Open Access

An inter-laboratory comparison of urinary 3-hydroxypropylmercapturic acid measurement demonstrates good reproducibility between laboratories

  • Emmanuel Minet1Email author,
  • Graham Errington1,
  • Gerhard Scherer2,
  • Kirk Newland3,
  • Mehran Sharifi4,
  • Brian Bailey5,
  • Mike McEwan1 and
  • Francis Cheung1
BMC Research Notes20114:391

DOI: 10.1186/1756-0500-4-391

Received: 16 November 2010

Accepted: 10 October 2011

Published: 10 October 2011

Abstract

Background

Biomarkers have been used extensively in clinical studies to assess toxicant exposure in smokers and non-smokers and have recently been used in the evaluation of novel tobacco products. The urinary metabolite 3-HPMA, a metabolite of the major tobacco smoke toxicity contributor acrolein, is one example of a biomarker used to measure exposure to tobacco smoke. A number of laboratories have developed liquid chromatography with tandem mass spectrometry (LC-MS/MS) based methods to measure urinary 3-HPMA; however, it is unclear to what extent the data obtained by these different laboratories are comparable.

Findings

This report describes an inter-laboratory comparison carried out to evaluate the comparability of 3-HPMA measurement between four laboratories. A common set of spiked and authentic smoker and non-smoker urine samples were used. Each laboratory used their in-house LC-MS/MS method and a common internal standard. A comparison of the repeatability ('r'), reproducibility ('R'), and coefficient of variation for 3-HPMA demonstrated that within-laboratory variation was consistently lower than between-laboratory variation. The average inter-laboratory coefficient of variation was 7% for fortified urine samples and 16.2% for authentic urine samples. Together, this represents an inter-laboratory variation of 12.2%.

Conclusion

The results from this first inter-laboratory comparison for the measurement of 3-HPMA in urine demonstrate a reasonably good consensus between laboratories. However, some consistent measurement biases were still observed between laboratories, suggesting that additional work may be required to further reduce the inter-laboratory coefficient of variation.

Background

Cigarette smoke contains thousands of chemicals, including toxicants, which can be categorized as either gases, semi-volatiles (gas/vapor phase), or particles ("tar" phase) [1]. Machine-measured cigarette yields under the ISO testing regimen do not provide an accurate estimate of human exposure to cigarette smoke toxicants [2]. These limitations have led to the development of methods to quantify biomarkers for specific toxicants in biological fluids such as urine, saliva, and plasma [3].

The gas phase, tobacco smoke toxicant acrolein [CAS:107-02-8] (Figure 1A) has been identified by the World Health Organization (WHO) study group on Tobacco Product Regulation (TobReg) as a major contributor to smoke toxicity [4]. This evaluation was based on the concentration of acrolein in smoke and its toxicity potency factor (cancer and non-cancer), established using various models. 3-hydroxypropylmercapturic acid (3-HPMA) is the major urinary metabolite of acrolein (Figure 1B) [5], and it can be quantified using LC-MS based methods [3, 5].
https://static-content.springer.com/image/art%3A10.1186%2F1756-0500-4-391/MediaObjects/13104_2010_Article_1152_Fig1_HTML.jpg
Figure 1

Chemical structure of acrolein ( A ), 3-HPMA ( B ), and 3-HPMA- 13 C 3 - 15 N ( C ). 3-HPMA and 3-HPMA-13C3-15N molecular weights are also indicated.

One critical element in the measurement and interpretation of biomarker data (including 3-HPMA) is the comparability in method analysis between different laboratories, which can use different methodologies. For instance, a study conducted by Biber and colleagues on a common set of urinary and plasma samples comparing nicotine and cotinine data from eleven laboratories concluded that individual values could vary significantly between laboratories [6]. In a more recent study, Bernert and colleagues showed that good measurement reproducibility for cotinine in a common set of samples could be achieved between six laboratories, when a standardized HPLC-UV method was used [7].

In this study we tested the reproducibility of 3-HPMA measurement between four laboratories using their in-house method and a common set of fortified and authentic urine samples. Each laboratory used a common reference compound and the internal standard 3-HPMA-13C3-15N.

