HNG DN XC NHN GI TR S DNG

PHNG PHP
THE MANUAL OF METHOD VALIDATION

DANH MC CC CH VIT TT

CRM

mu vt liu chun c chng nhn.

Fcritical

gi tr F tra bng ca chun F Fisher.

C spiked

nng mu thm chun.

gi tr t tnh ton ca chun t student.

nng trung bnh.

tcritical

gi tr t tra bng ca chun t student.

mc ngha 5% ng vi khong tin cy

95%.

LID

gii hn pht hin ca thit b.

LoD

gii hn pht hin.

(%)B

chch (tng i).

LoMD

gii hn pht hin ca phng php.

(%)CV

h s bin thin (tng i).

LoID

gii hn pht hin ca thit b.

%R

hiu sut thu hi.

LoQ

gii hn nh lng.

(%)RSD

lch chun (tng i).

LoMQ

gii hn nh lng ca phng php.

(%)RSM

gi tr hiu dng (tng i).

LoIQ

gii hn nh lng ca thit b.

%T

ng.

LoL

gii hn khong tuyn tnh.

(%)u

khng m bo o (tng i).

MRL

gii hn ti a cho php ca d lng (%) uc

mt cht.

khng m bo o tng hp (tng

MRPL

yu cu ca th trng v gii hn ti
ue
thiu ca d lng mt cht cm m (%)
phng php phi xc nh c.

khng m bo o m rng (tng

PL

yu cu ca th trng v gii hn ti (%) uB

a ca d lng mt cht.

khng m bo o (tng i) ca

S/N

t s tn hiu trn nhiu nn.

phng sai ca mt tp d liu.

SD

lch chun ca mt tp d liu.

gi tr F tnh ton ca chun F Fisher.

(%)

uC

i).

i).

chch.
ref

khng m bo o (tng i) ca
mu tham chiu.

I. i tng v phm vi p dng

Xc nhn gi tr s dng phng php l s khng nh bng vic kim tra v cung cp bng chng
khch quan chng minh rng phng php p ng c nhng yu cu t ra khi p dng ti phng
kim nghim.
i tng phng php phng kim nghim s dng thng c chia lm hai nhm:

Phng php tiu chun l phng php c cng b bi t chc tiu chun quc gia, khu vc,
quc t (TCVN, Codex, ISO,), hoc ban hnh bi cc t chc k thut chuyn nghin cu v
xc nh gi tr s dng phng php phn tch (AOAC, AFNOR,) ng thi cng b km theo
y cc thng s v xc nhn gi tr s dng ca phng php.

Cc phng php khng tiu chun hay phng php ni b l cc phng php do phng th
nghim t xy dng, phng php theo hng dn ca nh sn xut thit b, phng php theo
cc tp ch, ti liu chuyn ngnh...

Theo yu cu ca ISO 17025, phng kim nghim phi tin hnh xc nhn hoc xc nhn li gi tr
s dng ca phng php trc khi a vo s dng trong cc trng hp:

Phng php p dng khng phi l phng php tiu chun.

Phng php do phng kim nghim nghin cu v xy dng.

Thay i quy trnh, iu kin p dng phng php c s dng trc y.

Theo thng t 54/2011/TT-BNNPTNT ca B Nng nghip v Pht trin nng thn, trc khi mt
phng php c p dng thng quy cn phi tin hnh xc nhn gi tr s dng. i vi vic p dng
phng php tiu chun hay khng tiu chun, cc thng s cn nh gi c quy nh c th theo nh
yu cu ca thng t.
II. Gii thch thut ng
2.1. Khong tuyn tnh ca ng chun/ Working rank of calibration
L khong nng ca ng chun c xc nh l ph hp vi tuyn tnh, chm v
chnh xc.
2.2. tuyn tnh ca ng chun/ Linearity of calibration
tuyn tnh ca ng chun ca mt phng php phn tch l kh nng th hin c s tng
quan tuyn tnh gia cng tn hiu o c (tn hiu t thit b) v tham s phn tch (nng ).
2.3. Hiu ng nn/ Matrix effects
L hin tng tn hiu phn hi t thit b gim hoc gia tng cng khi c hoc khng c s
hin din ca nn mu.
2.4. chn lc/ Selectivity
L kh nng ca phng php phn bit c gia cht cn phn tch v nhng cht khc nh ng
phn, cht chuyn ha, sn phm phn hy, sn phm ni sinh, thnh phn trong nn mu...
2.5. nhy/ Sensitivity
L mc thay i ca tn hiu o tng ng vi s thay i ca nng cht phn tch.

