Determination of nicotine and cotinine in human serum and urine: An interlaboratory study

45

Toxicology Letters, 35 (1987) 45-52 Elsevier

TXL. 01711

DETERMINATION OF NICOTINE AND COTININE AND URINE: AN INTERLABORATORY STUDY*

(Smokers; non-smokers; variation)

radioimmunoassay;

IN HUMAN SERUM

gas chromatography;

interassay

ANTON BIBER”, GERHARD SCHERERb, ILLE HOEPFNERb, FRANZ ADLKOFERb**, DIETER HELLER’, JAMES E. HADDOWd and GEORGE J. KNIGHTd

WOLF-

“Forschungslaboratoriutn Prof. Schievelbein, Munich; bForschungsgesellschaft Rauchen und Gesundheit mbH; Frauenthal2, D 2000 Hamburg 13; ‘Institut fiir Statistik und Mathematische Wirtschaftstheorie der Universitiit Karlsruhe, Karlsruhe (F.R. G.), and dFoundation for Blood Research, Scarborough, ME (U.S.A.) (Received I5 September 1986) (Accepted 25 September 1986)

SUMMARY An interlaboratory study aimed at determining nicotine and cotinine in human serum and urine was carried out. 11 laboratories from 6 countries, all experienced in performing nicotine and cotinine determinations in biological fluids by radioimmunoassay (RIA) and/or gas chromatography (CC) were involved. Each of them received 18 serum and 18 urine samples. The specimens were obtained from 8 smokers and 10 non-smokers; 2 samples from non-smokers were spiked with defined amounts of nicotine and cotinine. All the laboratories distinguished perfectly between the smokers and the non-smokers and according to cotinine levels in serum the laboratories ranked the samples with good agreement. There were systematic differences in the absolute values between the laboratories. The ratios of urinary cotinine concentrations between active and passive smokers differed widely from laboratory to laboratory. The reasons for this are not yet known and necessitate further investigation.

*Presented at the International Experimental Toxicology Symposium on Passive Smoking, October 23-25, 1986, Essen (F.R.G.). **TO whom correspondence and reprint requests should be addressed. Abbreviations: c.v., coefficients of variation; chromatography; RIA, radioimmunoassay.

ETS,

environmental

tobacco

037%4274/87/$ 03.50 0 Elsevier Science Publishers B.V. (Biomedical Division)

smoke;

CC,

gas

46

INTRODUCTION

Nicotine and cotinine in body fluids are often used in order to quantify tobacco smoke uptake. For their determination, GC and RIA methods are normally applied. A controversial discussion in the literature gives rise to some doubt whether data published by different laboratories are comparable with one another [l, 21. The analytical difficulties appear to be most evident in the case of ETS exposure, as very low exposure levels coming close to the detection limit have to be measured. In order to gain further insight into this matter, we initiated an international interlaboratory study. 11 laboratories were involved being experienced in performing nicotine and cotinine determinations in biological fluids by RIA and/or GC.

TABLE

1

NICOTINE

IN SERUM

(ng/ml)

GASCHROMATOGRAPHY

AS MEASURED

X, S and C.V. are calculated -.__

without

the outlying

Laboratories Smoke

Subjects Non-smoker

BY 5 DIFFERENT

A-GC

measurements. -_

D-GC

F-CC

uptake

H-CC

K-CC


6.Sa

co.5

0.4

1.0

0


4.3a

< 0.5

0.2

1.2

3 4

1


?.4a

0.8

0.3

1.5

2


5.?

0.5

0.3

1.3

2 3


5.3 10.1*

0.9 0.6

0.6 0.5

1.8 2.1

3


3 Cig./day 15

Smoker

s

C.V.

-

0

8

x

ETS exp.b

1

6 1

USING

- Methods -.____

2

5

LABORATORIES

(CC)

nd 1

-

-

9.6a

<0.5

0.2

1.8

0.8

0.3

2.1

19

15.0

17.5

18.6

20.4

18.1

2.0

0.11

10

15

7

10.4

9.0

8.8

13.2

9.7

2.3

0.24

11

20

3

ls.oa

1.9

2.3

2.2

2.3

0.5

0.20

12

20

8

14.2

4.8

6.3

8.0

8.3

3.6

0.43

13 14

30 40

11 22

16.7

16.3

4.7

0.29

4.5 4.7

60

32

24.1

32.4

40.0

37.1 40.7

15.1 29.3

0.30

27

15.0 27.7

15.8

40

14.3 12.9 25.0

15.8

15 16

23.1 9.9 29.8

33.8

6.8

0.20

17c

39

26.8

35.4

44.9

44.1

38.0

7.4

0.19

18’

15

17.3

17.5

21.8 _

21.9

18.7

3.0

0.16

9

nd, not detectable. “Outlier. bETS exposure: 0, none; ‘Serum

spiked

1, low; 2, medium;

with 41 and 20 ngiml

nicotine,

3, high. respectively.

