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Lod Loq
Lod Loq
Ch bin:
DS. Trn Cao Sn
Nhm bin son:
DS. Trn Cao Sn
PGS. TS. Phm Xun
TS. L Th Hng Ho
CN. Nguyn Thnh Trung
Hiu nh:
PGS. Phm Gia Hu
KS. Phm Thanh Nh
LI GII THIU
Cng vi s pht trin ca khoa hc cng ngh, kinh t - x hi vic
chm sc v bo v sc kho, nng cao cht lng cuc sng ca con ngi
ngy cng c quan tm, trong c vn an ton thc phm. Bo m
ngun thc phm an ton s gp phn bo v v ci thin sc kho, cht
lng cuc sng v ging ni dn tc. Tuy nhin, cng tc bo m an ton
thc phm lun l mt thch thc khng ch vi cc nc ang pht trin
m ngay c vi cc nc pht trin. S a dng ca cc chng loi thc
phm, cng ngh, cc cht ph gia, cc cht h tr ch bin, cc cht
nhim thc phm lun c ci tin, b sung l thch thc i vi h
thng qun l, kim sot an ton thc phm, c bit l i vi cc phng
kim nghim. Do , vic nng cao nng lc h thng phng kim nghim
an ton thc phm ng vai tr c bit quan trng trong cng tc qun l
an ton thc phm ca mi quc gia, nht l trong giai on hi nhp kinh
t ton cu hin nay.
Ti Vit Nam, trong nhng nm gn y cng tc bo m an ton
thc phm c ng v Nh nc c bit quan tm v gt hi c
nhiu thnh tu ng khch l. H thng php lut, qun l c hnh
thnh v kin ton, trong c h thng kim nghim an ton thc phm
m in hnh l s ra i ca Vin Kim nghim an ton v sinh thc phm
quc gia (theo Quyt nh s 376/Q-TTg ngy 23/3/2009) vi chc nng
l n v trng ti quc gia trong kim nghim thc phm. Tuy nhin, do
nhiu yu t v ngun lc u t cha p ng, h thng kim nghim thc
phm ca Vit Nam cn gp rt nhiu kh khn, ngay c cp Trung ng.
Bi v i vi h thng kim nghim thc phm, khng ch cn u t tho
ng v c s vt cht, trang thit b, con ngi m cn phi u t cc h
thng qun l cht lng, k thut (phng php) phn tch ph hp.
Hin nay a s cc phng kim nghim thc phm ca cc a
phng s dng cc phng php th nghim khc nhau, cha c chun
LI CM N
Xin by t lng bit n su sc n nhng ngi thy PGS. Phm Gia
Hu v KS. Phm Thanh Nh - nhng ngi trc tip h tr v ng gp
kin sa cha hon thin cun sch.
Xin chn thnh cm n nhng ngi thy, ngi bn, ng nghip
trong hi ng khoa hc thng qua ni dung cun sch PGS.TS. Trn
Chng Huyn, PGS.TS. T Th Tho, Trng i hc Quc gia H Ni;
PGS.TS. Thi Phan Qunh Nh, TS. Trn Vit Hng, Vin Kim nghim
thuc trung ng; TS. Trn ng Ninh, Cc Qun l cht lng Nng lm
sn v Thy sn; ThS. L Vn Giang, Cc An ton v sinh thc phm v
KS. Nguyn Th Vn Lan, Vin Kim nghim an ton v sinh thc phm
quc gia v nhng kin ng gp qu bu trong qu trnh bin son cun
sch.
Xin chn thnh cm n cc bn ng nghip ti Vin Kim nghim an
ton v sinh thc phm quc gia to iu kin h tr xut bn cun
sch.