2.6. chnh xc/ accuracy

L mc sai lch gia kt qu th nghim v gi tr tham chiu c chng nhn. chnh xc
bao hm c ng v chm.
2.7. ng/ Trueness
L mc sai lch gia gi tr trung bnh ca lot th nghim ging nhau v gi tr tham chiu
c chng nhn.
2.8. chch/ Bias
L s khc bit gia gi tr trung bnh ca lot th nghim ging nhau v gi tr tham chiu c
chng nhn.
2.9. chm/ Precision
L mc tp trung gia cc kt qu ca lot th nghim ging nhau c thc hin trong cng
iu kin xc nh hoc cc iu kin xc nh khc nhau. chm c biu th thng qua lp li v
ti lp.
2.10. lp li/ Repeatability
L chm c xc nh di cng iu kin th nghim (cng phng th nghim, ngi thc
hin, thit b, thi gian).
2.11. ti lp/ Reproducibility
L chm c thc hin di cc iu kin th nghim khc nhau (khc phng th nghim,
ngi thc hin, thit b, thi gian).
2.12. thu hi/ Recovery
L phn trm nng thc ca cht phn tch thu hi c trong qu trnh phn tch. Thng s ny
c xc nh trong qu trnh ph duyt nu nh phng th nghim khng c vt liu tham chiu c
chng nhn.
2.13. Gii hn pht hin/ Limit of Detection
Gii hn pht hin (LoD) c chia lm hai thnh phn:

Gii hn pht hin ca phng php/ Limit of Method Detection (LoMD): l nng cht phn
tch ti thiu c trong mu m mt phng php c kh nng pht hin hoc phn bit.

Gii hn pht hin ca thit b/ Limit of Instrumental Detection (LoID): l nng cht phn tch
ti thiu c trong mu m mt phng php c kh nng pht hin hoc phn bit bng mt thit
b c th.

2.14. Gii hn nh lng/ Limit of Quantitation

Gii hn nh lng (LoQ) c chia lm hai thnh phn:

Gii hn nh lng ca phng php/ Limit of Method Quantitation (LoMQ): l nng cht
phn tch ti thiu c trong mu m mt phng php c kh nng pht hin hoc phn bit vi
mc chnh xc ng tin cy.

Gii hn nh lng ca thit b/ Limit of Instrumental Quantitation (LoIQ): l nng cht phn
tch ti thiu c trong mu m mt phng php c kh nng pht hin hoc phn bit bng mt
thit b c th vi mc chnh xc ng tin cy.

2.15. khng m bo o/ Mesurement Uncertainty

L thng s i km vi kt qu o v c trng cho mc phn tn ca cc gi tr o.
III. M hnh th nghim
3.1. tuyn tnh ca ng chun/ Linearity of calibration

Tn
hi
u

Xc nh khong lm vic trong qu trnh thit lp tuyn tnh ca ng chun, c m t nh

hnh sau:

Khong
lm vic
Vng
bn
nh
lng
LI L
L
D o
o
D
Q

L
o
L

Nng

Trong :

LID: l gii hn pht hin ca thit b.

LoD: l gii hn pht hin ca phng php.

LoQ: l gii hn nh lng ca phng php.

LoL: l gii hn khong tuyn tnh ca phng php.

xc nh khong tuyn tnh cn thc hin o cc dung dch chun c nng thay i v kho
st s ph thuc ca tn hiu vo nng . Dng ng cong ph thuc gia tn hiu v nng sau
gii hn s ph thuc trong khong tuyn tnh.
Sau khi xc nh khong tuyn tnh cn xy dng ng chun v xc nh h s tng quan.
ng chun phi tha mn cc yu cu sau:

Phi c t nht 6 im chun hoc nhiu hn (bao gm c im chun c nng bng 0).

ng chun phi cha ng t 0-200% hoc 50%-200% nng quan tm hoc nng thng
gp phi (MRL, MRPL).

Khong chia gia cc im chun phi ng u nhau v bao gm nng quan tm. Cc nng
chun khc nhau cn c chun b c lp, khng ly t chun tng t c nng cao hn.

ng chun phi c thc hin lp li t nht 2 ln, nn thc hin 3 ln hoc hn nhm th hin
tnh ngu nhin trong phn tch.

ng chun phi c th hin r rng s tng quan gia tn hiu v nng cc im chun
(tuyn tnh, phi tuyn tnh hay dng no khc).

3.2. Hiu ng nn/ Matrix effects

Chun b hai dy ng chun c v khng c s hin din ca nn mu, mi dy thc hin lp li
t nht 3 ln.
Khng nh s nh hng ca nn mu i vi ng chun. p dng kim nh t student cho hai
mu so snh h s gc ca ng chun da trn s c hoc khng hin din ca nn mu. T
quyt nh lp ng chun trn nn mu hay trn dung dch.
3.3. chn lc/ Selectivity
Phn tch mu trng v mu thm chun nhm pht hin v nh gi mc nh hng ca thnh
phn gy nhiu:

Phn tch t nht 6 mu trng i vi tng loi nn mu.

Phn tch t nht 6 mu thm chun i vi tng loi nn mu.

