NAT'L INST OF STAND & TECH PUBLICATIONS HEFEREiCE

NIST Special Publication 260-162

2006 Edition

Standard Reference Materials

Compilation of NIST Higher-Order Methods

for the Determination of Electrolytes in

Clinical Materials

Stephen E. Long

Karen E. Murphy

lOO Jll^Sl" National Institute of Standards and Technology • Technology Administration • U.S. Department of Commerce

NIST Special Publication 260-162

2006 Edition

Standard Reference Materials®

Compilation of Higher-Order Methods for

the Determination of Electrolytes in Clinical

Materials

Stephen E. Long

Karen E. Murphy

Analytical Chemistry Division Chemical Science and Technology Laboratory

National Institute ofStandards and Technology

Gaithersburg MD 20899 USA

September 2006

U.S. Department of Commerce

Carlos M. Gutierrez, Secretary Technology Administration Robert C. Cresanti, Under Secretaryfor Technology

National Listitute of Standards and Technology

William A. Jeffrey, Director
Certain commercial entities, equipment, or materials may be identified in this document in order to describe an experimental procedure or concept adequately. Such identification is not intended to imply recommendation or endorsement by the

National Institute of Standards and Technology, nor is it intended to imply that the

entities, materials, or equipment are necessarily the best available for the purpose.

National Institute of Standards and Technology Special Publication 260-162

Natl. Inst. Stand. Technol. Spec. Publ. 260-162, 101 pages (September 2006)

CODEN: NSPUE2

U.S. GOVERNMENT PRINTING OFFICE

WASHINGTON: 2006
Abstract

This manual comprises higher-order analytical methods developed and used at the National Institute of Standards and Technology (NIST) for the determination

of electrolytes in clinical materials. Higher order methods are used to certify Standard Reference Materials (SRM) such as the 909, 956 (blood serum) and 2670 (Urine) series for clinical analytes and to provide a basis for evaluating the accuracy and comparability of other reference methods. Several of these methods are listed in the Joint Committee for Traceability in Laboratory Medicine (JCTLM) database. The manual comprises comprehensive descriptions, evaluation and method performance data for the major electrolytes calcium,

chloride, lithium, magnesium, potassium and sodium.
I
Foreword

Higher order or "definitive" methods have been in use at NIST for several decades to establish certified values for electrolytes and other trace elennents in clinical Standard Reference Materials (SRMs). Although some of these methods

have been described in various NIST 260 series documents, there has not been,

until now, a single information resource providing comprehensive descriptions of the methods in a format which facilitates systematic updates for new methods, performance improvements to existing methods and removal of methods which

are considered obsolete. This manual attempts to address this issue and

provides a unified collection of all methods currently in use within the Analytical Chemistry Division for the measurement of electrolytes in clinical samples. This resource should prove useful not only for personnel within the Division but also to external customers who are involved in clinical testing. For example, the manual

provides comprehensive information on the traceability basis of methods used to

certify higher-order reference materials, which are an essential component of the European Union In-Vitro Diagnostic Medical Devices Directive (IVD), which became effective in December 2003.

Stephen E. Long, Ph.D.

Inorganic Chemical Metrology Group

September 2006

Please send any comments or notification of errors and omissions found in this document to selonq(S)nist.qov.
I
Glossary of Terms Used in This

IManual

Accuracy: The degree of agreement of the measured test result with the true value or accepted reference value. Certified Reference Material (CRIVI): A reference material, accompanied by

a certificate, one or more of whose property values are certified by a procedure which establishes its traceability to an accurate realization of the unit in which the

property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence [ISO Guide 30, Terms and Definitions Used in Connection with Reference Materials: 1992 and ISO International Vocabulary of basic and General Terms in Metrology

(VIM):1993]. Electrolyte: Positively and negatively charged species present in extra-cellular and intra-cellular systems, plasma and fluids. Electrolytes are responsible for maintaining various critical functions within the body. Inductively Coupled Plasma - Mass Spectrometry (ICP-MS):

Instrumental technique in which the analyte is ionized in an argon plasma and

the ions separated and measured by a mass spectrometer as a function of their mass to charge ratio. Isotope Dilution: A highly accurate method in which a separated isotope of the analyte of interest is added to the sample and the perturbed isotopic composition measured. The analyte concentration can be calculated from the perturbed

isotope ratios and the known mass of added spike. National Metrology Institute (NMI): Nationally designated laboratory considered to have the greatest competence in the measurement of physical and

chemical quantities using the highest-order metrological measurement methods

available. NIST: Is an agency of the Department of Commerce. Its role as the National Metrology Institute (NMI) for the United States is to develop, and maintain custody of the national standards of measurement, to provide the means and methods for making measurements consistent with those standards and to assure the compatibility of United States national measurement standards with those of other nations. NIST SRM Certificate: Document stating the intended purpose and

application of an SRM, its certified property value(s) with associated

uncertainty(ies), and any other technical information deemed necessary for its proper use. In accordance with ISO Guide 31:1996, a NIST SRM certificate bears the logo of the U.S. Department of Commerce, the name of NIST as

certifying body, and the name and title of the NIST officer authorized to accept

responsibility for its contents.
Plasma: The liquid component of blood, containing suspended blood cells and

platelets. Precision: A measure of the dispersion of the sample distribution. Procedure Blank: A measure of the amount of the analyte contributed to the sample aliquot from the measurement process. The procedure blank is an

absolute quantity. Reference Material: Material or substance, one or more of whose property values are sufficiently homogeneous to be used for the calibration of an

apparatus, the assessment of a measurement method, or for assigning values to materials [ISO VIM: 1993]. Serum: Liquid component of plasma with clotting factors removed. Standard Reference Material (SRM): A CRM issued by NIST that also meets additional NIST-specific certification criteria and is issued with a certificate or certificate of analysis that reports the results of its characterizations and

provides information regarding the appropriate use(s) of the material [NIST SP

260-136]. Thermal Ionization Mass Spectrometry (TIMS): Mass spectrometric technique in which the sample is loaded onto a metal filament (usually rhenium) and ionized by heating the filament to a high temperature. A very stable ion beam

is produced which can be processed by the mass spectrometer. Traceability: The property of the result of a measurement or the value of a

standard whereby it can be related to stated references, usually national or

international standards, through an unbroken chain of comparisons, all having

stated uncertainties [ISO VIM: 1 993].
NIST Standard Reference

Materials® (SRMs)

One of the primary functions of the Inorganic Chemical Metrology Group of the NIST Analytical Chemistry Division is to assign values for target analytes in Standard Reference Materials for chemical measurements. These

measurements are principally earned out by using higher-order or definitive methods. Reference methods are also used for some measurements. Values

appearing on certificates fall into three categories [NIST SP 260-136]: NIST Certified Value - Value and its uncertainty assigned by NIST in conformance with the NIST uncertainty policy. A NIST certified value is a value

for which NIST has the highest confidence in its accuracy in that all known or suspected sources of bias have been fully investigated and accounted for by

NIST. A certified value is obtained by one or more of the following measurement

modes:

1. Certification at NIST using a single primary method with confirmation by

other method(s).

