HOW CONFIDENT ARE WE?

Alain LeBlancSpecial projects’ manager,

support services and external quality assessment

Pierre DumasResearch & Development division

Alain LeBlancSpecial projects’ manager,

support services and external quality assessment

Pierre DumasResearch & Development division

Centre de toxicologie du Québec (CTQ)  INSPQ, Québec, CanadaCentre de toxicologie du Québec (CTQ)  INSPQ, Québec, Canada

ISBM, Manchester UK, 9th ‐ 11th September 2013 

www.inspq.qc.ca/ctq

Among its expertise, the Centre de toxicologie du Québec (CTQ)  offers analytical support in clinical, environmental and occupational health

The INSPQ is a provincial government body created to improve the coordination, development and use of expertise in public health

CENTRE DE TOXICOLOGIE DU QUÉBEC (CTQ)Claude Thellen

QUEBEC MINISTRY OF HEALTH AND WEALTHFARE (MSSS)INSTITUT NATIONAL DE SANTÉ PUBLIQUE DU QUÉBEC (INSPQ)

ENVIRONMENTAL HEALTH AND TOXICOLOGY DEPT. (DSET)

TOXICOLOGYLABORATORY BRANCH

Normand FleurySPECIAL PROJECTS, SUPPORT SERVICES and EQAS SECTOR

Alain LeBlanc

RESEARCHPierre Ayotte

METHOD DEVELOPMENTPatrick Bélanger

CLINICALMichel Lefebvre

ORGANICSEric Gaudreau

METALSCiprian‐Mihai

Cirtiu

PROJECT  MANAGEMENTand  SUPPORT

SERVICES

EXTERNAL QUALITY 

ASSESSMENT SCHEMES

QUALITY MANAGEMENTSergine Lapointe

To make scientists, researchers and epidemiologists aware of day‐to‐day issues that

laboratories encounter when confrontedwith testing new environmental contaminants

Triclosan

• To develop reliable, robust and accurate analytical methods

• To assure tracability of the metrology

• To assure tracability of the data

• To assure the comparability of the analytical results

• To develop and maintain competence of staff

Sample collection, integrity and storage• Time of collection (spot/24 hrs; day‐to‐day variability)

• Handling (freeze/thaw)

• Stability (+ > 5 yrs)

• Homogeneity: free/conjugated species

• Contamination (collection process, container, …)

• Sampling container (adsorption onto plastics) / Tubes

• Are we sure we are measuring the right biomarker? in the right matrix?

• Shipping considerations (overnight, deep freeze)

Analytical innovation• Keep costs down; compromise with multi‐analyte methods

• Tend to less invasive matrices, but are they the right ones?

Analytical standards • Availability of conjugated analytes

• Accuracy (certificates, multiple sources)

• Impact on biomonitoring data (cycle to cycle)• Availability of appropriate internal standards

13C labeled and unlabeled analogs are usually not available at the same time• Custom synthesis (TRC, CANSYN, CHIRON, CIL, etc)

High cost, time to delivery, lot‐to‐lot, purity (UV, NMR, IR and MS spectra)

Analytical methods• Classical versus experimental protocols

• Sensitivity (LODs) and specificity

• Reportable limits (no international consensus; study comparisons)

• Laboratory contamination (ex: BPA, Triclosan, Parabens, BDEs, …)

Analytical instrumentation• Comparability

Reference materials• Availability (certified?)

• Appropriateness (same matrix?)                commutability

• Internal RM                   2nd source of standard!!

External quality assessment schemes• How to compare study results?

• Collaboration among reference labs or research centers

• To help reduce vulnerability

SAMPLE INTEGRITY AND STORAGE

-30

-20

-10

0

10

20

30Te

mpe

ratu

re°C

Sample handling: Chronological events(worst case scenario)

Sample Temperature Tracking Chart« Freeze/thaw »

Freeze/Thaw ‐ Stability of total BPA in urine (14 samples)

80

85

90

95

100

105

110

115

120

125

cycle 0 cycle 1 cycle 2 cycle 3 cycle 4

% B

PA

-10%

-5%

+5%

+10%

88

90

92

94

96

98

100

cycle 0 cycle 1 cycle 2 cycle 3 cycle 4

%C

onju

gate

d B

PA

Freeze/Thaw ‐ Stability of conjugated BPA in urine (14 samples)

• Free Triclosan can be rapidly adsorbed onto plastic tips according to  type of material and % of acetonitrile in spike solution.

