kirk newland, max scherer, stephen hecht, valerie troude ... · kirk newland, max scherer, stephen...

Kirk Newland, Max Scherer, Stephen Hecht, Valerie Troude and G. L. Prasad

October 26, 2018

CORESTA Congress

Kunming, China

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Outline of the Presentation

• Background

• Selection of Total 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL)

• Study Plan Design

• Participating Laboratories

• Summary of Performance

• Quality Control Samples

• NIST Standard

• Multi-lot Samples from Smoker and Non-smoker subjects

• Conclusion

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Background• NNAL (4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol) is a major

metabolite of the tobacco-specific nitrosamine NNK

(4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone)

• NNK is a known lung carcinogen (IARC Group 1 carcinogen) and is

designated as a Harmful and Potentially Harmful Constituent by the US FDA

• Measurement of NNAL is a widely accepted biomarker of exposure to NNK

• NNAL is also considered as a carcinogen

• NNAL is enzymatically conjugated as a glucuronide and excreted in urine

• Urinary levels of total NNAL (free + glucuronidated NNAL) are widely used

as a biomarker of exposure to cigarettes and smokeless tobacco products

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A

N

OO

HO

OH

OH

COOH

NN

O

HO

N

NO

N

O

OH

HO

HOHOOC

N

OH

N

NO

NNAL

NNAL-O-Glucuronide NNAL-N-Glucuronide

Quantitation of the “Total” Excretion of NNAL in Human

Urine as a Measure of NNK exposure

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Inter-laboratory Testing Study Plan Design

• Shared Quality Controls at 3

concentrations

• 15.0 pg/mL (n=6)

• 70.0 pg/mL (n=6)

• 750 pg/mL (n=6)

• Individual Lots of Smoker Urine

from 9 Volunteers (n=3)

• NIST (SRM 3672) Smoker

Urine Standard (n=3)

• Control Blank Matrix (n=3)

• 1 Set of Imbedded Standards

• 9 Standard Concentrations

• Range: 5.00 to 1000 pg/mL

• NNAL reference material used

by the organizing lab was

synthesized and characterized by

Aptochem Synthèse.

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Inter-laboratory Participating Laboratories

• 9 Laboratories initially

included

• 5 Laboratories discontinued

• Issues with sample shipment to

some locations

• Issues with assay performance at

the lab

• 4 Laboratories provided final

results

• Celerion, Inc – Nebraska,

USA

• ABF GmbH – Munich,

Germany

• SEITA – Fleur les Aubrais,

France

• Univ. of Minnesota –

Minnesota, USA

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Participating Laboratories

• 9 Laboratories initially included

• 5 Laboratories discontinued

• Issues with sample shipment to some locations

• Issues with assay performance at the lab

• 4 Laboratories provided final results

• Celerion (USA), coordinating lab

• ABF GmbH (Germany)

• University of Minnesota Cancer Center (USA)

• SEITA (France)

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Summary of Performance – Quality Controls

• Analytical results were provided as concentrations in pg/mL

(quantitation with each laboratory’s standard set).

• The laboratories also provided the analyte and internal standard

area counts to allow quantitation with the imbedded standards.

• Analysis of the Quality Control sample data gives a clear

evaluation of the sample processing recovery, and detection

selectivity.

• The hydrolysis was performed on the samples however no

conjugated analytes were present in the samples.

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Inter-laboratory Comparison Summary Data – Total NNAL

Lab A Lab B Lab C Lab D

12

13

14

15

16

17

18

19

20

21

Ran

ge

QC A Statistics (15.0 pg/mL)


45

50

55

60

65

70

75

80

85

90

95

100

105

Ran

ge

QC B Statistics (70.0 pg/mL)


600

700

800

900

1000

1100

Ran

ge

QC C Statistics (750 pg/mL)

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QC Sample Data – Independent Standards

Quality Control A (15.0 pg/mL)

Laboratory A B C D

Rep 1 18.2 19.8 13.7 18.3

Rep 2 18.9 17.7 14.5 18.2

Rep 3 18.1 19.4 14.4 19.8

Rep 4 18.5 18.0 15.7 16.1

Rep 5 16.5 18.9 14.6 19.2

Rep 6 16.0 19.2 15.3 18.2

Mean 17.7 18.8 14.7 18.3

S.D. 1.2 0.8 0.7 1.3

% C.V. 6.6% 4.3% 4.8% 6.9%

% R.E. 21.3% 25.3% -2.0 22.0%

n 6 6 6 6

Quality Control B (70.0 pg/mL)