2. Certification at NIST using two independent critically-evaluated metht^ds.

3. Certification/value assignment using one method at NIST and different methods by outside collaborating laboratories. NIST Reference Value - Is a best estimate of the true value provided by NIST

where all known or suspected sources of bias have not been fully investigated by NIST. NIST Information Value - Is a value that will be of interest and use to the SRM/RM user, but insufficient information is available to assess the uncertainty associated with the value.

For additional information see

http://ts.nist.gov/ts/htdocs/230/232/ABOUT/definltions.htm
I
Contents

Title Revision Date

/l\/lethod sAbstract Foreword

Glossary of Terms ; NIST Standard Reference Matenals

Introduction 1 Calcium iVo 9/06 2

Total Calcium by Isotope Dilution Inductively Coupled Plasma Mass Spectrometry

1.0 9/06 CA-1

Chloride 1.0 9/06 3

1 Chloride by Isotope Dilution Thermal Ionization Mass

Spectrometry

1.0 9/06 CL-1 Lithium 1.0 9/06 4

! Lithium by Isotope Dilution Inductively Coupled Plasma -

i Mass Spectrometry

1.0 |9/06

1 LI-1 Magnesium 1.0 |9/06 5 Magnesium by Isotope Dilution Inductively Coupled Plasma - Mass Spectrometry

1.0 b/06 MG-1

iPotassium 1.0 9/06 6

i Potassium by Isotope Dilution Inductively Coupled Plasma -

Mass Spectrometry

1.0 9/06 K-1 Sodium To"""" 9/06 7

1 Sodium by Stable-Isotope Internal Standard Inductively

1 Coupled Plasma - Mass Spectrometry

1.0 9/06 NA-1
I
Section 1 - Introduction 1 Introduction

1.1 Clinical Electrolyte Measurements

Determinations of electrolytes in serunn and urine samples are among the most commonly ordered panels for clinical diagnosis and point-of-care

testing. Electrolytes in body fluids occur either as free ions or partly bound to proteins. The maintenance of correct electrolyte concentrations in body fluids

is vital and disorders in electrolyte balance are used to diagnose changes in health status (such as renal and metabolic function) and fluid balance. The

determination of the major electrolytes in body fluids within the clinical laboratory setting is therefore well established. Traditionally, inductively coupled plasma emission spectrometry (ICP-AES) or flame photometry have been used, but electrochemical systems, especially automated ion-selective electrode (ISE) systems which can rapidly measure a panel of electrolytes, have gained in popularity. Methods based on spectrophotometry are becoming less common than they once were but are still important for some

electrolytes such as the determination of total calcium and magnesium in serum.

1.2 The IVD Directive

In an effort to unify the regulatory status of the medical device and

diagnostics industry, the In-Vitro Diagnostics (IVD) Directive was adopted by

the European Union Council of Ministers on October 5, 1998, and formally published on December 7, 1998. This, the last of three directives in this industry, mandates a Conformite Europeene (CE) marking system for manufacturers of diagnostic medical devices which encompass reagents,

calibrators and instruments intended for ascertaining the status of patient

health, and which are destined for sale in the European Union. The Directive came into effect five years later in December 2003. One important requirement of the IVD Directive is the provision of a risk analysis. It must be

demonstrated that products are suitable for their intended purpose and that the risks associated with their use be fully evaluated and mitigated as far as

possible within the limits of the current state of the art knowledge. Another

important requirement is that "the traceability of values assigned to calibrators and/or control materials must be assured through available reference measurement procedures and /or reference materials of a higher

order." It is predicted that this requirement will increase demand for a variety of appropriate certified clinical reference materials from National Metrology

1-1
Section 1 - Introduction

Institutes (NMIs). For example, sales of the NIST material SRM 956a

(Electrolytes in Frozen Human Serum) increased dramatically at the time the IVD Directive came into effect.

1.3 JCTLM

The Joint Committee on Traceability in Laboratory Medicine (JCTLM) was

formed in June 2002. The aim of the Joint Committee is to meet the need for a worldwide platform to promote and give guidance on comparability,

reliability and equivalence of measurement results in Laboratory Medicine and traceability to appropriate measurement standards for the purpose of improving health care. The four principal promoters of the JCTLM are the

International Bureau of Weights and Measures (BIPM), the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC), the

International Laboratory Accreditation Cooperation (ILAC) and the World

Health Organization (WHO). Several other key stakeholders are also

involved including reference material producers, representatives of the IVD

industry, quality assurance organizations and the International Organization

for Standardization (ISO). The JCTLM is currently divided into two working

groups, one assigned to reference measurement methods and reference

materials, with the purpose of identifying reference materials and reference procedures and endorsing those appropriate to meet the requirements of the IVD Directive, the other responsible for collecting information on existing and

candidate reference measurement laboratories and to encourage and

facilitate the formation of networks of laboratories for different groups of measurable quantities.

1.4 NIST Clinical Measurement Methods - Philosophy and Trends

Where possible, NIST clinical measurement methods for electrolytes have

traditionally employed isotope dilution mass spectrometry [1]. When correctly used, this is a highly accurate analytical method with direct traceability to the mole. Before the advent of inductively coupled plasma mass spectrometry

(ICP-MS), isotope dilution thermal ionization mass spectrometry (TIMS) was

widely employed for these measurements [2]. While this offers extremely good isotope ratio measurement repeatability, sample throughput tends to be

rather slow when using the NIST designed solid-source mass spectrometers.

Therefore, in common with many other laboratories, NIST clinical measurement methods have gradually been transferred to ICP-MS systems

which offer higher sample throughput and more cost-effective measurement

options. The trade-off in slightly poorer isotope ratio measurement

1-2
Section 1 - Introduction

repeatability is not a significant disadvantage for clinical reference material

certification. The emergence of "cool" (shielded) plasma, and more recently,

collision cell ICP-MS systems have now permitted some of the more

problematic elements such as calcium and potassium to be accurately measured. All of the common clinical electrolytes are now measured by ICP- MS, with the exception of chloride, which currently still remains the domain of TIMS.

1 .5 About This Manual

As a principal stakeholder in the JCTLM, NIST has several assigned

responsibilities which include the maintenance of primary measurement

methods and reference materials of a higher metrological order, participation

in global reference laboratory networks, providing reference laboratory measurement services and working with other NMIs and organizations to establish equivalence of measurement services. This manual provides a

collection of higher-order methods currently used at NIST for the measurement of electrolytes in clinical materials. The manual has been

organized in such a way that the methods can be updated with revised

versions, newer methods added, and obsolete methods deleted as

necessary to meet these responsibilities. The manual is divided into a section

for each of the principal electrolytes measured at NIST. Each section comprises a brief summary of the electrolyte, the context of clinical measurement together with detailed descriptions of all analytical measurement methods currently in active use at NIST.

1.6 References

[1] Bowers G.N., Fassett J.D. and White V.E., "Isotope Dilution Mass

Spectrometry and the National Reference System."

Clin. Chem. 65, pp. 475R-479R (1993).

[2] Moore L.J. and Machlan L.A., "Higfi Accuracy Determination of Calcium

in Blood Serum by Isotope Dilution Mass Spectroscopy."

Anal. Chem. 44, p. 2291 (1972).

