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I HC QUC GIA H NI

TRNG I HC KHOA HC T NHIN

TH THU HNG

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TCH V XC NH MT S C T NHM ALKALOID

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TRONG THC PHM CHC NNG BNG PHNG PHP SC K

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LNG KHI PH

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LUN VN THC S KHOA HC

H Ni 2015

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I HC QUC GIA H NI
TRNG I HC KHOA HC T NHIN

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TH THU HNG

TCH V XC NH MT S C T NHM ALKALOID

TRONG THC PHM CHC NNG BNG PHNG PHP SC K

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LNG KHI PH

Chuyn ngnh: Ha phn tch

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M S: 60440118

Ngi Hng Dn Khoa Hc: PGS TS. L Th Hng Ho

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LUN VN THC S KHOA HC

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H Ni 2015

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LI CM N

B Y t tn tnh hng dn, ng gp nhng kin qu bu v to mi iu

PGS TS. L Th Hng Ho, Vin Kim nghim an ton v sinh thc phm Quc gia

hon thnh lun vn ny li u tin cho em gi li cm n su sc ti

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kin gip em trong sut qu trnh thc hin ti.

Em xin by t lng bit n ti cc thy c gio ging dy ti khoa Ho hc,


c bit l cc thy c trong b mn Ho Phn Tch, cho em nhng kin thc

qu gi, to iu kin cho em trong sut qu trnh hc tp v nghin cu.

Ti xin c gi li cm n chn thnh ti ban lnh o Vin kim nghim

An ton v sinh thc phm Quc gia to mi iu kin thun li ti c hc

tp v hon thnh ti ny.

Ti cng xin gi li cm n ti cc ng nghip ti labo Ha Vin kim


nghim An ton v sinh thc phm Quc gia gip ti rt nhiu trong qu trnh

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lm thc nghim.

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Cui cng ti xin gi li cm n ti gia nh v bn b lun ng vin,

H Ni, nm 2015
Hc vin

Th Thu Hng

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chia s mi kh khn cng ti.

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MC LC

BNG K HIU CH VIT TT

M U .....................................................................................................................1

LI CM N

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CHNG 1. TNG QUAN .......................................................................................3


1.1. Tng quan v nhm alkaloid ..............................................................................3
1.1.1. Lch s pht hin ..............................................................................................3

1.1.2. Khi qut v nhm alkaloid .............................................................................3

1.1.3. Cu to ca mt s alkaloid c ......................................................................6

1.1.4. Tc dng v c tnh ca mt s alkaloid ........................................................7

1.2. Cc phng php xc nh ...............................................................................13

1.2.1. Phng php in di mao qun ......................................................................13

1.2.2. Phng php sc k lng vi detector UV ....................................................14

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1.2.3. Phng php sc k lng vi detector khi ph ............................................16

1.3. Sc k lng khi ph ........................................................................................18

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1.3.1. Nguyn tc chung ca sc k lng .................................................................18

10

1.3.2. Detector khi ph (Mass spectrometry MS) ...............................................18

1.3.2.4. B phn pht hin ........................................................................................22

1.3.3. Phn tch nh tnh v nh lng bng LC-MS/MS .....................................22

1.4. Ly mu ............................................................................................................23

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1.5. nh gi phng php phn tch ......................................................................23

CHNG 2: NI DUNG V PHNG PHP NGHIN CU...........................26

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2.1 i tng v mc tiu nghin cu ...................................................................26

2.2 Phng php nghin cu.....................................................................................26

2.3 Thit b, dng c v ha cht dng trong nghin cu ......................................27


2.3.1 Thit b..............................................................................................................27
2.3.2 Dng c ............................................................................................................27
2.3.3 Cht chun ........................................................................................................28
2.3.4 Cc loi ha cht, dung mi khc ...................................................................29

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CHNG 3. KT QU V THO LUN ............................................................30


3.1. Ti u iu kin tch v xc nh cc alkaloid trn thit b LC/MS/MS .........30

3.1.2. Ti u cc iu kin chy sc k lng hiu nng cao (HPLC) ......................33

3.1.1. Ti u cc iu kin ca detector khi ph (MS/MS) ...................................30

Kho st dung mi chit...............................................................................43

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3.2.1.

3.2. Ti u qu trnh x l mu ...............................................................................42

3.2.2. Kho st dung dch kim ha .........................................................................45


3.2.3.

Kho st th tch dung dch kim ha ..........................................................46

3.3. nh gi phng php phn tch ......................................................................48

3.3.1. Tnh c hiu/chn lc ...................................................................................48

3.3.2. Kho st khong tuyn tnh v lp ng chun ...........................................49


3.3.3. Gii hn pht hin (LOD), gii hn nh lng (LOQ) ca phng php ...51

3.3.4. nh gi lp li v thu hi ...................................................................52

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3.4. Phn tch mu thc. ..........................................................................................57


KT LUN V KIN NGH...................................................................................59

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KT LUN ...............................................................................................................59

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KIN NGH ..............................................................................................................60

TI LIU THAM KHO .........................................................................................61

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PH LC ..................................................................................................................65

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DANH MC BNG

Bng3.1: Cc mnh ion nh lng v nh tnh ca cc alkaloid ...........................30

Bng 1.2. Cc gi tr LD50 ca cc cht phn tch.......................................................8

Bng 1.1. Cu trc ho hc ca mt s alkaloid c c xc nh trong ti. ......6

Bng 3.2: Cc thng s ti u MS/MS i vi ch ion dng ............................33

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Bng 3.3: Cc h dung mi pha ng kho st .........................................................34


Bng 3.4: Din tch cc alkaloid khi s dng cc h dung mi khc nhau ..............36

Bng3.5: Cc chng trnh gradient kho st ...........................................................37

Bng3.6: nh hng nng amoniacetat ti din tch pc cc alkaloid ................41

Bng 3.7 : nh hng ca dung mi chit n hiu sut chit cc alkaloid ............44

Bng 3.8 : nh hng ca dung dch kim ha n hiu sut chit cc alkaloid ....45

Bng 3.9: nh hng ca th tch dung dch kim ha n hiu sut chit cc

alkaloid ......................................................................................................................46

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Bng 3.10: ng chun cc alkaloid ......................................................................50

Bng 3.11: Gii hn pht hin v gii hn nh lng ca cc alkaloid ..................51

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Bng 3.12: lp li v hiu sut thu hi ca cc Alkaloid trn nn mu thc phm

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chc nng ti nng 50-500 g/kg (0,05-0,5 mg/kg) ...........................................53

Bng 3.13: lp li v hiu sut thu hi ca cc Alkaloid trn nn mu thc phm

chc nng ti nng 0,1-1 mg/kg ..........................................................................54

Bng 3.14: lp li v hiu sut thu hi ca cc Alkaloid trn nn mu thc phm

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chc nng ti nng 0,2-2 mg/kg ..........................................................................55

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Bng 3.15: Bng kt qu phn tch mu thc ...........................................................57

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Hnh 1.1: Ch ion ha phun in t ESI ................................................................................. 19

DANH MC HNH

Hnh 1.2: Cu to ca b phn tch khi t cc chp ba ....................................................... 21

Hnh 3.1: Sc t l cc ion ca koumin v atropin............................................................. 31

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Hnh 3.2: Sc ion tng khi s dng h dung mi pha ng 1 ....................................... 34


Hnh 3.3: Sc ion tng khi s dng h dung mi pha ng 2 ....................................... 35

Hnh 3.4: Sc ion tng khi s dng h dung mi pha ng 3 ....................................... 35

Hnh 3.5: Sc ion tng khi s dng h dung mi pha ng 4 ....................................... 35

Hnh 3.7: Sc ion tng khi s dng chng trnh gradient 2 ......................................... 37

Hnh 3.8: Sc ion tng khi s dng chng trnh gradient 3 ......................................... 38

Hnh 3.9: Sc ion tng khi s dng chng trnh gradient 4 ......................................... 38

Hnh 3.10: Sc ion tng khi s dng chng trnh gradient 5 ...................................... 38

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Hnh 3.11: Sc ion tng ti tc dng 0,6 mL/pht ....................................................... 40


Hnh 3.13: Sc ion tng ti tc dng 0,4 mL/pht ....................................................... 40

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Hnh 3.14: Biu s ph thuc din tch pc ca cc alkaloid vo nng

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amoniacetat trong pha ng .............................................................................................................. 42

Hnh 3.15: S quy trin h chi t mu d ki n ........................................................................... 43

Hnh 3.16 : Biu nh hng ca dung mi chit n hiu sut chit cc alkaloid

......................................................................................................................................................................... 44

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Hnh 3.17 : Biu nh hng ca dung dch kim ha n hiu sut chit cc

alkaloid......................................................................................................................................................... 46

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Hnh 3.18 : Quy trnh chit mu ti u .......................................................................................... 47

Hnh 3.19 : Sc chun alkaloid, mu trng v mu trng thm chun ...................... 48

Hnh 3.20: Mi tng quan gia din tch pic v nng Atropin trong khong 0,5-

100 ng/mL .................................................................................................................................................. 49


Hnh 3.21: Mi tng quan gia din tch pic v nng aconitin trong khong 51000 ng/mL ................................................................................................................................................ 49
Hnh 3.22: ng chun Scopolamin (R2 = 0,9999) .............................................................. 50

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Hnh 3.23: Sc ca nicotin ti gii hn pht hin LOD 1,0 g/kg (S/N = 3,6) .... 51
Hnh 3.24: Sc ca Brucin ti gii hn nh lng LOQ 10 g/kg (S/N = 11,5) 52

nng 0,1 mg/kg .................................................................................................................................. 55

Hnh 3.26: Sc atropin trong mu thc phm chc nng thm chun ti mc

Hnh 3.27: Sc scopolamin trong mu thc phm chc nng thm chun ti mc

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nng 0,2 mg/kg .................................................................................................................................. 56


Hnh 3.28: Sc mu pht hin brucin ....................................................................................... 58

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Hnh 3.29: Sc mu pht hin strychnin ................................................................................. 58

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BNG K HIU CH VIT TT


Ting Anh

Ting Vit

ACN

Acetonitrile

acetonitril

Atmospheric pressure chemical

Ch ion ha p sut kh

ionization

quyn

CE

Collision energy

Nng lng va chm

ESI

Eelectrospray ionization

Ch ion ha phun in t

EU

European Union

Chu u

HPLC

High performance liquid

Sc k lng hiu nng cao

International Union of Pure and

IUPAC

Limit of deterction

LOQ

Limit of quantity

MeOH

Methanol

ODS

Octadecylsilan

LOD

Lin minh quc t v ha hc


c bn v ng dng
Liu gy cht trung bnh
Gii hn pht hin

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Lethal Dose

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Applied Chemistry
LD50

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chromatography

APCI

K hiu

Gii hn nh lng

Methanol

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Relative standard deviation

lch chun tng i

Standard deviation

lch chun

SPE

Solid phase extraction

Chit pha rn

UPLC-

Ultral performance liquid

Sc k lng siu hiu nng kt

MS/MS

chromatography tandem mass

ni khi ph

RSD

spectrometry

TO

SD

Octadecylsilan

UV

Ultraviolet

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T ngoi

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Vit Nam l nc nhit i c kh hu nng m, rt thun li cho s pht

M U

trin ca thc vt v y c coi l mt kho tng v gi v ngun cy thuc v

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dc liu qu. Trong alkaloid l nhng thnh phn hot tnh chnh ca nhiu cy
thuc. Alkaloid l amin ngun gc t nhin do thc vt to ra, nhng cc amin do

ng vt v nm to ra cng c gi l cc alkaloid. Cu trc bao gm carbon,

hydro, nit, v thng c oxy. Cc alkaloid l baz hu c tng t nh kim (baz

v c); tn c ngha l nh kim. Alkaloid xut hin ch yu trong cc chi khc

nhau ca thc vt c ht, chng hn nh cy thuc phin v cy thuc l, m tin.

Chng c th c tm thy trong hu ht cc b phn ca cc loi thc vt ny, bao

gm c l, r, ht, v v cy. Mi mt phn ca thc vt thng c cha mt s

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cht ha hc lin quan n alkaloid. Chc nng ca alkaloids trong chuyn ha thc

vt cha c bit, nhng hu ht cc alkaloid c nh hng r rt n h thng

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thn kinh ca ngi v ng vt khc. Trong s hng trm alkaloid c tm thy

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trong t nhin, ch c khong 30 cht c s dng thng mi. Mt s alkaloid,

chng hn nh nicotin, c s dng trong thuc tr su, v mt s khc c s

dng lm thuc th ha hc. Tuy nhin, cc alkaloid c s dng ch yu trong y

hc, bi v chng c th tc ng mt cch nhanh chng trn cc khu vc c th ca

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h thn kinh. Alkaloid l nhng thnh phn hot tnh chnh ca nhiu thuc gy m,

thuc an thn, cc cht kch thch v thuc an thn. Chng c dng bng ng

TO

ung v tim. Ngoi tr di s gim st ca bc s, s dng cc alkaloid l nguy

him, bi v hu ht hnh thnh thi quen (v d, gn nh tt c cc cht ma tu l


alkaloid) v dng liu lng ln c th l c hi.
Trong dn gian ta thng s dng cc cy tho dc nh c c dc, m

tin, c u tu dng cha bnh nhng trong cc loi cy ny c cha mt hm


lng khng nh cc cht c nhm alkaloid. Hin nay, mt s loi thc phm chc

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

nng c ngun gc tho dc cng s dng cc loi cy ny hoc c thnh phn


c chit xut t cy thuc c cc hot cht

nh cc alkaloid, terpenoid,

chc nng ny c th l con dao hai li, dng vi liu lng va phi c tc dng

phenolic c bit n nh l cc hp cht th cp. S dng cc loi thc phm

cha mt s bnh song dng khng ng cch hoc qu liu c th dn n ng c

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hoc cc bin chng. V vy s dng nhng thc phm chc nng c cha cc
alkaloid c ny phi cn trng v phi c s qun l v cho php ca cc c
quan chc nng. Tuy nhin, Vit Nam vic xc nh ng thi cc alkaloid c

trong thc phm chc nng vn cha c nghin cu. Do vy, gp phn kim

sot cc nguy c tim n m bo an ton cho ngi tiu dng v gip c quan

chc nng qun l a ra cc khuyn co nhm gim nguy c ng c do s dng


cc thc phm chc nng c ngn gc tho dc. Chng ti tin hnh ti:

Tch v xc nh mt s c t nhm alkaloids trong thc phm chc nng

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bng phng php sc k lng khi ph.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

CHNG 1. TNG QUAN

Tng quan v nhm alkaloid

1.1.1. Lch s pht hin

Nm 1804- 1805, cc nh ha hc Php v c phn lp c morphin

1.1.

v iu ch c dng mui ca n. ng thi chng minh c morphin l

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hot cht chnh ca cy thuc phin c tc dng sinh l r rt. Nm 1980, t v cy


canhkina, chit v kt tinh c mt cht t tn l cinchonino sau hai nh

ha hc Php xc nh cinchonino l hn hp ca hai alkaloid l quinin v

cinchonin. Nm 1918, pht hin ra alkaloid ca ht m tin l strychnin v brucin;

pht hin ra cafein trong ch, c ph. Sau tip tc pht hin ra nicotin trong thuc

l, atropin trong c c dc, theobromin trong cacao, codein trong thuc phin,

cocain trong l coca. Gia nm 1973, ngi ta xc nh c 4959 alkaloid khc

nhau trong c 3293 cht xc nh c cng thc ha hc. Hin nay pht

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hin ra c rt nhiu alkaloid v cng c a vo ng dng trong y hc ngy

mt tng [15].

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Khi qut v nhm alkaloid

10

1.1.2.

1.1.2.1. Khi nim

Alkaloid c ngun gc t ch: alcali ting rp l kim. Alkaloid l:

- Nhng hp cht hu c c cha d vng nit, c tnh baz thng gp

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trong nhiu loi thc vt v i khi cn tm thy trong mt vi loi ng vt.

- C hot tnh sinh hc rt quan trng.


- C mt s phn ng chung l to ta cn thit cho s xc nh chng.

Chng l mt nhm hp cht thin nhin quan trng v nhiu mt. c bit trong
lnh vc y hc, chng cung cp nhiu loi thuc c gi tr cha bnh cao v c o
[15] [16].