Laboratory A B C D

Rep 1 75.9 70.2 68.3 91.4

Rep 2 74.9 72.6 67.8 97.9

Rep 3 75.3 71.2 69.9 97.5

Rep 4 74.8 73.0 70.0 99.3

Rep 5 73.5 82.9 68.7 87.7

Rep 6 74.5 77.8 70.9 90.7

Mean 74.8 74.6 69.3 94.1

S.D. 0.8 4.8 1.2 4.8

% C.V. 1.1% 6.5% 1.7% 5.0%

% R.E. 6.9% 6.6% -1.0% 34.4%

n 6 6 6 6

Quality Control C (750 pg/mL)

Laboratory A B C D

Rep 1 836 751 731 982

Rep 2 826 730 715 1060

Rep 3 813 770 752 959

Rep 4 805 664 769 936

Rep 5 823 769 759 990

Rep 6 831 783 745 933

Mean 822 745 745 977

S.D. 12 43 20 47

% C.V. 1.4% 5.8% 2.6% 4.8%

% R.E. 9.6% -0.7% -0.1% 30.3%

n 6 6 6 6

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Laboratory A B C D

Rep 1 18.2 19.8 13.7 18.3

Rep 2 18.9 17.7 14.5 18.2

Rep 3 18.1 19.4 14.4 19.8

Rep 4 18.5 18.0 15.7 16.1

Rep 5 16.5 18.9 14.6 19.2

Rep 6 16.0 19.2 15.3 18.2

Mean 17.7 18.8 14.7 18.3

S.D. 1.2 0.8 0.7 1.3

% C.V. 6.6% 4.3% 4.8% 6.9%

% R.E. 21.3% 25.3% -2.0 22.0%

n 6 6 6 6


Laboratory A B C D

Rep 1 75.9 70.2 68.3 91.4

Rep 2 74.9 72.6 67.8 97.9

Rep 3 75.3 71.2 69.9 97.5

Rep 4 74.8 73.0 70.0 99.3

Rep 5 73.5 82.9 68.7 87.7

Rep 6 74.5 77.8 70.9 90.7

Mean 74.8 74.6 69.3 94.1

S.D. 0.8 4.8 1.2 4.8

% C.V. 1.1% 6.5% 1.7% 5.0%

% R.E. 6.9% 6.6% -1.0% 34.4%

n 6 6 6 6


Laboratory A B C D

Rep 1 836 751 731 982

Rep 2 826 730 715 1060

Rep 3 813 770 752 959

Rep 4 805 664 769 936

Rep 5 823 769 759 990

Rep 6 831 783 745 933

Mean 822 745 745 977

S.D. 12 43 20 47

% C.V. 1.4% 5.8% 2.6% 4.8%

% R.E. 9.6% -0.7% -0.1% 30.3%

n 6 6 6 6

A B C D21.3% 25.3% -2.0% 22.0%

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Laboratory A B C D

Rep 1 18.2 19.8 13.7 18.3

Rep 2 18.9 17.7 14.5 18.2

Rep 3 18.1 19.4 14.4 19.8

Rep 4 18.5 18.0 15.7 16.1

Rep 5 16.5 18.9 14.6 19.2

Rep 6 16.0 19.2 15.3 18.2

Mean 17.7 18.8 14.7 18.3

S.D. 1.2 0.8 0.7 1.3

% C.V. 6.6% 4.3% 4.8% 6.9%

% R.E. 21.3% 25.3% -2.0 22.0%

n 6 6 6 6


Laboratory A B C D

Rep 1 75.9 70.2 68.3 91.4

Rep 2 74.9 72.6 67.8 97.9

Rep 3 75.3 71.2 69.9 97.5

Rep 4 74.8 73.0 70.0 99.3

Rep 5 73.5 82.9 68.7 87.7

Rep 6 74.5 77.8 70.9 90.7

Mean 74.8 74.6 69.3 94.1

S.D. 0.8 4.8 1.2 4.8

% C.V. 1.1% 6.5% 1.7% 5.0%

% R.E. 6.9% 6.6% -1.0% 34.4%

n 6 6 6 6


Laboratory A B C D

Rep 1 836 751 731 982

Rep 2 826 730 715 1060

Rep 3 813 770 752 959

Rep 4 805 664 769 936

Rep 5 823 769 759 990

Rep 6 831 783 745 933

Mean 822 745 745 977

S.D. 12 43 20 47

% C.V. 1.4% 5.8% 2.6% 4.8%

% R.E. 9.6% -0.7% -0.1% 30.3%

n 6 6 6 6

A B C D21.3% 25.3% -2.0% 22.0%

A B C D6.9% 6.6% -1.0% 34.4%

A B C D9.6% -0.7% -0.1% 30.3%

