NBSIR 73-406

Reference Materials for the

Determination of Trace Elements

in Biological Fluids

p. D. LaFleur

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington. D.C. 20234

December 1973

Final Report

Prepared for Division of Laboratories and Criteria Development

National Institute for Occupational Safety and Health

Cincinnati, Ohio 45202

NBSIR 73-406

REFERENCE MATERIALS FOR THE

DETERMINATION OF TRACE ELEMENTS

IN BIOLOGICAL FLUIDS

P. D. LaFleur

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

December 1973 "

Final Report

Prepared for Division of Laboratories and Criteria Development

National Institute for Occupational Safety and Health

Cincinnati, Ohio ^.5202

\ \^^-\ s \ >.

' /

U. S. DEPART^'iEl^iT OF COMMERCE, Frederick B. Dent, Secretary

IMATiONAL ?.UREAU GF STANDARDS. Richard W. Roberts, Director

TABLE OF CONTENTS

PAGE

1. INTRODUCTION 1

2. MERCURY IN URINE REFERENCE MATERIALS 1

2.1 Introduction 1

2.2 Experimental 3

2.3 Results and Discussion 4

3. SELECTED TRACE METALS IN FREEZE-DRIED URINE ... 6

3.1 Introduction 6

3.2 Experimental 6

3.3 Results and Discussion 8

4. FLUORINE IN FREEZE-DRIED URINE 11

4.1 Introduction 11

4.2 Experimental 11

4.3 Results and Discussion 12

5. LEAD IN BLOOD REFERENCE MATERIAL 14

5ol Introduction 14

So 2 Experimental . . .' 14

5.3 Results and Discussion 15

6. CONCLUSION ' o . . . ^ . 18

iii
LIST OF TABLES

TABLE NO. PAGE

1 Mercury in Urine - "Elevated" Sample

Number 1 » .... o ... . 5

2 Mercury in Urine - "Elevated" Sample

Number 2 6

3 Metals in "Normal" Freeze-Dried Urine

by Neutron Activation Analysis /. . 9

4 Metals in "Elevated" Freeze-Dried Urine

by Neutron Activation Analysis 10

5 Metals in Freeze-Dried Urine by Polarography . , 11

6 Fluoride Concentration in Freeze-Dried Urine . . 13

7 Elevated Lead Concentration in Porcine Blood . . 17

8 Normal Lead Concentrations in Porcine Blood . . 17

9 "Elevated Normal" Lead Concentration in

Porcine Blood 18

IV
REFERENCE MATERIALS FOR THE DETERMINATION

OF TRACE ELEMENTS IN BIOLOGICAL FLUIDS

ABSTRACT

The preparation of a number of reference materials for the

analysis of trace elements in biological standards is

described. The standards produced include mercury in

urine at three concentration levels, five elements

[Se, Cu, As, Ni, Cr] in freeze-dried urine at two

levels, fluorine in freeze-dried urine at two levels, and

lead in whole blood at two concentration levels. These

reference materials have been analyzed for the element (s)

of interest by one or more analytical techniques, and are

supplied with known concentration levels.

1 . INTRODUCTION

The National Bureau of Standards (NBS) has prepared a number

of working standards for the determination of trace metals

and fluoride in biological fluids. The samples were prepared

for the National Institute for Occupational Safety and

Health (NIOSH) for use in methods evaluation and related

investigations. The development and analytical approach

used for these standards is described.

2 . MERCURY IN URINE REFERENCE MATERIALS

T. E. Gills

2 . 1 Introduction

Urine is the biological fluid most often analyzed in cases

of possible inorganic mercury exposure. For organomercurial exposure, serum is probably superior to urine

since it gives a better measure of the body burden. The

serum concentration of inorganic mercury, however, drops

rapidly after the exposure; the kidney concentration stays

elevated for some time after the exposure.
The original plan was to prepare the mercury in the urine

standard by feeding mercury to experimental animals and

collecting the urine, either by catheterization or in a metabolism cage. Through the cooperation o£ Dr. Eugene

Miller at the U. S. Food and Drug Toxicology Laboratory

in Beltsville, Maryland, a number of "minipig" laboratory

pigs were fed inorganic mercury (as the nitrate) . The

samples were taken in a metabolism cage with a small

plastic bag tied to the animal to minimize contamination

from feces. The samples were filtered and acidified.