TO

- C phn ng kim cho cc mui vi acid v cc mui ny d kt tinh.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

1.1.2.2. Cu to ha hc
V mt ha hc, s phong ph v a dng ca alkaloid tr thnh mt

ch thng tin v ha hc. i vi vic nghin cu alkaloid cn quan trng hn bi

chuyn nghnh, chim mt v tr quan trng trong lnh vc nghin cu v trong tp

v chng c trong h thc vt nhit i phong ph l ngun cung cp alkaloid ch

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yu. V mt cu trc ha hc, chng c t nht 1 nguyn t N trong phn t, ch


yu nm trong vng. S c mt ca nguyn t ca N trong cu trc quyt nh tnh

baz ca alkaloid v l ch da rt quan trng cho cc nh ha hc trong nghin


cu alkaloid [15].

1.1.2.3. Phn loi

Cc alkaloid bit c n trn 250 dng cu trc khc nhau vi hn 5.500

hp cht alkaloid trong t nhin. V vy cch phn loi da vo cu trc nhn c

bn trc y (khong 20 nhm cu trc) cha p ng c nn ngy cng c xu

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hng chia chng thnh nhng nhm nh: nhm alkaloid Ecgotamia, Harmin,

cc

nhm:

Benzyliaoquinolin,

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thnh

Yohimbin, Strychnin, Echitamin. Nhm alkaloid c nhn isoquinolin c chia


Apocphin,

Protobecberin,

10

BenzophenanthridinC kin xp cc hp cht c N ngoi vng nh Colchicin,

Hordenin, l cc protoalkaloid [15] [27].

1.1.2.4. S phn b

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Cromwell (1995) c tnh alkaloid phn b trong khong 1 phn 7 loi thc
vt c hoa. Mt c tnh khc (Hegnauer, 1963) cho rng alkaloid c t 12%-20%

trong tng s cy c nha. Cn Willaman v Schubert (1955) th cho rng trong hn

TO

300 h ca nghnh ht kn th 1/3 h c cha alkaloid. Nhiu tng kt cho thy i


a s cy c cha alkaloid l cy hai l mm. Ch c mt s t cy cha alkaloid l

cy 1 l mm v nghnh ht trn. Theo thng k n nay th cy thuc tho v cy


bi c nhiu alkaloid hn cy g, v trng lng phn t ca alkaloid trong cy g
thng b hn trng lng phn t cy thuc tho. Cy mt nm c nhiu alkaloid
hn cy lu nin (Levin, 1976). Alkaloid khng c trong loi sng nc ngoi tr

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

h sen (Nympheaceae) (Mc.Nair.1943). Thc ra rt nhiu loi c alkaloid nhng ch


mc dng vt hoc t l phn vn, mi vn. gii hn vi ngha thc

dc liu kh. Mi lin quan gia alkaloid vi cc cht khc trong 1 cy cng

tin, mt cy c xem l c alkaloid phi cha t nht l 0.05% alkaloid so vi

c nghin cu. Cy c cha alkaloid u vng mt tinh du v ngc li

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(Trelibs,1955) [27].
1.1.2.5. Tnh cht

Alkaloid l cc baz yu, do s c mt ca nguyn t N. Nhng kim

ca alkaloid khng ging nhau do nh hng khc nhau ca lp in tch nguyn t

N gy ra v nh hng ca cc nhm chc khc. Ni chung tnh baz gim dn theo

th t amoni bc 4, amoni bc 1, amoni bc 2, amoni bc 3.

Hu ht cc alkaloid baz thc t khng tan trong nc nhng tan trong cc


dung mi hu c nh CHCl3, eter v cc ancol bc thp (methanol, ethanol,

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butanol). Mt s nhm alkaloid c thm cc nhm phn cc nn tan c mt phn

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trong nc hoc trong kim. V d: Morphine, Cephalin, do c nhm OH phenol

10

nn tan trong dung dch kim v cc baz ca chng th gn nh l khng tan trong
eter. Ngc li vi baz, cc mui alkaloid ni chung tan c trong nc v cn

nhng hu nh khng tan trong dung mi hu c nh: CHCl3, eter, benzene. C

mt s trng hp ngoi l nh Ephedrin, Cochicin, Ecgovonin, cc baz ca

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chng tan c trong nc nhng cng kh tan trong dung mi hu c v cc mui

ca chng th ngc li. Alkaloid c N Bc 4 v N oxide baz ca alkaloid c N


Bc 4 v N oxide khc tan trong nc v trong kim t tan trong dung mi hu
chng. V vy c th chit chng bng dung dch kim v kt ta di dng mui c

ha tan thp nht. i vi cc alkaloid N oxide th dng phn ng kh. Oxy


bng bt km trong mi trng HCl chng chuyn thnh alkaloid dng thng
sau kt ta bng kim v chit bng dung mi hu c [14].

TO

c. Cc mui ca chng c ha tan khc nhau ty thuc vo gc acid to ra

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Lun vn Thc s khoa hc

1.1.3.

Th Thu Hng- K23 Ha hc

Cu to ca mt s alkaloid c

C23H26N2O4

394,46

8,28

C21H22N2O2

334,41

pKa

Cng thc cu to

Khi
lng
phn t
(g/mol)

TP
.Q
U

Alkaloid

Cng thc
phn t

STT

Bng 1.1. Cu trc ho hc ca mt s alkaloid c c xc nh trong ti.

Brucin
1

10,11-

Dimethoxystrychnine

Strychnin

(4aR,5aS,8aR,13aS,15aS,

15bR)-

b,16-decahydro-2H-4,6-

8,26

4a,5,5a,7,8,13a,15,15a,15

methanoindolo[3,2,1-

TR

ij]oxepino[2,3,4de]pyrrolo[2,3-

Aconitin

10

00

h]quinoline-14-one

C34H47NO11

645,74

5,88

C17H23NO3

289,37

4,35

Atropin
(RS)-(8-Methyl-8-

azabicyclo[3.2.1]oct-3-yl)
3-hydroxy-2phenylpropanoate

TO

-L

Acetylbenzoylaconine

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Scopolamin

N
7,75

C20H22N2O

306,41

303,35

C17H21NO4

TP
.Q
U

()-(S)-3-hydroxy-2phenylpropionic
acid(1R,2R,4S,7S,9S)-9methyl-3-oxa-9azatricyclo[3.3.1.02,4]no
n-7-yl ester

Koumin
7,20(2H,19H)Cyclovobasan, 1,2,18,19-

tetradehydro-3,17-epoxy-

, (3R,7alpha,20alpha)-

Colchicin
N-[(7S)-1,2,3,10Tetramethoxy-9-oxo-

TR

C22H25N O6

8,5

Nicotin

pyridyl)pyrrolidine,(S)-3-

C10H14N2

-L

(1-Methyl-2-pyrrolidinyl)
pyridine

162,23

10

en-7-yl]acetamide

12,35

00

tetrahydrobenzo[a]heptal

()-1-Methyl-2-(3-

399,44

5,6,7,9-

TO

1.1.4.

Tc dng v c tnh ca mt s alkaloid

Cc cht trong nghin cu ny thuc nhm alkaloid chng c tc dng ln h

thn kinh, tim mch v h hp [3] [12]. Khi dng vi hm lng nh chng c tc
dng cha bnh nhng khi dng vi hm lng ln gy ng c c th dn n t
vong v y u l cc alkaloid c. Bng 1.2 gii thiu gi tr LD50 (liu lng
cht c gy cht cho mt na s c th trong nghin cu) [24].

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

TP
.Q
U

Brucin
Strychnin
Colchicin
Koumin
Scopolamin
Nicotin
Aconitin
Atropin

LD50 trn chut ng LD50 trn chut ng


ung (mg/kg)
tim (mg/kg)
150
12
2,2
0,16
5,886
100
1270
650
3
1
0,12
75
30

Tn cht

Bng 1.2. Cc gi tr LD50 ca cc cht phn tch.

1.1.4.1. Brucin v strychnin

Brucin v strychnin u l alkaloid ng, brucin c lin quan cht ch vi

strychnin. Hai cht ny xut hin mt s loi thc vt, c bit n nhiu nht l

TR

alkaloid chnh trong v, l, ht cy m tin c tm thy ng Nam . Ht cha

khong 1,5% strychnin, cn hoa kh cha khong 1,023 %, ngay c v cy cng

00

cha cc hp cht c, bao gm c brucin [5]. Strychnin l mt alkaloid khng

10

mu, t tan trong du la v trong nc, khng tan trong ethyl eter. Strychnin trch

H
-

dc in

t ht cy m tin hay nhn, rt c, cht strychnin c ghi vo bng A ca cc

-L

Trong y hc dn gian, m tin dng nh thuc b lnh bnh v kch thch s

thm n v l mt loi thuc vi lng ng cn, c dng cho nhng vn tiu

TO

ha thng thng, nhy cm vi nhit lnh, cu knh kh chu v s dng nh

Strychnin dng tinh th s dng lm thuc tr su, c bit l git cht


ng vt c xng sng nh nh cc loi chim v ng vt gm nhm. Strychnin
gy co git c bp v cui cng cht v ngt.

thuc b cho h thng tun hon trng hp b chng suy tim.

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

ng trn quan im dc l, strychnin l mt cht kch thch ch yu vo


ty sng, y l h thng thn kinh trung ng. N lm tng cm nhn gic quan

vo trung tm h hp nm trong hnh ty ( bulle rachidien ) v gia tng bin h

nh: v gic, khu gic, th gic. Vi lng va phi trung bnh, hot ng tc dng

hp. Cht strychnin c s dng trong cha tr nhng chng ri lon chc nng c

TP
.Q
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vng ( sphinctriens ) nht nh, s suy h hp v vim a thn kinh nghin ru.
Khi dng vi mt liu rt nh, theo Stille ( Systme de Madica et thrapeutique,
1864 ): dng trong ri lon tiu ha, gia tng bi tit dch v d dy, ming

gan v ty tng. Nhng khi dng qu liu c th dn n ng c. Khi ng c

strychnin, c th hin bi nhng cn ng kinh co git v i khi a n t

vong v ngt th.V vy, strychnin khng cn s dng trong y hc hin i, mc d


n c s dng rng ri trong y hc thi trc th chin th hai. Li u dung lam

thu c cua strychnin sulfat la 1 mg/mL [12] [13].

TR

Cht brucin gn ging nh strychnin trong chc nng, nhng nh hn, t c

hn, n ch lm t lit h thn kinh vn ng ngoi vi. Cho mc ch y t, brucin

00

c s dng ch yu trong vic iu tit huyt p cao v bnh tim tng i lnh

10

tnh khc. Brucin khng phi l cht c hi nhng mt ngi tiu th hn 2 mg

brucin tinh khit gn nh chc chn s b cc triu chng tng t nh ng c

strychnin v c th gy t vong liu lng ln [16] [27] [31].

-L

1.1.4.2. Aconitin

Aconitin l mt alkaloid c c trong c u tu, cn gi l c gu tu, l r

c ca cy u Vit Nam, tn khoa hc l Aconitum fortunei. Cy thng mc

TO

hoang cc vng ni cao bin gii pha Bc nc ta: Lo Cai, Tuyn Quang, H
Giang... u c xp vo loi thuc rt c (bng A). c tnh ca aconitin rt

mnh: Ch cn mt liu t 0,02 - 0,05 mg cho 1kg th trng l c th gy cht


ngi. nhit cao, aconitin b phn hy thnh benzoylaconin v sau l
aconin km c hn aconitin khong 1.000 n 2.000 ln. Loi c t ny thng
c dng tm vo u tn khi sn th rng, k cvoi.

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Trong ng y, u tu c dng ngm ru xoa bp khi b au nhc, t


mi chn tay. Ty y dng lm thuc ho, cha chng ra m hi nhiu. Thng ch

dng lm thuc ung khi qua ch bin cn thn v c dng vi liu nh, c s

ch nh v theo di thn trng ca thy thuc.

C u tu ngm ru ch dng xoa bp. Nguy c b ng c thng l

TP
.Q
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trong nhng trng hp ung nhm tng l ru thuc, trong tm vi ca tr, khi
dng qu liu ch nh ca thy thuc. mt s tnh vng cao nh Lo Cai, H

Giang, ngi dn thng nu cho c u tu dng nh mn c sn vng cao. Tuy

nhin nu n nhng mn n c c u tu ch bin cha ng cch s gy ng c.

Biu hin ca ng c : Sau khi n, aconitin ngm rt nhanh qua nim mc d dy,

rut vo mu gy nn cc triu chng nh t ming li, ni kh, t mi chn tay,

chut rt, au u, nhn m, bun nn, nn, a chy, nng hn c th lit c h hp,

lon nhp tim, Nu khng c iu tr kp thi bnh nhn s t vong [16] [17].

TR

1.1.4.3. Atropin

(Datura meterl), ging cy c

00

belladonna), cy c c d c

Atropin l mt alkaloid t nhin chit xut t cy ba ch anh

(Atropa

(Mandragora

10

officinarum) v cc b phn ca cy h c (Solanaceae). Atropin lm gin ng t,

tng nhp tim, v lm gim tit nc bt v dch tit khc. Atropin c xp vo

thuc c bng A, liu c atropin tc ng ln no lm say c khi pht in, h

hp tng, st, cui cng thn kinh trung ng b c ch v t lit.

-L

Atropin l mt trong cc alkaloid chnh trong cy c c dc c Vit

Nam, vi hm lng cao nht l v ht. Hm lng ton phn cc alkaloid t 0,2-

TO

0,5%, ch yu l scopolamin, hyoscyamin, atropin v cc saponin, flavonoid,

tanin... Tc dng dc l ca c c dc ch yu l do cc alkaloid: lm gin ph


qun, gin ng t, gim nhu ng rut v bao t nu nhng c quan ny co tht,
lm kh nc bt, dch v, m hi. Theo ng y, hoa c c dc c v cay, tnh
n, c c, c tc dng nga suyn, gim ho, chng au, chng co git, phong thp
au nhc. L l v thuc nga cn hen, gim au bao t, chng say tu xe. Ngoi ra
cn iu tr phong t thp, au dy thn kinh to, au rng... Ngi ta thng dng

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

l cun thnh iu hay thi nh vn thnh iu thuc ht (cha ho, hen suyn),
dng l h nng p iu tr au nhc, t thp, hoc phi kh tn bt mn.V cy c

hin tng gin ng t, m mt, tim p nhanh, gin ph qun, mi ming kh,

c tnh cao nn ch dng theo s hng dn ca thy thuc. Khi b ng c, c

kh c n mc khng nut v khng ni c. Cht c tc ng vo h thn kinh

TP
.Q
U

trung ng, c th gy t vong do hn m [27] [31].

Atropin l alkaloid khng th th i giao cm M (Muscarin), tc dng ln c

trung ng v ngoi bin. Atropin c dng c ch tc dng ca h thn kinh i

giao cm. Vi liu iu tr, Atropin c tc dng yu ln th th Nicotin [12] [13].

1.1.4.4. Scopolamin

Scopolamin hay cn gi l hi th ca qu hoc gi tn khc l


mt

loi

ma

ty

hay

ma

dc

bo

ch

Burundanga

cy Borrachero Colombia v c tc dng gy m ng thi c kh nng lm mt

TR

i thn tr ca con ngi v a con ngi vo trng thi b thi min. Cy

00

Borrachero thuc h c c dc v gn ging vi cy loa kn trng. Scopolamin

10

c c im l khng mu, khng mi v khng v nhng li c kh nng to ra


nhng gic m k l cho con ngi khi ht phi thuc ny. Do cu trc ho hc,

thuc c th gy ra tnh trng hoang tng o gic rt mnh. c bit, Scopolamin

s ngn chn ngay t giai on u, khng k c c hnh thnh, nhng s kin

-L

xy ra trong thi gian thuc nh hng ti thn kinh con ngi s khng c ghi

li n khi thuc ht tc dng, ngi ta vn khng ti no nh ni chuyn g xy


ra, n c th gy ra tnh trng mt tr nh mc tng t nh thuc an

TO

thn diazepam. Nu s dng vi liu cao th c th gy cht ngi, Scopolamin cn

lm con tim p nhanh hn v gy ra tnh trng kch ng. Scopolamin c th bin


ngi ta thnh dng khng c nhn thc ging nh cc thy ma sng/xc sngnn
ngi chu nh hng ca thuc khng th nh chuyn g xy ra [12] [13] [31].
Scopolamin l mt loi thuc khng cholinergic. Scopolamin c nhiu tc
dng trong c th bao gm gim s tit dch, lm chm d dy v rut, lm gin

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

n con ngi. Scopolamin c s dng lm gim bun nn, nn, chng mt


v lin quan n say tu xe v phc hi t gy m sau phu thut. Scopolamin

cng c th c s dng trong iu tr bnh Parkinson, hi chng rut kch thch,

vim ti tha [27].

