miOM s;3&e?

NBSIR 78-1585

Final Report

FDA Contract No. 74-58(0)

R SchaM#r R A V® ipo*di and 0 J Reeder

Cantmt for Analytcal Chemistry

Vi'orvai Measurement Laboratory

Satiorvai Bureau of Standards

W»sb*ngtoo 0 C 20234

October 1978

Report for Period

April 1 976 to October 1 97*

Issued January 1 979 QQ100

•U56

73-1535

Prepared for Bureau of Medical Devices and

Diagnostic Products

Food and Drug Administration

Rockville. Maryland 20852

Rational' Bureaa of S*ar.tf&rta DEC 12 19/9

aoi c - CU.P

NBSIR 78-1585

FINAL REPORT

FDA CONTRACT NO. 74-58(0)

R Schaffer R A Velapoldi, and D. J. Reeder

Center for Analytical Chemistry

National Measurement Laboratory

National Bureau of Standards

Washington. D C 20234

October 1978

Report for Period

April 1976 to October 1977

Issued January 1979

Prepared for Bureau of Medical Devices and

Diagnostic Products

Food and Drug Administration

Rockville, Maryland 20852

U S DEPARTMENT OF COMMERCE, Juanita M. Kreps, Secretary

Jordan J. Baruch, Assistant Secretary for Science and Technology

NATIONAL BUREAU OF STANDARDS. Ernest Ambler, Director

S« ••

Table of Contents

Page

Introduction 1

Task la. Glucose in Serum 2

A. Definitive Results and Inter laboratory Test

Results with the Reference Method 2

B. Additional Definitive Method Analyses 2

C. Definitive Method Analysis of New Pools .... 4

D. Definitive Method Analysis of WHO Reference

Serum 6

Preliminary Work on an Alternative Definitive

Method 6

Task lb. Lithium, Magnesium, Sodium, Potassium, and

Chloride 9

A. Lithium 9

B. Magnesium 14

C. Sodium 17

D. Potassium 17

E . Chloride 2 5

Task lc. Lead (Pb) in Blood 30

A. Inter laboratory Exercise IV 30

B. Interlaboratory Exercise V 30

C. Preparations for Use of an Alternative

Procedure 32

Task Id. Uric Acid in Serum 33

A. Description of the Definitive Method 33
Table of Contents (Continued)

Page

B. Initial Results by the Definitive Method .... 34

C. Study Group Meetings 37

Task le. Cholesterol 39

A. Cholesterol-dy for the Definitive Method .... 39

B. Presentation of the Definitive Method at NBS

Symposium 40

C. Comparison of Definitive Method Analyses with

ID-MS Values Obtained at the Karolinska

Institute 40

D. Study Group Meetings 42

Task If. Serum Iron 44

A. Definitive Method 44

B. Study Group Meetings 45

Task lg. Bilirubin in Serum 48

A. Isotope-labeled Bilirubin for Use in a Definitive Method 48

B. Study Group Meetings 48

Task lh. Urea 51

A. Preliminary Work on a Definitive Method .... 51

B. Description of the Definitive Method 52

C. Study Group Meetings 53

Acknowledgements 56
INTRODUCTION

This report covers work done at the National Bureau of

Standards (A'BS) between April 1976 and October 1977 under FDA

Contract ‘*4-58(0). The minutes of meetings held during this

reporti" connection with this work have been supplied

previously to the Food and Drug Administration (FDA), and therefore are not included here. However, results obtained through

pt 1978 are included to bring the information up to date.

We expect to keep the FDA informed of progress as this work

cont inues

.

1
Task la. GLUCOSE IN SERUM

A. Definitive Results and Inter laboratory Test Results

with the Reference Method

The statistical analysis of the serum- glucose values that

were determined by interlaboratory testing of the reference

method and by the ID-MS method at NBS was sent to the Food and

Drug Administration in September 1976. That analysis showed

that there was no bias between the methods with serum glucose

at a concentration of 0.8 g/L, but that relative to ID-MS the

reference method gave a higher result at a concentration of

0.4 g/L and lower results at concentrations greater than 1.3 g/L.

B . Additional Definitive Method Analyses

We sought to analyze additional samples from these serum

pools to enhance the precision of the ID-MS measurements, but

only one more set could be analyzed because the supply of these

samples was exhausted. These additional ID-MS results are

shown in Task la, Table 1, labeled as Set V. The table also

provides old and new results for Sets III and IV, listing the

results shown in Table 19 of the September 1976 statistical

report and giving new results for Sets III and IV which were

obtained when Set V was being measured. This was to assure

that the results for Set V could be reliably compared with the

old results. The new results for Sets III and IV are remeasurements by GC/MS of the specimens of isolated diacetone glucose

that had been originally measured.

2
'.ask la. Table 1. ID-MS Results for Glucose in Pools ]-5

(in g/L) ; Results for Set V and Previous

and New Results for Sets III and IV

1 3 4 5

Set III 0.4029 0.7765 1 . 320 1.928 2.929

previous 0.4027 0.7778 1 . 332 1.927 2.927

0.4028 0.7745 1.328 1 . 923 2 .927

0.4034 0.7784 1 . 327 1.923 2 .924

Set III 0.4005 0.7724 1.335 1 . 934 2 .919

new data 0.4025 0.7802 1.328 1.938 2.937

0.4030 0.7822 1.333 1.929 2.931

0.4023 0.7782 1 . 325 1.923 2.928

Set IV 0.3939 0.7632 1 . 314 1 .878 2.883

previous 0.3937 0.7624 1.313 1 . 891 2 . 867

0.3930 0.7626 1.314 1.890 2 . 883

0.3940 0.7632 1 . 314 1.894 2 . 867

Set IV 0.3935 0.7630 1.309 1.911 2.878

new data 0.3950 0.7632 1.318 1.902 2 .879

0.3944 0.7643 1.308 1 . 894 2.851

0.3926 0.7655 1.310 1.892 2.859

Set V 0.3908 0.7662 1.305 1 . 887 2.853

0.3955 0.7645 1 . 302 1 . 871 2.839

0.3968 0.7693 1.314 1.892 2.847

0.3969 0.7727 1.302 1.885 2.849

3
Task la, Figure 1 illustrates the results obtained on

reanalysis of the serum pools over a period of time, and are

shown as percent changes from the initially determined ID-MS

values. The data used for this figure are the averages of the

values listed in Table 19 of the September 1976 report and the

averages of the values for Set V, given in Task la, Table 1.

Note that non-uniform time intervals between the sets of

analyses are shown in the figure. The finding of lower results

upon later reanalyses of each of the serum pools raised the

possibility that the glucose levels in these serum pools were

declining. However, the similarity in the magnitudes of the

differences in concentration and the imprecision of measurement

obscured the evidence for sample instability.

C . Definitive Method Analysis of New Pools

To substantiate the finding of a concentration- dependent

bias in the reference method and to obtain additional data on

the precision of the ID-MS method, we planned to use the ID-MS

method on new serum pools that CDC proposed to prepare (using

human serum) and on which CDC would perform the reference method

analysis. At the close of the project reporting period, the

preparatory wet-chemical work-up for the ID-MS analysis of

these new samples had begun, but now at the time of writing

this present report, six sets of samples from these pools have

been analyzed. Although the results have not been intercompared

with reference method results nor examined statistically, a

4
PERCENT

CHANGE

ANALYSIS TIME (days) • 0

Task la, Figure 1. Percent changes in the glucose concentrations found in the five IE-III serum pools, as measured

at different times by ID-MS.

