• nalytical ( - nt Labors

National Bureau of Stand-. r:-; Washington, D.C. 2022 ,

August 1973

teport J

'

'

_

/

'

'

: MS'.' S-

"‘is; .-vloa ;

. S,..

Prepared fct

.

>"

: • '•• • h UKi' ‘ •• r :

• : S

Nj I . 79 I

:

' ",

I

'

F '.a.

R. Sci R- A V< I and D. J . .

uf for

Natio, Mens?. • -me:, Lab . Nation;-: Bureau of re r :'srv. Washington, D C. 20:

:t 1 ...

Report for Period

;

: 1 977 to E

R- - d August 19 . 9

Prepared fc~ ©au c •

: D ivl . 'inis . Roc!* -ylan

Stic

U.S. DEPART!

Luther H. Jr.,

Barud

• IAL Bl . FS

'Report on FDA Contract No. 74 - ssfCO)

^or- the period

O’ r c 0

T

1 0 v 7 to Sep t ein b e r 1078

I '

.

. ]

R. Schaffe#, R. A. Velapoidi and D. J. Reeder

P . epar . d tor

Bui ~ an of Medical Devices 'no

Diagnostic Products

’ t at ion

c^vil I

-

. Mar] land 208 2

Introduction .

r

-; at e o £ Coni it

Page

i 3. s k i. a

.

A,

B.

A.

Task If

A.

B

«

Task lg

Task ih

Glucose in Serum . .

• 1 h l ) I : erei ‘Serum . . Analyses of Hu n an Serum Pools

C. • 3

'

. D e f ini Me h

- for G1 uco: • 9

Task lb

.

-

1 :hium agnes rum. „ Sodium ? Potass

Chi or i ie .......

i i . and

1 !. A. Lithium ............ . . . • , • 11

B. Magnes • u .. .......... . . » . ft 11

C. Sodium ... ......... • * . • 11

D. Potas' .......... * * * ' •> C 11

E. Chloride ............ . . . 11

Task 1c. Lead (Pb) in Blood ........ . . . « 13

Task Id. Uric Acid in Serum ....... . . . 9 15

Task le

.

Cholesterol ............. c c * « C 16

- c . : .

.

. of II Result wi t h tl

Institute ............ ....

Serum Iron ....... . Definitive Measurements of Total Ire

A Spectrop] lalysi

Experi :

• tal Serum Pools ......

.0 J- i ... ~1. U l li • . . . Urea .

16

17

17

17

1 NTROD U C T 1 9 N

This

Bureau of

in the cor

74-58(0)

.

timer! b’.t

this : r

report describes work performed at the ha tic

Stoiiuo, rds from October 1977 through September 1978

tmuation of projects

Some or the ievelopmeats rep rted here we 7 me- - jiven m detail in 1

3

Task la. GLUCOSE IN SERUM

A G Lu i t he WHO Rei e r< n S e rum

Six vials of frozen scrum, se lected randomly among all

the vials were thawed and pooled. Eight . < . we ta

from this pool and : ig] - i . Fos were tr e r t

e

d wi t h weighed

port ions of an

1 a qv. o 0 u s g 1 u c ose-lJx~C solution of k :.own concentration , The other four were treated similarly bo

L separately pi pare known soluti< l

;

- glucose. Af v er mixing ar 1 -allowing approximately three

hours at room temperature for reaching : w: libri ,:t the

i op€ - c h d ' mple; n i freer e- dr i her we

tre ted to convert th gj c

-

- intc 1,2: ,6 ii-0 i

D-gli cos e Th la t e 2 \ ras s 0

! it e 5. b> s ub 2 i 1

' 1 on ai I ]

if-* b> TLC to remove the possibility of con t ami

by othe j d i - 0 - i s opr ot /l iden x -hexoces.

Gu/MS was performed by ini ec ring chlo ."ofor'.c solr tic

of pu 1 ifi < d ,

: uc ds . d 2

; \ a t i ve from th = sa

mixtt ir e

: in to a coli nun conts ing

sh Gas Chrom Q and monitor ing the entered to unlabeled (M- 15 ) ions a ?S 0

pe tiv 1 y 9 in a medium re : s

ip ] O] . a a 0 letic f i e

1 bin; a l i

le signals duo to the selected ions.