TP
.Q
U

1.1.4.5. Koumin

Koumin l mt trong cc alkaloid c c tm thy trong l ngn.c


tnh ca l ngn l do cc alkaloid cha trong ton b cy, trt t c gim t r,

l, hoa, qu v thn cy. Ti 17 n phn alkaloid c chit ra t l ngn

trong hm lng koumin l cao nht. Ngi b ng c l ngn c cc triu

chng kht nc, au hng, chng mt, hoa mt, bun nn sau b mi c,

thn nhit h, huyt p h, rng cn cht, si bt mp, au bng d di, tim p

yu, kh th, ng t gin v cht rt nhanh do ngng h hp. Ti Trung Quc, n

c s dng iu tr eczema, bnh tr, nhim trng rng, phong (hi), nht

TR

ngoi da, chng tn thng v co tht, nhng do c tnh cao nn ch hn ch

00

trong cc ng dng ngoi da [5] [27] [31].

10

1.1.4.6. Colchicin

Colchicin l cht c t nhin c chit xut t thc vt thuc chi

Colchicum (ngh ty) v t cy ti c. N c s dng iu tr cc bnh thp

khp, c bit l bnh gt.Ngoi bnh gt, colchicin c s dng iu tr st,

-L

vim mng ngoi tim. Hin nay ngi ta thy conchicin gy h nhit, tng huyt p,

tng nhu ng mt cch thi qu. Trn im ni thn kinh c (jonction neuro-

TO

musculaire), conchicin gy nghn biu hin bng t lit v nu ko di biu hin teo

c xng. Do tc dng cacolchicin nn dng ti c c th c nhng hin tng


ng c nh: nn ma, i lng, au bng. Liu cht trung bnh l 0.03mg i vi kg
th trng, 1centigam gy cho ngi nhng hin tng ng c, s bi tit cht
c ca conchicin chm do nhng ngi vim thn hay thiu nng thn khng
nn dng. Li u dung lam thu c cua colchicin la 1 mg, khi tim hang ngay li u cao

hn 1 mg thi colchicin se tic h tu m va co th dn n ng c .

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Colchicin c hp thu ng tiu ha v i vo vng tun hon rut-gan.


Nng nh huyt tng xut hin sau khi ung 2 gi. Colchicin ngm vo cc

c o thi ch yu theo phn v nc tiu (10-20%). Khi tim hng ngy liu

m, nht l nim mc rut, gan, thn, lch, tr c tim, c vn v phi. Colchicin

cao hn 1 mg th colchicin s tch t m v c th dn n ng c.

TP
.Q
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Colchicin c nhiu tc dng: chng bnh gout, chng vim khng c hiu,
chng phn bo, lm tng sc bn mao mch, kch thch tuyn v thng thn, phn

hy t bo lympho, c ch ph giao cm, kch thch giao cm, chng nga, gy a

chy, c ch in vitro kh nng ngng tp kt tp tiu cu [27] [31].

1.1.4.7. Nicotin

Nicotin l mt alkaloid c tm thy trong cc h cy ba ch anh , cy h C

(Solanaceae), ch yu l trong cy thuc l, v vi hm lng thp hn trong c


chua, khoai ty, c tm, v tiu xanh. Nicotin c to ra trong r v tch t trong l

TR

ca cy. N chim khong 0,6-3,0% trng lng kh ca cy thuc l. N c chc

00

nng nh mt cht c thn kinh rt mnh vi nh hng r rt n cc loi cn

10

trng, do nicotin c s dng rng ri nh mt loi thuc tr su. Cc cht


tng t nicotin nh imidacloprid hin ang s dng rng ri lm thuc bo v

thc vt. Vi liu lng nh hn (trung bnh khong 1 mg nicotin hp th), cht ny

hot ng nh mt cht kch thch trong ng vt c v, trong khi lng cao (30-60

-L

mg) c th gy t vong. Hiu ng kch thch nyc th l mt yu t chnh ng

gp vo cc thuc tnh ph thuc hnh thnh ca ngi ht thuc l (gy


nghin).Theo Hip hi tim mch M, nghin thuc l c lch s l mt trong nhng

TO

thi nghin ngp kh khn nht ph v, cc c im dc l v hnh vi xc


nh nghin thuc l cng tng t nh nghin heroin v cocain [24] [27] [31].
1.2.
1.2.1.

Cc phng php xc nh
Phng php in di mao qun
y l phng php tch cc cht phn tch l cc ion hoc cc cht khng

ion nhng c mi lin h cht ch vi cc ion trong mt ng mao qun hp cha

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

y dung dch m, t trong in trng. Do linh ng in di ca cc ion khc

xc nh 5 alkaloid c trong hai loi thuc tho dc ph bin bng phng php

Tc gi Hua-Tao Feng v Sam F.Y Li v cng s pht trin phng php

nhau, chng di chuyn vi tc khc nhau v tch ra khi nhau.

in di mao qun. Cc cht aconitin, mesaconitin, hypaconitin, benzoylaconin,

TP
.Q
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benzoylmesaconin c phn tch ch trong 15 pht bng m c cha amonium


acetat 40 mM v acid acetic 0,1% acid trong methanol 80%. Khong tuyn tnh t

2 200 mg/L. Gii hn pht hin ca phng php t 0,85 n 1,90 mg/L. thu

hi trong khong 95 108,8% [22].

Cng s dng phng php in di mao qun phn tch alkaloid nhng
vi k thut sc k in ng micell (micellar electrokinetic chromatography,

MEKC). y l phng php in di trong cc cht c tch ra bi s phn b

khc nhau ca chng trong cc micell (c xem nh l mt pha tnh gi) v dung

TR

dch m (pha ng). Trong MEKC, cc cht hot ng b mt c thm vo dung

dch m nng ln hn nng micell ti hn. Trong phn ln trng hp,

10

00

MEKC c tin hnh trong mi trng kim. Natri dodecyl sulfat (SDS) l cht

hot ng b mt thng s dng nht.

Nhm tc gi Yu L, Xu Y, Feng H xy dng phng php xc nh cc c

t alkaloid nhm pyrrolizidin trong thuc tho dc truyn thng Trung Quc. Cc

-L

cht senkirkin, senecionin, retrorsin v seneciphyllin c tch bng dung dch m

c cha borat 20 mM, SDS 30 mM v methanol 20 % pH 9,1. Gii hn pht hin

TO

ca cc cht trong khong 1,19 2,70 g/mL; gi tr RSD u nh hn 5 %.[34]

1.2.2.

Phng php sc k lng vi detector UV


Phng php sc k lng rt ph hp cho vic xc nh cc alkaloid 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 c detector UV. Nguyn l: cht phn tch
sau khi tch khi ct sc k bng h pha ng c dn vo flowcell o c chiu

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

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

c truyn Trung ng nghin cu thnh phn c c dc trong ch phm Camat

Nhm nghin cu Trn Quc Bnh v Th Oanh thuc bnh vin Y hc

Lambert-Beer.

ng dng trong h tr ct cn nghin ma ty. Nhm tc gi nh tnh atropin v

TP
.Q
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scopolamin trong c c dc bng sc k lp mng vi bn mng Silicagel G254


trng sn ca Merck v dung mi khai trin l ethylacetat methanol amoniac

m c (17:2:1). nh lng alkaloid ton phn trong mi b phn cy c c

dc tnh theo scopolamin bng phng php acid-baz. nh lng scopolamin

bng phng php HPLC s dng ct Bondapak C18 (150 x 4,6 mm; 5m), pha

ng gm c: tetrabutylamonium hydrogensulfat, acetonitril v m acetat.

Detector UV bc sng 257 nm [1].

Nhm tc gi Lng Th Phn, Nguyn Tin Vng, Nguyn Th Thun

TR

trng i hc Dc H Ni nghin cu nh lng ng thi strychnin v

brucin trong huyt tng bng phng php HPLC. Quy trnh x l mu 2 mL mu

10

00

huyt tng thm 400 L acid percloric 1N v 50 L dung dch NaOH 1M, lc
xoy trong 15 giy, ly tm 5000 vng/pht trong 10 pht. Lc dch thu c qua

mng 0,45 m. S dng ct tch Acclaim C18 (4,6x150; 5m), pha ng ACN :

m K2HPO4 1M pH9 (38:62;v/v), tc dng 0,8 ml/pht. Detector Diode Array

-L

bc sng 257 nm [7].

Tc gi Xie Y v cc cng s pht trin phng php xc nh 6 hp cht

TO

alkaloid nhm aconitum thnh phn trong thuc Trung Quc bng HPLC vi
benzoylmesaconin v benzoylhypaconin c tch trn ct ODS vi chng trnh
gradient. Pha ng gm acetonitril v m amoni bicacbonat (pH 10,0 0,2). Hiu
sut thu hi trong khong 90 103 %, gi tr RSD nh hn 3,28%.[32]

detector DAD. Cc hp cht aconitin, mesaconitin, hypaconitin, benzoylaconin,

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Lun vn Thc s khoa hc

1.2.3.

Th Thu Hng- K23 Ha hc

Phng php sc k lng vi detector khi ph


y l mt phng php nhanh, nhy xc nh ng thi cc alkaloid.

ion ho thnh cc ion phn t hay ion mnh ca phn t mang in dng hoc m,

Sau khi qua ct tch, cht phn tch c ha hi, cc hp cht hu c trung ho b

cc gc t do. Sau , cc ion c a sang b phn tch theo khi lng. T cc

TP
.Q
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tn hiu thu c, da vo khi lng ion phn t, da vo ng v, da vo cc


mnh ion phn t, da vo c ch tch v da vo ngn hng d liu cc ion v

mnh ion, ngi ta nh tnh v nh lng c cht phn tch mt cch chnh xc.
Nhm tc gi Trn Th Vn Thi v Trn Thanh Minh thuc trng i hc

Khoa hc, i hc Hu tin hnh nghin cu phn lp v nhn dng cu trc

alkaloid trong dch chit t l vng nem. Alkaloid ton phn c chit bng

ethanol 960 v NH4OH 12,5% v chit ngm kit bng ethanol 960. Dch chit c

ct loi dung mi, ha tan cn bng H2SO4 2%, lc ly dch lc (cc alkaloid lc

TR

ny dng mui, khng tan c trong CHCl3). em dch lc loi tp bng CHCl3,
ri a v pH = 9-10 bng NH4OH m c (cc alkaloid lc ny dng baz t

00

do, tan c trong CHCl3) v chit ly alkaloid bng CHCl3. Lc dch chit CHCl3

10

qua giy lc c Na2SO4 khan, ra giy lc v Na2SO4 khan bng CHCl3, ri bay
0

C) thu c alkaloid ton phn. S dng cc phng

hi CHCl3 (c kh 50

php: chit phn on trong cc mi trng pH khc nhau, chit phn on bng

cc dung mi c phn cc khc nhau, sc k ct v sc k lp mng phn lp

-L

cc cu t alkaloid t cn alkaloid ton phn. Xc nh tinh khit v nh

tnh bng sc k lng - khi ph (LC-MS); iu kin sc k: dung mi

TO

methanol - nc (85:15), thi gian 35 pht, tc dng 1 ml/pht, ct ODS-C18,

detector DAD, bc sng 284 nm; iu kin ghi ph khi: Ngun ion EFI, kh lm
kh N2 (nhit kh 350

C, p sut 30 psi, lu lng kh 15 lt/pht). Chn

khng 2,5.10-5 mmbar. in th (nng lng detector) 1118 V. [11]


Tc gi Peihong Qiu v cng s pht trin phng php xc nh ng
thi 5 alkaloid trong nc tiu v mu bng sc k lng khi ph. Trong c cc
hp cht brucin, strychnin, aconitin v colchicin. Quy trnh x l mu: 200 L mu

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

mu v nc tiu c trn vi 300 L dung dch 1 % acid trifluoroacetic trong


acetonitril, vortex trong 2 pht v rung siu m trong 10 pht. Mu sau c lm

vi ch ion dng ngun ESI. Ct tch XBridge C18 (5 m3.0 mm250mm),

sch bng SPE vi ct Oasis MCX v em i phn tch trn h thng LC-MS/MS

pha ng: acetonitril (knh A), amoni bicacbonat 10 mM chnh pH 10,5 (knh B)

TP
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theo chng trnh gradient. Gii hn nh lng ln lt l: brucin: 0,03 ng/mL,


strychnin: 0,05 ng/mL, ephedrin: 0,20 ng/mL, aconitin: 0,05 ng/mL v colchicin:
0,01 ng/mL. Hiu sut thu hi trn nn nc tiu t 96 110 % v trn nn mu l

94 113 %.[28]

Tc gi Huiqin v cc cng s ti trung tm nghin cu Quc gia Trung

Quc pht trin phng php xc nh ng thi 17 alkaloid c trong dch c

th ngi bng LC-MS vi ngun ESI dng ch MRM. Trong c 7 alkaloid

trong ti l : koumin, atropin, scopolamin, brucin, strychnin, aconitin v

TR

colchicin. Mu mu c chit lng lng nh sau: 0,5 mL mu c thm 1 mL

dung dch m borat pH 9, thm 6 mL chloroform v vortex trong 5 pht, ly tm

00

400 vng/pht trong 10 pht. Ly phn dung dch pha di, phn dung dch pha

10

trn c chit tip bng 12 mL hn hp chloroform : ether (2:1, v/v). Gp dch

chit v lm bay hi n kh bng nit 55 oC. Ha cn bng 1 mL methanol v

phn tch bng LC-MS. S dng ct tch Ultimate XB-C18 (4,6 x 250 mm, 5m)

chng trnh ra gii gradient hai knh A (dung dch acid focmic 0,2 %), knh B

-L

(methanol); tc dng 0,6 mL/pht. Khong tuyn tnh ca cc cht trong khong

0,5 50 g/L v 0,5 500 g/L, LOD trong khong 0,1 1 g/L. Hiu sut thu hi

Nhn xt: Cc phng php: sc k lng hiu nng cao HPLC (detector

UV); in di (CE) thng kh phc tp v km chnh xc do nh hng ca nn


mu. Vic tch v xc nh ng thi 8 cht trong i tng thc phm chc nng
c nn mu phc tp l kh khn nn chng ti tin hnh xc nh hm lng 8

TO

t 81,7 102,3%, gi tr RSD nh hn 10 % v 15 %.[23]

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

alkaloid c ny bng phng php sc k lng khi ph (LC-MS/MS). y l

phng php hin i, c nhy, chn lc v chnh xc cao.

Nguyn tc chung ca sc k lng

1.3.1.

1.3. Sc k lng khi ph

TP
.Q
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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 [6]. Sau cc cht phn tch c a vo

b phn pht hin v ghi nhn thnh tn hiu sc k detector. C rt nhiu loi

detector nh UV, RF, khi ph.

TR

Hin nay, detector c th cung cp thng tin nh tnh, nh lng v cu

00

trc ca cc cht phn tch l detector khi ph.

1.3.2.1. Nguyn tc

10

1.3.2. Detector khi ph (Mass spectrometry MS)

Khi ph (Mass spectrometry MS) l da vo cht nghin cu c ion

ha trong pha kh hoc pha ngng t di chn khng cao, to thnh cc ion c s

-L

khi khc nhau, cc ion ny c phn tch thnh cc mnh theo t s khi trn in

tch (m/z)[29]. Cc ion ny c th to ra bng cch thm hay bt in tch nh loi

TO

b electron, proton ha... Cc ion to thnh theo t s m/z v pht hin t cho

thng tin v khi lng hay cu trc phn t ca hp cht.


Cu to ca thit b khi ph gm 3 b phn chnh: ngun ion, thit b phn

tch v detector. Cc mu c ion ha trong ngun ion sau a vo b phn


phn tch khi tch cc ion theo t s m/z, cc tn hiu thu c s chuyn vo
my tnh x l v lu tr.