5
graphic representation of the percent changes found on sequential analysis, given in Task la, Figure 2, provides a preview

of these ID-MS results. Because the first samples taken for

the analysis of Pools A and E were lost, the percent changes

in determined values for all pools are based on changes from

the results found at 20 days. Again as in Task la, Figure 1,

the time frame in Task la, Figure 2 is non-linear. Our ID-MS

results on these new pools appear to suggest that loss of glucose

occurs with time. The concentrations of glucose in these pools

range from 3.5 to 20 mmol/L. The precision of these new analyses

is better than was obtained with the previous pools.

D . Definitive Method Analysis of WHO Reference Serum

During the project reporting period, the ID-MS method was

employed for the analysis of the so-called World Health Organization Reference Serum. The analysis was performed essentially

as a within-day precision study in that the additions of labeled

glucose to eight serum aliquots were made on one day. Possible

inter-vial inhomogeneities were eliminated by pooling the contents of several randomly selected vials and taking the eight

aliquots for analysis from the mixture. The result, based on

16 measurements (two on each of the eight aliquots) , was

5.444 ± 0.0093 mmol/L representing the mean and one standard

deviation, respectively.

E . Preliminary Work on an Alternative Definitive Method

As an independent means for establishing the accuracy of

results obtained by the ID-MS method involving diacetone glucose,

6
ANALYSIS

TIME

(days)

O

CJ>

QQ 7

POOL

Task

la,

Figure

2.

Percent

changes

in

the

glucose

concentrations

found

in

six

pools

of

human

serum,

as

measured

at

different

times

by

ID-MS.
r

preliminary experiments were begun with an ID-MS method that

employs the bis -butaneboronate acetate derivative of glucose

(J. Wiecko and W.R. Sherman, J. Amer. Chem. Soc . , 98 , 7631

(1976)). Use of this derivative for ID-MS was reported by

D. M. Bier, W. R. Sherman, W. H. Holland, and D. M. Kipnis,

(Proceedings of the First International Conference on Stable

Isotopes in Chemistry, Biology, and Medicine, May 9-11, 1973,

Argonne, II., P. Klein, Ed., p. 397). The method they reported

cannot be used as a definitive method because the butaneboronate

acetate derivatives of other hexoses present in serum are not

separated from that for glucose in the GC procedure the authors

describe. (In the diacetone glucose method, TLC is run in

several systems to remove other hexoses that may be present.)

Our preliminary experiments indicate that these other hexose

butaneboronate acetates may be resolved from the glucose derivative by use of capillary column gas chromatography. Work

beyond this preliminary stage has been held up until the backlog

of samples to be measured by GC/MS has been cleared up.

8
'.ask lb. LITHIUM, MAGNESIUM, SODIUM, POTASSIUM, AND CHLORIDE

Sunnary : Statistical analyses of the results from the

Interlaboratory Exercises (IE) for Lithium IV, Magnesium II,

Sodium II, Potassium II, and Chloride II were presented and

discussed at the Experts Committee meeting held at NBS on

November 22, 1976. The Committee members, NBS statisticians,

and other NBS participants present agreed that the goals set

original! Committee for precision and accuracy for the

L . , Na, K , and Cl reference methods had been achieved. The

• iti itical results are summarized in Task lb, Table 1.

The results are discussed in detail for each electrolyte.

Lithium: Statistical analysis of the results from IE-IV

was completed. The exercise consisted of tests on four samples

provided in separate vials) at each of four different concentrations (Pools 1, III, 5, V). Two vials at each concentration

ere anal vied on day 1 and the two other vials were analyzed on

dav 2. A summary of the results for the manual (M) and semiautomated (SA) procedures is given in Task lb, Table 2. The

number immediately after the 'M' or 'SA' is the lab identification and the last number (either 1 or 2) denotes the day of

analysis. The values in the columns are averages of the results

from the two vials for that day. Laboratory 7 (M 72X) did not

obtain results by the manual procedure for day 1. The data are

illustrated in Task lb, Figures la and lb, as percent deviations

from the ID-MS assigned values. The pools are designated in the

figure by 1, III, 5, and V. The same pool-sample sequence is

used for each laboratory's results.

9
Task

lb:

Table

1.

Imprecision

and

Bias

Goals

and

the

Values

Obtained

by

Statistical

Analysis

from

Inter

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Exercise

Testing

for

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10
Task lb, Table 2. Averages of Lithium Concentration.

Cooperating Laboratories' Results for IE- IV.

Lithium, mmol/L

Procedure Pool

Lab Dav 1 III 5 V

M 11 . 5430 . 9890 1.7580 2.9910

M 12 . 5225 . 9945 1.7550 2.9080

M 21 . 5050 . 9910 1.7805 2.9925

M

"y

. 5030 .9985 1.7670 2.9775

M 41 . 5170 . 9795 1.7465 2.8695

M 42 . 5205 . 9890 1.7685 2.9160

M SI . 5185 1.0080 1.7845 2.9460

M S2 .5115 1.0150 1.8060 2.9250

M 72X . 5330 1.0055 1.8090 2.9790

M 81 . 5360 1.0315 1.8435 3.0195

M 82 . 5360 1.0295 1.8260 3.0515

M 91 . 5225 . 9940 1.7810 2.9260

Nf 92 . 5230 . 9960 1.7725 2.9260

M 101 . 5260 1.0060 1.8395 2.9495

M 102 . 5265 .9990 1.7690 2.9535

M 131 . 5200 . 9795 1.7665 2.9145

M 132 . 5205 . 9840 1.7700 2.9000

M 141 . 5205 .9840 1.7630 2.8985

M 142 . 5230 . 9855 1.7635 2.9020

M 151 . 5200 . 9790 1.7625 2.8930

M 152 . 5210 . 9900 1.7605 2.8925

SA 41 . 5150 . 9835 1.7590 2.9035

SA 42 . 5165 . 9785 1.7425 2.8810

SA 61 . 5390 1.0145 1.8050 2.9645

SA 62 . 5225 .9950 1.8015 2.9320

SA 91 . 5235 . 9935 1.7795 2.9260

SA 92 . 5230 . 9975 1.7750 2.9240

SA 101 . 5275 1.0045 1.7890 2.9420

SA 102 . 5250 . 9915 1.7780 2.9675

SA 111 . 5185 . 9750 1.7590 2.8690

SA 112 . 5180 . 9930 1.7675 2.9010

SA 151 . 5210 . 9870 1.7580 2.9120

SA 152 . 5185 . 9855 1.7560 2.9350

ID-MS Values . 534 1.004 1.809 2.954

11
Lithium

IE-IV

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Although a bias of approximately two to three percent was

observed, the statistical analysis showed that the results from

IE-IV were within the imprecision and bias goals; therefore

further investigation was not necessary. Work leading to an

NBS 260 Special Publication was begun.