Unfortunately, the mercury concentration of the porcine

urine was only slightly above the normal background

concentration. After conversations with toxicologists , it

was determined that most, if not all, of the mercury

in urine is inorganic and is probably unbound or bound

very loosely. Because of these considerations, we

decided that direct spiking of a urine sample would not

be substantially different from a sample in which the

mercury was present from a metabolic process.

A number of experiments were performed to determine the

storage stability of mercury in urine. This was done

by adding ^°^Hg tracer to some samples of urine to which

various amounts of acid had been added. It was found

that most of the mercury would accompany the precipitate

which formed on standing in non-acidified urine. If

'^5% of either HNO3 or HCl were added to the urine,

however, the mercury concentration was stable over

several months.

Concurrently, experiments were performed to determine

the feasibility of lyophilizing the urine as an alternate

storage method. Several problems became obvious. The

2
mercury losses were higher than desired (>10^) and were

not constant. It would, therefore, have been impossible

to guarantee homogeneity from sample to sample. It was

decided on the basis of these experiments to provide

the urine as an acidified liquid for which the elevated

concentrations were obtained by direct spiking.

2 . 2 Experimental

Eighteen liters of human urine were collected at NBS to

be used as the mercury in urine standard. Six liters

provided a standard at the "normal" concentration. Another

six liters were doped to contain '^^90 mg Hg/1. The last

six liters were doped to contain '^200-250 mg/1.

The urine was collected from NBS personnel and stored in

6 liter Erlenmeyer flasks. Mercuric nitrate was used as

a spike for the elevated levels. All of the standards

were made 5% with high -purity nitric acid and stirred

for 6 hours with a magnetic stirring bar. This acidity

was chosen to prevent mercury adsorption on the

surface of the flask. After mixing, 50 ml aliquots of

the urine were decanted into 125-ml glass bottles. The

bottles have Teflon-lined caps. The bottles of each

standard were numbered sequentially so as to obtain

information on the homogeneity of each lot.

After the preparation and bottling procedure, it was

necessary to determine the homogeneity of both the

elevated and normal levels by analyzing for the mercury

content in each lot. Every tenth bottle was chosen

for the homogeneity determination.

3
One milliliter aliquots were taken from each bottle

and encapsulated in precleaned quartz vials. The

samples, along with solution standards of mercuric

nitrate, were irradiated for 1 hour in RT-3 of the

NBS Reactor at a thermal neutron flux of 5x10^ 'n»cm' ^s' *

After allowing the activity to decay for 1-1/2 days to

minimize personnel exposures, the ampoules were frozen

in liquid nitrogen, broken, and the sample solution

allowed to warm to room temperature. The solution was

weighed and transferred, along with mercury carrier, to

125-ml Erlenmeyer flasks. Each sample was then digested

with a 1:2 mixture of nitric acid and hydrochloric acid.

After digestion a sulfide precipitation was performed and

the mercuric sulfide was filtered onto glass'fiber filter

pads

Mercury- 197 produced by the thermal neutron bombardment

of mercury-196 was used as the measuring nuclide. The

counting was done on a Ge(Li) low-energy photon detector

in conjunction with a 1024 channel pulse height analyzer.

The concentration was determined by comparing the activity

in the samples to that of the standard.

2 . 3 Results and Discussion

The results for the elevated concentrations are given

in Tables 1 and 2. For the "normal" urine samples, a

concentration of <0.005 yg/g was found.

As an indication of the stability of these reference

materials, the .084 mg/1 samples were analyzed 6 months

after preparation and the amount found was in total

agreement with the first analysis.