• Time residence in the pipette tips can increase the degree of adsorption.

Tip Y  0% ACN

Tip Y 5% ACN

Tip Y  50% ACN

Tip Y  0% ACN 1 Min

Tip Y 5% ACN  1 Min

Tip Y  50% ACN 1 Min

Tip B  0% ACN

Tip B 5% ACN

Tip B 50% ACN

Tip R 0% ACN

Tip R 5% ACN

Tip R 50% ACN

86

93

103

75

82

98

90

91

101

95

97

100

% Recovery in urine

11.7

12.7

13.7

14.7

15.7

16.7

17.7

18.7

4391

0

4449

5

4508

1

4566

6

4625

1

4683

7

4742

2

4800

7

4859

2

4917

8

4976

3

# séquence

Con

cent

ratio

n (u

g/L)

1.250

2.250

3.250

4.250

5.250

6.250

7.250

4391

0

4449

5

4508

1

4566

6

4625

1

4683

7

4742

2

4800

7

4859

2

4917

8

4976

3

# séquence

Con

cent

ratio

n (u

g/L)

Internal QC from unspiked urine. Endogenous conjugated  form of trichlorophenol

External QC made from spiked urine using free trichlorophenol

Good stability

Poor stability

ANALYTICAL STANDARDS

Supplier MMP MEP MiBP MnBP MCHP MBzP MEHP MEHHP MEOHP MECPP MOP MCPP MNP

Certifiedsolutions

CIL‐2006 ‐25% ‐2% ‐ ‐47% ‐1% ‐63% ‐29% ‐11% ‐7% ‐ 12% ‐22% ‐39%

CIL‐2009 ‐9% ‐11% ‐5% ‐13% ‐12% ‐76% ‐14% ‐21% 4% 3% 2% ‐70% ‐16%

CIL‐2010 ‐3% ‐9% 0% ‐11% ‐7% 3% ‐11% ‐19% ‐9% ‐2% 3% ‐78% ‐10%

Chiron ‐ ‐ ‐3% ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

Accustandard ‐26% ‐31% ‐ ‐19% ‐ ‐4% ‐27% ‐ ‐ ‐ ‐ ‐ ‐

Neatstandards

Accustandard 8% ‐7% ‐ ‐5% ‐ ‐2% ‐27% ‐ ‐ ‐ ‐ ‐ ‐

Aldrich 9% ‐ ‐ ‐6% ‐ ‐5% ‐ ‐ ‐ ‐ ‐ ‐ ‐

Chiron ‐ ‐21% ‐ 0% ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐ ‐

Dr Ehrenstorfer 13% ‐ ‐ ‐2% ‐ ‐ ‐29% ‐ ‐ ‐ ‐ ‐ ‐

CanSyn 9% ‐5% 4% ‐1% ‐6% ‐1% ‐8% ‐6% ‐4% ‐1% 10% ‐7% ‐3%

CIL 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%

TRC 7% ‐3% 4% ‐6% ‐10% ‐2% ‐11% 9% 9% ‐70% 12% ‐2% ‐6%

Langlois E., LeBlanc A., Simard Y., Thellen C. Accuracy investigation of phthalate metabolite standardsJournal of Analytical Toxicology, 2012 ; 36 :270-279

compounds synthesized at CHULBPA‐13C12 and TCS‐13C12 were obtained from Cambridge Isotope Laboratories

BPA

Triclosan

ANALYTICAL METHODS

• Cadmium in urine by ICP MS

• Mo‐O interferencecorrected VS uncorrected

• Mean concentration can be       biased by 30 % with individual  error greater than 200%

• Comparability between studies   is strongly related to laboratory methodologies 130