1-3

Section 2 - Calcium 2 Calcium

2.1 Clinical Significance The total calcium body content in humans is approximately 1 kg, the majority

of which is localized in the skeletal system as hydroxyapatite. A small amount of calcium is present in both the intra-cellular and extra-cellular fluid compartments, with approximately 50 % of the extra-cellular calcium present

in blood plasma. About 40 % of plasma or serum calcium is bound to proteins (mostly to albumin), 10 % is complexed with inorganic ions and the remainder is so-called ionized calcium. Approximately 15 % of the total serum calcium is in a biologically active free form. Calcium has many

critically important functions aside from its structural role, among which are

the proper functioning of the neuromuscular system, myocardial contractility, the structure of cell membranes and the production of clotting factors. The

level of calcium in plasma, therefore, is under strict homeostatic control. Serum calcium levels are regulated by a complex mechanism involving renal excretion and tubular renal re-absorption. Parathyroid hormone (PTH)

stimulates re-absorption and therefore increases calcium activity. Calcitonin on the other hand, reduces the calcium activity by inhibiting the osteoclastic release of calcium and phosphate from bone to the extra-cellular fluid and

decreases renal re-absorption. In hypocalcemia, calcium serum levels are depressed. Two common causes are renal failure and pancreatitis. IHypercalcemia, a condition where serum calcium is elevated, is commonly

encountered in hyperparathyroidism, where too much PTH is produced by

the parathyroid gland.

2.2 Normal Range and Reporting Units The normal range for total calcium in serum or plasma is 9.2-1 1 .0 mg/dL in children aged 4 to 20, and 8.8-10.2 mg/dL in adults, with a mean of 9.4 mg/dL. The reference interval for ionized calcium in adults is about 4.7-5.3 mg/dL. Calcium clinical results are normally reported in mg/dL. To convert from mmol/L to mg/dL multiply by 4.00780. To convert from mg/dL to mmol/L multiply by 0.24951

2.3 Routine Clinical Measurement Methods

Blood and urine are the most commonly used media for assessing patient health status. For the measurement of total calcium, flame atomic absorption

2-1
Section 2 - Calcium

spectrometry (FAAS), using the 422.7 nm resonance line, has been the accepted reference method, however, in the routine clinical laboratory, colorimetnc methods adapted for use on automated analyzer systems are more commonly found. One of these involves reaction with o-cresolphthalein complexone in a buffered solution, which produces an intense red-purple

color at 570 nm. The method exhibits linearity up to about 20 mg/dL. A more

recent method utilizes the reaction with arsenazo III, which purportedly provides better specificity, sensitivity, stability and freedom from

interferences than the o-cresolphthalein method. The calcium complex

produces a blue-purple color at around pH 8, which is measured at 650 nm. The method is also linear up to approximately 20 mg/dL, which reduces the need for excessive sample dilutions.

2.4 Available Standard Reference Materials Two blood serum SRMs are available with certified values for calcium, which

are SRM 909b (Human Serum), a two-level lyophilized material, and SRM

956b (Electrolytes in Frozen Human Serum), which is a three-level frozen

material intended for standardizing and calibrating ion selective electrode

analyzers. There i& also a reference value for calcium in SRM 2670a (Toxic Elements in Freeze-Dried Urine), a two level, freeze-dried material. SRM

915b (Calcium Carbonate, Clinical Standard) is sold as a clinical assay

standard which is intended for the purpose of instrument calibration and

standardization. Certificate information for these SRMs is summarized in Table 2-1.

2-2
Section 2 - Calcium

Table 2-1. Clinical Standard Reference Materials for Calcium

SRM Description Value

(mg/dL)

Expanded

Uncertainty

(mg/dL) 909b Level 1 Human Serum 8.890 0.063 909b Level 2 Human Serum 14.16 0.11 956b Level 1 Electrolytes in Frozen Human Serum 11.82 0.08 956b Level 2 Electrolytes in Frozen Human Serum 9.844 0.059 956b Level 3 Electrolytes in Frozen Human Serum 7 911 0.053 2670a Level 1 Toxic Elements in Urine (Freeze-Dried) (2 9) (0.2) 2670a Level 2 Toxic Elements in Urine (Freeze-Dried) (3.0) (0.2) SRM Clinical Standard

Purity (% mass

fraction)

Expanded

Uncertainty

(%) 915b Calcium Carbonate (Clinical Standard) 99.907 0.021

(

) Values in Parentheses are Reference Values

2-3

CA-1 Total Calcium by ICP-MS

Version Number 1.0 Approved S E Long

Effective Date Sep 2006

CA-1

TOTAL CALCIUM BY ISOTOPE DILUTION

INDUCTIVELY COUPLED PLASMA - MASS

SPECTROMETRY

1. Applicability The method is applicable to the determination of total calcium at physiological levels in blood plasma, serum (frozen and lyophilized) and

urine.

2. Principle of Measurement

The method is based on isotope dilution mass spectrometry using a quadrupole inductively coupled plasma - mass spectrometer (ICP-MS)

operated in "cool plasma" (shielded) mode [1]. A stable isotopic spike of ''^Ca

is added to the sample, equilibrated by oxidation with nitric acid in a microwave digestion system, and the ^°Caf^^Ca isotope ratio measured using low power plasma conditions. The method is extremely accurate and

selective for calcium.

3. Measurement Strategy and Experimental Design

The determination of calcium in biological fluids by isotope dilution ICP-MS is not simple because of the presence of potential spectral interferences from

, ^'Na'^O'H and ^'Mg'^0 which interfere with ^°Ca, and ^°Ar^H2 and

^^Mg^^O, which interfere with '*^Ca. The use of "cool" or shielded plasma

conditions effectively attenuates the Ar-based spectral background

interferences. The other interferences are minor, except for the ^^Na^^O^H

interference which is significant because of the relatively high level of sodium

in biological fluids. For this reason, a chemical separation of calcium and sodium is necessary. For convenience, potassium (which is similarly

afflicted) and calcium can be separated, re-combined and measured

together. The isobaric interference from '*°K on ''°Ca is negligible for the potassium levels normally found in biological fluids. Treatment of the sample

with nitric acid in a microwave digestion system is necessary to oxidize

[CA-1] Page 1 of 13
CA-1 Total Calcium by ICP-MS

protein-bound calcium. Normally, ''^Ca is used as the spike, which is available in approximately 95 % isotopic purity.

The amount of spike added to the sample prior to digestion needs to be

optimized. The error propagation has been calculated and plotted in Figure

3-1 for a typical measurement system employing a '^^Ca spike of 95 % purity. The calculated error magnification factor (EM) is at a minimum (EM = 1 .04)

for a ''°Ca/^^Ca ratio of 2.8. However, although this is the ideal ratio, it is considered better to overspike the system to yield an isotope ratio closer to

unity. Under these conditions, the effects of detector dead-time and pulse counting statistics, which are important aspects of ICP-MS measurement

systems, are minimized. As is evident from Figure 3-1 , at a ratio of approximately unity, the EM factor is about 1.06, which is acceptable. An

additional approach to measurement is to closely match the ratios of the spike calibration mixtures to the sample measurement mixtures, and run them in the same temporal measurement space. This requires a fairly accurate knowledge of the concentration of calcium in the unknown sample

prior to spiking. FIGURE 3-1

[CA-1] Page 2 of 13
CA-1 Total Calcium by ICP-MS

4. Safety

Clinical fluids are biohazardous materials which may contain pathogenic

substances. The usual safety precautions should be taken to prevent exposure during handling, including the use of gloves at all times. Although most materials will have been screened for such substances, a clinical monitoring regimen, including screening for HIV 1 and 2 and vaccination against hepatitis B is recommended for personnel handling biological specimens on an extended basis.