4
TABLE 1

Mercury in Urine - "Elevated" Sample Number 1

Bottle No. mg/l Ave. mg/1

1 .090 .091

.092

10 .087 .085

. 084

20 .082 .083

.084

30 .081 .080

.080

40 .089 .088

. 087

50 .080 .083

.087

60 .086 .087

.088

70 .087 .089

.092

80 .076 .074

.072

90 .090 .089

.089

Average = .085

s = .005

5
TABLE 2

Mercury in Urine - "Elevated" Sample Number 2

Bottle Number mg/1

30 .216

10 .216; .218

20 ^ .218

40 .222

1

' : ] ^: ' -213

90 ' . .215

50 .209

80 .205

100 ' .208

60 .225

110 .210

Average = .214

B''. s = .006

3. SELECTED TRACE METALS IN FREEZE -DRIED URINE

L. T. McClendon, E. J. Maienthal, T. E. Gills

3 . 1 Introduction

The freeze-dried urine samples requested by the National

Institutefor Occupational Safety and Health, consisting

of a normal lot, a doped fluoride lot and a doped

trace metal lot [Se, Cu, As, Ni, Cr(VI)], were obtained

from the Lederle Laboratories and analyzed in the

Analytical Chemistry Division of the National Bureau of

Standards . ^

3 . 2 Experimental

The fluoride concentration of the urine samples was

determined with the fluoride specific- ion electrode and will

6
be discussed separately below. The trace-metal concentrations

were determined in both the normal lot and doped lot by the

neutron activation analysis technique. Nickel and copper were

also determined polarographically utilizing a sodium diethyldithiocarbamate extraction procedure to separate the copper and

nickel from the matrix and measured polarographically in a pyridine -pyridinium sulfate supporting electrolyte.

In order to determine the trace m.etal concentrations

in the freeze-dried urine by neutron activation analysis,

radiochemical separations had to be performed because of

the matrix radio-activity created in the urine samples

during the irradiation. The procedure used for the determination

of selenium, arsenic, and copper in the freeze-dried urine

samples was as follows: The urine sample was reconstituted

in 1 ml high-purity HNO3 plus 10-ml ultra-pure H2O (11 ml total),

instead of 50 ml H2O as prescribed by the suppliers, to obtain

optimum sensitivity for the elements of interest. The

concentrations found for the trace metals were corrected for

a 50-ml dilution. Aliquots of the reconstituted urine sample,

along with standards, were encapsulated in quartz vials,

irradiated in the XBS Reactor at a flux of '^^1x10 ^ ^n • cm ^'S ^ for 30-60 minutes and allowed to decay for 8 hours to reduce

short-lived interferences. For arsenic, copper, and selenium

determinations, the irradiated urine samples were weighed,

placed in 100-ml beakers, appropriate carriers added, and

a mixture of HClOi^-HNOs added. The solutions were then

heated to near dryness. After cooling the samiples, HCl

and KI were added and samples heated to reduce As"^^ to As"*"^.

The sulfides of As, Se, and Cu were then precipitated by the

addition of a 5% thioacetamide solution. After filtration

of each sample onto a 2.5 cm glass fiber filter pad, the samples

7
were counted on a high resolution Ge(Li) detector in conjunction

with a 2048 channel pulse height analyzer.

The nickel concentration in the irradiated urine samples

was determined by precipitation of nickel dimethylglyoximate

(after carrier addition and pH adjustment) , with filtration

and counting of the precipitate as described

previously. The chromium concentration in the irradiated

urine samples was determined by the solvent extraction of

the Cr(VI) tr ibenzylamine complex into chloroform" with

subsequent counting of an aliquot of the organic phase

on the detector previously mentioned. lt_ should be

noted that chromium is present as Cr (III) instead of

Cr (VI

)

in the freeze-dried urine samples as the technique

used to determine chromium allows distinction of the two

oxidation s tates . The commercial supplier assured us that

chromium was indeed added as Cr(VI) (dichromate) , but it

is apparently reduced to Cr(III) in subsequent manipulations

of the f reeze-drying process.

