HO

NBSIR 74-548,f)

Quarterly Report of In-Vitro

Diagnostic Products

FDA Contract No 74-58 (Oj

R. Schaffer

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C. 20234

August 1974

Interim Report for Period

April -- June 1974

Prepared for

Bureau of Medical Devices and

Diagnostic Products

Food and Drug Administration

Rockville, Maryland 20852

NBSIR 74-548

QUARTERLY REPORT OF IN-VITRO

DIAGNOSTIC PRODUCTS

FDA CONTRACT NO 74-58 (0)

R. Schaffer

Analytical Chemistry Division

Institute for Materials Research

National Bureau of Standards

Washington, D. C- 20234

August 1974

Interim Report for Period

April-- June 1974

Prepared for Bureau of Medical Devices and

Diagnostic Products

Food and Drug Administration

Rockville, Maryland 20852

U. S. DEPARTMENT OF COMMERCE. Frederick B. Dent, Secretary

NATIONAL BUREAU OF STANDARDS, Richard W. Roberts. Director

Quarterly Report of In-Vitro Diagnostic Products

FDA Contract No 74-58(0)

Quarterly Report April- -June 1974

to

Food and Drug Administration

by the

National Bureau of Standards

Progress toward the goal of establishing the

accuracy of several clinical reference methods is

reported. These are reference methods for serum

glucose, sodium, potassium, lithium, magnesium,

and chloride, and for blood lead. Progress is

reviewed on the development of or use of base-line

methods for assigning high accuracy target values

to samples needed for the study. The progress made

by expert teams for the several reference methods

is reported.

TASK la: SERUM GLUCOSE

Base-line Methods . 13 2 Preparations of U- C-D-glucose and 6,6- H-g-glucose were

carefully purified. The two isotope- labeled sugars and normal

g-glucose (NBS-SRM 917)* were separately converted into crystalline

1 , 2 : 5 , 6 -di-0- isopropyl idene-D - glucofuranose , and purified.

(This derivative, unlike glucose itself, is suited to gas

chromatography-mass spectroscopy [GC-MS].) A series of mixtures

composed of a variety of known proportions of either of the

isotopic derivatives with the normal glucose derivative were

prepared for use in calibrating GC-MS isotope-dilution

measurements. To examine for evidence of isotope effects that

may occur during the course of preparing that derivative from

the isotopic sugars, a series of mixtures having known proportions of either of the labeled glucoses and the non-labeled

glucose were converted into the di- isopropylidene derivative

for testing for evidence of alterations of isotopic proportions

from that predicted from the starting materials. Different

known amounts of either of the labeled glucoses were added to

^Available from National Bureau of Standards

Office of Standard Reference Materials

Washington, D. C. 20234
Page 2

aliquots of one serum containing glucose, and the isotopic

mixture of glucoses was isolated from these sera as the

di - isopropyl idene derivative. The precision and accuracy of

the isotopic dilution-mass spectrometry (ID-MS) method of

measurement for the original glucose content of the serum will

be determined from the data obtained in the last cited series.

Use of the two isotopic glucoses for this same kind of

measurement is to provide added assurance for the applicability

of ID-MS as a base-line method for glucose.

A gas chromatograph- -mass spectrometer system for

application to problems in organic isotope dilution mass

spectrometry has been designed. The major components of the

system are a Varian 2740* gas chromatograph, a Varian MAT CH7-A*

mass spectrometer and a Hewlett-Packard 9830 programmable

calculator. The gas chromatograph is capable of dual column

operation and has flame ionization detectors to allow comparison of effluent peak profiles with the total ionization trace

obtained from the mass spectrometer.

The mass spectrometer is capable of operation in electron

impact and chemical ionization modes. Dual sets of electronics

allow rapid switching between the two modes without refocusing

the ion beam. A multiple ion selection (MIS) unit allows the

monitoring of eight (8) mass - to- charge (m/e) ratios simultaneously, a feature of importance for isotope dilution analyses

of multiply labeled compounds. Switching between m/e values

is accomplished magnetically, avoiding the refocusing problems

and mass range limitations of accelerating voltage alternation.

An interface is being designed which will multiplex the

analog signal from the MIS unit of the mass spectrometer and

feed the data through a vol tage - to- frequency converter to the

programmable calculator. Software is being written for the

analysis of the data obtained.