Kim tra gin kt qu ca mu trng v mu thm chun:

Mu thm chun: tn hiu ca cht cn phn tch phi c lp vi cc tn hiu khc.

Mu trng: khng xut hin tn hiu nhiu ti vng xut hin tn hiu ca cht cn phn tch.

3.4. nhy/ Sensitivity

nhy c xc nh bng vic xy dng ng chun, nhy chnh l h s gc ca ng
chun.
3.5. chnh xc/ accuracy
chnh xc c xc nh thng qua ng v chm.
3.6. ng/ Trueness, chch/ Bias
ng, chch c th c xc nh qua nhiu cch khch nhau, bao gm vic so snh kt qu
thc nghim vi kt qu thc hin bi mt phng php tiu chun, so snh kt qu thc nghim vi kt
qu thc hin trn mu CRM hoc t vic xc nh thu hi.
3.6.1. Xc nh ng, chch thng qua so snh vi phng php tiu chun
Phng php tham chiu tt nht l phng php tiu chun ca mt t chc c uy tn hoc l
phng php qua thm nh cho kt qu tin cy trong di o ang thc hin. Tnh cc kt qu trung
bnh v lp li ca s liu t hai phng php.
Phn tch t nht 6 mu chun hoc mu th bng phng php kho st v phng php tham
chiu. nh gi tng ng v kt qu thu c t phng php kho st v phng php i chiu.
p dng ln lt cc chun thng k sau nh gi:

Chun F Fisher: dng so snh hai phng sai ca hai tp d liu. So snh hai phng sai
cng chnh l so snh chm ca tp d liu. chm ca hai tp d liu phi t s tng
ng mi c c s xc nh ng. Nu khng tm thy s tng ng ca chm gia hai

tp d liu, tin hnh r sot v lm li th nghim. Tm li, cn c kt lun chc chn v s khc
bit ca hai phng sai l c ngha hay khng c ngha v mt thng k ti mc ngha =
5% trc khi so snh n gi tr trung bnh ca hai tp d liu.

Chun t Student: dng so snh s khc bit ca gi tr trung bnh t phng php kho st v
phng php tham chiu sau khi c kt lun so snh hai phng sai.

3.6.2. Xc nh ng, chch thng qua vt liu chun c chng nhn (CRM)
Phn tch t nht 6 mu CRM. p dng chun t Student so snh d liu va phn tch vi gi
tr ng ca mu CRM.
3.6.3. Xc nh ng, chch thng qua thu hi
ng, chch c th c tin hnh xc nh trn mu thm chun nu khng c phng php
tiu chun tham chiu hoc mu CRM.
Phn tch lp li t nht 6 mu thm chun ti mi mc nng gp 1, 1.5, 2 ln gi tr MRPL hoc
6 mu thm chun ti mi mc gp 0.5, 1, 1.5 ln gi tr nng cho php PL. So snh kt qu thu c
vi yu cu ca cc t chc uy tn, t kt lun v ng ca phng php.
3.7. chm/ Precision
chm c xc nh thng qua lp li v ti lp.
3.7.1 lp li/ Repeatability
lp li c th c tin hnh trn mu vt liu tham chiu c chng nhn (CRM) hoc tin
hnh trn mu thm chun nu khng c mu CRM.
Phn tch lp li t nht 6 mu ti mi mc nng gp 1, 1.5, 2 ln gi tr MRPL hoc 6 mu thm
chun ti mi mc gp 0.5, 1, 1.5 ln gi tr gi tr nng cho php PL. Tin hnh trong cng iu kin
xc nh (cng phng th nghim, ngi thc hin, thit b, thi gian) v c biu din thng qua
lch chun hoc lch chun tng i (h s bin thin).
3.7.2 ti lp/ Reproducibility
ti lp c th c tin hnh trn mu vt liu tham chiu c chng nhn (CRM) hoc tin
hnh trn mu thm chun nu khng c mu CRM.
Phn tch lp li t nht 6 mu ti mi mc nng gp 1, 1.5, 2 ln gi tr MRPL hoc 6 mu thm
chun ti mi mc gp 0.5, 1, 1.5 ln gi tr nng cho php PL. Tin hnh trong cc iu kin xc nh
khc nhau (khc phng th nghim, ngi thc hin, thit b, thi gian) v c biu din thng qua
lch chun hoc lch chun tng i (h s bin thin).
3.8. thu hi/ Recovery
thu hi c th c tin hnh trn mu vt liu tham chiu c chng nhn (CRM) hoc tin
hnh trn mu thm chun nu khng c mu CRM.
Phn tch t nht 18 mu trng v 6 mu thm chun ti mi mc nng gp 1, 1.5, 2 ln gi tr
MRPL hoc 6 mu thm chun ti mi mc gp 0.5, 1, 1.5 ln gi tr gi tr nng cho php PL.
3.9. Gii hn pht hin/ Limit of Detection, Gii hn nh lng/ Limit of Quantitation
C th xc nh LoD bng cc cch sau:

nh gi trc quan: phn tch lp li t nht 6 mu mi mc thm chun ti cc mc nng

ngu nhin v xc nh mc nng thp nht c kh nng pht hin c.