Measurement; each 3 amp

I

Sample cone e n t r a. t i 0 n s w e r e c a leu • a t e d b v

c a 1 i

1.. ~

- ci 1. - x on in xtu

OV- I 7 on 100/1 2 0 :

s x t y

T* ^ d* I Ct c. i 0 of 1 ab

3 I i. d 245 ,

• cti

trot:. et er that e nip

for recording the

M urei n t s each of two day s •

1 :: ne a r i nterpo lat

.

calibration mixtmes that closely bra eke ;vd to imoto;'

0 f t ha t sa pie -

: that \ . run in Li

ately before and after each sample. The individual value.

. i t T . h 1 . t • •• oi t d

:

5

<

'

merits /

Q o O o .

: 1 Corree . :! Glucose Levels in the

Refer ice Serum [mg/I.)

e Number Da v 1 Dav 2

1 982 .

6

981.1

2 9 8 0.5 980.3

a 982 j 98 .

4 983.4 98 3.3

5 9 79 .7 980 . 2

6 978.0 98 0.8

7 9 79.3 9 7 8.4

8 9 79.4 978.8

mean = °30.S mg/I.

• 5.444 mmol/L

RSD = 0.191

If the mean of the values for the first four s

coi ired t o 1

1

1 m an c i t h 5 sect f our , a 1

difference s 3 rved Si h

o nm< )i p c the j i le c il Lbrj : io m ture

anal vs es > and the values obtained ...alJ in rather < ; . wine t roups

,

j

the solutions of the glucose-U- 1 appear to be th, scare* c the systematic- difference. This could bo due to an enor ii t

calculated concentrations of the two glucose -U

1

“C solutions

i: ght have arisen

iety in the glucose -U-^C, or bo a. Such cons .nt ration er wo ' i l t nsferr f sample

found

.

Th y

•

. t i n : ' 1 va 1 u e , i . e . , net t ho s e o r i g i n a 1 1 y r ep o r t e d t o t i * e 1V1 10

rrectio four ne y vd seric of cs

n i: t emplo; 1 in the! measui w e tesl

sistency . The series was comps ed of two s u: Her interlaced

se: - : . Th . 1

' gl 1 1 t Lowe st calil ion mixt

carte from only one of the scries . This consisten.c) .test was

done by bracketing each calibration mixture in turn with the

3

' i tio mi ing c - l

<

; and

\ i g . io th; n 1 1 of the ii ledi ixtur e ,

;

calci lating the weight-ratio of the intermediate ni

fi oi . 5

' red int nsi , i t s

; tl thr

assuming tin we t - t - h outer ]

i i ) 1

One of h :

' : b i

- i i - ; - s wa found 1 .n

e c e f i t nc h co r > ;:H uc c,t tiic: t con

values for tire WHO - e 1 err. nee pool. i h

"* co j red > n -

f oi . .

•.

to a lowering of 0.14 percent.

The WHO =

th; our mas s pe< t r

- n< - m .su

such high precision that sources of minor systems tic er

being made c

corn e c t i o n , t n e P

"

c c 1 'j

‘

' ‘

h i gh for accepts, b i 1 it . 1 ] 1

ki f: Even wi thou

•1 o f the t •

-

: and seen, to be correct a .. .... e

of the results is s

a definitive method

B . Analyses of Human S y'um .Pools

A< . scribed dui previous report i

«e> ' Pools « -

their . cone

.

' t ; cone tratic n dependence o

d fir itive a id lie - ; 5 ( is A :

results ? showing the decline in the gluccre . oncer..:

of all the serum pools with tim~ , s

p

1

' ' her * in Iasl 3 v

S r.aD I e s

i

.. ; _ v. p pi so analvs ed. by the t

•••• f these pools wen a.uu

j .r + m p which the c c )retho i at the CDC. The period of time -

lo, aer than the period through which

analyzed tr s 2 pool was J

i - -

. were ri at!

de fii il Lve methot

hc - that o

‘ 18-* P«

' he pool con

b lib -

1

, n A o n n 9 D6 1 C £

n

t . fell between 2.6 ana o . - p

- .