5. Required Reagents

5.1 '^^Ca Isotopic Spike A spike having an isotopic purity > 95 % can be obtained through Oak

Ridge National Laboratory (ORNL) or other stable-isotope supplier in a

form such as ''^CaCOa. A stock solution can be prepared by dissolving a

small amount of the '^^CaCOa in 2 % (volume fraction) high-purity nitric acid to yield a concentration of about 50 pg/g (1.2 pmol/g) and storing in a cleaned Teflon® fluoropolymer bottle. Tracking the tare weight of the

bottle is useful for long-term storage and monitoring the concentration of the spike on a multiple-use basis.

5.2 SRM 915b, Calcium Carbonate, Clinical Standard The material should be dried according to the directions on the certificate

in a laboratory oven at 200 °C - 210 °C for 4 h. The certified purity of the material is 99.907 % (mass fraction) with an expanded uncertainty of 0.021 %. The relative mass fraction of calcium

in SRM 915b adjusted for the purity is 0.400104.

Alternatively, Calcium Standard Solution (SRM 3109a), which has a

concentration of 10.025 mg/g ± 0.017 mg/g (0.2 % relative) can be used.

5.3 High-Purity Nitric Acid

High-purity acid is essential to minimize the extent of calcium

contamination from reagent sources. High-purity acid containing less than 100 pg/g calcium may be obtained from a number of commercial

suppliers.

5.4 High-Purity Hydrochloric Acid

For chemical separation of calcium from sample matrix. High-purity acid

is essential to minimize the extent of calcium contamination from reagent

sources. High-purity acid containing less than 100 pg/g calcium may be

obtained from a number of commercial suppliers.

[CA-1] Page 3 of 13
CA-1 Total Calcium by ICP-MS

5.5 High-Purity Water

De-ionized or quartz-distilled water, which has been tested and is low in calcium contamination. High-purity water is used for sample dilution. At a minimum, the water should meet or exceed the specifications of CAP/NCCLS Type I water [2].

5.6 Ion-Exchange Resin AG 50-X8, 100-200 mesh (75-150 pm) cation-exchange resin, or equivalent for chemical separation of calcium from the sample mathx. The resin should be bulk cleaned, packed into columns and batch cleaned twice by alternating between 25 mL, 5 mol/L hydrochloric acid and sufficient high-purity de-ionized water to achieve neutral pH.

6. Equipment

6.1 ICP-MS System

The method utilizes a standard ICP-MS system employing a quadrupole mass spectrometer and an automated shielded torch system operating at low RF forward power ("cool-plasma") for reducing argon-based spectral

interferences. The instrument should be capable of operating in the pulse counting detection mode, should be fitted with a low dead-volume,

cooled spray chamber and a high-efficiency pneumatic nebulizer system,

if available.

6.2 Microwave Digestion System A commercial system should be used which is capable of reaching at least 70 bar (7 MPa) vessel pressure and a maximum vessel temperature of at least 200 °C using either PTFE or quartz reaction vessels. The system should be fitted with industry-standard safety

interlocks. A continuous (un-pulsed) variable power delivery system is advantageous in maintaining optimum reaction control.

6.3 Teflon® Fluoropolymer Beakers

Clean 50 mL Teflon® fluoropolymer beakers for solution evaporation

prior to column separations.

6.4 Ion-Exchange Columns

Clean plastic or glass ion-exchange columns containing a cation- exchange resin bed (see Reagents) of approximate dimensions 10 cm

long X 0.7 cm wide. This bed should contain sufficient capacity and

column resolution for most clinical samples.

[CA-1] Page 4 of 13
CA-1 Total Calcium by ICP-MS

6.5 Sample Bottles Clean 30 mL polyethylene (Nalgene) sample bottles (or equivalent

material) for sample storage and analysis.

Note on Sample Bottles: Polyethylene or Teflon® fluoropolymer sample bottles formulated for low trace-metal content should be used. These contain very low levels of electrolyte contaminants. Containers such as polypropylene centrifuge tubes should not be used for this purpose, as they frequently contain appreciable levels of contaminants. Clean the sample bottles prior to use by soaking in dilute nitric acid for a minimum of 24 h.

6.6 MIcro-Pipette Automatic or manual micro-pipette for dispensing sample, spike and

standard aliquots. A capped plastic syringe fitted with a Teflon®

fluoropolymer uptake tube may also be used for this purpose, depending

on the weighing method employed.

6.7 Analytical Balance

Five-place analytical balance for weighing sample, spike and standard

aliquots. The balance should be calibrated and verified to be functioning

correctly prior to weighing measurements.

7. Sample Handling, Preparation and Quality Control

7.1 Analytical Sample

The analytical sample should be equilibrated to room temperature prior

to measurement. Follow supplier instructions for reconstitution of

lyophilized material or equilibration from freezer temperature. All sample

preparation should be carried out in a Class 10 clean environment with the use of protective clothing and gloves.

7.2 Aliquoting and Acidification Approximately 0.7 g of the liquid sample should be weighed accurately, using a five-place analytical balance, into a cleaned microwave digestion vessel (manufactured from quartz or Teflon® fluoroploymer) and spiked

with an aliquot of ''^Ca stock (approximately 1.3 g for normal blood serum) to yield a ""Ca/^^Ca ratio of approximately unity. Six grams of

high-purity nitric acid should be added to each vessel and the vessels

then sealed using the appropriate procedure specified by the manufacturer.

[CA-1]Page5of 13
CA-1 Total Calcium by ICP-MS

7.3 Digestion The prepared solutions should be placed Into a high-pressure microwave

digestion system and digested using the manufacturers recommended

conditions, or user-developed digestion protocol. A vessel pressure approaching 70 bar (7 MPa) for at least 30 min will usually ensure

complete oxidation of the sample, and conversion of all carbon in the sample to carbon dioxide.

7.4 Evaporation and Chemical Separations The digested samples should be evaporated to near dryness using the microwave digestion system (if this is a feature), or alternatively by

heating on a hot-plate in 50 mL Teflon® fluoropolymer beakers. Redissolve in a small quantity of dilute 2 % (volume fraction) nitric acid. Transfer to cation-exchange columns. Elute the sodium using approximately 40 mL of 0.3 mol/L hydrochloric acid. Elute the potassium

with approximately 20 mL of 0.5 mol/L hydrochloric acid, elute the magnesium with approximately 20 mL of 1 mol/L hydrochloric acid and

finally elute the calcium fraction with approximately 20 mL of 1.75 mol/L

hydrochloric acid. Evaporate the calcium fraction to dryness and re- dissolve in 2 % (volume fraction) nitric acid to a concentration suitable for ICP-MS measurement. Store in clean 30 mL polyethylene bottles.

7.5 Stability The prepared solutions are stable for several days. However,

determinations should be carried out as soon as possible to avoid the hsk of contamination, losses or stability issues.