B. Magnesium : IE-II for magnesium was completed with nine

laboratories performing the manual pipetting protocol and four

laboratories performing the semi - automated protocol on a total

of 12 serum samples consisting of four samples in separate vials

at each of three different concentrations (pools 1, 5, 7). Two

vials at each concentration were analyzed on day one and the

remaining vials were analyzed on a subsequent day. The results

are summarized in Task lb, Figures 2a and 2b, as percent

deviations from the definitive method values. These figures

reveal that if we exclude from the manual -pipetting procedure

results those obtained on both days in laboratory 8 and those

from one day in laboratories 7 and 13, the remaining manual

procedure values are generally within three percent of the

values determined by ID-MS. Furthermore, all of the results

obtained by the semiautomated procedure are well within two

percent of the definitive method values. However, further

interlaboratory testing for this electrolyte was discontinued

until the magnesium gluconate dihydrate SRM is made available,

since that is a basic requirement for clinical reference method

development. (The new supply of the material has been received.

It is undergoing certification. Interlaboratory testing will

resume when the certification of the SRM is completed.)

14
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15
Magnesium

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Sodium: Statistical analysis of the results obtained in

IF1

1 was completed. Four samples were analyzed at each of

five concentrations (Pools 1, 5, 4, 5, 7). As usual, two

vials of each concentration were analyzed on one day and the

remaining pairs were analyzed on another day. A summary of

the results is given in Task lb, Table 3. The designations

follow those given in Table 1. These data are graphically

depicted in Task lb, Figures 3a and 3b as percent bias from

the values assigned by the ion-exchange/gravimetry technique.

Laboratory 15 experienced blank problems with the manual

procedure. This laboratory also performed an extra semiautor.ated analysis (designated A151X and A152X) in which

samr'.es were diluted with 20 parts of diluent rather than

with 19 parts of diluent. These data are listed for comparison, but were not used in the statistical analysis.

The statistical analysis of these IE-II results showed

that they were within the preset accuracy and precision goals.

The final report, NBS Special Publication 260-60, was published

after a draft had been sent to the Experts Committee and

participating laboratories for comment. A copy of that publication is provided with this report.

D. Potassium : Statistical analysis of data from IE-II for

potassium in serum has been completed. For this exercise, four

samples in separate vials were supplied at each of three concentrations (Pools 1, 4, 6). Two vials at each concentration

were analyzed on day one and the remaining pairs were analyzed

17
Task lb. Table 3. Averages of Sodium Concentration.

Cooperating Laboratories' Results for I E - 1 1

.

Sodium, mmol/L

Procedure Pool

Lab Day 1 3 4 5 7

M 41 113.570 129.370 136.350 146.860 157.410

M 42 113.320 129.020 135.470 146.740 157.570

M 51 113.900 129.750 136.350 149.810 157.620

M 52 113.890 129.670 136.430 148.010 157 .870

M 71 113. 930 128.920 135.860 146.900 157.970

M 72 113.440 129.260 135.680 146.480 157 .720

M 81 113.690 129.730 136.360 147.330 158 .210

M 82 113.190 129.020 134.660 145.490 157.460

M 91 112.980 128.440 136.180 147.060 158 .210

M 92 113.010 128.320 136.700 146.590 156.970

M 111 113.890 129.440 136.340 147.360 157.805

M 112 113.680 129.575 137.000 145.780 157.720

M 131 113.540 130.130 137.685 148.600 158.350

M 132 113.290 129.300 136.500 147.500 158.080

M 1 51X 118.700 131.445 139.725 149.485 162 .220

M 1 52X 117.950 134.150 141.470 150.445 160 . 770

SA 11 113.840 129.220 135.980 147.200 157.810

SA 12 113.970 129.980 136.070 147.210 157.930

SA 21 113. 705 129.270 135.240 146.165 157.025

SA 22 114.095 130.135 136.855 147.770 158 . 245

SA 41 113.150 129.310 135.490 146.400 156 . 760

SA 42 113.210 129.510 136.090 147.310 157.570

SA 91 113. 590 128.470 137.710 146.450 157.410

SA 92 113.360 128.700 136.520 147.080 157 . 530

SA 101 113.710 129.400 135.690 147.290 158.200

SA 102 113.150 128'. 880 136.100 146.570 157.190

SA 111 115.230 130.475 137.205 148.645 159.495

SA 112 114.030 130.365 137.170 147.720 158.410

SA 151 113.680 129.070 134.745 146.380 157.485

S 152 114.720 129.840 136.080 145.665 156.030

A 1 51X 113.875 129.065 135.850 146.785 157.455

A 1 52X 113.360 130.205 135.635 146.065 157.605

Ion-Exchange/

Gravimetry 113.2 129.9 136.6 146.3 158.6

Values

18
Sodium

IE-11

Manual

£ 2

O

4-1 4h

CVJ O CVJ

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19

Laboratory

Number

and

Day

Run

Task

lb,

Figure

3a.

The

percent

deviations

of

the

sodium

reference

method

measurements

the

ion-exchange/gravimetry

values,

obtained

by

the

manual

pipetting

procedure

for

I

E

- 1

1.

The

pools

are

identified

by

the

numbers

1,

3,

4,

5,

and

7

near

the

results

laboratory

4

—

identification

of

the

pools

are

the

same

for

the

remaining

results.
Sodium

IE- 1

1

Semi-automated

CsJ o OJ

i 0n|DA |96jdi uio-y uo^DjAeQ *u90J9d

Z3

cn

>%

a

o

20

Task

lb.

Figure

3b.

The

percent

deviations

of

the

sodium

reference

method

measurements

from

the

ion-exchange/gravimetry

values,

obtained

by

the

semi

-

automated

pipetting

procedure

for

IE-II.

The

pools

are

identified

by

the

numbers

1,

3,

4,

5,

and

7

near

the

results

from

laboratory

1

—

identification

of

the

pools

are

the

same

for

the

remaining

results.
on dav two. The averages of the results for the manual (M) and

semi - automated (SA) pipetting procedures are summarized in

Task lb, Table 4. Lab, procedure, day run, etc., designations

are used as before. The percent deviations from the ID-MS values

are graphically shown in Task lb, Figures 4a and 4b.

laboratory 4 repeated the manual and semi - automated procedures M4 1

X

, M42X, SA41X, SA42X) because, due to instrumental

problems, the diluted samples and standards had to be stored

overnight in a refrigerator. Although shown in the table,

these values are not included in the statistical analysis.

Results from Laboratory 15 were quite variable. A repeat of

the semi - automated pipetting procedure (SA151R, SA152R) provided values with less scatter and, in general, less bias from

the ID-MS values.

Statistical analysis of these results showed no intrinsic

problem with the reference method and it was decided to proceed

with I E - 1 1 , and accordingly IE-II samples were sent to the

participating laboratories.

IE-II consisted of four samples at each of five different

potassium concentrations (Pools 1, 2, 4, 5, and 7). Statistical

analysis of the results showed that 0.1 mmol/L imprecision at

the 2.5 and 6.5 mmol/L potassium levels (which correspond to

coefficients of variation of 4.0 percent and 1.5 percent,

respectively) and of 0.2 mmol/L bias at these levels (8.0%

and 3.0%, respectively) were reached. These precision and

accuracy values were like those obtained in IE-II. The data

21
Task lb, Table 4.

Procedure

Average of

Cooperating

Potassium Concentration.