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

1.3.2.2. B ngun ion (Ion sources)

LC/MS, nng lng ca my trc tip tc ng vo pha lng hoc rn to thnh

phi chuyn cht phn tch t pha lng sang pha hi. Theo k thut ion ho ca

Cht phn tch ra khi ct sc k dng lng, kh khn ln nht y l

ion th hi. C nhiu k thut ion ha khc nhau nhng hai k thut hay c s

TP
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dng trong sc k lng khi ph bao gm:

a) Ch ion ha phun in t (Electrospray Ionization - ESI)

K thut ion ha phun in t bao gm ba qu trnh c bn sau:

- To thnh cc git mang in tch.

- Lm gim kch thc ca cc ht, v phn nh cc ht.

10

00

TR

- Qu trnh hnh thnh pha hi cc ion.

Hnh 1.1: Ch ion ha phun in t ESI

Ty theo loi in tch ca ion nghin cu m ngi ta chn kiu bn vi ch

-L

ion dng (+) hoc ion m (-). Kiu bn ph ch ion dng thng cho nhiu

TO

thng tin hn v ion nghin cu nn c dng ph bin hn.

o Ph hp nht cho phn tch cc loi cht phn tch c tnh baz.
o Thng hnh thnh nn ion [M+H]+.
o Cng c th hnh thnh ion [M+nH]n+ , [M+Na+]+

- Loi hnh thnh ion dng.

Loi hnh thnh ion m.


o Thch hp nht cho s phn tch cc loi cht phn tch c tnh acid.
o Hnh thnh nn ion [M-H]-, [M-nH]n-

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

ESI l k thut ion ha mm, c nhy cao, ESI - MS thch hp cho c


phn t c phn t khi nh (khong 100-150 amu) cng nh phn t khi ln ca

cc phn t sinh hc, cc hp cht kh bay hi, khng bn nhit, phn cc v khng

phn cc.

Trong nghin cu ny, chng ti s dng k thut ion ho phun in t (ESI)

TP
.Q
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bn ph vi ch ion dng [29].

b) Ch ion ha ha hc p sut kh quyn (Atmospheric Presure Chemical

Ionization-APCI)

S ion ha trong APCI ph thuc vo s va chm ca dng ion dng hay m

vi phn t, mu b ion ha bi phn ng vi cc ion c to ra t cc cht kh

1.3.2.3. B phn tch khi ph (analyser)

nh methan ( dng CH5+), amoniac ( dng NH4+)...

TR

B phn tch khi ph c nhim v tch cc ion c tr s m/z khc nhau

thnh tng phn ring bit. Sau khi c ion ho, cc ion c a n b phn

00

tch khi nhm loi b nhng ion khng cn thit, la chn cc ion phn t, thc

10

hin bn pha thm thu c cc ion con. Mt s b phn tch khi ph :

B phn tch by ion.

B phn tch thi gian bay.

-L

B phn tch t cc.

Nguyn tc: to ra mt trng tn s v tuyn bin i (trng t cc).B phn

tch t cc bao gm 2 loi: B phn tch t cc n, b phn tch t cc chp ba.


B phn tch t cc n (Single focusing magnetic analyser): C 4 cc bng
kim loi t song song v theo hng ca chm ion. Trng tnh in c to ra
do th mt chiu (DC) v tn s radio (RF) c t vo cc thanh. Cc t cc nh

TO

a) B phn tch t cc (Quadrupole analyser)

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

b lc khi, khi c in trng th ion chuyn ng trong n s dao ng ph

bao gm ba b t cc ghp ni vi nhau. Trong , t cc th nht (Q1) c nhim

B phn tch t cc chp ba (Triplequad mass spectometry): B phn tch khi

thuc vo t s m/z v t trng RF. Ch nhng ion ph hp mi i qua b lc ny.

v tch cc ion, la chn ion m vi m/z nht nh t ngun ion chuyn n

TP
.Q
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chuyn n Q2. t cc th hai (Q2) cc ion phn t m b phn li do va chm vi


kh tr c mt nh kh N2, Ar, He, b phn mnh tip to ra cc ion nh hn, ion

con. Q2 khng ng vai tr l b lc ion m n chp nhn tt c cc ion do Q1


chuyn n. Sau tt c cc ion con c chuyn qua b tch Q3. B t cc th

10

00

TR

ba (Q3) lm nhim v tch cc ion c chuyn t Q2 i ti b phn pht hin.

Hnh 1.2: Cu to ca b phn tch khi t cc chp ba

-L

K thut MS mt ln c nhc im l khng nghin cu c c ch phn

mnh, s khc bit gia cc ng phn, chu nh hng r rt ca nn mu cht

TO

phn tch, trn ph ch cho thy ion phn t. K thut MS/MS khng ch khc

phc c nhng nhc im trn m cn tng nhy phn tch ti hm lng

femtogram, tng chnh xc ca kt qu phn tch, loi b nh hng ca nn


mu. H MS/MS l h hai my khi ph c lp c ni vi nhau cch nhau
bung va chm (Collision Cell) [29].
b) B phn tch thi gian bay (Time of flight analyser TOF)

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Cc ion ra khi bung ion ho c gia tc nh th 10-20 kV bay qua mt ng


phn tch (khng c trng in t) c chiu di n 2m vi cng ng nng. Tuy

m z ca cc ion. Cc ion s

gian bay ht ng ny n detector t l thun vi

nhin, ty thuc vo t s m/z m cc ion c tc khc nhau ti detector. Thi

c pht hin cc thi im khc nhau. B phn tch ny cho ph tuyn tnh vi

TP
.Q
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thang m/z.

c) B phn tch t cc by ion (Quadrupole Ion-Trap Mass Analyser)

By t cc hot ng theo nguyn l b phn tch khi t cc, ch c mt

im khc l cc ion c lu gi v a dn ra khi by. Bng cch thay i th

xoay chiu p vo cc cc cc ion c t s m/z khc nhau c th vt qua khong


khng n detector. Cc ion ny cng c th b bn ph trong by thu c

cc ion con. V nguyn tc, loi phn tch khi ph by ion c th lm n MS

nhiu ln.

TR

B phn tch khi ba t cc c s dng ph bin trong k thut LC-

10

1.3.2.4. B phn pht hin

00

MS/MS v y chnh l k thut c chng ti s dng trong nghin cu ny.

Sau khi i ra khi thit b phn tch khi, cc ion c a ti phn cui ca

thit b khi ph l b phn pht hin ion. B phn pht hin cho php khi ph to

ra mt tn hiu ca cc ion tng ng t cc electron th cp c khuch i

-L

hoc to ra mt dng do in tch di chuyn. C hai loi b phn pht hin ph

bin: b phn pht hin nhn electron v b phn pht hin nhn quang.

TO

Trong nghin cu ny chng ti s dng b phn pht hin nhn electron.

1.3.3. Phn tch nh tnh v nh lng bng LC-MS/MS


i lng c trng cho s tch sc k l thi gian lu ca cc cht do vy
da vo thi gian lu ca cht phn tch v s phn mnh ph c th nh tnh tng
cht trong hn hp. Dng phng php ng chun v thm chun nh lng
cht phn tch trong mu, ng thi kim sot thu hi ca phng php.

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

1.4. Ly mu

Ly mu: Lng mu ly phi m bo nhu cu phn tch, ph hp phn tch

nh lng, gi nguyn hin trng v ng thnh phn

Cch bo qun mu: Qu trnh bo qun mu phi m bo sao cho khng lm


tch do cc hin tng tng tc ha hc, t phn hy cht [30].
1.5. nh gi phng php phn tch

Tnh c hiu, tnh chn lc

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nhim bn hoc mt cht phn tch. Mc ch gi v bo ton c cht phn

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

cht khc nh cc tin cht, cc cht chuyn ha, cc cht tng t, tp cht.

Tnh chn lc: L khi nim rng hn tnh c hiu, lin quan n vic phn

tch mt s hoc nhiu cht chung mt qui trnh. Nu cht cn xc nh phn bit r

vi cht khc th phng php phn tch c tnh chn lc.

TR

C nhiu cch xc nh tnh c hiu, tnh chn lc, trong nghin cu ny do

thit b sc k c kt ni detercter MS nn chng ti s dng cc phng php:

00

So snh ph ca cht phn tch trn 3 mu: mu trng, mu chun v

10

mu thm chun. Mu trng khng c ln tn hiu cht phn tch, mu thm

chun phi c tn hiu cht phn tch ti thi gian lu tng ng thi gian lu

trn mu chun.

-L

Phng php xc nhn (confirmation method). Theo AOAC im


nhn dng IP (identification point) i vi k thut sc k lng khi ph 2 ln

(LC-MS/MS) l 4, tc l cn 1 ion m bn ph ra 2 ion con.


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

c th pht hin c nhng cha th nh lng c.

TO

Gii hn pht hin, gii hn nh lng

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


th m ta c th nh lng v cho kt qu c chm mong mun.

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Xc nh LOD, LOQ da trn t l tn hiu nhiu ng (S/N): Phn tch


mu thm chun nng thp cn c th xut hin tn hiu ca cht phn tch

(n=4). Xc nh t l tn hiu chia cho nhiu (S/N = Signal to noise ratio).

LOD l nng m ti S/N = 3, LOQ l nng m ti S/N = 10.

TP
.Q
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Trong : S l chiu cao tn hiu ca cht phn tch, N l nhiu ng nn


Khong tuyn tnh v ng chun

Khong tuyn tnh ca mt phng php: l khong nng c s ph

thuc tuyn tnh gia i lng c o v nng cc cht phn tch.

ng chun: l ng biu din s ph thuc tuyn tnh gia i lng

c o v nng cc cht phn tch.

xc nh khong tuyn tnh ngi ta thc hin o cc dung dch chun c

TR

nng thay i v kho st s ph thuc ca tn hiu vo nng . Sau v


ng cong s ph thuc gia din tch pic thu c vo nng , quan st s ph

00

thuc cho n khi khng cn tuyn tnh [10][18].

10

C th xy dng ng chun trn nn mu thc, nhm mc ch loi tr

nh hng ca nn mu n kt qu phn tch.

H s hi quy tuyn tnh (R): Ch s u tin ca mt ng chun t yu

-L

cu v h s tng quan hi quy (Coefficient of corelation), gi tr R phi t yu

cu sau: 0,995 R 1 hay 0,99 R2 1.

lp li (nh gi chm) l mc gn nhau ca cc gi tr ring l ca

cc php o lp li v c biu din bng lch chun SD v lch chun


tng i RSD (%):

TO

lp li v thu hi.

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Trong :

Nng trung bnh tnh c ca n ln th nghim.

x:

xi: Nng tnh c ca ln th nghim th i.

TP
.Q
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n: S ln th nghim.

ng l mc gn nhau ca gi tr phn tch vi gi tr thc hoc gi tr


c chp nhn. ng l khi nim nh tnh v c biu din nh lng di

dng thu hi (recovery). thu hi (nh gi ng) l t l phn trm gia

f
.100 (3).
Ca

R(%) =

gi tr thu c so vi gi tr l thuyt [10][18].

Trong :

TR

R: thu hi (%)

10

00

Cf : Nng cht phn tch trong mu trng xc nh c (ng/ml).

TO

-L

Ca : Nng chun thm vo mu trng (ng/ml).

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

CHNG 2: NI DUNG V PHNG PHP NGHIN CU

ngun gc tho dc ang t ra cho c quan thanh tra, kim tra thc phm cn c

Nguy c tim n ng c do cc alkaloid c trong thc phm chc nng c

2.1 i tng v mc tiu nghin cu

phng php xc nh chnh xc cc alkaloid c qun l v cnh bo m bo

TP
.Q
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an ton v sc khe ngi tiu dng. Ph hp vi thc tin, chng ti tin hnh xc
nh hm lng mt s alkaloid c bao gm (Brucin, Strychnin, Aconitin,

Colchicin, Atropin, Scopolamin, Nicotin, Koumin) trong thc phm chc nng c

Mc tiu tin hnh nghin cu ca ti:

ngun gc tho dc bng sc k lng ghp ni khi ph LC-MS/MS.

Ti u cc iu kin chy my LC-MS/MS tch v xc nh ng thi

cc alkaloid.

Kho st, ti u ha qu trnh x l mu.

TR

Thm nh phng php phn tch.

10

00

2.2 Phng php nghin cu

ng dung phng php phn tch trn mt s mu thc t.

Phng php nghin cu ca lun vn ny bao gm:

Phng php tch chit mu:

Qua tham kho cc nghin cu trn chng ti la phng php chit lng

-L

lng. Mu c thm dung dch kim chuyn cc alkaloid v dng baz sau

Phng php LC-MS/MS:

Mu sau khi tch chit c em i phn tch trn h thng LC-MS/MS vi

ch ion dng ngun ESI, c s l thuyt c nu trong phn tng quan.


Cc kt qu c tnh ton t ng theo phn mm phn tch ca thit b (phn
mm Analyst 1.5.1, ABSciex).

TO

cc alkaloid c chit vo pha dung mi hu c.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Phng php thm nh:

nh gi phng php chng ti tin hnh theo quy nh ca AOAC.

Gii hn pht hin v gii hn nh lng

Khong tuyn tnh v ng chun

lp li v thu hi

Tnh c hiu, tnh chn lc

TP
.Q
U

Phng php thm nh bao gm:

Phng php ly mu:

Phng php ly mu: Ngu nhin

i tng mu: Mt s thc phm chc nng c ngun gc tho dc

a im: Mt s hiu thuc trn a bn ni thnh H Ni

TR

2.3 Thit b, dng c v ha cht dng trong nghin cu


2.3.1 Thit b

H thng sc k lng khi ph khi ph LC/MS/MS bao gm:

00

10

+ My sc k lng ca Shimadzu. Model 20 AD-UFLC.

+ My khi ph. Model AB sciex Triplequard 5500


Ct sc k InertSustain C18 (150mm 4,6mm 5m). Nc sn xut Nht

-L

Bn

My lc xoay ( vortex) VELP.

TO

My ng nht mu.

My ly tm MIKRO 22R c th t c tc ti thiu 4000 rpm vi ng ly


tm 50 mL.

Cn phn tch (c chnh xc 0,1mg v 0,01mg) (Metter Toledo).

My ct quay chn khng c b iu nhit EYELA

2.3.2 Dng c
-

Cc c m dung tch 50, 100, 200mL.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

ng ong dung tch 10,50, 100 mL.

Autopipet loi 100 L, 1000 L, 5000 L,v u cn tng ng.

ng ly tm 50 mL.

Mng lc 0,2 m.

Vial loi 1,8 mL.

Bnh nh mc dung tch 10, 25, 50, 100, 500 mL.

ng nghim thy tinh c np 6mL

TP
.Q
U

2.3.3 Cht chun

Cc cht chun: Nicotin c cung cp bi hng Sigma Aldrich dng rn

tinh khit 98,7 % l 1 g; Koumin cung cp bi hng Chengdu Biopurify

dng rn tinh khit 99,72 % l 10 mg; Atropin c cung cp bi hng


Sigma Aldrich dng rn tinh khit 99 % l 1 g; Scopolamin cung cp

bi hng Sigma Aldrich dng rn tinh khit 99,3 % l 1 g; Brucin v


Strychnin cung cp bi hng Sigma Aldrich dng rn tinh khit 98%

TR

l 5g; Aconitin cung cp bi hng Sigma Aldrich dng rn tinh khit

00

95 % l 5 mg; Colchicin cung cp bi hng Sigma Aldrich dng rn

10

tinh khit 95 % l 100 mg

Dung dch chun gc 100 g/mL: cn chnh xc 0,01g tng cht chun trn

cn phn tch c c 0,0001g, ha tan v nh mc 100 mL bng

Dung dch chun hn hp trung gian ca nicotin, scopolamin v atropin (10

-L

methanol. Bo qun trong t lnh 40C, s dng trong 1 nm.

g/mL): ht 1 mL dung dch chun gc ca mi cht vo bnh nh mc 10

TO

mL, pha long v nh mc n vch bng methanol. Bo qun trong t lnh

40C, s dng trong 1 thng.

Dung dch chun hn hp trung gian (1-10 g/mL): ht 1 mL dung dch


chun gc ca brucin, strychnin, koumin, aconitin, colchicin v 1 mL dung
dch chun hn hp trung gian ca nicotin, scopolamin, atropin (10 g/mL)
vo bnh nh mc 10 mL, pha long v nh mc n vch bng methanol.
Bo qun trong t lnh 40C, s dng trong 1 tun.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Pha long dung dch chun hn hp trung gian 1-10 g/mL trong methanol
thu c cc dung dch chun lm vic nng 0,5-5; 1-10; 2-20; 5-50;

10-100; 20-200; 50-500; 100-1000; 200-2000; 500-5000 ng/mL.