7.6 Spike Calibration Mixtures The concentration of the "^Ca spike stock solution must be measured by

preparing at least four independent spike calibration mixtures. Prepare two different stock calibration solutions of natural calcium from SRM

915b or SRM 3109a. Prepare two spike calibration mixtures from each of the natural calcium calibration solutions by aliquoting known amounts of the calibration stock and "^^Ca spike into sample bottles and diluting with 2 % (volume fraction) nitric acid to a concentration suitable for measurement by ICP-MS. The aliquots should be adjusted to yield ""Ca/^^Ca isotope ratios as similar as possible to the analytical samples. A five-place analytical balance should be used for all weighing

operations.

[CA-1] Page 6 of 13
CA-1 Total Calcium by ICP-MS

7.7 Procedure Blanks

Prepare a minimum of three procedure blanks by adding approximately 100 ng of the ""^Ca spike to microwave digestion vessels and carrying them through the entire preparation and analysis procedure.

7.8 Composition Sample

Prepare a composition sample by aliquoting approximately 0.7 g of sample into a microwave digestion vessel and carrying it through the same preparation procedure as the samples. The composition sample is not spiked with '*^Ca and is used to assess the presence or absence of spectral interferences in the ICP-MS measurement process.

7.9 Controls To assess the accuracy of the measurements, prepare at least two

different SRM control materials having certified values for calcium,

preferably of the same matrix type as the samples, and carry the controls through the same sample preparation and measurement procedures as

the samples.

8. Analytical Measurement

8.1 Instrument Preparation

Prior to analytical measurements, the ICP-MS system should be

equilibrated for 30 min. The sample introduction system should be

rigorously cleaned before use, including the sampler and skimmer cones,

peristaltic pump tubing, plasma torch and spray chamber. These

procedures are designed to mitigate the background from calcium as much as possible. It is recommended that new peristaltic pump tubing be

installed prior to measurements. A dilute nitric acid solution (2 % volume

fraction) should be pumped through the tubing for a while to remove any

leachable calcium from the tubing.

8.2 Instrument Optimization The instrument should be optimized for maximum ion transmission and minimum background at m/z 40 (maximum signal to background ratio) using a standard solution of calcium and adjusting ion lens voltages, plasma gas flows, torch position (relative to the sample cones) and rf forward power.

[CA-1] Page 7 of 13
CA-1 Total Calcium by I CP-MS

8.3 Run Sequence

Software acquisition menus should be prepared for measuring the '*°Ca and ''^Ca isotopes. The run order for the samples should be established according to the following protocol:

Analytical procedure blanks should be run at the beginning of the sequence, followed by the composition sample and then the analytical samples (including control materials) in randomized order. Instrument mass discrimination measurement standards (see note) should be run

after every three to four samples depending on the stability of the

instrument.

Note on Mass Discrimination Measurement: The measured

'*°Ca/^^Ca ratios should be corrected for the ICP-MS mass

discrimination. This can be achieved by measurement of a calibration standard (SRM 915b) consisting of natural calcium. However, the

natural ratio of '*°Ca/^^Ca is approximately 150, which is very different from the measured sample ratios. A better way to assess

discrimination and measurement run discrimination drift is to carefully calibrate a solution matched more closely to the samples. A spike

calibration solution can conveniently be used for this purpose.

8.4 Isotope Ratio Acquisition The isotope ratio ''°Ca/^^Ca should be acquired in peak jumping mode. A

typical acquisition scheme would be five to seven sets of ratios with a

total acquisition time of 60 s per set, using a dwell-time of 10 ms per

isotope. The isotope ratio data should be dead-time corrected and downloaded to a spreadsheet program such as Microsoft Excel. Inspect the ratios and remove any considered to be statistical outliers.

9. Data Reduction

9.1 Mass Discrimination Corrections

Convert the experimentally measured '*°Ca/^^Ca isotope ratios to absolute ratios using the following equation:

40,'Ca

40,'Ca

42,^CaJtrue 42,=Ca 40,'Ca 'JcORR 42,Ca MEAS

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CA-1 Total Calcium by ICP-MS

where, is the discrimination corrected sample ratio

is the true ratio for the isotopic standard

'Ca is the measured ratio for the isotopic standard

The measured ratios for the isotopic standard can be applied to sample

discrimination corrections in a number of ways, which will depend on the

discrimination drift as a function of time: (1) the ratio can be calculated by averaging the two standard measurements bracketing the samples of

interest, (2) a more sophisticated approach is to mathematically model

the drift in the standard ratio as a function of time and interpolate the

ratio for each sample. This may require more frequent measurement of the isotopic standard to define the drift function adequately.

.2 Spike Calibration

Calculate the concentration of the '^^Ca spike in each spike calibration mixture using the following equation:

^SPK ~ Mstd(BR - A)

K(A3-B3R)

where, Cspk is the concentration of calcium in the spike in pg/g

IVlsTD is the mass of calcium added to the spike mix (pg) B is the natural abundance of the spike isotope R is the corrected '*°Ca/'^Ca ratio in the spike mix A is the natural abundance of the reference isotope

IVIs is the mass of '^^Ca spike aliquot added to the mix (g) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike

Calculate the mean and standard deviation of the spike calibration mixtures. Blank corrections to the spike calibration are not normally necessary because they are simple mixtures prepared in dilute acid with minimal sample processing.

9.3 Calculation of Calcium Sample Concentrations

Calculate the calcium concentration (pg/g) in the sample using the

following equation:

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CA-1 Total Calcium by ICP-MS

c = rM3K(A,-B3R) 1 1 X —

(BR-A) ^ W

where, C is the concentration of calcium in the sannple (pg/g) Ms is the absolute mass of '^^Ca spike added (pg) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike R is the corrected ""Ca/'^Ca ratio B is the natural abundance of the spike isotope A is the natural abundance of the reference isotope Sb is the absolute mean measured blank (pg) W is the mass of sample aliquot taken (g) An alternative approach, if desired, is to calculate the spike and sample

concentrations in mol/g. In this case the equations are slightly modified and the relative atomic masses of natural calcium and the spike are not required for the calculations.

10. Uncertainty Analysis

The expanded uncertainty for the set of samples should be calculated according to ISO guidelines [3] by combining both Type A and Type B

uncertainties. Typical Type A uncertainty components for this measurement

might be the variability of the measured concentrations of calcium in the samples, the measurement of the spike concentration and the variability of the correction for the blank. Typical Type B uncertainty components might be

the uncertainty of the assay of the primary calibrant, weighing measurements, conversion correction for the density of the sample and

instrumental parameters such as detector dead-time correction, instrument mass discrimination correction and instrument background correction.

For a 95 % confidence level, combine the Type A and Type B components

and calculate the total expanded uncertainty using the following equation: U= k [Si^/dfi + ...+ Sn^/dfn + Bi^ +...+ Bn' ]

where, U is the expanded uncertainty k is the coverage factor s is the observed standard deviation of (n) Type A components

df is the degrees of freedom associated with each component

[CA-1] Page 10 of 13
CA-^ Total Calcium by ICP-MS

B is the Type B component (n connponents) with infinite degrees

of freedom.

Report the expanded uncertainty in the same units as the sample

concentrations.

