‘ ’if ' J‘ ?! t w*‘ ‘i^.’

rPfOTt:""

te « i. « -i--J U1 V5lr’’^'"‘>)rt » '*> • • : •^' ^ ^

A' 7’^-, /f*S*'

Li i-;.7^ '>-^jB<^ v.>' 2 -' Vi. 43

""T’ n

<'"• ' g' I '-!»* '

. '*

. J' ..; -eiH

/

R. Schaffer, R. A. Vel-ipoldi, arid D. J. Reeder

f

Cr>nter fcr Ana^licai Chcmbtry

Nalionai MoasurernGnt Laboratory

H3tio:''.al Bureau of Standards

Washington, D.C. 20234

August 1C7S

Report for Peyad

(,^clcn3^~T97B to fv'or^l9{9:N

issued AuvjU'j'i TS/'J

Pfcpiited for Surc-u of Mcdicn! Oevio.»s ood Uiagnc^tic Prod'i<-:s

food and Duv^i Adminltifr^tfon

r.cdiviuc, fdaryl<.nd 20352
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NBSIR 79-1 7S8

FL'^AL )T:^?Olir

FDA CONTRACT NO.

A

Sct-.afj'sr, R. A. V.Japoldi, and D. s) Rcodor

Center for Ariaiytica! Chrinistry

ffationa! r/ioacuremerit Lt.loratoi'/

Nationa! Bureau of Standards

VJashincj'or:, D.C. 202 'll

Augu 1979

Ffeport for Period

Octo':'er 1978 to r-/!arch \Q7'J

Issued August 1 979

Prepared for Bureau of Medicai Det'ice'i end CFagn ;stic Products

Pood and Drug Ac’mir.ist! aticn

Rockville, Maryland 20B52

Co.

'iiij

’c I

'V M

UX. DEPAmvlcN'r' OP GOrnAiEACF. d' Krc.’.;

Luther H. Hedgof:, Jr,, Ur.'-Ji-rSs^n .."y

Jordr n J. P’AT'CNALBarijch. /2<5s;if.9nJ SscfHsiy for S^lviiC:) i^ncl Tochn-. : SUr^EAU CF E.-n-'JSt Arnk'.:f, /cthic/ D;r,?/..
I

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RepoT i: on FLA ContraC': No. 74- 58 (03

for tl'.e pcrioc

October 197 T' to March 197'

Isri;.:d

R. Schaffer, A. VelapoldS , D

Prepared for

Bui'eau of Medical Devices and

Diagnostic Products

Food and Drug Adminis t ration

Silver Spring, Maryland 20910

J. Ree.ler

Table o

Introduction .......

TasL. la. Glucose iji Seru:::

A

B.

Ta sT. lb

B.

C.

D.

E.

Glucose in WHO Serun by the Lut looboroiiate

Acetate/ ID -MS Method

Pas

1

2

2

4

G orip a r a t i e 5 1, ii d y o f D i a c e r . i ’ e Glucose aJt d

Butaneborenate Ace tat'"* Gluccr'c ED/I!S Methods

Lithium^ Magnesium, Sociu;:;', Potassiiijn and

Chloride

Lithiuni . , . . 5

Magnesiuin 5

Sodi’.un

Potassium .... ......... S

Chloride .......... ..... 5

T o 1-

1 a s iv Task

Task

Task

Task

1C.

l d.

Aa .

B.

le.

1

ai-c IJ^.

A.

lend (Pb) in Blood

Uric Acid in Seriira

Uric Acid in the IVHO Reference Serum by

ID/MS ........... 11

Uric Acid in the Serum Pools Used in

• Interlaboratoi y Exercise IV . 12

Cholesterol lb

Serum Ir-. i . . 1'-^

Bilirubin ........ lu

Urea lb

Ratio Moasurement s . , . ,jo

IN^TRODUCTION

This report describes work perfornod ; t the 1

Bureau of Standards froir. October 1978 through Mar

in the continuation of projects initiated uncc-r 1*

74 - 58 (0).’ The previous report was for the peric 1

October 1977 through September 1978.

