TS.

L Th Hng Ho

Cc phng php phn tch ha hc 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)
vng (n nh) ca phng php;
(Robustness/Ruggedness

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....
Trong php phn tch nh tnh: chng minh
dng tnh khi c mt cht phn tch, m tnh
khi khng c mt n, ng thi kt qu phi l
m tnh khi c mt cc cht khc c cu trc gn
ging cht phn tch.
Trong php php phn tch nh lng: xc nh
chnh xc cht phn tch trong mu khi b nh
hng ca tt c cc yu t khc, nhm hng
n kt qu chnh xc.
Tnh c hiu thng lin quan n vic xc nh
ch mt cht phn tch.

L khi nim rng hn tnh c hiu, lin quan

n vic phn tch mt s hoc nhiu cht chung
mt quy trnh. Nu cht cn xc nh phn bit
r vi cc cht khc th phng php phn tch

c tnh chn lc.

Nh vy, tnh chn lc c th bao trm c tnh

c hiu. Do cc phng php phn tch thng
c nhiu cht cng xut hin nn khi nim tnh
chn lc thng mang tnh khi qut hn.

 Cch xc nh

Phn tch cc mu trng, lp li ti thiu 6 ln i vi tng loi

nn mu. Mu trng phi khng c cho tn hiu phn tch. Nu

mu trng c hn 10% dng tnh hoc xut hin tn hiu th cn
phi thay i phng php loi tr cc nh hng.

Trong trng hp nhng ch tiu phn tch khng th c mu

trng (sample blank) xc nh tnh chn lc/c hiu, c th
thc hin cc th nghim trn cc mu trng thuc th (reagent
blank), tc l thc hin phn tch cc bc tng t nh khi
phn tch mu nhng khng c mu.

 Cch xc nh
Sc k lng s dng detector DAD (chui diod)

Tnh chn lc trong sc k lng c th t c

thng qua vic vic la chn ct ti u, iu kin
sc k ti u, nhit ct v bc sng. Ngoi
vic thay i iu kin sc k, th giai on x l
mu cng phi ti u t c tnh chn lc
cao nht. Trong sc k, cn xc nh rng pc sc
k c phi l mt pc n hay c ln cc tp cht
khc. i vi cc h thng sc k c detector
chui diod (DAD) c th xc nh c tnh chn
lc thng qua xc nh tinh khit (peak purity)
ca pc, hay so snh ph ca pc vi th vin ph
sn c.

Sc k khi ph
Cc thit b sc k kh c gn detector MS, thng so snh ph ca cht phn tch vi
ph chun c sn trong th vin i km hoc ph ca cht chun tng ng.
S dng cc phng php xc nhn (confirmation method) l mt cch rt tt m
bo tnh c hiu ca phng php. Hi ng chu u quy nh cch tnh im IP (im
nhn dng identification point) i vi cc phng php khc nhau khng nh chc
chn s c mt ca mt cht. Cch tnh im IP i vi cc k thut khi ph khc nhau
c quy nh nh sau:
K thut khi ph

S im IP t c vi 1 ion

MS phn gii thp (LR-MS)

1,0

LR-MS (ion m)

1,0

LR-MS (ion con)

1,5

MS phn gii cao (HR-MS)

2,0

HR-MS (ion m)

2,0

HR-MS (ion con)

2,5

K thut khi ph

S im IP t c vi 1 ion

K thut

S ion

S im IP

GCMS (EI hoc CI)

GCMS (EI v CI)

2 (EI) + 2 (CI)

GCMS (EI hoc CI) 2 dn xut

2 (dc A) + 2 (dc B)

LC-MS

GC-MS-MS

1 ion m, 2 ion con

LC-MS-MS

1 ion m, 2 ion con

GC-MS-MS

2 ion m, mi ion m c 1 ion con

LC-MS-MS

2 ion m, mi ion m c 1 ion con

LC-MS-MS-MS

1 ion m, 1 ion con v 2 ion chu

5,5

HRMS

2n

GC-MS v LC-MS

2+2

GC-MS v HRMS

2+1

si
ps ,
y
ct n
si
pt s , e

Max. 2.0e4 cps.

XIC of +MRM (10 pairs): 331.000/245.000 Da ID: G2 from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray)

0.0
0.0

p t s , e sI i

ctnn

5.0e4

6.43
0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

+MRM (10 pairs): 6.537 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray)

5.0
Time, min

5.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5
Max. 5.1e4 cps.