Laboratories' Results for IEPotassium, mmol/L

Pool

Lab Day 1 4 6

M 41 1.3180 4.3340 6.1110

M 42 1.3100 4.3610 6.1345

M 4 IX 1.3085 4.3515 6.1625

M 42X 1.3045 4.3080 6.1500

M 51 1.2820 4.3950 6.0680

M 52 1.3410 4.3720 6.1980

M 71 1.4150 4.4190 6.1390

M 72 1.3360 4.3710 6.0710

M 7 IX 1.7260 4.4190 6.1390

M 81 1.3630 4.6520 6.3970

M 82 1.3720 4.4290 6.1560

M 111 1.3040 4.3460 6.0845

M 112 1.2960 4.3715 6.1630

M 131 1.3425 4.3395 6.1625

M 132 1.3260 4.3400 6.1275

M 15 IX 1.3645 4.4050 6.0925

M 152X 1.5405 4.5660 6.6250

SA 41 1.3460 4.3015 6.1250

SA 42 1.3200 4.4045 6.2260

SA 4 IX 1.3320 4.3810 6.0895

SA 42X 1.3020 4.4015 6.1755

SA 101 1.3000 4.2590 6.1840

SA 102 1.3420 4.3520 6.1350

SA 111 1.2640 4.4050 6.3345

SA 112 1.3915 4.5485 6.2560

SA 15 IX 1.4980 4.5000 6.2645

SA 1 52X 1.3020 4.3085 6.1540

SA 151R 1.3405 4.3730 6.1590

SA 152R 1.3675 4.3695 6.1470

A 15 IX 1.3050 4.3330 6.1380

A 152X 1.3425 4.3785 6.1315

ID-MS Values 1.3191 4.3230 6.0921

22
Potassium

IE-1

Manual

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24
for IT - II are presented in Task lb, Figures 5a and 5b in percent

deviations from the definitive method values. After discussion

of these results with the Experts Committee and statisticians,

a draft of the final report as an NBS 260 Special Publication

has been started.

E. Chloride: Results for I E - 1 1 have been statistically

analyzed. I E - 1 1 involved the analysis of two samples at each

of five concentrations (Pools 1, 3, 4, 5, 7) on two different

days. The results for the micro (MI) and macro (MA) pipetting

procedures are summarized as averages in Task lb. Table 5. The

usual pattern of procedure, lab number, and day-run identifications is used. The results are presented also as percent

deviations from the ID-MS values in Task lb, Figure 6. Statistical analysis showed that their precision and their bias from

D-MS values were comparable to those obtained in RRI

.

At the Experts Committee Meeting it was decided that

t icial a nalysis si

the ID- MS values

At the Expe

the res ults from

goa Is

.

A draft 1

260 was started

.

25
Potassium

IEII

Manual

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I I I I I I I I 1 I I 1 I I I I I I 1 I O00O<frC\JO CVJ t CO GO — I

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26
Percent

Deviation

From

The

Definitive

Method

Value

10

8

Potassium IE-11

Semi-automated

-8

1 1

1

1 1

1

1 1

1

1 1

1

1 1

1

i i*i

1 3 1 3 1 3 1 3 1 3 1 2

1 2 10 II 15 9

Laboratory Number and Day Run

Task lb, Figure 5b. Percent deviations of the IE-II measurements

using semi -automated pipetting from the definitive method

values. The analyzed pools are identified by the numbers 1,

2, 4, 5, and 7 near the results from laboratory 1. The

designations are similar for the remaining results. The numbers

1 and 3 placed directly above the laboratory number, designate

the first day and subsequent day test results, respectively.

27
Task lb, Table 5. Averages of Chloride Concentration.

Procedure

Cooperating Laboratories'

Chloride, mmo

Results for

1/L

IE - 1 1 .

Pool

Lab Day 1 3 4 5 7

MI 11 79.790 94.735 102.515 108 . 520 117.475

MI 12 80.230 94.510 102.755 108.035 117.460

MI 21 76.875 91.150 100.650 105.760 115.000

MI 22 77.150 92.380 99.950 105.310 114.600

MI 41 79.660 94.580 101.860 107.820 117.755

MI 42 79.580 95.050 102.290 107.955 118.445

MI 71 78.980 94.890 100.980 107 . 105 117 . 585

MI 72 79.905 94.485 101.910 107.440 116.805

MI 81 78.575 94.645 101.660 106.800 116.905

MI 82 79.015 94.200 101.685 107 . 235 115.975

MI 91 79.850 94.465 101.000 106.880 114.015

MI 92 79.480 93.935 100.570 106.185 116.515

MI 101 78.440 94.265 100.100 106.050 114.800

MI 102 78.950 93.365 100.955 106.715 116.970

MI 131 79.850 94.835 102.350 107. 150 116.850

MI 132 79.650 95.000 101.950 106.765 117.100

MI 141 80.850 96.550 104.950 109.750 118 . 100

MI 142 79.850 94.700 102.050 107.400 116 . 900

MI 151 79.500 94.675 101.960 107.495 116.905

MI 152 79.450 94.745 101.990 107.315 116.925

MA 51 79.100 94.440 100.670 106.660 115.860

MA 52 79.065 93.805 101.810 106.410 116.670

MA 61 78.435 94.870 101.500 106.335 117.040

MA 62 78.430 93.545 101.485 106.345 116.235

MA 111 79.360 94.310 102.345 107.075 115.165

MA 112 79.855 94.675 101.990 107.820 116.790

ID-MS Values 79.20 94.00 101.8 107.20 116.80

28
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E29
Task lc. LEAD (Pb) IN BLOOD

A. Interlaboratory Exercise IV

Based on the fact that two-thirds of the analyses in IE-IV

(June 1975-December 1975) were within 20 percent of the ID-MS

target value, we planned another Interlaboratory Exercise so

that laboratories could use a newly revised protocol. This

revised protocol was sent to the Food and Drug Administration

and all participating laboratories in April 1976.

B . Interlaboratory Exercise V

Porcine blood, obtained from the USDA, Beltsville, MD

,

was processed into three pools for IE-V. In June 1976, each

laboratory received three samples of porcine blood containing

Pb at low, medium, and high concentrations. Results of IE-V

are shown in Task lc, Tables 1 and 2.

In summary, the results were disappointing, unexpected,

and outside the accepted goal for analytical accuracy and

precision. Statistical analysis of the results of IE-V were

presented and discussed at the Experts Committee Meeting

at NBS on September 17, 1976. Topics discussed were 1) sources

of error and differences between labs, 2) digestion procedures

3) background correction procedures, and, 4) other possible

reference methods. Anodic stripping voltammetry was considered,

but was rejected as a reference method. A graphite furnace

method, developed by T. Rains, at NBS, was discussed as a potential reference method. Other committee members offered

modifications of other methods that they hoped to try to perfect. Minutes of that meeting were sent to FDA.

