Robert L. Jones, PhD and Kathleen L. Caldwell, PhD Robert L. Jones, PhD and Kathleen L. Caldwell, PhD

Inorganic and Radiation Analytical Toxicology Branch

Use & Misuse of Medical Chelation Therapy Conference

2/29/2012

CONSIDERATIONS IN THE CHOICE

OF LABORATORY AND

ANALYTICAL METHODS

CONSIDERATIONS IN THE CHOICE

OF LABORATORY AND

ANALYTICAL METHODS

National Center for Environmental Health

Division of Laboratory Sciences

DisclaimerMention of company or product

names does not constitute

endorsement by the National

Center for Environmental Health

(NCEH), Centers for Disease

Control (CDC), or the Public

Health Service.

Criteria in Selecting a Clinical Laboratory

• Currently analyzing for the analyte of concern (e.g. Pb) in the

matrix of choice utilizing a validated method.

• Currently CLIA certified.

• Active participation in a proficiency testing program for the

analyte in the biologic matrix (e.g. Pb in blood).

• The laboratory has an ongoing QA/QC program for the

analyte/matrix combination.

• Turnaround time

• Costs per sample

• Will the Laboratory supply “pre-screened” collection

materials

• Sample volume requirements

• Sample rejection requirements

Analytical Method Quality Factors

• Specificity – are you measuring the correct analyte (metal,

radionuclide, pesticide, VOCs)?

• Precision –What is the error in the measurement over the

range of reportable results (is it “fit for purpose”)?

• Accuracy – how “true” is the answer (true concentration or

activity)?

• Linearity – how linear is the activity range that you are

measuring?

• Range (analytical/reporting) – what is the

analytical/reporting range of the measurements (how is that

determined)?

• Recovery - The proportion of the analyte present in the test

material which is extracted and available for measurement

Analytical Method Quality Factors

• LOD – what is the minimum level (activity) that you have a

100% error within 95% Confidence interval (95% sure result

is not zero)?

• Robustness –What is the day to day variability on the

accuracy and precision on the analyte/matrix combination?

• Routine QC – Is there daily Quality Control (QC) for the

analyte/matrix combination near the relevant concentration

levels?

• Calibrators – Are there analyte/matrix samples of known

“activity” used to calibrate the instrument?

• Check Standards for all analytes – Are there analyte/matrix

samples of known “activity” shown to be within the pre-

determined QC range BEFORE measuring unknown

samples?

Multiples of So

Re

lati

ve

Un

ce

rta

inty

(%

)

John Taylor, “Quality Assurance of Chemical Measurements”, Lewis Publishers,1987, page 82.

Short versus Long Term QCLong-Term Bench QC Plot (Low Pool)

Urine Beryllium by ICP-MS

Analysis DatesFrom 9/11/2002 Through 4/4/2005

Ob

se

rve

d C

on

ce

ntr

atio

n (

µg

/L)

0.2

0.3

0.4

0.5

0.6

0.7

0.8

+ 3 SD

+ 2 SD

Mean

- 2 SD

- 3 SD

Average: 0.493 µµµµg/L

SD: 0.049 µµµµg/L

N: 629

Method validation may be defined as “ … the

process of proving that an analytical method is

acceptable for its intended purposes.”

J.M. Green Anal Chem 68:305A 1996

ICP-MS Basics

• Measures elemental ions: e.g. Pb-207

• Uses a plasma to ionize elements

• Hi sensitivity: e.g. low LODs

• High dynamic range: 4 to 12 orders of magnitude

• Very linear calibration

• Must methods are “dilute and shoot”: limited sample

preparation

• Limited interferences: isobaric and polyatomic

• Can be coupled to HPLC or GC for species detection: e.g.

As species

• Isotope ratio abilities: e.g. Pb (204, 206, 207 and 208 ratios)

Schematic and Layout of ICP-DRC-MS

Detector Analyzing

Quadrupole

Dynamic

Reaction

Cell

IonLens

ICP-MS

Interface

ICP

Reaction Gas InletPlasma

Development History of 13-Element

Biomonitoring Urine Method

12 Element

ICP-MS

Ba, Be, Cd, Co,

Cs, Mo, Pb, Pt,

Sb, Tl, U, W

1 element

ICP-DRC-MS

Cd

1 element

ICP-DRC-MS

As

1 element

GFAAS

As

13 Element

ICP-DRC-MS

As

Cd

Ba, Be, Co,

Cs, Mo, Pb, Pt,

Sb, Tl, U, W

13 Element

ICP-MS

As

Ba, Be, Cd, Co,

Cs, Mo, Pb, Pt,

Sb, Tl, U, W

Importance of Cadmium

to Public Health

• Health Concerns: Kidney damage, Low bone-mineral density

• Commercial Uses: batteries (78%), pigments (12%), coatings and plating (8%), plastic stabilizers (1.5%), nonferrous alloys and other uses (0.5%)

• Non-Occupational Exposures:

– Non-Smokers, no occupational exposure: Food

– Smokers: Smoking

Cd Isotopes ICP-MS Interferences

106Cd (1.3%) Pd, ZrO SrO, YO

108Cd (0.9%) MoO, Pd, ZrO

110Cd (12.5%) MoO, Pd, ZrO

111Cd (12.8%) MoO

112Cd (24.1%) MoO, Sn, ZrO

113Cd (12.2%) MoO, In

114Cd (28.7%) MoO, Sn

116Cd (7.5%) MoO, Sn, Th++

CDC Biomonitoring

Urine Cadmium

Method Initially

Corrected 114Cd

Only for Sn overlap

- (0.027250 * 118Sn)