n r, i n a JL

rh 1979 .

b \ (orvrrct

f torn

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Task la. GLUCOSk IN SERUM

A. Glucose in V/HO Serum by the Rutaneboron.ate

^ ‘'12

This nev/ metliod for ID/MS \\^as carried out on sample •, o the WHO reference serupi pool. In order to study ihe precision of the metJiod, duplicate samples v/ere tested, ard i.n

order to observe v;hether the time allovved for equil ibra*. ion

between the labeled and unlabeled molecules of glucose v;ou.ld

inf]uence the results, the duplicate sainples were allowed to

stand at room temperature with the added glucose -U-^^C for

either 2 , 6, or 20 hours before continuing with the procedure. The results are given in Task la. Table I. The

interesting fact emerged that the glucose levels were found

to be larger W'ith the increase of time allowed for equilibration. i

It v/as later learned tliat, by use. of the refer tree I method, CDC had also found increases in the glucose concentrations *

in their -20 '^C-stored serum pools if the serum was allo‘wed i longer periods of time at room temperature, up to two or

I

I three days, before proceeding with the method (inst-.'ad of ;

aTiov/ing the serum to stand at room temperature for one ?

l liour) , CDC then found a maximum increase in gluccie of

about 1 to 2 percent. But only serum stored at -20 °C for a ^r long time showed this effect; newly froten serum does not

i shovv it. CDC is studying the temperature of storage as a possible control parameter to overcome tlie problem. ;

I

(-S

3 k 1 a j T ci ij 1 c 1. Giucos e Levol s in WiiO Reference S?ru

S amp 1

c

3 by the But n e b 0 i' u n a t e c e t i t ID /MS Me tliod

,

Mo a 5. u r c d After D i i f e r

- Equilibration Times

Equilibration A1 iquo

t

Glucose (mq/Ej

T ime

Hour3 Nuinbt r _ Day 1 Da'-’" 2 Day 3

2 1 (905.8) (910.2)^

9 959 .

5

960.8-

'fO . 961 .

8

962.6

6

4- 9 34.7 965.1 958.1

5 961.2 9 6 4.

3

6 961.8 962.0

2 4

/ 958.2 964.4 960.8

n0 967.1 . 966.2

9 965.2 967. 4

10 966 .

5

968.2

‘^Outlisr Ti.ot included in av^ragG.

..'i

G1 JCo<

Ave.' 'if, ng / 1.

961 .

1

95 1.3

955.4
I

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. From, those obscrviit i oris ^ it s nocossr.x:)’' to cxo:':’

;

lurthcr control over the time of soLiplin o- than was though

initiaj.lv necessary for the reference and definitive •v.v.hr^ds

to be proper.!'/ compared. •

,

B, Comparative Study c f D i a a ? t o n e G 1 u c. o s e and

Bu t a ru ; b o r o n a t e Ac c l; 3, te Glucose TD/MS Me tliod s New serum samples c^ ntaining glucose at 3 different

concentrat ion levels were received for this StMdy fvO::. ti

CDC = The testing pattern to be follov/ed is to analyr; 1

pools once per week for 3 'weeh.

analyse would be decided rf i.OT

adifcd.) ' The GDC would also do '

alio^uot s of eaclj of 1

t, J . . c

(The need Vr.-,' r. r> P-'i -: Lena

these res ults could D 0

re.fe rones method a

L

the

Dools 'were to be stored at -S0° and for analvsis kev

room temperature for a total of 22t2 hours be.^ore the

an.alvses were to rrcceed.

At N3Sj each week, s number of vi

poo!. \^ere combined into 5: eparat e p 0 o 1. s

for the aliquots needed for both ID/Md

w-re taken between 1 to 2 hours a. .f t c r

had been allowed to thaw and mixed ari combined . The

labeled glucose for each method was then addend. For rlie

remainder of the 20-plus riours^ the aliquots were lew: at

room t emperauure . Then the individual procedures were

begun. The study v.aas begun, .hn the latter part r

resu! - s were available in tiite for this report.

X Cj

"= -H

I !

