B Y T

TRNG I HC DC H NI

NGUYN TH VN ANH

XC NH ADENOSINE
TRONG THC PHM CHC NNG
C CHA NG TRNG H THO
BNG PHNG PHP HPLC
KHA LUN TT NGHIP DC S

H NI - 2014

B Y T
TRNG I HC DC H NI

NGUYN TH VN ANH

XC NH ADENOSINE
TRONG THC PHM CHC NNG
C CHA NG TRNG H THO
BNG PHNG PHP HPLC
KHA LUN TT NGHIP DC S
Ngi hng dn:
ThS. Bi nh Sn
Ni thc hin:
Vin KNVSATTP Quc gia

H NI - 2014

LI CM N

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

giao ti v tn tnh hng dn, gip trong sut qu trnh thc hin kha lun.
Ti xin chn thnh cm n cn b, cng nhn vin Vin kim nghim V sinh
an ton thc phm Quc gia to mi iu kin thun li ti hon thnh ti.
Ti cng xin chn thnh cm n Ban gim hiu nh trng, cc phng ban,
cng cc ging vin, nhn vin B mn Ha phn tch - c cht v cc b mn
khc trong Trng i hc Dc H Ni gip ti trong sut qu trnh hc tp
ti trng.
Cui cng, ti xin chn thnh cm n gia nh, bn b quan tm ng vin
ti trong qu trnh hc tp v thc hin kha lun.
Ti xin chn thnh cm n!
H ni, ngy 09 thng 5 nm 2014
Sinh vin

Nguyn Th Vn Anh

MC LC
Trang
DANH MC CH VIT TT
DANH MC CC BNG
DANH MC HNH V V TH
T VN

Chng 1. TNG QUAN.

1.1. Gii thiu chung v ng trng h tho......................................................

1.1.1. c im ca ng trng h tho..................................................

1.1.2. Phn loi ng trng h tho.........................................................

1.1.3. Thnh phn ca ng trng h tho..

1.1.4. Tc dng ca ng trng h tho..

1. 2. Gii thiu v adenosine..

1.2.1. Cu to ca adenosine

1.2.2. Chuyn ha v tc dng ca adenosine..........

1.3. Phng php xc nh adenosine

1.3.1. Phng php sc k bn mng TLC..

1.3.2. Phng php in di mao qun (CE).........................

1.3.3. Phng php sc k lng...

1.4. Phng php sc k lng hiu nng cao (HPLC)........................

11

1.4.1. Nguyn tc chung ca sc k lng

11

1.4.2. Phn tch nh tnh v nh lng bng HPLC..

13

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

15

2.1. i tng ...................................................................................................

15

2.2. Ha cht v dng c ..................................................................................

15

2.2.1. Thit b...........................................................................................

15

2.2.2. Dng c.........................................................................................

15

2.2.3. Ha cht..........................................................................................

14

2.3. Phng php nghin cu.............................................................................

16

2.3.1. Phng php ly mu......... 16

2.3.2. Phng php phn tch......... 16
2.4. Thm nh phng php phn tch............. 17
Chng 3. THC NGHIM, KT QU V BN LUN.............................. 19
3.1. La chn phng php phn tch.................................................................... 19
3.2. Ti u iu kin xc nh adenosine trn h thng HPLC..............19
3.2.1. La chn ct tch sc k 19
3.2.2. Kho st thnh phn pha ng....... 21
3.2.3. Kho st chng trnh ra gii gradient............................................ 23
3.2.4. iu kin ti u ca qu trnh sc k................................................. 26
3.3. Kho st quy trnh x l mu.......................................................................... 27
3.3.1. Kho st dung mi chit 27
3.3.2. Kho st dung mi loi bo....... 28
3.3.3. Kho st nhit chit....... 29
3.3.4. Kho st thi gian chit ..... 30
3.3.5. Kho st s ln chit...... 30
3.4. nh gi phng php phn tch............ 32
3.4.1. Kho st khong tuyn tnh v lp ng chun....... 32
3.4.2. Gii hn pht hin (LOD) v gii hn nh lng (LOQ) ca
phng php 33
3.4.3. nh gi c hiu ca phng php............................................. 35
3.4.4. nh gi lp li ca h thng........................................................ 36
3.4.5. lp li v thu hi ca phng php......................................... 37
3.5. nh gi cht lng ca TPCN c cha THT..............................................40
3.6. Bn lun .......................................................................................................... 41
3.6.1. i tng nghin cu........................................................................ 41
3.6.2. Xy dng quy trnh phn tch............................................................ 41
3.6.3. Kho st quy trnh x l mu............................................................ 41
3.6.4. Thm nh phng php.................................................................... 42

3.6.5. Kt qu phn tch mu thc............................................................... 42

KT LUN V KIN NGH.............................................................................. 43
TI LIU THAM KHO
PH LC

DANH MC CH VIT TT
T vit tt
ACN
AOAC

Ting Anh hoc tn khoa hc

Acetonitrile
Association of Official Analytical

Hip hi cc nh Ha phn

Chemists

tch

AMP

Adenosine monophosphat

CE

Capillary electrophoresis

CMC

Carboxyl methyl cellulose

THT
EtOH
HPLC

Ting Vit

in di mao qun

ng trng h tho
Ethanol
High performance liquid

Sc k lng hiu nng cao

chromatography

LOD

Limit of detection

Gii hn pht hin

LOQ

Limit of quantification

Gii hn nh lng

MeOH

Methanol

MS

Mass spectrometry

Khi ph

PDA

Photo diode array

Dy diod quang

RSD

Relative standard deviation

lch chun tng i

Standard deviation

lch chun

SD
TB
TLC

Trung bnh
Thin layer chromatography

TPCN

Sc k lp mng
Thc phm chc nng

The united states food and drug

Cc qun l thc phm v

administration

dc phm M

USP

The united states pharmacopeia

Dc in M

UV

Ultra violet

T ngoi

Vis

Visible

Kh kin

USFDA

DANH MC CC BNG
Trang
Bng 3.1. Kho st thnh phn pha ng..... 22
Bng 3.2. Kho st s ln chit mu................

30

Bng 3.3 . Kho st khong tuyn tnh. 32

Bng 3.4. LOD v LOQ ca adenosine........................................................ 34
Bng 3.5. Kho st lp li ca h thng ................................................. 36
Bng 3.6. lp li v thu hi trn nn mu dng vin nang mm.......

38

Bng 3.7. lp li v thu hi trn nn mu dng vin nang cng ....... 38

Bng 3.8. lp li v thu hi trn nn mu dng lng......................... 39

DANH MC HNH V V TH
Trang
Hnh 1.1. Hnh nh ca Cordyceps sinensis.3
Hnh 1.2. Hnh nh ca Cordyceps militaris3
Hnh 1.3. Cu to ca mt s nucleoside trong ng trng h tho.. 4
Hnh 1.4. Cng thc cu to ca adenosine 7
Hnh 1.5. S cu to h thng HPLC..13
Hnh 1.6. Sc k trong phn tch sc k..13
Hnh 2.1. Quy trnh phn tch mu d kin. 17
Hnh 3.1. Ph hp th ca adenosine...........19
Hnh 3.2. Sc phn tch trn ct X-Brigde C18 20
Hnh 3.3. Sc phn tch trn ct Symmetry C18. 20
Hnh 3.4. Sc phn tch trn ct IntertSustain C18 21
Hnh 3.5. Sc phn tch s dng pha ng H2O v ACN...22
Hnh 3.6. Sc phn tch s dng pha ng H2O v MeOH............22
Hnh 3.7. Sc phn tch theo chng trnh gradient 1.23
Hnh 3.8. Sc phn tch theo chng trnh gradient 2.24
Hnh 3.9. Sc phn tch theo chng trnh gradient 3.24
Hnh 3.10. Sc phn tch theo chng trnh gradient 4...25
Hnh 3.11. Sc phn tch theo chng trnh gradient 5.. 26
Hnh 3.12. Sc phn tch vi iu kin ti u.............27
Hnh 3.13. Kho st dung mi chit ....27
Hnh 3.14: Kho st nhit chit...29
Hnh 3.15: Kho st thi gian chit 30
Hnh 3.16. Quy trnh phn tch mu thc ...31
Hnh 3.17. Kho st khong tuyn tnh....32
Hnh 3.18. ng chun ca adenosine. 33
Hnh 3.19. Sc nn mu TPCN dng vin nang ti LOD............................... 34
Hnh 3.20. Sc nn mu TPCN dng lng ti LOD...................................... 34

Hnh 3.21. c hiu ca chun adenosine.......................................................35

Hnh 3.22. c hiu ca phng php trn nn mu dng nang cng............35
Hnh 3.23: c hiu ca phng php trn nn m dng nang mm..............36
Hnh 3.24: c hiu ca phng php trn nn mu dng lng.....................36
Hnh 3.25: nh gi cht lng mu TPCN c cha THT...............................40

-1-

T VN
Y hc c truyn Trung Quc t xa xa coi ng trng h tho l v thuc
c tc dng B ph ch can, b tinh in tu, ch huyt ho m, B ph ch
thn, h dng tng ph, T m trng dng, kh bnh kin thn, l loi thuc
c th cha c Bch h bch tn. Trn lm sng, cc nghin cu c truyn
cng nh cc thc nghim hin i u xc nh ng trng h tho hu nh khng
c tc dng ph i vi c th ngi v ng vt. Trong thnh phn ha hc ca
ng trng h tho, adenosine l mt trong nhng hot cht chnh c tc dng.
Adenosine c vai tr quan trng trong qu trnh chuyn ho nng lng ca c
th. N gip ci thin tun hon ngoi bin v tim mch, ci thin nng lc c bp,
gim sinh trng ca t bo xu, tng lng oxy trong mu. V vy, vic b sung
adenosine hm lng cao gip c th lun di do nng lng hot ng hiu
qu v nhanh chng xo i cc triu chng mt mi.
Tuy nhin, hin nay ti Vit nam cha c phng php chun xc nh hm
lng hot cht chnh trong ng trng h tho nn vic pht trin phng php
phn tch cc hot cht chnh ni chung v adenosine ni ring trong nguyn liu
hay cc sn phm c cha ng trng h tho l v cng cn thit. N l cng c
gip cho cc nh qun l kim sot tt cht lng v gip cho ngi tiu dng c
s dng cc sn phm m bo cht lng.
V vy, chng ti tin hnh nghin cu ti: "Xc nh adenosine trong
thc phm chc nng c cha ng trng h tho bng phng php HPLC"
vi cc mc tiu sau:
+ Xy dng phng php xc nh adenosine trong TPCN c cha THT
bng phng php sc k lng hiu nng cao HPLC
+ ng dng phng php phn tch sn phm TPCN c cha THT trn
th trng. T , nh gi cht lng sn phm TPCN c cha THT.