Da trn lch chun: phn tch lp li t nht 6 mu trng v 6 mu thm chun ti mc nng
thp nht cho php.

Da trn t s tn hiu v tn hiu nhiu (S/N): phn tch lp li t nht 6 mu trng v 6 mu thm
chun v xc nh mc nng c t s S/N l 2/1 hoc 3/1.

IV. Hng dn tnh ton

4.1. tuyn tnh ca ng chun/ Linearity of calibration
Xc nh nng gii hn trn ca ng chun trn c s cc gi tr MRL, MRPL hoc nng
quan tm ca cht cn phn tch (nng gii hn trn ca ng chun gp hai ln cc gi tr ny). T
lp khong chia gia cc im chun v tin hnh.
Thc hin t nht 3 ng chun v thit lp s tng quan gia tn hiu o v nng cc im
chun. S dng phng php bnh phng cc tiu s cho mt m hnh tuyn tnh dng y = ax + b. Tnh
h s tng quan Pearson.
n

( x i x ) ( y i y )
R=

i=1

(
n

i=1

x i x )

( y i y )2
i=1

Trong :

yi: tn hiu ca im chun th i o c t thit b.

y : gi tr trung bnh cc tn hiu.
xi: nng ca im chun th i.
x : gi tr trung bnh cc nng .

a: h s gc ca ng chun.
b: tung gc ca ng chun.
R: h s tng quan Pearson.
Nu 0.995 < R 1 hoc 0.99 < R 2 1chng t c mi lin h cht ch gia cc tn hiu o v cc
nng kho st, nh vy m hnh tuyn tnh trn c chp nhn.
4.2. Hiu ng nn/ Matrix effects
4.2.1. nh gi hiu ng nn da trn kim nh thng k
4.2.1.1. So snh hai phng sai ca h s gc t hai dy ng chun
Kim nh F Fisher c xy dng da trn gi thuyt ban u (H o:
ca n (H1:

S 21 >S 22 ). Tnh gi tr F thc nghim v so snh vi gi tr F critical (F - ti hn, tra bng)

a ra kt lun chp nhn gi thuyt ban u hay gi thuyt i.

Xc nh phng sai (S2) ca hai dy d liu theo cng thc:
n

( aia )2

S 2= i=1

S 1=S2 ) v gi thuyt i

n1
Trong :

ai: gi tr h s gc ti kt qu th i.
a : gi tr trung bnh t nhng h s gc ang xt.

n: s ln th nghim.
Tnh gi tr F thc nghim theo cng thc:
S21
F= 2
S2
Trong :

S 1 , S2 : phng sai ca h s gc t hai dy ng chun.

F: gi tr F thc nghim tnh ton.

Quy c 1: F 1
Nu F Fcritical (, k1, k2). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia chm

ca hai dy ng chun khng c ngha mc ngha .

Nu F > Fcritical (, k1, k2). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia chm

ca hai dy ng chun c ngha mc ngha .

Quy c 2: F < 1
Nu F > Fcritical (, k1, k2). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia chm

ca hai dy ng chun l khng c ngha mc ngha .

Nu F Fcritical (, k1, k2). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia chm

ca hai dy ng chun l c ngha mc ngha .

Trong :

Fcritical (, k1, k2): gi tr F tra bng (ph lc I).

k1, k2: bc t do (k1 = n1 1, k2 = n2 1).
n1, n2: s ln th nghim ca hai dy ng chun.
: mc ngha 5% ng vi tin cy 95%.

4.2.1.2. So snh hai gi tr trung bnh ca h s gc t hai dy ng chun

Kim nh t student cho hai mu c lp c xy dng da trn gi thuyt ban u (H o:
a1= a2
a a
) v gi thuyt i ca n (H1: 1 2 ). Tnh gi tr t thc nghim v so snh vi gi tr tcritical (t
- ti hn, tra bng) a ra kt lun chp nhn gi thuyt ban u hay gi thuyt i.
Tnh gi tr t thc nghim theo cng thc:
t=

|a1 a2|

S 21 S 22
+
n1 n2
Trong :

S 21 , S22 : phng sai ca d liu t h s gc hai dy ng chun.

a1

a2

: gi tr trung bnh ca h s gc t hai dy ng chun.

n1, n2: s ln th nghim tng ng vi hai dy ng chun.

t: gi tr t thc nghim tnh ton.

Quy c:

Nu s khc bit ca hai phng sai khng c ngha thng k mc ngha , bc t do tnh
bng cng thc k = n1 + n2 2.