Material and methods

Reagents and samples

Synthetic 3-HPMA (reference compound, Figure 1B) and 3-HPMA-13C3-15N (internal standard, Figure 1C) were obtained from AptoChem (Montreal, Canada). 3-HPMA-13C3-15N was ordered as a custom synthesis and the same lot was used by each laboratory. 3-HPMA-d3 was supplied by Toronto Research Chemicals (North York, Canada). Pooled non-smoker urine samples were supplied fortified with 3-HPMA by RECIPE Chemicals (Munich, Germany). Four concentrations of synthetic 3-HPMA were used: unspiked (background ≈30-50 ng/ml), 400 ng/ml, 1200 ng/ml, and 3600 ng/ml 3-HPMA. Prior to distribution, the samples were quantified using one of the participating laboratories (Lab 2) in order to ensure the quality of the preparation. The samples were then portioned into 5-ml-aliquots, lyophilized and shipped to the laboratories in triplicates of each sample (3 × 4 vials with lyophilized urine). The laboratories were advised to reconstitute the samples with 5 ml water.

Five authentic urine samples, covering a 3-HPMA concentration range which reflects typical levels in non-smokers to heavy smokers, were aliquoted in triplicates and sent to the participating laboratories (3 × 5 vials). The lyophilized non-smoker urine samples were supplied by RECIPE® (Munich, Germany), a supplier of samples used for quality assurance testing. The smoker samples were obtained as part of a biomarker study conducted previously by BAT. The corresponding study protocol and informed consent forms were approved by the Ethics Committee of the Bayerische landesarztekammer Munich, Germany (v. 18.02.2008), which contained a provision for revisiting the samples for the purpose of biomarker method development. The clinical study was conducted in accordance with the World Medical Association Declaration of Helsinki (World Medical Association, 2004) and International Conference on Harmonisation (ICH) Guidelines for Good Clinical Practice (GCP) (International Conference on Harmonization, 1996).

Analytical procedure

The samples were labelled with a code number and randomized prior to distribution. 10 mg of each reference compound and the internal standard were dispatched as a dried powder. Samples were received in one batch by each laboratory in September 2009, and all analyses were completed by November 2009. A summary of the analytical methods is given in Table 1, which is based on the performance of QC samples measured prior to the initiation of the study. All four laboratories used their in-house protocol, which included a solid phase extraction step and analysis by LC-ESI-MS/MS. The methods were validated according to FDA guidelines.
Table 1

Analytical method characteristics

Laboratories

1

2

3

4

Method

LC-ESI-MS/MS

LC-ESI-MS/MS

LC-ESI-MS/MS

LC-ESI-MS/MS

mode

positive

negative

positive

negative

SPE (column)

Phenomenex Strata-X

Isolute ENV+

Waters OASIS

Waters OASIS

SPE recovery (%)

74

68

75

78

HPLC column (make)

Waters Xterra MS C18

Waters HILIC-Silica

Waters Acquity Phenyl

Thermo BioBasic AX

HPLC column (size)

50 × 2.1 mm, 2.5 μm

150 × 2.1 mm, 3 μm

100 × 2.1 mm, 1.7 μm

50 × 3 mm, 5 μm

Quantifier ion (mass)

m/z 222 - 163

m/z 220 - 89

m/z 222 - 117

m/z 220 -91

Qualifier ion (mass)

m/z 222 - 117

m/z 220 - 91

  

precision intra-day (%)

1.1 to 5.9

1.4 to 8.6

1.1 to 1.5

1.2 to 6.5

precision inter-day (%) a

5.1 to 5.3

3.3 to 7

1.7 to 3.9

3.3 to 7.5

accuracy (%)

93.2 to 102

83.9 to 102

97.6 to 102

96.8 to 101

LOD (ng/ml)

2.21

NDd

ND

NDd

LOQ (ng/ml)

7

25

50f

35f

Linearity (ng/ml)

7 to 5400

25 to 10000

50 to 5000

35 to 5000

Matrix effect (%)

ND

7.4 to 17

-1.7 to 19.6

-5.5 to 6.3

aValues represent the precision range obtained for low, medium, and high concentrations at the time the methods were developed, except for Lab 1 where only low and high concentrations were tested.

bBased on calibration standards.

cLOD was an estimate based on spiked water, water being used as the SPE solvent.

dNot determined

eLOQ was established in spiked water, water being used as SPE solvent.

fMatrices used were either diluted non-smoker urine or non-smoker urine with very low 3-HPMA background.