Nhm bin son
MC LC
MC LC.................................................................................................. 7
DANH MC CH VIT TT................................................................... 9
Chng I: CC YU CU CHUNG........................................................ 10
1. Khi nim v thm nh phng php ............................................... 10
2. Thm nh phng php tiu chun (method verification)................. 11
3. Thm nh phng php khng tiu chun (method validation) ........ 13
4. Thm nh li.................................................................................... 14
Chng II: THM NH PHNG PHP PHN TCH HA HC ..... 16
1. Tnh c hiu/chn lc ...................................................................... 19
1.1. nh ngha ................................................................................. 19
1.2. Cch xc nh............................................................................. 20
1.3. Tnh c hiu/chn lc i vi phng php chun .................... 24
2. Khong tuyn tnh v ng chun ................................................... 25
2.1. nh ngha ................................................................................. 25
2.2. Cch xc nh khong tuyn tnh................................................ 26
2.3. Xy dng ng chun .............................................................. 26
2.4. Cc lu khi xy dng ng chun ......................................... 30
2.5. Gii hn chp nhn ca ng chun ......................................... 31
3. Gii hn pht hin............................................................................. 32
3.1. nh ngha ................................................................................. 32
3.2. Cch xc nh............................................................................. 32
4. Gii hn nh lng .......................................................................... 36
4.1. nh ngha ................................................................................. 36
4.2. Cch xc nh............................................................................. 37
5. chnh xc ( ng v chm).................................................. 39
5.1. chm .................................................................................... 40
5.2. ng (trueness) ..................................................................... 47
6. n nh (hay vng/ chc chn) ca phng php................. 58
6.1. nh ngha ................................................................................. 58
DANH MC CH VIT TT
AOAC (Association of Official
Analytical Chemists)
ASTM (American Society for Testing
of Materials)
CRM (Certified reference material)
DAD (Diod array detector)
GC-MS (Gas chromatography mass
spectrometry)
HPLC (High performance liquid
chromatography)
HR (High resolution)
ICH (International Conference on
Harmonization)
IP (Identification point)
ISO (International Standard
Organization)
LC-MS (Liquid chromatography
mass spectrometry)
QC (Quality control)
S/N (Signal to noise ratio)
SOP (Standard Operation
Procedure)
TCVN
USFDA (United States Food and
Drug Administration)
Dc in M
10
CHNG I: CC YU CU CHUNG
Chng I: CC YU CU CHUNG
1. Khi nim v thm nh phng php
Thm nh 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 cc yu cu t ra (fitness for the purpose). Kt qu ca thm nh
phng php c th c s dng nh gi cht lng, tin cy ca kt
qu phn tch. Thm nh phng php phn tch l mt phn khng
th thiu nu mun c mt kt qu phn tch ng tin cy.
Hin nay nhiu thut ng khc nhau c s dng ch khi nim
trn, nh nh tr phng php, nh gi phng php, xc nhn gi tr s
dng ca phng php, ph duyt phng php. Tt c cc thut ng ny
u l cch gi khc nhau ca thm nh phng php (method validation).
Phng th nghim thng s dng nhiu phng php khc nhau.
Da vo ngun gc c th phn loi cc phng php thnh hai nhm:
- Cc phng php tiu chun: cc phng php th theo tiu chun
quc gia, quc t, hip hi khoa hc c chp nhn rng ri trn th gii
nh TCVN, ISO, ASTM, AOAC
- Cc phng php khng tiu chun hay phng php ni b (nonstandard/alternative/in-house method): 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 php phn tch phi c thm
nh hoc thm nh li khi:
11
- Phng php p dng khng phi l phng php tiu chun (nonstandard method)
- Phng php do phng th nghim t xy dng mi trc khi a
vo s dng thnh thng quy.
- C s thay i v i tng p dng nm ngoi i tng p dng
ca phng php thm nh hoc phng php tiu chun.
- C s thay i cc iu kin thc hin phng php c thm
nh (v d: thit b phn tch vi cc c tnh khc bit, nn mu, ngi
phn tch ).
2. Thm nh phng php tiu chun (method verification)
Trc khi p dng mt phng php phn tch cn c cc chng minh
rng phng php p ng yu cu t ra, tc l phng php phi c
thm nh. Yu cu ny khng ch cho cc phng php th ni b m cn
cn cho cc phng php tiu chun. Vic thm nh phng php tiu
chun v phng php ni b c s khc nhau, do cn ch khi lp k
hoch thm nh.
C hai yu cu ch yu ca vic thm nh phng php tiu chun:
- Phi c kt qu thm nh ca phng php tiu chun, v kt qu
ny phi ph hp vi yu cu ca phng th nghim.
- Phng th nghim cn m bo c th t c cc thng s c
m t trong phng php tiu chun.
Theo yu cu ca ISO 17025, khi cc phng th nghim p dng cc
phng php tiu chun cn c h s nh gi cc iu kin c bn, cc
ngun lc theo yu cu ca phng php th v vic t c kt qu th
nghim c chnh xc nh phng php yu cu hoc nh mong mun
12
CHNG I: CC YU CU CHUNG
13
14
CHNG I: CC YU CU CHUNG
a. Cc php th thm nh s b
b. Thay i cc thng s ca phng php (nu cn)
c. Thc hin thm nh hon thin
5. Hon thin SOP ca phng php
6. Bo co thm nh: cn c cc thng tin sau
a. Tn ngi thm nh, thi gian thm nh
b. Tm tt phng php: nguyn l, thit b, ha cht, quy trnh,
cc lu ...
c. Cc kt qu thm nh
d. Cc yu cu cn p ng a phng php vo thc hin
thng xuyn (routine): kim tra tnh tng thch ca h
thng, mu QC, c lng khng m bo o ca kt
qu...