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Ta si-.: ib LITHIUM,

CHLORIDE

MUM, SODIUM, POTASSIUM AND

A. Li thi uvg : The N3S-260 is in preparation,

B, ^iRRTies -Uim ; The magnesium gluconave * ZihjO SPOT has

been certified. The program for. developing a refcrrcnc

method for magnesium needs to be reviewed.

C

,

D.

E .

So diu.m: Work is completed,

Po_^assJuin-. The NBS-260 is bei

C h 1 o r i d e : T Ii e N 3 S - 2 6 0 is un d

e

ng prir.ited.

rgoing final review

,

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Task Ic. LEAD (Pb) IN LLOOD

Isotope d i but ion-mass spectroinct r ic measureii'er.tr were

1 C-. r e d. £ o r s amp let o £ the p u i' c ,i. n e b 1 o o d . Th. ( . s e v a 1 u e s

v;cre used as target values for comprrisen of data from, the

five labo-^ratories report-ing results using the Rains prcvod--

dure .. In sd;l:lticn; three other laboratories using a variety

of techniques reported values for coinparison

,

Results were reported to the laboratories in correspondey.ee sent in Mar'"]i 1979 with data plotted graphically

as percent deviation from the ID/MS targe" value, ' These are

show'n i]i Task Ic, figure 1 , The sequenc e of analytical

results a r:q from left to right . the J.owq medium j mediumhigh, a7id high levels of lead in blood . More d. e v i a i o n f

the targe s V c;. J. li e \i s seen 1 n t ] i. e 1 V. o el values ^ as

expected. There appears 1 0 b e a

.. T 4 -f...

' b X ii c- overall negcuive

bias for t’-e method. x\7ith the lo V value estiiaates being

higher thav.. the ID/ME target value. The ID/MS values are

summarised in Task ICj Table 1.

The criteria e s tabi islied by the CDC Proficiency Testi.ng

Division for accept; ble results in blood lead analysis are

as follows: when the targe i: value is greater- than 40'iig/100 mb,

the acceptable range is defined as rhe mean, ±1:> p,.’vcej.t,

when the target value is equal to or less than 40 yg/100 mL

6

r

t

[

t/t '.) o o

p 'H

r"H •!-* /•' O re; Cj C3 O

p. j-.i ^ r ' -^ r;

+-> f-i O C o c

^-1 - V. r-*

C'j -^4 lA ‘f-*

'—' o ^

t'} f.-i 4--^ C/5 '.0 • ^ ^

2T, O 'O '..y o

• ! rP o u

C-i rt L'j l-A O O ’-r- •P' O ^ - w r ^ s.^ C:. r-'i ' b o p; ^ "d 'r~l 5^ C

<-;•! r: o O Cb C

rH D b'5 *f-'

t/) o -ij-y fp I.P !-• O ^

o r

, CiO ’j". ,“.N y\ r^'

CJ 'f-< K -/j o

•ri -''i 'H

>' ci c; b 'T, p-; -:-J c o

’i;:; -1-^ ;c! '>

r*H o ,C1 >0

•i-j p

C4 tn P b 1 « o / N >-S c

(J p '“J ;

C.1

0,1 t-.-J <J-, :::b. c:

P., o p w' c5 rC

(.U ^"1

b-0

y"". r'i

C'i

(

rJi

zawi. (SKCl) J.savvx KOSJ SOIXVIAXC I.SSD'did

I
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the acceptable range is the m an value '±6 yg/100 ml. Results from the 197 8 exercise uere compared to tl)e CRC

criteria and are shown in Task Ic , Table ?

The exercise showed the need f^r continued work cn •

method developaien and experience vx-. tli the tecj'iniques

involved.

T-'-.-l' I." TjUI 7 i cl dJ i. V . , j. ct t.-' -L - 4 •

ID/MS lev«

yg/ino ml

"Ac c e p t a b 1 e R a n p; e

’

bv GDC DefinltiOi;

No. Taboratorie:

t IT!