Statistical analysis

Basic statistical analysis was carried out with MINITAB v15.1. Individual value plots were produced to test inter-laboratory variation of 3-HPMA concentrations. A non-parametric Wilcoxon paired t-test was performed to compare preliminary test analyses conducted with different internal standards. Analysis of covariance was used to compare the analytical methods at the four laboratories using the same internal standard (3-HPMA-13C3-15N). Precision statistics, as defined in ISO 5725-2 [8], were used as a measure of random errors, and expressed as repeatability ('r') and reproducibility ('R'). For the purposes of this study, in which each laboratory used its own method, 'R' refers to inter-laboratory variation.

Results and discussion

Data for both fortified and authentic urine, using 3-HPMA-13C3-15N as internal standard, were reported by each lab and the corresponding 3-HPMA concentrations (ng/ml) are shown in Table 2. In addition, laboratory 1 repeated the measurements using two different internal standards - 3-HPMA-d3 and 3-HPMA-13C3-15N - which were prepared and analyzed on the same day, in order to investigate the potential confounding effects of using different standards under the same analytical conditions. A non-parametric paired t-test showed that the use of 3-HPMA-d3 gave consistently higher concentrations than 3-HPMA-13C3-15N (Figure 2), highlighting the importance of standardizing the use of internal standards across each laboratory throughout the study.
Table 2

All urinary 3-HPMA data in ng/ml

   

3-HPMA (ng/ml)

Samples

Lab1

Lab2

Lab3

Lab4

1

a

pooled NSa urine

40.6

31.9

< LOQb

< LOQ

 

b

pooled NS urine

29.8

30.8

< LOQ

< LOQ

 

c

pooled NS urine

40.0

30.7

< LOQ

< LOQ

2

a

fortified NS urine

466

492

396

405

 

b

fortified NS urine

402

470

400

403

 

c

fortified NS urine

431

471

401

402

3

a

fortified NS urine

1302

1340

1180

1070

 

b

fortified NS urine

1230

1270

1160

1140

 

c

fortified NS urine

1140

1340

1150

1090

4

a

fortified NS urine

3624

3780

3470

3220

 

b

fortified NS urine

3714

3820

3420

3210

 

c

fortified NS urine

3504

3970

3370

3240

5

a

NS urine

39.8

48.3

< LOQ

< LOQ

 

b

NS urine

48.2

46.1

< LOQ

36.6

 

c

NS urine

62.4

46.9

< LOQ

35.1

6

a

smoker urine

371

376

269

321

 

b

smoker urine

376

382

294

269

 

c

smoker urine

371

370

300

258

7

a

smoker urine

870

842

613

556

 

b

smoker urine

960

874

673

659

 

c

smoker urine

840

830

721

619

8

a

smoker urine

1080

1200

862

969

 

b

smoker urine

1122

1160

925

914

 

c

smoker urine

1044

1180

878

929

9

a

smoker urine

1482

1390

1200

1100

 

b

smoker urine

1296

1370

1210

1130

 

c

smoker urine

1260

1390

1100

894

Samples are numbered from 1 to 9. Samples 1 non-smoker urine, samples 2-4 = fortified samples, and samples 5-9 = authentic urine samples. Each sample was aliquoted in triplicates labeled a, b, and c.

aNS: non smokers

b< LOQ: below limit of quantification

https://static-content.springer.com/image/art%3A10.1186%2F1756-0500-4-391/MediaObjects/13104_2010_Article_1152_Fig2_HTML.jpg
Figure 2

Boxplot of non-parametric differences between 3-HPMA measured in all samples using 3-HPMA-d 3 and 3-HPMA- 13 C 3 - 15 N internal standards. The hypothesis (Ho) is based on no difference (0) between the 3-HPMA-d3 measures minus the 3-HPMA-13C3-15N measures. The box plot shows a clear positive difference with p = 0 based on a Wilcoxon paired t-test with a 95% confidence interval for the mean difference (x).

