Measurement Uncertainty for Drugs worked example for

9-THC

The calculations relate to this laboratory procedures defined in the associated

standard Operation Procedure

1. Contents
1.

Contents_____________________________________________________________ 2

2.

Summary ____________________________________________________________ 3

3.

Principle _____________________________________________________________ 3

4.

Method Procedure_____________________________________________________ 3

5.

Standards____________________________________________________________ 3

6.

Identified sources of uncertainty _________________________________________ 4

7.

Uncertainty of the analysis of 9-THC in blood by GC/MS.____________________ 5

Uncertainty of stock solution of

9-THC __________________________________________ 5

Uncertainty of dilution - adding 900 L of methanol to 100 L of Stock 9-THC __________ 5

Uncertainty of purity of 9-THC _________________________________________________ 5
Combined Uncertainty of Working Solution of THC UWorkingStd _________________________ 6

8.

The Standard Uncertainty of the calibration curve __________________________ 7

Estimation of uncertainty from calibration curves for this assay UPred __________________ 7
Estimation of precision uncertainty from Quality Control Material UQC _________________ 11
Uncertainty associated with mass of test sample Um _______________________________ 11
Combining Uncertainties______________________________________________________ 12

Part II. Top Down Approach _______________________________________________ 13

Performance of Quality Controls _______________________________________________ 13

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 2 of 14

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.

Add 1 mL blood (or control or standard) to silanised glass extraction tubes.

Add 25 ng d3-THC (25 L of IS2) to each tube, mix.
Add 1.0 mL 1.0 M ammonium sulphate, vortex.
Add 7 mL hexane whilst vortexing and extract for 60 min on a wheel. Ensure blood is
mixing with solvent, if not agitate tubes.
5. Centrifuge at 3250 rpm, 10 min.
6. Place tubes in an alcohol freezing bath. When aqueous layer is frozen decant solvent to a
fresh glass extraction tube.
7. Include a ``mix' by adding 100 ng of THC (100 L WS3) and 25 ng IS (25 L IS2) to a
separate tube.
8. Evaporate to dryness in a Savant Concentrator (or in heating block under nitrogen).
9. When dry, add 25 L pentafluoro-1-propanol, vortex, then add 50 L pentafluoropropionic
anhydride. Stopper and mix on a vortex mixer.
10. Incubate in reaction block at 65 C for 25 min, vortex briefly at 10 min. Evaporate to
dryness under nitrogen.
11. Reconstitute with 100 L anhydrous ethyl acetate and transfer to MS injection vials
(screw-on with teflon lined septa).
12. Inject 1 L into the GC/MS.
See- Determination of D9-THC in Whole Blood using Gas Chromatography-Mass
Spectrometry Hok Chi Chu M and Drummer O.H. Journal of Analytical Toxicology, Volume
26, Number 8, November/December 2002, pp. 575-581(7).

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

Date Effective: 16/11/2005

Page 3 of 14

6. Identified sources of uncertainty

Result

GC Determination

Extraction

Sample Homogeneity

Sample Mass

Precision

GC Calibration

Calibration Standards

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 4 of 14

7. Uncertainty of the analysis of

9-THC in blood by GC/MS.

Uncertainty of stock solution of 9-THC

The uncertainty of the stock solution of 9-THC can be determined from the positive
displacement pipette which is used to dispense the initial volume of 100 L of stock.
100 L 0.21 % (from manufacturers specifications)
= 0.21 L or 0.00021 mL
assuming rectangular distribution
USpecification 100 L = 0.00021
3
= 0.0001212 (V1)

Uncertainty of dilution - adding 900 L of methanol to 100 L of Stock 9THC

The uncertainty of the dilution of the stock solution of 9-THC can be determined from the
positive displacement pipette which is used to dispense a working volume of 900 L.
1000 L 0.32 % (from manufacturer specifications)
= 3.2 L or 0.0032 mL
assuming rectangular distribution
USpecification 1000 L = 0.0032
3
= 0.001847 (V2)
Also the uncertainty of the pipetter can be calculated from laboratory calibration data which
over the last 6 months has shown the relative standard deviation at 1000 L to be 0.50%.
So U V1 +V2 = U V1 + U V2
2

U V1 +V2 = 0.00012122 + 0.0018472

U V1 +V2 = 0.000000014 + 0.000003411
U V1 +V2 = 0.00185

Uncertainty of purity of 9-THC

Purity of 9-THC is 98.5% min
100 1.5 %
1.0 0.015
assuming rectangular distribution
Upurity 9-THC = 0.015