30
Task lc, Table 1. Measurements of Lead in IE-V. Percent

Deviation from Target Value

Lab Low Medium High

A 234 51 74

B 8 15 2

C 4 -12 -5

D 78 41 40

E 38 10 3

F 38 15 13

G 50 22 27

H 27 36 56

Average 60 22 26

Task lc, Table 2. Measurements

yg/mL

ID-MS Target Values

Mean values from laboratories

using protocol (±1 SD) , n = 8

Mean values from laboratories

using "other methods", n = 4

of Lead in IE-V. Results

Low Medium High

.1738 . 4184 .4946

. 32± . 21 . 54± .14 . 62± .11

.18±. 02 o +i o . 50+ .02

31
Although additional samples of porcine blood were sent to

several laboratories in December 1976, for use in developing

better reproducibility and accuracy in their atomic absorption

procedures, only two laboratories reported their findings. One

laboratory offered improvements in the prior protocol; the other

gave instructions for wet digestion of blood.

C . Preparations for Use of Alternative Procedure

After discussions with committee members, it was decided

to explore the use of the graphite furnace procedure that was

suggested by Rains as a reference method. Initial committee

reluctance to consider the Rains’ procedure as a reference

method rested on the fact that it would employ an instrument

that was available from one manufacturer only. However,

several instrument manufacturers now offer suitable equipment

for use with the procedure. Several additional laboratories,

equipped with graphite furnaces, are to be added to the list

of participants and an Interlaboratory Exercise using the Rains'

procedure was planned.

A new batch of porcine blood was processed during May and

June of 1977 to provide 9 pools of 100 vials of blood. The Pb

concentration of the pools ranged from 4 to 90 yg/mL. In addition, a single pool of 500 vials was prepared with a Pb level of

35 yg/mL.

32
Task Id. URIC ACID IN SERUM

A . Description of the Definitive Method

In our ID-MS method, [ 1? N,] uric acid is added in a 7- to

10-fold proportion to the natural uric acid in the sample. The

size of aliquot of serum used for analysis is adjusted according

to its approximate uric acid content so that, after adding the

labeled uric acid, a total of about 2 mg of uric acid is present

in the sample.

In performing the method, a Li-,CO, solution of the

labeled uric acid is added to a weighed aliquot of serum;

t r iethylanilinium carbonate is then added. The solution is

freeze-dried, and the residue obtained is extracted with methanol.

The extract is concentrated and in a sublimation apparatus the

residue is heated to 150 °C for five minutes under a pressure

7 150 N/m (1 mm of Hg on a vacuum gauge) so that the volatile

products, which include the tetraethyl uric acid isomers, collect

on the cold condenser surface. The two tetraethyl uric acid

isomers that predominate in yield (several tetraethyl uric acid

isomers form) are isolated from the sublimate by TLC. Although

for determining the uric acid content of a serum sample only

one of these tetraethyl uric acid isomers needs to be analyzed,

the two are measured to enhance the validity of measurement.

For use as standards, mixtures of weighed quantities of SRM

uric acid and isotope- labeled uric acid are ethylated under similar conditions, and the same two isomeric products are isolated.

Bracketing is used. The corresponding isomeric products from

33
the serum and the standard mixtures are compared by ID-MS for

assigning values. This is necessary because the two isomers

obtained on ethylating a standard mixture of uric acid do not

have an identical non-labeled to labeled relative molecular ion

intensity. (This isotope effect results primarily from the

different rates of ethylation of the and atoms located

at the same structural positions of the otherwise identical

labeled and unlabeled uric acid molecules.)

B. Initial Results by the Definitive Method

ID-MS uric acid results on five serum pools are shown in

Task Id, Table 1. The values for the two individual isomers

isolated from the first sample of pool 1 are labeled as 1A and

1C, from the second sample as 2A and 2C, etc. Task Id, Table 2

is a summary of the analytical results of ID-MS measurements

and early inter laboratory measurements by the candidate reference method. The same samples were analyzed by both methods.

The uric acid ID-MS values shown here are somewhat different

from the values that appear in the minutes of this Study Group.

The data had been submitted to the Study Group in the units of

mg/100 g of serum, and were correlated with the preliminary

inter laboratory data without correction for the specific gravity

of the sera. This error has not been brought to the attention

of the Study Group, since it is a relatively minor one.

Further, we have discovered another minor source of error that

probably affected the results as well. The scaler, a part of

the ID-MS data handling instrumentation, was used at the time

34
Task Id, Table 1. Uric Acid in Five Serum Pools by ID-MS.

Averages in mg/L

Analysis

and Isomer

U1 Cl

1A 20.68* 43.41

1C 20.82 43.40

2A 20.82 44.38

2C 20.70 44.27

3A

3C

Average 20.76 43.86

Each value listed is an average

ments

.

Pool

U2 U3 C2

59.90 80.45 101.03

60.34 81.30 101.22

59.78 81.02 101.02

60.47 81.94 101.67

60.35 81.30

61.08 81.86

60.32 81.31 101.24

of between 6 and 12 measure35
Task Id, Table 2. Summary of Uric Acid Results Obtained by

ID-MS and the UV-Uricase Methods (mg/dL)

Cl

ID-MS X 4.386

S 0.053

CV,% 1.21

UV-Uricase X 4 .

2

at CDC Lab. s 0.16

cv,% 3.8

UV-Uricase X 4.3

IE1s 0.25

i cv,% 5.8

UV-Uricase X 4.2

IE- 1

1

s 0.3

cv,% 7.1

Pools

C2 U1 U2 U3

10. 124 2.076 6.032 8.131

0.03 .008 .046 .055

0.30 0.4 0.76 0.68

9.8 2.1 6.0 8.0

0.29 0.14 0.18 0.23

2.9 6.7 3.0 2.9

9.9 2.1 6.0 8.0

0.25 0.27 0.20 0. 20

2.5 12.8 3.3 2.5

9. 5 2.0 5.9 7.9

0.4 0.2 0.2 0.3

4.2 10.0 3.4 3.8

36
with an improper setting. This would cause an inaccuracy in

the absolute ratios observed for ratios of labeled to nonlabeled species that are far removed from 1:1, and we had used

ratios between 7 to 1 and 10 to 1. However, bracketing would

undoubtedly have reduced the error. Hence, we judge the error

in our results, due to the defective scaler, to have been small.

C . Study Group Meetings

The Study Group Meeting held on July 23, 1976, dealt with

the data from IE-II. Attention was given: (a) to the quality

of the inter laboratory data that were obtained on the standard

solutions by direct spectrophotometry, and by assay for calibration and control and test samples; and, (b) to some of the

methodologic details. The review was directed mainly at evaluating the criteria by which each laboratory can judge itself

while performing the reference method.

The June 27, 1977, meeting was a review of IE- III

results which were run on the same serum pools as had been

previously analyzed by the candidate reference method. The

difference for IE-III was in the use of reagents that the

laboratories purchased independently and prepared for themselves. The data showed that interlaboratory precision was

not adversely affected. However, data from some of the laboratories did not meet all of the criteria set for the method;

as a consequence, such data were excluded from the evaluation.