e. Xc nh cc thng s v thi gian cn thm nh li
f. Ti liu tham kho (nu c)
g. Kt lun v xut (nu c)
h. K tn, ngy ca ngi lm bo co
i. K ph duyt ca ngi c thm quyn
4. Thm nh li
Cng vic thm nh khng phi ch thc hin mt ln khi pht trin
phng php ban u m cn thc hin trong sut qu trnh p dng. V a
s cc iu kin thc hin phng php c s thay i trong sut qu trnh
15
16
ng; (Trueness)
chm; (Precision)
17
Cc thng s thm nh
Phn tch
Phn tch
Phn tch
Xc nh gii
nh tnh
vi lng
a lng
hn tp cht
+/-
ng
(Trueness)
chm
(Precision)
c hiu, chn lc
(Specification/Selectivity)
LOD
18
(Limit of detection)
LOQ
+/-
(Limit of quantitation)
tuyn tnh
(Linearity/range)
vng (n nh)
(Ruggedness/Robustness)
(+) Cn thc hin thm nh (-) Khng cn thc hin thm nh
Cc thng s nh gi
Xc nh
Phn tch
Phn tch
Phn tch
nh tnh
vi lng
a lng
gii hn tp
cht
ng
(Trueness)
chm
(Precision)
c hiu, chn lc
(Specification/Selectivity)
LOD
(Limit of detection)
19
LOQ
(Limit of quantitation)
tuyn tnh
(Linearity/range)
vng (n nh)
(Ruggedness/Robustness)
(+) Cn thc hin thm nh (-) Khng cn thc hin thm nh
1. Tnh c hiu/chn lc
1.1. nh ngha
* Tnh c hiu: L kh nng pht hin c cht phn tch khi c mt
cc tp cht khc nh cc tin cht, cc cht chuyn ha, cc cht tng t,
tp cht.... C th, trong php phn tch nh tnh l phi chng minh
c kt qu l 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 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, nhm hng n kt qu chnh xc.
Tnh c hiu thng lin quan n vic xc nh ch mt cht phn
tch.
* Tnh chn lc: 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.
20
1.2. Cch xc nh
S dng phng php thm chun sau chun b mu (cochromatography), cch ny thng p dng i vi cc phng php
sc k. Sau khi chun b mu (mu trng hoc mu thc) v phn
tch mu trn thit b sc k thu c cc pic sc k, ta thm chun
vo mu chit xut v phn tch mu ny. So snh sc k ca
hai mu nh gi tnh c hiu/chn lc.
Phn tch mu khng c cht phn tch nhng c cht cu trc tng
t cht phn tch (nu c): Phi cho kt qu m tnh (i vi phng
php nh tnh) v khng c nh hng n kt qu nh lng
ca cht phn tch (i vi phng php nh lng).
21
22
23
S im IP t c vi 1 ion
1,0
LR-MS (ion m)
1,0
1,5
2,0
HR-MS (ion m)
2,0
2,5
S ion
S im IP
2 (EI) + 2 (CI)
2 (dc A) + 2 (dc B)
LC-MS
GC-MS-MS
LC-MS-MS
GC-MS-MS
LC-MS-MS
LC-MS-MS-MS
5,5
24
HRMS
2n
GC-MS v LC-MS
2+2
GC-MS v HRMS
2+1
25
26
27
( x X )( y
(x X )
i
Y)
(yi Y )
28
29
Khi phn tch mu, ni chun cng phi c thm vo (tt nht l t
u, sau khi cn, ong) sao cho to c nng cui cng bng nng
ni chun trong cc dung dch chun. Vi cch tin hnh nh th ny c
th hn ch c hu ht cc nh hng trong qu trnh phn tch (bao gm
30
13
C,
15
chun l chloramphenicol, s dng thm cht ni chun l chloramphenicold5; clenbuterol th s dng clenbuterol-d9, melamin th s dng melamin13
nh tng t cht phn tch do i din tt cho cht phn tch trong c
qu trnh. Ngoi ra, trong mt s trng hp c th chn cc cht ni chun
khc, vi iu kin l cc cht ny phi c mt s tnh cht c bn ging
cht phn tch, v c th phn tch c bng phng php ang thc hin.
2.4. Cc lu khi xy dng ng chun
-
31
0,99 R2 1
lp ng chun xong cn kim tra bng phng php tnh ngc li nng
ca cc im chun s dng xy dng ng chun, t tnh cc gi
tr chch theo cng thc sau:
i =
C t Cc
100
Cc
32
V d:
Bng 5: V d cch tnh LOD ca phng php nh tnh
Nng
(ppm)
S ln
th
T l %
200
10
10/0
100
100
10
10/0
100
75
10
5/5
50
50
10
1/9
10
25
10
0/10
33
LOD = x 0 + 3SD 0
Tnh LOD:
Vi
Trong :
SD0 =
(x
0i
x0 )
n 1
34
LOD = 3 x SD
SD =
(x
x)
n 1
35
3,3 SD
a
36
4. Gii hn nh lng
4.1. nh ngha
LOQ l nng ti thiu ca mt cht c trong mu th m ta c th
nh lng bng phng php kho st v cho kt qu c chm mong
mun.
