycophenolic acid and mycophenolic acid glucuronide in human plasma utilizing

liquid chromatography tandem mass spectrometry, Journal of Chromatography

B, 859, 42-51.

14. Thermo Fisher Scientific, Developing a GC-MS/Ms method for the analysis of

steroid using mass frontier, Austin, TX, USA, Application Note 10050.

15. Valerie A.Frerichs, Kathleen M. Tornatore (2004), Determination of the

glucocorticoids prednisone, prednisolone, dexamethansone and cortisol in human

serum using liquid chromatography coupled to tandem mass spectrometry, Journal

of Chromatography B, 802, 329-338.

16. Wei Gao, Qiaozhen Xie, Jing Jin, Ting Qiao, Haiyang Wang, Liqin Chen,

Huihua Deng, Zuhong Lu (2010), HPLC-FLU detection of cortisol distribution in

human hair, Clinical Biochemistry , 43, 677-682.

PH LC

Ph lc A: Xy dng ng chun v khong tuyn tnh

Hnh A.1. th biu din mi tng quan gia din tch v nng prednison

Hnh A.2. th biu din mi tng quan gia din tch v nng prednison

Hnh A.3. th biu din mi tng quan gia din tch v nng

methylprednisolon

Hnh A.4. th biu din mi tng quan gia din tch v nng betamethason


hydrocortison acetate


cortison acetate

Ph lc B: Sc phn tch hn hp chun

Hnh B.1. Sc dung dch chun hn hp mc nng 1ppm



Ph lc C: Sc cc mu thm chun xc nh lp li v thu hi

Hnh C.1 Sc mu thm chun mc thp

Hnh C.2. Sc mu thm chun mc trung bnh

Hnh C.3. Sc mu thm chun mc cao

Ph lc D: Sc phn tch mu thc

Hnh D.1. Sc phn tch mu tc dng tng cng sinh lc1

khng pht hin corticosteroid

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

50

100

150

200

250

300

350

400

450

500

550

600


Hnh D.2. Sc phn tch mu tc dng tng cng sinh lc 2


Hnh D.3. Sc phn tch mu tc dng tng cng sinh lc 3 khng pht hin

corticosteroid

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

5

10

15

20

25

30

35

40

45

50

55

60


Hnh D.4. Sc phn tch mu tc dng mc tc khng pht hin corticosteroid

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

25

50

75

100

125

150

175

200

225

250

275

300

325mV

Detector A Ch1:254nm

Hnh D.5. Sc phn tch mu b thn 1 khng pht hin corticosteroid

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

10

20

30

40

50

60

70

80

90

100

110

120

130mV

Detector A:254nm

Hnh D.6. Sc phn tch mu b thn 2 khng pht hin corticosteroid

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

5

10

15

20

25

30

35

40

mVDetector A:254nm

Hnh D.7. Sc phn tch mu h tr iu tr xng khp 1


0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

5

10

15

20

25

30

35

40

45

50

mVDetector A:254nm

/2.91

7/32

246

/3.56

3/116

6355

Hnh D.8. Sc phn tch mu h tr phng nga bnh Gout khng pht hin

corticosteroid

Hnh D.9. Sc phn tch mu h tr iu tr xng khp 2 pht hin c

prednisone

Bia.pdfMuc luc.pdfDanh muc viet tat.pdfDanh muc bang.pdfBng 3.2. Hiu sut qua ct chit pha rn C18 vi dung mi ra gii MeOH 31 Bng 3.3. lp li dung dch chun hn hp 6 corticosteroid 33

Danh muc hinh.pdf Trang Hnh 1.1. Cu trc khung c bn ca steroid 2 Hnh 3.12. Sc phn tch dung mi v dung dch chun 6 corticosteroid 32

Noi dung.pdfT VN CHNG 1. TNG QUAN 1.1. Gii thiu mt s corticosteroid trong nghin cu Hnh 1.1. Cu trc khung c bn ca steroid