G1

0.0

Ii

ctnn

5.0e4

4.5

0.0

B2

tnn

5.0e4

Ii

ts , e

331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000
Q1/Q3 Masses, Da
+MRM (10 pairs): 6.666 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray)
Max. 5.5e4 cps.

ts

331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000
Q1/Q3 Masses, Da
+MRM (10 pairs): 6.769 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray)
Max. 3.3e4 cps.

B1

0.0

nn

3.3e4

331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000
Q1/Q3 Masses, Da
+MRM (10 pairs): 6.433 min from Sample 17 (Std 5) of DataSET1.wiff (Turbo Spray)
Max. 2.0e4 cps.

G2

0.0

1.0e4

2.0e4

331.000/245.000 331.000/285.000 329.000/243.000 329.000/215.000 315.000/259.000 315.000/287.000 313.000/241.000 313.000/269.000 329.000/273.000 329.000/259.000
Q1/Q3 Masses, Da

t
c

c
t

si

si

p s

p s

tn
c

tn
c

pt s
tnn
c

8.0

8.5

9.0

6.69

2.5

3.0

3.5

4.0

4.5

5.0
5.5
Time, min
XIC of +MRM (8 pairs): 315.000/259.000 Da ID: B2 from Sample 7 (Blank) of DataSET1.wiff (Turbo Spray)

8.09 8.34

6.0

6.5

7.0

7.5

8.0

8.5

9.0

1.5

2.0

2.5

3.0

3.5

4.0

7.49

4.5

5.0
5.5
Time, min
XIC of +MRM (8 pairs): 329.000/243.000 Da ID: G1 from Sample 7 (Blank) of DataSET1.wiff (Turbo Spray)

6.0

6.5

7.0

7.5

7.73

8.64

8.0

8.5

9.0

9.5
Max. 185.0 cps.

ts

7.05 7.16

0.0
0.0

1.5

2.0

2.5

3.0

3.5

4.0

5.0
5.5
Time, min
XIC of +MRM (8 pairs): 331.000/245.000 Da ID: G2 from Sample 7 (Blank) of DataSET1.wiff (Turbo Spray)

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5
Max. 165.0 cps.

7.0

7.5

8.0

8.5

9.0

9.5
Max. 4.1e4 cps.

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5
Max. 4.7e4 cps.

B1
5.92

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
Time, min
XIC of +MRM (8 pairs): 331.000/245.000
G2 Da ID: G2 from Sample 5 (Std Af-5ppb) of DataSET1.wiff (Turbo Spray)

6.0

6.5

7.0

7.5

8.0

8.5

9.0

9.5
Max. 1.3e4 cps.

G2

6.5

nn

7.49 7.81 8.23 8.83 8.91 9.43 9.59

5.52

4.5

6.0

nn

1.0

0.5

4.7e4

0.5

5.03

0.77 0.92 1.51 1.77 2.02 2.35 2.74 3.12 3.42 3.77 3.91 4.32

9.5
Max. 3.2e4 cps.

6.06

B2

5.0
5.5
Time, min
XIC of +MRM (8 pairs): 329.000/243.000 Da ID: G1 from Sample 5 (Std Af-5ppb) of DataSET1.wiff (Turbo Spray)

5.85
0.30
I

5.84

6.93

165

1.0e4
n

100

0.27 0.38
0
0.0
0.5

1.61
1.0

1.5

3.06
2.0

2.5

3.0

3.61 4.05 4.29 4.70

3.5

4.0

4.5

5.36

7.38

5.69 5.83

5.0
5.5
Time, min

6.0

6.5

7.0

7.5

8.20 8.58
8.0

8.5

9.08
9.0

Sc mu lc khng c cht phn tch

9.5

0.0
0.0

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
Time, min
XIC of +MRM (8 pairs): 315.000/259.000 Da ID: B2 from Sample 5 (Std Af-5ppb) of DataSET1.wiff (Turbo Spray)

B1

185
100

9.0

1.0

6.27

5.55 5.82

3.83

0.0
0.0

4.1e4

6.66

0.5

2.492.54

1.62

8.5

tnn

y
tnn
Ii

0
0.0

7.16
5.98

0.49

9.5
Max. 265.0 cps.

B2
200

8.0

, e

2.0

7.5

ts

1.5

7.0

6.11

1.0

5.85

ts , e

0.5

5.36

2.90

6.5

G1

3.2e4

ts

1.94 2.05

6.0

ctnn

6.10

7.35

ctnn
Ii

Max. 140.0 cps.