LOQ ch p dng cho cc phng php nh lng.
Ging nh LOD, c nhiu cch khc nhau xc nh LOQ, ph
thuc vo tng phng php c th m la chn cho ph hp.
Vic xc nh LOQ cn tnh n cc yu t nh hng trong mu phn
tch, do cn thc hin trn nn mu tht.
LOQ trong nhiu trng hp c th l im thp nht ca khong
tuyn tnh. Hnh 8 m t mi quan h gia LOD, LOQ v khong tuyn
tnh.
37
4.2. Cch xc nh
Vic b tr th nghim xc nh LOQ thng kt hp vi tnh LOD.
C nhiu cch khc nhau tnh LOQ nh sau:
Da trn lch chun:
C hai trng hp nh trong phn tnh LOD l thc hin trn mu
trng v thc hin trn mu th. Cc cng thc tnh ton nh sau:
LOQ = x 0 + 10SD 0
LOQ = 10 SD
Ln
Abs ti
575nm
HL metanol
(mg/l)
Trung bnh
(mg/l)
SD (mg/l)
0,021
12,85
13,61
1,75
0,024
15,12
0,022
13,61
0,024
15,12
38
0,019
11,34
0,025
15,88
0,018
10,58
0,024
15,12
0,022
13,61
10
0,021
12,85
Nh vy,
Nng
(mg/kg)
0,01
0,02
0,05
Nh vy
39
Ln
S/N
1765
1231
2,87
1723
1429
2,41
1598
1364
2,34
1628
1151
2,83
3021
1351
4,47
3109
1491
4,17
2919
1502
3,89
2934
1385
4,24
6531
1421
9,19
6691
1323
10,1
6567
1299
10,1
6519
1313
9,93
Da trn ng chun:
Cch tnh tng t nh trong phn LOD nhng theo cng thc sau:
LOQ =
10 SD
a
40
nh l hai khi nim khc nhau. Trong ti liu ny chng ti s dng hai
thut ng ng v chm din t chnh xc ca mt phng php
phn tch theo quan im mi nht ca tiu chun quc t (ISO 5725 16:1994) v tiu chun quc gia (TCVN 6910 1-6:2005). 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 9
m t s mi quan h gia chm, ng v chnh xc.
chnh xc (accuracy) = chm (precision) + ng (trueness)
5.1. chm
5.1.1. nh ngha
Trong nhiu trng hp cc php th nghim trn nhng i tng v
vi nhng iu kin khc nhau thng khng cho kt qu ging nhau. iu
ny do cc sai s ngu nhin ca mi quy trnh gy ra, ta khng th kim
41
lp li (repeatability)
ti lp (reproducibility)
S khc nhau gia cc khi nim lp li, chm trung gian v
ti lp c tm tt trong bng 6.
Bng 6: 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
Ging
Khc
Khc
Dng c, ha cht
Ging
Khc
Khc
iu kin mi
trng
Ging
Khc
Khc
Phng th nghim
Ging
Ging
Khc
42
5.1.2. Cch xc nh
Cch 1. B tr th nghim
Tin hnh lm th nghim lp li 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.
Tng phng th nghim, c th b tr th nghim tnh lp li
hoc chm trung gian. Trong mt s trng hp tham gia so snh vi cc
phng th nghim khc (v d trong chng trnh th nghim thnh tho, so
snh lin phng).
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 =
(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
43
X=
s=
n
x i1 + x i 2
2
d tb
1,118
RSD % =
s
100
X
44
D tb =
di
xi
RSD% =
D tb
100
1,118
5.1.3. Tiu ch nh gi
i chiu gi tr tnh c vi gi tr mong mun hay gi tr yu cu
hoc so vi RSD% lp li cho trong bng 7 (RSD% tnh c khng c
ln hn gi tr 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 7: lp li ti a chp nhn ti cc nng khc nhau (theo AOAC)
Hm lng %
T l cht
n v
RSD (%)
1.
100
100%
1,3
2.
10
10-1
10%
1,8
3.
10-2
1%
2,7
4.
0,1
10-3
0,1 %
3,7
TT
45
5.
0,01
10-4
100 ppm
5,3
6.
0,001
10-5
10 ppm
7,3
7.
0,0001
10-6
1 ppm
11
8.
0,00001
10-7
100 ppb
15
9.
0,000001
10-8
10 ppb
21
10.
