QUARTERLY REPORT

FDA CONTRACT NO. 74-58(0)

R. Schaffer

Analytical Chemistry Dii^ision

Institute for Materials Research

National Bureau of Standards

Washington, D.C. 20234

February 1975

Interim Report for Period

October- -December 1974

Prepared for

Bureau of Medical Devices and

Diagnostic Products

Food and Drug Administration

Rockville, Maryland 2 08 52

FDA Contract No. 74-58(0)

Quarterly Report October- -December 1974

to the

Food and Drug Administration

by the

National Bureau of Standards

Progress is reported tov/ard the goal of establishing

the accuracy of several clinical reference methods, namely,

those for glucose, sodium, potassium, lithium, magnesium,

and chloride in serum, for lead in blood, and for uric acid.

The work performed during the interval October-December 1974

is reviewed, method by method.

TASK la: SERUM GLUCOSE

Base-line Method

Glucose analysis by the method of isotope-dilution mass

spectrometry (ID-MS) has begun. Samples (see Quarterly Report

April-June 1974) being studied are 1) known mixtures of the

1 , 2 : 5

, 6-di -0- isopropylidine derivatives of glucose-6 ,6-d^

13 (dideuteroglucose) or U- C-glucose (uniformly labeled

C-glucose) and natural glucose to determine the accuracy of

recovering by MS the known proportions of the isotopic molecules

in the molecular form measured; 2) known mixtures of either

labeled glucose with natural glucose; these mixtures of the

isotopic and the natural glucose are converted into the diisopropylidene derivative, and then the proportions are measured by MS

to determine whether changes in the proportions of the labeled

to unlabeled forms might occur on synthesizing and separating

the derivative; and 3) multiple portions of a single serum

containing glucose to which different proportions of either

one of the labeled glucoses are added and then the derivative

is synthesized, isolated and tested by MS to assay for the

glucose content of the serum.

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The results from 1) and 2) provide estimates of attainable

accuracy. The results from 3) provide an estimate of precision.

The overall result that has emerged from analyses performed

thus far is that the ID-MS approach will afford base-line

accuracy and precision within 1% for the glucose content of

serum.

The receipt and installation of the improved multiple ion

selection unit for the mass spectrometer (referred to in Quarterly

Report July- September 1974) are expected momentarily.

Analytical Work

Second round results obtained by use of the candidate

reference method (hexokinase) were reviewed with participants

at a meeting held in November 1974.

For this second round in addition to analyzing for glucose

in serum samples, participating laboratories were asked to report

the spectrophotometric absorbances of a set of aqueous solutions

of acid dichromate that were supplied; these measurements were

sought to enable the committee to evaluate the influence of the

spectrophotometers being used on the slopes of the glucose

calibration curves obtained in the individual laboratories.

(In the first round it was found that these calibration curves

differed from lab to lab.) The additional spectrophotometric

data were considered statistically by examining 1) the slopes

of the lines obtained by correlating each laboratory's reported

dichromate absorbance values against those measured by NBS

and 2) the slopes of the lines connecting the set of absorbances

measured in each laboratory in setting up its glucose calibration

curve. Despite the small range of values over which correlations

were calculated, it could be concluded that the differences

between the slopes of the glucose calibration curves are

reflected by the corresponding differences in slopes measured

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on the dichromate solutions; hence, the characteristics of

the different spectrophotometers largely determine the differences

observed in glucose calibration curves.

The serum samples were analyzed in the second round in two

separate ways: 1) by the previous protocol using manual

pipetting and, where laboratories were appropriately equipped,

2) by an alternative protocol in which an automatic pipetter,

whose precision could meet necessary specifications, was employed.

The results obtained by the method employing manual

pipetting only are as follows:

Between Lab CV Between Lab CV

Average Within Lab CV (Single Test/Lab) (4 Test Avg/lab)

mg/dl %% % 40.4 1.49 5.71 5.56

75.4 1.05 2.66 2.50

134.3 1.89 2.10 1.32

205.9 1.44 1.65 1.08

450.0 0.62 1.33 1.22

Results for the method employing the automatic pipettor

are as follows:

Average Within Lab CV Between Lab CV Between Lab CV

mg/dl % (Single test/lab) (4 Test Avg/lab)

40.8 1.91 3.60 3.20

76.0 1.02 1.90 1.68

133.9 1.13 1.43 1.04

204.1 0.70 1.04 0.85

450.3 0.52 0.92 0.80

The second round results reveal a measureable improvement

in the between- laboratory coefficient of variation (CV) over

the results from the first round. However, the results

continue to show a high between- laboratory coefficient of

variation for samples containing glucose at the lowest concen-

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tration levels examined.