11. Data Reporting

Convert data from a mass/mass basis to a mass/volume basis using the measured density of the serum. For clinical use, report calcium data in units of mg/dL. To convert data from Mg/mL to mg/dL divide by 10. To

convert from mg/dL to mmol/L multiply by 0.24951

.

12. Performance Statement

12.1 Measurement Repeatability The isotope ratio measurement repeatability is usually limited by

detector counting statistics. Typical measurement repeatability on a quadrupole ICP-MS system for a '*°Ca/^^Ca ratio approaching unity s

0.1-0.2 %.

12.2 Detection Limit The instrument detection limit is influenced by the instrument background. A typical instrument detection limit for a well-optimized quadrupole ICP-MS instrument is 5 pg/mL. For a 0.7 g sample aliquot

this equates to a matrix detection limit of approximately 0.04 ng/mL, which is far below the calcium concentrations typically found in clinical

fluids.

12.3 Instrument Background and Procedure Blank The ICP-MS instrument background tends to be very high for calcium owing to the direct isobaric interference of argon species. This can be

very effectively reduced by means of "cool plasma" technology. It should be possible to reduce the background for '*°Ca to around 100

counts per second by judicious choice of operating conditions. A typical procedure blank using this method is in the range 50-100 ng

(1-2 nmol), depending on the purity of the reagents, cleanliness of containers and processing conditions. This is low in relation to the calcium content in clinical fluids. For example, using this method for the determination of calcium in blood serum, the relative blank

correction would be < 0.1 %.

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CA-I Total Calcium by ICP-MS

12.4 Performance Summary A summary of typical method performance parameters expected for use of this method are provided in Table 12-1.

Table 12-1: Method Performance Summary

Parameter Typical Value or Range

Isotope ratio measurement repeatability 0.1-0.2 %

Applicable concentration range

All ranges found in clinical specimens

Instrument background / sample ratio = 0.01 %

Procedure blank = 50-100 ng Sample throughput (measurement) 10 samples per hour

13. Reference Citations

1. Murphy K.E., Long S.E., Rearick M.S. and Ertas O.S., "The Accurate

Determination of Potassium and Calcium using Isotope Dilution Inductively Coupled "Cold" Plasma Mass Sectrometry."

J. Anal. At. Spectrom. 17, pp. 469-477 (2002).

2. Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline - Third Edition, NCCLS Document C3-A3, NCCLS,

Wayne, Pennsylvania (1997).

3. Guide to the Expression of Uncertainty in Measurement, ISBN 92-67- 10188-9, 1st Ed. ISO, Switzerland, 1993.

[CA-1]Page12of 13
CA-1 Total Calcium by ICP-MS

CA-1 Revision History

Revision Date Author Record of Changes and Comments

1.0 9/2006 SELong Initial draft method

[CA-1] Page 13 of 13

Section 3 - Chloride

Chloride

3.1 Clinical Significance

Chloride is the most abundant anion in extra-cellular fluid. The principle function is to provide charge neutrality. Therefore, the concentration of chlohde is closely related to that of sodium as the major cation in extracellular fluid. Homeostatic maintenance of chloride is achieved through

dietary intake and renal excretion. An elevation in the chloride concentration

in serum (hyperchloremia) over normal levels may be caused by excessive

intake, dehydration, renal failure and renal tubular acidosis. Correspondingly, a reduction in chloride activity over normal levels (hypochloremia) may be caused by excessive loss through sweating or vomiting or other conditions, such as metabolic alkalosis and Addison's disease.

3.2 Normal Range and Reporting Units The normal range for chloride in serum or plasma is about 96-106 mmol/L.

Clinical data for chloride are normally reported in mmol/L. The unit mEq/L Is also sometimes used. To convert from mmol/L to mg/dL multiply by 3.54527. To convert from mg/dL to mmol/L multiply by 0.28207.

3.3 Routine Clinical Measurement Methods

Chloride is commonly measured in serum, plasma, urine and sweat. Several methods are used for the determination of chloride. Electrochemical methods

are commonly employed which are based predominantly on

amperometry/coulometry and ion-selective electrodes. Another common

method is mercurimetric titration in which the chloride reacts with mercuric

nitrate to form insoluble mercuric chlohde. Diphenylcarbazone, which complexes with free Hg^"" to form a blue/violet color, is used as an indicator

of the end-point. A spectrophotometric method, based on complexometry,

utilizes a reaction of the chloride with mercuric thiocyanate to quantitatively release free thiocyanate. The free thiocyanate is reacted with Fe^"" to form a

red complex at 480 nm.

3-1
Section 3 - Chloride

3.4 Available Standard Reference Materials Two primary blood serum SRMs are available with certified values for chloride which are SRM 909b (Human Serum), a two-level lyophilized

material, and SRM 956b {Electrolytes in Frozen Human Serum), which is a

three-level frozen material intended for standardizing and calibrating ion selective electrode analyzers. Two clinical assay standards may be used for chloride: SRM 919a {Sodium Chloride, Clinical Standard) and SRM 918a

(Potassium Chloride, Clinical Standard) which are sold as clinical primary standards intended for instrument calibration and standardization. Certificate information for these SRMs is summarized in Table 3-1

.

Table 3-1. Clinical Standard Reference Materials for Chloride

SRM Description Value

(mmol/L) Expanded

Uncertainty (mmol/L) 909b Level 1 Human Serum 89.11 0.57 909b Level 2 Human Serum 119.43 0.85 956b Level 1 Electrolytes in Frozen Human Serum 99.01 0.66 956b Level 2 Electrolytes in Frozen Human Serum 111.88 0.82 956b Level 3 Electrolytes in Frozen Human Serum 126.85 0.81 SRM Clinical Standard

Purity (% mass

fraction)

Expanded

Uncertainty (%) 919a Sodium Chloride (Clinical Standard) 99.89 0.03 918a

Potassium Chloride (Clinical Standard) 99.9817 0.0084

(

) Values in Parentheses are Reference Values

3-2
CL-1 Chloride by Isotope Dilution TIMS

Version Number 1.0 Approved S E Long

Effective Date Sep 2006

CL-1

CHLORIDE BY ISOTOPE DILUTION

THERMAL IONIZATION MASS SPECTROMETRY

1. Applicability The method is applicable to the determination of chloride at physiological and

therapeutic levels in blood plasma, serum (frozen and lyophilized), sweat and

urine.

2. Principle of Measurement

The method is based on isotope dilution mass spectrometry using a solid source magnetic sector thermal ionization mass spectrometer (TIMS). A

stable isotopic spike of ^'^Cl is added to the sample and the chloride

precipitated as silver chloride, which is deposited onto a rhenium filament

after re-dissolving in ammonia. The ^^CI/^'^CI isotope ratio is measured in negative ion mode. The method is extremely accurate and very selective for chloride.