Methanol (Merck 99,9%), Acetonitril (Merck 99,9%), acid formic (Merck

2.3.4 Cc loi ha cht, dung mi khc

TP
.Q
U

98%), acid acetic bng (Merck), amoniacetat (Merck), ethylacetat (Merck),


ether (Merck), chloroform (Merck), NaOH (Merck), acid boric,

Nc ct hai ln
Dung dch NaOH 0,1M: Cn 2g NaOH ha tan v nh mc bng 500 mL

Dung dch KCl 0,1M: Cn 7,455g KCl ha tan v nh mc bng 1000 mL

nc ct hai ln.

Dung dch m borat pH 9: Cn 3,09 g acid boric ha tan ha tan bng 500

nc ct hai ln.

Dung dch NH4OH 5%: ht chnh xc 5 mL dung dch NH4OH vo bnh 100

TR

mL dung dch KCl 0,1M. Thm tip 210 mL dung dch NaOH 0,1M.

TO

-L

10

00

mL v nh mc n vch bng nc ct.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

CHNG 3. KT QU V THO LUN

3.1. Ti u iu kin tch v xc nh cc alkaloid trn thit b LC/MS/MS

Kho st ion m v ion con

3.1.1.1.

3.1.1. Ti u cc iu kin ca detector khi ph (MS/MS)

TP
.Q
U

Qua tham kho ti liu chng ti tin hnh kho st xc nh cc alkaloid


bng k thut ion ha phun in t ESI vi ch bn ph ion dng. ti u ha

iu kin khi ph, dng kim bm hn hp cc cht chun 50 ng/mL tim trc tip

vo detector khi ph kho st. Chn ch kho st t ng i vi tng cht

chn c ion m, ion con dng nh lng v nh tnh i vi tng cht v

cc iu kin bn ph tng ng ca tng mnh nh trong bng :

CE (eV)

CXP (V)

Ghi ch

41

34

nh lng

47

34

nh tnh

184,2

51

18

nh lng

156

53

20

nh tnh

586,2

43

34

nh lng

526,2

51

28

nh tnh

124

29

20

nh lng

93

37

14

nh tnh

138

29

18

nh lng

156

21

20

nh tnh

180

57

22

nh lng

204

63

24

nh tnh

358

27

46

nh lng

310

33

20

nh tnh

132

19

18

nh lng

130

29

24

nh tnh

324

395

244

Aconitin

00

335,2

10

Strychnin

Brucin

Mnh con
(m/z)

Mnh m
(m/z)

TR

Tn cht phn
tch

290

-L

Atropin

646,2

304

Koumin

307

Colchicin

400

Nicotin

163

TO

Scopolamin

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

Bng3.1: Cc mnh ion nh lng v nh tnh ca cc alkaloid

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Trong :
+ CXP (Collision Cell Exit Potential): Th p gia b t cc Q2 v Q3.

+ CE (Collision Energy): L nng lng va chm c to ra do th p vo b

t cc Q2, to ra nng lng phn mnh ion m.

Mnh ion con m/z c cng ln nht dng nh lng, mnh con th 2

-L

10

00

TR

TP
.Q
U

c cng thp hn dng xc nhn cht phn tch.

Hnh 3.1: Sc t l cc ion ca koumin v atropin

Kt lun: Sau khi tin hnh kho st, chng ti chn c ion m v ion con

TO

thch hp cho cc c t alkaliod ti cc iu kin MS nh bng 3.1.


3.1.1.2.

Ti u ha cc iu kin MS

ti u ha iu kin MS cho tng cht, chng ti s dng k thut FIA,


ngha l hn hp chun c bm trc tip vo my MS cung vi dong pha ng
m khng qua c t s c ky vi iu kin nh sau : Pha ng l dung dch amoniacetat

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

10 mM trong nc : methanol (80:20), hn hp chun c nng 50 ng/mL, th

tng cht.

Chn ch kho st t ng i vi tng ion con nh lng v nh tnh ca

tch bm 10 L, thi gian 1 pht.

TP
.Q
U

Cc thng s cho b phn to ngun ion.

+ IS (IonSpray Voltage): Th ion ha, th ny c p ln u phun v mn


chn ca b phn phn tch ion. Th ny s quyt nh loi ion chuyn n b phn

phn tch khi. i vi loi ion dng 4000 5500V.

+ GS1 (Ion Source Gas 1): To p sut kh hai bn u phun, c tc dng

lm cho s hnh thnh nn cc git c d dng hn. Tc kh ca Gas 1 thng

cao hn so vi Gas 2.

+ TEM ( Temperature): Nhit ca ngun kh nng thi vo (Gas2). N

TR

thc y qu trnh ha hi cc git cht phn tch khi i ra khi u phun.

+ GS2 (Ion Source Gas 2): To p sut ca lung kh nng, h tr qu trnh

00

lm bay hi dung mi, tng hiu qu ca qu trnh ion ha.

10

+ CUR (Curtain Gas): Lung kh N2 tinh khit c thi vo khe gia 2 mn

chn ca b phn ion ha v b phn phn tch ph. N c tc dng y cc git

dung mi v cc phn t trung ha, gi cho Q0 (ngun ion m ) sch hn.

-L

Cc thng s ca b phn phn phn tch khi:

+ DP, CE, CXP nh gii thch trn.

TO

+ EP (Entrance Potential): Th p vo ngun ion m Q0.

thc y qu trnh phn mnh th cp, ngoi ra cn c tc dng lm mt cc ion con


v hng chng n b t cc Q3.
T ng kho st cc thng s cho tng cht trn vi cc gi tr nh sau:

+ CAD (Collision Gas Pressure): Kim sot p sut kh N2 trong b t cc Q2,

IS ch ion dng (V):5000,0; 4500,0; 4000,0; 3500,0

TEM (oC) : 250,0; 300,0; 350,0; 400,0;450,0

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

GS1 (psi): 25,0; 30,0; 10,0;15,0

GS2 (psi): 8,0; 9,0; 10,0; 5,0; 2,0;

CUR (psi): 15,0;20,0; 5,0; 10,0;

DP (V) :110,0; 120,0; 100,0; 90,0; 80,0

EP (V) : 0;7,0; 8,0; 9,0; 10,0; 11,0

CAD (psi): 7,0; 8,0; 9,0; 5,0; 6,0

TP
.Q
U

Qua kho st ta thu c gi tr ti u ca tng thng s trn cho c cc cht


nghin cu. Gi tr c lit k trong bng 2
Ga tr ti u

IS (V)

4500

TEM (oC)

500
25

GS1 (psi)

TR

GS2 (psi)

20
20
130

00

CUR (psi)
DP (V)

Thng s

Bng 3.2: Cc thng s ti u MS/MS i vi ch ion dng

10

EP (V)

CAD (psi)

Kt lun: Chng ti tin hnh kho st cc iu kin MS v a ra c cc

-L

iu kin ti u nh trong bng trn.

TO

3.1.2. Ti u cc iu kin chy sc k lng hiu nng cao (HPLC)


3.1.2.1.

La chn ct tch

Ct tch l b phn quan trng ca h thng sc k, n ng gp mt phn

quan trng trong vic quyt nh qu trnh tch.Qua tham kho cc bi bo nn


chng ti chn ct tch l ct pha o. Hn na, cht nhi pha o s dng h dung
mi phn cc c tnh kinh t cao, ph hp vi iu kin ca phng th nghim.
Trong nghin cu ny, chng ti chn ct pha o C18 tch cc cht . bo v

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

ct, chng ti s dng thm tin ct . Loi ct s dng l InertSustain C18 (5 m;

4,6 x 150 mm) v tin ct tng ng ca hng GL Sciences (Nh t Ban).

Kho st thnh phn pha ng

3.1.2.2.

Trong phng php sc k lng khi ph, pha ng khng ch nh hng

TP
.Q
U

ti qu trnh tch cc cht m n cn nh hng ti qu trnh ion ha v tn hiu ca

cht phn tch. Vi k thut ion ha phun in t bn ph ch ion dng, qu


trnh ion ha tng khi c thm cc cht nh acid acetic, acid formic, amoni acetate

do vy cc cht ny c s dng trong pha ng. Bn cnh , ct tch s dng l

ct C18, t phn cc nn pha ng phi l h dung mi phn cc nn chng ti

chn dung mi pha ng l acetonitrile v methanol. C nh cc iu kin sc k:


Ct C18 (5m; 4,6 x 150mm) v tin ct tng ng

Tc dng: 0,5 mL/pht

Mu phn tch: hn hp chun nng : 5-50 ng/mL

TR

v tin hnh kho st theo ch gradient hai knh A v B theo cc h sau:

10

00

Bng 3.3: Cc h dung mi pha ng kho st

H 2

Methanol

CH3COOH 0,1 %

Methanol

HCOOH 0,1 %

Methanol

CH3COONH4 10 mM

Acetonitril

CH3COONH4 10 mM

-L

H 3
H 4

Knh B

H 1

Knh A

XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 10 (Std Mix 5-10ppb-MeOH+0.1%CH3COOH) of DataSET1valid.wiff (T...

Max. 1.0e6 cps.

5.0e6
4.8e6
4.6e6

TO

4.4e6
4.2e6
4.0e6
3.8e6
3.6e6
3.4e6

Intensity, cps

3.2e6
3.0e6
2.8e6
2.6e6
2.4e6
2.2e6
2.0e6
1.8e6
1.6e6
1.4e6
1.2e6

10.75

1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
0.0
0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Time, min

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

Hnh 3.2: Sc ion tng khi s dng h dung mi pha ng 1

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 8 (Std Mix 5-10ppb-MeOH+0.1%HCOOH) of DataSET1valid.wiff (Turbo...

Max. 1.1e6 cps.

4.8e6
4.6e6
4.4e6
4.2e6
4.0e6
3.8e6
3.6e6
3.4e6

3.0e6
2.8e6
2.6e6
2.4e6

Intensity, cps

3.2e6

2.2e6
2.0e6

1.8e6
1.6e6
1.2e6

11.34

1.0e6
8.0e5
6.0e5

1.4e6

2.0e5
0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Time, min

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

TP
.Q
U

0.0
0.0

4.0e5

12.5

Hnh 3.3: Sc ion tng khi s dng h dung mi pha ng 2


XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 14 (Std Mix 5-10ppb-MeOH+CH3COONH4 10mM) of DataSET1valid.wi...

5.0e6

4.5e6

4.0e6

3.5e6

3.0e6

2.5e6

Intensity, cps

Max. 1.8e6 cps.

5.5e6

2.0e6

1.5e6

1.0e6

11.07

5.0e5

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0.0
0.0

Time, min

9.0

10.0

11.0

12.0

13.0

TR

Hnh 3.4: Sc ion tng khi s dng h dung mi pha ng 3


XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 16 (Std Mix 5-10ppb-ACN+CH3COONH4 10mM) of DataSET1valid.wiff ...

3.8e6

00

3.6e6
3.4e6
3.2e6
3.0e6

10

2.8e6
2.6e6
2.4e6
2.2e6
2.0e6
1.8e6

10.26

Intensity, cps

Max. 1.8e6 cps.

4.0e6

1.6e6

1.4e6
1.2e6
1.0e6

8.0e5
6.0e5
4.0e5

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Time, min

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

-L

0.0
0.0

2.0e5

Hnh 3.5: Sc ion tng khi s dng h dung mi pha ng 4

TO

Ghi ch:

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Bng 3.4: Din tch cc alkaloid khi s dng cc h dung mi khc nhau
Din tch pc
Anconitin

Atropin

Brucin

Colchicin

Koumin

Nicotin

Scopolamin

Strychnin

H 1

5930000

39800000

31800000

20600000

24200000

38400000

33400000

34100000

H 2

7180000

40200000

28600000

21700000

24700000

44900000

29100000

H 3

15700000

52000000

36700000

47600000

38800000

9000000

14400000

H 4

10600000

27800000

18600000

22900000

15600000

2460000

15300000

H pha
ng

32800000

TP
.Q
U

41800000
20900000

Nhn xt: Khi s dng h pha ng l methanol v acid formic 0,1 % hoc
h pha ng methanol v acid acetic 0,1 % th cc cht tch ra khi nhau kh tt tuy

nhin mt s pc b ch. S dng h pha ng gm acetonitril v amoniacetat 10

mM cc pc nhn v cn i nhng din tch cc pc nh, do vy la chn h pha

ng gm methanol v amoniacetat 10 mM cho pc cn i v din tch pc l ln

Kho st chng trnh gradient

TR

3.1.2.3.

nht cho a s cht phn tch tr nicotin v scopolamin.

00

Thc hin tch v xc nh ng thi 8 hp cht s dng ch ra gii

10

ng dng (isocractic) l khng ph hp. Hn na c th va tch c cc cht,


va tit kim c thi gian phn tch v dung mi, chng ti tin hnh chy pha

ng vi ch gradient. Chng ti tin hnh kho st mt s chng trnh ra

Tc dng: 0,5 mL/pht

Ct C18 (5m; 4,6 x 150mm) v tin ct tng ng

-L

gii gradient. C nh cc iu kin sc k:

TO

Dung mi pha ng knh A: CH3COONH4 (10mM), knh B: MeOH

Kho st mt s chng trnh gradient nh trong bng:

Mu phn tch: hn hp chun nng : 5-50 ng/mL

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Bng3.5: Cc chng trnh gradient kho st

10

% knh
A
80
80
0
0
80
80

% knh
B
20
20
100
100
20
20

% knh
A
80
80
20
20
0
0
80
80

Grad5
% knh
B
20
20
80
80
100
100
20
20

t
(pht
)
0,01
1,5
6,5
10,5
10,51
13,0

% knh
A
80
80
0
0
80
80

TR

t
(pht)
0,01
1,0
3,5
4,5
5,51
10,0
10,01
13,0

% knh
A
80
80
20
20
80
80

00

t (pht)
0,01
1,5
6,5
10,5
10,51
13,0

Grad4
% knh
B
20
20
80
80
20
20

%
knh B
20
20
100
100
20
20

t
%
%
(pht
knh B knh A
)
5
95
0,01
5
95
1,5
100
0
8,0
100
0
13,0
5
95
13,01
5
95
16,5

t
(pht)
0,01
1,5
8,0
13,0
13,01
16,5

Grad3

TP
.Q
U

Grad2

Grad1

X
IC of +M
R
M (18 pairs): 646.200/586.200 D
a ID
: Anconitine1 from Sam
ple 2 (Std M
ix5-50ppb-G
rad2) of D
ataSET1.w
iff (Turbo Spray)

M
ax. 1.2e6 cps.

5.0e6

4.8e6
4.6e6
4.4e6
4.2e6

4.0e6
3.8e6
3.6e6
3.2e6

Intensit y, cps

3.4e6
3.0e6
2.8e6
2.6e6
2.4e6
2.2e6

2.0e6
1.8e6
1.6e6
1.4e6
1.2e6
8.0e5
6.0e5
4.0e5
2.0e5

1.0

2.0

11.66

12.70
3.0

4.0

5.0

6.0

7.0

8.0
Tim
e, m
in

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

Hnh 3.6: Sc ion tng khi s dng chng trnh gradient 1

TO

0.0
0.0

-L

1.0e6

XIC of +M
RM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sam
ple 3 (Std M
ix5-50ppb-G
rad3) of DataSET1.w
iff (Turbo Spray)

M
ax. 1.3e6 cps.

5.5e6

4.5e6

4.0e6

Intensit y, cps

5.0e6

3.5e6

3.0e6

2.5e6

2.0e6

1.5e6

10.98

1.0e6

5.0e5

0.0
0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0
Tim
e, m
in

9.0

10.0

11.0

12.0

13.0

14.0

15.0

16.0

Hnh 3.7: Sc ion tng khi s dng chng trnh gradient 2

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

XIC of +M
RM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sam
ple 10 (Std M
ix5-50ppb-Grad6) of DataSET1.wiff (Turbo Spray)

M
ax. 1.1e6 cps.