Although only about one-half of the number of laboratories

that performed the manual method also performed the method using

the automatic pipettor, the results from the latter method clearly

reveal better between- laboratory coefficients of variation over

results from the manual procedure.

TASK lb: SODIUM POTASSIUM, LITHIUM, MAGNESIUM AND CHLORIDE

Base-line Methods

Application of the base-line methods for these electrolytes

is contingent on NBS receiving serum samples from the CDC. NBS

has contracted for the needed bulk supply of serum and its

delivery to CDC where the serum is chemically treated and vialed

as samples for the study of the reference methods for each of

the electrolytes. A minor technical difficulty in processing

the serum has slowed the production of these samples. As a consequence at this time, only the samples for the lithium study

have been received, and those having magnesium are expected

within the next week or so. Sodium and potassium samples are

expected by early March and chloride samples will follow one

month later. Base-line methods are to be performed on these

samples as they are received.

Lithium

The protocol for the candidate lithium reference method

is appended to this report. The first test round of the

protocol for lithium has been carried out but NBS base-line

values are not yet available; hence, the results received from

the participating laboratories are summarized only in terms of

precision:

Sample Average Standard Deviation CV

Number mmol/1 mmol/1 %

1 1.005 0.023 2.32

2 1.497 0. 030 1.98

3 1.975 0. 031 1.57

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Magnesium

The protocol for this reference method has been prepared

following the format of the lithium protocol. Participating

laboratories are about to be supplied with the protocol, and with

magnesium gluconate to use as the standard and serum samples

to use for practice runs. As soon as the magnesium serum samples

are received from CDC, they will be sent to the laboratories

for the first round of study to begin.

Sodium-Potassium and Chloride

The writing of candidate protocols for each of these

electrolytes is near completion. Analytical work awaits

the arrival of samples.

TASK Ic: LEAD IN BLOOD

An extraction-atomic absorption procedure for determining

lead in blood was used by six participating labs as a first

round of study of a protocol for the reference method. A meeting

to review the results of that round of study and to criticize

the protocol was held in November 1974. The best reported

results (from only 2 of the participants) were within 201 of

the ID-MS target values; however, three labs reported values that

were off by more than 50%. Possible sources of difficulty and ambiguity and recommended changes in the protocol were discussed

for use in a revised protocol. A highly detailed protocol

has been prepared. Further detail may need to be added to the

protocol, but this v/ill become obvious on consideration of the

results from the next round of study.

Because of the uncertainty of the accuracy of analyses

for lead by methods other than ID-MS, samples to be used for

later rounds of study are to be analyzed by the NBS ID-MS

method before being sent to participants.

To ensure freedom from lead contamination, all materials

that come into contact with the blood samples (syringes,

extraction tubes. Teflon tape, etc.) are being tested by

ID-MS. ID-MS values are now being obtained on the samples

for round two.

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TASK Id: URIC ACID

Base-line Method

The ID-MS approach to definitive values for uric acid in

serum is being pursued. Because uric acid is present in serum

at concentrations of about 0.1 mg/ml, we expect to use gas

chromatography in the last stage of purification of the mixture

of isotopic molecules. However, uric acid is insufficiently

stable to be passed through a gas chromatograph to the MS.

Hence, a volatile derivative of uric acid is needed. (This

is somewhat like the situation with glucose, for which the

diisopropylidene derivative was needed to provide sufficient

volatility .

)

Methylation has been used to obtain volatile derivatives,

but a number of different derivatives are produced. Now we

find the same situation on use of ethylation procedures.

Ethylation would be preferable as it may avoid possible

difficulty with interferences due to methylated substances

(e.g., drugs) that may be present in serum samples.