3. Measurement Strategy and Experimental Design

Thermal ionization mass spectrometry (TIMS) has been used at NIST since

the late 1960s to determine a variety of analytes, including chloride, in clinical reference materials. Eventually TIMS will be superceded by inductively coupled plasma - mass spectrometry (ICP-MS) methods when suitable methodology is established. However, TIMS is still the principal analytical method for certification of chloride in NIST SRMs at the present time. The

use of isotope dilution analysis for the determination of chlonde is straightforward, except that, because of the relatively high first ionization

potential of chlorine (13.01 V), the measurement is made using negative ion mode. The attainable analytical measurement repeatability is, however,

slightly poorer than for positive ion measurements. The principal component

of uncertainty in the method is the process blank, which is relatively high. The blank comes principally from reagent sources, but stringent precautions

also need to be taken to reduce external contamination from the laboratory

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CL-1 Chloride by Isotope Dilution TIMS

environment. In order to maintain a higin sample to blanl< ratio, the amount of chloride in the sample aliquot needs to be high. For clinical samples such as serum, a minimum of 0.3 g is recommended. As is typical with TIMS

methods, the chloride needs to be chemically separated from the rest of the sample matrix, which is achieved by precipitating any protein from the

material, and then adding silver chloride to precipitate the chloride as silver chloride. The silver chloride is re-dissolved in ammonium hydroxide and

deposited onto rhenium filaments. The filaments are then mounted into the mass spectrometer as a triple filament assembly.

The amount of spike added to the sample prior to separation, needs to be

optimized. The error propagation has been calculated and plotted in Figure

3-1 for a typical measurement system employing a ^''Cl spike of 98 % purity. The calculated error magnification factor (EM) is at a minimum (EM = 1.18)

for a ^^CI/^''CI ratio of approximately 0.25. However, ratios in the range 0.06

to about 1 .2 would also yield valid measurements. FIGURE 3-1

Isotope Dilution Error Propagation Plot ^*CI / ^^Cl

2.40 ,

»_ 2.20

1.00 -I ,

, . I .

, I ,

,

^ n '

: ^

0.01 0.1 1 10 "ci/"ci

The TIMS method described here is written specifically for use with the NIST

12 in series 90° sector solid source mass spectrometers. These mass

spectrometers permit only single sample loading and measurement, and

therefore limit the sample throughput to about 5 to 6 samples per day. Although this is relatively slow, this is not too restrictive for small sample

[CL-1] Page 2 of 13
CL-1 Chloride by Isotope Dilution TIMS

loads such as those associated with SRM certification measurements or

international comparisons.

4. Safety

Clinical fluids are biohazardous materials which may contain pathogenic

substances. The usual safety precautions should be taken to prevent exposure during handling, including the use of gloves at all times. Although most materials will have been screened for such substances, a clinical monitoring regimen, including screening for HIV 1 and 2 and vaccination against hepatitis B is recommended for personnel handling biological specimens on an extended basis.

5. Required Reagents

5.1 ^^Cl Isotopic Spike A spike having an isotopic purity > 98 % can be obtained through Oak

Ridge National Laboratory (ORNL) or other stable-isotope supplier in a form -uch as Na^^CI. A stock solution can be prepared by dissolving a

small amount of the spike in high-purity de-ionized water to yield a

concentration of about 800 pg/g (22 pmol/g) and storing in a cleaned PTFE bottle. Tracking the tare weight of the bottle is useful for long-term storage and monitoring the concentration of the spike on a multiple-use

basis.

5.2 SRM 919a, Sodium Chloride, Clinical Standard The material should be dried according to the directions on the SRM

certificate, which specify 3 h at 1 10 °C in a convection oven. Allow to

cool. Store in a desiccator at room temperature when not in use. The

purity of the material is 99.89 %. The mass fraction of chloride in the sodium chloride, adjusted for the purity, is 0.60596.

5.3 SRM 918a, Potassium Chloride, Clinical Standard The matenal should be dried according to the directions on the SRM

certificate, which specify 2 h at 110 °C in a convection oven. Allow to

cool. Store in a desiccator at room temperature when not in use. The

purity of the material is 99.9817 %. The mass fraction of chloride in the potassium chloride, adjusted for the purity, is 0.47546.

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CL-1 Chloride by Isotope Dilution TIMS

5.4 SRM 975a, Isotopic Standard for Chlorine

Isotopic standard having a natural ratio ^^CI/^^CI = 3.1279 ± 0.0047

(Relative Atomic Mass 35.45265) for assessing mass fractionation during sample measurements.

5.5 Ammonium Molybdate

Obtain from a commercial supplier as ammonium heptamolybdate

tetrahydrate. High-purity reagent is essential to minimize the extent of chloride contamination. Prepare a fresh solution containing 50 g/L In high-purity de-ionized water.

5.6 Silver Nitrate Obtain from a commercial supplier. High-purity reagent is essential to minimize chloride contamination. Prepare a fresh solution containing 20

g/L in high-purity de-ionized water. When not in use, store in a dark

place.

5.7 Ammonium Hydroxide

High-purity ammonium hydroxide is essential to minimize chloride contamination. It may be obtained from a number of commercial

suppliers. Prepare a 500 mUL (50 % volume fraction) solution in deionized water and store in a clean bottle.

5.8 Nitric Acid

High-purity acid is essential to minimize the extent of chloride contamination from reagent sources. High-purity acid may be obtained from a number of commercial suppliers.

5.9 High-Purity Water

De-ionized or quartz-distilled water, which has been tested and is low in chloride contamination, is used for sample dilution. The water should meet or exceed the specifications of CAP/NCCLS Type I water [1].

6. Equipment

6.1 Thermal Ionization Mass Spectrometer The method utilizes a NIST designed 12 in, 90° sector field solid source mass spectrometer, operating in the negative ion mode. A modified open

ion source designed specifically for use with the negative ion mode is beneficial in producing a stable ion beam. The instrument is equipped

with a manual triple-filament control, optical pyrometer for setting

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CL-1 Chloride by Isotope Dilution TIMS

filament temperature and a computer control system for measuring and

recording ^^CI/^^CI isotope ratios.

6.2 Filament Degasser Vacuum chamber system fitted with a current source and readout, ion collector and pressure readout for degassing filaments.

6.3 Centrifuge A general purpose laboratory centrifuge capable of reaching at least 1000 g relative centrifugal force (RCF). The system should be fitted with

industry standard safety interlocks and a rotor which is capable of holding several 1 5 mL capacity centrifuge tubes.

6.4 Centrifuge Tubes

Polystyrene (or similar material with low chloride contamination)

centrifuge tubes of 15 mL capacity for use in centrifuge system. Tubes

must be cleaned prior to use by soaking in dilute nitric acid (2 % volume

fraction), and rinsing thoroughly with de-ionized water.

6.5 l\/licro-Pipette Automatic or manual micro-pipette for dispensing sample, spike and

standard aliquots. A capped plastic syringe fitted with a Teflon®

fluoropolymer uptake tube may also be used for this purpose.

6.6 Analytical Balance

Five-place analytical balance for weighing sample, spike and standard

aliquots. The balance should be calibrated and verified to be functioning

correctly prior to weighing measurements.

7. Sample Handling, Preparation and Quality Control

7.1 Analytical Sample Preparation The analytical sample should be equilibrated to room temperature prior to measurement. Follow supplier instructions for reconstitution of

lyophilized material or equilibration from freezer temperature. All sample

preparation should be carried out in a Class 10 clean environment with mandatory use of protective clothing and gloves.