• . ... nf the data for a two month period ,d time

A comp anson or. n.

du i . hichboti th ds - e b in I

- -

at 1 . I- - ; :1 ' T ~

’ ... ....... „ values the bias bebg gmtt-vlowei : the del ini

at the highest concentration levels.

?

TabK 1. Table 2. Gli •cose Levels (IgA0 in Six Hue si Sv rum Pools. by ID/J

Analysis Measur

a

Prr

. arti ment 3077 3177 3277 -t *7 ZO / / 3477

Date Day

10/ 20/77 1 1179.6 1684.2 2190.

i

567

8

. 6

2 1177.7 1681.6 2189,6 3679.8

11/15/77 1 670.4 1181 .

2

1( 2186.5 2371.

5

36.° a 7 9u 6 MM 7 1181.

2

16 2185.

1

2874.0 5679.

5

1 2/6/7

7

1 667.

6

1180.8 1684 . 0 2156 o 2873 8 3685 q

2 668.2 117r 6 1681.9 2192.5 2372.

3

3679.1

1/17/78 1 666.2 1170.5 . 2182.2 2859.2 3659.9

2 666.2 1169.

7

] 6 .. 1 S 2136.

6

2858.5 3657.

2

3/27/78 1 666.0 1175. 8

.

1630.0 2176.9 2854.4 3662.4

2 664 .

3

1172.9 1679.0 2177.5 90 7 7 f' Z O 7 .J . y 2660.9

4/18/78 663 ,

4

1164 .

7

1672.2 2165.8 2835.

7

365''- . 6

2 661.7 1161.4 1668.4 2167.0 2830.2 36 .

V

0

r-j

',o

Task la, ''’able 3 Comparison cf th Do Cini rive (DM] r

• Refere (RM) eth

{ I urn an S e r i a Poo

1

Poc 1.

DM , Mean 1

mg/L

RM ? Mean

mg/L

DM - RM

irg/L

—DM-RM irA

; - x 100

Perc nt

30 77 660.5 665.6 z . s 0.43

5177 1175.

S

1168.0 7 . S 0 . 66

^ O ^ *7

/ / 1 6

c; ?: . o 1667.2 14.8 0.83

5377 2189.

2

21 58.5 50.7 1.40

34 7 7 28" . .0 2330.7 34.3 1.20

- - r =7 0 0/, 3671 ,

6

3617.7 C ~ 0 1 .47

Pool

DM

12/6/77

my /L

12/5/77

mg/L

DM- ?M

it . /

-.

r)M-P ' nr x 100

Pe r r r '

3077 6 6 7 . 7 665.

6

2 .

1

0.31

3177 1180.7 1 1 7 1 .

9

8.8 0 .75

3277 1682,5 1672.1 10.2 0.64

3377 219.4 .

5

2171 . 7 O'

-

'

- 6 • 1.03

3477 2872.7 2843.0 24 .

2

0.84

3577 3683.5 3629.1 C 4 1 •P *T • . 1.48

Mean value

December 6,

th

’

15 7 7 , ana J anuar^. 1

or an: lyses tar

197S .

Mean \aiue by - is re.. i period December 5, .0 , for each pool.

ce met red ter _ ur r

- s ov .

' y= ; 2

Task la, Table 3. (Continued)

Pool

DM

1/17/78

RM

1 / 5 6 2 a / 7 8 DM - RM

mg/ ]'.

~DM RM

i

•

' X 10

mg /I, m.r ' / 1. JJri Percent

30 7 7 666.

0

665. 1 0.9 0.14

3177 1171 .0 3. 1 6 6 ,

8

4. 2 0 . 36

32 7 7 1681.

C

1685.8 15.2 0.90

337 7 2 1 3 j . 1 2154. 2 29 .

9

1.37

3 4 7 7 2 8 5 7 .