0.5
1.0
1.5
2.0
2.5
3.0
3.5
4.0
4.5
5.0
5.5
Time, min
XIC of +MRM (8 pairs): 313.000/241.000 Da ID: B1 from Sample 5 (Std Af-5ppb) of DataSET1.wiff (Turbo Spray)

p ts
y

G1

9.5

5.84

sI i

7.5

0.0
0.0

,e

7.0

9.08

Ii

6.5

8.20 8.58

Ii

3.06
3.0

,e

sI i

1.61
1.5
2.0

p ts

6.93 7.38 7.52

3.61 4.05 4.29 4.70 5.36 5.69 5.83

1.0
2.5
3.5
4.0
4.5
5.0
5.5
6.0
Time, min
XIC of +MRM (8 pairs): 313.000/241.000 Da ID: B1 from Sample 7 (Blank) of DataSET1.wiff (Turbo Spray)

0
0.0

Max. 1.3e4 cps.

4.7e4

0.27 0.38
0
0.0
0.5

140
100

XIC of +MRM (8 pairs): 331.000/245.000 Da ID: G2 from Sample 5 (Std Af-5ppb) of DataSET1.wiff (Turbo Spray)

tnn
c

1435
1000

,e

si

si
,e

Max. 165.0 cps.

pt s

XIC of +MRM (8 pairs): 331.000/245.000 Da ID: G2 from Sample 7 (Blank) of DataSET1.wiff (Turbo Spray)

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

4.5

5.0
5.5
Time, min

6.0

6.5

7.0

7.5

8.0

8.5

Sc mu thm hn hp chun nng 5 ppb

9.0

9.5

L khong nng c s ph thuc tuyn tnh gia i lng o c v nng

cht phn tch.
Khong lm vic: l khong nng gia gii hn trn v gii hn di ca cht phn
tch (bao gm c cc gii hn ny), ti c chng minh l c th xc nh c bi
phng php nht nh vi ng, chnh xc v tuyn tnh nh nu.

Tn
hiu

Khong lm vic
Khong tuyn tnh

Nng

Ch tiu chp nhn ca ng chun:

H s hi quy tuyn tnh (R): Ch tiu u tin ca mt ng chun
t yu cu l h s tng quan hi quy (Coefficient of correlation). R
phi t theo yu cu sau:
0,995 R 1
Ct C c
Hay 0,99 R2 1

Cc

100

Trong :
i chch ca tng im chun dng xy dng ng chun
Ct : Nng tnh ngc theo ng chun ca cc im chun
Co: Nng ca cc im chun
Theo quy nh ca nhiu t chc ca M, Canada, chu u, gi tr
khng c vt qu 15% cho tt c cc nng , ring nng
LOQ c th chp nhn gii hn 20%.

1500000
1000000

ng chun AFB1

ng chun AFB2

150000

y = 837.44x - 424.73
100000
R = 0.9997

y = 7278.7x + 18437
R = 1

50000

500000

0
0

50

100

150

200

1200000
3000000

100

150

200

ng chun AFG2

1000000
y = 6818.9x + 3533.9
800000

ng chun AFG1

2000000

50

y = 15578x - 9048.4

600000

R = 1

400000

1000000

R = 0.9999

200000
0

0
0

50

100

150

200

50

100

150

200

L nng m ti gi tr xc nh c ln hn khng
m bo o ca phng php. y l nng thp nht ca
cht phn tch trong mu c th pht hin c nhng cha
th nh lng c (i vi phng php nh lng).
Cch xc nh
LOD ca phng php nh tnh:
Cn xc nh c nng no m ti vic xc nh chc
chn s c mt ca cht phn tch.
Phn tch cc mu trng thm chun cc nng nh khc
nhau, mi nng phn tch lp li 10 ln. Xc nh t l
phn trm s ln pht hin (dng tnh) hoc khng pht hin
(m
tnh)
Protocole
pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Cch tnh LOD ca phng php nh tnh