Nu s khc bit ca hai phng sai c ngha thng k mc ngha , bc t do tnh bng

)[

2
S 21 S22
( S21 / n1 ) ( S22 /n2 )
cng thc k = n + n / n + 1 + n +1 2
1
2
1
2

Nu |t| tcritical (, k). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia kt qu ca hai
h s gc khng c ngha mc ngha . Kt lun nn mu nh hng khng ng k n
cng tn hiu ca cht cn phn tch nn c th xy dng ng chun trn nn mu hoc
dung dch.

Nu |t| > tcritical (, k). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia kt qu ca hai
h s gc c ngha mc ngha . Kt lun nn mu nh hng ng k n cng tn hiu
ca cht cn phn tch nn phi xy dng ng chun trn nn mu.
Trong :

tcritical (, k): gi tr t tra bng (ph lc II).

k: bc t do tng ng khi so snh hai phng sai.
n1, n2: s ln th nghim ca hai dy ng chun.
: mc ngha 5% ng vi tin cy 95%.

4.2.2. nh gi hiu ng nn da trn chch tng i

Tnh chch tng i (%B) gia h s gc ca hai dy ng chun, theo cng thc:
%B slop=

|a ma p|
a p

100

Trong :

a m

: gi tr h s gc trung bnh ca ng chun trn nn mu.

a p

: gi tr h s gc trung bnh ca ng chun dung dch.

%B slop

: chch tng i ca h s gc.

nh gi hiu ng nn c nh hng n ng chun hay khng:

Nu

%B slop

> 10%, kt lun nn mu c nh hng n cng tn hiu ca cht cn phn

tch nn phi xy dng ng chun trn nn mu.

Nu %B slop < 10%, kt lun nn mu nh hng khng ng k n cng tn hiu ca cht

cn phn tch nn c th xy dng ng chun trn nn mu hoc dung dch.

4.3. nhy/ Sensitivity

Thc hin t nht 3 ng chun, tnh gi tr trung bnh t cc h s gc ly t ng chun:
a=

y b
x

Tnh gi tr trung bnh t cc h s gc. nhy c nh gi l t khi |a |>1 .

Trong :

a: h s gc ca ng chun.
x: nng ca im chun.
y: tn hiu o c t thit b.
b: tung gc ca ng chun.
a : gi tr trung bnh t cc h s gc.

4.4. ng/ Trueness, chch/ Bias

4.4.1. Xc nh ng (%T), chch (%B) thng qua so snh vi phng php tiu chun
4.4.1.1. So snh hai phng sai ca d liu t hai phng php
Kim nh F Fisher c xy dng da trn gi thuyt ban u (H o:
ca n (H1:

S 21=S22 ) v gi thuyt i

S 1 >S 2 ). Tnh gi tr F thc nghim v so snh vi gi tr F critical (F - ti hn, tra bng)

a ra kt lun chp nhn gi thuyt ban u hay gi thuyt i.

Xc nh phng sai (S2) ca hai dy d liu theo cng thc:
n

2
(C iC)

S 2= i=1

n1
Trong :

Ci: gi tr nng ti kt qu th i.

C
: gi tr trung bnh t nhng kt qu o c ti nng ang xt.
n: s ln th nghim.

Tnh gi tr F thc nghim theo cng thc:

2

F=

S1
2
S2
Trong :

S 21 , S22 : phng sai ca d liu t hai phng php.

F: gi tr F thc nghim tnh ton.

Quy c 1: F 1

Nu F Fcritical (, k1, k2). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia chm
ca hai phng php khng c ngha mc ngha .

Nu F > Fcritical (, k1, k2). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia chm
ca hai phng php c ngha mc ngha .
Quy c 2: F < 1

Nu F > Fcritical (, k1, k2). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia chm
ca hai phng php khng c ngha mc ngha .

Nu F Fcritical (, k1, k2). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia chm

ca hai phng php c ngha mc ngha .

Trong :

Fcritical (, k1, k2): gi tr F tra bng (ph lc 1).

k1, k2: bc t do (k1 = n1 1, k2 = n2 1).
n1, n2: s ln th nghim ca hai phng php.
: mc ngha 5% ng vi tin cy 95%.

4.4.1.2. So snh hai gi tr trung bnh ca d liu t hai phng php

Kim nh t student cho hai mu c lp c xy dng da trn gi thuyt ban u (H o:
C 1 =C 2 ) v gi thuyt i ca n (H : C 1 C 2 ). Tnh gi tr t thc nghim v so snh vi gi tr t
1
critical
(t - ti hn, tra bng) a ra kt lun chp nhn gi thuyt ban u hay gi thuyt i.
Tnh gi tr t thc nghim theo cng thc:
t=

|C 1 C 2|

S 21 S 22
+
n1 n2
Trong :

S 1 , S2 : phng sai ca d liu t hai phng php.

