B Y T

TRNG I HC DC H NI

DNG TH MINH HNG

XC NH SIBUTRAMIN TRONG

THC PHM GIM BO BNG HPLC

KHA LUN TT NGHIP DC S

H NI 2014

B Y T

TRNG I HC DC H NI

DNG TH MINH HNG

XC NH SIBUTRAMIN TRONG

THC PHM GIM BO BNG HPLC

KHA LUN TT NGHIP DC S

Ngi hng dn:

1. TS. L nh Chi

2. TS. L Th Hng Ho

Ni thc hin:

Vin Kim Nghim An Ton V

Sinh Thc Phm Quc Gia

H NI 2014

LI CM N

Li u tin, ti xin by t lng bit n chn thnh v su sc ti:

TS. L nh Chi

TS. L Th Hng Ho

ThS. Cao Cng Khnh

l nhng ngi thy du dt ti t nhng ngy u lm nghin cu khoa

hc v cng l nhng ngi trc tip hng dn, tn tnh ch bo, gip ti

hon thnh kha lun tt nghip ny.

Xin chn thnh cm n ban lnh o, cc anh ch trong Vin Kim

Nghim An Ton V Sinh Thc Phm Quc Gia, 48B Tng Bt H - H Ni

quan tm, gip ti trong thi gian va qua.

Ti cng xin cm n Ban gim hiu, cc phng ban, cc thy c gio

v cn b nhn vin trng i hc Dc H Ni nhng ngi dy bo

v gip ti trong sut 5 nm hc tp ti trng.

Xin chn thnh cm n cc thnh vin trong Nhm Ti Bo hng

dn v gip ti tm ti liu hon thnh kha lun ny.

Cui cng, ti xin by t lng bit n su sc ti gia nh, bn b, anh

ch em dnh cho ti s gip v ng vin qu bu trong sut thi gian

qua

H Ni, ngy 25 thng 4 nm 2014

Sinh vin

Dng Th Minh Hng

MC LC

DANH MC CC K HIU- T VIT TT

DANH MC CC BNG

DANH MC CC HNH

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

Chng 1. TNG QUAN .............................................................................. 2

1.1. Vi nt chung v cht gim bo ............................................................ 2

1.2. Tng quan v sibutramin ...................................................................... 2

1.2.1. Cu trc- tnh cht l ha ............................................................... 2

1.2.2. Tnh cht dc ng hc, tc dng dc l .................................... 3

1.2.3. Mt s phng php phn tch sibutramin ...................................... 5

1.3. Tng quan v HPLC ............................................................................. 8

1.3.1. Khi nim chung ............................................................................ 8

1.3.2. Mt s khi nim c bn trong sc k ............................................ 8

1.3.3. Thit b sc k lng ...................................................................... 10

Chng 2. I TNG V PHNG PHP NGHIN CU (NGUYN

LIU, THIT B, NI DUNG, PHNG PHP NGHIN CU) ............. 14

2.1. i tng nghin cu ......................................................................... 14

2.2. Nguyn vt liu - thit b .................................................................... 15

2.2.1. Nguyn vt liu ............................................................................ 15

2.2.2.Thit b .......................................................................................... 15

2.3. Ni dung nghin cu .......................................................................... 16

2.4. Phng php nghin cu .................................................................... 16

2.4.1. Kho st cc iu kin phn tch sibutramin bng HPLC.............. 16

2.4.2. Kho st iu kin x l mu ....................................................... 18

2.4.3. Thm nh quy trnh ..................................................................... 19

2.5. Phng php x l s liu .................................................................. 19

Chng 3. THC NGHIM, KT QU V BN LUN .......................... 20

3.1. Thc nghim v kt qu ..................................................................... 20

3.1.1. Thit lp iu kin sc k phn tch sibutramin ....................... 20

3.1.2. Xy dng quy trnh x l mu chit sibutramin trong thc phm

gim bo ................................................................................................ 24

3.1.3. Thm nh phng php ............................................................... 28

3.1.4. Kt qu p dng phng php xc nh sibutramin trong mt s sn

phm ...................................................................................................... 36

3.2. Bn lun ............................................................................................. 37

Chng 4: KT LUN V KIN NGH .................................................... 38

4.1. Kt lun .............................................................................................. 38

4.2. Kin ngh ............................................................................................ 39

DANH MC CC K HIU- T VIT TT

K hiu Ting anh Ting vit

AOAC Association of Official

Analytical Community

Hip hi cng ng phn tch

chnh thc

ESI Electrospray Ionization Ion ha phun in t

FDA Food and Drug Adminstration Cc qun l thc phm v

dc phm Hoa K

GC-MS Gas Chromatography Mass

Spectrometry

Sc k kh khi ph

HPLC High Performance Liquid

Chromatography

Sc k lng hiu nng cao

HPTLC High Performance Thin Layer

Chromatography

Sc k lp mng hiu nng

cao

KN ATVSTP Kim nghim an ton v sinh

thc phm

LC-MS/MS Liquid Chromatography tandem

Mass Spectrometry

Sc k lng ghp khi ph

hai ln

LOD Limit of Detection Gii hn pht hin

LOQ Limit of Quantification Gii hn nh lng

R(%) Recovery Hiu sut thu hi

SD Standard Deviation lch chun

DANH MC CC BNG

Tn bng Trang

Bng 1.1 Tm tt cc quy trnh phn tch sibutramin c

cng b 5

Bng 2.1 Danh mc cc mu phn tch 14

Bng 2.2 Danh mc cc pha ng kho st 17

Bng 3.1 Gradient h pha ng 1 21

Bng 3.2 Gradient h pha ng 2, 3 v 4 22

Bng 3.3 Kt qu nh lng sibutramin trong nang cng NC07

theo dung mi dng chit mu 25

Bng 3.4 Kt qu nh lng sibutramin trong nang mm NM07

theo dung mi dng chit mu 27

Bng 3.5 Kt qu nh gi thch hp h thng 30

Bng 3.6 Kt qu nh gi tuyn tnh 30

Bng 3.7 lch ca tng im chun dng xy dng ng

chun 31

Bng 3.8 thu hi ca phng php vi vin nang mm 32

Bng 3.9 thu hi ca phng php vi vin nang cng 33

Bng 3.10 lp li ca phng php vi vin nang cng 34

Bng 3.11 lp li ca phng php vi vin nang mm 34

Bng 3.12 Kt qu phn tch sibutramin trong cc thc phm

chc nng gim bo. 36

DANH MC CC HNH

Tn hnh Trang

Hnh 1.1 Cng thc cu to ca sibutramin 3

Hnh 1.2 S khi ca mt my sc k lng hiu nng cao 10

Hnh 3.1 Sc k dung dch chun sibutramin vi pha

ng 1 21

Hnh 3.2 Sc k dung dch xc nh phn gii vi pha

ng 1 21

Hnh 3.3 Sc k dung dch xc nh phn giivi h

pha ng 2 22

Hnh 3.4 Sc k dung dch chun sibutramin vi h pha

ng 3 23

Hnh 3.5 Sc k dung dch xc nh phn gii vi h

pha ng 3 23

Hnh 3.6 Sc k dung dch chun sibutramin vi h pha

ng 4 23

Hnh 3.7 Sc k dung dch xc nh phn gii vi h

pha ng 4 24

Hnh 3.8 Sc k dung dch chun sibutramin 100 g/mL . 25

Hnh 3.9 Sc k dung dch mu nang cng chit bng

methanol. 25

Hnh 3.10 Sc k dung dch chun sibutramin 100 g/mL 26

Hnh 3.11 Sc k mu nang mm chit bng dung mi

methanol 26

Hnh 3.12 Sc k dung dch nh gi phn gii. 28

Hnh 3.13 Sc k dung dch chun sibutramin 200 g/mL 29

Hnh 3.14 Sc k ca mu thc phm chc nng khng

cha sibutramin. 29

Hnh 3.15 Sc k ca mu thc phm chc nng khng

cha sibutramin c thm chun sibutramin. 29

Hnh 3.16 ng chun ca sibutramin 31

Hnh 3.17 Sc k dung dch chun sibutramin 0,5 g/mL 35

Hnh 3.18 Sc k dung dch chun sibutramin 0,2 g/mL 35

1

T VN

Bo ph l mt vn sc khe trn ton th gii, n c th tc ng

khng tt n cht lng cuc sng. Bo co gn y nht ca WHO ch ra

rng khong 1,5 t ngi trng thnh c tui trn 20 c xp vo lp

tha cn; trong s hn 200 triu nam gii v gn 300 triu ph n bo

ph. Theo c tnh n nm 2015 s c khong 2,3 t ngi tha cn v trn

700 triu ngi bo ph. Mt s liu khc cho thy gn 43 triu tr em di 5

tui b tha cn vo nm 2010 [25]. Chnh v th nhu cu gim bo cng

theo m tng ln. Phng php iu tr gim bo chnh l n king kt hp

hot ng th lc, v thuc iu tr bo ph cng c s dng tng cng

tc dng. Thc phm chc nng gim bo t nhin ra i nh mt la chn

thay th cho thuc gim bo tng hp v tr thnh bin php tt hn bi l

theo mt cch tng qut th chng an ton hn. Song tc dng ca nhng sn

phm li thp hn nhng tc nhn gim bo tng hp, do vy tng hiu

qu, chng thng c cho thm nhng cht ha hc gim bo hiu qu

khc mt cch bt hp php, trong c sibutramin l cht b cm s dng

[4], [12], [16]. Do vic pht hin c sibutramin c mt tri php trong

thc phm chc nng gip gim bo l mt yu cu thc tin trong kim sot

tnh an ton ca loi sn phm ny. Xut pht t yu cu thc tin k trn,

ti Xc nh sibutramin trong thc phm gim bo bng HPLC c tin

hnh nhm:

- Xy dng phng php pht hin sibutramin c mt tri php trong thc

phm chc nng gip gim bo.