-2-

Chng 1. TNG QUAN

1.1. Gii thiu chung v ng trng h tho
1.1.1. c im ca ng trng h tho [10], [14], [21], [22], [23]
Cordyceps (ng trng h tho) l mt loi nm thuc ngnh Ascomycotina,
lp Asiomycetes, c coi l mt thn dc c gi tr cao trong y hc Trung Quc
trong nhiu th k. l mt dng cng sinh gia mt loi nm ti c tn khoa hc
l Cordyceps sinensis vi u trng ca mt loi cn trng thuc chi Hepialus.
Thng gp nht l loi Hepialus armoricanus, ngoi ra cn 40 loi khc thuc chi
Hepialus cng c th b Cordyceps sinensis k sinh.
Cordyceps xut pht t ting Latinh "Cord" = "chy", "ceps" = "u". Nm c
dng ging con su, vi ui l mt cnh nh, mc l. Khi sy kh c mi tanh nh
c, t ln c mi thm. Phn "l" hnh dng ging ngn tay, di khong 4 11 cm,
trng lng khong 0,06g do si nm mc dnh lin vo u su non m thnh. u
su non ging nh con tm, di chng 3 5 cm, ng knh khong 0,3 - 0,8 cm.
Bn ngoi c mu vng sm hoc nu vng vi khong 20 - 30 vn kha, vn kha
gn u nh hn, c 8 cp chn, nhng 4 i gia l r nht. Cht m nm hnh
que cong mc ra t mnh su non, di hn su non mt cht. Su non d b gy,
rut bn trong cng y, mu trng hi vng, cht m nm kh dai v bn trong
rut hi rng, c mu trng ng. Khi nm t n trng thnh nht, n tiu th
n 99% cht dinh dng t thn su v su bin thnh xc kh. Vo ma ng, su
b nhim bo t nm do n phi hoc ht phi bo t nm. Vo ma h, nm pht
trin thnh cy mc ra t u su vn ra khi mt t. ng trng h tho ch
c pht hin vo ma h mt s cao nguyn cao hn mt bin t 3500 n
5000m nh: Ty Tng, T Xuyn, Thanh Hi, Cam Tc, Vn Nam...
1.1.2. Phn loi ng trng h tho [14]
Chi nm Cordyceps c ti 680 loi khc nhau, ring Trung Quc tm thy
60 loi. Tuy nhin cho n nay ngi ta mi ch nghin cu nhiu nht c 2 loi
Cordyceps sinensis v Cordyceps militaris.

-3-

Hnh 1.1. Hnh nh ca Cordyceps sinensis

Hnh 1.2. Hnh nh ca Cordyceps militaris

1.1.3. Thnh phn ca ng trng h tho [7], [14], [18], [19], [21], [23]
Trong ng trng h tho cha nhiu hp cht c hot tnh sinh hc qu: cc
hp cht nucleoside, cc polysaccharide, protein v cc hp cht nitragenous, sterol,
vitamin, khong cht...
* Cc nucleoside
y l thnh phn chnh c gi tr c bit trong ng trng h tho, gm mt
s hp cht nh: deoxyuridine, 2-3-dideoxyadenosine, hydroxyethyladenosine,
cordycepin [3-deoxyadenosine], uridine, guanidine, deoxyguanidine, cordycepin
triphosphate. Trong , adenosine l mt nucleoside c hot tnh sinh hc chnh
nh gi cht lng ca ng trng h tho.

-4-

. Hnh 1.3. Cu to ca mt s nucleoside trong ng trng h tho

* Cc nhm hp cht khc
Nakamura v cng s [18], [19] nghin cu cho thy cc polysaccharide
trong THT c hiu qu trong vic iu tit ng trong mu, chng di cn, c
hiu qu chng ung th v tng cng min dch.
Cordyceps cha protein, peptide v 17 acid amin thit yu khc. Ngoi ra,
Cordyceps cn cha mt s dipeptide cyclic khng ph bin nh cyclo-[Gly-Pro],
cyclo-[Leu-Pro], , lng nh cc polyamin nh 1,3-diamino propane,. Cc hp
cht ny c chng minh l c tc dng khng sinh [14]
Mt s cc sterol c trong ng trng h tho nh ergosterol, ergosterol
peroxide,.... c tc dng chng ri lon tnh dc.
Cc vitamin cn thit cho c th: trong 100g ng trng h tho c 0,12mg
vitamin B12; 29,19mg vitamin A; 116,03mg vitamin C, ngoi ra cn c vitamin
B2, vitamin E, vitamin K.
Ngoi ra cn c hp cht hydrocarbon, alcohol v aldehyde,
1.1.4. Tc dng ca ng trng h tho [7], [14], [16], [23]
ng trng h tho c s dng kh ph bin trong khong 200 nm trc
y Trung Quc v cc nc Phng ng. n nm 1976, n c bit n
v s dng rng ri ti cc nc phng ty.

-5-

ng trng h tho c tc dng b phi, thn, cm mu, tan m, iu tr ho

mn tnh, iu tr ra m hi trm v m bo phc hi sc khe nhanh cho ngi b
bnh v tng cng sc khng.
Ci thin chc nng gan: chng x gan, vim gan mn tnh v cc bnh lin
quan n gan khc. Th nghim lm sng cho thy khi s dng TPCN c cha
THT trn 33 bnh nhn vim gan B v 8 bnh nhn x gan cho thy trn 71%
bnh nhn c ci thin tnh trng bnh l.
Ci thin chc nng thn: nghin cu Trung Quc cho thy 51% s bnh
nhn ci thin c bnh thn mn tnh khi s dng 1 thng cc sn phm t
Cordyceps.
Tng kh nng hp th O2, c kh nng lm gim bnh phi mn tnh
Ci thin chc nng tim: iu tr ri lon nhp tim v suy tim mn tnh
Bo v c th chng li cc gc t do
Gim kch thc ca khi u v chng li ung th: dng polysaccharide chit
xut t Cordyceps vi liu 1-10mg/kg trng lng mi ngy cho chut c khi u
cho thy c tc dng chng khi u sarcoma 180. Mizuno [16], nm 1999 nghin
cu dng -(1,3)-D-glucan, phn on CO-N, dn xut t THT C.ophioglossoides
chng li chui t bo sarcoma 180 vi liu 0,5mg/kg trn chut. Kt qu, khi u
b hn ch pht trin vi t l t n 98%. Trong mt nghin cu iu tr 50 bnh
nhn b ung th phi vi liu iu tr 6g/ngy cng vi ho tr, khi u gim xung
46%. Khi iu tr cc loi ung th khc nhau vi liu 6g/ngy trong vng hai thng
cho thy bnh c ci thin ng k, kim tra t bo bch cu thy tng cao cc
bnh nhn c khi u gim 50%.
iu tit kh nng min dch: khi cc bnh nhn b thiu kh nng min dch,
nh bnh nhn ung th, vim gan B v HIV c s dng ng trng h tho h
tr iu tr, kt qu cho thy s lng v tc ng ca bch cu trong mu tng.
Ngc li, vi bnh nhn tng min dch qu cao nh bnh bch cu lympho hoc
thp khp khi dng ng trng h tho cho thy s lng v tc ng ca bch cu
trong mu gim v s lng hng cu li tng.

-6-

Duy tr lng cholesterol tt: Theo mt s nghin cu C.sinensis c kh

nng gim lng cholesterol tng xung 10-21% v triglycerid xung 9-26% ng
thi lm tng hm lng cholesterol c t trng cao (HDL - High density
Lipoprotein) t 27 - 30%
H tr cc triu chng lo ha: Nghin cu lm sng trn mt s bnh nhn
ln tui b mt mi v cc triu chng lin quan n lo ha sau khi s dng cc sn
phm t ng trng h tho trong 30 ngy. Mt mi ca h gim 92%, cm gic
ca h v lnh gim 89% v hin tng chng mt gim 83%, mt s bnh nhn c
vn h hp c ci thin r rt.
Ci thin chc nng tnh dc
Mt s hp cht c hot tnh sinh hc c s dng nhiu trong lnh vc y hc
nh 2,3- dideoxyadenosine (c coi l cht antiretroviruses) dng cho iu tr ly
nhim HIV (cc bit dc: DidannosineTM hay VidexTM)
Cc nghin cu c truyn cng nh hin i u xc nh ng trng h
tho hu nh khng c tc dng ph i vi c th ngi v ng vt. Liu ung
THT an ton i vi chut th nghim l trn 45g/1kg th trng.
Vi nhng tc dng nh trn, ng trng h tho c coi l mt thn dc
c s dng rng ri trong y hc. Trong , adenosine l thnh phn hot cht
chnh nh gi cht lng ca ng trng h tho.
1. 2. Gii thiu v adenosine
1.2.1. Cu to ca adenosine [13], [20], [22]
Adenosine l mt nucleoside nhn purin. y l hp cht cha N c cu trc
phn t hai vng gm mt phn t adenine lin kt vi mt ng ribose nh lin
kt -N9-glycosidic.
Cng thc phn t C10H13N5O4, khi lng phn t 267,241g/mol
Tn thng mi: adenocard
Cng thc cu to ca adenosine:

-7-

Hnh 1.4. Cng thc cu to ca adenosine

Adenosine dng bt tinh th mu trng, tan nhiu trong nc. ha tan tng ln
khi nhit tng v pH gim.
1.2.2. Chuyn ha v tc dng ca adenosine [1], [7], [13], [20]
Trong c th, adenosine c to thnh do s thy phn AMP bi enzym 5'nucleotidase, mt phn b chuyn ha ngc tr li to thnh AMP bi enzym
adenosine kinase, mt phn chuyn ha thnh inosine nh enzym adenosine
deaminase, sau chuyn ha thnh hypoxanthine nh enzym nucleosidase v
thnh xanthin nh enzym xanthin oxydase, sn phm cui cng ca s chuyn ha
l to thnh acid uric.
Adenosine l mt nucleoside ni sinh nh hng nhiu qu trnh sinh l ca
c th, n c mt khp cc c quan trong c th v ng vai tr quan trng trong
qa trnh trao i cht ca c th nh chuyn ha nng lng cng nh truyn tn
hiu cho AMP vng.
* c tnh khng vim
Adenosine c chng minh l mt cht c kh nng khng vim ti th th
A2A. Nng adenosine ngoi bo t bo bnh thng l khong 300nM. Tuy
nhin, p ng vi tn thng t bo nng ny c nhanh chng nng ln
600 - 1200nM. V vy, chc nng chnh ca adenosine l ngn nga tn thng m
trong trng hp thiu oxy, thiu mu cc b, co git, tng cng tun hon mch
vnh, mch no v iu ha nhp tim

-8-

* Tc dng trn tim

Adenosine trc tip kim sot cc chc nng ca m tim, ng thi n c kh
nng bo v tim chng li mt s chuyn ha c hi bng cch gim s trao i cht
ti c tim v tng lu lng mu ng mch vnh.
Adenosine c coi l thuc iu tr hiu qu ri lon nhp nt nh tht, ri
lon nhp trn tht cp. Hi phc c nhp xoang ngi b ri lon nhp nhanh
trn tht kch pht.
* Tc dng trn phi
Adenosine c kh nng iu chnh chc nng ca nhiu t bo khc nhau
lin quan n vim ng h hp nh bch cu trung tnh, bch cu a acid, t bo
lympho v i thc bo.
* Tc dng trn h thng thn kinh trung ng
Adenosine c tc dng tt vi ri lon thn kinh nh: thiu no cc b, ng
kinh, cc bnh thoi ha thn kinh v au dy thn kinh. V vy, adenosine c
s dng nhiu trong y hc sn xut thuc dng iu tr ri lon nhp tim,
thuc chng lon nhp tim, bnh lin quan n h thng thn kinh...
* Tc dng trn tc
Adenosine c chng mnh c kh nng kch thch s pht trin ca tc
nhng ngi c tc tha
1.3. Phng php xc nh adenosine
1.3.1. Phng php sc k bn mng TLC [8], [15]
Ma Yan v cng s [15] tin hnh xc nh cc nucleoside trong Cordyceps
sinensis bng phng php TLC. Dch chit c phn tch trn bn mng silica
GF254 c cha 1% CMC-Na, dung mi gm cloroform-ethylacetate-isopropanolnc-amoniac t l 8 2 6 0,5 0,12 (theo th tch), pht hin cht phn tch ti 2
bc sng 254nm v 300nm. Phng php rt n gin, lch chun tng i
trong khong 0,73 - 0,98% v thu hi cao t 95,270% - 96,366%.
L.Wuqing v cng s [8] xc nh adenosine trong vin nang Jinshuibao.
Adenosine c tch trn bn mng silica GF254 CMC - Na2HPO4 vi dung mi

-9-

tng t nh trn, pht hin ti 2 bc sng l 260nm v 300nm. ng chun

c xy dng trong khong 0,65 - 5,2g vi r = 0,9995, thu hi ca phng
php l 99,5% v h s bin thin 3,7 %.
1.3.2. Phng php in di mao qun (CE) [6], [12]
Li Hai Ying v cng s [12] tch v nh lng adenosine, adenin, uridine
bng dung dch m dinatri tetraborat 15mM v natridihydrophosphat 14mM (pH =
9,5) vi 5% MeOH theo th tch, in th 18kV v detector UV bc sng 254nm.
H s tng quan ca phng php R 0,9985.
Feng-Qing Yang v cng s [6] tin hnh xc nh 12 nucleoside v
nucleobase

(cytosine,

adenine,

guanine,

cytidine,

cordycepin,

adenosine,

hypoxanthine, guanosine, inosine, 2-deoxyuridine, uridine v thymidine) trong mt

s loi ng trng h tho bng phng php in di mao qun khi ph (CE-MS).
Mu c ha tan bng H2O 950C, rung siu m nhit 75oC trong 30 pht. Sau
a dch chit v nhit phng, lc qua mng 0,25m. Dch chit c bm
vo h thng CE-MS vi pha ng l m formic 10mM cha 10% methanol, tc
dng 3L/pht. Phng php cho kt qu hm lng nucleoside v nucleobase
trong cordyceps sinensis l 9138 4823g/g cao hn so vi C.militaris l 3722
1446g/g.
1.3.3. Phng php sc k lng [9], [11], [17], [24], [25]
Lan -Fang Huang v cng s [9] tin hnh xc nh thnh phn hot tnh
ca Cordyceps sinensis v Cordyceps militaris gm adenin, hypoxanthin,
cordycepin v cc sn phm chuyn ha ca n bng phng php sc k lng khi
ph LC/ESI-MS. Phng php s dng pha ng gm H2O, methanol v acid
formic vi t l 85 : 14 : 1 (theo th tch), ct VP - ODS (2,0 x 150mm) ca hng
Shimadzu, la chn ion [M + H]+ ti m/z l 136, 137, 268, 252, 302 phn tch
nh lng cho 4 thnh phn hot tnh. ng chun c xy dng cho adenin vi
nng t 1,4 - 140g/mL, hypoxanthine t 0,6 - 117,5g/mL, cho adenosine t
0,5 - 128,5g/mL v cordycepin t 0,5 - 131,5g/mL. Gii hn pht hin v gii
hn nh lng ca adenin l 0,5 v 1,4g/mL, hypoxanthin l 0,2 v 0,6g/mL,

-10-

adenosine v cordycepin l 0,1 v 0,5g/mL. thu hi ca phng php trong

khong 93,7 - 107%.
Xiao Feng Xue v cng s [25] tin hnh phn tch adenosine trong sa ong
cha. Adenosine c chit bng ethanol : H2O t l 80 : 20 theo th tch. Dch
chit mu c bm vo h thng HPLC vi ct sc k C18 v dung mi gm 2
knh: knh A l acid phosphoric 0,4% v knh B l MeOH. Detector UV bc sng
257nm. thu hi ca phng php trong khong 91,6 - 98,3% vi lch chun
nh hn 5,3%. Gii hn pht hin v gii hn nh lng tng ng l 0,017 v
0,048g/mL.
Li chen v cng s [11] xc nh adenosine v cordycepin trong ng trng
h tho. Mu c chit vi H2O, sau dch chit c phn tch trn ct Eclipse
XDB -CN. Pha ng gm MeOH : H2O theo t l 7 : 93, s dng detector PDA ti
bc sng 260nm. Phng php c thu hi trong khong t 93,8 - 102,9%, vi
lch chun tng i nh hn 3,62%. Gii hn nh lng ca adenosine v
cordycepin l 0,21 v 0,083mg/lt. Phng php n gin, nhanh, chnh xc cao
v gi thnh thp.
Hemanth Kumar, M.Spandana [17] phn tch ng thi adenosine, cordycepin
v sn phm chuyn ha ca cordyceps sinensis bng phng php HPLC. iu
kin phn tch: ct C18 ProtoSIL (250nm x 4,6mm x 5m), pha ng MeOH : H2O
t l 20 : 80 theo th tch, tc dng 1mL/pht, detector UV bc sng 254nm,
nhit bung ct 300C. Hm lng ca adenosine v cordycepin khong 0,288 v
0,346mg/g. thu hi 101,1%, lch chun tng i l 1,33%.
Theo dc in Trung Quc [24], adenosine c chit tch t ngun
nguyn liu ng trng h tho nh sau: cn chnh xc 0,5g mu cho vo bnh cu,
thm 10mL MeOH 90%, y np, lc u, gia nhit v un hi lu trong 30 pht,
lm ngui, thm methanol 90% n lng chnh xc, lc u, lc. Chy sc k vi
iu kin nh sau: pha ng l m phosphat (pH = 6,5) v MeOH t l 17 : 3, ct
pha tnh octandecylsilane, pht hin ti bc sng 260nm.

-11-

1.4. Phng php sc k lng hiu nng cao (HPLC)

1.4.1. Nguyn tc chung ca sc k lng [2], [3], [4]
Sc k lng l qu trnh tch xy ra trn ct tch vi pha tnh l cht rn hoc
cht lng v pha ng l cht lng (sc k lng - rn, lng - lng). Mu phn tch
c chuyn ln ct tch di dng dung dch. Khi tin hnh chy sc k, cc cht
phn tch c phn b lin tc gia pha ng v pha tnh. Trong hn hp cc cht
phn tch, do cu trc phn t v tnh cht l ho ca cc cht khc nhau, nn kh
nng tng tc ca chng vi pha tnh v pha ng khc nhau, chng di chuyn vi
tc khc nhau v tch ra khi nhau.
1.4.1.1. Pha tnh trong sc k lng
Tu theo bn cht ca pha tnh, trong phng php sc k lng pha lin kt
thng chia lm 2 loi: sc k pha thng (NP-HPLC) v sc k pha o (RPHPLC).
+ Sc k pha thng: pha tnh c b mt l cc cht phn cc ( l cc silica
trn hoc cc silica c gn cc nhm ankyl c t cacbon mang cc nhm chc
phn cc: -NH2, -CN)
+ Sc k pha o: pha tnh thng l cc silica c ankyl ho, khng phn
cc, loi thng dng nht l C18H37
u im ca sc k pha o l tch v phn tch cc cht c phn cc rt a
dng: t rt phn cc, t phn cc ti khng phn cc. Hn na, trong rt nhiu
trng hp th thnh phn chnh ca pha ng li l nc nn rt kinh t.
1.4.1.2. Pha ng trong sc k lng
Pha ng trong sc k lng l thnh phn c cho i qua ct lin tc phn
tch cc hp cht trong mt hn hp, c nhng yu cu sau:
+ Pha ng phi tr vi pha tnh
+ Bn vng, n nh v khng b phn hu trong qu trnh chy sc k
+ Ho tan c mu
+ Phi c tinh khit cao, c nht thp v ph hp vi detector
C th chia pha ng lm hai loi:

-12-

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

phn tch cc cht hu c, cn thm cc dung mi gim phn cc. Pha ng
loi ny c dng trong sc k pha ngc.
+ Pha ng c phn cc thp: l cc dung mi t phn cc nh cyclopentan,
n-pentan, n-heptan, n-hexan, 2-chloropropan, cacbondisulfua (CS2), CCl4, toluene
tch mt nhm cht thng thng pha ng mt thnh phn i khi khng
p ng c kh nng ra gii, ngi ta thng phi hp 2 hay 3 dung mi c
c dung mi c phn cc t thp n cao ph hp vi php phn tch. S thay
i thnh phn pha ng i khi din ra theo thi gian, trng hp ny ngi ta gi
l gradient pha ng.
1.4.1.3. Detector trong sc k
Detector l b phn quan trng quyt nh nhy ca phng php. Tu thuc
bn cht l ho ca cht phn tch m la chn detector cho ph hp
+ Detector quang ph hp th phn t UV-Vis: p dng cho cc cht c kh
nng hp th nh sng trong vng t ngoi (UV) hoc vng kh kin (Vis).
+ Detector hunh quang: s dng pht hin cc cht c kh nng pht hunh
quang. i vi nhng cht khng c kh nng nh vy, cn phi dn xut ho cht
phn tch, gn n vi cht c kh nng pht hunh quang hoc cht phn tch phn
ng vi thuc th to thnh sn phm c kh nng pht hunh quang.
+ Detector dn: ph hp vi cc cht c hot tnh in ho: Cc ion, cc hp
cht c tnh dn in
+ Detector PDA: da vo s thay i cng bc x do s hp th bc x
ca hp cht c trong hn hp m chm tia bc x chiu qua. Da vo s thay i
cng bc x, xc nh c cht phn tch.
1.4.1.4. Cu to ca h thng sc k lng hiu nng cao
Cng nh tt c cc thit b sc k khc, thit b HPLC gm 3 phn chnh:
- Phn u vo cp pha ng c thnh phn mong mun v mu phn tch.
- Phn tch l phn trung gian ca h sc k bao gm ct tch, i khi c ct ph
tr.

-13-

- Phn pht hin v x l s liu gm cc detector, phn khuch i, computer v

phn mm x l s liu, b phn ghi tn hiu.

Hnh 1.5. S cu to h thng HPLC

1.4.2. Phn tch nh tnh v nh lng bng HPLC [2], [3]
Phn tch nh tnh: i lng c trng cho s tch sc k l thi gian lu
ca cc cht. Da vo thi gian lu ca cht phn tch trong chun v trong dung
dch phn tch nh tnh tng cht trong hn hp.

Hnh 1.6. Sc k trong phn tch sc k

nh lng cht phn tch, dng phng php ng chun, so snh hoc
thm chun.
* Phng php ng chun
- Pha ch mt s dung dch chun i chiu c nng khc nhau
- o p ng Si ca cc dung dch chun v lp ng cong ph thuc ca S
vo C:
S = f(C)
- Chn khong nng ph thuc tuyn tnh
- o tn hiu p ng Sx v ni suy nng Cx t ng chun

-14-

* Phng php so snh

- em o hp th quang ca dung dch chun
- o mt quang ca dung dch cn xc nh X
- Tnh nng ca dung dch X theo cng thc sau:
CX

D X . C ch
Dch

* Phng php thm chun

- o cng p ng ca mu phn tch Sx.
- Pha ch mt s dung dch c nng Cx
- o nng cht chun i chiu CR khc nhau:

Cx + CR, i

- o p ng ca cc dung dch Si
- Xy dng th s ph thuc Si = f(CR,i)
- Chn khong ph thuc tuyn tnh. Tnh nng Cx

-15-

Chng 2. I TNG V PHNG PHP NGHIN CU

2.1. i tng
i tng nghin cu: TPCN c cha THT
2.2. Ha cht v dng c
2.2.1. Thit b
-

H thng sc k lng hiu nng cao ca Shimadzu

My lc vortex VELP, my ng nht mu, my li tm lnh Hermle, my rung

siu m c ch gia nhit, cn phn tch (c chnh xc 0,1mg v 0,01mg), cn

k thut (c chnh xc 0,01g)
2.2.2. Dng c
-

ng ly tm, ng ong, cc loi pipet, bnh nh mc

ng nghim,....

2.2.3. Ha cht
Cc loi ha cht dng trong phng php u thuc loi tinh khit phn tch
* Chun adenososine t sigma aldrich tinh khit 99%
-

Dung dch chun gc 1000g/mL: cn chnh xc khong 0,1000g adenosine

chun trn cn phn tch c chnh xc 10-4, ha tan v nh mc 100mL
bng H2O. Bo qun trong t lnh 40C, s dng trong 1 thng.

Dung dch chun trung gian (200g/mL): ly 2mL dung dch chun gc vo
bnh nh mc 10mL v nh mc ti vch bng H2O.

Cc dung dch chun lm vic nng 0,1; 0,5; 1,0; 5,0; 10,0; 20,0g/mL
c pha trong H2O.

* Cc loi ha cht, dung mi khc:

-

Methanol (Merck 99,9%), n-hexan (Merck 99,9%), ethanol (Merck 99,9%),

diethyl ether (Merck 99,9%), ether du ha (Merck 99,9%), ethyl acetate
(Merck 99,9%)

Nc ct dng cho sc k lng

-16-

2.3. Phng php nghin cu

2.3.1. Phng php ly mu
- i tng mu: TPCN c cha THT
- Phng php ly mu: ngu nhin
- a im ly mu: thnh ph H ni
- Khi lng mu ly: hai n v mu (hp, gi, l)/mu
- Bo qun v lu tr mu: iu kin thng
2.3.2. Phng php phn tch
Nguyn l: Adenosine c ha tan trong dung mi ph hp, tin hnh rung
siu m, ly tm. Dch chit c nh mc 50mL, lc v bm vo h thng sc k
lng hiu nng cao vi detector PDA.
* Quy trnh tm tt:
- Mu c ng nht k
- Cn mu vo ng ly tm, vi nn mu cha nhiu bo cn loi bo trc khi phn
tch
- Thm dung mi chit mu vo ng ly tm
- Lc vortex mu v dung mi c o trn u
- Rung siu m tng hiu sut ha tan cht phn tch vo dung mi chit
- ngui n nhit phng
- Ly tm vi tc cao, phn tch phn dung dch v lng phn b
- Chit lp mu vi dung mi trn
- Gp dch chit mu v nh mc n vch
- Lc
- Bm vo h thng HPLC

-17-

Cn mu
Loi bo (nu cn)
Dung mi chit
Lc vortex

Siu m
Chit lp
Ly tm
Cn
Dch chit
nh mc
Lc
HPLC
Hnh 2.1. Lc quy trnh phn tch mu d kin
2.4 Thm nh phng php phn tch [5]
Sau khi xy dng phng php phn tch cn phi c thm nh theo quy
nh ca USFDA, AOAC, USP cc thng s cn thm nh bao gm:
- Tnh c hiu, tnh chn lc (Specifility/Selectivity)
- Khong tuyn tnh v ng chun (Linearity and Calibration)
- Gii hn pht hin (Limit of Detection LOD)
- Gii hn nh lng (Limit of Quatification LOQ)
- ng (Trueness)
- chm (Precision)
- n nh ca phng php (Robustness/Ruggeness)

-18-

Vic la chn cc thng s thm nh ty thuc vo: K thut p dng, yu cu

ca phng php, iu kin v ngun lc ca phng th nghim. Trong phm vi v
thi gian nghin cu, chng ti thc hin thm nh phng php thng qua vic
nh gi cc thng s: Tnh c hiu, khong tuyn tnh v ng chun, gii hn
pht hin v gii hn nh lng, ng (thng qua hiu sut thu hi), chm
(thng qua lp li)

-19-

Chng 3. THC NGHIM, KT QU V BN LUN

3.1. La chn phng php phn tch
Chng ti tin hnh phn tch adenosine trn h thng sc k lng hiu nng
cao, detector PDA vi cc iu kin nh sau:
-

Ct sc k C18 pha o

Detector PDA ti bc sng 260 nm

Tc dng: 1mL/pht

Nhit bung ct: 40oC

Th tch bm mu: 20L

Adenosine l hp cht c kh nng hp th ti bc sng trong vng t

ngoi nn c th s dng detector UV thay th. Tuy nhin, detector PDA c u
im l cho bit hnh dng ph ca cht phn tch. Khi kt hp gia thi gian lu v
hnh dng ph hp th c th khng nh chnh xc cht cn phn tch. Do , trong
nghin cu ny chng ti quyt nh s dng detector PDA.
Cht phn tch sau khi c tch khi ct sc k, c dn vo flowcell ca
u o v c chiu bi mt chm tia t ngoi. S hp th tia bc x bi cc cht
tan tun theo nh lut Lambert-Beer.