78 9.523

32

21

0

5

10

15

20

25

30

35

0 50 100 150 200 250 300

Labo

ratorie

sCV

 %

Ratio conc. Mo/Cd

Cd nmol/L

‐Mo in sample‐Mo in calibration curve

Cadmium Urine (nmol/L)QMEQAS 2011-2

Lab A

Lab B

Comparing United States and Canadian population exposures from National Biomonitoring Surveys: Bisphenol A intake as a case studyJudy S. LaKind, Johanne Levesque, Pierre Dumas, Shirley Bryan, Janine Clarke and Daniel Q. Naiman

Journal of Exposure Science and Environmental Epidemiology (2012) 22, 219‐226

X 1.6

X 1.6

QC Low QC High

Target value 2.90 ±0.23 10.17 ±0.58

CTQ 1 2.98 10.6

CTQ 2 2.82 10.2

PCB 153 (1µg/L) + PCB 132 (0.3 µg/L)Complete overlap of both congeners

NCI EI

0.000.200.400.600.801.001.201.401.601.802.00

35800 36800 37800 38800 39800

Blan

k level  µg

/L

Sequence #

None Specific MetaboliteDiethyl thiophosphate (DETP)

Specific Metabolite3,5,6‐trichloro‐2‐pyridinol

Free BPA

G‐BPA

SO3‐BPA

(SO3)2‐BPA

y = 0.9149x - 0.0207R² = 0.9946

0

5

10

15

20

25

30

35

40

0 5 10 15 20 25 30 35 40

BP

A to

tal

µg

/L (G

C)

BPA free + BPA-glucuronide + BPA-sulfate + BPA-disulfate in µg BPA eq./L (LC)

LC‐MS/MS VS GC‐MS/MS

BPA

y = 1.0229x + 0.1818R² = 0.9954

0

100

200

300

400

500

600

700

800

900

0 100 200 300 400 500 600 700 800 900

TC

S t

ota

lµg

/L (

GC

)

TCS free + TCS-glucuronide + TCS-sulfate µg in TCS eq./L (LC)

LC‐MS/MS VS GC‐MS/MS

Triclosan

67.6

77.6

87.6

97.6

107.6

117.6

127.6

137.6

Con

cent

ratio

n (u

g/L)

# séquence

Setting up Single analyst + single instrument “Rush” multi analysts and instruments  New standard batch

External Quality Assessment Schemesand Reference Materials

EQASBlood lead (17) AAFP CAP DigitalPt G‐EQUAS LAMP NEQAS NYSDH OELM PCI Penn QMEQAS RCPA SEKK SKZL TEQAS WIV‐ISP WSLHUrine BPA (1) G‐EQUASUrine Triclosan (0)Urine Triclocarban (0)

RMBlood lead (6) IAEA IRMM NIST RECIPE SERONORM UTAKUrine BPA (1) RECIPE [NIST]Urine Triclosan (0) [NIST]Urine Triclocarban (0)

More than 30 countries

250 laboratories

External Quality Assessment SchemesEQAS

Since 1979

• 3 continuing programs in blood, urine, serum and hairMetals: PCI, QMEQAS      POPs: AMAP

• Material from human origin; unexposed volunteers

• 2 developing programs: MDA, Organic compounds in urine

• Voluntary participation

• Funded by the participants

• Reference material virtual store

36

REFERENCE MATERIALS

ISO/CEI 17043

www.inspq.qc.ca/ctq/sales

• Laboratories involved in HBM face challenges at all levels of operation

• Competence of staff must be developped and maintained

• Method validation must be brought to a higher level(stability, matrix effect, comparability of standards, …)

• Importance of External quality assessment schemes and referencematerials is obvious adapted QA strategy (work to do)

• A laboratory facility with a scientific and administratrive infrastructure ismandatory

• The term « new contaminant » alone shouldn’t set the rationale for biomonitoring

• A reference laboratory must have the capabilities and experience to tackle these recurring issues (not necessarily compatible with everylaboratory structure)

QUESTIONS?