- ng dng phng php xy dng vo kim tra sibutramin trong mt s

mu thc phm gim bo thin nhin.

2

Chng 1. TNG QUAN

1.1. Vi nt chung v cht gim bo

Nhng cht gim bo s dng u tin gm c nhm cc cht cng

adrenalin (phentermin, benzphetamin, phendimetrazin, mazindol,

diethylpropion, phenylpropanolamin) v nhm cht cng serotonin

(fenfluramin, dexfenfluramin). Tuy nhin, phenylpropanolamin - mt tc

nhn thuc nhm cng adrenalin b cm lu hnh ti th trng M vo

thng 10 nm 2000 do bo co tng nguy c bin c mch mu no nh xut

huyt t qu khi n c s dng nh mt cht gim cn ph n [14].

Cng nh vy, cc cht cng serotonin mc d cng c hiu qu trong vic

h tr gim cn nhng chng cng b cm lu hnh trn th trng M t

thng 9 nm 1997 do cc bo co v thay i van tim v tng huyt p ng

mch phi [15]. Sibutramin (thuc nhm cht c ch ti thu hi serotonin-

noradrenalin) chnh thc c lu hnh ti M t thng 2 nm 1998, sau

n c s dng cho iu tr bo ph trong khong 40 quc gia trn th

gii.

Sibutramin c chng minh l c hiu qu trong vic iu tr bo

ph. Cc d liu lm sng trong mt v hai nm chng minh rng

sibutramin, cng vi mt ch n ung t calo, gy ra tc dng gim cn

ban u v bn vng [21]. Tuy nhin sibutramin li c nhng tc dng ph

trm trng lin quan n cc nguy c tim mch nh tng nhp tim, tng huyt

p thm ch l nhi mu c tim [24], do n b cm lu hnh trn nhiu

th trng nh th trng chu u [12], M [16] v c th trng Vit Nam

[4].

1.2. Tng quan v sibutramin

1.2.1. Cu trc- tnh cht l ha

- Cng thc phn t: C17H26ClN (Khi lng phn t: 297,5)

3

- Cng thc cu to:

Hnh 1.1: Cng thc cu to ca sibutramin [5]

- Tn khoa hc: (+/-)-1-(p-chlorophenyl)--isobutyl-N,N-dimethyl-

cyclobutan-methylamin [5].

- Dng thng dng: Sibutramin hydroclorid monohydrat.

- tan: tan ca sibutramin (C17H26ClN) trong nc (pH 7,4) l 0,04

mg/mL, ca sibutramin hydrochlorid monohydrat l 5,2 mg/mL.

Sibutramin base tan nhiu hn trong alcol, tan tng dn theo di

mch C t methanol n octhanol [5].

- Nhit nng chy: Sibutramin base c nhit nng chy l 55.15oC v

enthalpy l 60,75 J/mol; Sibutramin hydroclorid monohydrat c nhit

nng chy 1190C [5].

- Sibutramin base hp th nh sng trong vng UV-VIS. Cc i hp th ti

bc sng 225nm [23].

1.2.2. Tnh cht dc ng hc, tc dng dc l

Dc ng hc [13].

- Hp thu: Sibutramin hp thu tt qua ng tiu ha (77%). Thi gian

thuc t nng nh trong huyt tng l 1 gi v thi gian bn thi l 1,1

gi.

- Phn b: phn b nhanh chng v rng ri n cc m.

4

- Chuyn ha: Khi dng ng ung sibutramin b chuyn ha ng k khi

qua gan ln u. Sibutramin c chuyn ha bi cytochrom P450 CYP3A4

thnh hai amin (gi l cht chuyn ha c hot tnh 1 v 2) vi chu k bn r

tng ng l 14 v 16 gi. Nng nh trong huyt tng ca cht chuyn

ha c hot tnh 1 v 2 t c sau 3-4 gi.

- Thi tr: Cc cht chuyn ha thi tr ch yu qua nc tiu.

Tc dng dc l:

- Sibutramin c nh gi nh mt thuc chng trm cm tim tng do

cc c ch c chng minh l tc ng cng tng t nh cc thuc

chng trm cm ba vng, chng hn nh amitriptylin. Tuy nhin, sibutramin

khng c tc dng ph ca thuc chng trm cm ba vng bao gm c bun

ng, tc dng khng acetylcholin th ng [21].

- Sibutramin l mt cht c ch ti thu hi serotonin - noradrenalin, thc y

gim cn ngi bo ph. N lm gim cm gic thm n, cho cm gic no

v gy ra s sinh nhit [21].

- Trong c th, sibutramin chuyn ha nhanh chng thnh cc cht chuyn

ha desmethyl: M1 (mono-desmethyl sibutramin) v M2 (di-desmethyl

sibutramin) v sibutramin tc ng dc l ch yu thng qua 2 cht chuyn

ha ny gy ra tc dng gim cn [11], [8].

- Th nghim lm sng chng minh sibutramin c hiu qu nh mt cht

gim cn vi liu lng khc nhau, t 10 n 20 mg/ngy [21].

- Tc dng khng mong mun:

Sibutramin ng thi cng kch thch qu mc h thn kinh trung ng

v nh hng mt vi kha cnh nh bn chn, kh ming, au u, t lit v

nhng d cm (cm gic khc thng nh b chm chch, kin b) c th xy

ra. Hn th na n cn lin quan n tng nguy c tim mch nh tng p lc

mu, nhp tim v thng tng nguy c au tim cng nh t qu [20]. Do

5

nhng nguy c ny sibutramin b cm lu hnh trn th trng chu u t

ngy 21/1/2010 [12].T thng 8 nm 2010, M thm mt chng ch nh

mi cho cc bnh nhn trn 65 tui do cc tc dng ph thc t qua cc

nghin cu lm sng ca sibutramin [17]. Ti Vit Nam, cc ch phm cha

sibutramin b rt s ng k lu hnh t thng 4/2011 [4].

1.2.3. Mt s phng php phn tch sibutramin

c rt nhiu nghin cu c cng b a ra phng php xc nh

sibutramin s dng nhiu k thut phn tch khc nhau nh: GC-MS [22], [9];

HPLC [23], [7], [6], [26]; HPTLC [7]; LC-MS/MS [10]; IR [18].

Bng 1.1. Tm tt cc quy trnh phn tch sibutramin c cng b

K

thut

phn

tch

Mu

phn tch iu kin phn tch

iu kin

x l mu

Ti

liu

tham

kho

HPLC

Vin

nang

(Reductil,

10 mg, 15

mg,

Abbott

lab,

Brazil)

-Ct Phenyl Hypersil C18 (250 x 4,6

mm, 5 m)

-Pha ng: acetonitril - amoni

dihydrophosphat 50 mM, pH 6,0 (35 :

65)

-Tc dng: 1 mL/pht

-Nhit ct: 30oC

-Detector PDA: 225 nm

-Th tch tim mu: 20L

Chit bng

methanol-

nc

(65:35)

trc khi

tim sc k

[23]

HPLC

Vin

nang

gim bo

thin

nhin

-Ct ACE Phenyl

(150 4,6 mm, 5 m)

-Pha ng: acetonitril - nc - acid

formic 0,19 M, pH 3,0 (45 : 55 : 0,78)

-Tc dng: 1 mL/pht

Chit bng

acetonitril -

nc (1:1)

v pha long

trc khi

[7]

6

-Nhit ct: 25oC

-Detector hunh quang (Kch thch

225 nm, bc x 316 nm)

-Th tch tim mu: 5L

tim sc k

HPLC

Vin

nang

cng

gim bo

-Ct CN Nucleosil 100-5 CN (125 x

4,6 mm, 5 m)

-Pha ng: acetonitril - kali

dihydrophosphat 0,025M, pH 4,0 (35 :

65)

-Tc dng: 2,5 mL/pht

-Nhit ct: 25oC

-Detector PDA: 225 nm

-Th tch tim mu: 20L

Chit bng

methanol

trc khi

tim sc k

[6]

HPLC

Vin

nang

cng

gim bo

-Ct Thermo C18 (2504,6 mm, 5 m)

-Pha ng: acetonitril acid

phosphoric 0,02M, pH 3,0 (t l theo

gradient)

-Tc dng: 1,0 mL/pht

-Nhit ct: 300C

-Detector PDA: 222 nm

-Th tch tim mu: 20 L

Chit bng

methanol,

rung siu

m 600C,

pha long,

lc th.

[26]

HPTLC

vin nang

gim bo

thin

nhin

-Bn mng: Silica gel 60 F254 (8mm

20mm)

-Pha ng: n-hexan - aceton - amoniac

(10 : 1 : 0,1)

-Qut sc 225 nm, tc 10

mm/giy

-Hin vt: Phun thuc th Dragendoft

Chit bng

acetonitril -

nc (1 : 1)

s dng my

lc xoy.