8

2824.6 33.2 1.16

3577 365 9 6 3610.4 49 . 2 1 . 34

C . An Al l

i idi cat

on another definitive method that could be use’ in parallel

t i ie origin

ion. term definitive method stability , for which vidence

presently lacking. (That the C"-C had iso found evident.

;

for the long term instability of the re pool? vn u .

to us until recently.)

:] : 1 1 sfiniti1 a .

-v- glucose-U- 1 :'C is being used. It has '• higher 'C-er : in . . and glucose labelled with six J C atoms is present : i : "t

ercen i abundance Ai

' ivati tion \

Id i c t abund ma s s is five mas

for ordinary glucos . because of the :. over isotope- . i

n

method involves the fold ov/ir i

: About i mg of t e glu os : - j

13

c is :

. 3d iqu - t

-

glucose. After time is alio ed for the labeled and un

'

:

. c equili b te etl lcoho.l i

:e in. Ti .

. .. is free

then de ion: :

. a n on-excl

f 0

-

- - : i e d the -

; reate

butaneboronic acid, and f heated

0 . 5 hour I lall

The residue is dissolved in iso-cc ne for GC,..o.

to the original definitive nr’ uiod, - J1 w ^ ^

seen to require much less; effort to prepare s .

Gu/r.-o is performed b y injecting aliquots of solutio. 3

of simi! ar

mixture

of

- - f* K n i tc: bis (bu

. 1 0 a X. ill ary

3 dc- riva t i v e i

5 true re tha

or mannos? r

‘

a an - . - v s i S, ;

e in j

: : ed b e f(

!< s so t hat th:

ion in a c T ngl*

s „ This s i

d da7 * (Dupl:A

rf

-1 m C- -* -At, r s

-

in t he .. ro res

ur em ent is niad.

:ompleteiy separated fn.r;i derivatives

formed if fructose, ,, I be -Pr

up 2 e in a cilose

s rape? ted

first ID/::-S method.) A 0.5-pc. ce. t

is required; if not rchie -d , r

agre ent. ar: d the 5

results art used.

10

Task lb. LITHIUM, MAGNESIUM, , PC tSSIUM,

S ummary : Although our last report covered th t

'

i ] 1977, it was mentioned

duct ion that results obtained through September 1978 dli o h d

been included to "bring the information up to date". Thus,-

the information- reported here may be somewhat duplicate /. i

nature. The goals and selected data from the final sta

'

analyses are summarized in Task lb, Table 1.

A. Lit! ium: Data i f rmation, collection, and analyses are

complete and writing of the NES-260 h< s begun.

Int c

i :atorj exerci

until the magnesium, gluconate dihydrate SRim is issued. A

resolution of different interpretations ox. th star a mac - 1

analyses of certification data has to be mad' be: or t :

m

v ork c n so (See discussion in cover letter.)

tion 260-60, "A Reference

minat ion of Sodium i Serum* • has a

a Sodium NB3 soecxs . Puc ' l c a •

Method for t!;e Deter:

issued

.

D. pot assi Sj

M he for th a> - ic 11 ' -

completed (copy attached) and i. being sent to th p: ir t- ’s.

E. Chloride: The NES Special Publication 260 has bee

wr i 1 1 e n a n a is un c e r

g

r going internal review pricy to r

11

I

r-i C3' j

•r-i -i—*

’/) H

+-> oo

C3 ; M O

C/j

H

•vr-“ cd

rT.

i*-«

G 0 £ ‘

•ftC3 CO

4~>

G. H

O •*~f in

IT, O- O c— Pr ; O

rj

a

G

U

r-'GG

M

X

CO 'fi o

A) i~J r-i ri cd f~ O C

e: .o

00 r-i CO G

•r-i

G

PG

C

1

Cd £ . O '-/) G h O G '—i -P •H X VJ 00 r—i •H -H o U oo P G 4*3

?•< .-“1 (J

C: CO

O £ c' <~i *-* < ;g G

r-i

CO

•3

Tlrr

Task 1c. LEAD (Pd) IN BLOOD

The graphite furnace procedure developed by T. C. Fa : ns

at MBS was compared to a chelation- extract ion method i ;ed by

Dr. E. Berman in routine clinical analysis The print y pun

poses of this exercise , which took several mom:' 5 to cony et ,

'1) :o .. the ] di g stability of the mate:

Li s 1 in 1 . ' i 1 it r corny 11 is >n . idi< ( 2 )

t

me hod: hen 1 :d on hi 3 F t son : i hi

( y . provi d < within- ind be tween- day d; f 0 at

i

s

t

v d i e s 0 f r e p e a t a b ility

.