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 5 of 14

3
Upurity

9-THC

= 0.00866 (S)

Combined Uncertainty of Working Solution of THC UWorkingStd

Concentration = W / V
Where combining all the uncertainties at concentration 100 ng/mL:
U 100
= 100
100

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

(0.001212)2 + (0.0866)2 + (0.008849)2

U 100 = 0.000001468 + 0.00749956 + 0.000078304

U 100 = 0.007579

U 100 = 0.08705
Therefore Combined Uncertainty of Working Solution of THC is 100 g/mL 0.09
g/mL

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 6 of 14

8. The Standard Uncertainty of the calibration curve

Estimation of uncertainty from calibration curves for this assay UPred
Eleven different assays run on two different GC/MS systems gave the following results
Table 1. Calculation of response ratios (Y) from known concentrations of 9-THC from 11 calibration
curves.

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

Concentration of 9THC (ng/mL)

5 ng/mL 10 ng/mL 25 ng/mL 50 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

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 7 of 14

Linear Regression calculations for uncertainty associated with calibration curves.

Table 2. Calibration data compiled from 11 calibration curves constructed for the determination of 9-THC (using 2, 25, 100
ng/mL points).
Amount X

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

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 8 of 14

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

Document ID: Uncertainty example for THC.DOC

Date Effective: 16/11/2005

Page 9 of 14

To calculate relative uncertainty associated with calibration curves:

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 1 (23.425 1.68)

+
+
1.0635189 1 33
107.06105

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

Uncertainty example for THC.DOC

Page 10 of 14

Estimation of precision uncertainty from Quality Control Material UQC

Quality control data produced from several analysts give data on precision uncertainty (UQC) of the method.
From our QC reporter the following results have been obtained from the last 24 months of assays:

QC name

Batch no. and

number of
points

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

So 6.2 % is average CV over 24 months.

Uncertainty associated with mass of test sample U m

A blood sample (1 mL) weighed 10 times will enable the uncertainty to be determined for the volume of sample
dispensed for analysis.

Replicate Mass of test samples (g)

1
1.08067
2
1.07679
3
1.07882
4
1.07511
5
1.07556
6
1.07313
7
1.07684
8
1.06637
9
1.07661
10
1.07366
Mean
1.075356
SD
0.0038694
The mean was and 1.075 g the SD 0.0038694.

UPipettor 1000 L = ( USpecification 1000 L)2 + (ULab repeatability 1000 L)2

= (0.001847)2 + (0.0045)2
= (0.0000034) + (0.002025)
= 0.045037
UMassSample = ( UMassSample)2
= (0.001847)2
Uncertainty example for THC.DOC
Page 11 of 14

= (0.0000034)
= 0.045037

UCombinedMTSample = ( Upipettor 1000 L)2 + (UMassSample)2

= (0.001847)2 + (0.0038694)2
= (0.0000034) + (0.0000149)
= 0.00428

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

0.0009 + 0.00021 + 0.003844 + 0.000010

0.004979
= 0.07 ng/mL

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

k =2 for a 95% confidence interval

UExpanded = expanded uncertainty of the measure

Uncertaintycombined

= k x UExpanded

= 2 x 7 ng/mL

= 14 ng/mL
So at 100 ng 14 ng

Uncertainty example for THC.DOC

Page 12 of 14

Um
+
Mass test

Part II. Top Down Approach

Performance of Quality Controls
Since most of the uncertainty is assessed through quality controls [preparation of standards, variation in peak
area ratio it is apposite to also use QCs to estimate uncertainty].
Validation data for intra-assay variability CV of quality controls. From our QC reporter the following results
have been obtained from the last 24 months of assays:

QC name

Batch no. and

number of
points

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

The average CV is 6.2 % over 24 months.

Long Term precision = 6.2 %

Bias

= 1.44 @ 19.71

Bias = 19.71(observed) 20.21(expanded)

= -0.5
Where U bias =

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

ODonnell, G & Hibbert D. Anlayst 130, 721-29(2005)

Uncertainty example for THC.DOC

Page 13 of 14

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

k =2 for a 95% confidence interval

UExpanded = expanded uncertainty of the measure

Uncertaintycombined

= k x UExpanded

= 2 x 6.2 ng/mL

= 12.4 ng/mL

Dr Dimitri Gerostamoulos PhD Monash BSc(Hons)

Manager - Toxicology
Victorian Institute of Forensic Medicine
57-83 Kavanagh St., Southbank 3006
Victoria, AUSTRALIA
dimitrig@vifm.org

Uncertainty example for THC.DOC

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