[7]

GC-MS Vin nn, -Thit b : sc k kh HP-6890, Mu c [9]

7

vin

nang, tr

ti

detector HP 5973

-Ct: HP-5-MS (30m0,25mm, b

dy phase : 0,25mm)

-Chng trnh nhit : bt u

110oC, tng 10 oC/pht n 280 oC v

duy tr trong 3 pht

-Kh mang: Heli vi tc 0,6

ml/pht

-Ph c thu thp trong khong m/z

t 40 - 400

ha trong

nc, kim

ha bng

Natri

carbonat n

pH 10. Chit

bng methyl

chlorid, sau

khi tch cc

lp, lm kh

bng natri

sulphat khan

ri phn tch

bng GC-

MS.

LC-

MS/MS

Huyt

tng

-Ct Waters Xterra MS C18 (502,1

mm, 3,5 m)

-Pha ng: acetonitril - acid

trifluoroacetic 0,1% (55:45, v/v).

-Tc dng: 0,3 mL/pht

-Nhit ct: 200C

-Th tch tim: 5 L

-Detector: S dng k thut ESI (ion

ha tia in) ion ha cht phn tch

v phn tch khi ph 2 ln MS/MS.

Mu c

chit bng

tert-butyl

ether, thm

vo cht

chun ni

propranolol

hydroclorid

ri phn tch

bng HPLC-

ESI/MS/MS

[10]

Trong cc k thut phn tch c ng dng phn tch sibutramin,

HPLC l k thut ph bin nht, d p dng do iu kin thit b sn c. V

8

vy, trong nghin cu ny, HPLC c la chn lm k thut xy dng

quy trnh phn tch sibutramin trong thc phm chc nng gim bo.

1.3. Tng quan v HPLC

1.3.1. Khi nim chung

Sc k lng hiu nng cao (High performance Liquid Chromatography

HPLC) l k thut tch sc k trong cc cht phn tch ha tan trong pha

ng l cht lng v di chuyn qua ct cha cc ht pha tnh. Ty thuc vo

i lc ca cht phn tch vi pha ng v pha tnh m cc cht di chuyn vi

tc khc nhau, do th t ra gii khc nhau. Thnh phn pha ng a

cht phn tch ra khi ct c thay i ra gii cc cht vi thi gian hp

l [1].

1.3.2. Mt s khi nim c bn trong sc k

Thi gian lu:

Khong thi gian t lc bm mu vo ct n khi pic n detector l

thi gian lu tR.

Thi gian cht: thi gian tM ca cht khng lu gi (tc di chuyn ca n

bng tc di chuyn trung bnh ca cc phn t pha ng).

Thi gian lu hiu chnh: tR = tR - tM

H s phn b K:

K = M

S

C

C

CS, CM: nng mol ca cht tan trong pha tnh, pha ng

K cng ln, s di chuyn ca cht tan qua pha tnh cng chm. Cc cht trong

hn hp c h s K khc nhau cng nhiu, kh nng tch din ra cng d dng

hn.

H s dung lng k:

9

k = M

S

MM

SS

V

VK

VC

VC.

.

. =

M

MR

t

tt

Trong : VS, VM tng ng l th tch ca pha tnh, pha ng.

k ph thuc vo bn cht cht phn tch, bn cht 2 pha vo h s VS/VM

Thng thng chn k = 1-5.

H s chn lc :

H s chn lc c trng cho tc di chuyn t i ca 2 cht A v B:

= MAR

MBR

A

B

A

B

tt

tt

k

k

K

K

'

'

Trong :

KB, KA ln lt l h s phn b ca cht B, A (A l cht ra trc).

kA, kB tng ng l h s dung lng ca cht A, B.

(tR)A, (tR)B tng ng l thi gian lu ca cht A, B.

Hai cht A v B ch c th tch c khi nhau nu > 1. Trong phn tch

sc k, thng la chn iu kin phn tch c c = 1,05 - 2. Nu

qu ln, thi gian phn tch ko di.

S a l thuyt:

S a l thuyt l i lng c trng cho hiu lc ct sc k

2

2/1

2

55,516

W

t

W

t

H

LN RR

Trong :

L: chiu di ca ct c chia thnh N a l thuyt;

H: chiu cao ca a l thuyt;

W: chiu rng ca pic sc k;

W1/2: chiu rng pic ng vi mt na chiu cao ca pic.

phn gii ca ct:

phn gii (RS) ca ct nh gi kh nng tch nh lng hai cht trong

10

hn hp trn ct sc k:

RS =

BA

ARBR

BA

ARBR

WW

tt

WW

tt

,2/1,2/1

.18,1

.2

1

Trong :

WA, WB tng ng l chiu rng pic sc k ca cht A, B;

W1/2,A, W1/2,B tng ng l chiu rng pic sc k ca cht A, B ng vi mt

na chiu cao ca pic.

1.3.3. Thit b sc k lng [1], [2]

Bung ct

Hnh 1.2. S khi ca mt my sc k lng hiu nng cao [1].

a. H thng cung cp pha ng:

- Ngun cp pha ng: pha ng trong sc k lng thng l 2 dung mi

ha tan vo nhau c kh nng tch vi phn gii ph hp, c cha

trong bnh thy tinh hoc thp khng r.

- B phn loi kh (degasser): trc khi s dng cn lc (mng lc 0,45 m)

v ui kh ha tan trong pha ng trnh vic kh ha tan c th lm bin

dng pic, gim hiu lc ct, lm nhiu ng nn. C th loi kh ha tan

bng cch: chy siu m, sc kh tr.

H thng cp

dung mi Bm B phn tim mu

Ct sc k Detector

H thng thu

nhn v xa l

s liu (my

ghi, my tnh)

11

- B phn trn pha ng (mixer): trn cc dung mi p sut thp hoc p

sut cao

Chng trnh dung mi p sut thp: mi bnh cha dung mi c mt

van ring ly lng dung mi xc nh a vo bnh ha trn, sau

ch dng mt bm a pha ng vo van tim mu.

Chng trnh dung mi p sut cao: Mi dung mi c mt bm ring,

vic ha trn c thc hin p sut cao.

- Bm: v mt kt cu c 3 loi thng gp: Bm y mt pittong, bm lm

y nhanh v bm kp y ko.

b. B phn tim mu:

- Mu lng hoc dung dch c tim thng vo pha ng cao p ngay u

ct m khng cn dng dng bng mt van tim c vng cha mu. Vng

cha mu c dung tch khc nhau: thng dng loi 0,5020 L. C vng

cha mu ln hn.

- Ngoi ra, i khi ngi ta tim mu bng bm tim qua tm m u ct.

Khi tim phi dng dng v p sut trong ct khng cao. Cch tim ny c

lp li thp, sai s ln hn so vi khi dng van tim.

- Hin nay hay dng van tim mu v c u im l s d dng t ng ha,

ct khng b tc hay b lm bn bi cc mnh ca vch ngn, th tch a vo

ct hng nh nn lp li cao.

c. Ct sc k

- Ct sc k lng hiu nng cao thng c ch to bng thp khng g,

thy tinh hoc cht do c chiu di 10-30 cm, ng knh trong 4-10 mm.

Ct nhi thng c ht c 5-10 m. u im ca ct c ct nhi c ht c

nh l chy tn t dung mi v t thi gian v s a l thuyt ln.

- bo v ct sc k, ngi ta s dng ct bo v c t trc ct sc k

loi cc cht c mt trong pha ng v trong mu phn tch lm gim tui

12

th ct. Ct bo v ngn hn ct sc k, c nhi ht cng loi nhng kch

thc ht ln hn.

- Sc k lng phn b l k thut s dng ph bin nht hin nay. Loi pha

tnh ph bin nht c ch to t silic dioxid (silica). Nhm OH trn b mt

silica phn ng vi dn cht clorosilan to ra dn cht siloxan.

B mt silica Dn cht clorosilan Dn cht siloxan

Da vo gc R ca dn cht siloxan, ngi ta chia ra 2 nhm:

- Pha tnh khng phn cc c R l:

Gc octadecyl (C18)

Gc octyl (C8)

Gc phenyl propyl

- Pha tnh phn cc c R l:

Cyano

Amino

Diol

Da vo phn cc tng i ca pha tnh v pha ng hnh thnh hai

loi sc k phn b l sc k phn b pha thun v sc k phn b pha o:

- Sc k phn b pha thun: thng dng pha tnh lng phn cc nh

triethylen glycol, nc. Cn pha ng l dung mi t phn cc hn nh hexan.

- Sc k phn b pha o: pha tnh khng phn cc nh hydrocacbon (C18

hoc C8) cn pha ng phn cc hn pha tnh nh nc, acetonitril.

d. Detector

C nhiu detector c s dng trong HPLC v thng s dng 6 loi sau:

13

- Detector hp th UV-VIS

- Detector hunh quang

- Detector ch s khc x

- Detector tn x bay hi

- Detector o dng

- Detector dn

Trong nghin cu ny, quy trnh xy dng c c kh nng ng dng

rng, chng ti la chn detector PDA, loi detector ph bin trong cu hnh

tiu chun ca thit b HPLC hin ti. V bn cht, y l mt detector UV-

Vis cho php ng thi ghi nhn tn hiu hp th ng thi trn ton di ph

UV gn v Vis. Di bc x UV-Vis (thng cc detector PDA c di bc

sng trong khong 190 800 nm) sau khi i qua t bo o c a n mt

cch t phn thnh cc tia n sc i n mt mng diod quang. Mi diod

quang n nhn mt phn di bc x tng ng vi mt khong bc sng

hp. Nh vy, mi mt diod c th pht hin mt s hp thu mt bc sng

nht nh. Ton b dy diod c qut nhiu ln trong 1 giy bi b phn vi

x l. Kt qu ph c th hin trn mn hnh my tnh hoc c lu tr in

ra di dng bn ph bng my in.

u im ca detector ny l to c ph UV ca cc cht phn tch trong

khong bc sng chn, kim tra s tinh khit ca sn phm v nh danh

c sn phm bng cch so snh ph tng ng ca nh sc k vi ph ca

mt ngn hng d liu hoc vi ph ca cht chun bit trc.

e. H thng thu nhn v x l d liu

B phn lu tr d liu ca cc thit b HPLC hin nay thng l cc

my vi tnh hin i c kh nng ghi nhn, lu gi, bin tp, x l cc thng

tin ht sc hiu qu.