Hnh 3.1. Ph hp th ca adenosine

3.2. Ti u iu kin xc nh adenosine trn h thng HPLC
3.2.1. La chn ct tch sc k
Ct tch c vai tr quan trng trong vic tch cc cht phn tch ra khi
nhau, n c v nh tri tim ca h thng sc k. Hin nay, sc k pha o c

-20-

s dng rng ri, do tnh ph bin, tnh kinh t v c hiu qu cao. Do , chng ti
la chn h sc k pha o, kho st kh nng tch ca cht phn tch trn mt s
ct C18 v c nh cc iu kin phn tch khc vi nng adenosine 10,5ppm:
- Ct X-Bridge C18 (150mm 2,1mm 3,5m)
- Ct Symmetry C18- Waters (150mm 4,6mm 5m)
- Ct IntertSustain C18 (250mm 4,6mm 5m)
/3.303/735082

mAU
260nm4nm (1.00)
50

25

0
0.0

1.0

2.0

3.0

4.0

5.0

6.0

Hnh 3.2. Sc phn tch trn ct X-Brigde C18

Hnh 3.3. Sc phn tch trn ct Symmetry C18

7.0 min

-21-

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100
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50
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2.5

5.0

7.5

10.0

12.5

min

Hnh 3.4. Sc phn tch trn ct IntertSustain C18

Vi ct X-Brigde C18, thi gian lu ngn (khong 3,5 pht), do khi phn
tch trn nn mu thc s b nh hng bi tp cht do khng tch c cht phn
tch v tp cht. Hn na, ct X-Brigde C18 (150mm x 2,1mm x 3,5m) c kch
thc ht nh nn p sut ca ct cao c hi cho bm v h thng sc k. Nu hot
ng lin tc p sut cao nh vy, bm d xy ra hin tng p sut khng n
nh dn n tc dng khng n nh.
Khi s dng ct Symmetry C18 (150mm 4,6mm 5m) hnh dng peak t,
chn peak dong, thi gian lu ngn (khong 6,1 pht). Nh vy, khi phn tch trn
nn mu thc c th cht phn tch b ln peak tp hoc s b dnh peak.
Khi s dng ct IntertSustain C18 (250mm 4,6mm 5m) tn hiu peak
nhn, cn i, rng chn peak nh khong 0,2 pht, thi gian lu 10,3 pht. V
vy khi phn tch trn nn mu thc, cht phn tch c th c tch tt, trnh c
hin tng chng peak, dnh peak vi cc tp cht khc. Do , trong nghin cu
chng ti s dng ct IntertSustain C18 (250mm 4,6mm 5m) tch v nh
lng adenosine ng thi tin hnh cc kho st tip theo.
3.2.2. Kho st thnh phn pha ng
Sau pha tnh th pha ng l yu t th hai quyt nh n hiu qu tch sc
k. Nhn chung, pha ng c th nh hng n chn lc ca h pha, thi gian
lu gi, hiu lc ca ct tch, phn gii, rng ca peak sc k...

-22-

Chng ti tin hnh nghin cu cc pha ng khc nhau gm: Knh A l

H2O, knh B l dung mi ACN hay MeOH, c nh cc iu kin phn tch khc.
Qu trnh kho st cho kt qu nh sau:
Bng 3.1. Kho st thnh phn pha ng
Thnh phn pha ng

Thi gian

Knh A

lu (pht)

Knh B

Kt qu
Peak dong, rng chn peak ln, tn

H2O

ACN

9,5

hiu cht phn tch thp, tn hiu nhiu

ng nn cao hn.
Peak rt nhn, cn i, rng chn

H2O

MeOH

10,3

peak nh, tn hiu cht phn tch cao

hn, tn hiu nhiu ng nn rt thp.

mAU
260nm,4nm (1.00)

/9.540/721987

35
30
25
20
15
10
5
0

-5
0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

min

Hnh 3.5. Sc phn tch s dng pha ng H2O v ACN

/10.341/751169

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100
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50
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5.0

7.5

10.0

12.5

Hnh 3.6. Sc phn tch s dng pha ng H2O v MeOH

min

-23-

T kt qu trn, chng ti nhn thy:

C th s dng c 2 dung mi l MeOH v ACN tch v nh lng
adenosine. Tuy nhin, khi s dng MeOH tn hiu peak rt nhn, cn i, nhy
cao, nhiu ng nn thp hn so vi dung mi ACN. Hn na, gi tr kinh t
mang li khi s dng dung mi MeOH ln hn rt nhiu so vi dung mi l ACN.
V vy, trong nghin cu ny chng ti s dng MeOH lm thnh phn pha ng
phn tch adenosine.
3.2.3. Kho st chng trnh ra gii gradient
Trong thnh phn cu to ca ng trng h tho c cha rt nhiu cc cht
thuc nhm nucleoside c tnh cht tng t nhau, nu ch ra gii cht phn tch
theo chng trnh c nh pha ng, cc cht trong nhm c th c cng thi gian
lu dn n hin tng chng peak hoc dnh peak. Do vy, tng nhy ca tn
hiu peak, tng kh nng tch sc k, trnh hin tng dong peak, chng peak,
dnh peak, chng ti kho st chng trnh gradient tch v nh lng
adenosine trong ng trng h tho v c nh cc thng s khc.
Kt qu thu c khi chy chng trnh gradient nh sau:
Thnh phn pha ng %
Gradient 1

Knh A = H2O

Knh B = MeOH

92

Thi gian lu
(pht)
13,2
/13.204/741704

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15

10

0.0

2.5

5.0

7.5

10.0

12.5

Hnh 3.7. Sc phn tch theo chng trnh gradient 1

15.0

min

-24-

Thnh phn pha ng %

Gradient 2

Knh A = H2O

Knh B = MeOH

(pht)

80

20

9,5

mAU
260nm,4nm (1.00)

/9.540/721987

35

Thi gian lu

30
25
20
15
10
5
0
-5
0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

min

Hnh 3.8. Sc phn tch theo chng trnh gradient 2

Thi gian
(pht)

Gradient 3

Thnh phn pha ng (%)

Knh A = H2O

Knh B = MeOH

0,01

100

95

80

20

11

80

20

12

100

16

100

Thi gian
lu (pht)

15,2

/15.289/356904

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30

20

10

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2.5

5.0

7.5

10.0

12.5

Hnh 3.9. Sc phn tch theo chng trnh gradient 3

15.0 min

-25-

Thi gian (pht)

Thnh phn pha ng (%)

Thi gian lu

Knh A = H2O Knh B = MeOH

(pht)

0,01

100

95

80

20

10

90

10

12

95

15

100

Gradient 4

12,9

/12.996/715976

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260nm,4nm (1.00)

50

25

0
0.0

2.5

5.0

7.5

10.0

12.5

min

Hnh 3.10. Sc phn tch theo chng trnh gradient 4

Gradient 5

Thi gian

Thnh phn pha ng (%)

Thi gian lu

(pht)

Knh A = H2O Knh B = MeOH

(pht)

0,01

100

85

15

70

30

10

55

45

12

85

15

15

100

10,3

-26-

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100
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5.0

7.5

10.0

12.5

min

Hnh 3.11. Sc phn tch theo chng trnh gradient 5

Khi s dng thnh phn pha ng gm H2O v MeOH theo chng trnh
gradient 1 thi gian lu di (13,2 pht) v gim cn 9,5 pht khi tng t l MeOH
ln 20% chng trnh gradient 2. Tuy nhin, c 2 chng trnh peak u dong,
rng chn peak ln. V vy, khi phn tch hiu qu tch sc k khng tt.
Nu s dng chng trnh gradient 3 v 4 thi gian lu ca cht phn tch rt
mun (12,9 - 15,2 pht) dn n tn dung mi, ko di thi gian phn tch, nh
hng n hiu qu kinh t. Hn na, tn hiu cht phn tch thp lm gim LOQ
ca phng php. Khi tng t l MeOH (theo chng trnh gradient 5), thi gian
lu gim v nhy ca cht phn tch ln hn, peak nhn, cn i hn. Do ,
chng ti s dng chng trnh 5 tch v nh lng cht phn tch.
3.2.4. iu kin ti u ca qu trnh sc k
Sau khi kho st, chng ti la chn c iu kin ti u cho qu trnh
sc k nh sau :
-

Ct IntertSustain C18 (250mm x 4,6mm x 5m)

Detector PDA bc sng 260 nm

Pha ng gm 2 knh: knh A l H2O v knh B l MeOH theo chng trnh

ra gii gradient 5

Tc dng: 1mL/pht

Nhit ct: 40oC

Th tch bm mu: 20L

-27-

/10.361/752306

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100
75
50
25
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5.0

7.5

10.0

12.5

min

Hnh 3.12. Sc phn tch vi iu kin ti u

3.3. Kho st quy trnh x l mu
Quy trnh x l mu d kin theo hnh 2.1. Theo quy trnh d kin ny chng
ti tin hnh kho st mt s yu t nh hng ti qu trnh chit nh: dung mi
chit mu, dung mi loi bo, nhit chit, thi gian chit, s ln chit mu trn
nn mu TPCN c cha THT.
3.3.1. Kho st dung mi chit
Chng ti s dng dung mi chit l hn hp H2O - MeOH v hn hp H2O
- EtOH vi cc t l khc nhau la chn c dung mi chit ph hp nht.
Trn cng mt nn mu TPCN c cha THT chng ti tin hnh chit theo
lc d kin. S dng dung mi trn vi cc t l khc nhau. Vi mi loi dung
mi phn tch lp li 2 ln, tnh kt qu trung bnh. Kt qu phn tch c ch ra
trong hnh sau:

Hnh 3.13. Kho st dung mi chit

-28-

Khi s dng dung mi l EtOH hoc t l gia H2O : EtOH = 9 : 1 hm

lng adenosine thu c trong nn mu phn tch rt thp (khong 12,9 28,5mg/100g). Trong khi , vi dung mi chit MeOH, hiu lc chit tt hn so
vi EtOH, hm lng adenosine thu c trong mu l 34,7mg/100g. Tuy nhin,
khi tip tc gim t l MeOH v tng t l H2O hm lng adenosine tng ln ng
k v t hiu lc chit cao khi s t l H2O : MeOH = 9 : 1 (65,5mg/100g) v thay
i khng ng k khi dung mi l 100% H2O (65,6mg/100g). Tuy nhin, trong nn
mu thc phm chc nng rt a dng v phc tp, khi s dng dung mi H2O c
rt nhiu cht c kh nng ha tan khin cho dung dch chit thu c ln tp nhiu,
khi phn tch gy bn ct v h thng sc k. Trong khi , vi t l H2O : MeOH =
9:1 s hn ch c s ha tan ca mt s cht lm cho dch chit mu sch, m
bo an ton cho ct v h thng khi phn tch. Do , chng ti s dng hn hp
H2O : MeOH theo t l 9 : 1 chit adenosine ra khi nn mu phn tch.
3.3.2. Kho st dung mi loi bo
i vi cc sn phm TPCN, nu thnh phn bo khng c loi b s nh
hng n kh nng ha tan ca cht phn tch trong dung mi chit. Hn na, dch
chit mu bn khi phn tch trn h thng sc k d gy bn ct v h thng, sau
mt thi gian phn tch, p sut ct tng, hnh dng peak xu, tui th ct ngn.
ng thi, tp nhiu cng lm gim tnh chn lc i vi cc cht phn tch. V
vy, chng ti kho st mt s dung mi hu c khng phn cc gm: n-hexan,
diethyl ether, ether du ha loi bo trong cc TPCN.
Trn nn mu TPCN c cha THT dng vin nang v dng dch lng c
cha nhiu bo, tin hnh so snh gia nn mu khng loi bo v nn mu c loi
bo bng cc dung mi trn. S dng 20mL dung mi mi loi, tin hnh lc xoy,
rung siu m, ly tm, thnh phn bo s c ha tan trong cc dung mi hu c v
tch lp trn, dng pipet pasteur ht phn dch trn, sau tin hnh chit theo
quy trnh d kin, dch chit sau c phn tch trn h thng sc k. Kt qu
trong dch chit ca mu khng loi bo rt c, khi phn tch trn h thng HPLC,
peak sc k c cha nhiu tp cht nh hng n ct sc k v h thng. Hn na,

-29-

n lm gim kh nng ha tan ca cht phn tch trong dung mi chit. Do , lm

gim hiu sut chit.
Khi s dng dung mi diethyl ether hay ether du ha, s phn lp gia 2
pha rt kh quan st, kh nng loi bo km, hm lng adenosine thu c thp.
Nu s dng dung mi loi bo l n-hexan dch chit mu thu c rt trong, kh
nng tch gia 2 pha r rng nn vic loi bo thun tin hn. Mt khc, hm lng
adenosine thu c khi s dng dung mi ny l ln nht. V vy, chng ti la
chn n-hexan loi bo trong mu.
3.3.3. Kho st nhit chit
Trn cng mt nn mu TPCN c cha THT, chng ti tin hnh chit
cc nhit khc nhau: nhit thng, 45oC, 60oC, 75oC, 100oC kt hp vi rung
siu m. mi mc nhit tin hnh lm lp 2 ln, ly kt qu trung bnh.
Kt qu kho st thu c nh sau:

Hnh 3.14. Kho st nhit chit

Nh vy, hm lng adenosine thu c khi chit tng dn theo nhit , t
hm lng cao nht nhit 100oC l 60,8mg/100g. Do khi nhit tng, ha
tan ca cht phn tch trong dung mi chit mu tng; ng thi thc y qu trnh
chuyn ha t AMP thnh adenosine. V vy, hm lng adenosine thu c ln.
Do , trong nghin cu ny chng ti s dng nhit 100oC chit cht phn
tch ra khi nn mu.

-30-

3.3.4. Kho st thi gian chit

Trn nn mu TPCN, chng ti tin hnh chit mu theo quy trnh d kin
vi thi gian thay i: 15 pht, 30 pht, 60 pht, 90 pht v 120 pht, s dng cc
iu kin c ti u, kt qu thu c nh sau:

Hnh 3.15. Kho st thi gian chit

Hm lng adenosine thu c trong mu tng khi thi gian chit mu tng
t 5 pht n 30 pht. Tuy nhin, nu tip tc tng thi gian chit ln 60 pht, 90
pht, 120 pht th hm lng adenosine gim dn do s chuyn ha ca adenosine
sang adenin hoc inosin trong qu trnh trao i cht. Do , chng ti la chn
thi gian 30 pht chit adenosine ra khi nn mu.
3.3.5. Kho st s ln chit
Trn cng mu TPCN c cha THT chng ti tin hnh chit lp 4 ln
kho st kh nng chit. Ti mi ln chit chng ti lm lp 2 ln song song, ly kt
qu trung bnh. Kt qu thu c nh sau
Bng 3.2. Kho st s ln chit
S ln chit (ln)

Hm lng adenosine (mg/100g)

43,7

69,0

69,3

70,1

Kt qu phn tch cho thy: hm lng ca adenosine tng theo s ln

chit. Tuy nhin, ln chit 3 v 4 hm lng tng khng ng k so vi ln 2. V
vy chng ti tin hnh chit lp 2 ln vn m bo hiu sut v tit kim dung mi.

-31-

Sau khi kho st, chng ti quyt nh s dng phng php chit adenosine
t TPCN theo quy trnh c trnh by sau y:
Cn, ong mu (cn 0,1-0,5g mu rn hay ly
5mL mu lng) vo ng ly tm 50mL
20mL n-hexan
Loi bo (nu cn)
30mL H2O:MeOH = 9:1
Lc vortex (10 pht)

Chit lp ln 2

Siu m (1000C, 15 pht)

Ly tm ngui (6000rpm, 5 pht)

Cn
Gn dch chit vo bnh nh mc 50 mL

nh mc

Lc

HPLC
Hnh 3.16. Quy trnh phn tch mu thc

-32-

3.4. nh gi phng php phn tch

3.4.1. Kho st khong tuyn tnh v lp ng chun
Khong nng cht phn tch ph thuc tuyn tnh vo chiu cao hay din
tch peak gi l khong tuyn tnh. Mi quan h tuyn tnh gia nng cht phn
tch vi tn hiu o c gi l ng chun. Ch tiu u tin ca mt ng
chun t yu cu l h s tng quan hi quy R phi t theo yu cu sau:
0,995 R 1 hay 0,99 R2 1
Vi cc iu kin chy HPLC chn, chng ti tin hnh kho st s ph
thuc tuyn tnh ca din tch peak sc k vo nng ca adenosine. T dung dch
chun gc, tin hnh pha long thnh dung dch chun trung gian v dy chun lm
vic cc nng khc nhau. Sau tin hnh bm vo h thng HPLC, thu c
kt qu nh sau:
Bng 3.3 . Kho st khong tuyn tnh
Nng (g/mL)

Din tch (mAU)

Nng (g/mL)

Din tch (mAU)

350

17920193

10,5

744575

263

16570462

5,03

371489

210

15379030

2,10

149170

189

13843488

1,05

74495

105

7135000

0,500

37186

50,3

3616964

0,210

15565

21,0

1493088

0,105

7771

20000000
18000000
16000000
14000000
12000000
10000000
8000000
6000000
4000000
2000000
0
0

100

200

300

Hnh 3.17. Kho st khong tuyn tnh

400

-33-

T th cho thy, khi tng dn nng th din tch peak cng tng, th
tuyn tnh trong khong t 0,105g/mL n 210g/mL. Nh vy, khong tuyn tnh
ca adenosine t 0,105 - 210g/mL.
Tin hnh xy dng ng chun: Trong khong tuyn tnh va xc nh,
thit lp phng trnh tng quan gia nng v din tch peak theo phng trnh
hi quy tuyn tnh, thu c phng trnh ng chun:
Y = 72871X - 47211 vi R2 = 0,9992

Hnh 3.18. ng chun ca adenosine

3.4.2. Gii hn pht hin (LOD) v gii hn nh lng (LOQ) ca phng php
Gii hn pht hin (LOD): l nng thp nht ca cht phn tch c th xc
nh c nhng khng nht thit phi nh lng c trong iu kin th nghim
c th. Trong sc k, LOD c th c xc nh l nng ca cht phn tch m
ti t l gia tn hiu ca cht phn tch v tn hiu nn bng 3 (S/N =3).
Gii hn nh lng (LOQ): l nng ti thiu ca mt cht c trong mu
th m ti c th nh lng c bng phng php phn tch v cho kt qu c
chnh xc mong mun. LOQ c th c xc nh l nng ca cht phn tch
m ti t l gia tn hiu ca cht phn tch v tn hiu nn bng 10 (S/N =10).
Do , c th tnh LOQ t LOD theo cng thc: LOQ

10
LOD
3

-34-

Cch tin hnh: S dng nn mu thc (TPCN c cha THT dng vin
nang, dng dung dch) tin hnh pha long v bm vo h thng sc k cho ti khi
tn hiu peak sc k gp 3 ln tn hiu nhiu ng nn. T xc nh c LOD
v LOQ nh sau:
Bng 3.4. LOD v LOQ ca adenosine
Nn mu

LOD (g/kg, g/lt)

LOQ (g/kg, g/lt)

Dng vin nang

20

66,67

Dng dung dch

14

46,67

/9.894/39

1.0

0.5

/10.383/121

mAU
260nm4nm (1.00)

0.0

-0.5

0.0

2.5

5.0

7.5

10.0

12.5

15.0

Hnh 3.19. Sc nn mu TPCN dng vin nang ti LOD

Hnh 3.20. Sc nn mu TPCN dng lng ti LOD

min

-35-

3.4.3. nh gi c hiu ca phng php

Tnh c hiu: l kh nng pht hin c cht phn tch khi c mt cc tp
cht khc nh tin cht, cc cht chuyn ha, cc cht tng t tp cht C th,
trong php phn tch nh lng, l kh nng xc nh chnh xc cht phn tch
trong mu khi b nh hng ca tt c cc yu t khc.
Khi s dng detector PDA, tnh c hiu ca phng php c c trng
qua tinh khit ca peak (peak purity). Trn sc phn tch chun v thc chng
ti tin hnh xc nh c hiu ca phng php thu c kt qu nh sau:

Hnh 3.21. c hiu ca chun adenosine

Hnh 3.22. c hiu ca phng php trn nn mu dng nang cng

-36-

Hnh 3.23. c hiu ca phng php trn nn mu dng nang mm

Hnh 3.24. c hiu ca phng php trn nn mu dng lng

Kt qu cho thy, tinh khit ca peak trong cc trng hp u ln hn
0,999, chng t phng php c c hiu tt.
3.4.4. nh gi lp li ca h thng
lp li ( chm) th hin s gn nhau ca cc kt qu o so vi gi tr
trung bnh, l mc thng nht ca cc kt qu th ring bit khi quy trnh phn
tch c p dng lp li trn cng mt mu. lp li c th hin bng lch
chun tng i RSD%. S dng dung dch chun 10,5g/mL tin hnh tim lp 10
ln vo h thng HPLC, tnh lp li ca thi gian lu v din tch peak.
Bng 3.5. Kho st lp li ca h thng
Ln

Thi gian lu (pht) Din tch peak (mAU)

10,321

746099

10,603

752840

10,361

752306

10,341

751169

-37-

Ln

Thi gian lu (pht) Din tch peak (mAU)

10,335

753232

10,324

753154

10,322

752909

10,332

752767

10,328

753088

10

10,332

753030

Trung bnh

10,360

752059

RSD %

0,83

0,29

Kt qu cho thy lp li ca thit b rt tt, lch chun tng i ca

thi gian lu l 0,83% v din tch peak l 0,29% - u nm trong gii hn cho
php ca AOAC. Nh vy, khi phn tch adenosine h thng cho cc kt qu rt n
nh.
3.4.5. lp li v thu hi ca phng php
y l hai yu t quan trng nh gi hiu qu ca phng php phn tch.
Mt phng php phn tch tt ngoi vic c sai s nh cn yu cu c lp li
cao. Theo l thuyt thng k, cc i lng c trng cho lp li l lch
chun SD v lch chun tng i RSD%.
thu hi c tin hnh xc nh da trn k thut thm chun. thu hi
R% c tnh nh sau:

R%
Trong :

(C m c C m )
100 %
Cc

Cm+c: Nng ca mu thm chun

Cm: Nng ca mu
Cc: Nng ca chun thm vo mu

Cch tin hnh: Trn nn mu thc chng ti tin hnh phn tch lp li 6 ln
v thm chun 3 mc nng khc nhau (mc thp, trung bnh v mc cao), sau
tin hnh phn tch lp 3 ln. Kt qu phn tch c ch ra trong cc bng sau:

-38-

Bng 3.6. lp li v thu hi trn nn mu dng vin nang mm

Mu thc

m (g)

0,4001
0,4102
0,4608
0,4045
0,4103
0,4505
0,3828
0,3561
0,4826
0,4093
0,3983
0,3526
0,3829
0,3808
0,4282

2
3
4
5
6
1a
1b
1c
2a
2b
2c
3a
3b
3c

mc (mg)

0,0525

0,105

0,210

mm + c (mg/g)

0,3991
0,4172
0,4082
0,3955
0,4222
0,4174
0,5359
0,5268
0,5033
0,6499
0,6537
0,6729
0,9572
0,9585
0,9068

TB

SD

RSD%

R%

0,4099 0,0108

2,65

0,5220 0,0168

3,22

96,99

0,6588 0,0123

1,87

95,30

0,9408 0,0295

3,13

104,80

Bng 3.7. lp li v thu hi trn nn mu dng vin nang cng

mc

Mu thc

m (g)

0,3716

0,3048

0,3526

0,3003

0,4278

0,3423

0,3056

0,3536

0,3135

0,4102

0,2984

1a

0,3762

0,4365

1b

0,3743 0,0525

0,4274

1c

0,3895

0,4662

(mg)

mm + c (mg/g)

0,2898

TB

SD

RSD%

0,3021

0,0080

2,64

0,4434

0,0203

4,58

R%

100,13

-39-

mc

Mu thc

m (g)

2a

0,3332

2b

0,3789

2c

0,3283

0,6003

3a

0,3913

0,8024

3b

0,3522

3c

0,3396

(mg)

mm + c (mg/g)

TB

SD

RSD%

R%

0,5861

0,0228

3,89

96,22

0,8516

0,0443

5,20

96,24

0,5981
0,105

0,210

0,5598

0,8640
0,8884

Bng 3.8. lp li v thu hi trn nn mu dng lng

Mu thc V (mL)

mc
(mg)

mm + c (mg)

0,0796

0,0801

0,0806

0,0803

1a

0,1094

1b

1c

0,1034

2a

0,1331

2b

2c

0,1313

3a

0,1969

3b

3c

0,0746
0,0750

0,0315

0,063

0,126

0,1037

0,1342

0,1997
0,2042

TB

SD

RSD%

R%

0,0784

0,0028

3,56

0,1055

0,0034

3,19

85,95

0,1329

0,0015

1,11

86,45

0,2003

0,0037

1,86

96,72

-40-

Nh vy, lch chun tng i ca adenosine trong cc nn mu thc

phm chc nng nm trong khong 1,11% - 5,20% v thu hi ca phng php
khong 85,95% - 104,80%. Nh vy, kt qu t yu cu ca AOAC, chng t
phng php c lp li v thu hi ph hp.
3.5. nh gi cht lng ca TPCN c cha THT
Chng ti tin hnh phn tch hm lng adenosine ca mt s sn phm
TPCN c cha THT. T , nh gi cht lng ca mt s sn phm TPCN c
cha THT lu thng trn th trng H Ni.
Tin hnh thu thp ngu nhin mt s sn phm chc nng ng trng h
tho hin ang lu thng trn th trng H Ni. Sau tin hnh phn tch hm
lng adenosine theo quy trnh kho st. So snh kt qu phn tch c ti
phng th nghim vi kt qu cng b trn nhn ca sn phm. T nh gi cht
lng ca cc sn phm ny. Kt qu phn tch c ch ra nh sau:

Hnh 3.25. nh gi cht lng mu TPCN c cha THT

Thc nghim cho thy c 57,1% mu c hm lng adenosine t hm
lng ghi nhn, s sn phm khng t l 28,5%, s sn phm cn li khng c
cng b (14,3%).
Mt s sn phm ny ch cng b hm lng cordyceps b sung vo m
khng cng b c th hm lng adenosine, mc d trong cc loi cordyceps khc
nhau hm lng cc nucleosides khc nhau. C th, trong cordyceps sinensis hm

-41-

lng adenosine cao v hm lng cordycepin thp. Ngc li vi cordyceps

militaris hm lng cordycepin l ch yu. V vy, khuyn co cc nh sn xut
nn cng b c th ngun nguyn liu b sung l cordyceps sinensis hay cordyceps
militaris vo sn phm c th nh gi cht lng thc ca sn phm.
3.6. Bn lun
3.6.1. i tng nghin cu
ng trng h tho l mt dc liu qu, c gi tr cao, do vic tiu
chun ha cht lng ca chng l rt quan trng. Dc in Vit Nam cha cp
n dc liu ny v cc ch phm ca n, v vy gp phn qun l cht lng
cho cc sn phm c cha ng trng h tho chng ti la chn ti ny
nghin cu.
Tuy nhin, do iu kin v thi gian nn chng ti mi ch thc hin c
vi trn 20 mu thc t v ly mu ngu nhin.
3.6.2. Xy dng quy trnh phn tch
Adenosine l hp cht c kh nng hp th ti bc sng trong vng t
ngoi nn c th s dng detector UV phn tch. Tuy nhin, detector PDA c u
im l cho bit hnh dng ph ca cht phn tch. Khi kt hp gia thi gian lu v
hnh dng ph hp th c th khng nh chnh xc cht cn phn tch. Do , trong
nghin cu ny chng ti quyt nh s dng detector PDA.
Kt qu ca ti ny chng ti tm c cc iu kin ti u cho qu
trnh phn tch trn h thng HPLC kt ni detector PDA vi h dung mi thng
dng, r tin, ct tch thng dng.
3.6.3. Kho st quy trnh x l mu
Chng ti kho st v tm c cc iu kin chit adenosin t mu thc
phm chc nng cho hiu sut chit cao v lp tt. Thi gian chit tng i
ngn, tn t dung mi.
i vi cc mu c cha nhiu bo nh dng vin nang, ch phm cn c
loi bo trc khi tin hnh x l nh cc mu khc.

-42-

3.6.4. Thm nh phng php

Sau khi xy dng quy trnh phn tch, chng ti tin hnh thm nh phng
php theo AOAC. Cc tiu ch thm nh u t theo yu cu ca AOAC, khong
tuyn tnh ca phng php rng, cho php nh lng adenosin trong cc i
tng a dng, hn ch sai s khi phi lm giu mu hoc pha long mu.
3.6.5. Kt qu phn tch mu thc
Do cha c tiu chun cho cc sn phm ca ng trng h tho nn trong
nghin cu ny chng ti tm thi chp nhn ch phm t yu cu khi hm lng
nm trong khong 90 110% so vi hm lng ghi nhn. Vi iu kin , c
57,1% ch phm t yu cu v hm lng.
Nh vy, mt lng khng nh sn phm khng t yu cy hoc khng
cng b hm lng, v vy chng ti cho rng cn phi kim sot cht ch cc sn
phm thc phm chc nng hn na.

-43-

KT LUN V KIN NGH

Trn c s cc kt qu thc nghim nghin cu xc nh hm lng
adenosine trong TPCN c cha THT bng phng php sc k lng hiu nng cao
kt ni detector PDA, chng ti thu c cc kt qu sau:
1. Ti u ha c cc iu kin chy sc k lng hiu nng cao cho xc nh
adenosine trong TPCN bao gm:
- Kho st v tm c cc iu kin ti u cho phn tch sc k: ct sc k
Intersustain C18 (250mm 4,6mm 5m); pha ng gm 2 knh: knh A l H2O,
knh B l MeOH; chng trnh ra gii gradient, detector PDA bc sng 260nm.
- Xy dng c quy trnh chit mu: Mu c loi bo bng n-hexan (nu
mu cha bo). Sau , chit trong hn hp H2O : MeOH t l 9 : 1, s dng my
rung siu m c ci t nhit 100oC, chit lp 2 ln, vi tng thi gian 30 pht.
2. Xy dng c ng chun t 0,105 - 210g/mL, phng php c gii
hn pht hin 14 - 20g/kg hoc g/L v gii hn nh lng tng ng t 46,67
66,67g/kg hoc g/L ty tng nn mu, lch chun tng i t 1,11 5,20%
v thu hi 85,95 104,80%.
3. phn tch trn 20 mu TPCN c cha THT ang lu thng trn th
trng H Ni. Kt qu phn tch cho thy c 57,1% mu c hm lng adenosine
t so vi cng b trn nhn. Cc sn phm cn li khng t hoc khng cng b
c th hm lng cht chnh ny.
T cc kt qu thu c, chng ti nhn thy phng php phn tch n
gin, c nhy t yu cu, phn tch nhanh v chnh xc, c th p dng kim
nghim rng ri ti cc phng th nghim c thit b HPLC vi tin cy cao.
Trong tng lai, chng ti s tip tc m rng phng php phn tch ng
thi nhiu cht khc thuc nhm nucleosides. V tip tc m rng i tng phn
tch, khng ch trong phm vi TPCN m trn cc i tng sn phm khc.