0,0000001
10-9
1 ppb
30
Ln
10
x i1 21,3
26,5
24,1
25,7
24,6
27,8
26,6
25,4
20,1
18,5
x i 2 21,5
25,9
24,1
25,3
24,5
27,6
26,5
25,1
20,5
18,3
21,4
26,2
24,1
25,5
24,55
27,7
26,55 25,25
20,3
18,4
di
0,2
0,6
0,0
0,4
0,1
0,2
0,4
0,2
0,1
0,3
Di 0,0093 0,0229 0,0000 0,0157 0,0041 0,0072 0,0038 0,0119 0,0197 0,0109
46
Ln
11
12
13
14
15
16
17
18
19
20
x i1
27,1
25,3
24,6
25,1
27,6
22,4
26,3
19,8
22,6
24,5
xi2
27,4
25,2
25
25,8
26,9
22,4
26,1
20,4
23,1
24,9
25,45 27,25
22,4
26,2
20,1
22,85
24,7
0,0
0,2
0,6
0,5
0,4
27,25 25,25
x
di
0,3
Di
24,8
0,1
0,4
0,7
0,7
0,0110 0,0040 0,0161 0,0275 0,0257 0,0000 0,0076 0,0299 0,0219 0,0162
D tb =
RSD% =
0,265
= 0,0133 ;
20
Dtb
0,0133
100 =
100 = 1,19 %
1,118
1,118
47
Vic tham gia th nghim thnh tho, so snh lin phng th nghim
l iu kin rt quan trng trong nh gi phng php. Cc phng th
nghim tham gia phi c kt qu th nghim lin phng trc khi c
cng nhn t ISO 17025 v duy tr cng nhn ISO 17025.
5.2. ng (trueness)
5.2.1. nh ngha
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).
=
Trong :
X tb
100
: chch (bias), %
Xtb: Gi tr trung bnh ca kt qu th nghim
: Gi tr thc hoc gi tr c chp nhn l ng
5.2.2. Cch xc nh ng
Mun xc nh ng cn phi tm c gi tr ng, c nhiu cch
khc nhau xc nh ng, bao gm vic so snh kt qu thc nghim
vi kt qu thc hin bi mt phng php i chiu hoc s dng mu
48
bit nng (mu kim tra hoc mu chun c chng nhn) v phng
php xc nh thu hi ( tm li).
Cch 1: So snh vi phng php chun/i chiu
Phn tch mu chun hoc mu th, thc hin 10 ln bng phng
php kho st v bng mt phng php i chiu. Phng php i chiu
tt nht l phng php tiu chun ca cc t chc c uy tn, nu khng
phng php i chiu l phng php qua thm nh cho kt qu tin cy
trong di o ang thc hin. Tnh ton cc kt qu trung bnh v lp li
(h s bin thin) ca hai phng php.
nh gi tng ng v chm ca hai phng php bng cch
so snh phng sai s2 ca hai phng php , dng tiu chun F (Fisher)
v so snh hai tr gi trung bnh bng tiu chun t (Student). Vic b tr cc
th nghim phi c thc hin theo phng php tham chiu mt cch
nghim ngt v cc php o phi c tin hnh di iu kin lp li.
-
phng php khc nhau. Vi tp s liu nh, tnh ton gi tr Ftn (F thc
nghim) theo cng thc sau y v so snh vi gi tr Fc (F tra bng).
Ftn =
Trong :
S1
S2
>1
49
(n 1 1)S12 + (n 2 1)S 2 2
n1 + n 2 2
50
t tn =
x1 x 2
1
2 1
S c +
n1 n 2
k = n1 + n2 - 2
Trong :
x1 x 2
2
S1
S
+ 2
n1
n2
S1 2 S 2 2
n + n
1
2
k=
2
2
2
S1
S2 2
n 1
n 2
+
n 1 + 1
n2 +1
51
S2 2
= 1,3671
S12
Tra bng ta c: F (0,05; 9; 9) = 3,18 > Ftn : Hai phng sai khng c
s khc nhau c ngha, hai phng php c lp li tng ng..
- So snh hai gi tr trung bnh:
Sc 2 =
10 + 10 2
10,5 10,9
t tn =
= 1,265
1
1
0,50
+
10 10
3,8025 + 5,1984
= 0,50
18
Tra bng c t(0,05; 18) = 2,101 > ttn , do khng c s khc nhau
v kt qu ca hai phng php.
52
x
100
: chch (bias), %
x : Gi tr trung bnh ca kt qu th nghim
: Gi tr thc hoc gi tr c chp nhn l ng
53
Trong :
x
S2
n
54
50 46,5
4,52
10
= 2, 46
Cch 3: Xc nh thu hi
Cc phng php tnh ng theo cch 1 hay cch 2 u gp nhng
kh khn nht nh. Trong nhiu trng hp khng th tm hoc p dng
mt phng php tiu chun so snh kt qu, cng nh khng th d
dng c c cc mu chun hoc mu chun c chng nhn ph hp vi
phng php. Vic xc nh ng do c th thc hin thng qua xc
nh thu hi (cn gi l tm li) ca phng php.