Relationship of Observed Urine Cd

and Urine Mo

0 200 400 600 8000

1

2

3

4

5

Uri

ne

Cd

, u

g/L

(Me

asu

red

as 1

14C

d)

Urine Mo, ug/L

N = 5881

NHANES

1999 – 2003

Y=0.00181x – 0.02310

Quadrupole ICP-MS

The Estimated Effect of MoO

N =

5881

Cd Observed *(Cd + MoO)

Cd ESTIMATED **

(After Subtracting MoO)% Diff

Max 36.8 36.8 0%

95th 1.30 1.14 -13%

75th 0.55 0.40 -27%

50th 0.33 0.19 -42%

25th 0.19 0.09 -54%

10th 0.11 0.03 -70%

* Cd observed = standard ICP-MS, corrected for Sn overlap, non-weighted,

non-creatinine corrected, LOD = 0.06 ug/L

** Cd estimated = Cd observed – (0.00181[Mo] – 0.02310)

(from NHANES Cd vs. Mo data)

Proficiency Testing Run #

96-1

96-2

96-3

96-4

96-5

96-6

97-1

97-2

98-1

98-2

99-1

99-2

00-1

00-2

00-3

01-1

01-2

01-3

02-1

02-4

02-7

03-1

03-2

03-3

Cd,

µg/L

0

10

20

30

40

50

60

CDC Cd Result

Target Value

Urine Cd Proficiency Testing (PT)

No consistent bias observed in PT.

In most PT samples,

the MoO interference contributed

< 5% to the cadmium concentration.

Historical Cd Proficiency Testing

Results

Mo

, µ

g/L

0

20

40

60

80

100

120

140

160

M olybdenum Conc.

Profic iency Testing Run #

96-196-296-396-496-596-697-197-298-198-299-199-200-100-200-301-101-201-302-102-402-703-103-203-3

Cd z

-score

-1.5

-1.0

-0.5

0.0

0.5

1.0

1.5

Cadm ium Z-score

Corrective Options• Mathematical Correction: Not desirable since MoO interference may vary with

– variations of instrumental parameters

– variations of sample matrix

• Magnetic Sector ICP-MS: Resolution (> 37,000) not achievable

• DRC: eliminate MoO with oxygen cell gas

MoO Interference Removed By DRC

y = 0.00175x + 0.01360 (N = 208 DRC analyses)

y = 0.00181x – 0.0231 (N = 5881 Cd vs. Mo graph)

Urine Cadmium Difference, ug/L

(STD m

ode -DRC m

ode)

Urine Molybdenum (ug/L)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

0 100 200 300 400 500 600 700

N = 208

ICP-DRC-MS, oxygen cell gas

-0.60

-0.40

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

0.0 1.0 2.0 3.0 4.0 5.0

N = 99 (random NHANES samples)

Blood Cadmium Difference, ug/L

(STD m

ode -DRC m

ode)

Blood Molybdenum (ug/L)

MoO Interference Not Significant in

Blood

Impact of Method Enhancements

• National Exposure Report (NHANES) data (1999 – 2002)

was mathematically adjusted for estimated bias from

MoO interference.

• Re-analysis of NHANES 2003 urine samples using new

ICP-DRC-MS method for consistency of method within a

2 year reporting cycle.

• Incorporation of the DRC method into the 13 element

urine metals panel for all future work.

• Ranked #1 on the Priority List of Hazardous

Substances by the Agency for Toxic Substances

and Disease Registry (ATSDR) since 1997.

• Arsenic has been found in at least 63% of current

or former National Priority List (Superfund) sites.

Arsenic Exposure: a World-Wide

Problem

U.S. Geographical Occurrence

of

Arsenic Contaminated Wells

Source: U.S. Geological Survey Water Resources Investigation Report 99-4279

21.2 . . .13.7 . . .4.6 . . .17.0 . . .32.8 . .

. . .8.1 . . .12.8 . . .22.6 . . .18.9 . . .

10.8 . . 4.4 . . . 393.6 . . .48.2 . .57.0

. . .3.0 . . .5.8 . . .12.0 . . .11.6 . . .10.3 18.7 . .

.4.8 . . .13.3 . . .8.3 . . .38.4 . . .

. . .98.7 . . .12.5 . . .10.5 . . .19.4 . .

59.8 . . .33.3 . . .4.6 . . .1.9 . . .88.3 . .

. . . 2.6 . . .38.1 . . .26.2 . . .65.3 . .

• Rapid conversion of As(III) to As(V)

in aqueous solution.

• Slower As(III) ―> As(V) conversion in

biological matrices.

• Flash freezing the urine will inhibit

the inter-conversions.

• Sample collection, processing, and

shipping conditions are critical.

Arsenic Species Instability

Chromatogram of 7 arsenic species

Data from Exposed Subjects

Conclusions

• Carefully consider the laboratory criteria when

selecting a laboratory.

• Understanding the “Quality Factors” of an Analytical

Method as implemented, will assist with lab selection.

• Analytical method general issues will help you in

evaluating a lab’s claims for analyte/matrix methods.

• ICP-DRC-MS has great advantages as well as limitations.

• Interferences must be accounted for in the method.

• Elemental “speciation” analysis can provide the health

care provider with valuable information

Questions

Discussions

Questions

Discussions

National Center for Environmental Health

Division of Laboratory Sciences

Contact

Robert L. Jones, PhDCenters for Disease Control and Prevention

4770 Buford Hwy

Mailstop F-50

Atlanta, GA 30341-3724

RLJones@cdc.gov

“The findings and conclusions in this presentation have not been formally

disseminated by the Centers for Disease Control and Prevention/the Agency for Toxic

Substances and Disease Registry and should not be construed to represent any

agency determination or policy.”