Nng s ln th

(ppm)
200
100
75
50
25

10
10
10
10
10

T l dng tnh/m tnh

T l %

10/0
10/0
5/5
1/9
0/10

100
100
50
10
0

Vi nng di 100 ppm kt lun dng tnh khng cn chc

chn 100%, gii hn pht hin trong trng hp ny l 100 ppm
Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

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).
Tnh gi tr nng trung bnh xtb, lch chun
SD (S)
N
2
LOD = 3.S
xi x
S

i 1

N 1

nh gi LOD tnh c ta kim tra qua gi

tr R = Xtb/LOD
Nu 4 < R < 10 th nng dung dch em th l
ph hp v gi tr LOD tnh c l ng tin cy.
LOQ = 10/3 * LOD

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Kt qu

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

0,49

0,45

0,42

0,45

0,48

0,42

0,46

0,42

0,49

0,44

0,44

0,39

0,48

0,47

0,48

0,47

0,38

0,39

0,42

0,42

0,47

0,48

0,38

0,47

0,48

0,44

0,39

0,43

0,49

0,42

0,45

0,49

0,48

0,41

0,49

0,41

0,48

0,40

0,42

0,47

xtb(ng/ml)

0,47

0,43

0,44

0,44

SD

0,03

0,03

0,04

0,03

LOD (ng/ml)

0,1

0,1

0,1

0,1

LOQ (ng/ml)

0,3

0,3

0,3

0,3

4,7

4,9

4,0

4,5

RSD(%)

6,4

7,0

9,1

6,8

xi(ng/ml)

ng ch mc gn nhau gia gi tr trung bnh ca kt qu

th nghim v gi tr thc hoc gi tr c chp nhn l ng .
chm ch mc mc dao ng ca cc kt qu th nghim
c lp quanh tr gi trung bnh. Hnh 10 m t s mi quan h

gia chm, ng v chnh xc.

chnh xc (accuracy) = chm (precision) + ng (trueness)

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

 chm c th c phn ra thnh 3 trng hp sau:

lp li (repeatability)
chm trung gian (intermediate precision)
ti lp (reproducibility)
S khc nhau gia lp li, chm trung gian v ti lp

iu kin

lp li

chm trung gian

ti lp

Nn mu

Khc

Khc

Khc

Nng

Khc

Khc

Khc

Thit b

Ging

Khc

Khc

Ngi phn tch

Ging

Khc

Khc

Dng c, ha cht

Ging

Khc

Khc

iu kin mi trng

Ging

Khc

Khc

Phng th nghim

Ging

Ging

Khc

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Cch 1. B tr th nghim.
Tin hnh lm th nghim lp 10 ln (t nht 6 ln) trn cng mt
mu (mi ln bt u t cn hay ong mu). Mu phn tch c th
l mu chun, hoc mu trng c thm chun, tt nht l lm trn
mu th hay mu th thm chun.
Nn tin hnh nng khc nhau (trung bnh, thp, cao) trong
khong lm vic, mi nng lm lp li 10 ln (t nht 6 ln).
Tnh lch chun SD v lch chun tng i RSD hay h s
bin thin CV theo cc cng thc sau:
SD

i x

n 1

RSD% CV%

SD
100
x

Trong : SD: lch chun

n: s ln th nghim
xi: Gi tr tnh c ca ln th nghim th i
x : Gi tr trung bnh ca cc ln th nghim
RSD%: lch chun tng i
Protocole pourla
validation
methode
danalyse en chimie, 2009, Quesbec Canada.
CV%:
H dune
s bin
thin

Cch 2. Tnh ton trn cc kt qu phn tch mu thc lm.

Da trn kt qu phn tch lm trn mu thc trong nhiu tun

t nht l 10 mu, c th l cc nn mu khc nhau, nng khc
nhau nhng phi c kt qu lm lp 2 ln .
Trng hp cc mu c nng , hm lng gn nh nhau. Tnh
lch gia hai kt qu lp ca mi mu di ri tnh lch trung
bnh dtb, sau tnh lch chun s:

di x i1 x i2

d tb

xi

x i1 x i 2
2

x
X

d tb
1,118

RSD%

s
100
X

Nu cc mu c nng xi khc nhau nhiu th thay cho

lch di , tnh lch tng i Di, v lch tng i trung bnh
Dtb v sau tnh lch chun tng i:
Di

di
xi

D tb

RSD %

D tb
100
1,118

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Nng cht cng thp th kt qu cng dao ng

nhiu (khng chm) ngha l RSD cng ln.
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