Thm mt lng cht chun xc nh vo mu th hoc mu trng,
phn tch cc mu thm chun , lm lp li ti thiu bn ln bng phng
php kho st, tnh thu hi theo cng thc sau y:
-
i vi mu th:
R% =
55
C m+c C m
100
Cc
i vi mu trng:
R% =
C tt
100
Cc
56
Mu
(ml)
HL SO2
(mg/100ml)
20
7,72
20
7,69
20
7,76
20
7,73
20
12,4
20
12,5
20
12,6
20
12,9
20
10
17,6
10
20
10
17,8
11
20
10
17,8
12
20
10
17,7
13
20
25
32,4
14
20
25
32,1
15
20
25
32,9
16
20
25
31,9
TB
R
(%)
7,73
12,6
97,5
17,7
100
32,3
98,4
57
12,6 7,73
100 = 97,5%
5
17,7 7,73
100 = 100%
10
32,3 7,73
100 = 98, 4%
25
5.2.3. Tiu ch nh gi
Sau khi nh gi thu hi, so snh kt qu vi cc gi tr cho trong
bng 8. thu hi cc nng khc nhau c k vng khc nhau. Trong
trng hp phn tch cc cht hm lng vt c th tham kho tiu chun
ca hi ng chu u (bng 9).
Bng 8: thu hi chp nhn cc nng khc nhau (theo AOAC)
TT
Hm lng
T l cht
n v
thu hi [%]
[%]
1.
100
100%
98-102
2.
10
10-1
10%
98-102
3.
10-2
1%
97-103
4.
0,1
10-3
0,1 %
95-105
5.
0,01
10-4
100 ppm
90-107
6.
0,001
10-5
10 ppm
80-110
7.
0,0001
10-6
1 ppm
80-110
8.
0,00001
10-7
100 ppb
80-110
9.
0,000001
10-8
10 ppb
60-115
10.
0,0000001
10-9
1 ppb
40-120
58
n v
thu hi [%]
1.
1 ppb
50-120%
2.
> 1 n < 10
1-10 ppb
70-110%
3.
10
10ppb
80-110%
TT
59
60
61
62
c hiu, nhy
Gii hn nh lng
lp li
chnh xc ( ng)
ti lp
63
c. Tnh kt qu
LOD ca mi loi thc phm c tnh nh sau:
64
b. Cch xc nh
Vi mi loi i tng, chun b cc mu khc nhau, chun b cc
mu ny thnh hai nhm, trong c mt nhm mu m tnh v mt nhm
mu c gy nhim mc nng khong bng 10 ln LOD (khng thng
bo cho kim nghim vin). B tr cc kim nghim vin phn tch c lp
theo cng mt phng php ang nghin cu.
c. Tnh kt qu
Tnh kt qu theo cc cng thc sau:
Kt qu
Mu chng (+)
Mu chng (-)
TP
FP
Mu c kt qu m tnh (-)
FN
TN
Trong :
65
AC =
TP + TN
100
N
c hiu
SP =
TN
100
TN + FP
nhy
SE =
TP
100
TP + FN
lch dng
PD =
FP
100
FP + TP
66
ND =
lch m
FN
100
FN + TN
d. nh gi kt qu
Cc thng s cn c nh gi theo cc chun mc sau:
chnh xc (AC) 90 %
c hiu (SP) 90 %
nhy (SE) 90 %
lch m (ND) 10 %
TP
37
100 =
100 = 95 %
TP + FN
37 + 2
- c hiu: SP =
TN
38
100 =
100 = 93 %
TN + FP
38 + 3
- lch dng: PD =
- lch m: ND =
67
FP
3
100 =
100 = 7,5 %
FP + TP
3 + 37
FN
2
100 =
100 = 5 %
FN + TN
2 + 38
- chnh xc: AC =
TP + TN
37 + 38
100 =
100 = 94%
n
80
68
c. Tnh kt qu
Tnh gii hn pht hin v gii hn nh lng theo cng thc sau:
S0 =
LC
1,65
LOD = 3,3 S 0
LOQ = 10 S0
Trong :
2.2.2. Xc nh chm ( lp li v ti lp ni b)
a. nh ngha
Nh nu trong chng 2, thm nh phng php ha hc, khi
nim chm c s dng ch mc gn nhau gia cc kt qu phn
tch.