14

Chng 2. I TNG V PHNG PHP NGHIN CU

(NGUYN LIU, THIT B, NI DUNG, PHNG PHP NGHIN

CU)

2.1. i tng nghin cu

Trong ti ny, chng ti tin hnh nghin cu phng php xc nh

hm lng ca sibutramin.

i tng mu phn tch l cc thc phm chc nng di mt s dng

bo ch: vin nang cng, vin nang mm. Cc mu phn tch c ly t vin

KN ATVSTP QG c ngun gc t cc cng ty sn xut khc nhau v c

m ha nh bng 2.1

Bng 2.1 Danh mc cc mu phn tch

Mu Tn sn phm Nh sn xut Hn dng

NC01 Pretty woman Dc phm Jemo-Pharm

A/S 7/2014

NC02 Vin nang gim bo Phc

Linh

Cng ty Bch An- Trung

Quc 31/8/2014

NC03 Vin nang thanh qu o nh

Yunan Baian Medicinal

Science & Technology co.,

LTD

25/3/2015

NC04 Green Oake USA O&K trading 12/2014

NC05 Vin nang khng nhn mc Khng r 23/6/2014

NC06 Vin nang khng nhn mc Khng r 15/5/2014

NC07 Eva beauty Khng r 1/9/2015

NM01 Doctors Choice Slim&slim Dramerico group LLC 21/12/2016

NM02 Vin nang khng nhn mc Khng r Khng r

NM03 Slim Fit Bacai Inc 12/2016

NM04 Beauty Arkopharma 4/2016

NM05 Vin nang mm khng nhn Khng r Khng r

15

mc

NM06 Beauty slim NHK laboratories (USA) 6/2016

NM07 Vin nang khng nhn mc Khng r 25/8/2014

2.2. Nguyn vt liu - thit b

2.2.1. Nguyn vt liu

- Cht i chiu sibutramin hydroclorid monohydrat (Sigma - Adrich) (S

kim sot: MFCD04039806; hm lng: 98% (HPLC)).

- Cht i chiu furosemid (S kim sot: MFCD00010549; hm lng:

99%).

- Cht i chiu phenolphthalein (S kim sot: MFCD00070132; hm lng:

85% (TLC) ).

- Dung mi loi tinh khit cho HPLC: methanol, acetonitril, tetrahydrofuran

(Merck)

- Dung mi loi tinh khit phn tch: ethanol, aceton (Merck)

- Ha cht tinh khit phn tch: kali dihydrophosphat (Merck), amoni acetat

(Merck).

- Nc ct 2 ln

2.2.2.Thit b

- H thng sc k lng hiu nng cao HPLC Shimadzu (LC 20AD) trang b

detector PDA.

- Ct sc k C18 Symmetry Waters (250 mm x 4,6 mm; 5 m).

- Tin ct C18 Symmetry Waters (20 mm x 3,9 mm; 5 m).

- Cn k thut XT1200c.

- Cn phn tch Mettler Toledo c chnh xc 0,0001 g v 0,00001 g.

- My o pH: pH Meter 744.

- My lc siu m Elma.

16

- My ly tm Hermle Z383K

- My lc Vortex IKA

- Dng c thy tinh cc loi: Bnh nh mc 20 mL, 50 mL, cc c m, pipet

cc loi, autopipet 1000 L, 200 L.

- ng ly tm 50 mL

- Phu lc, giy lc, mng lc 0,45 m, 0,2m.

2.3. Ni dung nghin cu

- Kho st xy dng iu kin chy my HPLC phn tch sibutramin.

- Kho st xy dng quy trnh x l mu tch chit sibutramin trong thc

phm chc nng.

- nh gi quy trnh phn tch v x l mu.

- p dng quy trnh phn tch v x l mu vi mt s sn phm trn th

trng.

2.4. Phng php nghin cu

2.4.1. Kho st cc iu kin phn tch sibutramin bng HPLC

Nghin cu phn tch sibutramin chng ti tin hnh l mt nhnh trong

mt ti ln hn nhm xy dng quy trnh phn tch cc cht c tc dng

lm gim cn c mt tri php vo thc phm chc nng. Trong s cc cht

ny c phenolphthalein (do tc dng nhun trng) v furosemid (do tc dng

li tiu) l hai i tng cng tng b pht hin c mt tri php trong thc

phm chc nng, ng thi li rt kh tch khi sibutramin bng HPLC nu

c mt trong cng nn mu (t cc kt qu nghin cu s b). Chnh v vy,

trong khi xy dng quy trnh phn tch sibutramin trong nghin cu ny,

chng ti t mc tiu cn kho st cc iu kin sc k cho php tch ring

sibutramin khi phenolphthalein v furosemid nng cao hiu qu ng dng

thc t ca quy trnh.

17

- Pha tnh: chng ti chn pha tnh l ct C18 Symmetry Waters (250mm x

4,6mm; 5m.)

- Pha ng: kho st ch gradient nng vi cc pha ng sau:

Bng 2.2: Danh mc cc pha ng kho st

Thnh phn pha ng (%) Ch

chy my A B

Pha ng 1 KH2PO4 0,05M (pH 4,0) Methanol

Gradient Pha ng 2 KH2PO4 0,05M (pH 4,0) Acetonitril

Pha ng 3 CH3COONH4 0,05M (pH 3,5) Acetonitril

pha ng 4 CH3COONH4 0,05M ( pH 3,5) Methanol

- Tc dng: Da trn nhiu bi bo nghin cu [23], [7], [26], chng ti

kho st vi tc dng 1 mL/pht.

- Th tch tim mu: 50 L.

- Detector: PDA qut di ph t bc sng 200 nm n 450 nm.

- Bc sng pht hin: 225 nm (do sibutramin c bc sng hp th cc i

l 225 nm) [23].

- Nhit ct: 300C.

- Dung dch chun sibutramin gc 500 g/mL: Cn chnh xc mt lng

cht i chiu sibutramin hydroclorid monohydrat tng ng khong

50,0 mg sibutramin vo cc c m 50 mL, ha tan bng 20 mL methanol

v chuyn vo bnh nh mc 100mL, dng methanol trng cc, chuyn

dch trng cc vo cng bnh nh mc, thm methanol va n vch,

lc k. Bo qun trong t lnh.

18

- Dung dch chun lm vic: ht chnh xc 40,0 mL dung dch chun gc

vo bnh nh mc 100 mL, thm methanol va (dung dch chun lm

vic c nng sibutramin 200 g/mL).

- Cc dung dch chun sibutramin c nng nh hn c pha t dung

dch chun lm vic.

- Dung dch nh gi phn gii: chun b dung dch hn hp sibutramin,

furosemid v phenolphthalein trong methanol c nng mi cht l

sibutramin 200 g/mL, furosemid 100 g/mL, phenolphthalein 100

g/mL.

- Dung dch m amoni acetat 0,05 M (pH 3,5): cn chnh xc khong 1,93

g mui amoni acetat ho tan vo khong 400 mL nc, chnh n pH 3,5,

nh mc 500 mL, lc qua mng lc 0,2 m.

- Dung dch m kali dihydrophosphat 0,05M (pH 4,0): cn chnh xc

khong 3,4 g mui kali dihydrophosphat, ha tan vo khong 400 mL

nc, thm 5mL tetrahydrofuran, chnh n pH 4,0 bng dung dch acid

phosphoric 5%, nh mc bng nc ct 2 ln ti 500 mL, lc qua mng

lc 0,2 m.

2.4.2. Kho st iu kin x l mu

Da kh nng ha tan ca sibutramin v tham kho nhiu bi bo,

nghin cu [23], [7], [16], [26], chng ti kho st kh nng chit sibutramin

t nn mu bng cc loi cc dung mi sau y: methanol, ethanol, nc,

acetonitril, aceton.

Quy trnh chit sibutramin cho i tng phn tch l thc phm chc

nng dng nang cng v nang mm d kin gm cc bc sau:

- Xc nh khi lng trung bnh thuc trong nang, ng nht mu.

- Cn chnh xc mt lng mu sau khi ng nht (cn khong 0,3 - 0,4 mg)

vo ng ly tm 50 mL.

19

- Thm khong 20 mL dung mi chit vo ng ly tm.

- Lc xoy ng ly tm 5 - 10 pht, sau lc siu m 15-30 pht nhit

phng.

- Ly tm 5 pht, tc 6000 vng/pht

- Gn ly phn dch, nh mc 25 mL bng cng dung mi v lc qua mng

lc 0,45 m.