An alternative route to the needed volatile derivative is

acylation. Such reactions of uric acid are poorly understood.

However, our examination of such methods has led to the isolation

of a possibly useful derivative having isobutyryl residues.

The structure of this product is being studied.

Expenditures October- -December 31, 1974

Cfi^om NBS Accounts Division Statement)

Labor 2283 hours

Labor, leave, etc.

Other objects

$68,245. 00

53,200. 00

$121,445. 00

^

PROTOCOL:

The Determination of Lithium in Serum

I . Scope

The method is designed to provide accurate values of the

lithium concentration in serum or aqueous solutions as submitted

for analysis, provided strict adherence to this protocol is

. .maintained. The method does not assure adequacy or integrity

of sampling per se. The method has applicability, at the

specified accuracy for lithium, in the range from 0.5 to 2.5

imnol/1 and is an adaptation of a procedure by Pybus and Bowers [1]

.

II . Reagent Specifications

1. At the time of preparation, distilled and/or deionized water

should exhibit a specific resistance of at least 1.0 Mohm cm

at 25 _+ 2 °C. At the time of use, aspiration of this reagent

grade water should show no detectable signal under the instrumental settings used for this analysis. The water should

be available in large quantity for use as a diluent and for

the final rinse operation on all glassware and apparatus

coming in contact with the solutions involved.

2. Lithium standard solutions are to be prepared from Standard

Reference Material Li^CO^ (SRiM 924) [2] issued and certified by the National Bureau of Standards (NBS) . The SRM

Li2C02 should be stored at room temperature in a desiccator

containing silica gel or equivalent. ( NOTE : The lithium

in the SRiM is depleted in the ^Li isotope; thus the

molecular weight for this Li2C02 is 73.94855, and calculations should be based on this value.)

3. Sodium and potassium chlorides, hydrochloric and nitric

acids, and 95^ ethanol CV:V) meeting ACS [3] (or equivalent)

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specifications are to be used.

4. Dilute nitric acid is prepared by making a twenty-fold

dilution of concentrated IINO^ (15.4 M) with water.

5. Dilute hydrochloric acid is prepared by making a fivefold dilution of concentrated FICl (11.6 M) with diluent

solution (see IV-1). Prepare approximately 25 ml.

III. Glassware Specifications

All volumetric glassware (pipets and volumetric flasks)

should meet NBS Class A specifications [4] (or equivalent)

.

All glass or plastic surfaces that come into contact with

• reagents, water, diluent, or sample must have been previously

cleaned as follow's (see also: Section V-3):

1. Use routine cleaning procedure (warm water with detergents,

plus usual rinses)

2. Soak glassware for 50 min in dilute HNO^, (NOTE: Concentrated HNO^ will etch glass and produce sites for ion

adsorption)

.

3. Rinse six times with a volume of water equal to or greater

than 10^ of the container volume and

4. Use apparatus immediately or' air dry (inverted) in a dustfree environment for future use.

IV. Preparation of Reagents

1. Diluent (Blank) Solution : Contains NaCl at 140 mmol/1 and

KCl at 5.0 mjnol/1. Add 16.56 g of NaCl and 0.746 g of

KCl to a 2-liter volumetric flask, dissolve in water, and

add water to belov; the calibration mark. At 1^ 2 °C,

fill to the calibrated volume with water. Mix by inverting

the flask 30 times.

2. Lithium Standard Stock Solution: Weigh accurately to the

nearest one tenth milligram 0.3698 g of Li2C0. (NBS SRM 924)

and transfer it quantitatively to a 1-liter volumetric flask.

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To dissolve the Li2C02, just cover the bottom of the flask

v/ith diluent solution and slowly add 10 ml of dilute HCl

(See Section II-5). Fill to the mark at 25 + 2 °C using

diluent solution to give the 10.0016 mmol/1 lithium

standard stock solution.

3. Lithium Standard Diluted Solutions : Transfer 5-, 10-,

15-, 20-, 25-, and 30-ml aliquots of the lithium standard

stock solution to six -100 ml volumetric flasks in that

order. Fill each to the mark at 25 2 °C using diluent

solution to give the diluted standards of 0.50-, 1 . 00-

,

1.50-, 2. 00-, 2. 50-, and 5.00- mmol/1 of Li, respectively.

