Beruflich Dokumente
Kultur Dokumente
9-THC
1. Contents
1.
Contents_____________________________________________________________ 2
2.
Summary ____________________________________________________________ 3
3.
Principle _____________________________________________________________ 3
4.
Method Procedure_____________________________________________________ 3
5.
Standards____________________________________________________________ 3
6.
7.
9-THC __________________________________________ 5
8.
2. Summary
This report describes an uncertainty estimate for the measurement of 9-THC in blood by
GC/MS.
3. Principle
Specimens of blood are analysed for the determination of 9-THC by GC/MS.
4. Method Procedure
1.
2.
3.
4.
5. Standards
Stock solutions of 9-THC are prepared as follows:
1. Prepare dilutions by adding 100 L STOCK (1 mg/mL) to 900 L methanol in silanised
glass vials (WS1, 1/10; 100 g/mL); then
add 100 L WS1 to 900 L methanol in silanised glass vials (WS2, 1/100, 10 g/mL),
add 100 L WS2 to 900 L methanol in silanised glass vials (WS3, 1/1000, 1 g/mL), and
add 100 L WS3 to 900 L methanol in silanised glass vials (WS4, 1/10,000, 100 ng/mL),
Working Internal Standard 9-THC-D3
2. Prepare dilutions by adding 100 L STOCK (100 g/mL) to 900 L methanol in silanised
glass vials (IS1, 1/10; 10 g/mL), and add 100 L IS1 to 900 L methanol in silanised
glass vials (IS2, 1/100, 1 g/mL).
Document ID: Uncertainty example for THC.DOC
Result
GC Determination
Extraction
Sample Homogeneity
Sample Mass
Precision
GC Calibration
Calibration Standards
3
Upurity
9-THC
= 0.00866 (S)
U 100 =
U 100 =
Us
S
0.00866
0.1
U
+ v1
V1
2
U (V1 +V2 )
(V1 + V2 )
0.0001212
+
0.1
where U V +V = U V 2 + U V
1
0.00185
+
(1.0)
2
2
U 100 = 0.08705
Therefore Combined Uncertainty of Working Solution of THC is 100 g/mL 0.09
g/mL
Replica
te
Numbe
r
1Y
2
3
4
5
6
7
8
9
10
11
Sum
Mean
n
SD
Respon
se
Ratio X
1 ng/mL
2 ng/mL
100
ng/mL
Response ratio Y
0.062560
621
0.049182
712
0.373704
528
0.203368
982
0.048377
686
0.050155
592
0.111210
007
0.045470
522
0.046593
264
0.043136
229
0.050474
712
1.084234
855
0.098566
805
11
0.103125
071
0.128410
915
0.092482
309
0.423910
183
0.221607
09
0.093456
743
0.093162
854
0.277918
549
0.092571
855
0.081692
509
0.089714
551
0.088308
587
1.683236
144
0.153021
468
11
0.110464
086
0.228555
167
0.201950
036
0.463017
527
0.337645
914
0.229941
63
0.232216
041
0.288673
92
0.221270
298
0.229806
972
0.209960
181
0.217562
753
2.860600
441
0.260054
586
11
0.077985
709
0.464852
795
0.420435
057
0.606303
245
0.528717
476
0.418635
89
0.426423
363
0.516486
273
0.440230
504
0.455622
129
0.434483
445
0.420844
784
5.133034
961
0.466639
542
11
0.059878
251
1.026603
852
0.999186
141
1.190583
652
1.258000
153
1.130049
647
1.133886
119
1.201099
446
1.100896
861
0.975635
17
1.068005
632
1.081991
313
12.16593
799
1.105994
362
11
0.087984
28
2.009213
006
1.995677
901
2.198491
79
2.145557
351
2.248224
005
2.232503
864
2.320320
62
2.229787
387
2.162878
176
2.147182
608
2.131746
564
23.82158
327
2.165598
479
11
0.097810
019
4.641011
772
3.769606
03
4.238783
703
4.097202
842
4.440535
942
4.280899
363
4.355789
43