- Tim vo h sc k HPLC (pha long nu cn).

2.4.3. Thm nh quy trnh

Phng php x l mu v iu kin phn tch sibutramin bng HPLC c

thm nh v cc tiu ch:

- c hiu

- thch hp ca h thng

- Khong tuyn tnh

- chnh xc

- ng

- Gii hn pht hin (LOD), gii hn nh lng (LOQ).

2.5. Phng php x l s liu

-Cc kt qu thu c trong qu trnh tin hnh nghin cu u c x l

bng cc phn mm c sn trong thit b HPLC

- S dng cc phng php x l thng k trong phn tch nh cc hm ton

hc, thng k c sn trong phn mm tin hc Microsoft Office Excel tnh

ton: kt qu trung bnh, lch, lch chun, phng sai, lch chun

tng i, h s bin thin, thu hi khi x l cc kt qu thc nghim v

nh gi thm nh phng php.

20

Chng 3. THC NGHIM, KT QU V BN LUN

3.1. Thc nghim v kt qu

3.1.1. Thit lp iu kin sc k phn tch sibutramin

xy dng quy trnh phn tch sibutramin bng HPLC, chng ti s

dng cc iu kin sc k thng dng di y c nh trong ton b nghin

cu:

- Ct C18 Symmetry Waters (250 mm x 4,6 mm; 5 m) v tin ct cng loi.

- Tc dng: 1,0 mL/pht

- Th tch tim mu: 50 L

- Detector: PDA qut di ph t bc sng 200 nm n 450 nm

- Bc sng pht hin: 225 nm.

- Nhit ct: 300C

c c quy trnh phn tch cho p ng pic tt vi sibutramin v c

kh nng tch sibutramin khi furosemid v phenolphthalein, thnh phn pha

ng c kho st ti u ha thng qua kt qu phn tch thu c khi tim

dung dch nh gi phn gii vo h thng HPLC mi iu kin pha

ng. Nhng kt qu nghin cu s b cho thy phn tch ch ng dng

khng cho php tch c sibutramin khi furosemid v phenolphthalein. V

vy, vic s dng ch gradient dung mi l bt buc c c phn

gii nh mong mun, ng thi cho thi gian phn tch va phi (khong 16-

17 pht). C tt c 4 h dung mi c kho st:

-H pha ng 1: Kali dihydrophosphat (0,05M, pH 4,0) methanol vi

chng trnh gradient nh bng 3.1. Kt qu l kh nng tch cc cht tt

nhng pic sibutramin khng cn i, ko ui (Hnh 3.1, 3.2).

21

0.0 2.5 5.0 7.5 10.0 12.5 min

0

50

100

150

200

mAU225nm,4nm (1.00)

/13.4

22/3

600172

Hnh 3.1: Sc k dung dch chun sibutramin vi pha ng 1

0.0 2.5 5.0 7.5 10.0 12.5 min

0

500

1000

mAU225nm,4nm (1.00)

/10.4

08/7

506226

/11.5

67/2

2165545

/13.3

64/3

441493

Hnh 3.2: Sc k dung dch nh gi phn gii vi pha ng 1

Bng 3.1 Gradient h pha ng 1

Thi gian (pht) Thnh phn pha ng (%)

KH2PO4 (0,05 M, pH 4,0) Methanol

0,1 80 20

1 80 20

3 50 50

9 20 80

11 20 80

12 80 20

16 80 20

-H pha ng 2: Kali dihydrophosphat (0,05 M, pH 4,0) acetonitril vi

chng trnh gradient nh bng 3.2. Kt qu phn tch trn dung dch nh

gi phn gii cho thy pic sibutramin thu c gn, cn xng hn so vi

sibutramin

sibutramin furosemid

phenolphtalein

22

h pha ng 1, ng thi sibutramin cng c tch ring khi furosemid v

phenolphthalein (Hnh 3.3).

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

250

500

750

1000mAU

225nm,4nm (1.00)

/9.4

62/7

171036

/12.6

29/2

2074439

/14.7

56/3

320950

Hnh 3.3: Sc k dung dch nh gi phn gii vi h pha ng 2

Bng 3.2 Chng trnh gradient ca h pha ng 2, 3 v 4

Thi gian (pht)

T l pha ng (%)

Kali dihydrophosphat

(hoc amoni acetat)

Acetonitril

(hoc methanol)

0,1 80 20

1 80 20

3 60 40

11 50 50

12 50 50

13 80 20

17 80 20

-H pha ng 3: Amoni acetat (0,05 M, pH 3,5) - acetonitril vi gradient theo

bng 3.2. Kt qu phn tch thu c vi dung dch nh gi phn gii cho

thy tuy sibutramin c tch tt khi furosemid v phenolphthalein nhng

tn hiu ng nn khng c tt, pic sibutramin b dong chn v ko ui

(Hnh 3.4, 3.5)

sibutramin furosemid

phenolphtalein

23

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

-100

0

100

200

mAU225nm,4nm (1.00)

/12.1

42/5

195574

Hnh 3.4: Sc k dung dch chun sibutramin vi h pha ng 3

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

500

1000

1500mAU

225nm,4nm (1.00)

/8.5

89/1

4460208

/9.8

55/1

8460828

/12.1

51/6

869873

Hnh 3.5: Sc k dung dch nh gi phn gii vi h pha ng 3

-H pha ng 4: Amoni acetat (0,05 M, pH 3,5) - methanol vi gradient theo

bng 3.2. Kt qu phn tch thu c vi dung dch nh gi phn gii

cho thy tuy sibutramin c tch tt khi furosemid v phenolphthalein

nhng tn hiu ng nn khng c tt, pic sibutramin b dong chn v

ko ui (hnh 3.6 v 3.7)

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

100

200

mAU225nm,4nm (1.00)

/14.6

68/6

094832

Hnh 3.6: Sc k dung dch chun sibutramin vi h pha ng 4

sibutramin

sibutramin

sibutramin

phenolphtalein

furosemid

24

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

250

500

750

1000

mAU225nm,4nm (1.00)

/10.6

54/1

703

362

5

/12.1

36/2

009

269

9

/14.8

00/7

2335

54

Hnh 3.7: Sc k dung dch nh gi phn gii vi h pha ng 4

Nh vy, trong bn h pha ng c kho st, h pha ng 2: Kali

dihydrophosphat (0,05 M, pH 4,0) - acetonitril vi chng trnh gradient nh

bng 3.2 cho pic sibutramin sc k gn v cn xng hn c,hn na tch tt

khi furosemid v phenolphtalein. T kt qu ti u ha pha ng, chng ti

i ti quy trnh phn tch sibutramin bng HPLC vi iu kin c th nh sau:

- Ct C18 Symmetry Waters (250 mm x 4,6 mm; 5 m) v tin ct cng loi.

- Pha ng: Kali dihydrophosphat (0,05 M; pH 4,0) acetonitril, chng trnh

gradient nh bng 3.2.

- Tc dng: 1,0 mL/pht

- Th tch tim mu: 50 L

- Detector: PDA qut di ph t bc sng 200 nm n 450 nm

- Bc sng pht hin: 225 nm

- Nhit ct: 300C

3.1.2. Xy dng quy trnh x l mu chit sibutramin trong thc

phm gim bo

Nm dung mi khc nhau, gm nc, methanol (MeOH), ethanol (EtOH),

aceton v acetonitril (ACN) c kho st la chn ra dung mi ph

hp nht cho vic chit sibutramin t nn mu l thc phm gim bo dng

nang cng v nang mm. Vi mi dng bo ch, mt mu sn phm qua kho

st s b bit c cha sibutramin tri php c x l bng cch chit ln

lt vi 5 loi dung mi v phn tch bng cc iu kin HPLC thit lp

mc 3.1.1. Cc bc ca qu trnh chit tun theo trnh t mc 2.4.2.

sibutramin

phenolphtalein

furosemid

25

a.Kho st dung mi chit vi vin nang cng

S dng tng dung mi chit sibutramin t mu nang cng NC07

xc nh s b c cha sibutramin. Dung dch th c phn tch sc k v so

snh vi dung dch chun sibutramin 100 g/mL pha trong methanol. Kt qu

hm lng sibutramin thu c trn cng mu nang cng khi chit bng cc

dung mi khc nhau c th hin bng 3.3.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

0

50

100

150

200

mVDetector A:225nm

/5.9

86

/48

33

258

Hnh 3.8: Sc k dung dch chun sibutramin 100 g/mL.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

0

50

100

150

mVDetector A:225nm

/6.1

33

/31

62

13

4

Hnh 3.9: Sc k dung dch mu nang cng chit bng methanol.

Bng 3.3. Kt qu nh lng sibutramin trong vin nang cng NC07 theo

dung mi dng chit mu

Dung mi

Lng cn mu (g)

Th tch

dung mi (ml)

H s pha

long

Din tch pic

(mAU.s)

Nng sibutrami

n trong dung dch

th (g/mL )

Hm lng

sibutramin (mg/g)

Hm lng

TB (mg/g)

Aceton-01 0,3161 25 5 2432033 50,3 19,9 21,6

Aceton-02 0,3489 25 5 3130751 64,8 23,2

MeOH-01 0,2978 25 5 3162134 65,4 27,5 26,4

MeOH-02 0,3001 25 5 2933162 60,7 25,3

ACN-01 0,3432 25 5 2616370 54,1 19,7 20,8

26

ACN-02 0,3599 25 5 3038800 62,9 21,8

EtOH-01 0,3159 25 5 2984704 61,8 24,4 22,2

EtOH-02 0,3597 25 5 2767511 57,26 19,9

Nc-01 0,3048 25 5 981681 20,31 8,3 8,5

Nc-02 0,3227 25 5 1081464 22,38 8,7

b.Kho st dung mi chit vi vin nang mm

Tin hnh kho st tng t s dng tng loi dung mi trn mu nang mm

NM07 xc nh s b c cha sibutramin qua kho st s b thu c kt

qu nh bng 3.4.