69
log a i + log b i
2
70
RSD r =
Trong :
[(log a
i =1
log b i ) x i ]
2n
log a i + log b i
2
[(log a
n
RSD R =
Trong :
i =1
log b i ) x i ]
2n
71
Ln Ln x ai =
x bi =
Hiu
TB xi =
Hiu/
(Hiu/
x ai + xbi
2
Trung
Trung
x ai xbi
bnh
bnh)2
93
86 1,968 1,934
0,034
1,951
0,017415 0,000303
36
28 1,556 1,447
0,109
1,502
0,072679 0,005282
34
30 1,531 1,477
0,054
1,504
0,036135 0,001306
70
64 1,845 1,806
0,039
1,826
0,021318 0,000454
98
73 1,991 1,863
0,128
1,927
0,066365 0,004404
0,034
2,401
0,014363 0,000206
72
89
83 1,949 1,919
0,030
1,934
0,015671 0,000246
0,112
2,077
0,054083 0,002925
0,043
2,043
0,021181 0,000449
10
54
49 1,732 1,690
0,042
1,711
0,024658 0,000608
11
0,032
2,209
0,014568 0,000212
12
86
68 1,934 1,833
0,102
1,884
0,054149 0,002932
13
62
56 1,792 1,748
0,044
1,770
0,024970 0,000623
14
35
28 1,544 1,447
0,097
1,496
0,064796 0,004199
15
38
28 1,580 1,447
0,133
1,513
0,087630 0,007679
16
71
64 1,851 1,806
0,045
1,829
0,024650 0,000608
Tng KNV A
0,015222
Tng KNV B
0,017214
Tng A+B
0,032436
lp li ca KNV A:
RSDr(A) =
0,015222
= 0,0308 ;
2*8
lp li ca KNV B:
RSDr(B) =
0,017214
= 0,0328 ;
2*8
ti lp ni b:
RSDR=
0,032436
= 0,045
2 16
73
74
2. Cc ngun gy ra khng m bo o
Trong mt php th c rt nhiu ngun gy ra khng m bo o:
-
75
3. Cc cch nh gi khng m bo o
Ti liu ny gii thiu hai cch nh gi khng m bo o hin
nay ang c p dng, u tin l c lng khng m bo o t tt
c cc yu t cu thnh n (theo hng dn ca EURACHEM v cch th
hai l c lng khng m bo o tng t vic b tr th nghim v xc
nh theo phng php thng k. Trong phm vi cc phng th nghim hin
nay Vit Nam th cch tip cn th hai thun li hn v c tnh kh thi
cao.
3.1. Cch 1: c lng khng m bo o theo hng dn ca
EURACHEM
3.1.1. Bc 1: Xc nh cc i lng o
Kt qu o thu c cui cng l gi tr s hc ca i lng o, gi
tr ny ph thuc vo gi tr cc i lng u vo v gi tr cc i lng
trung gian.
Lit k cc i lng o c nh hng n kt qu o cui cng,
thng thng da vo quy trnh thao tc chun (SOP) v cng thc tnh ton
xc nh cc i lng ny.
76
77
Tin hnh cc php o trn cht chun: Khi nim cht chun c
ngha l mt c tnh no ca cht c xc nh chnh xc, s
dng hiu chun thit b hay thm nh phng php. Cht
chun phi c chng nhn c cung cp bi cc t chc uy tn c
nng lc thc hin.
78
-
(x
u % = RSD% =
Trong :
n 1
SD
x
u: khng m bo o
SD: lch chun
xi: Gi tr th i
x : Gi tr trung bnh
n: S ln lm lp li
RSD: lch chun tng i
79
u=
99,7%)
- i vi mu ln (n 10):
u=
80
0,2
3
= 0,12 ml
81
u=
0,2
6
= 0,08 ml
u a , u b , u c : khng m bo o ca cc u vo a, b, c
82
U TH
u u u
= a + b + c
y
a b c
m P
1000 =
V
2,498 mg/l
C ba ngun gy ra khng m bo o ca nng :
- Qu trnh cn: u a = 0,266 mg (theo tnh ton v d trc)
- tinh khit mu th: ub =
0,001
3
=0,00058
- Th tch: uc = 0,1 ml
Vy khng m bo o tng ca nng c tnh theo dng y =
(a.b)/c:
2
83
84
: S ln phn tch lp li
SD
x
100 =6,12
85
uR
CVR
t ,k
: Bc t do (k = n - 1)
: S ln th nghim
SD
x
100 = 6,87
4. Cng b khng m bo o
Phi thng bo cc thng tin sau y khi trnh by kt qu trong
cc bo co thm nh hoc khi c yu cu.
86
a. Kt qu o.
b. khng m bo o.
c. Mc tin cy c s dng trong vic xc nh khong ca
khng m bo o m rng.
4.1. Cch vit khng m bo o chun tng hp
Khi s dng h s ph k = 1 th khng m bo o chnh l
khng m bo o chun v c vit nh sau:
Kt qu (n v) vi khng m bo o chun U (n v)
Ch : Khng s dng du khi bo co khng m bo o chun v
du ny thng gn vi tin cy cao nh 95% v 99%.