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10

11

12

13

14

15

16

17

18

19

20

Range

QC A Statistics (15.0 pg/mL) Shared Stds


50

60

70

80

90

Ra

nge

QC B Statistics (70.0 pg/mL) Shared Stds


450

500

550

600

650

700

750

800

850

900

950

1000

1050

Ran

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QC C Statistics (750 pg/mL) Shared Stds

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QC Sample Data – Embedded Standards


Laboratory A B C D

Rep 1 16.6 17.0 13.7 13.5

Rep 2 17.2 15.0 14.5 15.8

Rep 3 16.5 16.7 14.4 15.1

Rep 4 16.9 15.3 15.7 15.1

Rep 5 15.1 16.1 14.6 15.1

Rep 6 14.7 16.4 15.3 16.2

Mean 16.2 16.1 14.7 15.1

S.D. 1.0 0.8 0.7 0.9

% C.V. 6.3% 4.9% 4.8% 6.1%

% R.E. 8.0% 7.3% -2.0% 0.7%

n 6 6 6 6


Laboratory A B C D

Rep 1 69.0 64.5 68.3 76.0

Rep 2 68.1 66.7 67.8 67.5

Rep 3 68.5 65.4 69.9 69.9

Rep 4 68.0 64.5 70.0 69.8

Rep 5 66.8 66.7 68.7 75.3

Rep 6 67.7 65.4 70.9 74.4

Mean 68.0 65.5 69.3 72.2

S.D. 0.7 1.0 1.2 3.5

% C.V. 1.1% 1.5% 1.7% 4.9%

% R.E. -2.9% -6.4% -1.0% 3.1%

n 6 6 6 6


Laboratory A B C D

Rep 1 759 706 731 707

Rep 2 750 686 715 741

Rep 3 738 770 752 705

Rep 4 731 664 769 743

Rep 5 747 769 759 800

Rep 6 754 783 745 720

Mean 747 730 745 736

S.D. 10.4 50.6 19.6 35.3

% C.V. 1.4% 6.9% 2.6% 4.8%

% R.E. -0.4% -2.7% -0.6% -1.9%

n 6 6 6 6

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QC Sample Data – Embedded Standards


Laboratory A B C D

Rep 1 16.6 17.0 13.7 13.5

Rep 2 17.2 15.0 14.5 15.8

Rep 3 16.5 16.7 14.4 15.1

Rep 4 16.9 15.3 15.7 15.1

Rep 5 15.1 16.1 14.6 15.1

Rep 6 14.7 16.4 15.3 16.2

Mean 16.2 16.1 14.7 15.1

S.D. 1.0 0.8 0.7 0.9

% C.V. 6.3% 4.9% 4.8% 6.1%

% R.E. 8.0% 7.3% -2.0% 0.7%

n 6 6 6 6


Laboratory A B C D

Rep 1 69.0 64.5 68.3 76.0

Rep 2 68.1 66.7 67.8 67.5

Rep 3 68.5 65.4 69.9 69.9

Rep 4 68.0 64.5 70.0 69.8

Rep 5 66.8 66.7 68.7 75.3

Rep 6 67.7 65.4 70.9 74.4

Mean 68.0 65.5 69.3 72.2

S.D. 0.7 1.0 1.2 3.5

% C.V. 1.1% 1.5% 1.7% 4.9%

% R.E. -2.9% -6.4% -1.0% 3.1%

n 6 6 6 6


Laboratory A B C D

Rep 1 759 706 731 707

Rep 2 750 686 715 741

Rep 3 738 770 752 705

Rep 4 731 664 769 743

Rep 5 747 769 759 800

Rep 6 754 783 745 720

Mean 747 730 745 736

S.D. 10.4 50.6 19.6 35.3

% C.V. 1.4% 6.9% 2.6% 4.8%

% R.E. -0.4% -2.7% -0.6% -1.9%

n 6 6 6 6

A B C D

-2.9% -6.4% -1.0% 3.1%

A B C D

-0.4% -2.7% -0.6% 1.9%

A B C D

8.0% 7.3% -2.0% 0.7%

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Comparison of Algycone Only QCs

• With QCs prepared with aglycone only the impact of

differential hydrolysis is removed.