Experts Team

The AACC Standards Committee's Subcommittee on a Glucose

Reference Method (Dr. R. Schaffer, Chairman) serves as the

*brand name waiver
Page 3

Experts Team. Subcommittee member, Dr. Jane Neese, undertook

the clinical development of the protocol for the hexokinase

plus glucose 6-phosphate dehydrogenase method on which the

experts team chose to concentrate its attention as the candidate

reference method. Neese* s work, performed at the Center for

Disease Control (CDC) , was reviewed at the meeting of the Experts

Team held in Washington, 28 March 1974. Subsequent to that

meeting, reagents and samples were prepared for a first-round

performance of the candidate method; this was initiated in early

June 1974. The assembled data, which looks encouragingly good,

is undergoing statistical examination (at the time of this writing).

TASK lb: SODIUM, POTASSIUM, LITHIUM, MAGNESIUM, AND CHLORIDE

Base-line Methods

Portions of analytical reports pertaining to base-line

determinations are reproduced as illustrations of isotopedilution applications.

Lithium in Blood Serum:- -A 5 gram sample was taken from

each of six ampoules for each of the three lots of serum. The

samples were spiked with Li, K, Mg and ^Ca solutions.

The serum was decomposed by adding HNO^ and HCIO^, and heating.

The samples were then evaporated to dryness, taken up in ^0

and the four elements, Li, K, Mg, and Ca were separated using

cation exchange columns. The Li and K were eluted with 0.5

mol/liter HC1 and the Ca was eluted with 6 mol/liter HC1. The

fractions were evaporated to dryness, heated with a small amount

of HNO^ to destroy organic matter, and the Li and K were converted

back to the chloride by adding a small volume of HC1.

A 6- inch radius of curvature 60° analyzer tube instrument

was used for the isotopic ratio measurement. The mass spectrometric technique for lithium is similar to a tantalum triplefilament procedure developed for potassium analysis. The

estimated lithium sample size per analysis is approximately
Page 4

5 ug. Isotopic compositional analyses for each serum concentra6 7 tion level gave Li/ Li ratios that were experimentally identical

to the isotopic ratio of the natural reference standard. The

6 7 isotopic abundances (atom %) for •• Li and Li of the serum samples

are 7.4555 and 92.5445, respectively. Concentration values for

a single analysis of each of six serum subsamples at three different concentration levels, are given in table 1.

The data has been corrected for an average blank of three

nanograms which is insignificant with respect to the total amount

of lithium in each subsample. The indicated uncertainty is a 95% limit of error for a single analysis and is based upon a

6 7 careful study of Li/ Li measurements for the tantalum triplefilament technique. This study has revealed that the ability

to reproduce the same isotopic fractionation between analyses

limits the precision of the ratio measurement to 0.251-0.50%.

The larger uncertainty is being used as the 95% limit of error

for the concentration measurements.
Page 5

Table 1. Concentration of Lithium in Blood Serum

Sample

1-2

1-170

1-235

1-287

1-356

1-443

Average

3-64

3-116

3-184

3-256

3-401

3-495

Average

5-63

5-96

5-266

5-320

5-398

5-500

Average

Ug/g

6.8168

6.7832

6. 8095

6. 8088

6. 8039

6.7877

= 6.816 ± 0.034

13. 331

13. 364

13.366

13.357

13.363

13. 378

= 13.36 ± 0.07

20. 058

20.005

20. 072

20.002

20.020

20. 020

= 20.03 ± 0.10
Page 6

Chloride in Blood Serum: --The samples consisted of six

randomly selected ampoules from each of the three lots of

serum. Each sample was treated as follows:

37 A 1 gram sample was spiked with CI and, after equilibration, the protein was precipitated by ammonium molybdate

solution. The chloride in the "protein- free" filtrate was

precipitated as silver chloride which was removed by centrifuging the solution. The AgCl was then dissolved in concentrated

ammonium hydroxide solution so that 1 ml of solution contained

5 mg CI.

Two different mass spectrometers were used for chlorine

isotopic ratio measurements. One instrument was dedicted to

37 blanks and enriched CI samples and the other to spiked samples

and the natural isotopic standard. This arrangement is

absolutely essential in order to avoid serious memory problems

caused by analysis of enriched and natural chlorine ratios

using a single instrument. In addition, the maximum range in

isotopic ratio without removing and thoroughly cleaning the

source was restricted to 3%.

Isotopic composition analyses, made for each lot of serum,

were found to be identical to the absolute isotopic ratio of

the natural standard. Concentration values for a single analysis

of each sample of the three lots of serum are given in table 2.