IE-IV, the final testing of the candidate reference method, was

planned. New serum pools will be involved. Samples of the

37
pools have been sent to NBS for ID-MS analysis. The preparatory chemical work-up of these samples was completed at NBS

before the close of the project reporting period, but the

spectroscopy runs were not. At the time of this writing,

GC/MS is about to be started.

mass

the

38
Task le. CHOLESTEROL

A. Cholesterol-cU for the Definitive Method

Cholesterol, containing deuterium in place of hydrogen at

all positions on carbon atoms 26, 27, and 28, was obtained from

Applied Science Laboratories (State College, PA). The preparation received was purified by sublimation and recrystallization,

'lass spectrometry then revealed only the presence of a small

proportion (^2 percent) of a non-labeled C-25 steroid. (Presumably it is related to the precursor from which the labeled

cholesterol was synthesized.) The molecular weight of the

impurity was such that it would not interfere with the mass

spectrometry. Its presence in the labeled cholesterol remained

a source of concern to us only in that it might not be uniformly

distributed, and, hence to that extent, could affect the proportions of cholesterol in quantities taken from the supply of the

crystalline labeled material. Since removal of this impurity

fro -' the labeled cholesterol was not considered feasible, only

one crop of this recrystallized, labeled cholesterol was collected

for use in ID-MS analyses, with the assumption that in the single

crystalline crop the impurity would be homogeneously distributed

and therefore without effect on the analyses. Further, the use

of bracketing with ID-MS analysis, where a single preparation

of labeled cholesterol is employed in both the unknown samples

and the standards, would obviate the effects of the presence of

an impurity unless the impurity both chromatographs like the

analyte and gives ions in the mass spectrometer with the same

39
mass to charge ratio as are used for measuring the labeled

or unlabeled analyte.

B . Presentation of the Definitive Method at NBS Symposium

A paper prepared for presentation at NBS on 10 April 1978,

as part of a symposium on trace organic analysis, summarizes

the status of the work on this definitive method as of the

project reporting period. A copy of this paper, "A Candidate

Definitive Method for the Determination of Total Cholesterol

in Serum" was previously submitted to the Food and Drug

Administration

.

C . Comparison of Definitive Method Analyses with ID-MS

Values Obtained at the Karolinska Institute

Since the close of the project reporting period, Schaffer

met with Dr. Ingemar Bjorkhem of the Karolinska Institute (KI)

in Stockholm and suggested that the cholesterol ID-MS method

in use at the KI laboratory be used to analyze the serum pools

that had been run by the ID-MS method at NBS. That has now

been done. The method used in Stockholm [I. Bjorkhem,

R. Blomstrand, and I. Svensson, "Determination of Serum

Cholesterol by Mass Fragmentography , Chim . Chim. Acta . , 54,

185 (1974)] provides for GC/MS analysis of the isolated

cholesterol directly rather than analysis of the TMS ether

derivative as is done at NBS. The results of the two laboratories are compared in Task le, Table 1. The results from

the two laboratories differed by from 0.8 to 2.3 percent, and

all the values from the Karolinska Institute were lower than

40
Task le, Table 1. Comparison of Results from Two Laboratories

doing ID-MS Cholesterol Measurements

KI NBS NBS/KI

Serum

Pool

mean ± S.E.M.

(mmol/L)

mean

(mmol/L) ratio

I 3.394 ± 0.039 3.430 1.011

II 4.679 ± 0.076 4.719 1.009

III 6.101 ± 0.039 6.149 1.008

IV 7.346 ± 0.047 7.456 1.015

1/V 8.588 ± 0.066 8.788 1 .023

MBS values. (The Karolinska Institute group found an error

in pipeting aliquots of Serum Pool V and suggested that its

difference from our value should be more like the others.)

Because of the systematic difference between their results and

ours, we investigated their standard reference material, a cholesterol preparation that they had purified themselves. A

problem with their standard (or even possibly with our cholesterol, SRM 911a) could account for the more or less regularly

observed differences between the results. By analysis, we

found that their cholesterol standard was only 98.2 percent

pure. We determined this value by the ID-MS method with our

labeled cholesterol used in a series of weighed mixtures with

their standard material and in a similar series with SRM 911a.

furthermore, we found the impurity in their cholesterol to be

lathosterol, a steroid that has the same mass as cholesterol.

41
By electron- impact mass spectrometry, lathosterol gives its

molecular ion in relatively greater abundance than does

cholesterol. There can be little question as to the origin

of the systematic error, or that the systematic error occurred

in the Karolinska Institute's analyses.

D. Study Group Meetings

At meetings of the Study Group held 10 November 1975 and

20 July 1976, the several clinical methods for the determination

of total cholesterol that had been recognized as potential candidates for the reference method were reviewed. Work done at

the CDC on the semi - automated Abell -Kendall method and the

cholesterol esterase/cholesterol oxidase enzymatic method was

reported. The Study Group recommended a continued study of

those methods. The GLC methods in Dr. Kuksis' and Dr. Martin's

laboratories were also reported; however, neither laboratory

was attempting to upgrade its method to be of reference method

quality. The Parekh-Jung method in Dr. Edward's laboratory also

continued to provide very good results. The Study Group decided

to await the outcome of the studies at CDC, rather than encourage

further intensive study of any of these other methods. The

remaining problems with the optimization of the enzymatic method

and with cholesterol solution standards used with that method

were to be studied, as were the causes of small unexplained

drifts in the Abell - Kendall method. Also, a comparison of these

optimized methods with the ID-MS method was to be made before

the next meeting was to take place.

42
In order to facilitate comparison of the data obtained by

the several methods with ID-MS data,' NBS at first provided CDC

with an estimate of the overall imprecision of the ID-MS results.

Thereafter, actually in October 1977, the CDC and NBS exchanged

results. It was then seen that a systematic difference of one

to two percent existed between the ID-MS values and the AbellKendall values. Differences between the ID-MS and the enzymatic

methods were even larger. Cooper and Schaffer discussed whether

to bring these comparative results before the Study Group at

once, but Cooper declined to do this until he had all of his

data assembled. Meanwhile, with knowledge of the ID-MS results,

Cooper undertook to investigate further sources of bias in the

methods being used at the CDC. '

43
Task If. SERUM IRON

A. Definitive Method

In order to use ID-MS to provide high accuracy iron analysis

at the concentration levels present in serum, it was necessary

to minimize the possible contamination that could occur during

the wet-chemical processing of samples. In the class-100,

filtered-air , clean room where the chemical work would be performed, it was necessary to replace all the steel laboratory

furniture and the many implements present made with iron. The

specially purified reagents used for this ID-MS analysis were

meticulously monitored for iron.

It was recognized at the start that the ID-MS method as

applied would provide total iron measurements, which includes

hemoglobin iron, and not just serum iron (or more specifically,

transferrin-bound iron) as the reference method is expected to

do. The Study Group thought it would be possible to correct

the total iron values by measuring the hemoglobin in the samples.

However, the hemoglobin content needed to be measured very

accurately in order to apply meaningful corrections. Since

total iron can be measured by ID-MS with an estimated inaccuracy

of 0.4 percent and 1.0 mg of hemoglobin corresponds to 3.47 yg

of iron, a several milligram or larger error in the estimation

of hemoglobin would produce a significant loss of attained

accuracy in the analysis of a sample with a total iron content

of, for example, 1000 yg/L.

44
Although the direct determination of serum iron by ID-MS

has not been attempted, the direct analysis may be a better

approach since it obviates the need to correct for the overestimation of iron. For direct analysis, the serum and the

isotope enriched iron would be combined and the mixture would

be treated with a coraplexing agent to liberate all the iron

(except the hemoglobin iron) from its binding sites. Then this

ionic iron would be reduced to Fe II to ensure equilibration

of the isotopic forms. Finally, the protein precipitation

would be carried out. The isotope ratio measurements would be

erformed on the iron isolated from the supernatant liquid.