3 . 3 Results and Discussion

The results of the trace metal analyses of the freeze-dried

urine samples are shown in Tables 3, 4, and 5. The copper

concentrations as shown in Table 4 are higher than

specified due to the relatively high normal- copper values

in the freeze-dried urine (Table 3). The difference in

elevated nickel values between activation analysis

(ave.=1.01 mg/1) and polarography (ave.=0.82 mg/1) is

not believed to be significant in view of the estimated

overall uncertainties of ±10 percent for each method.

Accordingly, a recommended value for nickel of 0.9 mg/1

±0.1 is consistent with both measurements.

8
TABLE 3

Metals in "Normal" Freeze-Dried Urine

by Neutron Activation Analysis

Metal mg/1 Average (mg/1)

Cu 0 . 028 0.028

0.029

As 0.022 0.023

0 . 023

0.025

be U . U i U n m n u . U 1 u 0.011

Ni <0 .100 <0.100

Cr 0.0261 0.026

0.0257

The samples as supplied to the National Institute for

Occupational Safety and Health are labeled as Freeze-Dried

Urine - "Normal" (285 total); "w/Fluoride" (95 total);

"w/Metals" (84 total) . The samples should be reconstituted

with 50 ml of quartz-distilled water and are guaranteed

to be as stable as fresh urine after recons titut ion . The

urine samples may be reconstituted with volumes of distilled

water less than 50 ml if it is desired to have a solution

more concentrated with the trace metals; however, the

50 ml volume is recommended. The urine samples should

be refrigerated at all times.

9
TABLE 4

Metals in "Elevated" Freeze-Dried Urine

by Neutron Activation Analysis

Specified

Average Concentrations

mg/1 mg/1 mg/1

0.068 0.062 0.030

0.061

0.067

0.063

0.060

0.054

0.060

0.98 0.96 1.00

0.99

0.91

0.027

0.025

1.15

0.92

0.93

1.06

0. 047

0. 051

0.046

0.043

0.026

1.01

0. 047

0.025

1.00

0.050

10
Metals

TABLE 5

in Freeze-Dried Urine by Polarography

Concentrations (mg/1)

Metal Normal Elevated

Cu 0.026

0. 026

0.026 average

0.0608

0.0626

0.0617 average

Ni <0.02 0.81

0.84

0.82 average^

^Estimated uncertainty = ±10%

4. FLUORINE IN FREEZE-DRIED URINE

R. A. Durst

4 . 1 Introduction

The determination was carried out by the method of

standard additions, using a Gran plot extrapolation to the

end point. A combination fluoride electrode was used to

monitor fluoride concentration during the analysis.

4 . 2 Experimental

The procedure consisted of diluting 10 ml of the

reconstituted urine with 10 ml of a total ion strength

adjustment buffer (TISAB) and adding three aliquots

of a standard fluoride solution. The TISAB (57 ml/1

glacial acetic acid, 58 g/1 NaCl, 0.3 g/1 sodium citrate

and adjusted to pH 5.0-5.5 with NaOH) was used to maintain

the ionic strength and pH constant. The standard fluoride

solution contained 0.306 g KF/1 for a fluoride concentration

of 0.1 mg F"/ml.

11
4 . 3 Results and Discussion

For the urine samples with elevated fluoride, nominally

7 mg/1 (ppm) , three 1 ml aliquots of the fluoride

standard were used and the data were plotted on 10 percent

volume-corrected Gran paper. Five samples were run in

duplicate and the results are given in Table 6. An

average of 7.8 ± 0.1 mg f"/1 was found with no indication

of inhomogeneity between samples.

The normal urine samples were spiked with three

100 ul aliquots of the standard fluoride solution and

the data were plotted on zero volume-corrected Gran

paper. Three samples were run in duplicate and the

results are given in Table 6. An average of 0.41 ± 0.02

mg F"/l was found.

A duplicate blank determination on the TISAB gave a

value of 0.01 mg F~/l. The above values and the data

in Table 6 are not corrected for this small blank.

The freeze-dried urine samples should be handled as

specified in section 3.3.