0.16

41

MATERIALS

AND

METHODS

Urine obtained from 8 non-smokers and 8 smokers was collected for 24 h, and blood was drawn between 4 and 6 pm at the Forschungslaboratorium Prof. Schievelbein. 6 of the non-smokers reported exposure to ETS. The urine and serum specimens were aliquoted and stored at -20°C. In addition, 2 serum and 2 urine samples derived from non-exposed non-smokers were spiked with nicotine and cotinine. In total, there were 18 urine and 18 serum samples. The frozen specimens (4 ml serum and 10 ml urine) were dispatched on dry ice. The participating laboratories were sent the samples under a code number. Each laboratory could choose its own analytical method. They were asked to return one single value for nicotine and cotinine from each sample.

TABLE

II

NICOTINE

IN URINE

(ng/mI)

AS MEASURED

GASCHROMATOGRAPHY

(CC)

X, S and C.V. are calculated

without

the outlying

Laboratories Smoke

Subjects

BY 5 DIFFERENT

LABORATORIES

USING

measurements.

- Methods

A-CC

D-W

I-GC

F-GC

K-GC

X

S

C.V.

uptake Non-smoker

ETS exp.b

1

0

4

nd

8.3

<5

7.1

2

0

7

5

4.0

16.0

3

6.2

3

1

nd

4.5

26 <5

4

2

<3

nd

4.9

450a

5

2

<3

nd

2.4

<5

6

3

<3

nd

4.9

38

15.9

I

3

9

nd

5.6

8

3

32

nd

19.9

28 540a

34.0

2.3 8.4 8.2

Cig./day

Smoker 9

15

149

124

150

190

153

27

0.18

10

15

1191

907

1190

345

1189

11

20

255

186

154

118

204

964 183

367 51

0.38 0.28 0.04 0.16

51Sa

12

20

861a

615

609

650

650

631

22

13

30

519

459

450

338

500

453

70

14

40

1333

1155

1092

1320

627

1105

286

0.26

15

40

3218

2967

3331

3198

3125

3168

134

0.04

16

60

3279

3417

3486

2813

3391

3277

270

0.08

2687 1404

2483 1112

2599

1365= 1025

2601 1269

2592

83

0.03

1233

161

0.13

11’ 18’ aOutlier.

nd, not detectable.

bETS exposure: ‘Urine

1356

spiked

0, none;

1, low; 2, medium;

with 2500 and 1200 ng/ml

3, high.

nicotine,

respectively.

48 RESULTS AND DISCUSSION

Only 5 laboratories were able to measure nicotine in serum and in urine, all of them using GC. Cotinine in serum and urine were each determined by 10 laboratories. The results are summarized in Tables I to IV. The recovery rates calculated from the spiked serum and urine samples ranged from 55 to 117% for NICOTINE

IN SERUM (NC/ML.)

‘ik

'ik

NICOTINE

IN URINE (NC/ML)

7000

2000

f-

k-lx D-SC F-6C H-BC

4.5 4.8 . 0 1.4

K-SC

. 0

3000

F-GE I-GC x-Gc

-0 403 230

1500

/ ’

cI IO

20 COTININE

‘ik

A-RIA

IN SERUX

‘k

40

30 (NG,‘ML)

^

”

(NG/M,,)

48

600 E-RIA

COTININE IN URINE

X ,k

iooo

42

8-RIA C-RIP.

50 26

E-RIA

349

5000 300

0

0

200

400

600

80

3000

6000

9000

Fig. 1. Linear model (according to Jaech [3]) based on the samples from smokers and on the spiked samples (n = 10). Model, Xik7

Pk, @i, I%), Eik,

u,

Xik = ai + pi . pk + EI~;i = 1, . . . , 11; k = 9, ..* , 18. measurement value for sample k, derived by laboratory i. true but unknown value for sample k. bias for laboratory i. error term with expectation 0 and variance bi2 diagonal line in plots.