Method

jibC Crite_ria

"Other"

Methods

4. 3-v 0 - 10.3 4/5 4/4

36. G1 30.01 - 42.01 4/S \/'\

51.59 4 5.9 - 59.3 3/5 4/4

85,37 73.0 -98.3 3/'

5

i/4

Consisten ' difference in repcf::ed high lead levels in hums.n

blood we .10 noted when the Rain.s ' method and the chelation

extraction procedu 'e of Berman, w'ero compared. it w-as though

that the differences may be explained by the existence- o 1 a lead-bindinv7 protein in 1 ead exposed workers as described in

nd Gonick , Soc. Exper. Biol

.

To resolve this inte re Stine difto obt a i

n

blood samp If- 3 from lead9
1II i
ex].!r,sod workers. These samples will theii be analysed in CTi

intorcompa.rison protocol that will require all samples to be

analyzed by the graphite - fu7'nace aiid chelation extraction

methods v'/ithin the same 7veek that ID/MS values are obtainea.

lida.ti.cn of the grt phite - furnace method vn.1.1

Ive feel tlii^ va

be a ?"ecossary step in e s tab 1 :l shin g the utility ox the

procedure as a reference method for human serum sam.ples

f TasU 3d. UI3IC ACID IN ST3RUM

Acid in the Rt ;f e - ence Seru.iii by ID /MS

N.ine vials, raridoinly se-locted from the serum pool,

v.'ere thawed, mixed, and combi Tied. Eight weighed al i quo ts

of this C(;mbined sample were analyzed. Two independeii:

1

5

stock solutions of N..-uxic acid v/cre prepared. Four

of t.he samples were treated w'ith one stock sol.ution, and

the remaining samples were treated with the other. The

ratio of labeled to unlabeled uric acid in, the mixtures

ranged between 7 to 1 and 10 Lo i. Calibration mixtures

composed of the labeled uric acid and of Uric .icid, SRM 91.3

were also prepared in this range.

The isotope -enriched serum sa'^plss and standard hiixtures were similarly treated to ethylate the uric ac.id..

The same tw'o tetraethyl uric acid i.5omers were isolated

from each reaction mi.xti’re, GC/MS ^.as used to de t . r:,-ine

the obsvjrved isotopic radios in earTi standard mixture and

unk.nov;n. Intencity ratios were measured at m/ z 282 and

280 in a medium resolution mass spectromev' r that employs

magnetic field switching and is equipped for multiple ion

monitoring.

Measureiuents were made in. duplicate ov each of tv.'o

days. Sample concent rat ions were calculated by linear

interpolation hez\.een the incensity ratios of two standards

that closely bracketed the isotopic cor.ij^os it io.i of the

1.1

saitiplc and were run iiarriediatel / be Tor e and a.fter t hat

sample. The individual values in Tas.k Id, Table 1 are

averages of the daily duplicate measarements . Each

RSr> is based on 7 average values. The averages fcr

Sai.rple S are significaTitly different from the others,

and. have been, therefore exciudcil from the calculations.

Tne correspondence of the values for Isomers .1 and C is

verv* >**> rood.

B. Uric Acid in the Serum Pools Used in Interlaboratory

Exercise TV

The results we obtained are shewn in Task Id, Table 2

Only th./ values --f tlie aveiages £

an c liquot in a set (2 on one day

day) are shoven. Til. e pairs of me

a

our 1 % agreement re qui'^ement. Un

bet'.-eeij. the results for Isomers ,1 and C is not as good a.s

V.' C would like, to have for a definitive method. We believe

we kiiow its cause. Theiefore v;e ha.ve requested addit i.ona.l

sa.mples of IF. • IV for .7:.;rther analyses.

V/e received from tlio CDC the av'erage values obtained i.n

IH-IY. Our values and. those values ace shown in Task Id,

Table 3.