(NOTE: The concentrations of the lithium standard diluted

solutions must be calculated and reported to four decimal

places as in Table 1, Section VI-4).

V. Procedure for Pipetting and Diluting

All solutions should be at 25 +_ 2 °C.

All blank, diluted standard and unknown sample solutions

are diluted ten-fold using a 5-ml Class A volumetric pipet in

the "to contain" mode and 50-ml volum.etric flasks. Only one

volumetric pipet is used throughout to avoid errors that would

be caused by differences in drainage betw-een the aqueous or

dilute acid and serum solutions, using different pipets. If

50 ml of a standard is insufficient for the total test, prepare

a second 50-ml portion.

1 . Pipetting and Diluting, General : Transfer approximately 15 ml of water to a 50-ml volumetric

flask and then add 5 ml of the appropriate solution by the

following procedure. Fill the 5 ml Class A pipet to

approximately 1.0 cm above its calibration mark. Place

the index finger over the top end of the vertical pipet,

contact the tip of the pipet with the side of the container

and deliver excess solution until the meniscus is at the

calibrated mark on the pipet. Remove the pipet from

. 4 -

contact with the container and tilt the pipet to an

approximately horizontal position. Then remove excess

liquid from the exterior of the delivery end of the

pipet by wiping with clean absorbent paper. Direct the

delivery end of the pipet into the receiver while bringing

the pipet back to a vertical position. The sample should

be delivered by contacting the pipet tip with the receiver

wall and allowing the pipet to drain. After drainage

of the pipet stops, gently blow out the residual liquid.

Rinse the pipet tip into the volumetric flask with

4 ml of water delivered, for example, from a disposable

Pasteur or similar pipet. (Caution: New pipets need

to be cleaned.) Rinse the 5-ml volumetric pipet three

times by filling with v/ater, each time delivering the

contents into the receiver by drainage against the inner

wall of the flask above the liquid level. Then fill to

the calibrated volume with water at 25 _+ 2 °C and mix

thoroughly with 30 inversions.

2 . Pipetting and Diluting, V.'orking Blank and Lithium Standard

Working Solutions : Prepare the working blank and the 0.50-, 1.00-, 1.50-,

2. 00-, 2. 50-, and 3.00-mmol/l lithium standard working

solutions in that order. Fill the 5-ml pipet with the

diluent solution (blank) or lithium standard diluted

solution to be transferred and discard that portion of the

solution. Repeat twice. Then fill the pipet, adjust

to volume, deliver to the receiver flask, rinse the pipet,

dilute using water and mix a s described in Section V-1.

3. Pipetting and Diluting Sera (The volume of the serum

specimens provided is insufficient for use of the pipetconditioning technique described in Section V-2; hence,

the pipet must be cleaned and dried for each serum

sample . )

Fill the clean, dry 5-ml pipet with the serum sample,

adjust to volume, deliver and rinse into the receiver,

and dilute as described in Section V-1. After transfer

of serum, wash the pipet with dilute IINO^ and rinse with

a minimum of four portions of water and then twice with 95%

ethanol. Allow pipet to drain. Apply minimum suction to

the top end of the pipet for one minute to assure the absence

of ethanol. The pipet should then be ready for use with the

next serum sample. (NOTE: If the dilute HNO^ washings

do not drain cleanly from the pipet, clean the pipet by

rinsing it several times with hexane. Then repeat the

cleaning and drying steps in Section III, followed by

ethanol drying as described in this paragraph.

4. At the conclusion of the dilution procedures there should

be:

a. One 50-ml volumetric flask containing a ten-fold

dilution of the diluent (blank) solution, labeled

"blank ten-fold dilution,"

b. Six 50-ml volumetric flasks containing ten-fold

dilutions of each of the lithium standard diluted

solutions, labeled working solutions, 0.50-, 1.00-,

1.50-, 2. 00-, 2. 50-, and 3.00-mmol/l of lithium,

respectively, and

c. As many additional 50-ml volumetric flasks containing ten-fold dilutions of solutions of unknov;ns as

there are samiples to be analyzed, each labeled

appropriately.