A background level of 40 to 60 ng/ml 3-HPMA was observed in the non-smoker samples selected for this study. This is expected given that acrolein is also the product of lipid peroxidation, fossil fuel combustion, and is found in cooked food [9].

As a quality control check, data from the fortified urine samples (Table 2) were plotted to generate a regression line and the corresponding equation. Using this, the values for the urine samples (Table 2) were recalculated based on the 3-HPMA peak area and the 3-HPMA-13C3-15N internal standard. The calculated concentrations were consistent with the reported concentrations from each lab (Additional file 1).

Individual value plots constructed using the sample data indicate a close similarity in the measurements, across the broad range of 3-HPMA concentrations, for all four laboratories using 3-HPMA-13C3-15N (Figure 3). However, an analysis of covariance (ANOVA) indicated a significant variation between laboratories still existed (Table 3). A closer fit could be observed between lab 1 and 2, and between lab 3 and 4. The coefficients of variation, giving an estimate of the imprecision for repeated measures at different concentration ranges, are also reported in Table 3. The imprecision for each concentration range should be interpreted carefully since the replicate measures were obtained from three aliquots from a single solution rather than a triplicate measure of a unique sample. The fortified samples (samples 2 to 4), were used as an internal calibration reference to calculate accuracies (Table 4).
https://static-content.springer.com/image/art%3A10.1186%2F1756-0500-4-391/MediaObjects/13104_2010_Article_1152_Fig3_HTML.jpg
Figure 3

Individual value plots for 3-HPMA (blue circles) (ng/ml). A. Value plot for the fortified samples in four laboratories. B. Value plot for authentic urine samples in four laboratories. Missing values were below the LOQ.

Table 3

One way ANOVA for 3-HPMA vs laboratories (lab1, 2, 3, and 4) for each set of samples

  

Mean 3-HPMA (ng/ml)

StDev

CoV

P value (one-way Anova)

Sample 1

lab1

36.8

6.1

16.5

0.186

 

lab2

31.13

0.7

2.1

 
 

lab3

< LOQ

NA

NA

 
 

lab4

< LOQ

NA

NA

 

Sample 2

lab1

433

32.2

7.4

0.002

 

lab2

477.7

12.4

2.6

 
 

lab3

399

2.6

0.7

 
 

lab4

403.3

1.5

0.4

 

Sample 3

lab1

1224

81.2

6.6

0.004

 

lab2

1316.7

40.4

3.1

 
 

lab3

1163.3

15.3

1.3

 
 

lab4

1100

36.1

3.3

 

Sample 4

lab1

3614

105.4

2.9

0.000

 

lab2

3856.7

100.2

2.6

 
 

lab3

3420

50

1.5

 
 

lab4

3223.3

15.3

0.5

 

Sample 5

lab1

50.1

11.4

22.8

0.182

 

lab2

47.1

1.1

2.4

 
 

lab3

< LOQ

NA

NA

 
 

lab4

35.8

1.1

3

 

Sample 6

lab1

372.4

2.77

0.7

0.000

 

lab2

376

6

1.6

 
 

lab3

287.7

16.4

5.7

 
 

lab4

282.7

33.6

11.9

 

Sample 7

lab1

890

62.4

7

0.000

 

lab2

848.7

22.7

2.7

 
 

lab3

669

54.1

8.1

 
 

lab4

611.3

51.9

8.5

 

Sample 8

lab1

1082

39

3.6

0.000

 

lab2

1180

20

1.7

 
 

lab3

888.3

32.7

3.7

 
 

lab4

937.3

28.4

3

 

Sample 9

lab1

1346

119.1

8.85

0.006

 

lab2

1383.3

11.5

0.8

 
 

lab3

1170

60.8

5.2

 
 

lab4

1041.3

128.5

12.3

 

gNA: not applicable, fewer than three data points due to at least one measure < LOQ.