A blank correction of 1.5 yg which is less than 0.03% of the

total amount of chlorine present in a 1 g sample has been

made. The indicated uncertainty is an estimated 95% limit

of error and is slightly larger than the 95% confidence limits

for a single determination (to) calculated from the concentration

data. The additional allowance is made for possible error

contributions from background signals, trace impurities and

loss of chlorine during the sample mounting procedure.
Table 2. Concentration of Chlorine in Blood Serum

Sample Pg/g

1-1

1-2

1-3

1-4

1-5

1-6

Average

3173.30

3175.44

3177. 06

3184 . 18

3172.43

3174.83

3176.2 ± 15.9

3-1

3-2

3-3

3-4

3-5

3-6

Average

3554. 27

3554.17

3552 . 50

3548.84

3541.98

3542.83

3549.1 ± 17.7

5-1

5-2

5-3

5-4

5-5

5-6

Average

3948. 37

3956.44

3942.33

3945. 62

3951. 22

3946. 53

3948.4 ± 19.7
Page 8

For base-line sodium values, gravimetry and activation

analysis are being applied, but the results of these efforts

are not yet completed.

Experts Team

On 21 March 1974, the Experts Team (Drs. George N. Bowers, Jr.

Bradley E. Copeland, Denis 0. Rodgerson, and James M. White)

assembled at NBS. The team selected Dr. Bowers as chairman.

Base-line methods, candidate reference clinical methods and

accuracy goals were discussed. NBS would proceed with its work

on base-line methods for the electrolytes. Individual members

of the Expert Team would take the principal lead in working with

the NBS coordinator to prepare drafts of candidate reference

protocols for assigned electrolytes. Recommendations for

cooperating laboratories were discussed, and lists of proposed

laboratories to work with the reference protocols were prepared.

TASK lc: LEAD IN BLOOD

Base-line Methods

The quality of isotope dilution-mass spectrometr ic

analyses for lead at all concentration levels, including trace

levels, was established in previous programs involving analysis

for this element, and hence the method is ideal for providing

base-line values for the samples studied for the candidate

reference method. Samples are being prepared from animals

receiving lead acetate in their diets and from other animals

whose blood lead content is at very low levels as proved by

similar application of ID-MS.

Experts Team

This team was organized prior to begining of the FDA-NBS

interagency agreement. Eight experts in lead analysis were

invited to participate on the team. Initially, the chairman

was Dr* Philip D. LaFleur, who is now Acting Chief of the
Page 9

Analytical Chemistry Division; Mr. Donald A. Becker is now

leading the effort. The results of two rounds of analyses

were needed before the experts team was able to concentrate

on the development of a single method as the candidate clinical

reference method. Details of a procedure for a proposed

reference method were discussed at the Experts Team meeting

and then integrated in a candidate protocol. This protocol

was distributed to all members of the experts team for comment

and criticism.

N3S-114A (KUV. 7-70

' ; i

• Of- comm. |. |>| hi |, \ | ok K| |>ok 1 NO. 2. C.v'i An t-s-.n n BIBLIOGRAPHIC DATA No. shfet NBS I R 74-548

3. K. i ipicnt A, i '.ii in No.

4. Mill \.\i> m;h n 1 Quarterly Report of In-Vitro Diagnostic Products

FDA Contract No 74-58(0)

5. I'uMii .it. on Date August 1974

6. Performing Organization * o.Je

7. AT IHORfSl

Robert Schaffer

8. Performing Organ. Report No.

9. PERFORMING ORGANIZATION X A Ml- AND ADDRESS

NATIONAL BUREAU OF STANDARDS

DEPARTMENT OF COMMERCE

WASHINGTON, D.C. 20234

10. Project Ta.sk 'Work tinit No.

11. ( ontr.ut Grant No.

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

Bureau of Medical Devices and Diagnostic Products

Attn: Dr. Eloise Eavenson, Rm. 330 HFK-200

Food and Drug Administration

1901 Chapman Avenue, Rockville, Maryland 20852

13. Type of Report & Period Interim April-June

1974

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. ABSTRACT (A 200-word or less tactual summary oi most significant information. H document includes a significant

bibliography or literature survey, mention it here.) Progress toward the goal of establishing the accuracy of several

clinical reference methods is reported. These are reference methods

for serum glucose, sodium, potassium, lithium, magnesium, and chloride,

and for blood lead. Progress is reviewed on the development of or use

of base-line methods for assigning high accuracy target values to

samples needed for the study. The progress made by expert teams for

the several reference methods is reported.

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

)

Clinical chemical accuracy; clinical chemical analysis; electrolytes

in serum; glucose in serum; lead in blood; reference clinical methods.

18. AVAILABILITY [ Unlimited

For Official Distribution. Do Not Release to NT1S

[_ Order From Sup. of Doc, U.S. Government Printing Office Washington, D.C. 20 502. SD Cat. No. ( H

L Order From National Technical Information Service (NTIS)

Springfield, Virginia 22151

19. SECURITY CLASS

(THIS REPORT)

UNCLASSIFIED

21. NO. OF PAGES

9

20. SECURITY CLASS

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UNCLASSIFIED

22. Price

USCOMM.DC Z9042-P74
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