C 1 , C 2 : gi tr trung bnh ca d liu t hai phng php.

n1, n2: s ln th nghim tng ng vi hai phng php.

t: gi tr t thc nghim tnh ton.
Quy c:

Nu s khc bit ca hai phng sai khng c ngha thng k mc ngha , bc t do tnh
bng cng thc k = n1 + n2 2.

Nu s khc bit ca hai phng sai c ngha thng k mc ngha , bc t do tnh bng

)[

2
S 21 S22
( S21 / n1 ) ( S22 /n2 )
k
=
+
/
+
2
cng thc
n 1 n2
n1 + 1
n 2+1

Nu |t| tcritical (, k). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia kt qu ca hai
phng php khng c ngha mc ngha . Phng php kho st c ng t yu cu.

Nu |t| > tcritical (, k). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia kt qu ca hai
phng php c ngha mc ngha . Phng php kho st mc sai s h thng.
Trong :

tcritical (, k): gi tr t tra bng (ph lc 2).

k: bc t do tng ng khi so snh hai phng sai.
n1, n2: s ln th nghim ca hai phng php.
: mc ngha 5% ng vi tin cy 95%.

4.4.2. Xc nh ng (%T), chch (%B) thng qua mu vt liu tham chiu c chng nhn
(CRM)

Kim nh t student cho mt mu c xy dng da trn gi thuyt ban u (H o: C=CCRM )

v gi thuyt i ca n (H1: C CCRM ). Tnh gi tr t thc nghim v so snh vi gi tr t critical (t - ti

hn, tra bng) a ra kt lun chp nhn gi thuyt ban u hay gi thuyt i.
Tnh gi tr t thc nghim theo cng thc:

|CC
CRM|

t=

S2
n
Trong :

S 2 : phng sai ca d liu t phng php kho st.

C
,: gi tr trung bnh ca d liu t phng php kho st.
CCRM

: gi tr c chng nhn ca mu CRM.

n: s ln th nghim ca phng php kho st.

t: gi tr t thc nghim tnh ton.
Quy c:

Nu |t| tcritical (, k). Ta chp nhn Ho, bc b H1. iu ny ch ra s khc bit gia kt qu ca
phng php kho st v gi tr tham chiu khng c ngha mc ngha . Phng php kho
st c ng t yu cu.

Nu |t| > tcritical (, k). Ta bc b Ho, chp nhn H1. iu ny ch ra s khc bit gia kt qu ca
phng php kho st v gi tr tham chiu c ngha mc ngha . Phng php kho st
mc sai s h thng.
Trong :

tcritical (, k): gi tr t tra bng (ph lc 2).

k: bc t do k = n - 1.
n: s ln th nghim ca phng php kho st.
: mc ngha 5% ng vi tin cy 95%.

Tnh ng v chch qua cng thc:

C
%T = CRM 100
CCRM
C |
|C CRM
CRM

%B=

CCRM

100=100%T

Trong :

CCRM: gi tr c chng nhn ca mu CRM.

CCRM
: gi tr trung bnh t nhng kt qu o c ca mu CRM.

4.4.2. Xc nh ng (%T), chch (%B) thng qua thu hi

C
%T = spike 100
C spike
C spike|
|C spike

%B=

C spike

100=100%T

Trong :

Cspike: gi tr nng trong mu thm chun.

C spike
: gi tr trung bnh t nhng kt qu o c ca mu thm chun.

4.5. lp li/ Repeatability, ti lp/ Reproducibility, chm/ Precision

Xc nh lp li (r) v ti lp (R w) thng qua lch chun (SD) hoc lch chun tng
i (%RSD) - h s bin thin (%CV).

SD=

2
(Ci C)
i=1

n1

%RSD=%CV =

SD
100

Trong :

Ci: gi tr nng ti kt qu th i.

C
: gi tr trung bnh t nhng kt qu o c ti nng ang xt.
n: s ln th nghim.

chm trn mi nn mu c th hin thng qua cc lch chun tng i ca tng mc

nng kho st, cng thc pooled:

%RSD pooled =

[ ( ji1 ) %RSDi2 ]
i=1

( ji1 )
i =1

Trong :

ji

: s ln o mc nng i.

n: s mc nng .
RSD i

: lch chun tng i ca

ji

ln o mc nng i.

chm tng hp ca phng php c th hin thng qua cc chm ca mi nn mu.

%RSD pooled ,c =

i=1

%RSD 2pooled ,i

Trong :

%RSD pooled ,i

: thng s th hin chm ca nn mu i.

n: s nn mu kho st.
4.6. thu hi/ Recovery
Xc nh nng hin din thc v hiu sut thu hi (%R) trong mi mu:
)
(CC
o
R=
100
C spiked
Trong :

C: gi tr nng thc o trong mu thm chun.

Co: gi tr nng o c trong mu trng.
Cspiked: gi tr nng trong mu thm chun.