B . Study Group Meetings

Our attempt to organize an experts committe to develop a reference method for serum iron was merged with a similar

: : rt of the AACC Standards Committee which was being organized

at that time. Dr. Eleanor Berman had been charged with the

responsibility for organizing a study group for the Standards

Committee. Dr. Richard Carter was asked to head the Study

Group. Dr. Schaffer arranged to fund the following for participating in this study:

Dr. Eleanor Berman, Cook County Hospital, Chicago, IL

Dr. George N. Bowers, Jr., Hartford Hospital, Hartford, CT

Dr. Thomas J. Giovanniello , Veterans Administration

Hospital, 3oston, MA

Dr. Philip J. Garry, University of New Mexico,

Albuquerque, NM

45
Dr. Theodore Peters, The Mary Imogene Bassett Hospital,

Cooper st own, NY

Dr. Robert Carter, MD, Bowman-Gray School of Medicine,

Winston-Salem, NC

Other members of the Study Group were:

Dr. Richard Carter

,

CDC, Atlanta, GA

Dr. I

.

Lynus Barnes

,

NBS

Dr. R. Schaffer, NBS

The Study Group met for the first time on 16 September 1976

and again 19 July 1977, during the project reporting period.

Subsequent meetings were held 28 October 1977 and 28 May 1978.

Although a considerable body of information already existed

on optimum methods for the determination of serum iron (see

"Modern Concepts in Hematology", G. Izak and S. M. Lewis, Eds.,

Academic Press, NY, 1972, pp. 69-129), the Study Group considered it worthwhile to test various methods in common use as

well as the method tentatively recommended by the International

Committee for the Standardization of Hematology (ICSH), which is

given on pages 126-7 of the reference just cited, and to test

minor variations of the methods thought to be of possible benefit.

The methods were performed using samples prepared at the CDC,

some of which (deliberately) contained substances that might

interfere with iron analysis. These analyses were to be done

with a standard for iron to be supplied by NBS and with newer and

possibly better spectrophotometers than previously were available,

and thus the results were expected to afford some fresh insights.

46
NBS would also provide total- iron, ID-MS measurements on the

sane samples. Hemoglobin would be measured in laboratories

other than NBS.

High purity electrolytic iron was judged most appropriate

for the iron SRM, and samples of an electrolytic iron preparation of high purity were sent to the members of the Study Group

for use with their iron determinations. By the 28 October 1977

meeting, the quality of the results obtained in the interlaboratory study of the test sera that CDC prepared led the

Study Group to decide to concentrate its work on testing a minor modification of a tentatively proposed ICSH reference

method. Consequently, the same serum pools were reanalyzed,

and at the conclusion of that round of study some minor changes

in the ICSH procedure were discussed. Meanwhile, the ICSH published an approved method for the determination of serum iron

[3r. J. Hematol. 38, 291 (1978)]. Nevertheless, some of the

procedural details still seemed worth further study, and they

are still being done.

47
Task lg. BILIRUBIN IN SERUM

A. I sotope- labeled Bilirubin for Use in a Definitive Method

An ID-MS method that might be developed for bilirubin was

considered the best available route to a definitive method.

Bilirubin with isotopically tagged atoms attached to the first

two and also the last two of the linearly linked, tetrapyrrole

rings appeared to be the type of isotope-labeled material needed

for our purpose. It would leave open the option of analyzing

for bilirubin by use of dipyrrole fragments or analyzing for the

intact molecule. Such a mult iply- labeled bilirubin might be

prepared by biosynthesis (through feeding experiments) with a

2 , 2

, 3 , 3 -d^ - 5 - aminolevul inic acid as a precursor. The bilirubin

produced would then have deuterium atoms in the two propionic

acid side chains in positions where the labeled atoms could be

expected to be stable. The labeled precursor (also labeled

bilirubin) was not commercially available. We tried to synthesize it by an alkaline-D 20 treatment of 5 -aminolevul inic

acid, but this was unsuccessful. A much more involved synthetic

procedure will be needed to produce this intermediate, but work

on it was deferred.

B . Study Group Meetings

Our interest in a bilirubin reference method complemented

that of an AACC Standards Committee Study Group being set up for

the same purpose. Dr. Basil Doumas (Medical College of Wisconsin,

Milwaukee) was the head of the small group composed of

48
Dr. R. Carter (CDC) , Dr. Robert McComb (Hartford Hospital,

Hartford, CT) and Dr. R. Schaffer (NBS) .

Bv use of a carefully prescribed procedure for the

Jendrassik-Grof analysis for bilirubin, very high interlaboratory precision was obtained; however, a wide-ranging study was

then carried out of potential metal ion interferences, and the

results, which revealed a previously unknown but significant

absorptivity -enhancing effect of zinc ions, may require some

change in the reagents for the Jendrassik-Grof method.

Before working on these changes, the group is awaiting the

results of some very important experiments now underway in the

laboratory of Dr. Harry Pardue (Purdue University, Lafayette, IN)

on the so-called bilirubin glucuronides . (These are esters of

bilirubin and properly referred to as glucuronyl bilirubinates.)

These experiments are expected to provide previously inaccessible information because these bilirubin esters had not been

isolated in sufficient quantity and carefully studied previously.

Thus, the molar absorptivity of bilirubin, which is important

for the direct measurements of bilirubin and the nature of the

products formed in the Jendrassik-Grof assay, may finally become

known. The Jendrassik-Grof method is standardized with bilirubin; one therefore assumes that the glucuronic acid residues

are cleaved in forming the diazotized dipyrrole reaction products

or, if they remain attached, that the products have the same

molar absorptivity as the products of the reaction which do not

have the groups. These answers will determine the future work

49
of the Study

developing a

Group and may impact on the possibility of

definitive method.

50
Task lh. UREA

A. Preliminary Work on a Definitive Method

During our search for a suitable procedure for performing

ID-MS analyses for urea, a paper on this subject appeared in

Clin. Chim. Acta , 71, 199 (1976) by I. Bjorkhem et al on the

"Determination of Serum Urea by Mass Fragmentometry" . In this

method, urea is treated with diallyl -malonic acid diethyl ester

to form 5 , 5 - dial lyl -barbituric acid which is then converted

into the dimethyl ester for analysis by ID-MS. The authors

reported a 3.6 percent RSD for the method based on duplicate

analyses of 50 serum samples, and analytical recoveries were

as low as 97 percent. The disturbing fact in this method is

their use of a urea derivative that is a known medicinal with

no provision for correcting for that as a potential source of

error. We did not follow up that method.

In considering the use of a derivative form of urea for

our work, we explored the possible use of the reaction of

urea with diacetyl and with xanthydrol. However, our study

of the reaction of urea with diacetyl and diacetylmonoxime

was not productive. Other diketones were also considered but

these too did not prove useful. Reaction with xanthydrol

[R. Fosse, Compt. rend . 158, 1076, 1588 (1914); ibid, 159,

253 (1914)] showed more promise. The product obtained from

the reaction of two molecules of xanthydrol and one of urea,

X ,N " -di - 9H-xanthen- 9 -y lure a , is readily separable and yields

are quite high. But it was difficult to work with this compound

51
because of its extreme insolubility in a variety of solvents.