12
TABLE 6

Fluoride Concentration in Freeze-Dried Urine

"Elevated" Fluoride mg/1

Sample 1 7.9

7.9

Sample 2 7.8

7.7

Sample 3 7.7

7.8

Sample 4 7.7

7.7

Sample 5 7.7

7.7

"Normal" Fluoride mg/1

Sample 1 0.39

0.42

Sample 2 0.41

0.40

Sample 3 0.43

0.42

Blank mg/1

Sample 1 0.01

Sample 2 0.01

13
5. LEAD IN BLOOD REFERENCE MATERIAL

L. T. McClendon, J. W. Gramlich, L. A. Machlan,

T. J. Murphy, E. J. Maienthal, D. A. Becker

5.1 Introduction

The determination of lead in blood is of interest both

to occupational health laboratories and clinical

laboratories. Because of the importance of this analysis

in inner-city areas for the determination of lead in

blood of children, considerable work has been completed

in the areas of analytical technology and sample

preparation. As a result of the considerable effort

for these samples, much information has been obtained.

Most workers in the field feel that for the analysis of

the blood samples containing elevated lead concentrations,

the lead should be present from normal metabolic processes,

as opposed to a direct spiking technique. For this reason,

the elevated lead in blood samples prepared for the National

Institute for Occupational Safety and Health through the

National Bureau of Standards, were obtained by feeding lead

acetate to pigs. This feeding was accomplished through

the courtesy of Dr. E. Miller of the United States Food

and Drug Administration Toxicology Laboratory.

5 . 2 Experimental

After a several-week feeding regimen, the pigs were sacrificed

and blood samples were obtained by way of heart puncture on

the animals. Approximately 1.5 liters of blood from these

animals were obtained. In this particular case the scientists

in the FDA laboratory used ethylenediaminetetraacetic

acid (EDTA) as the ant icoagulent . A second two-liter sample

of pig blood was obtained from animals which had not been

fed lead other than that from normal dietary sources. For

this blood, sodium heparin was used as the anticoagulent

14
Upon arrival at NBS, samples were taken of the two blood

sources and were analyzed polarographically for

confirmation of proper approximate concentrations of the

starting material and to detect any changes in the lead

concentration that might occur in the processing

sequences. In preparing the material for use as a standard

it was desired to have material which was as homogeneous

as possible and which had been hemolyzed. To hemolyze

the blood it was frozen at a temperature of -12 °C

for 24 hours, thawed and refrozen and rethawed twice at

the same temperatures and times. After this treatment some

cryoproteins had apparently been precipitated as two

phases were obviously present in the sample.

The samples were then treated with an ultrasonic cell

disrupter to break up as many of these remaining unlysed

cells as possible. After sonif icat ion , the blood samples

were centrifuged, and the supernatant liquid and precipitate

were then analyzed by polarographic techniques. At this

point it was demonstrated that the lead concentration in

the liquid phase was essentially the same as the starting

material, with only a minute amount of the lead in the

solid phase. After centr ifugat ion , all of the supernate

liquid was combined and mixed well. All of the operations

with both the high concentration and low concentration

blood samples were carried out in preleached (with high

purity HNO3) and presterilized Teflon or polypropylene

containers

5 . 3 Results and Discussion

After careful mixing, the blood samples were packaged in

12 -ml sterile polycarbonate sample containers and

random samples were picked for assay by isotope dilution

mass spectroscopy. Seven ampoules of the blood were

15
analj^zed for this purpose; five ampoules of blood at the

elevated concentration and two background concentration

samples. Each ampoule of elevated blood was sampled

with one five -gram and two three-tenths -gram aliquots;

for the background blood, only five -gram aliquots were

used. The results of these analyses are shown in Tables 7

and 8. Three analytical blank determinations were made

which averaged 5.5 nanograms of lead. This gave a blank contribution of approximately 0.1%, 1.61, and 2.9%

to the five gram aliquots, the three -tenths -gram elevated

blood aliquots and the five -gram background aliquots,

respectively.