4.500810
779
4.390148
996
4.386608
706
4.689465
552
47.79086
312
4.344623
92
11
0.255233
348
0.04
0.08
0.2
0.4
Amount Y
X-X
(X-X)
Y-Y
(Y-Y)
(x-x)(y-y)
Predicted Y
Residuals
Standard Residuals
0.04
0.1284109
-1.739469
3.0257524
-1.739469
3.0257527
3.0257526
0.0016
0.1237088
0.0047021
0.0283907
0.04
0.0924823
-1.775398
3.1520367
-1.775398
3.152037
3.1520368
0.0016
0.1237088
-0.031227
-0.188543
0.04
0.4239102
-1.44397
2.0850486
-1.44397
2.0850488
2.0850487
0.0016
0.1237088
0.3002013
1.8125867
0.04
0.2216071
-1.646273
2.7102142
-1.646273
2.7102145
2.7102144
0.0016
0.1237088
0.0978982
0.5911002
0.04
0.0934567
-1.774423
3.1485776
-1.774423
3.1485779
3.1485777
0.0016
0.1237088
-0.030252
-0.182659
0.04
0.0931629
-1.774717
3.1496207
-1.774717
3.1496209
3.1496208
0.0016
0.1237088
-0.030546
-0.184434
0.04
0.2779185
-1.589961
2.5279772
-1.589961
2.5279774
2.5279773
0.0016
0.1237088
0.1542097
0.9311033
0.04
0.0925719
-1.775308
3.1517187
-1.775308
3.151719
3.1517189
0.0016
0.1237088
-0.031137
-0.188002
0.04
0.0816925
-1.786187
3.1904655
-1.786187
3.1904658
3.1904656
0.0016
0.1237088
-0.042016
-0.253691
0.04
0.0897146
-1.778165
3.1618721
-1.778165
3.1618724
3.1618722
0.0016
0.1237088
-0.033994
-0.205254
0.04
0.0883086
-1.779571
3.1668741
-1.779571
3.1668744
3.1668743
0.0016
0.1237088
-0.0354
-0.213743
1.0266039
-0.841276
0.7077454
-0.841276
0.7077456
0.7077455
1.144687
-0.118083
-0.712975
0.9991861
-0.868694
0.7546289
-0.868694
0.754629
0.7546289
1.144687
-0.145501
-0.87852
1.1905837
-0.677296
0.4587302
-0.677296
0.4587303
0.4587303
1.144687
0.0458966
0.2771194
1.2580002
-0.60988
0.3719533
-0.60988
0.3719534
0.3719534
1.144687
0.1133131
0.6841737
1.1300496
-0.73783
0.5443935
-0.73783
0.5443936
0.5443936
1.144687
-0.014637
-0.088379
1.1338861
-0.733994
0.5387469
-0.733994
0.538747
0.538747
1.144687
-0.010801
-0.065215
1.2010994
-0.66678
0.4445962
-0.666781
0.4445963
0.4445963
1.144687
0.0564124
0.3406127
1.1008969
-0.766983
0.588263
-0.766983
0.5882631
0.5882631
1.144687
-0.04379
-0.264401
0.9756352
-0.892245
0.7961007
-0.892245
0.7961008
0.7961008
1.144687
-0.169052
-1.020719
1.0680056
-0.799874
0.6397989
-0.799874
0.639799
0.6397989
1.144687
-0.076681
-0.462995
1.0819913
-0.785889
0.6176209
-0.785889
0.617621
0.617621
1.144687
-0.062696
-0.378551
4.6410118
2.7731319
7.6902603
2.7731318
7.6902598
7.6902601
16
4.3352439
0.3057679
1.846197
3.769606
1.9017261
3.6165622
1.901726
3.6165619
3.6165621
16
4.3352439
-0.565638
-3.415267
4.2387837
2.3709038
5.6211848
2.3709037
5.6211844
5.6211846
16
4.3352439
-0.09646
-0.582417
4.0972028
2.2293229
4.9698807
2.2293228
4.9698803
4.9698805
16
4.3352439
-0.238041
-1.437269
4.4405359
2.572656
6.618559
2.5726559
6.6185586
6.6185588
16
4.3352439
0.1052921
0.6357433
4.2808994
2.4130194
5.8226628
2.4130194
5.8226624
5.8226626
16
4.3352439
-0.054345
-0.328127
4.3557894
2.4879095
6.1896937
2.4879094
6.1896933
6.1896935
16
4.3352439
0.0205456
0.1240521
4.5008108
2.6329309
6.9323249
2.6329308
6.9323245
6.9323247
16
4.3352439
0.1655669
0.9996769
4.390149
2.5222691
6.3618413
2.522269
6.3618409
6.3618411
16
4.3352439
0.0549051
0.3315118
4.3866087
2.5187288