5.5e6

5.0e6

4.5e6

4.0e6

3.0e6

2.5e6

Intensity, cps

3.5e6

2.0e6

1.5e6
10.48

5.0e5
11.52

0.0
0.0

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Tim
e, m
in

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

TP
.Q
U

0.5

1.0e6

Hnh 3.8: Sc ion tng khi s dng chng trnh gradient 3


XIC of +M
RM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sam
ple 8 (Std M
ix5-50ppb-Grad5) of DataSET1.wiff (Turbo Spray)
4.7e6
4.6e6
4.4e6
4.2e6
4.0e6

3.8e6
3.6e6
3.4e6
3.2e6
3.0e6
2.8e6
2.6e6

Intensity, cps

M
ax. 1.4e6 cps.

2.4e6
2.2e6
2.0e6
1.8e6

1.6e6
1.4e6
1.2e6

1.0e6
8.0e5
6.0e5
4.0e5
2.0e5
0.0
0.0

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Tim
e, m
in

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

0.5

11.63

TR

Hnh 3.9: Sc ion tng khi s dng chng trnh gradient 4


XIC of +M
RM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sam
ple 5 (Std M
ix5-50ppb-Grad7) of DataSET2.wiff (Turbo Spray)

1.05e6
1.00e6
9.50e5
9.00e5

8.50e5
8.00e5
7.50e5

00

6.50e5
6.00e5
5.50e5
5.00e5

10

Intensity, cps

7.00e5

4.50e5
4.00e5
3.50e5
3.00e5
2.50e5

9.70

2.00e5
1.50e5

5.00e4
1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Tim
e, m
in

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

0.5

1.00e5

0.00
0.0

M
ax. 2.0e5 cps.

-L

Hnh 3.10: Sc ion tng khi s dng chng trnh gradient 5

TO

Ghi ch:

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Nhn xt: Khi s dng chng trnh chy gradient 1 th thi gian cht phn
tch ra mun, cc cht phn tch bt u ra gii 11 pht. Do vy chng ti tin

gradient 2 v 3. Khi tng nhanh nng methanol th cc cht phn tch ra sm hn

hnh ngt bt qu trnh gradient giai on u, tng nhanh nng methanol trong

tuy nhin cc pic sc k b vai, khng sc nhn, r nt. Chng ti tip tc kho st

TP
.Q
U

gradient 4 gim nng methanol cho kt qu cc pic sc k nhn r nt hn tuy


nhin thi gian cht phn tch ra li gim. Tip tc kho st ko di thi gian tng
nng methanol ln theo chng trnh gradient 5. Kt qu cho tn hiu pic p,

cc cht c tch ra khi nhau kh tt , pic thu c khng b t, khng b ch pc,

v thi gian lu ca cc cht khng qu di. Nh vy, vic thay i thnh phn

ACN trong pha ng cn phi tin hnh chm. Chng ti quyt nh chn chng
trnh ra gii gradient 5. Cc alkaloid c ra gii trong khong t 8-10 pht.

Kho st tc dng pha ng

TR

3.1.2.4.

Ton b qu trnh tch v xc nh c thc hin trong 13 pht.

Tc pha ng nh hng n thi gian ra gii v lng dung mi tiu

00

tn. c bit, vi k thut LC-MS/MS, tc dng nh hng rt nhiu. Nu tc

10

dng ln th qu trnh bay hi dung mi to ion s km hiu qu nh hng

n nhy ca cht phn tch gim. Nu tc dng qu nh, thi gian phn tch

ko di hn na chng trnh gradient c th km n nh nh hng n lp li

ca phng php. Do vy chng ti tin hnh kho st tc pha ng 0,4

-L

mL/pht; 0,5 mL/pht; 0,6 mL/pht v c nh cc iu kin:


Ct C18 (5m; 4,6 x 150mm) v tin ct tng ng

Dung mi pha ng knh A: CH3COONH4 (10mM), knh B: MeOH

Chng trnh gradient nh trn

Mu phn tch: hn hp chun nng : 5-50 ng/mL

TO

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 11 (Std Mix 5-50ppb-CH3COONH4-TF 0.6ml/phut) of DataSET2.wiff (T...

Max. 1.5e5 cps.

9.3e5
9.0e5
8.5e5
8.0e5
7.5e5
7.0e5
6.5e5

5.5e5
5.0e5

Intensity, cps

6.0e5

4.5e5
4.0e5

3.5e5
3.0e5

2.5e5
2.0e5
8.78

1.5e5

5.0e4
0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Time, min

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

TP
.Q
U

0.0
0.0

1.0e5

Hnh 3.11: Sc ion tng ti tc dng 0,6 mL/pht


XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 5 (Std Mix5-50ppb-Grad7) of DataSET2.wiff (Turbo Spray)

Max. 2.0e5 cps.

1.05e6
1.00e6
9.50e5
9.00e5
8.50e5

8.00e5
7.50e5

6.50e5
6.00e5

Intensity, cps

7.00e5

5.50e5
5.00e5

4.50e5
4.00e5
3.50e5
3.00e5
2.50e5

9.70

2.00e5
1.50e5
1.00e5
5.00e4
1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

0.00
0.0

Time, min

9.0

10.0

11.0

12.0

13.0

TR

Hnh 3.12: Sc ion tng ti tc dng 0,5 mL/pht


XIC of +M
RM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sam
ple 12 (Std M
ix 5-50ppb-CH3COONH4-TF 0.4m
l/phut) of DataSET2.wiff (T...

M
ax. 2.0e5 cps.

1.10e6

1.05e6
1.00e6

00

9.50e5
9.00e5
8.50e5
8.00e5

7.00e5

10

Intensity, cps

7.50e5

6.50e5
6.00e5
5.50e5
5.00e5
4.50e5

4.00e5
3.50e5
3.00e5

2.50e5
2.00e5
1.50e5

5.00e4
0.5

1.0

1.5

2.0

2.5

3.0

11.00

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Tim
e, m
in

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

0.00
0.0

1.00e5

-L

Hnh 3.13: Sc ion tng ti tc dng 0,4 mL/pht

TO

Ghi ch:

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Nhn xt: Khi tng tc dng ca pha ng, cc pic sc k c xu hng nhn v
r nt nhng din tch pc li gim. Do qu trnh bay hi dung mi to ion km

cht ln nht nhng cc pic b ko chn v pha sau. Do , chng ti quyt nh

hiu qu khi tng tc dng. Ti tc dng 0,4 mL/pht din tch pc ca cc

la chn tc dng 0,5 mL/pht. Ti tc ny, cc alkaloid bt u c ra

TP
.Q
U

gii t 8 pht.

Kho st thnh phn amoniacetat trong pha ng

3.1.2.5.

Trong phng php sc k lng khi ph, pha ng khng ch nh hng ti

qu trnh tch cc cht m n cn nh hng ti qu trnh ion ha do nh hng

n tn hiu ca cht phn tch. Pha ng trong phng php s dng amoniacetat

gip qu trnh ion ha ca cht phn tch hiu qu hn tuy nhin vi hm lng

amoniacetat qu cao c th lm gim tui th ct phn tch. Do vy chng ti kho

st nng amoniacetat trong knh A, vi hm lng 5 mM, 10 mM, 20 Mm. C

TR

nh cc iu kin sc k:

Ct C18 (5m; 4,6 x 150mm) v tin ct tng ng

Tc dng: 0,5 mL/pht

Chng trnh gradient nh trn

Mu phn tch: hn hp chun nng : 5-50 ng/mL

10

00

-L

Bng3.6: nh hng nng amoniacetat ti din tch pc cc alkaloid

TO

Nng

Anconitin

Din tch pc
Atropin

Brucin

Colchicin

Koumin

Nicotin

Scopolamin

Strychnin

1370000

7910000

3260000

5710000

7650000

749000

3280000

8310000

10 mM

1440000

7770000

4390000

6640000

7340000

359000

3810000

9100000

1370000

6740000

6320000

6110000

6080000

319000

3180000

9040000

5 mM

20 mM

Trng i hc KHTN

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Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh 3.14: Biu s ph thuc din tch pc ca cc alkaloid vo nng


amoniacetat trong pha ng

Nhn xt: Kt qu cho thy nng 10 mM cho din tch pc sc k cc

TR

akaloid ln nht, nng 5 mM cng cho din tch pc sc k ca mt s cht

phn tch ln tuy nhin hnh dng cc pc sc k khng cn i. Do vy chng ti

10

00

la chn nng amoniacetat l 10 mM.

3.2. Ti u qu trnh x l mu

Alkaloid l cc baz yu, do s c mt ca nguyn t N. Nhng kim ca

alkaloid khng ging nhau do nh hng khc nhau ca lp in tch nguyn t N

-L

gy ra v nh hng ca cc nhm chc khc. Tnh baz gim dn theo th t

amoni bc 4, amoni bc 1, amoni bc 2, amoni bc 3. Tnh baz phn nh pKa

TO

khc nhau ca cc alkaloid, cc baz yu c tr s pKa thp. Mt s alkaloid c tnh

baz mnh c th tn ti dng mui trong mi trng acid. Cc mui alkaloid ni


chung tan c trong nc v cn nhng hu nh khng tan trong dung mi hu c
nh: chloroform, eter, benzen. Ngc li hu ht cc alkaloid dng baz thc t
khng tan trong nc nhng tan trong cc dung mi hu c nh CHCl3, eter v cc
ancol bc thp (MeOH, EtOH, PrOH, BuOH). Da vo c im ny c th tch
chit cc alkaloid.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Qua tham khao chung ti l a chn chit mu theo phng php lng lng,
da trn nguyn tc: Cc alkaloid c kim ha sau c chit bng dung mi

Cn 0,5 g mu/ng ly tm 50 ml

TP
.Q
U

+ 1 ml m acid boric pH 9

hu c v em i phn tch, qu trnh chit mu theo qui trnh d kin nh sau:

+ 10 ml dung mi chit

Lc votex 5 pht

Ly tm 5 pht, tc 6000 vng/pht

Gn dch vo bnh c, c quay 500C

00

TR

Ha cn bng 5ml methanol

Vial, LC-MS/MS

-L

10

Lc qua mng 0,2 m

Hnh 3.15: S quy trinh chi t mu d ki n

T qui trnh d kin trn, chng ti thy cn phi kho st dung mi chit

TO

v dung dch kim ha ph hp thu c hiu sut chit ti u. Chng ti tin

hnh kho st mt s iu kin chit nh sau:


3.2.1. Kho st dung mi chit
Dung mi chit l mt yu t v cng quan trng quyt nh hiu sut ca
qu trnh x l mu. Do cc alkaloid y c a v dng baz bng dung dch
m borat pH 9 nn chng ti tin hnh kho st trn cc dung mi hu c t phn

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

cc: eter, ethylacetat, chloroform, methanol v acetonitril. C inh


cac bc chi t
khc nh quy trnh trn s dng nn mu thc phm chc nng c ngun gc tho

hn hp 1-10 g/mL cc alkaloid ( thu c nng alkaloid trong dch cui l 5

dc khng pht hin cac alkaloid. Sau khi cn mu, thm 25 L dung dch chun

ng/mL i vi nicotin, scopolamin, atropin v 50 ng/mL i vi brucin, strychnin,

TP
.Q
U

koumin, aconitin, colchicin trong dch chit cui cng trc khi bm vo my
LC/MS/MS). yn 5 pht. Tin hnh chit lp hai ln theo qui trnh trn thu c
k t qua trung bnh nh bng sau:

Atropin

Brucin

Colchicin

97,8

4,08

1,52

22,1

79,9

25,0

49,3

76,2

19,4

64,5

49,3

8,1

59,6

47,4

Koumin

Nicotin

Scopolamin

Strychnin

47,9

50,4

42,6

19,3

72,9

106

71,8

63,2

30,4

41,9

109

55,9

31,4

28,5

30,5

84,5

50,4

25,8

7,8

34,6

29,7

21,7

27,7

63,8

46,0

20,1

TR

Anconitin

Hnh 3.16 : Biu nh hng ca dung mi chit n hiu sut chit cc

TO

-L

10

00

Hiu sut (%)


eter+1mL
m pH9
ethylacetat+
1mL m H9
CHCl3+1mL
m pH9
ACN+1mL
m pH9
MeOH+1mL
m pH9

Bng 3.7 : nh hng ca dung mi chit n hiu sut chit cc alkaloid

alkaloid

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Nhn xt: S dng dung mi chit l ethylacetat cho hiu sut chit cao i
vi a s cc alkaloid tr atropin trong khi s dng dung mi chit l chloroform th

mi chit ny c th thu c hiu sut chit cao nht i vi tt c cc alkaloid

cho hiu sut chit ca atropin cao nht. Do vy chng ti s dng c hai loi dung

nghin cu. Nh vy mu sau khi c kim ha c chit bng 10 mL

TP
.Q
U

ethylacetat, thu phn dung mi hu c pha trn, phn dch pha di c chit lp
nh trn vi 10 mL chloroform. Gp dch chit hai ln em c quay chn khng v

phn tch.

3.2.2. Kho st dung dch kim ha

Dung dch kim ha ng vai tr chuyn cc alkaloid v dng baz do vy

nh hng n hiu sut chit ca cc alkaloid vo pha hu c. Do vy chng ti

tin hnh kho st mt s dung dch kim ph bin dng trong chit alkaloid t

dc liu: dung dch NaOH 0,1 N, dung dch NH4OH 5% v dung dch m borat

TR

pH 9. C inh
cc bc chit khc nh quy trnh trn s dng nn mu thc phm

chc nng c ngun gc tho dc khng pht hin cac alkaloid . Sau khi cn mu,

00

thm 25 L dung dch chun hn hp 1-10 g/mL cc alkaloid ( thu c nng

10

alkaloid trong dch cui l 5 ng/mL i vi nicotin, scopolamin, atropin v 50

ng/mL i vi brucin, strychnin, koumin, aconitin, colchicin trong dch chit cui

cng trc khi bm vo my LC/MS/MS). yn 5 pht. Thm 1 mL cc dung dch

kim ha. Tin hnh chit lp hai ln theo qui trnh trn thu c k t qua trung bnh

-L

nh bng sau:

TO

Bng 3.8 : nh hng ca dung dch kim ha n hiu sut chit cc alkaloid
Anconitin

Atropin

Brucin

Colchicin

Koumin

Nicotin

Scopolamin

Strychnin

NaOH 0,1N

33

45

54

80

56

50

73

39

83

74

80

116

112

103

120

95

79

60

68

106

103

115

112

80

NH4OH 5%
Acid boric
pH9

Hiu sut
(%)

Trng i hc KHTN

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Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh 3.17 : Biu nh hng ca dung dch kim ha n hiu sut chit cc

alkaloid

TR

Nhn xt: So snh hiu sut chit ca cc cht phn tch cho thy hiu sut
chit ca hu ht cc alkaloid tr nicotin u cao hn khi s dng dung dch kim

10

dung dch NH4OH 5%.

00

ha l dung dch NH4OH 5%. Do vy chng ti la chn dung dch kim ha l

3.2.3. Kho st th tch dung dch kim ha

khao sat anh hng ca th tch dung dch kim ha NH4OH 5% ln

-L

hiu sut chit cc cht phn tch chng ti c nh cc iu kin chit trn v

thay i th tch dung dch NH4OH 5% thm vo ln lt l 0,5; 1,0 v 2 mL. Tin

TO

hnh lm lp hai ln v ly kt qu trung bnh thu c nh sau:

Bng 3.9: nh hng ca th tch dung dch kim ha n hiu sut chit cc alkaloid

Hiu
Anconitin Atropin Brucin Colchicin Koumin Nicotin Scopolamin Strychnin
sut (%)
0,5 L
32
83
48
79
52
55
84
38
1 mL
83
112
72
108
108
97
108
89
2 mL
29
60
51
73
52
49
76
39

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Nhn xt: T bng trn cho thy khi s dng 0,5 mL dung dch kim ha cho
hiu sut chit mt s cht thp do cha c kim ha hon ton. Khi s dng 2

ny tng ln lm gim hiu sut chit vo pha hu c. Khi dng 1 mL dung dch

mL dung dch kim ha th hiu sut chit gim i r dt do th tch pha nc lc

kim ha cho hiu sut chit ca tt c cc cht l cao nht do vy chng ti s

TP
.Q
U

dng 1mL dung dch NH4OH 5% kim ha.