Table 4

Accuracies calculated for each laboratory based on 3-HPMA fortified samples at 400 ng/ml (samples 2), 1200 ng/ml (samples 3), and 3600 ng/ml (samples 4)

 

Accuracies (%)

Fortified sample (ng/ml)

Lab1

Lab2

Lab3

Lab4

400 ng/ml

108

119

99

101

1200 ng/ml

102

110

97

92

3600 ng/ml

100

107

95

90

aAverage calculated over three independent measures

A comparison of the repeatability ('r'), reproducibility ('R'), and coefficient of variation for 3-HPMA demonstrated that within-laboratory variation was consistently lower than between-laboratory variation. The average intra-laboratory CoV was 5%, while the average inter-laboratory CoV was 12.2% (Table 5). The average inter-laboratory coefficient of variation was 7% for the fortified urine samples and 16.2% for the authentic urine samples. These results show close comparability with those observed by Biber and colleagues, where samples spiked with cotinine had an inter-laboratory CoV ranging from 3 to 19%, while a CoV range of 4 to 59% was reported for authentic urine samples of smokers [10].
Table 5

Repeatability, reproducibility, and intra, inter-laboratory coefficient of variation for 3-HPMA between the four participating laboratories

Samples

Mean 3-HPMA (ng/ml) a

r b

R c

CoV within (%) d

CoV between (%) e

1

34

5.8

13.9

6.1

NAf

2

428

50

100.8

4.2

8.4

3

1201

142.7

261.8

4.2

7.8

4

3528

268.4

460.3

2.7

4.7

5

45

7.6

24.2

5.9

NAf

6

330

54.9

138

6

15

7

755

143.3

363.4

6.8

17.2

8

1021

89.1

346.4

3.1

12.1

9

1235

223.1

710.1

6.5

20.5

aMean of individual 3-HPMA values for all participating laboratories and for the corresponding sample set

bReproducibility

cRepeatability

dcomposite intralaboratory coefficient of variation

einter-laboratory coefficient of variation for the four participating laboratories

fNA: not applicable due to data < LOQ

The overall average inter-laboratory coefficient of variation for all samples in this study was 12.2%. A CoV value higher than 10% might indicate that there is still some room for improvement; however, this seems to be in line with WHO standardized clinical methods, which in previous studies have reported average inter-laboratory coefficients of variation (CoV) above 10% [1113].

The results from this first inter-laboratory comparison for the measurement of 3-HPMA in urine demonstrate a reasonably good consensus between laboratories, with an average CoV of 12.2%. However, some consistent measurement biases were still observed between laboratories, suggesting that additional work may be required to reduce the inter-laboratory CoV even further.

Abbreviations

3-HPMA: 

3-hydroxypropylmercapturic acid

CoV: 

coefficient of variation

ESI: 

electrospray

LC-MS: 

liquid chromatography-mass spectrometry

r: 

repeatability

R: 

reproducibility

TobReg: 

tobacco product regulation

UV: 

ultraviolet

WHO: 

world health organization

Declarations

Authors’ Affiliations

(1)
British American Tobacco, Group Research and Development
(2)
Analytisch-Biologisches Forschungslabor GmbH
(3)
Celerion
(4)
Labstat International Inc
(5)
Covance Laboratories Ltd