• A bias between laboratories is observed when each laboratory

uses it’s own source of reference material to prepare standard

calibrators (~20% R.E.)

• When a single set of calibrators is used by all laboratories this

bias is significantly reduced (-6 to 8% R.E.)

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Summary of Performance – Smoker Urine Lots

• Smoker lots will have present unconjugated NNAL and the N-and O-glucuronides of NNAL.

• An enzymatic hydrolysis is typically performed so that a total NNAL concentration may be measured

• As there is no known concentration for the lots of urine only a comparative analysis may be performed.

• The urine lots were classified for comparison purposes

• low concentration (10 to 45 pg/mL)

• mid concentration (90 to 300 pg/mL)

• high concentration (400 to 600 pg/mL)

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Lab A Lab B Lab C Lab D Lab A Lab B Lab C Lab D

10

20

30

40

50R

an

geLot 1 Lot 2

Low Level Smoker Urine Lots (pg/mL)

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10

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50R

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Lot 1 Lot 2

52% difference36% difference

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A B C D A B C D A B C D A B C D A B C D

0

50

100

150

200

250

300

350

400

450

Ran

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Mid Level Smoker Urine Lots (pg/mL)

Lot 3 Lot 4 Lot 5 Lot 6 Lot 9

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0

50

100

150

200

250

300

350

400

450

Ran

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Mid level Smoker Urine Lots (pg/mL)

Lot 3 Lot 4 Lot 5 Lot 6 Lot 9

31% 46% 42%

38% 40%

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A B C D A B C D

200

300

400

500

600

700

800

900

1000

Ra

nge

High Level Smoker Urine Lots (pg/mL)

Lot 7 Lot 8

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A B C D A B C D

200

300

400

500

600

700

800

900

1000

Ra

nge

High Level Smoker Urine Lots (pg/mL)

Lot 7 Lot 8

39% difference

46% difference

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A B C D A B C D

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15

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25

30

35

40

Ran

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Low Level Smoker Lots (pg/mL) Shared Stds

Lot 1 Lot 2

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A B C D A B C D

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15

20

25

30

35

40

Ran

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Low Level Smoker Lots (pg/mL) Shared Stds

32% difference12% difference

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50

100

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200

250

300R

ange

Lot 9Lot 6Lot 5Lot 3 Lot 4

Mid Level Smoker Lots (pg/mL) Shared Stds 2018

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50

100

150

200

250

300R

ange

Mid Level Smoker Lots (pg/mL) Shared Stds

15% 7%

6% 11%

8%

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A B C D A B C D

300

400

500

600

700R

ange

High Level Smoker Lots (pg/mL) Shared Stds

Lot 7 Lot 8

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A B C D A B C D

300

400

500

600

700R

ange

High Level Smoker Lots (pg/mL) Shared Stds

Lot 7 Lot 8

18% difference

13% difference

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A B C D A B C D

140

160

180

200

220

240

260

280

300

320

Ran

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NIST Urine Standard (pg/mL)

Separate Stds Imbedded Stds

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A B C D A B C D

140

160

180

200

220

240

260

280

300

320

Ran

ge

NIST Urine Standard (pg/mL)

Separate Stds Imbedded Stds

39% difference

24% difference

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16 35 90 110 120 270 280 390 600

Lab Stds 17 12 11 16 14 15 12 16 13

Embedded Stds 10 4 5 6 4 4 3 7 4

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Variance of Measurements

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Conclusion

• The assays employed by the participating laboratories provide consistent results within standard bioanalytical acceptance when a single source of reference material is used

• Increased bias was observed when standard calibrators prepared at each laboratory were used for quantitation

• It is important to use a well characterized single source of reference standard for comparison of bioanalytical results in inter-laboratory studies.

• BMK-SG has published a guideline for the proper characterization of reference materials.

➢CORESTA Guide N° 20 – Biomarker studies – Requirements for the Certification of Analytical Reference Standards

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Special thanks to contributors

• Aptochem Synthèse• Francois Deschamps

• ABF GmbH• Max Scherer

• Nikola Pluym

• Philip Morris International• Mark Bentley

• British American Tobacco• Krishna Prasad

• Reynolds American Inc.• Eckhardt Schmidt

• G. L. Prasad

• University of Minnesota• Steve Carmella

Study Sponsors

• Altria Client Services

• British American Tobacco

• Imperial Tobacco

• Japan Tobacco

• Philip Morris International

• RAI Services Company

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