Kt lun: Vy quy trnh chit ti u ca cc alkaloid nh sau:

Cn 0,5g mu/ng ly tm 50 ml

+ 1 ml NH4OH 5%

Lc votex 5 pht

B + 10 ml CHCl3

00

TR

Ly tm 5 pht, tc 6000 vng/pht

Chit ln 2

+ 10 ml ethylacetat

Ha cn bng 5ml methanol

TO

-L

10

Gn dch vo bnh c, c quay 500C

Lc qua mng 0,2 m

Vial, LC-MS/MS

Hnh 3.18 : Quy trnh chit mu ti u

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

3.3. nh gi phng php phn tch

ti tin hnh phn tch cc mu trng. Kt qu cho thy mu trng khng c tn

khng nh phng php nghin cu c tnh c hiu/chn lc cao, chng

3.3.1. Tnh c hiu/chn lc

hiu ti thi gian lu ca cht phn tch. Mt khc t bng 3.1 cho thy tt c cc

TP
.Q
U

alkaloid nghin cu u c 1 ion m bn ph ra 2 ion con nn s im IP l 4 t theo


yu cu ca AOAC v im nhn dng IP i vi k thut sc k lng khi ph 2 ln.

TO

-L

10

00

TR

Nh vy, phng php nghin cu c tnh c hiu p ng c yu cu.

Hnh 3.19 : Sc chun alkaloid, mu trng v mu trng thm chun

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

3.3.2. Kho st khong tuyn tnh v lp ng chun

Pha long dung dch chun hn hp trung gian 1-10 g/mL trong methanol

thu c cc dung dch chun lm vic nng 0,5-5; 1-10; 2-20; 5-50; 10-100; 20-

Kho st khong tuyn tnh

3.3.2.1.

200; 50-500; 100-1000 ng/mL; 200-2000 ng/mL; 500-5000 ng/mL. Tin hnh chy

TP
.Q
U

my vi cc iu kin ti u trn. V th mi tng quan tuyn tnh gia din

TR

tch pic v nng chun, thu c cc kt qu sau :

00

Hnh 3.20: Mi tng quan gia din tch pic v nng Atropin trong khong 0,5-100

TO

-L

10

ng/mL

Hnh 3.21: Mi tng quan gia din tch pic v nng aconitin trong
khong 5-1000 ng/mL
Kt lun : Khong tuyn tnh t 51000 ng/mL vi cc cht brucin,
strychnin, koumin, aconitin, colchicin; khong tuyn tnh t 0,5 100 ng/mL vi

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

cc cht nicotin, atropin v scopolamin. Tt c u cho h s tng quan R >


0,9990, khong tuyn ca cc alkaloid l rng. Do , chng ti tin hnh lp

ng chun

3.3.2.2.

ng chun cc alkaloid nm trong khong tuyn tnh ca cc cht.

c kt qu sau:

Khong nng (ng/mL)

Phng trnh hi quy

H s tng quan R2

Cht phn tch

Bng 3.10: ng chun cc alkaloid

TP
.Q
U

Lp mi tng quan tuyn tnh gia din tch pc v nng cc alkaloid, thu

10 500

y = 15200x + 70700

1,0000

Strychnin

10 500

y = 15100x + 9930

0,9999

Aconitin

10 500

y = 1640x +2670

0,9996

Atropin

1 50

y = 12600x - 5780

0,9999

Scopolamin

1 50

y = 72500x +59300

0,9999

y = 14200x + 27800

0,9999

y = 9250x - 8460

0,9997

y = 40400x + 35000

0,9998

10 500

N
TR

Colchicin

10 500

10

00

Koumin

Brucin

Nicotin

1 50

Nhn xt : Din tch Alkaloid t l tuyn tnh vi nng trong khong tuyn

TO

-L

tnh vi h s tng quan R2 > 0,999 i vi t t ca cac ch t .

Hnh 3.22: ng chun Scopolamin (R2 = 0,9999)

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

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

(mu thc khng pht hin cht phn tch cho n khi thu c t s tn hiu/nhiu

Thm cc nng nh dn ca hn hp 8 Alkaloid vo dch chit mu trng

phng php

TP
.Q
U

(S/N) = 3, thu c kt qu LOD, LOQ ca cc Alkaloid nh sau:


Bng 3.11: Gii hn pht hin v gii hn nh lng ca cc alkaloid
LOQ

trn dch chit

phng php

trn dch chit

phng php

(ng/mL)

(g/kg)

(ng/mL)

(g/kg)

Strychnin

1,0

10

Aconitin

1,0

10

Atropin

0,1

Scopolamin

0,1

Koumin

1,0

Colchicin

10

3,3

33

3,3

33

1,0

0,3

3,3

1,0

0,3

3,3

10

3,3

33

1,0

10

3,3

33

1,0

0,3

3,3

N
TR
B

00

0,1

Nicotin

LOQ ca

1,0

3,0

0,3

Brucin

LOD ca

10

Cht phn tch

LOD

Nhn xt: Gii hn pht hin ca cc Alkaloid tng i thp (1 10 g/kg)


50

ca cc alkaloid (bng 1.20) v

gi tr LOD ca cc cht l nh so vi gi tr LD

-L

nh hn liu dng lm thuc i vi colchicin va strychnin do v y phng php c

TO

nhy p ng yu cu phn tch.

Hnh 3.23: Sc ca nicotin ti gii hn pht hin LOD 1,0 g/kg (S/N = 3,6)

Trng i hc KHTN

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Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh 3.24: Sc ca Brucin ti gii hn nh lng LOQ 10 g/kg (S/N = 11,5)

3.3.4. nh gi lp li v thu hi

nh gi hiu qu ca phng php phn tch chng ti tin hnh xc

nh lp li v thu hi ca phng php. tin hnh xc nh hai yu t


ny, chng ti tin hnh thm chun trn nn mu thc phm chc nng khng pht

hin Alkaloid, thm chun ti 3 mc nng 5-50 ng/mL, 10-100 ng/mL v 20-200
ng/ml tng ng trn nn mu l: 0,05-0,5 mg/kg; 0,1-1 mg/kg; 0,2-2 mg/kg. Mi

TR

mc chng ti tin hnh lm lp li 6 ln. thu hi tnh theo cng thc (3) v

00

lp li c xc nh theo cc i lng SD v RSD theo cng thc (1),(2) kt qu

TO

-L

10

thu c nh bng di y.

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Bng 3.12: lp li v hiu sut thu hi ca cc Alkaloid trn nn mu thc phm

Scopolamin

92,1

83,8

90,6

87,1

87,9

86,9

42,3

43,7

45,8

43,1

42,8

44,5

80,6

83,4

91,6

86,2

85,6

89

501,0

482,5

481,2

490

468,1 401,0

100,2

96,5

96,2

98,0

93,6

474,0

405,0

426,8 481,3 457,3 475,5

94,8

81,0

85,4

507,0

496,1

490,4 487,1 480,8 485,4

101,4

99,2

98,1

97,4

96,2

97,1

46,0

43,4

40,5

41,6

43,5

44,5

92,0

86,8

81,0

83,2

87,0

89,0

48,1

41,1

40,0

43,2

44,4

81,0

96,2

82,2

80,0

86,4

88,8

480,6

458,5

400,7 415,5 437,5 483,2

96,1

91,7

80,1

40,5

87,5

H
N

Y
TP
.Q
U

4,5

470,6

35,8

7,6

453,3

30,8

6,8

491,1

9,3

1,9

43,3

2,0

4,6

42,9

3,1

7,1

446,0

34,0

7,6

95,1

96,6

TO

-L

83,1

2,0

N
91,5

3,3

80,2

96,3

RSD

43,0

M5

M4

14,7

Strychnin

440,4

TR

Nicotin

453,1 435,7 439,7 434,5

460,5

M3

Koumin

418,9

00

Colchicin

SD

M2

10

Brucin

Trung
bnh

M1

Atropin

M6

Anconitin

Thng
s
C
(g/kg)
H (%)
C
(g/kg)
H (%)
C
(g/kg)
H (%)
C
(g/kg)
H (%)
C
(g/kg)
H (%)
C
(g/kg)
H (%)
C
(g/kg)
H (%)
C
(g/kg)
H (%)

Cht PT

chc nng ti nng 50-500 g/kg (0,05-0,5 mg/kg)

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Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh 3.25: Sc brucin trong mu thc phm chc nng thm chun ti mc nng

500 g/kg

Bng 3.13: lp li v hiu sut thu hi ca cc Alkaloid trn nn mu thc phm

Thng
Trung
M1 M2
M3
M4 M5 M6
SD RSD
s
bnh
C
1,03 0,86 0,91 0,95 0,94 0,81
0,92
0,08 8,5
(mg/kg)
H (%) 103,0 85,9 91,0 95,2 94,0 80,6
C
0,088 0,093 0,090 0,096 0,098 0,098 0,09 0,004 4,5
(mg/kg)
H (%) 88,0 93,0 90,0 95,7 98,4 97,5
C
1,06 1,05 1,00 1,03 0,94 0,81
0,98
0,09 9,6
(mg/kg)
H (%)
106
105
100
103 94,2 81,0
C
1,09 0,99 1,04 0,99 0,99 0,85
0,99
0,08 8,1
(mg/kg)
H (%)
109 98,5
104 99,0 98,8 84,9
C
0,84 1,02 0,92 0,98 0,93 0,92
0,93
0,06 6,6
(mg/kg)
H (%) 83,9
102 92,0 98,0 92,5 91,7
C
0,095 0,095 0,087 0,087 0,100 0,084 0,09
0,01 6,7
(mg/kg)
H (%) 94,7 95,3 87,2 87,1 100 84,2

-L

Atropin

Anconitin

10

00

Cht PT

TR

chc nng ti nng 0,1-1 mg/kg

TO

Brucin

Koumin

Colchicin

Nicotin

Trng i hc KHTN

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Th Thu Hng- K23 Ha hc

0,01

5,5

1,00

0,08

7,9

0,10

TR

TP
.Q
U

C
0,105 0,101 0,092 0,098 0,104 0,094
Scopolamin (mg/kg)
H (%)
105
101 91,9 97,6 104 93,6
C
1,10 0,90 0,99 0,99 1,07 0,92
Strychnin (mg/kg)
H (%)
110 90,0 99,0 99,4 107 92,0

Lun vn Thc s khoa hc

Hnh 3.26: Sc atropin trong mu thc phm chc nng thm chun ti mc nng

00

0,1 mg/kg

10

Bng 3.14: lp li v hiu sut thu hi ca cc Alkaloid trn nn mu thc phm

Thng
M1 M2
M3
M4 M5 M6
s
C
1,89 1,96 1,84 1,96 2,02 1,79
(mg/kg)
H (%) 94,7 98,0 92,1 97,9 101 89,3
C
0,168 0,163 0,166 0,188 0,168 0,171
(mg/kg)
H (%) 84,0 81,3 83,0 94,0 83,9 85,7
C
2,06 2,00 2,16 1,96 2,00 1,89
(mg/kg)
H (%)
103 99,8 108 98,0 100 94,7
C
1,74 1,82 1,71 1,94 2,16 1,86
(mg/kg)
H (%) 87,0 91,0 85,3 97,0 108 92,8

-L

Cht PT

chc nng ti nng 0,2-2 mg/kg

TO

Anconitin

Brucin

Atropin

Colchicin

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

Trung
bnh

SD

RSD

1,91

0,09

4,5

0,171

0,01

5,3

2,01

0,09

4,5

1,87

0,16

8,8

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Th Thu Hng- K23 Ha hc

0,13

6,8

0,171

0,01

4,0

0,192

0,01

1,86

TP
.Q
U

C
1,75 2,08 1,80 1,92 1,75 1,87
(mg/kg)
Koumin
H (%) 87,4 104 90,0 96,1 87,7 93,4
C
0,162 0,166 0,179 0,178 0,167 0,172
(mg/kg)
Nicotin
H (%) 81,1 83,0 89,5 89,0 83,6 86,1
C
0,184 0,198 0,202 0,212 0,172 0,182
Scopolamin (mg/kg)
H (%) 91,9 99,2 101
106 85,8 91,0
C
1,64 2,08 1,96 1,80 1,76 2,00
Strychnin (mg/kg)
H (%) 81,8 104
98
89,8 87,9 99,8

Lun vn Thc s khoa hc

0,17

9,0

10

00

TR

1,87

7,8

ti mc nng 0,2 mg/kg

Nhn xt: Theo qui nh ca AOAC, ng v chm ph thuc nng

cht bng P3.1 v P3.2 (ph lc): ti cp t 100 1000 g/kg, h s bin thin
CV 15% v ti cp 1000 g/kg, h s bin thin CV 10%; hiu sut thu
hi ti cp 10 g/kg phi nm trong khong 80 110%. Nh vy, phng php
c ng v chm t yu cu ca AOAC [19].

TO

-L

Hnh 3.27: Sc scopolamin trong mu thc phm chc nng thm chun

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

3.4. Phn tch mu thc.

hiu thuc ti H ni. Cc mu c ng nht v phn tch theo quy trnh chit

ng dng phng php phn tch mt s mu thc phm chc nng mua cc

Sau khi hon thnh qu trnh kho st v nh gi phng php, chng ti

TP
.Q
U

mu c ti u, kt qu thu c trong bng 3.15:

Atropin

Brucin

Colchicin

Koumin

Nicotin

Scopolamin

Strychnin

mg/vin

mg/vin

mg/vin

mg/vin

mg/vin

mg/vin

mg/vin

mg/vin

Gout Kingphar

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Gout Hong Tin an

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Gout Tm Bnh

KPH

KPH

0,12

KPH

KPH

KPH

KPH

0,23

Gout Thanh Bnh

KPH

KPH

0,019

KPH

KPH

KPH

KPH

0,035

Artrex DS

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Jex

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Ct thoi vng

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Xng khp Su Tong

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Kin ct hon

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Xng khp Makong

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Thp khp Tu Linh

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Xng khp Bch X

KPH

KPH

KPH

KPH

KPH

KPH

KPH

KPH

Vin khp Tm Bnh

KPH

KPH

0,079

KPH

KPH

KPH

KPH

0,14

TR

00

10

Mu

Aconitin

Bng 3.15: Bng kt qu phn tch mu thc

TO

-L

Ghi ch: k hiu KPH l khng pht hin cht phn tch

Trng i hc KHTN

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Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

TO

-L

10

00

TR

Hnh 3.28: Sc mu pht hin brucin

Hnh 3.29: Sc mu pht hin strychnin

Nhn xt: Trong 13 mu thc phm chc nng c ngun gc tho dc c

cht ca cy m tin. Hm lng cao nht l trong vin guot Tm Bnh vi hm


lng brucin 0,12 mg/vin v strychnin 0,23 mg/vin vi liu dng 2 ln/ ngy mi
ln 3 vin th hm lng ny mc an ton.

mua ti cc hiu thuc ti H Ni th c 3 mu c cha brucin v strychnin l hai

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

KT LUN V KIN NGH

Alkaloid c trong thc phm chc nng cm bng phng php sc k lng khi

Trn c s cc kt qu thc nghim nghin cu xc nh hm lng nhm

KT LUN

TP
.Q
U

ph, chng ti thu c cc kt qu sau:

1. Nghin cu v ti u c cc iu kin chy sc k lng khi ph xc


nh 8 Alkaloid bao gm:

- Ti u cc iu kin khi ph (xc nh th u phun, nng lng bn,

tc kh...) thu c cc mnh ph c trng ca tng Alkaloid.

- Ti u cc iu kin sc k lng: ct sc k Ct sc k InertSustain

C18 (150mm 4,6mm 5m); pha ng: knh A amoniacetat 10 mM trong

nc, knh B l acetonitril; chng trnh ra gii gradient; tc dng 0,5

TR

ml/pht.

2. Ti u iu kin tch chit cc Alkaloid ra khi nn mu: dung mi chit

00

ethylacetat v chlorofom, kim ha bng 1mL NH4OH 5%

10

3. Xy dng khong tuyn tnh t 51000 ng/mL vi cc cht brucin, strychnin,

koumin, aconitin, colchicin, khong tuyn tnh t 0,5 100 ng/mL vi cc

cht nicotin, atropin, scopolamin v ng chun xc nh 8 Alkaloid u c

h s tng quan tuyn tnh R2 > 0,99. Phng php c gii hn pht hin

-L

nh 1 10 g/kg, thu hi t 80,1 110 % v RSD t 1,9 9,6 u t yu

cu ca Chu u.