1.2. Vai tr ca nhm corticosteroid [1], [2] 1.3. Tnh hnh s dng Corticosteroid 1.4. Cc phng php xc nh corticosteroid. 1.4.1. Phng php ELISA (enzyme-linked immunosorbent assay) [11] 1.4.2. Phng php in di mao qun [9] Phng php phn tch cortisol t do trong nc tiu c Lokinendi V.Rao v cng s pht trin nm 1999 [9]. Mu c lm giu bng k thut chit pha rn s dng ct SPE C18 bin i trn nn cht tr ca polytetrafluoroethylane. Mu nc tiu c a nhanh qua ct c hot ha trc vi 250l methanol v 1ml nc ct. Sau , ct c ra 2 ln, mi ln 1ml hn hp aceton : nc t l 1 : 9 (v/v). Cui cng, ra gii vi 80l ACN v phn tch trn h thng in di mao qun. S dng h m 75mmol/l SDS, 20% ACN (v/v) v 20mmol/l MES (pH 6,0). ng mao qun ng knh trong 50m x 375m x 37cm. Mu c bm vo h thng di p sut 5psi trong 20s v nhit ct c duy tr 16 0,10C, detector UV bc sng 254 nm v th 10kV. Phng php cho thu hi 89 - 94%, gii hn pht hin l 10g/l. 1.4.3. Phng php sc k kh [14] Theo ti liu hng dn ca hng Thermo Fisher Scientific [14], phng php GC-MS/MS c th phn tch c cc steroid. Thng s thit b gm: th tch bm mu l 1l, s dng ct Rtx-CLP 0,32mm x 20m x 0,5micron v tc kh l 1ml/pht. Chng trnh gradient nhit ci t 600C trong 1 pht sau tng ln 2200C tc 400C/pht, tng tip ln 2600C tc 10o/pht v gi 1 pht nhit 2600C, tip tng ln 3000C vi tc 400C/pht v gi 10 pht nhit ny. Hng kho st thy phng php c nhy cao, khong tuyn tnh t 4pg n 4ng. Gii hn pht hin trong mu nc tiu thm chun l 50ng/ml. Cc steroid c n 70% c mnh con tng ng nhau khi qut ph. Cc steroid l cc hp cht c khi lng phn t tng i cao, do mun s dng phn tch bng phng php sc k kh, yu cu phi c dn xut trc vi cc thuc th to hp cht d bay hi ri mi tin hnh phn tch. Do , ha cht cng nh thao tc thc hin phc tp, qu trnh dn xut c th khng hon ton gy ra sai s phn tch. 1.4.4. Phng php sc k lng Sc k lng l phng php tch c s dng ph bin nht trong lnh vc phn tch hin nay v phm vi ng dng rt rng ri. 1.4.4.1. Phng php sc k lng khi ph LC-MS [7], [13], [15] 1.4.4.2. Phng php sc k lng vi detector hunh quang [16] phn tch tn d dexamethasone v prednisolon trong sa b, Eszter Desi v cng s [8] s dng phng php chit pha rn x l mu v phn tch trn sc k lng vi detector UV. Phng php s dng ct C18, pha ng gm ACN v nc ct vi chng trnh gradient, detector 240nm, th tch bm mu 100l. Phng php phn tch c th ng dng phn tch dexamethason v prednisolon trong sa t yu cu cho php ca Chu u vi gii hn pht hin ca dexamethason v prednisolon ln lt l 0,075g/kg v 0,05 g/kg. Qiang Fu v cng s [12] phn tch 26 hp cht lin quan vi betamethason bng sc k lng hiu nng cao pha o. Betamethason v cc hp cht lin quan c chun b trong hn hp dung mi axit acetic v ACN t l 0,1:100 (v/v). Phng php s dng ct C18 ACE3 (15cm x 4,6mm x 3m), pha ng gm 2 knh: knh A l 0,1% methanesulfonic acid, knh B l hn hp tert-butanol v 1,4 dioxane t l 7:93 (v/v) vi chng trnh gradient pha ng, tc dng 1ml/pht, th tch bm mu l 50l, detector UV 254nm. Tng thi gian phn tch l 68 pht. Phng php tch c cc chun r rng, peak cn i, gii hn nh lng v gii hn pht hin thu c ca betamethason l 0,05mg/kg v 0,02mg/kg, vi cc hp cht khc l 0,01mg/kg v 0,02mg/kg. 1.5.1. Nguyn tc chung ca sc k lng [3], [4], [5] 1.5.2. Phn tch nh tnh v nh lng bng HPLC [3], [4] Hnh 1.3. Cc bc tin hnh ca qu trnh chit pha rn