TI LIU THAM KHO

Ting Vit
1. B Y t (2012), Dc l hc tp 2, Nh xut bn Y hc, H ni.
2. Trn T Hiu, T Vng Nghi, Nguyn Vn Ri, Nguyn Xun Trung (2007),
Ha hc phn tch phn 2, Nh xut bn Khoa hc v k thut, H ni.
3. Cao Minh Quang (2006), Sc k lng hiu nng cao, Nh xut bn Y hc,
Thnh ph H Ch Minh.
4. 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.
5. Vin Kim nghim An ton V sinh thc phm quc gia (2010), Thm nh
phng php trong phn tch ha hc v vi sinh, Nh xut bn Khoa hc v k
thut, H ni.
Ting Anh
6. Feng-Qing Yang, Liya Ge, Jean Wan Hong Yong, Swee Ngin Tan, ShaoPing Li (2009),"Determination of nucleosides and nucleobases in different species
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Cheng (2003), "Simultaneous separation and determination of active components
in Cordyceps sinensis and Cordyceps militarris by LC/ESI-MS", Journal of
Pharmaceutical and Biomedical Analysis, 33, 1155-1162.
10. Lei Huang, Qizahang Li, Yiyuan Chen, Xuefei Wang and Xuanwei Zhou
(2009), "Determination and analysis of cordycepin and adenosine in the products
of Cordyceps spp.", African Journal of Microbiology Research, Vol. 3, 957-961.

11. Li Chen, Yan Aiguo, Cai Chunyan, Liu Zhiping (2012), "Fast
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(2001), "Determination of the active components in artificial cordyceps by
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and applications", European Journal of

Pharmacology, 533, 77-88.

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Cordyceps sinensis of Nepal by new validated HPLC method, Journal of
Pharmacognosy and Phytochemistry, Volume 2, Issue 4.
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115(2), 715-719.

PH LC
PH LC A: MT S HNH NH V SC THU C
Ph lc A1: Mt s loi THT

Hnh A1.1. Hnh nh ca Cordyceps capitata

Hnh A1.2. Hnh nh ca Cordyceps ditmarri

Hnh A1.3. Hnh nh ca Cordyceps gracilis v Cordyceps sphecophata

Ph lc A2: Sc kho st khong tuyn tnh v xy dng ng chun

Adenosine/10.316/16570462

mAU
2500 260nm4nm (1.00)
2000
1500
1000
500
0
0.0

2.5

5.0

7.5

10.0

12.5

min

12.5

min

Hnh A2.1. Sc chun 263ppm

Adenosine/10.311/15379030

mAU
260nm4nm (1.00)
2000
1500
1000
500
0
0.0

2.5

5.0

7.5

10.0

Hnh A2.2. Sc chun 210ppm

Adenosine/10.331/3616964

mAU
260nm4nm (1.00)
500

250

0
0.0

2.5

5.0

7.5

10.0

Hnh A2.3. Sc chun 50,30ppm

12.5

min

Adenosine/10.301/1493088

mAU
260nm4nm (1.00)
200
150
100
50
0
0.0

2.5

5.0

7.5

10.0

12.5

min

Hnh A2.4. Sc chun 21,00ppm

Adenosine/10.321/744575

mAU
125 260nm4nm (1.00)
100
75
50
25
0
0.0

2.5

5.0

7.5

10.0

12.5

min

Hnh A2.5. Sc chun 10,50ppm

Adenosine/10.309/371489

mAU
260nm4nm (1.00)
50

25

0.0

2.5

5.0

7.5

10.0

Hnh A2.6. Sc chun 5,03ppm

12.5

min

Adenosine/10.310/149170

mAU
260nm4nm (1.00)
20
15
10
5
0
-5
0.0

2.5

5.0

7.5

10.0

12.5

min

Hnh A2.7. Sc chun 2,10ppm

Adenosine/10.311/74495

mAU
12.5 260nm4nm (1.00)
10.0
7.5
5.0
2.5
0.0
-2.5
0.0

2.5

5.0

7.5

10.0

12.5

min

Hnh A2.8. Sc chun 1,05ppm

Adenosine/10.307/37186

mAU
260nm4nm (1.00)
5.0

2.5

0.0

0.0

2.5

5.0

7.5

10.0

Hnh A2.9. Sc chun 0,50ppm

12.5

min

Adenosine/10.302/15565

mAU
2 260nm4nm (1.00)

-1

0.0

2.5

5.0

7.5

10.0

12.5

min

Hnh A2.10. Sc chun 0,21ppm

Adenosine/10.318/7771

mAU
260nm4nm (1.00)
0.5

0.0

-0.5

-1.0

0.0

2.5

5.0

7.5

10.0

Hnh A2.11. Sc chun 0,105ppm

12.5

min

Ph lc A3: Sc phn tch mt s mu thc

Adenosine/10.177/345876

mAU
400 260nm4nm (1.00)

300

200

100

0.0

2.5

5.0

7.5

10.0

12.5

15.0

min

15.0

min

Hnh A3.1. Sc mu 08131791nk

Adenosine/10.175/77607

mAU
300 260nm4nm (1.00)
250
200
150
100
50
0

0.0

2.5

5.0

7.5

10.0

12.5

Hnh A3.2. Sc mu 11131425dv

Adenosine/10.171/368538

mAU
260nm4nm (1.00)
400
300
200
100
0

0.0

2.5

5.0

7.5

10.0

Hnh A3.3. Sc mu 2116nk

12.5

15.0

min

Adenosine/10.179/84352

mAU
260nm4nm (1.00)
75

50

25

0.0

2.5

5.0

7.5

10.0

12.5

15.0

min

Hnh A3.4. Sc mu 83-2dv

mAU
260nm4nm (1.00)

Adenosine/10.208/203402

75

50

25

0.0

2.5

5.0

7.5

10.0

Hnh A3.5. Sc mu 776nk

12.5

15.0

min

PH LC B: MT S TIU CHUN V CCH NH GI

Ph lc B1: Tiu chun xc nh lp li
i lng o

ngha

Gi tr trung bnh

x l gi tr trung bnh s hc ca tp

hp cc gi tr xi, dng c lng xu

xi

i 1

hng ca gi tr trung tm (gi tr

thc).
lch chun SD
SD

SD ni ln mc dao ng ca cc kt
qu o xi xung quanh gi tr trung bnh,

( xi xtb ) 2
n 1

th hin s sai lch phn tn ca mt

dy kt qu th nghim so vi gi tr
trung bnh ca n.

lch chun tng i RSD (%)

RSD %

SD
100 %
x tb

Biu th s bin ng, phn tn ca cc

kt qu th nghim.

nh gi chm:
Nu theo 1 tiu chun hoc 1 quy trnh chnh thng c cng b RSD (%) th
RSD (%) tnh c phi nh hn hoc bng RSD (%) cho php.
Nu theo 1 tiu chun, 1 quy trnh chnh thng khng cng b th ngi ta
thng so snh RSD (%) tnh c so vi RSD (%) cho php nng cht tng
ng theo AOAC. RSD (%) tnh c khng c ln hn tr gi trong bng hm
lng cht tng ng, chm thay i theo nng cht phn tch. Nng cht
cng thp th kt qu cng dao ng nhiu (khng chm) ngha l RSD cng ln.
Bng B1.1. lp li ti a chp nhn ti cc nng khc nhau (theo AOAC)
TT

Hm lng %

T l cht

n v

RSD (%)

100

100%

1,3

10

10-1

10%

1,8

10-2

1%

2,7

TT

Hm lng %

T l cht

n v

RSD (%)

0,1

10-3

0,1%

3,7

0,01

10-4

100ppm

5,3

0,001

10-5

10ppm

7,3

0,0001

10-6

1 ppm

11

0,00001

10-7

100ppb

15

0,000001

10-8

10ppb

21

10

0,0000001

10-9

1ppb

30

Ph lc B2: nh gi thu hi
Thng thng thu hi tnh c phi nm trong khong cho php ca
AOAC nng cht tng ng. Trong trng hp phn tch cc cht hm lng
vt c th tham kho tiu chun ca hi ng chu u.
Bng B2.1. thu hi chp nhn cc nng khc nhau theo AOAC
TT

Hm lng (%)

T l cht

n v

thu hi (%)

100

100%

98-102

10

10-1

10%

98-102

10-2

1%

97-103

0,1

10-3

0,1%

95-105

0,01

10-4

100ppm

90-107

0,001

10-5

10ppm

80-110

0,0001

10-6

1 ppm

80-110

0,00001

10-7

100ppb

80-110

0,000001

10-8

10ppb

60-115

10

0,0000001

10-9

1ppb

40-120

Cn chu u th thu hi cho php theo bng nh sau:

Bng B2.2. Quy nh v thu hi ca hi ng chu u

TT

Hm lng cht

n v

thu hi (%)

1 g/kg

1ppb

50-120

>1 g/kg n <10 g/kg

1-10ppb

70-110

10g/kg

10ppb

80-110

Ph lc B3: Gii hn pht hin (LOD).

LOD c chp nhn ti nng m ti tn hiu ln gp 2-3 ln nhiu
ng nn, thng thng thng ly S/N =3

Hnh B2.1: Xc nh LOD bng cch tnh S/N