After shipment to NIOSH of the lead-in-blood samples, it

became obvious that the normal levels for porcine blood

were too low to be easily determined. Therefore, a second

set of normal levels was produced, from mixed norm.al

and elevated levels of porcine blood. This mixture was

thoroughly agitated and homogenized, then packaged and

delivered under conditions similar to the earlier

samples. The analyses of this second set of normal

samples are found in Table 9.

The samples as supplied to the National Institute for

Occupational Safety and Health are in '^IZ gram polycarbonate

sample containers labeled as either high level or low

level lead. We are satisfied as to their homogeneity and

the concentration value of the lead obtained using

isotope dilution mass spectroscopy. This is an absolute

technique demonstrated to have an accuracy of better

than 0.5% absolute. Prior to analysis, the sample should

be shaken vigorously for 60 seconds and, if desired, may

be treated by sonif ication. The samples should be

refrigerated at all times.

16
TABLE 7

Elevated Lead Concentration in Porcine Blood

Vial Number Aliquot Number ^ ppm Pb ^ ppm Pb ^

1 2 1.021 1.042

3 1.036 1.042

18 ^2 0.987 0.992

3 0.985

33 2 1.001 0.990

3 0.985

47 2 0.995 0.992

3 0.990

61 2 0.986 0.990

3 0.988

Average = 0. 997 1.001

s = 0.017 0.023

fSample size = 0.300 g

Sample size = 5.00 g

TABLE 8

Normal Lead Concentrations in Porcine Blood

Vial Number ppm Pb^

B22 0.0335

B117 0.0294

Average = 0.0315

^Sample size = 5.00 g

17
TABLE 9

"Elevated Normal" Lead Concentration in Porcine Blood

Sample Aliquot ppm Pb

5 1

2

0.1438

0.1436

6 1

0

0.1367

0.1352

6. CONCLUSION

The various reference materials produced and supplied,

along with known concentration levels for the elements

of interest, should be extremely useful for the evaluation

of field and laboratory analytical methods for the analysis

of toxic elements. In particular, the use of at least

two calibration points (i.e., "normal" and "elevated"

levels) for a given matrix should provide a more

positive calibration over the range of interest for

occupational toxicological levels of exposure.

18
NBS-114A (REV. 7-73)

U.S. DEPT. OF COMM. BIBLIOGRAPHIC DATA

SHEET

1. imULK Al ION OR Ki;i'OK r NO. NBSIR 73-406

2. Gov't Accession

No.

3. Recipient's Accession No.

4. TlTLl- AND SUBTITI.l- REFERENCE MATERIALS FOR THE DETERMINATION OF

TRACE ELEMENTS IN BIOLOGICAL FLUIDS

5. Publication Date December 1973

6. Performing Organization ( ode

7.AUTH0R(S) J). LaFleur 8. Performing Organ. Report No. NBSIR 75-406

9. PERFORMING ORGANIZATION NAME AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project/Task/Work Unit No.

11. Contract/Grant No.

12. Sponsoring Organization Name and Complete Address (Street, City, State, ZIP)

National Institute for Occupational Safety and Health

Division of Laboratories and Criteria Development

1014 BroadwayCincinnati, Ohio 45202

13. Type of Report & Period Covered

Final

14. ^>ponsoring Agency v^odc

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.) The preparation of a number of re analysis of trace elements in bio

The standards produced include me

tration levels, five elements [Se

dried urine at two levels, fluori

levels, and lead in whole blood a These reference materials have be

of interest by one or more analyt

supplied with known concentration

ference materials for the

logical standards is described,

rcury in urine at three concen-

, Cu, As, Ni, Cr] in freezene in freeze-dried urine at two

t two concentration levels,

en analyzed for the element(s)

ical techniques, and are

levels

„

17. KEY WORDS (six to twelve entries; alphabetical order, capitalize only the first letter of the first key word unless a proper name; separated by semicolons) Arsenic; blologlcal fluids; chromium; copper;

fluorine; lead; nickel; reference materials; selenium; trace elements;

urine

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