III

IN SERUM

Smoke

2

2

3

3

3

4

5

6

7

8

30

40

40

60

13

14

15

16

‘Serum

0, none;

249

478 42.Sa

95.0”

120.6”

105.8”

54.2a

53.3”

41.0a

11.7

12.0=

39.7

nd

0.4

0.2

nd

nd

nd

nd

nd

C-RIA

respectively.

3, high.

cotinine,

1, low; 2, medium;

224

463

697

631

706

670

741

325

234

63

90

211

620

289

229

75

107

260

334

spiked with 420 and 210 rig/ml

bETS exposure:

“Outlier.

nd, not detectable.

1.9 4.9

1

4

6.4

0.9

0.8

2.0

0.6

0.6

B-RIA

6

<1



<1

<1

A-GC

- Methods

measurements.

220

460

481

680

260

370

197

40

84

196

5.1

nd

7.7

3.0

2.3

6.4”

3.0”

8.6

D-CC

BY 10 DIFFERENT

354

357

330

338

223

20

12

60

457

20

11

80

228

4

2

6

2

2

1

17c

15

IO



18’

15

9

Cig./day

1

3

Smoker

0

0

A-RIA

Laboratories

the outlying

AS MEASURED

without

(GC)

(ng/ml)

ETS exp.b

uptake

2

Non-smoker 1

Subjects

x, s and C.V. are calculated

CHROMATOGRAPHY

COTININE

TABLE

208

388

382

449

263

289

199

40

65

119

nd

nd

nd

nd

nd

nd

nd

nd

E-RIA

2

3

4

4

2

185

398

527

587

261

250

157

31

73

179


<1


F-CC

LABORATORIES

165

330

480

168

351

347 198

399 196

183

525 467

529

205

407

542

585

544

24

54

104

92

50

51

278 278

54

202

19

14 42

58 76

s

176

x

(RIA) AND/OR

231

251 498

255

228

14

51

175



<1



<1



L-RIA

555

251

222 510

242

172

42

45 179

67

171

3.5

2.4

5.5

0.5

0.6

1.4

0.8

0.5

K-CC

75

198

2.1

0.4

2.5


0.2

1.4



H-CC

RADIOIMMUNOASSAY

210

155

47

67

16.5

nd

nd

15

nd

28’

nd

nd

62’

G-GC

USING

0.12

0.13

0.19

0.16

0.18

0.18

0.27

0.45

0.19

0.33

C.V.

GAS

IV

IN URINE

3

3

3

8

‘Urine

0, none;

3.9

14.5

75.0

16.0

4970

respectively.

795a

1645”

3573

7464

3193

10240

2253

486 671a

3490 4120

700

1145 1393

232

302

205

512

173 66

1150

268

nd

nd

nd

nd

nd

nd

43

nd

D-GC

347

2550

2015

2630

425

140

45.9

nd

36.1

2.6 45.0

3.1

nd

4.0

18.2

4.4

4.5

13.0

1635

3, high.

cotinine,

1, low; 2, medium;

spiked with 8700 and 4400 ng/ml

bETS exposure:

nd, not detectable. “Outlier.

8623

4120

3512

2536

8478

5245

4040

4088

60

16

1736

1124

1749

324

1014

1298

38

2

2

17c

40

15

3726

3726a

5196”

626

1002

2153

150a

11

17a

5

18’

30

20

12

40

20

11

14

15

10

13

15

9

Cig./day

19

31

2

5

6 I

Smoker

38

76

2

4

17a

2

1

13

3

7

0

25

0

B-RIA

C-RIA

measurements.

BY 10 DIFFERENT

- Methods

A-GC

Laboratories

A-RIA

2

ETS exp.b

uptake

Smoke

1

Non-smoker

Subjects

the outlying

AS MEASURED

without

(GC)

(rig/ml)

x, s and C.V. are calculated

CHROMATOGRAPHY

COTININE

TABLE

3834

8774

2960

2355

1823

2919

301

1080

1541

61

nd

44

nd

nd

nd

nd

nd

E-RIA

4159

8236

2833

2091

703

732

1002

129

460

878

15

9

21

2

7

4

10

18

F-GC

LABORATORIES

4270

8710

3280

2430

1570

940

1590

190

720

1370

H-GC

USING

4108

3278

1565

1750

690

1420

178

903

880

61

12

19

3oa

8

26 7

18

I-GC

1.0

8500 4242

7997 4018

3718

2604

2416

1474

2580

260

538

1236

29.1

3.8

12.6



4.0

<1

L-RIA

2920

2270

745

899

1091

201

605

1050

21.4

8.5

30.6

3.6

2.9

4.9

15.7

3.4

K-GC

RADIOIMMUNOASSAY

4138

8442

3566

2429

1754

1063

1648

264

704

1223

x

357

798

742

934

979

554

847

154

219

484

s

(RIA) AND/OR

0.09

0.09

0.21

0.38

0.56

0.52

0.51

0.59

0.40

0.40

C.V.