CHNG 2. I TNG V PHNG PHP NGHIN CU 2.2. Ni dung nghin cu 2.3. Phng php nghin cu 2.3.2. Phng php phn tch Chng 3. THC NGHIM, KT QU V BN LUN 3.1. La chn phng php phn tch 3.2. Ti u iu kin xc nh corticosteroid trn thit b HPLC 3.2.1. La chn ct tch 3.2.2. Kho st pha ng 3.3. Kho st qu trnh x l mu 3.3.1. La chn dung mi chit Theo cc ti liu tham kho [5], [8], [13], chng ti quyt nh la chn dung mi chit l methanol v hiu qu chit tt, ph bin v gi thnh ph hp. 3.3.2. Kho st quy trnh chit pha rn SPE Cc sn phm thc phm chc nng thng c nn mu rt phc tp. Nu khng c lm sch, khi phn tch trn h thng sc k d gy bn ct, sau mt thi gian lm tng p sut ct, nh hng n hiu lc tch v lm gim tui th ct. ng thi, tp nhiu cng lm gim tnh chn lc i vi cc cht phn tch. Do cn phi lm sch mu trc khi bm vo h thng HPLC. Bn cht ca cc hp cht corticosteroid l t phn cc, do chng ti la chn ct chit pha rn C18 cho mc ch ny. ng thi khi a mu qua ct chit pha rn cn c kh nng lm giu mu, to iu kin thun li cho phn tch lng vt v siu vt. 3.3.2.1. Kho st dung mi np mu qua ct SPE Pha chun hn hp trong hai dung mi l nc v methanol. Sau cho ton b dung dch chun hn hp qua ct SPE, tc 1ml/pht, hng dch chy qua ct trong qu trnh np mu, ra tp, ra gii. Sau , cc dch ny c phn tch trn h thng sc k. Kt qu thu c: + Vi hn hp chun c pha trong dung mi methanol: dch qua ct thu c cht phn tch, dch ra tp khng pht hin cht phn tch, dch ra gii ch thu c tn hiu rt thp ca cht phn tch c nng gii hn pht hin, c cht khng c tn hiu trong sc . Nh vy, vi dung mi methanol kh nng lu gi cht phn tch trn ct SPE rt km. + Vi hn hp chun c pha trong nc: dch qua ct v ra tp khng thu c tn hiu cht phn tch, ch thu c tn hiu ca cht phn tch trong dch sau qu trnh ra gii. Nh vy, trong trng hp ny kh nng lu gi ca ct SPE l rt tt. Do , trong nghin cu ny cht phn tch c ha tan trong nc trc khi tin hnh lm sch (lm giu) trn ct chit pha rn. 3.3.2.2. Kho st dung mi ra gii Cho 20ml hn hp chun pha trong nc qua ct SPE C18, tin hnh ra gii bng 3 dung mi: + Ethylacetat, + Hn hp methanol : ethylacete t l 50 : 50 (v/v) + Methanol Dch ra gii sau c bm vo h thng sc k, phn tch ng thi chun v dch ra gii qua ct SPE. Tnh hiu sut thu hi qua ct. + Vi dung mi ra gii l ethyl acetat: tn hiu cht phn tch thu c thp. c bit, khng c tn hiu ca hydrocortison acetat. Hiu sut qua ct t 20-35% ty tng cht. C th kt lun kh nng ra gii ca ethyl acetat km. + Vi dung mi ra gii l hn hp methanol - ethyl acetat: h s thu hi qua ct khi s dng h dung mi ny t 68 - 84% ty tng cht. Nh vy, h dung mi c th ra gii c hn hp cht phn tch nhng hiu sut cha cao. + Vi dung mi ra gii l methanol: kt qu thu c nh sau: Bng 3.2. Hiu sut qua ct chit pha rn C18 vi dung mi ra gii MeOH + Hot ha ct: 5ml n-hexan, 5ml methanol, 5ml nc + Np mu: Cho ton b dung dch cht phn tch pha trong nc qua ct, tc 1ml/pht + Ra tp: 5ml nc, 5ml n-hexan + Ra gii: 5ml methanol 3.4. nh gi phng php phn tch 3.4.1. nh gi tnh chn lc ca bc sng phn tch Bm hn hp chun 6 corticosteroid vo h sc k vi cc iu kin ti u, pht hin bng detector UV 254nm, sc thu c tn hiu ca 6 peak tng i cn xng, vi thi gian lu t 5 - 