VI . Atomic Absorption Spectrometry (AAS) Measurement Procedure

It is assumed that the operator is fully familiar v;ith the

instrument to be used. It is not possible in this method to

give detailed instruct ions necessary to assure instrument

stability, linearity, flame conditions, etc. In general,

the accuracy of the method cannot be attained unless the

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instrument is in optimum operating condition and meets all

the . specifications set forth by the manufacturer.

1. Instrument and Electrical Adjustment . Prepare the atomic

absorption spectrometer for operation according to instructions provided in the operator's manual. Place the lithium

hollow cathode lamp in the lamp housing receptacle. Turn

the pov.-er supply switch to "On." Select the optimum

current for the lamp, and allow ample "warm-up" time for

the lamp to become stable. Adjust the monochromator

slit and set the wavelength selector to the lithium

o resonance line at 670.8 nm (6708 A). Adjust the photomultiplier dynode voltage to give optimum current output

with minimum dark current.

2. Flame Condition . Open the tank valves on the air and

acetylene supplies. Adjust the secondary regulators as

recommended by manufacturer. Check the burner to make

sure the prem.ixing chamiber and nebulizer are clean and

free of any foreign obstructions. Light the burner and

adjust the air - acetylene flow rates recomm.ended for

the instrument. To stabilize the temperature of the

burner head, aspirate water into the flame for 10 min

before proceeding to the next step. (NOTE: A fuelrich air- acetylene flame gives optimum sensitivity for

the measurement of lithium; however, it may be- difficult

to obtain the precision specified in this method with

a fuel-rich flame. Therefore, it is suggested that a stoichiometric or only slightly fuel-rich flame be

used to obtain the highest precision for lithium in serum.)

Detcrinina t ion of Optimum Absorbance . Determine the

stability and repeatability of tlie instrument as well as

the calibration curve as follows:

a. Adjust the instrument to zero absorbance while

nebulizing water. (NOTE: Always nebulize water

when measurements of standard, blank, or unknown

solutions are not being made.

b. Nebulize the 3.00-mmol/l lithium standard working

solution and measure the absorbance.

c. Adjust the ins trum.ental gain such that 90^ full

scale for recording instruments or 2.000 units

for digital read-out instruments is observed.

d. Readjust the instrument to zero reading with working

blank*

e. Nebulize the working solutions of blank, 0.50-, 1.00-,

1.50-, 2. 00-, 2.50- and 5.00-mmol/l lithium standards

and record the instrum.ent al readings.

f. Repeat nebulization of the working blank and the 3.00

nmiol/l lithium standard 'working solution until stable

conditions are achieved.

Unknown Measurements . a. Nebulize all the dil- . ed unknown solutions and determine v;hich of the two working standards bracket each

of the unknowns

.

b. For the analyses of each unknown and its two bracketing standards, reduce or increase scale expansion

until tlie expanded absorbance for the high standard

of the bracketing pair of standards reads 2.000

expanded absorbance units for digital readout instruments or approximately 90^ full scale for recording

. instruments

.

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c. Nebulize the low working standard, diluted unknown,

high working standard, and diluted unknown in that

order. These four measurements constitute a "set".

d. Repeat step 4c until a valid group of 5 "sets" is

obtained as illustrated in step 5 and Table 2.

Table 1

Example of Steps 3e and 4c, Section VI

Lithium Standard Diluted

Solutions, inmol/1 0. 5001 1. 0002 1. 5005 2 . 0004 2 . 5005 3.000

Expanded Absorbance Measurements

1. Standards 0.335 0.660 1.000 1.300 1.665 2.000

2. Unknowns, A,B,C 0.473(A) 1.288(B) 1.699(C)

^* Valid Sets of Measurements . A set of measurem.ent s is

considered valid if the following conditions are met:

a. the two expanded absorbance values measured for the

diluted unknown in a given set must not differ by

more than 1?;; and

b. the expanded absorbance values for the diluted unknown

and the working standards in a given set must not

differ by more than II from the values measured in

the previous valid set.