TO

4. ng dng phng php phn tch 13 mu thc phm chc nng trn a
bn H Ni pht hin 3 mu c cha brucin v strychnin khng c mu no
nhim cc Alkaloid cn li.
T cc kt qu thu c, chng ti nhn thy phng php phn tch c

nhy cao, phn tch nhanh v chnh xc, c th p dng phn tch hm lng 8

Trng i hc KHTN

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

Alkaloid vi tin cy cao. Chng ti s tip tc m rng phng php phn

tch c thm cc c t nhm Alkaloid.

Tip tc nghin cu thm quy trnh phn tch ng thi cc alkaloid c

KIN NGH

khc trong thc phm chc nng c ngun gc tho dc trn h thng LC-

TP
.Q
U

MS/MS.

Hin nay ti Vit Nam cha c quy nh v hm lng cng nh gii hn

cc alkaloid c ny. C quan qun l nn c cc quy nh v hm lng cc

TO

-L

10

00

TR

alkaloid c ny trong thc phm chc nng.

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

60

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

TI LIU THAM KHO

1. Trn Quc Bnh, Th Oanh (2011), "Gp phn nghin cu xy dng tiu

Ting Vit

chun nguyn liu v bn thnh phm c c dc s dng trong ch phm

TP
.Q
U

Camat", tp ch Dc hc,421,tr.45-49.

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

Ha hc phn tch, phn 2: Cc phng php phn tch cng c, Nh xut bn


Khoa hc v K thut, H Ni.

3. Khoa dc (2008), "Gio trnh dc liu", tp II, NXB H Y Dc TP.HCM.

4. Phm Thanh K (1998), "Bi ging dc liu", tp II, NXB i hc Dc H

Ni.

5. Tt Li (2004), Nhng cy thuc v v thuc Vit Nam, NXB Y hc.

TR

6. Phm Lun (1998), C s l thuyt phn tch sc k lng hiu nng cao, H
Quc Gia H Ni.

00

7. Lng Th Phn (2014), Nghin cu nh lng ng thi strychnin v brucin

10

trong huyt tng bng phng php HPLC, kha lun tt nghip, Trng

i hc Dc H ni.

8. Nguyn Vn Ri (2009), Gio trnh Cc phng php tch, khoa Ha hc

trng i hc Khoa hc T Nhin, i hc Quc Gia H Ni.

-L

9. Nguyn nh Thnh (2011), C s cc phng php ph ng dng trong ha

hc, Nh xut bn Khoa hc v K thut, H Ni.

TO

10. T Th Tho (2010), Gio trnh Thng k trong Ha hc phn tch, khoa Ha
hc trng i hc Khoa hc T Nhin, i hc Quc Gia H Ni.

11. Trn Th Vn Thi, Trn Thanh Minh (2011), "Nghin cu phn lp v nhn
dng cu trc alkaloid trong dch chit t l vng nem Tha Thin Hu", tp
ch Khoa hc i hc Hu, 65, tr.225-230.
12. Mai Tt T, Nguyn Th Trm (2007), Dc l hc tp 1, NXB Y hc.
13. Mai Tt T, Nguyn Th Trm (2007), Dc l hc tp 2, NXB Y hc.

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

61

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

14. Trng i hc Dc H Ni (2001), Dc liu, Nh xut bn Y hc


15. TS. Th Tuyn Vin CNSH Vit Nam , Bi ging cng ngh tch chit cc

16. Vin dc liu (2004), Cy thuc v ng vt lm thuc Vit Nam, tp I,

hp ch t th sinh.

NXB khoa hc k thut.

TP
.Q
U

17. Vin dc liu (2004), Cy thuc v ng vt lm thuc Vit Nam, tp II,


NXB khoa hc k thut.

18. Vin kim nghim an ton v sinh thc phm quc gia (2010), "Thm nh

phng php trong phn tch ha hc & vi sinh vt", NXB Khoa hc v K

thut.

Ting Anh

19. AOAC International (2007), How to meet ISO 17025 requirements for method

verification, USA

TR

20. Borbla Boros, Agnes Farkas, Silvia Jakabova, Ivett Bacskay (2000), LC-MS
Quantitative Deterrmination of Atropine and Scopolamine in the Floral Nectar

00

of Datura Species, Journal of Chromatographia, 58, 137.

10

21. Dirk Steinmann, Markus Ganzera (2011), Recent advances on HPLC/MS in

medicinal plant analysis, Journal of Pharmaceutical and Biomedical

Analysis, 55, 744-757.

22. Hua-Tao Feng, Sam F.Y Li (2002), Deterrmination of five toxic alkaloids in

-L

two common herbal medicines with capillary electrophoresis, Journal of

Chromatography A, 1-2, 243247

TO

23. Huiqin Wu, Xiaoting Xiong, Xiaolan Huang, Zhixin Zhu (2013),
Simultaneous deterrmination of 17 toxic alcaloids in human fluids by liquid
chromatography coupled whith electrospray ionization tandem mass
spectrometry, Journal of Liquid Chromatography & Related Technologies,
36:9, 1149-1162.
24. Goodman, L. (2006), Goodman & Gilman's the pharmacological basis of
therapeutics (11 ed.), New York: McGraw-Hill

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

62

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

25. Jos Diana Di Mavungu, Svetlana V. Malysheva, Melanie Sanders, Daria


Larionova (2012), Development and validation of a new LCMS/MS method

corresponding epimers. Application to some food and feed commodities,

for the simultaneous deterrmination of six major ergot alcaloids and their

Food Chemistry, 135, 292303.

TP
.Q
U

26. Miao Li, Xiao-Fang Holi, Jie Zhang, Si-Cen Wang, Qiang Fu, Lang-Chong He
(2011), Applications of HPLC/MS in the analysis of traditional Chinese
medicines, Journal of Pharmacertical Analysis, 1:2, 81- 91.

27. Lounasmaa M.; Tamminem T.(1993), The Alkaloids, Academic Press: New

Xiaohong

Chen,

Xiang

Chen

(2008),

Qiu,

Simultaneous

28. Peihong

York, 114.

deterrmination of five toxic alkaloids in body fluids by high-performance

liquid chromatography coupled with electrospray ionization tandem mass


spectrometry, Journal of Chromatography B, 875(2), 471477

TR

29. R.E. Ardrey (2003), Liquid chromatography-mass spectrometry: an

00

introduction, New York, J. Wiley.

10

30. Somenath Mitra (2003), Sample Preparation Techniques in Analytical

Chemistry, John Wiley & Sons.

31. Tadeusz Aniszewski (2012), Alcaloids - Secrets of Life, Alkaloid Chemistry,

Biological Significance and applications and ecological Role, Elsevier

-L

Science.

32. Xie Y, Jiang ZH, Zhou H, Xu HX, Liu L (2005), Simultaneous

high-performance liquid chromatography, Journal Chromatography A,


1093(1-2), 195-203.

33. Kang XQ1, Fan ZC, Zhang ZQ (2010), Simultaneous deterrmination of three
Aconitum alkaloids in six herbal medicines by high-performance liquid
chromatography, Journal Chromatography Science, 48(10),860-865.

TO

deterrmination of six Aconitum alkaloids in proprietary Chinese medicines by

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

63

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WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

34. Yu L, Xu Y, Feng H, Li SF (2005), Separation and deterrmination of toxic


pyrrolizidine alkaloids in traditional Chinese herbal medicines by micellar

electrokinetic chromatography with organic modifier, Electrophoresis,

TO

-L

10

00

TR

TP
.Q
U

26(17), 397-404.

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

64

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

PH LC

TR

TP
.Q
U

Ph lc 1: Kho st pha ng

TO

-L

10

00

Hnh P1.1: Kt qu din tch pc ca cc alkaloid khi s dng cc h pha ng khc nhau

Hnh P1.2: Kt qu din tch pc ca cc alkaloid khi nng amoniacetat trong pha
ng khc nhau

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

65

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WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

XIC of +M
RM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sam
ple 7 (Std M
ix5-50ppb-CH3COONH4-5m
M
) of DataSET2.wiff (Turbo Spray...

M
ax. 1.7e5 cps.

1.09e6
1.05e6
1.00e6
9.50e5
9.00e5
8.50e5
8.00e5
7.50e5

6.50e5
6.00e5

Intensity, cps

7.00e5

5.50e5
5.00e5

4.50e5
4.00e5
3.50e5

3.00e5
2.50e5
2.00e5

9.63

1.50e5
1.00e5
5.00e4
0.00
0.0

2.0

3.0

4.0

5.0

6.0

Tim
e, m
in

7.0

8.0

9.0

10.0

11.0

12.0

13.0

TP
.Q
U

1.0

Hnh P1.3: Sc cc alkaloid ti nng amoniacetat trong pha ng l 5mM


XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 5 (Std Mix5-50ppb-Grad7) of DataSET2.wiff (Turbo Spray)

1.05e6
1.00e6

9.50e5
9.00e5
8.50e5
8.00e5
7.50e5

7.00e5
6.50e5
6.00e5
5.50e5
5.00e5

Intensity, cps

Max. 2.0e5 cps.

4.50e5
4.00e5

3.00e5
2.50e5
2.00e5
1.50e5
1.00e5
5.00e4
0.00
0.0

2.0

3.0

4.0

5.0

6.0

Time, min

7.0

9.70

8.0

9.0

10.0

11.0

12.0

13.0

1.0

3.50e5

TR

Hnh P1.4: Sc cc alkaloid ti nng amoniacetat trong pha ng l 10mM


XIC of +MRM (18 pairs): 646.200/586.200 Da ID: Anconitine1 from Sample 9 (Std Mix 5-50ppb-CH3COONH4-20mM) of DataSET2.wiff (Turbo Spra...

9.0e5

00

8.5e5
8.0e5
7.5e5
7.0e5

10

6.0e5
5.5e5
5.0e5
4.5e5

Intensity, cps

6.5e5

4.0e5
3.5e5

3.0e5
2.5e5

9.81

2.0e5
1.5e5
1.0e5
5.0e4
1.0

1.5

2.0

0.5

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

6.5
7.0
Time, min

7.5

8.0

8.5

9.0

9.5

10.0

10.5

11.0

11.5

12.0

12.5

-L

0.0
0.0

Max. 2.0e5 cps.

9.5e5

TO

Hnh P1.5: Sc cc alkaloid ti nng amoniacetat trong pha ng l 20mM

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

66

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WWW.DAYKEMQUYNHON.UCOZ.COM

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Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

00

TR

TP
.Q
U

Ph lc 2: Ti u qu trnh x l mu

TO

-L

10

Hnh P2.1: Sc atropin khi kho st dung mi chit

Hnh P2.2: Sc brucin khi kho st dung mi chit

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

67

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.4: Sc koumin khi kho st dung mi chit

TO

-L

10

00

TR

Hnh P2.3: Sc colchicin khi kho st dung mi chit

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

68

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.6: Sc strychnin khi kho st dung mi chit

TO

-L

10

00

TR

Hnh P2.5: Sc scopolamin khi kho st dung mi chit

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

69

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.8: Sc atropin khi kho st dung dch kim ha

TO

-L

10

00

TR

Hnh P2.7: Sc aconitin khi kho st dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

70

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.10: Sc colchicin khi kho st dung dch kim ha

TO

-L

10

00

TR

Hnh P2.9: Sc brucin khi kho st dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

71

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

TO

-L

10

00

TR

Hnh P2.11: Sc koumin khi kho st dung dch kim ha

Hnh P2.12: Sc nicotin khi kho st dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

72

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.14: Sc strychnin khi kho st dung dch kim ha

TO

-L

10

00

TR

Hnh P2.13: Sc scopolamin khi kho st dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

73

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.16: Sc atropin khi kho st th tch dung dch kim ha

TO

-L

10

00

TR

Hnh P2.15: Sc aconitin khi kho st th tch dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

74

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.18: Sc colchicin khi kho st th tch dung dch kim ha

TO

-L

10

00

TR

Hnh P2.17: Sc brucin khi kho st th tch dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

75

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.20: Sc nicotin khi kho st th tch dung dch kim ha

TO

-L

10

00

TR

Hnh P2.19: Sc koumin khi kho st th tch dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

76

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P2.22: Sc strychnin khi kho st th tch dung dch kim ha

TO

-L

10

00

TR

Hnh P2.21: Sc scopolamin khi kho st th tch dung dch kim ha

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

77

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

TP
.Q
U

Ph lc 3: nh gi phng php phn tch

TO

-L

10

00

TR

Hnh P3.1: Sc aconitin trong mu thm chun ti mc nng 500 g/kg

Hnh P3.2: Sc atropin trong mu thm chun ti mc nng 50 g/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

78

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P3.4: Sc koumin trong mu thm chun ti mc nng 500 g/kg

TO

-L

10

00

TR

Hnh P3.3: Sc colchicin trong mu thm chun ti mc nng 500 g/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

79

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P3.6: Sc scopolamin trong mu thm chun ti mc nng 500 g/kg

TO

-L

10

00

TR

Hnh P3.5: Sc nicotin trong mu thm chun ti mc nng 50 g/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

80

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

TO

-L

10

00

TR

Hnh P3.7: Sc strychnin trong mu thm chun ti mc nng 500 g/kg

Hnh P3.8: Sc aconitin trong mu thm chun ti mc nng 1 mg/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

81

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

TO

-L

10

00

TR

Hnh P3.9: Sc brucin trong mu thm chun ti mc nng 1 mg/kg

Hnh P3.10: Sc colchicin trong mu thm chun ti mc nng 1 mg/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

82

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

-L

10

00

TR

Hnh P3.11: Sc koumin trong mu thm chun ti mc nng 1 mg/kg

TO

Hnh P3.12: Sc scopolamin trong mu thm chun ti mc nng 0,1 mg/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

83

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P3.14: Sc aconitin trong mu thm chun ti mc nng 2 mg/kg

TO

-L

10

00

TR

Hnh P3.13: Sc strychnin trong mu thm chun ti mc nng 1 mg/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

84

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

TO

-L

10

00

TR

Hnh P3.15: Sc brucin trong mu thm chun ti mc nng 2 mg/kg

Hnh P3.16: Sc colchicin trong mu thm chun ti mc nng 2 mg/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

85

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

Hnh P3.18: Sc strychnin trong mu thm chun ti mc nng 2 mg/kg

TO

-L

10

00

TR

Hnh P3.17: Sc koumin trong mu thm chun ti mc nng 2 mg/kg

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

86

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

T l cht

n v

RSD (%)

100

100%

1,3

10

10-1

10%

1,8

10-2

1%

0,1

10-3

0,1 %

0,01

10-4

100 ppm

0,001

10-5

10 ppm

0,0001

10-6

1 ppm

11

0,00001

10-7

100 ppb

15

0,000001

10-8

10 ppb

21

0,0000001

10-9

1 ppb

30

TP
.Q
U

Hm lng %

Bng P3.1: lch chun tng i lp li ti a theo tng khong nng

2,7

TR

3,7
5,3
7,3

00

Bng P3.2: Khong chp nhn ca thu hi cc nng khc nhau


T l cht

n v

thu hi [%]

100%

98-102

10-1

10%

98-102

10-2

1%

97-103

10-3

0,1 %

95-105

0,01

10-4

100 ppm

90-107

0,001

10-5

10 ppm

80-110

0,0001

10-6

1 ppm

80-110

0,00001

10-7

100 ppb

80-110

0,000001

10-8

10 ppb

60-115

0,0000001

10-9

1 ppb

40-120

10

Hm lng [%]

-L

10

100

TO

0,1

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

87

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON

Lun vn Thc s khoa hc

Th Thu Hng- K23 Ha hc

TP
.Q
U

Ph lc 4: Phn tch mu thc

TO

-L

10

00

TR

Hnh P4.1: Kt qu mu khng pht hin alkaloid

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

88

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TP
.Q
U

Lun vn Thc s khoa hc

TR

Hnh P4.2: Sc mu Gout Hong Tin an khng pht hin alkaloid

Hnh P4.4: Kt qu mu pht hin brucin

TO

-L

10

00

Hnh P4.3: Kt qu mu pht hin strychnin

Trng i hc KHTN

ng gp PDF bi GV. Nguyn Thanh T

89

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM

WWW.DAYKEMQUYNHON.UCOZ.COM

WWW.FACEBOOK.COM/DAYKEM.QUYNHON
Th Thu Hng- K23 Ha hc

TO

-L

10

00

TR

TP
.Q
U

Lun vn Thc s khoa hc

ng gp PDF bi GV. Nguyn Thanh T

WWW.BOIDUONGHOAHOCQUYNHON.BLOGSPOT.COM