GAS

nicotine, and from 79-I 19% for cotinine. There were no differences in recovery rates between the urine and serum samples. The data from laboratory C were excluded from this calculation, as they show a recovery rate for cotinine of about 20% only. In the samples obtained from smokers the interlaboratory C.V. ranged from 4 to 59%. They were not influenced by increasing cigarette consumption. With regard to nicotine, the coefficients of variation were similar when the measurements were made in serum or urine, whereas for cotinine the mean coefficient of variation in urine was twice as high as found in serum. This is due to the fact that cotinine determinations in urine by RIA are less precise than in serum. For the spiked samples the coefficients of variation ranged from 3 to 19% being much lower than in the samples from smokers. As yet we have no explanation for this. The coefficients of variation for the samples obtained from non-smokers should not be calculated, since most of the values are below the detection limit and no numerical figures are available. The ratios of urinary cotinine concentrations between active and passive smokers differed widely ranging from 21 in laboratory C to 294 in laboratory L. In order to describe the results of the interlaboratory study in a more condensed form, a linear model [3] based on the samples obtained from smokers and on the spiked samples was used (Fig. 1). A comparison of the model line obtained from each laboratory with the ideal line (diagonal) indicates the degree of deviation of the laboratory’s results. A low u-value means high precision. Cotinine values in serum are comparable whether determined by RIA or GC, whereas in urine the RIA values are higher than the GC values. The data obtained in laboratory C have been disregarded. The results of our interlaboratory study indicate that data on nicotine and cotinine concentrations in serum and urine from smokers published so far are certainly comparable on a relative basis (coefficient of correlation: 0.6-0.9). In general, the laboratories ranked the samples according to cotinine levels in serum with good agreement. The absolute values, however, show large interlaboratory variations. These are particularly high in the samples obtained from subjects exposed to ETS. ACKNOWLEDGEMENTS

The study was carried out in the following laboratories: Dr. N. Benowitz, Clinical Pharmacology Unit, General Hospital Center, San Francisco (U.S.A.); Dr. A. Biber, Forschungslaboratorium Prof. Schievelbein, Munich (F.R.G.); Dr. M. Curvall, Swedish Tobacco Co, Stockholm (Sweden); Dr. C. Feyerabend, Dr, M.A.H. Russell, Institute of Psychiatry, University of London, London (U.K.); Dr. J.E. Haddow, Dr. GE. Knight, Foundation for Blood Research, Scarborough, ME (U.S.A.); Dr. N.J. Haley, American Health Foundation, New York (U.S.A.); Dr. S. Matsukura, Miyazaki Medical College, Kiyotake (Japan); Dr. H. Muranaka, Kyoto Senbai Hospital, Kyoto (Japan); Dr. H. Nau, Institut fur Toxikologie und

52

Embryonalpharmakologie, Freie Universitat Berlin, Berlin (Germany); Dr. K. Engstrom; Dr. M. Sorsa, Institute of Occupational Health, Helsinki (Finland); Dr. N. Wald, Medical College of St. Bartholomew’s Hospital, London (U.K.). The authors are indebted to all the scientists participating in this study. Without their spontaneous cooperation it would not have been possible to carry out this investigation.

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Jaech,

0.

K. Norikazu,

of environmental

for passive

2 F. Adlkofer, 719-720. 3 J.L.

T. Tomohiko, Effects

smoking,

Scherer

Statistical

S. Yutaka,

tobacco

N. Engl.

J. Med.,

and U. von Hees,

Analysis

smoke

M. Uchihashi, cotinine

H. Nakajima

excretion

and

in nonsmokers.

311 (1984) 828-832.

Passive

of Measurement

H. Hamada, on urinary smoking

Errors,

(Letter),

Wiley,

N. Engl.

New York,

J. Med.,

1985.

312 (1985)