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

0

50

100

150

200

250mV

Detector A Ch1:225nm

SIB

UTR

AM

INE

/6.1

01/5

1537

42

Hnh 3.10: sc k dung dch chun sibutramin 100 g/mL

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 min

0

50

100

150

200

250

300mV

Detector A Ch1:225nm

/2.5

93/

240

951

/6.0

91/

608

222

6

Hnh 3.11: sc k mu nang mm chit bng dung mi methanol

27

Bng 3.4. Hm lng sibutramin trong vin nang mm NM07 theo dung mi

dng chit mu

Dung mi

Lng cn mu (g)

Th tch

dung mi

(mL)

H s pha

long

Din tch pic (mAU.s

)

Nng sibutrami

n trong dung dch

th (g/mL)

Hm lng

sibutramin (mg/g)

Hm lng

TB (mg/g)

EtOH-01 0.4324 25 5 4650782 88,7 25,7 26,3

EtOH-02 0.4546 25 5 5138888 98,1 26,9

MeOH-01 0.4505 25 5 5478145 104,5 29,0 30,7

MeOH-02 0.4476 25 5 6082226 116,1 32,4

ACN-01 0.4613 25 5 5206367 99,4 26,9 26,6

ACN-02 0.4472 25 5 4922718 93,9 26,3

Aceton-01 0.4379 25 5 4698056 89,7 25,6 26,4

Aceton-02 0.4325 25 5 4928119 94,0 27,2

Nc-01 0.4730 25 5 3316977 63,3 16,7 17,6

Nc-02 0.4543 25 5 3525240 67,3 18,5

Nh vy, kt qu kho st thc nghim (Bng 3.3, 3.4) cho thy c vin

nang cng v vin nang mm khi chit vi methanol cho hm lng

sibutramin cao nht. Hn na trong qu trnh chit vi methanol, mu cng

trong nht v t cn nht. Do vy dung mi chng ti la chn chit mu l

methanol v quy trnh x l mu trc khi phn tch sibutramin gm cc bc

nh sau:

- Xc nh khi lng trung bnh thuc trong nang, ng nht mu.

- Cn chnh xc mt lng mu sau khi ng nht (vi vin nang cng cn

khong 0,3 g, cn vi nang mm cn khong 0,4 g) vo ng ly tm 50 mL.

- Thm khong 20 mL methanol vo ng ly tm.

- Lc xoy ng ly tm 5 pht, sau lc siu m 15 - 30 pht nhit

phng.

- Ly tm 5 pht, tc 6000 vng/pht

28

- Gn ly phn dch, nh mc 25,0 mL bng methanol v lc qua mng lc

0,45 m.

- Tim vo h thng sc k HPLC (pha long nu cn).

3.1.3. Thm nh phng php

a. c hiu

nh gi c hiu ca quy trnh phn tch v x l mu, chng ti

tin hnh phn tch trong iu kin HPLC nh mc 3.1.1. cc dung dch

chun sibutramin, dung dch nh gi phn gii, mu thc phm chc nng

khng cha sibutramin v mu thc phm chc nng khng cha sibutramin

c thm chun sibutramin (hai mu sau c x l theo quy trnh mc

3.1.2). Kt qu phn tch cho thy, sc k ca dung dch nh gi phn

gii cho 3 pic ca sibutramin, furosemid v phenolphthalein c tch ring

bit (Hnh 3.12 ); trn sc k ca mu thc phm chc nng khng cha

sibutramin khng xut hin pic no c cng thi gian lu vi pic ca

sibutramin trn sc k ca dung dch chun sibutramin (Hnh 3.13, 3.14).

ng thi trn sc k ca mu thc phm chc nng thm chun

sibutramin xut hin pic sibutramin c tch ring r (Hnh 3.15). Nh vy,

quy trnh thit lp p ng c yu cu v c hiu khi phn tch

sibutramin.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

500

1000

mAU225nm,4nm (1.00)

/9.1

56/9

594887

/12.4

68/2

6377525

/14.4

39/7

519153

Hnh 3.12: Sc k dung dch nh gi phn gii.

sibutramin

phenolphtalein

furosemid

29

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

100

200

300

mAU225nm,4nm (1.00)

/14.4

15/

8079449

Hnh 3.13: Sc k dung dch chun sibutramin 200 g/mL

0.0 5.0 10.0 15.0 min

0

50

100

150

200

250

mAU225nm,4nm (1.00)

Hnh 3.14: Sc k ca mu thc phm chc nng khng cha sibutramin.

0.0 5.0 10.0 15.0 min

-25

0

25

50

75

100

125

150

mAU225nm,4nm (1.00)

/14.290

/250129

3

Hnh 3.15: Sc k ca mu thc phm chc nng khng cha sibutramin

c thm chun sibutramin.

b. thch hp h thng

Tim 6 ln dung dch chun sibutramin 200 g/mL vo my HPLC. Kt qu

phn tch (Bng 3.5) cho thy lp li tt vi pic sibutramin c v thi gian

lu (RSD = 0,01%) v din tch pic (RSD = 0,02 %), trong khi yu cu ca

AOAC l nh hn 5,3% [3]. Nh vy, h thng sc k vi cc iu kin

thit lp thch hp cho vic phn tch sibutramin.

sibutramin

sibutramin

30

Bng 3.5: Kt qu nh gi thch hp h thng

Ln phn

tch

Thi gian lu

(pht)

Din tch pic

(mAU.s)

Ln 1 14,415 8079449

Ln 2 14,419 8079268

Ln 3 14,418 8079027

Ln 4 14,416 8079052

Ln 5 14,417 8078427

Ln 6 14,418 8075040

TB 14,417 8078377

SD 0,0015 1670,884

RSD (%) 0,01 0,02

c.Khong tuyn tnh

Mi quan h gia nng sibutramin trong dung dch tim sc k v p

ng pic c kho st trong khong nng t 1 g/mL n 200 g/mL.

Kt qu nh gi thc nghim (Bng 3.6) cho thy trong khong nng ny

c tng quan tuyn tnh cht ch (vi R =0,9995) gia nng sibutramin

v p ng din tch pic ca sibutramin khi phn tch bng HPLC trong iu

kin thit lp 3.1.1.

Bng 3.6. Kt qu nh gi tuyn tnh

Nng

(g/mL

)

1 2 5 10 20 50 100 200

Din

tch pic

(mAU.

101474

190029

445807

802296

1619762

3941470

7980841

15731022

31

s)

PT

ng

chun

Y=78625x+ 25766

R 0,9995

Hnh 3.16 ng chun ca sibutramin

Ngoi ra kho st thm ph hp ca ng chun, t ng chun thu

c v t din tch pic chng ti tnh li nng tng ng ca

sibutramin, t xc nh cc gi tr lch so vi im chun ban u. Kt

qu nh bng 3.7.

Bng 3.7: lch ca tng im chun dng xy dng ng chun

Nng ban u (g/mL)

Din tch pic (mAU.s)

Nng tnh ngc theo ng chun ca cc im

chun (g/mL)

lch ca tng im chun dng xy dng ng

chun (%) 200 15731022 199,75 -0,1 100 7980841 101,18 1,2 50 3841470 48,53 -2,9 20 1619762 20,27 1,4 10 802296 9,88 -1,2 5 445807 5,34 6,9 2 190029 2,09 4,5 1 101474 0,96 -3,7

32

Nhn xt: T cc kt qu bng v hnh v trn ta thy: ng chun c

R2=0,999, cc gi tr lch so vi im chun ban u cng u nm trong

gii hn 15% theo yu cu ca nhiu t chc M, Canada, chu u [3]. Nh

vy, ng chun biu din s ph thuc tuyn tnh ca din tch pic vi

nng sibutramin tng ng l t yu cu v c chp nhn theo AOAC

[3].

d. ng ca phng php

ng ca phng php c nh gi bng cch thm chun sibutramin

vo nn mu thc phm gim bo khng cha sibutramin qua nh gi s b

v xc nh t l (%) thu hi ca chun sibutramin khi x l mu v phn tch

theo quy trnh xy dng. ng ca phng php c thm nh trn 1

nn mu nang mm NM01 v 1 nn mu nang cng NC01. Kt qu phn tch

thc nghim c tp hp trong Bng 3.8 vi nn mu nang mm v Bng

3.9 vi nn mu nang cng.