1

[ Tash Id, Table 1. Uric Acid in mg/g in the WHO Se rum Pool by ID/MS.

ft i

, Sample

Isomer A Isomer C

Run 1 Run 2 Avert ge Run T Ru.n 2 Average

1 .0450/8 .044839 .044968 . 0450'3 . C'4 5 0 3 8 .043043

1 2 . 0 44 5 38 .044726 .044632 , 044749’ .044757 , 0447S3

; 3 .04454? .044820 . 0 4 4 7 3 i- . 044709 . 044754 .044 '; 32

.045142 .045247 .045194 .045235 . 043230 . 04 5 24 2

r .045004 .044832 .044913 . 044 303 . 04476x . 044752

6 .044866 . 0 4434 8 .044853 . 044849 .044793 .044 8 2 2

7 . 044906 .044720 .044813 . 044975 .045019 . O

' 4 99

7

8 .047049 . 0 4 / / 8 .047514 .048401 '.043461 . 048 4 31

Average [excluding

Devi a lion c£

RSD [excluding #S)

041873 r, .-1 A :•

. U '1-

s R Q f0.40^

^7.84%

0 . 42%

Diilerence Betveen C 8 Averages = 0.08%

Average o"' C 8 A [excluding tS) - .04489 2 Average corrected for purity cf Uric Acid SRK .044 76'

* This concentration of uric acid per gram of

^ R .1 f-T-r* (1 o-7->'; ...v,., ... 1 ./ the WHO scrum

corresponds to 45. S4 mg/L or 0.2727 mmol/L,

gravic)^ of the serum v;as 1,0242 g/L

lie specif i'

X J

L
I

A]
Exercise IV

I

ID /MS of the In ter laboratory

Seriiiu Pools 0 Resulcs on fl'C A- and

C:-I:.oni6rs of Tetrae thy luric Acid.

1 5 0 n fi'T A (in rag/

S'.:t Pool 3 Pool 4 Pool 1 Pool S Pool 2

1 0.0233/9 0. 035920 0.041013 0.055375 0 ,100 520

2 0. 023091 0 . 055461 0 . 04240S 0.055606 0 .099981

3 0. 02369S 0 . 0 a a 0 1

5

0. 041521 0.055176 0 . 09 8 58 2

0 , 025713 0.035409 0.041501 0. 0 3 -. 99

7

0 .099071

S' 0.025166 0. 054 38

2

0. G40S99 0.054898 0 .098654 tra^c 0.023409 0 , 055453 0 , 041 588 0 , 05 3210 0.099520 r-

- 1 . 24 1.04 1.44 0 . 5 2 0 . 9 2

I scr er C (in mg/ g)

Set Fool 5 Pool 4 Pool 1 I' 0 0 1 5 Pool 2

£

1 0. 022964 0, 0551 65 0.040954 0 . 054673 0 .098615

7 0. 025015 0.035219 0.042013 0.055282 0 . 0993 93

r

3 0 . 025302 0. 035207 0.041187 0 .055052 0 .0980.65 ,

.

4 . 0.025483 0, Oil'5382 0.041365 0.054933 0 . 098873

5 0.023312 0.0349 5 2 0. 04 097

7

0 .054935 0.098615 ri

Average 0.025215 0.035135 0 . 04 1299 0.054985 0 . 098672 fr.

{.vj 0.94 0.44 1 . 04 0.40 0. :2

li

Task Id, Table 3 Average Value

C Isomer Obtained

ile Terence Met

s of A '1 bv

somer arc ciia

the ID/MS and

Pod

4

2

A Isoiuer

2.3409

3. 54 5 3

4 r 1 3 S 0

5.5210

9.9320

C I sorrier

(th g / 1 0_0 c )

2.3213

3.5185

4.1295

5.4985

9.8672

lEdV

nig/ 10 0 I

2.39

3.59

4 . S 9

5.56

9 . 98

15

Tabk Xe, aiOI,GSTEROL

A manuscript on the definitive method, to bo submitted

for publication in C linic al Cho ir! stry, is undergoing final

examination by the authors before being entered, in the

Bureau's prepu:; licat i on revieu process. A preprint of the

manuscript uill be submitted, to the Food and Drug Administration at the completion of its processing at NBS

.

Task 1£. SERUM IRON

No \vork has been dont. during th^’s period.