When we found it possible to isolate urea itself from serum,

and use it directly for the ID-MS analysis, further attention

to urea derivatives was abandoned.

B . Description of the Definitive Method

Urea was found to be separable from lyophilized serum by

an alcohol extraction. This was followed by a sublimation of

the dried extract, and the urea thus separated could be further

purified by extraction. The method involves the following steps

addition of stable- isotope labeled urea to a serum sample;

lyophi 1 i zat ion ; extraction of the residue with methanol; concentration of the extract and sublimation; dissolution of the sublimate in water for removal of nonpolar impurities by extraction

with CIIC1-. Crystalline urea is obtained on evaporation of the

water phase. Mass spectrometry is performed after a direct

probe insertion of the urea sample into the mass spectrometer.

As a trial of the method, four serum samples were analyzed

in duplicate. The bracketing technique with standard mixtures

of SRM urea and labeled urea, was used for converting observed

intensity ratios for the labeled and unlabeled urea into weight

ratios in the sera. The values found by ID-MS as compared to

the expected values (i.e., the labeled values given on the

vials, supplied by the CDC) in mg/L are 340 vs 346; 470 vs 475;

600 vs 589; and 970 vs 975. Given this degree of agreement,

further work with the ID-MS method was deferred until the

52
serum pools to be used for the round robin studies would be

ready for analysis.

C . Study Group Meetings

The organization of the reference method work was dovetailed with a counterpart effort being set up under the Standards Committee of the American Association for Clinical

Chemistry. To a large extent the identical clinical laboratory

resources would have been called upon if there were two separate

efforts. Dr. N. Gochman (Veterans Administration Hospital,

San Diego) had been appointed by the Standards Committee of the

AACC to head the effort to develop reference methods for nitrogencontaining compounds. Schaffer arranged to fund the following

laboratories for participation. From these laboratories the

Study Group was formed. The members are:

Dr. G. N. Bowers, Jr. (Hartford Hospital)

Dr. N. Gochman (VA Hospital, San Diego)

Dr. G. Kessler (The Jewish Hospital, St. Louis)

Dr. R. E. Sterling (U.S.C. Medical Center, Los Angeles)

Dr. R. Vanderlinde (N.Y. State Department of Health)

Dr. C. F. Willis (Cleveland Clinic)

Dr. D. L. Witte (U. of Iowa)

participants, not funded:

Dr. C. Burtis CCDC)

Mr. P. Duncan (CDC)

Dr. G. Ert ingshausen (Union Carbide)

Dr. F. Ibbott (Bio-Science, Los Angeles)

53
Mr. W. Ryan (Beckman Inst., Fullerton, CA)

Dr. A. H. Smith (DuPont)

At the first meeting of the Urea Study Group, held on

22 September 1975, some of the desired general characteristics

of the reference method were discussed:

1. The reference method would employ high-quality,

general purpose equipment for which written specifications

could be provided.

2. The method would use reagents that are generally

available and for which written specifications could be

provided

.

3. The method would be applicable to serum.

4. Interferences would be tested by adding potential

interferences to serum base.

5. Precision should be 0.25 percent RSD in the normal

range

.

6. Bias should be within 3 percent of the definitive

method

.

7. Uncertainty of the definitive method should be

within 1 percent of the True Value.

The Study Group agreed that two methods should be studied

intensively: manual diacetyl monoxime method and a urease/

glutamate dehydrogenase (G1DH) end-point method. CDC volunteered to work to optimize these two methods, compare them

with other methods, and examine them for potential interferences .

54
Dr. Burtis was asked to serve as Study Group Chairman.

The second meeting of the Study Group occurred on

22 July 1977. Because of other duties assigned to Dr. Burtis,

Dr. Witte took over as Chairman.

Dr. E. Sampson (CDC) presented a report on the urease/

G1DH method. The optimization of this method appeared to

be complete and accepted as complete by the Study Group.

Very little was done with the diace tylmonoxime method for

the reason that it seemed unwarrented to devote much energy

to a method involving basic chemistry that is poorly understood. Dr. R. Thibert (U . of Windsor, Ontario) attended the

meeting to discuss the chemistry of the diacetyl monoxime

method, which he is actively studying. The consensus after

this discussion was to relegate that method to a secondary

position relative to the enzymic method.

Plans were made to have three of the participants set

up an automated version of the enzymic method in their labs

to screen abnormal serum samples for interferences. Each lab

would use its routine method also. Differences in the two

results were expected to give evidence for interfering

substances.

55
ACKNOWLEDGEMENTS

We would like to recognize and thank each one of the

investigators at NBS and institutions other than NBS who have

contributed toward the completion of the tasks described in

this report. Since there are more than 40 at NBS and a greater

number than that elsewhere to be recognized, we have taken the

liberty of deferring recognition of individuals until the time

when each task is completed and appropriate acknowledgements

can be given on publication of that work.

At this time however, we wish to recognize the scientific

interest and attention given to this program by Drs. Eloise

Eavenson and Charles S. Furfine of the Division of In Vitro

Diagnostic Device Standards of FDA. Major support for this

program was provided by FDA through an Interagency Agreement.

The work at NBS was carried out in the Center for Analytical

Chemistry of the National Measurement Laboratory. Additional

support for this program was provided by Dr. Philip D. LaFleur,

Director, Center for Analytical Chemistry and by Mr. J. Paul Cali,

Chief, Office of Standard Reference Materials. We thank them

for past support and for their current support and encouragement

for continuing this important work.

56
— -

U S. DEPT OF comm BIBLIOGRAPHIC DATA

SHEET

1. PUBLICNATION OR REPORT NO.

NBSIR 78-1585

2. Gov’t Accession

No.

3. Recipient’s Accession No.

4. n n r AND SUHTITI 1

Quarterly Report FDA Contract No. 74-58(0)

5. Publication Date January 1979

6. Performing Organization (ode

7. AUTHORlS)

R. Schaffer. R. VelaDoldi. and D. Reeder

8. Performing Organ. Report No.

9. PERFORM NG 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. >p i sor nc Organization Name and Complete Address (Street, City, State, ZIP) 13. Type of Report & Period Covered

14. Sponsoring Agency Code

15. SUPPLEMENTARY NOTES

16. 1 1 4 _ - -i or less factual summary of most significant information. If document includes a significant

bibliography or literature survey, mention it here.)

This report covers tasks performed at NBS between April, 1976 and October, 1977

under FDA contract No74-58(0). Definitive and reference methods are discussed for the following organic constituents in serum: glucose, cholesterol, uric acid, urea and irubin. ables include comparisons of analytical results obtained by the definitive and the reference methods.

Clinical reference methods for selected inorganic constituents of serum are reported. Included are statistical analyses of round robin tests completed for lithium magnesi u , sodium, potassium,chloride, iron, and lead.

17. KE'i WORDS ’'six to twelve entries; alphabetical order; capitalize only the first letter of the first key word unless a proper stamtlMT'antl’Tes”^

Defin1tive methods l reference methods; round robin testing;

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