15pht. Trong khi , khi bm dung mi methanol, khng thu c tn hiu peak trong khong thi gian 5 - 15 pht. Nh vy, ti bc sng 254nm c th m bo tnh chn lc cho phn tch cc corticosteroid trong ti ny. Hnh 3.12. Sc phn tch dung mi v dung dch chun 6 corticosteroid 3.4.2. nh gi lp ca h thng Bm hn hp dung dch 6 cht chun ti nng 3 - 5ppm tng ng tng cht vo h thng HPLC, tim lp 6 ln v tnh lp li ca thi gian lu (tR) v din tch peak (S) i vi tng cht. Bng 3.3. lp li dung dch chun hn hp 6 corticosteroid Qua kt qu trn cho thy lp li ca thit b tt, lch chun tng i ca din tch peak t 0,12 - 0,40% v ca thi gian lu t 0,14 - 0,32%. Nh vy, h thng n nh tt cho phn tch 6 hp cht corticosteroid. 3.4.3. Lp ng chun 3.4.4. Gii hn pht hin, gii hn nh lng ca phng php. 3.4.5. lp li v thu hi 3.6. Bn lun 3.6.1. i tng nghin cu Hin tng thm cc cht c vo thc phm ni chung v thc phm chc nng ni ring ngy cng tr thnh hin tng ph bin, gy nhiu bc xc cho ngi s dng. V vy, chng ti la chn ti ny nhm xy dng phng php xc nh corticosteroid trong thc phm chc nng, t gip cc c quan chc nng kim sot tt vn an ton v sinh thc phm nht l i vi thc phm chc nng, v bo v quyn li ca ngi s dng. Tuy nhin trong ti ny chng ti cha c iu kin ly mu i din m mi thc hin ly mu ngu nhin trn a bn H ni vi s lng mu cn nh. 3.6.2. Xy dng quy trnh phn tch Qua tham kho ti liu, chng ti la chn methanol l dung mi chit corticoid t nn mu thc phm chc nng. Tuy nhin, cc sn phm thc phm chc nng, thnh phn a dng t ngun nguyn liu sn xut ban u, do nn mu phn tch rt phc tp. Nu khng c lm sch, khi phn tch trn h thng sc k d gy bn ct, sau mt thi gian phn tch, p sut ct tng, hnh dng pic xu, tui th ct ngn. ng thi, tp nhiu cng lm gim tnh chn lc i vi cc cht phn tch. Do , cn c bc lm sch qua SPE. ng thi, khi mu c x l vi k thut chit pha rn, quy trnh ny s lm giu mu ln mt h s thch hp, thun li cho phn tch lng vt. Qua thc nghim, chng ti chn c cc iu kin cho qu trnh chit pha rn vi hiu sut thu hi khong 90%. 3.6.4. Thm nh phng php Sau khi xy dng quy trnh phn tch, chng ti tin hnh thm nh phng php theo AOAC. Cc tiu ch thm nh u t theo yu cu ca AOAC, khong tuyn tnh ca phng php rng, cho php nh lng corticoid trong cc i tng a dng. 3.6.5. Kt qu phn tch mu thc Do iu kin hn ch v thi gian nn chng ti mi phn tch c 9 mu thc t v pht hin 1 mu c s xut hin ca corticoid. Tuy nhin, hm lng thu c thp nn c th l dng tnh gi. Hn na, cch ly mu khng i din nn cha th kt lun chnh xc v mu thc phm chc nng ny. KT LUN V KIN NGH

Tai lieu tham khao.pdfTI LIU THAM KHO 9. Lokinendi V.Rao, John R.petersen, Michael G.Bisell, Anthony O.Okorodudu, Amin A. Mohammad (1999), Development of a urinary free cortisol assay using solid-phase extraction-capillary electrophoresis, Journal of Chromatography B, 730, 123-128. 10. Magali Hay, Piere Mormede (1997), Improved determination of urinary cortisol and cortisone, or corticosterone and 11-dehydrocorticosterone by high-performance liquid chromatography with ultraviolet absorbance detection, Journal of Chromatography B, 702, 33-39.

Phu luc.pdf

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