4.7. Gii hn pht hin/ Limit of Detection

4.7.1. Xc nh LoD da trn lch chun

SD=

2
(Ci C)
i=1

n1
LoD=C o +3 SD

Trong :

Ci: gi tr nng ti kt qu th i.

C
: gi tr trung bnh t nhng kt qu o c ti nng ang xt.
C o : gi tr nng trung bnh ca cc mu trng.

n: s ln th nghim
4.7.2. Xc nh LoD da trn t l S/N
S / N=

R
Ro
Trong :

R: tn hiu ca cht phn tch trong mu thm chun ti nng ang xt.
Ro: tn hiu xut hin trong mu trng tng ng vi tn hiu ca cht phn
tch trong mu thm chun.

4.8. Gii hn nh lng/ Limit of Quantitation

4.8.1. Xc nh LoQ da trn lch chun

SD=

2
(Ci C)
i=1

n1
LoQ=C o +10 SD
Trong :

Ci: gi tr nng ti kt qu th i.


C
: gi tr trung bnh t nhng kt qu o c ti nng ang xt.
C o : gi tr nng trung bnh ca cc mu trng.
n: s ln th nghim
4.8.2. Xc nh LoQ da trn LoD
LoQ=3 LoD

V. c lng khng m bo o
Theo yu cu ca ISO 17025, khng m bo o l mt thng s cn c thc hin cho tt c
cc phng php th trong phng kim nghim. Phng kim nghim c th phi hp xc nh
khng m bo o nh mt phn trong qu trnh thm nh gi tr s dng phng php.
khng m bo o c tnh thng qua cc khng m bo o thnh phn l khng m
bo o ca chm (th hin qua ti lp) v khng m bo o ca ng (th hin qua
chch). Tt c u da trn s liu thc nghim t qu trnh xc nhn gi tr s dng phng php.
%uc = %u 2R + %u2B
w

Vic bo co khng m bo o cui cng thng c quy v khng m bo o m rng.

%ue =k %uc
%uc

Trong :

%u R

: khng m bo o tng hp.

w

%u B

: khng m bo o ca ti lp trong phng kim nghim.

: khng m bo o ca chch.

k: h s ph tng ng khong tin cy 95% (k=2).

%ue

: khng m bo o m rng.

5.1. Xc nh khng m bo o ca ti lp trong phng kim nghim (

uR

Ngun gy ra khng m bo o ca ti lp l khi nghin cu ti lp cc mc nng

v trn mi nn mu. Tnh lch chun tng i t cc thnh phn ca cc mc nng kho st trn
tng nn mu, s dng cng thc pooled:

R SD R =%RSD pooled =
w

Trong :

[ ( ji1 ) %RSD 2i ]
i=1

( ji1 )
i=1

ji

: s ln o mc nng i.

n: s mc nng .
RSD i

: lch chun tng i ca

%RSD R , RSD pooled

w

ji

ln o mc nng i.

: lch chun tng i kt hp trn mi nn mu.

Kt hp cc lch chun tng i chung trn mi nn mu chnh l khng m bo o ca

ti lp trong phng kim nghim.

%u R =
w

i=1

%RSD 2R

w ,i

Trong :

%RSD R

w,i

: khng m bo o ca nn mu i.

n: s nn mu kho st.
5.2. Xc nh khng m bo o ca chch (

uB

khng m bo o ca chch c tnh thng qua cc thnh phn ca n, tnh theo cng
thc:
u B= %RSM 2B + u2C

ref

RSM B

Trong :

%uC

ref

: gi tr hiu dng tng i ca chch.

: khng m bo o ca mu tham chiu.

Gi tr hiu dng tng i ca chch c tnh l cn bc hai ca trung bnh bnh phng cc
chch, tnh theo cng thc:

%RSM B=
Trong :

%Bi

%B2i
i=1

: chch ca i tng th nghim i.

n: s i tng th nghim chch.

khng m bo o ca mu tham chiu (

%uC

ref

) c th hin qua gi tr tham chiu m

phng th nghim s dng. c th l gi tr ca mu vt liu chun c chng nhn (CRM), gi tr t

kt qu lin phng v gi tr ca mu thm chun. Mi trng hp s c cch tnh khc nhau ca
khng m bo o ca mu tham chiu.
5.2.1. Phng kim nghim s dng mu CRM tham chiu
Trng hp 1: nu phng kim nghim ch s dng mt mu CRM, khng m bo o ca
chch phi bao gm lch chun tng i ca cc ln th nghim, c tnh theo cng thc:

%RSDB 2 2
u B= %B +
+ uC
n
2

CRM

khng m bo o ca mu tham chiu c tnh thng qua khng m bo o m rng ca

mu CRM, gi tr ny c cung cp t nh cung cp mu CRM.
uC =
CRM

%u e
k
Trong :

%B: chch ca cc ln th nghim mu CRM.