6.3439947
2.5187287
6.3439943
6.3439945
16
4.3352439
0.0513648
0.3101359
4.6894656
2.8215856
7.9613455
2.8215856
7.961345
7.9613453
16
4.3352439
0.3542217
2.1387562
Sum
55.44
61.640037
-4.44E-15
107.06105
-2.75E-06
107.06105
Average
1.68
1.8678799
33
33
Table 3. Summary of statistical outputs associated with calibration curves for determination of uncertainty for 9-THC
SUMMARY OUTPUT
Regression Statistics
r=
0.995892
b=
1.063519
Multiple R
0.9958922
a=
0.081168
R Square
0.9918013
Six/y
0.1682705
Adjusted R Square
0.9915368
Sb
0.017367
Standard Error
0.1682705
Sa
0.041344
Observations
33
Sxx
107.06105
ANOVA
df
SS
MS
Significance F
Regression
106.18328
106.18328
3750.0768
6.569E-34
Residual
31
0.8777638
0.028315
Total
32
107.06105
Coefficients
Standard Error
t Stat
P-value
Lower 95%
Upper 95%
Lower 95.0%
Upper 95.0%
Intercept
0.0811681
0.0413437
1.9632512
0.0586422
-0.003153
0.1654892
-0.003153
0.1654892
X Variable 1
1.0635189
0.017367
61.237871
6.569E-34
1.0280987
1.0989392
1.0280987
1.0989392
UPredC =
S
M
1 1 (Cunknown Ccal )
+ +
P n
Sxx
Where Sxx =
(Cj C ) 2
j =1
And where:
UConcentration = Uncertainty associated with calibration model
S = Std Error (residual standard deviation) = 0.1682705
M = Slope of calibration curve = 1.0635189
P = no of measurements taken to determine unknown = 1
n = total number of measurements for calibration = 33
CUnknown = determined concentration of unknown from equation = 23.425 *
Ccal = mean value of calibration concentration = 1.68
Sxx = 107.06105
*This assumes that if 25 ng/mL of 9-THC was added then the resultant expected (y)
concentration would be 23.425ng/mL.
Hence
UPredC
0.1682705
1 + .03 + 4.41659
1.0635189
0.1682705
5.44659
1.0635189
UPredC
= 0.369 ng/mL
UPred =
0.369 ng/mL
25
= 0.01476 ng/mL
QC name
Target
Concentration
Batch
mean
(ng/mL)
(ng/mL)
05/2005
20.21
19.71
7.3
QC THC
CV (%)
n=27
QCTHCSAL
10/2004, n=18
10
10.6
8.2
QCTHCSAL
12/2004, n=20
10
10.4
4.2
QCTHCSAH
10/2004 n=19
20
21.04
6.9
QCTHCSAH
12/2004 n = 20
50
50.8
4.4
= (0.0000034)
= 0.045037
Combining Uncertainties
UCombined
C
=
=
=
UCombined
C
0.03
1
UWorkingStd
UPred
+
Calibratio nGC
Working std
0.369
25
6.2
100
3.2
1000
UQC
+
PrecisionQC
If C = 100 ng/mL
Uncertaintycombined = C x 0.07 ng/mL
= 100 x 0.07 ng/mL
= 7 ng/mL
Uncertaintycombined = k x UExpanded
Where
Uncertaintycombined
= k x UExpanded
= 2 x 7 ng/mL
= 14 ng/mL
So at 100 ng 14 ng
Um
+
Mass test
QC name
Target
Concentration
Batch
mean
(ng/mL)
(ng/mL)
05/2005
20.21
19.71
7.3
n=27
(observed)
(expected)
S=1.44
QCTHCSAL
10/2004, n=18
10
10.6
8.2
QCTHCSAL
12/2004, n=20
10
10.4
4.2
QCTHCSAH
10/2004 n=19
20
21.04
6.9
QCTHCSAH
12/2004 n = 20
50
50.8
4.4
QC THC
= 1.44 @ 19.71
U bias =
S
n
1.44
27
U bias = 0.28
Bias 2U b , so bias is not significant.
U c = 1.442 + 0.282
U c = 2.0736 + 0.0784
1
CV (%)
U c = 1.5
U (95%CI ) = 3
So C = 20 ng 3 ng for qc data.
So using the top-down approach
If concentration = 100 ng/mL, then the CV of 6.2 % can give an estimated uncertainty of
UncertaintyQC
= C x 6.2%
= 6.2 ng/mL
UncertaintyQC = k x UExpanded
Where
Uncertaintycombined
= k x UExpanded
= 2 x 6.2 ng/mL
= 12.4 ng/mL