Results of this exercise showed that Pb If no - in tht

frozen porcine blood were stal le over several months,

with the human blood samples was started but forth..

and am a lyse s are needed. The r elati ve

the within- 3. ay 1 J s 1

1

, 1 5 r ai g

e d from 1.1%

level to 13 .53 at the 4 yg/100 ml, level

A roun 1 s in ire

evalua so

and precision of the Rails s method was init :.a i.e<' by cc '

2 ji ...

;

: i pari i : i ;

- Sa mp 1 < 0 f i vial co linir

various levels of. lead in blood were s n' to 11 labor

cm May 15; 1978. The samples contained one via.I of bio

be u ’’ cl during familiarization ith tus p

• oc u

othc 1 v ials containing b < wi 1 I

!

about 4 to about 80 p :

' 100 mb.

Results from several labs were reported witnin three

weeks Hoi e\ ei , se veral I i\ ; I i d not follow

13

ane

1 several others did n '" L ies ?°nd to follow-up calls or

letters . Labs v. ere again conr • - • : . contacted m September, 1973, for a status report. Final results were requested.

while -

-

' precision, the variances between 'ab: dictated the necessity

ha ' in s isotope dilution-mass spectrometry (vn/MS) t

• ValU6S '

: f0 the = s - pla ; late 1978,

Task Id. URIC ACID IN SERUM

The samples cf six serum pools to be analyzed by the ' definitive method as part of interlaboratory exce

put through the wet cherical processing steps and then ct .hmeasurement b) :

.

beei lar: - 1

me nr was scheduled to begin in No -'e. her 1 '"S.

l r ± ^

Task le. CHOLESTEROL

'

. f IP/M

1 hi-5 subject was discussed in detail i. although the work was done during the present

\

olinska Institut e o"- previous repor

report 'nr period..

16

Task If. SERUM IRON

Definitive Measu ;i ts

Iron in CDC Sera Lot Nos. 1777, 2277 , and 2377 v/a d . mined by isotope dilution mass spectrometry. Three bottl

;h lot were samp] each <

' i i i

C aliquots were spiked with ' Fe The organic ma.' rix .•/ cp ith perchloric nd i i t icid ( separated by anion exchange chroma tog phy. In a,. att( .t tc

coi t leal

were further purified by ion exchange ch: nt. . tograpkv. Th

. . at ions w t hen .determine usi

me :• s sp - trome

t

ry

.

The data are shown in Task If, Table 1. The a nl/tf'l

blank contribution for this analysis was go nano:. -Ti

unc Lnt c< >mp or " c. d v i t t s

lat ion (C 1% ) s

: t s ike .

estimated accuracy of 0.4 percent ror the an. > e i s

.

B. A Spectropho tome trie Analysis Iron i n E

Serum Pools

Samples from seven different sets of serum we e ana y :ci r .. i s in - light i

'

requested by X. L. Barnes (MBS member of Fe study S’- cup' .

After allowing the frozen samples tc cose to voo:.

erature the samples were genMv mixed and 2 ml al iquo

:

were

transferred to 15 inL ground glass centrifuge sub.-;.

-17 -

Task If Table 1. Iron in CDC Serum Samples

Lot. No ug Fe/j? pm o.l s F-:/L

a 177 7 A 0.9370 • 17.17

17 7 7 B 0.9270 16 . 96

Av. 17.08

2277k 0 . 5387 9.788

2 277 >3 0.5562 9.742

Ay , 9.765

2 3 77A 1.662 30 . 45

2377B 1.688 30.93

Ay

.

30.69

Calculated using the atomic

following : e ru n den

weight of iron as 55.847 L

Lot No

.