0.1

10-3

0.1 %

3.7

0.01

10-4

100 ppm

5.3

0.001

10-5

10 ppm

7.3

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

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

ng ca phng php l khi nim ch mc gn

nhau gia gi tr trung bnh ca kt qu th nghim v gi
tr thc hoc gi tr c chp nhn l ng ().
i vi a s mu phn tch, gi tr thc khng th bit
mt cch chnh xc, tuy nhin n c th c mt gi tr quy
chiu c chp nhn l ng (gi chung l gi tr ng).
Ging nh chm, ng l mt khi nim nh tnh.
ng thng c din t bng chch (bias).

X tb
100

Trong :

: chch (bias), %
Xtb: Gi tr trung bnh ca kt qu th nghim
: Gi tr thc hoc gi tr c chp nhn l ng

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Mun xc nh ng cn phi tm c gi
tr ng, c nhiu cch khc nhau xc nh gi
tr ny, bao gm vic so snh kt qu vi kt qu
thc hin bi mt phng php i chiu hoc s
dng mu bit nng (mu kim tra hoc
mu chun c chng nhn) v phng php
xc nh thu hi ( tm li).
USFDA quy nh chch ca cc phng php xc
nh d lng phi khng c ln hn 15% v khng
ln hn 20% ti LOQ.
Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Th t
1
2

3
4

Aflatoxin B1

Aflatoxin B2

Aflatoxin G1

Aflatoxin G2

(ng/ml)

(ng/ml)

(ng/ml)

(ng/ml)

2,9
2,5

2,6
2,8

2,5

2,7

2,8
2,6

2,9

3,1

3,3
2,5

2,2

2,9

3,1

3,1

2,7

2,6

Trung bnh

2,6

2,8

R%

87

93

SD

0,30

0,23

RSD (%)

11

8,1

3,0

3,2
2,4
2,7
3,0
2,7
90
0,24
8,9

2,3
2,9
97
0,34
12

n nh phng php l kh nng cung cp cc kt

qu c chnh xc ( ng v chm) chp nhn
c di nhng iu kin c s thay i v mt s iu
kin thc hin phng php.
Ngi ta phn bit hai khi nim vng (robustness) v
chc chn (ruggdness) nh sau:
Robustness: L mc lp li cc kt qu phn tch khi
c s thay i nh mt s thng s ca phng php
(method parameters). V d i vi phng php sc k
lng c th k ra cc yu t ny bao gm pH pha ng,
tc dng, nhit ct, th tch tim mu, thnh phn
Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.
pha ng, bc sng pht hin

Ruggedness: L mc lp li cc kt qu phn tch khi

c s thay i mt s yu t thuc v iu kin phn tch
(test conditions). Cc iu kin ny c th l ngi phn
tch, thit b phn tch, ct sc k do cc nh cung cp
khc nhau, l ha cht thuc th, thi im thc hin th
nghim... Ni chung, chc chn thng lin quan n
vic tnh chm trung gian hay ti lp ni b phng
th nghim.
Trong ti liu ny chng ti ni n n nh bao gm
c hai yu t vng v chc chn ca phng php.
Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

V d, gia ngi A vi ngi B, gia my A v my B, gia

iu kin A v iu kin B... Cch tin hnh so snh ging
nh trong phn xc nh ng khi so snh hai phng
php nu trn.
Trong trng hp c mu CRM c th b tr cc th nghim
(c s thay i iu kin phn tch hay cc thng s ca
phng php) tnh kt qu mu CRM v so snh vi kt
qu chun nh gi n nh ca phng php.

Protocole pourla validation dune methode danalyse en chimie, 2009, Quesbec Canada.

Php th nghim lp li

Php th nghim ti lp

Php th nghim trn mu lu

Php th nghim trn mu trng

Php th nghim trn mu chun

Php th nghim trn mu thm chun

S dng cc phng php khc nhau

nh gi s ph hp ca h thng

Tham gia cc chng trnh th nghim lin phng

Cc gi tr chung

nh gi

Protocole
pourlabiu
validation
dunesot
methode danalyse en chimie, 2009, Quesbec Canada.
S dng
kim