%RSD B

: lch chun tng i ca cc ln th nghim trn mu

CRM.
n: s ln th nghim trn mu CRM.
%ue

: khng m bo o m rng ca mu CRM.

k: h s ph tng ng vi khong tin cy 95% (k=2).

Trng hp 2: nu phng kim nghim s dng nhiu mu CRM, khng m bo o ca
chch tnh theo cng thc:
u B= %RSM 2B + u2C

CRM ,c

Gi tr hiu dng tng i ca chch tnh theo cng thc:

%RSM B=

%B2CRM ,i
i=1

n
Trong :

%BCRM ,i

: chch ca mu CRM th i.

n: s mu CRM th nghim.
khng m bo o tng hp ca mu tham chiu c tnh thng qua cc khng m bo o
thnh phn ca cc mu CRM.

%uC

uC

CRM ,c

CRM ,i

%u 2C
i=1

CRM ,i

%u e ,i
k
Trong :

%RSM B

: gi tr hiu dng tng i ca chch.

%uC

CRM ,c

: khng m bo o tng hp ca cc mu CRM.

%uC

CRM ,i

: khng m bo o ca cc CRM th i.

n: s mu CRM th nghim.
%ue ,i

: khng m bo o m rng ca mu CRM th i.

k: h s ph tng ng vi khong tin cy 95% (k=2).

5.2.2. Phng kim nghim s dng gi tr o t kt qu lin phng tham chiu
Cc thng s nhn c t kt qu lin phng c th dng tnh khng m bo o ca
chch theo cng thc:

u B= %RSM 2B + u2C

lab ,c

Gi tr hiu dng tng i ca chch tnh theo cng thc:

%RSM B=

%B2lab ,i
i=1

n
Trong :

%Blab ,i

: chch t kt qu ca phng kim nghim th i.

j: s phng kim nghim tham gia lin phng.

khng m bo o tng hp ca gi tr lin phng c tnh thng qua lch chun tng i
ca kt qu t cc phng kim nghim tham gia.
%uC =

%R SD lab

lab ,c

Trong :

%RSD lab

: lch chun tng i gia cc phng kim nghim.

n: s phng kim nghim tham gia cho kt qu lin phng.

%uC

lab ,c

: khng m bo o tng hp ca gi tr lin phng.

5.2.3. Phng kim nghim s dng mu thm chun tham chiu

Trng hp phng kim nghim ch s dng mu thm chun tham chiu, khng m bo o
ca chch c tnh da trn thu hi, theo cng thc:
u B= %RSM 2B + u2C

R ,c

Gi tr hiu dng tng i ca chch tnh theo cng thc:

%RSM B=

%B2R , i
i=1

n
Trong :

%B R ,i

: chch t kt qu th nghim thu hi i.

n: s ln th nghim thu hi.

khng m bo o tng hp ca mu thm chun c c tnh da trn cc thnh phn gy ra
l khng m bo o ca cht chun v th tch thm chun.
%uC = %u2C
R,c

standard

+ u 2volume

khng m bo o ca cht chun c tnh thng qua khng m bo o m rng ca cht

chun, gi tr ny c cung cp t nh cung cp cht chun.
uC

standard

%u e
k

%ue

Trong :

: khng m bo o m rng ca cht chun.

k: h s ph tng ng vi khong tin cy 95% (k=2).

khng m bo o ca th tch thm chun c tnh thng qua khng m bo o ca dng
c s dng. Cc thnh phn ny gm chch v lp li ca dng c dng thm chun.
%u volume= %u2B

pipette

+ u 2r

pipette

Trong :

%u B

%ur

pipette

pipette

: khng m bo o ca chch ca pipette.

: khng m bo o ca lp li ca pipette.

khng m bo o ca chch ca pipette c th tnh thng qua cc ln th nghim chch

ca pipette hoc s dng thng s m nh sn xut cung cp sn. p dng quy lut phn phi hnh ch
nht:
%u B

pipette

%B pipette

3
Trong :

%B pipette

: chch ti a ca pipette do nh sn cut cung cp.

khng m bo o ca lp li ca pipette c th tnh thng qua cc ln th nghim lp li

ca pipette hoc s dng thng s m nh sn xut cung cp sn.
%ur

pipette

=%RSD r
Trong :

%RSD r

: lp li ti a ca pipette do nh sn cut cung cp.

5.3. Cng b khng m bo o

Phi thng bo cc thng tin sau khi trnh by kt qu trong cc bo co thm nh hoc khi c yu
cu:

Kt qu o.

khng m bo o.

Mc tin cy c s dng trong vic xc nh khng m bo o m rng.

Kt qu o ( n v ) ue ( ) ( khong tincy t
Trong :

ue

ngng h s ph)

: khng m bo o m rng.

PH LC
k
( 1 , k 2)
I. Bng phn phi Fisher:
F

(k)

II. Bng phn phi t - student: t