1777

2.7 7 7

2 57 7

Den • t

1.0234

1.0147

1.023.

F

' g reage

F I s . e h 2 te I to S°C ii

ad^-d and, after mixing, the

a water bath for IS minutes

6 d

After remixing,

an d c e n t r i fu s e d

t h o s up e rna t ant

tne samp es were copied to room tempt

ai v i ‘S

0

rpm 1 s for 10 minutes. Alio/ots of

were then transferred to other ground glass

20 g trichloroacetic

thiogl ycolic odd (S

with H 7 0

.

acid. (G. Frederick

gua) + 12.2 piL liCL

Smith)

d i luted

6 mL

to 200 mL

r.>

centrifuge tubes. Ferrozine chromogen solution/ was s' b • d d

after centrifuging again the ab: orbancc of the cole. ,o ut ion was read at 162 run. Iron standards were prep;; ' A -r TO"

SRM 937 a zording to directions obtained from T. J. Mur y

Results are given in Task If, Table 2.

F h e o n1 y d i £ e r e nc < s b e t. v.

: een the pi oc o d u ;

. s u ~ r -s to

and the p roc ui actual )

i

'

. were used throughout rather than the plastic tu! e • for the

secc t ep and an a li oi c ifug

just before reading the absorbance, this was found to be

necessary since any small p .rtic l.s of protein material

which may get into the supernatant aliquot can camabsorbance values if not centrifuged out.

Most of the results obtained in these analyses were lower

than previous ones. We find the t the isotope diluti ‘ r am; yses were indeed lower than the initial work done by this procedure, and these are lower still. We feel that -hi.

indicate that there is a problem with the samp' s , ti it Tic.

ually lo; : rc i l c o t i :

- thr<

some other step and that perhap this should be sen.

cons ids red

.

n sodium it ate -*

1 d i luted to 100 m L w i t. h H 7 0 .

- 19

Task If, Tabl

Se i urn Number

177 7

2577

? ?ill7

1877

: 7

2177

/. 0 7 7

Spe :1 ' °P'iOtOi..etric Method

Normal Iron

n - 10

High Iron

n =* 6

how Iron

n = 6-

• I ol yze

n - 0

r.

. Kemolyze

n = 6

High Bilirubin

n “ 6

• i Turbidi y

n = 6

U

-

T ron Values

TJ g/mL

’ umol/l

X

. 94 7 16.96

a =

x = 1.310 32.41

cr = . 020

x =

. 594 10.64

0 = .006

X = o o

1

—

i

18.62

a = .005

x = 1.120 20. : a = .011

x ~ . 890 15.94

a = . 0

1

5

x = . 950 7 . C i a = .005

r

Task lg. BILIRUBIN

No work ( on this analyi

Task lh. UREA

In our preliminary work on an ID/MS met 1, . urea ecu] be iso] s it

ratio o£ labeled to unls.be led molecules of ur a in the

I mat could 1 asured

tion cf the specii

ur i the

the

iso -

inser

results

obtained in the

of the expected

original experiment.- were within one percent

values, and for the limited amount of time and

effort put into the measurement we four 1 reasonable p -ecis Ion

(±1%) in the results; hence, no further study seemed to be

needed prior to applying the method to samples requ’-ing

ID/MS analyses.

On attempting the mea' : . it : : ii . boratorj

c i i pc Is pies I t h HO ] , i precis i . eyond is t att ined previc

the rratic i could

over the temperature of th s amg' le when it is in the p

of the mass spectrometer and hence over th-.. ev •.pora.tiu o.

the urea sample. Provision was therefore uncert. user tc

water-cool the probe for these measure :n s.

21

'DA Contract No. 7

4

-5 8 ( 0)

:[:ob..~ 1977 to Scpfo’-ber 1979

1

. 1 . S 0 K p T . OF COMM.

ciliOGR- HUC DATA

SHEET

1. PUBLICATION OR RE! .NT NO

N3SIS 79-1797

4. TITL E Till &

• 1 J . :' • -

?. AUTLOL'l)

-

' ' ’

• r 71, A . T

?

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