4.2. Cch vit khng m bo o m rng
Thng thng, khng m bo o m rng vi h s ph k = 2 c vit
nh sau:
Kt qu (n v) U (n v)
87
88
89
1. Php th nghim lp li
Cho cng mt nhn vin thc hin vic phn tch mt ch tiu no
xc nh trn cng mt mu th ng nht, trn cng mt thit b, cng
phng php trong mt khong thi gian tng i ngn v nh gi mc
lp li cc kt qu kim tra.
Trong phn tch hng ngy, cn thc hin song song ti thiu hai ln
nhm trnh c cc sai s ngu nhin gp phi. nh gi mc chnh
lch gia hai ln lm vi gi tr trung bnh, s chnh lch ny phi tha mn
theo yu cu ca tng phng php.
2. Php th nghim ti lp
Cc phng th nghim nn b tr cho nhn vin thc hin cc php
th nghim ti lp, bng cch cho thc hin trn cc thit b khc nhau hoc
cho cc nhn vin khc nhau thc hin cng mt php th nghim (i tay
nhn vin).
3. Php th nghim trn mu lu
C th b tr nhn vin thc hin cc php th nghim trn mu
lu, tuy nhin nh gi c kt qu cn m bo n nh ca mu
theo thi gian v trong iu kin bo qun ca phng; ng thi ch nn p
dng i vi cc cht c bit l tng i bn vng trong iu kin nht
nh no .
4. Php th nghim trn mu trng
Mu trng l mu tng t nh mu th nhng khng c cht phn
tch. Vic thc hin php th trn mu trng l yu cu cn thit i vi a
90
91
chm ca thi gian lu, din tch pc sc k: Tim trc tip dung
dch chun nhiu ln lp li vo h thng sc k v xc nh lch
chun tng i (RSD). S ln bm ti thiu 5 ln phi cho RSD
nh hn 2%. Nu RSD ln hn 2% cn s dng 6 im;
H s ko ui;
92
93
94
95
0,10
6,314
2,920
2,353
2,132
2,015
1,943
1,895
1,860
1,833
1,812
1,796
1,782
1,771
1,761
1,753
1,746
1,740
1,734
1,729
1,725
1,721
1,717
1,714
1,711
1,708
1,706
1,703
1,701
1,699
1,697
1,645
0,05
12,71
4,303
3,182
2,776
2,571
2,447
2,365
2,306
2,262
2,228
2,201
2,179
2,160
2,145
2,131
2,120
2,110
2,101
2,093
2,086
2,080
2,074
2,069
2,064
2,060
2,056
2,052
2,048
2,045
2,042
1,960
0,01
31,82
6,965
4,541
3,747
3,365
3,143
2,998
2,896
2,821
2,764
2,718
2,681
2,650
2,624
2,602
2,583
2,567
2,552
2,539
2,528
2,518
2,508
2,500
2,492
2,485
2,479
2,473
2,467
2,462
2,457
2,326
0,005
63,66
9,925
5,841
4,604
4,032
3,707
3,499
3,355
3,250
3,169
3,106
3,055
3,012
2,977
2,947
2,921
2,898
2,878
2,861
2,845
2,831
2,819
2,807
2,797
2,787
2,779
2,771
2,763
2,756
2,750
2,576
0,001
318,3
22,33
10,21
7,173
5,893
5,208
4,785
4,501
4,297
4,144
4,025
3,930
3,852
3,787
3,733
3,686
3,646
3,610
3,579
3,552
3,527
3,505
3,485
3,467
3,450
3,435
3,421
3,408
3,396
3,385
3,090
Ph lc 2 (tip theo)
k2
k1
97
Ti
sng
Ch
bin
ng
lnh
Khc
Ti
sng
Ch
bin
ng
X
X
X
Mn
n,
nc
st
X
Pat
Sn phm tht
X
X
X
X
X
X
X
X
C v thy sn
X
X
lnh
Hun
khi
Khc
Ti X
sng
Ch
bin
ng
lnh
Kh
Ln
men
p
Nc
p
m
thp
Khc
Sn phm t rau qu
X
X
X
X
X
X
99
Ph lc 3 (tip)
Sn phm
Yersi- Cl.
L.
E. coli Staph. S.aureus Campy- Salmo- Bacillus Men VK
Tng Coliforms E.
nia
Perfr- monocy- O157
aureus entero- lobacter nella
cereus mc Lactic khun
coli
spp.
ingens togenes &VTEC
toxins
spp.
spp.
lc
Ti sng
Ch bin
ng lnh
Ln men
Kh
Khc
X
X
X
X
X
X
X
Ng cc v sn phm
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
X
m thp
Kh
Khc
X
X
X
X
Trng
sa
Sn phm khc
Bia
Nc st
Gia v
Mayonnaise
M
Trng
Go
Thc
n
chn nui
X
X
X
X
X
X
X
X
2.
3.
4.
5.
6.
ICH (1996),
Validation of
Analytical
Procedures:
Text
and
8.
9.
101
Bin tp:
Trnh by ba:
103