Bng 3.8 thu hi ca phng php vi vin nang mm

Ln phn tch

Lng chun

sibutramin thm vo

(g)

Din tch (mAU.s)

Lng chun

sibutramin tm li c

(g)

thu hi (%)

01 10,00 897905 9,9 99,3 02 10,00 902320 9,98 99,8 03 10,00 903748 9,99 99,9 04 10,00 902215 9,97 99,7 05 10,00 903151 9,98 99,8 06 10,00 853924 9,44 94,4 07 10,00 859866 9,51 95,1 08 10,00 841711 9,30 93,1

Trung bnh 9,76 SD 0,29

RSD (%) 3,0

33

Bng 3.9 thu hi ca phng php vi vin nang cng

Ln phn tch

Lng chun

sibutramin thm vo

(g)

Din tch (mAU.s)

Lng chun

sibutramin tm li c

(g)

thu hi (%)

01 10,00 926473 10,12 101,2 02 10,00 943482 10,29 103,0 03 10,00 943339 10,29 102,9 04 10,00 937114 10,23 102,3 05 10,00 897459 9,82 98,3 06 10,00 891379 9,76 97,6 07 10,00 977657 10,64 106,5 08 10,00 964331 10,51 105,1 09 10,00 930577 10,16 101,6 10 10,00 893733 9,79 97,9

Trung bnh 10,16 SD 0,3

RSD (%) 3,0

Nhn xt: Theo AOAC, ti mc nng 100 ppb-10 g/mL, thu hi ca

phng php phi t l 80-110% [3]. T bng kt qu trn ta thy sibutramin

c hiu sut thu hi u nm trong gii hn cho php. Nh vy phng php

c thu hi t yu cu ca AOAC khi phn tch sibutramin trong vin

nang.

e. chnh xc ca phng php

Kho st trn 1 nn mu nang cng (NC07) v 1 nn mu nang mm (NM07)

c pht hin sibutramin. X l mu v tim mu vo h thng HPLC cc

iu kin chn. lp li ca phng php c nh gi thng qua

lch chun v lch chun tng i. Kt qu phn tch thc nghim c

tp hp trong Bng 3.10 vi nn mu nang cng v Bng 3.11 vi nn mu

nang mm.

34

Bng 3.10 lp li ca phng php vi vin nang cng

Ln

phn

tch

Khi

lng

cn

(g)

Th tch

dung mi

chit (mL)

H s

pha

long

Din tch pic

(mAU.s)

Nng trong dung

dch (g/mL)

Hm

lng

(mg/g)

01 0,2666 25 5 5406378 71,4 33,5

02 0,3148 25 5 6298576 83,7 33,3

03 0,2629 25 5 5256054 69,4 33,0

04 0,2650 25 5 5397384 71,3 33,6

05 0,2622 25 5 5032518 66,3 31,6

06 0,2404 25 5 4681806 61,5 32,0

TB (mg/g) 32,8 SD 0,8

RSD (%) 2,6

Bng 3.11 lp li ca phng php vi vin nang mm

Ln

phn

tch

Khi

lng

cn (g)

Th tch dung mi chit (mL)

H s pha

long

Din

tch

(mAU.s)

Nng

trong

dung dch

(g/mL)

Hm

lng

(mg/g)

01 0,4498 25 5 11772725 159,2 44,2 02 0,4577 25 5 12752861 172,7 47,2 03 0,4409 25 5 11614992 157,0 44,5 04 0,4420 25 5 11816802 159,8 45,2 05 0,4573 25 5 12845036 173,9 47,6 06 0,4703 25 5 13027837 176,5 46,9 TB (mg/g) 45,9

SD 1,5 RSD(%) 3,2

Nhn xt: Theo AOAC, ti mc nng 100 g/mL, lp ti a chp nhn

c gi tr RSD(%) l 5,3, cn ti mc 0,1% th gi tr ti a ca RSD(%) l

35

3,7 [3]. T bng kt qu trn ta thy sibutramin c lp u nm trong gii

hn cho php. Nh vy phng php c lp t yu cu ca AOAC khi

phn tch sibutramin trong vin nang.

f. Gii hn pht hin, gii hn nh lng

Trong phng php phn tch ny ph hp vi mc ch pht hin

sibutramin trong cc thc phm gim bo ta tin hnh xc nh LOD nh sau

pha long dung dch chun hn hp lm vic nhiu ln bng dung dch

methanol cho n khi khng cn thu c p ng pic ca sibutramin trn sc

k . Kt qu thc nghim cho thy, mc nng sibutramin ln lt l

0,2 g/mL v 0,5 g/mL (nng dung dch tim sc k), trn sc k cho

pic sibutramin c p ng cao khong gp 3 ln (Hnh 3.18) v 10 ln (Hnh

3.17) dao ng ng nn. Nh vy, phng php xy dng c gii hn

pht hin (LOD) l 0,2 g/mL, cn gii hn nh lng (LOQ) l 0,5 g/mL

vi sibutramin, tnh theo nng dung dch tim sc k.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

-10

0

10mAU

225nm,4nm (1.00)

/14.5

41/4

8677

Hnh 3.17 Sc k dung dch chun sibutramin 0,5 g/mL.

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

-10

-5

0

5

mAU225nm,4nm (1.00)

/14.5

83/1

9893

Hnh 3.18 Sc k dung dch chun sibutramin 0,2 g/mL

36

3.1.4. Kt qu p dng phng php xc nh sibutramin trong mt s

sn phm

Chng ti tin hnh nghin cu trn 14 thc phm chc nng gim bo

trn th trng theo phng php trn v thu c kt qu nh bng 3.12.

Bng 3.12 Kt qu phn tch sibutramin trong thc phm chc nng gim bo

Stt M sn

phm

Dng bo

ch Nh sn xut

Hm lng

sibutramin

(mg/vin)

1 NM01 Nang mm Dramerico Group

LLC -

2 NC01 Nang cng Dc phm Jemo-

Pharm A/S -

3 NC02 Nang cng Cng ty Bch An-

Trung Quc 26,6

4 NC03 Nang cng

Yunan Baian

Medicinal Science&

Technology co.,

LTD

-

5 NM02 Nang mm Khng r 13,7

6 NC04 Nang cng USA O&K trading 5,28

7 NM03 Nang mm Bacai Inc 20,23

8 NC05 Nang cng Khng r 17,0

9 NC06 Nang cng Khng r 28,2

10 NM04 Nang mm Arkopharma -

11 NM05 Nang mm Khng r -

12 NC07 Nang cng Khng r 11,0

37

13 NM06 Nang mm NKH Laboratories

(USA) -

14 NM07 Nang mm Khng r 14,51

Trong - : khng pht hin

3.2. Bn lun

Cng vi vic tham kho ti liu v thc nghim, chng ti la chn

c quy trnh phn tch sibutramin trong cc vin nang thc phm chc nng

gim bo, cc thng s chy my quan trng nh sau: th tch bm mu l 50

L; bc sng pht hin v nh lng i vi sibutramin l 225 nm; ct sc

k C18 Symmetry Waters (250 mm x 4,6 mm; 5m) vi tin ct cng loi;

tc pha ng l 1 mL/pht; nhit bung ct l 300C; thnh phn pha

ng gm acetonitril v m kali dihydrophosphat 0,05M, pH 4,0 chy theo

chng trnh gradient nng . Cc thng s ph hp vi iu kin phng th

nghim v vi h thng HPLC ca chng ti. im khc trong ti ca

chng ti l la chn c chng trnh gradient gip ra gii c

sibutramin mt cch hiu qu trn mu v thi gian cho mi ln phn tch ch

mt 16 pht.

Quy trnh x l mu ca chng ti la chn n gin, c kh nng lm sch

c mu, gip cho vic xc nh v nh lng cc cht trn sc d dng.

Phng php ca chng ti c thm nh ph hp vi cc yu cu ca

AOAC. Khong tuyn tnh ca sibutramin rng, gii hn pht hin v gii

hn nh lng cng tng ng vi cc nghin cu s dng HPLC trn.

38

Chng 4: KT LUN V KIN NGH

4.1. Kt lun

Sau khi hon thnh nghin cu ny, trn c s nghin cu cc iu kin

thc nghim, vi mc ch ng dng phng php HPLC xc hm lng

sibutramin, chng ti thu c kt qu nh sau:

Xy dng c phng php nh lng sibutramin trong vin nang thc

phm chc nng gim bo tri qua 2 giai on:

Giai on x l mu: mu c chit bng methanol, lc xoy, rung

siu m, nh mc, lc ri tin hnh chy sc k.

Giai on phn tch bng HPLC vi iu kin:

Pha tnh: Ct C18 Symmetry Waters (250 mm x 4,6 mm; 5 m) v tin

ct cng loi.

Pha ng: chy theo chng trnh gradient nng

Knh A: Kali dihydrophosphat 0.05M pH 4,0

Knh B: Acetonitril

Thi gian (pht) 0 1 3 11 12 13 17

Thnh phn pha ng

(%)

A 80 80 60 50 50 80 80

B 20 20 40 50 50 20 20

Nhit ct: 300C

Tc dng: 1 mL/pht

Th tch tim mu: 50 L

Detector PDA vi bc sng pht hin l 225 nm

Phng php xy dng xc nh c khong tuyn tnh v xy

dng c ng chun cho sibutramin, xc nh c LOD, LOQ ca

39

sibutramin, c chn lc, lp li, thu hi tt. lp li v thu hi

p ng yu cu ca AOAC.

p dng quy trnh phn tch trn xc nh hm lng sibutramin trong

14 mu thc phm chc nng gm c vin nang cng v nang mm. Kt qu

cho thy, c nhiu mu pht hin sibutramin trong nghin cu. y l mt kt

qu ng bo ng v tnh trng lm dng sibutramin trong cc thc phm

chc nng gim bo, t ra mt yu cu cn phi kim sot cht ch hn cc

thc phm ny.