A non-valid set must be eliminated and enougli sets made to

obtain a group of five valid sets. For example Set 2 in

Table 2 is a valid set since the differences between the

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expanded absorbance measurements for the Low Standard

(Set2-Set^ = +0.004), Unknown B (Set2-Set2 = -0.004),

(Set2-Set^ = +0.006) and High Standard (Set2-Set^ = +0.002)

are all less than 1^ of the expanded absorbance values.

However, the measurements made in Set 5 are not valid

because the differences between the expanded absorbance

measurements for the unknowns (Unknown B set 5 minus

Unknown B set 4 and Unknown B set 5 minus Unknown B set

5) are greater than the 1% limit; i.e., [(1.993-2.032)/

2.032](100) = 1,4% or [ (2 . 032- 1. 995) /1 . 995] (100) = 1.31.

Thus, Sets 1, 2, 3, 4 and 6 comprise the group of 5

valid sets and Set 5 is excluded.

Table 2

Valid Measurements for Unknown B (Steps VI-4b,c,d,5)

PiXPANDED ABSORBANCE READINGS

Lo Std Unk Hi Std

Set Li ,mmol/l 1. 5001 B 2. 00 02

1 Lo Std - Unk

- Unk

B

- B

- Hi Std. 1. 536^ 1.983^

1.987

2. 000^

2 Lo Std - Unk

- Unk

B

- B

Hi Std 1. 540 1. 981

1.985

2. 002

3 Lo Std - Unk

- Unk

B

- B

- Hi Std 1. 541 1.987

1. 989

2. 004

4 Lo Std - Unk

- Unk

B

- B

- Hi Std 1. 545 1. 991

1.993

2. 007

5 Lo Std - Unk

- Unk

B

- B

- Hi Std 1. 550 2. 032

1. 995

2. 009

6 Lo Std - Unk

- Unk

B

- B

- Hi Std 1, 556 1. 997

1.999

2. Oil

a Calculate and report Li,mmol/l to four decimal places,

b Record and report all expanded absorbance values with four

figures on data sheets provided.

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6. Data Recording ar.d Calculations . Please record the lithium

standard diluted solution concentrations in mmol/1 of Li to

four decimal places and the expanded absorbance readings to

four figures on the attached form labeled "Data Sheet" and

as illustrated in Tables 1 and 2. No calculations are

required for this round robin test. Data evaluation ivill

be performed at NBS and equations for the calculations

v/hich give the best expanded absorbance values and subsequent

mmol/l of Li will be determined.

1. Pybus, J., Bowers, G.N. Jr., "Measurement of Serum Lithium

by Atomic Absorption Spectroscopy," Clin. Chem. 1_6_, 139-143

(1970)

.

2. Office of Standard Reference Materials, The National Bureau

of Standards, V/ashington, D.C. 20234.

3. Reagent Chemicals, Am.erican Chemical Society Specifications

ACS Publications, Washington, D.C. (1968).

4. Hughes, J. C, "Testing of Glass Volumetric Apparatus,"

NBS Handbook 77, Volume III, 726/1-10, (1961). "Precision

Measurement and Calibration," A compilation by S.F. Booth

of selected papers on Optics, Metrology and Radiation.

Figure 1. Typical Calibration Curve for the

Atomic Absorption Spectrometric

Determination o£ Lithium. Data

Given in This Graph Kindly Supplied by Mr. Michael Epstein and

Mr. Tlieodore Rains, NBS.

NBS - Electrolytes in Serum - Clinical Reference Method

Lithium

Laboratory Analyst v Exercise No. RRI

Date Samples Received^ Dates Analyzed (1) (2)

Instrument Manufacturer _^ Model

Wavelength nm Slit Width ym

' Type Hollow Cathode Lamp

Current mA

Burner Type

Oxidant Flow rate 1/min

Fuel^ Flow rate 1/min

Instrument Time Constant s Scale Expansion

Recorder Time Constant s Readout: Recorder , Digital , Other

Laboratory Temperature °C to °C (Variation during Round Robir.

Background Correction? How?

Comments

DATA SHEET

Round Robin

Date:

EXPANDED ABSORBANCE

Lo Std Unknov\;n Hi Std

Li,inmol/l Valid Remark

Set^

2

3

5

Only 5 valid sets needed.

I.