References

  1. Rogdman A, Perfetti TA: The chemical components of tobacco and tobacco smoke. 2008, Baton Rouge, CRC PressGoogle Scholar
  2. WHO Scientific Advisory Committee on Tobacco Product Regulation (SacTob): Conclusions and recommendations on health claims derived from ISO/FTC method to measure cigarette yield. World Health Organization. 2002, GenevaGoogle Scholar
  3. Scherer G, Engl J, Urban M, Gilch G, Janket D, Riedel K: Relationship between machine-derived smoke yields and biomarkers in cigarette smokers in Germany. Regul Toxicol Pharmacol. 2007, 47: 171-183. 10.1016/j.yrtph.2006.09.001.PubMedView ArticleGoogle Scholar
  4. Burns DM, Dybing E, Gray N, Hecht S, Anderson C, Sanner T, O'Connor R, Djordjevic M, Dresler C, Hainaut P, Jarvis M, Opperhuizen A, Straif K: Mandated lowering of toxicants in cigarette smoke: a description of the World Health Organization TobReg proposal. Tob Control. 2008, 17: 132-141. 10.1136/tc.2007.024158.PubMedPubMed CentralView ArticleGoogle Scholar
  5. Carmella SG, Chen M, Zhang Y, Zhang S, Hatsukami DK, Hecht SS: Quantitation of acrolein-derived (3-hydroxypropyl)mercapturic acid in human urine by liquid chromatography-atmospheric pressure chemical ionization tandem mass spectrometry: effects of cigarette smoking. Chem Res Toxicol. 2007, 20: 986-990. 10.1021/tx700075y.PubMedPubMed CentralView ArticleGoogle Scholar
  6. Biber A, Scherer G, Hoepfner I, Adlkofer F, Heller WD, Haddow JE, Knight GJ: Determination of nicotine and cotinine in human serum and urine: an interlaboratory study. Toxicol Lett. 1987, 35: 45-52. 10.1016/0378-4274(87)90084-1.PubMedView ArticleGoogle Scholar
  7. Bernert JT, Jacob P, Holiday DB, Benowitz NL, Sosnoff CS, Doig MV, Feyerabend C, Aldous KM, Sharifi M, Kellogg MD, Langman LJ: Interlaboratory comparability of serum cotinine measurements at smoker and nonsmoker concentration levels: a round-robin study. Nicotine Tob Res. 2009, 11: 1458-1466. 10.1093/ntr/ntp161.PubMedPubMed CentralView ArticleGoogle Scholar
  8. ISO 5725-2: Accuracy (trueness and precision) of measurement methods and results: Part 2. Basic method for the determination of repeatability and reproducibility of a standard measurement method. International Organization for Standardization. 1994, Geneva, SwitzerlandGoogle Scholar
  9. Stevens JF, Maier CS: Acrolein: sources, metabolism, and biomolecular interactions relevant to human health and disease. Mol Nutr Food Res. 2008, 52 (1): 7-25. 10.1002/mnfr.200700412.PubMedPubMed CentralView ArticleGoogle Scholar
  10. Biber A, Scherer G, Hoepfner I, Adlkofer F, Heller WD, Haddow JE, Knight GJ: Determination of nicotine and cotinine in human serum and urine: an interlaboratory study. Toxicol Lett. 1987, 35: 45-52. 10.1016/0378-4274(87)90084-1.PubMedView ArticleGoogle Scholar
  11. Coucke W, Devleeschouwer N, Libeer JC, Schiettecatte J, Martin M, Smitz J: Accuracy and reproducibility of automated estradiol-17beta and progesterone assays using native serum samples: results obtained in the Belgian external assessment scheme. Hum Reprod. 2007, 22: 3204-3209. 10.1093/humrep/dem322.PubMedView ArticleGoogle Scholar
  12. Siekmann L: Requirements for reference (calibration) laboratories in laboratory medicine. Clin Biochem Rev. 2007, 28: 149-154.PubMedPubMed CentralGoogle Scholar
  13. Thorpe SJ, Heath A, Blackmore S, Lee A, Hamilton M, O'broin S, Nelson BC, Pfeiffer C: International Standard for serum vitamin B(12) and serum folate: international collaborative study to evaluate a batch of lyophilised serum for B(12) and folate content. Clin Chem Lab Med. 2007, 45: 380-386. 10.1515/CCLM.2007.072.PubMedView ArticleGoogle Scholar

Copyright

© Minet et al; licensee BioMed Central Ltd. 2011

This article is published under license to BioMed Central Ltd. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.