T cc kt qu thu c, nhn thy phng php HPLC l ph hp xc

nh sibutramin trong cc vin nang thc phm chc nng gim bo.

4.2. Kin ngh

- Phng php cho kt qu xc nh sibutramin c tin cy tng i cao.

Cn tip tc m rng nghin cu trn cc i tng khc ngoi vin nang

nh: cc loi tr tho dc dng lng, dng ti, dng bt, mt s loi thuc,

trong mu, nc tiu...

- Nghin cu ny ch thc hin trn mt cht gim bo l sibutramin v cn

rt nhiu cc cht gim bo khc m nghin cu cha cp ti. V vy, cn

tip tc nghin cu su hn, tm ra phng php xc nh hm lng cc cht

gim bo khc trn nhiu i tng thc phm.

- Phng php ny tin hnh trn sibutramin dng mui hydroclorid. Ngoi

ra, sibutramin cn di nhiu dng khc nh cc cht chuyn ha ca n. Do

, cn m rng ngin cu trn nhiu dng khc nhau.

40

TI LIU THAM KHO

Ti liu ting Vit

1. Trn T An v cs. (2007), Ha phn tch- tp 2- Phn tch dng c, Nh

xut bn Y hc, H Ni.

2. Trn T An v cs. (2010), Kim nghim dc phm, Nh xut bn Y hc,

H Ni.

3. Trn Cao Sn v cs. (2010), Thm nh phng php trong phn tch ha

hc & vi sinh vt, Nxb Khoa Hc v K Thut.

4. B Y T, Quyt nh s 120/Q-QLD: Quyt nh v vic rt s ng k

thuc do thng tin v phn ng c hi.

Ti liu ting Anh

5. Aceves-Hernndez Juan M., Ins Nicols Vzquez , Jaime Hinojosa-

Torres, Guillermo Penieres Carrillo , Gabriel Arroyo Razo, Ren Miranda

Ruvalcaba (2013), Sibutramine characterization and solubility, a

theoretical study, Journal of Molecular Structure 1038, 163169.

6. Ancuceanu R., Mihaela Dinu, Corina arama, Weight loss food

supplements: Adulteration and multiple quality issus in two products of

Chinese origin, Farmacia (2013), Vol.61(1), 28-42.

7. Ariburnu Etil, Mehmet Fazli Uludag, Huseyin Yalcinkaya, Erdem

Yesilada (2012), Comparative determination of sibutramine as an

adulterant in natural slimming products by HPLC and HPTLC

densitometry , Journal of Pharmaceutical and Biomedical Analysis 64

65 (2012) 77 81.

8. Bailey C.J., Turner S.H., Jones R.B (2000). Sibutramine metabolites

increase glucose transport by cultured rat muscle cells, International

Journal of Obesity (2001), 25, 478-485.

41

9. Blachut D., A.Siwinska-Ziolkowska, B.Szukalski, K.Wojtassiewicz, Z.

Czarnocki, A.Kobylecka, M. Bykas-Strekowska (2007), Identification of

N-desmethylsibutramine as a new ingredient in Chinese herbal dietary

supplements, Problems of Forensic Sciences LXX , 225235.

10. Chen Jun, Wei Lu, Qizhi Zhang, Xinguo Jiang (2002), Determination of

the active metabolite of sibutramine by liquid chromatography-

electrospray ionization tandem mass spectrometry, Journal of

Chromatography B, 785(2003) 197-203.

11. Colectta D.K, Bates S.H., Jones R.B., Bailey C.J. The sibutramine

metabolite M2 improves muscle glucose uptake and reduces hepatic

glucose output: preliminary data, Diabetes Vasc Dis Res (2006), 3, 186-

188.

12. European Medicines Agency (EMEA), EMEA recommends suspension of

marketing authorizations for sibutramine.

(http://www.ema.europa.eu/docs/en_GB/document_library/Press

release/2010/01/WC500069995.pdf).

13. FDA,http://www.fda.gov/downloads/Drugs/DrugSafety/PublicHealthAdvi

sories/UCM130745.pdf

14. FDA, Phenylpropanolamin information page.

(http://www.fda.gov/drugs/drugsafety/informationbydrugclass/ucm150738

.htm)

15. FDA announces withdrawal of fenfluramine and dexfenfluramine.

(http://www.fda.gov/Drugs/DrugSafety/PostmarketDrugSafetyInformation

forPatientsandProviders/ucm179871.htm).

16. FDA, Meridia (sibutramine): Market Withdrawal Due to Risk of Serious

Cardiovascular Events.

42

(http://www.fda.gov/safety/medwatch/safetyinformation/safetyalertsforhu

manmedicalproducts/ucm228830.htm).

17. The FDA August 2010 drugs safety update.

(http://www.fda.gov/Safety/MedWatch/SafetyInformation/ucm225956.ht

m).

18. Feng Lu, Li Shu, Le Jian, Chen Guiliang, Cao Yan, Qi Yunpeng, Chai

Yifeng, Wu Yutian (2007), A new method for testing synthetic drugs

adulterated in herbal medicines based on infrared spectroscopy, Analytica

Chimica Acta 589, 200-207.

19. Guyton A.C., Hall J.E. (2006), Textbook of Medical Physiology, eleventh

ed., W. B. Saunders company, Philadelphia, Pennsylvania.

20. Julia Jung, Maren Hermanns-Clausen, Wolfgang Weinmann (2006),

Anorectic sibutramine detected in Chinese herbal drug for weight loss,

Forensic Science International 161 (2006) 221-222.

21. Luque Carla A., Rey Jose A., (2001), The discovery and status of

sibutramine as an anti-obesity drug, European Journal of Pharmacology,

440 (2002) 119-128.

22. Sabina Strano-Rossi, Cristiana Colamonici and Francesco Botre (2006),

Detection of sibutramine administration: a gas chromatography/ mass

spectrometry study of the main urinary metabolite, Rapid

communications in Mass spectrometry (2007) 79-88.

23. Suthar A.P., Dubey S.A. and Patel S.R., A validated Specific Reverse

Phase Liquid Chromatographic Method for the estimation of Sibutramine

Hydrochloride Monojydrate in bulk drug and capsule dosage forms,

International Journal of Chemtech Research, Vol.1, No.4, 793-801.

24. Scheen Andr j. et al, Sibutramine on Cardiovascular Outcome,

Diabetes Care vol. 34, 114-119.

43

25. World Health Organization (WHO), 2010. Obesity and overweight

(http://www.who.int/mediacentre/factsheets/fs311/en/index.html).

26. Zhou Yu, Qiang Wei, Qingsheng Fan, and Chunhua Wan (2010), A

simple and convenient method for simultaneous determination of four

major species of illegal additives in slimming health food, Journal of

Liquid Chromatography & Related Technologies 33, 452461.

44

PH LC

200 300 nm

0

50

100

150

200

250

300

350

mAU

14.16/ 1.00

212

257 273 284 390

199

223

268 276 302

Ph lc 1: Ph hp th UV-VIS ca sibutramin

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

50

100

150

200

mAU225nm,4nm (1.00)

/14.9

23/9

0315

1

Ph lc 2: Sc k ca mu thc phm chc nng NM01 thm chun

10g sibutramin

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

100

200

mVDetector A Ch1:225nm

/13

.62

4/8

91

37

9

Ph lc 3: Sc k ca mu thc phm chc nng NC01 - thm chun

10g sibutramin

45

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

100

200

mAU225nm,4nm (1.00)

/14.

171/

5397

384

Ph lc 4: Sc k xc nh lp li trn nn mu thc phm chc

nng NC07

0.0 2.5 5.0 7.5 10.0 12.5 15.0 min

0

250

mAU225nm,4nm (1.00)

/14.

142/

1275

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Ph lc 5: Sc k xc nh lp li trn nn mu thc phm chc

nng NC07

DANH MC CC K HIU- T VIT TTDANH MC CC BNGDANH MC CC HNHT VN Chng 1. TNG QUAN1.1. Vi nt chung v cht gim bo1.2. Tng quan v sibutramin1.2.1. Cu trc- tnh cht l ha1.2.2. Tnh cht dc ng hc, tc dng dc l1.2.3. Mt s phng php phn tch sibutramin

1.3. Tng quan v HPLC1.3.1. Khi nim chung1.3.2. Mt s khi nim c bn trong sc k 1.3.3. Thit b sc k lng [1], [2]

Chng 2. I TNG V PHNG PHP NGHIN CU (NGUYN LIU, THIT B, NI DUNG, PHNG PHP NGHIN CU)2.1. i tng nghin cu2.2. Nguyn vt liu - thit b2.2.1. Nguyn vt liu2.2.2.Thit b

2.3. Ni dung nghin cu2.4. Phng php nghin cu2.4.1. Kho st cc iu kin phn tch sibutramin bng HPLC2.4.2. Kho st iu kin x l mu2.4.3. Thm nh quy trnh

2.5. Phng php x l s liu

Chng 3. THC NGHIM, KT QU V BN LUN3.1. Thc nghim v kt qu3.1.1. Thit lp iu kin sc k phn tch sibutramin3.1.2. Xy dng quy trnh x l mu chit sibutramin trong thc phm gim bo3.1.3. Thm nh phng php3.1.4. Kt qu p dng phng php xc nh sibutramin trong mt s sn phm

3.2. Bn lun

Chng 4: KT LUN V KIN NGH4.1. Kt lun4.2. Kin ngh