Task Ig. BILIKUBhi

No v/ork v;as done durin.g this nsriod.

Task Ih. UREA

Ra tio MaasuT'entont

s

Provision was made for cooling tlie probe of the

mass spectroitete r for the purpose c£ better con trolli:'g

the volatilization of the urea when the picbt; is inserteti

into tlie ness S|jec trometer for tlie ineasurement . Hov/ever,,

•this effected only a small improvement, and it was realized

that for the ratio measuremort of the quality desired, it

would be necessary to us-e a derivative of ure.a and GC/M3

r a t li e r t h an. d i r c c t p robe i n s e r t i l' n .. T h c u r e s v/ o ii 1 d b e

10

' > .

isolated as we have been doing; then the derivative would be

made. A search for suitable derivatives was begun!

,v‘;

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B'iLiOC.'. '.?HiC DATA . 5HPET

\\7lt AND 5Ut>Ti iLc

< I, r'lJBLICATiON' CR "cPORT*fio.

I NBSIR 79-179S

j

FM Contr5cl: Me- 74-58 (.0) October 1978 to March 1979

liUTriOWiSj

R. Schcffc", R. A. Velapoldi cind D. J

^FORMING OPnAr-.i/-/»TlC':. fMME AND ADORE5A

national Blf'.DAU OF 5T0,yOARDS

0;:r.\,CTMHNT OF COM.Uh'RCL'

o c 20.>3-4

F Uo/t /-io. t. i' .No;'

5. Putilication DiMa August 1979

Perfori:n:ri£ Org.’jnisstior; Cods

Reed or

a. Perfui'rr.iog O.-gan. Repa.'t No.

iO. F';aJ;-Ci/T£.5k/^0fk' tiUtNc.

ix. C.';ntfact/Gfa:ii No.

SFCNSOPi.'iG C;-'.'' NiZ.". :iON NAME AN? oC?’iPL£TE ADOPEFS fSfr-.-;, City, st.)!e, ZIP)

Luttau cf Mfcdical Dsuices aud Diagnostic Products

Food and Drr.g Aciuiuist'xati-'u

Silver Spring , Maryl.-'nd 20910

S[jiO',.r;,.a!TAr!Y NO 1 E

j

13. Type of Ropcfi & Period Cove, .i

. Spcr.soi'i.nt ;"£eao;.' ^.ods' i .

describe r a ."OTruter progra;.-.; S'“-185, FIPS Sofr.va:? SuTiinar/, is ai'.'2c.‘’od.

Vv. . 2C0-^:trd or /e - • .’aynta) »iro..nix.y o'.tT-.oot ai^niOesr.'. inlur r'i.cior.. li coctjtir.(.nt tneU-dys * si ^ni li r^\7

'Heier.'f 3u:* v, n'itni. jr. i:

This xfepcr.t .-'uscrihes u’ork perfrrited a. the National Bureau o£ S riindard.x

I'rcu 0r.tol.--r 1979 through M. ~cu 1979 it the contiiiuatioix of project.? irJ-ti

under IDA Contract 74-oo(0). The previous report was for the period iroia

Cc"ober 1977 thro.rgh S'epteraber 1978.

I'Fl' iiORDj . SK. to (iv.'Us .nf (

• f ipX./iicr-cj/ oco- r; ca}jt;alirs onty the lust lotto: .>/ ;h« first - y xocd anU'oi ,y:,f.ee ntunr.;

!>l>owiei 'ey stnucc-lons)

.diwCfficiji n slribfiion. t.5 Not Hciejse d) NT'.v

"* = A Picuing ON.ee, ’.'<’5s.'’,ington, DC: wjiQCX N o. c

Frc.n Nation ai ;

. ifc:-, 31:2,1 Fjivice tNTlS). S

^ ??.ibl

lA S£CURnS'~CLASS'

'THIS .REPOKT.t

21. Nij. OF

l-R!iNTE0 PAGES

UNCLASSIFIED

20. SECURITY CL-'-SS (THIS PAGE)

Uf'JCL AS3IFIED

2 >. PiTce

ui^o .../. :7c