7.2 Sample Aliquoting and Spiking The amount of analytical sample to be taken depends on the level of chloride present. In order to maintain a high sample to blank ratio, the sample aliquot needs to contain at least 1000 pg of chloride. For

[CL-1] Page 5 of 13
CL-1 Chloride by Isotope Dilution TIMS

example, in a serum material, which typically contains about 100 mmol/L

chloride, a sample size of 0.3 g should suffice. Weigh the sample aliquot accurately by difference into a clean 1 5 mL centrifuge tube by means of a pipette or syringe using a five-place analytical balance. Add an

accurately known amount of ^''Cl spike (approximately 1 g of the spike

solution prepared in section 5.1). Dilute mixture to 6 mL with dilute nitric acid (1% volume fraction).

7.3 Protein Separation

If protein material is present in the sample, it must be removed by

precipitation. Add ammonium molybdate to the cooled sample mixture (1 mL of a 50 g/L solution) and mix. Separate the precipitate by

centrifugation at approximately 2000 rpm (500 RCF) for 10 min and remove the supernatant containing the chloride by decantation. Transfer

to a clean 15 mL centrifuge tube.

7.4 Cliloride Separation Add silver nitrate to the sample (1 mL of a freshly prepared 20 g/L

solution) and allow to stand in the dark for 10 min. Centrifuge at approximately 2000 rpm (500 RCF) for 10 min. Discard the supernatant by decantation. Wash the precipitate with a small amount of dilute nitric acid (1 % volume fraction) and re-dissolve the precipitate in ammonium

hydroxide solution (1 mL of a 500 mL/L solution). Re-precipitate the

chloride by drop-wise addition of nitric acid (10 % volume fraction) and

dilute back to 6 mL with high-purity de-ionized water. Centrifuge again and discard the supernatant by decantation. Wash the precipitate with

dilute nitric acid (1 % volume fraction), remove, and dissolve the

precipitate in ammonium hydroxide (0.75 mL of a 500 mL/L solution). Store the prepared samples at freezer temperature (-20 °C) in the dark.

7.5 Stability The prepared solutions are stable for at least two weeks. However,

determinations should be carried out as soon as possible to avoid the

risk of contamination, losses or stability issues. Silver chloride solutions are unstable, particularly in the presence of light.

7.6 Spilte Calibration Mixtures The concentration of the ^'^Cl spike stock solution must be accurately measured by preparing at least four independent spike calibration mixtures. Prepare one stock calibration solution of natural chloride from SRM 919a (Sodium Chloride, Clinical Standard). Prepare two spike

calibration mixtures from the natural stock by aliquoting known amounts

of the calibration stock and ^'^Cl spike into sample bottles and process as

[CL-1] Page 6 of 13
CL-1 Chloride by Isotope Dilution TIMS

for the samples. Prepare two more mixtures using SRM 918a (Potassium

Chloride, Clinical Standard). A five-place analytical balance should be used for all weighing operations.

7.7 Procedure Blanks

Prepare a minimum of three procedure blanks by adding approximately

1 0 pg or less of the ^'^Cl spike to a clean 1 5 mL centrifuge tube and

processing them through the entire sample preparation procedure.

7.8 Composition Sample

Prepare a composition sample by aliquoting approximately 0.3 g of sample into a 15 mL centrifuge tube and carrying it through the same

preparation procedure as the samples. The composition sample is not spiked with ^''Cl and is used to assess the isotopic composition of the

material being measured and to demonstrate the validity of isotopic measurements by the mass spectrometer.

7.9 Controls To assess the accuracy of the measurements, prepare at least two

separate SRM control materials having certified values for chloride, preferably of the same matrix type as the samples, and carry the controls through the same sample preparation and measurement procedures as

the samples. An additional control material, consisting of an accurately known amount of standard, should be prepared and carried through the whole measurement and calculation process. The gravimetric recovery

of the standard facilitates assessment of the accuracy of the measurement process and veracity of the calculations.

8. Analytical Measurement

8.1 Instrument Preparation

If not already configured, switch the TIMS instrument from positive ion to negative ion mode. Prior to analytical measurements, warm up the

electronic components of the instrument for a minimum period of 30 min.

8.2 Filament Preparation

For each sample prepare a set of rhenium filaments, consisting of two

side filaments and one center filament. Outgas the filaments by placing them in a vacuum chamber degasser and passing a current of 4 A

through them for a period of 30 min. Allow to cool to ambient

temperature, remove them from the vacuum chamber and transfer to a

closed container which will protect them from dust and damage.

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CL-1 Chloride by Isotope Dilution TIMS

8.3 Filament Loading Remove the sample from the freezer and transfer to a Class 10 clean

area. Load approximately 10 pg of chloride onto each of two rhenium

side filaments using a suitable micro-pipette. Dry the filaments under an

infra-red heat-lamp for 30 min, after which a thin film of silver chloride should be present on each filament.

8.4 Filament IVIounting and Isotope Ratio Measurement

Assemble the two loaded side filaments in a suitable filament block former along with a center filament. Place in the source-can of the mass

spectrometer, connect the electrical leads and evacuate the chamber.

After a suitable vacuum has been attained, add liquid nitrogen to the source-can cold-finger and open the beam valve. Heat the center

filament to 1500 °C, and then slowly ramp the side filaments to produce a

stable ion beam. Optimum signals are usually obtained for a relatively narrow temperature range for the side filaments, corresponding to approximately 0.5-0.6 A filament current. After approximately 30 min

conditioning and background stabilization, acquire several blocks of

^^CI/^''CI isotope ratios. Assess the mass fractionation of the measurements by measuhng 3RM 975a, Isotopic Standard for Chlorine,

at periodic intervals.

8.5 Run Sequence

To reduce the effects of memory carry over, which are of significant concern in the measurement of chlohde by thermal ionization, all samples, standards, blanks and control materials should be divided into separate batches having essentially similar isotope ratios. Memory

effects should be assessed at the end of each measurement run by

turning off the side filaments and acquiring a final block of data with reduced signal. The measured ratios should not differ significantly from

the initial sample ratios.

9. Data Reduction

9.1 Mass Fractionation Corrections

Convert the experimentally measured ^^C\/^^C\ isotope ratios to absolute

ratios using the following equation:

35,CI

35,CI

37,CI TRUE 37,CI 35,CI J CORR 37,CI MEAS

[CL-1] Page 8 Of 13
CL-1 Chloride by Isotope Dilution TIMS

where, is the fractionation corrected sample ratio

is the true ratio for the isotopic standard

is the measured ratio for the isotopic standard

The measured ratios for the isotopic standard can be applied to sample

fractionation corrections in a number of ways, which will depend on the

fractionation drift as a function of time: (1 ) the ratio can be calculated by averaging the two standard measurements bracketing the samples of

interest, (2) a more sophisticated approach is to mathematically model

the fractionation in the standard ratio as a function of time and interpolate the ratio for each sample. This may require more frequent measurement

of the isotopic standard to define the fractionation adequately.

.2 Spike Calibration

Calculate the concentration of the ^'^Cl spike in each spike calibration mixture using the following equation:

M3,^(BR - A)

K(A,-B,R)

where, Cspk is the concentration of chloride in the spike in pg/g

IVIsTD is the mass of chloride added to the spike mix (pg) B is the natural abundance of the spike isotope (37) R is the corrected ^^CI/^'^CI ratio in the spike mix A is the natural abundance of the reference isotope (35) Ms is the mass of ^''Cl spike aliquot added to the mix (g) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike Sb is the absolute mean measured blank (pg)

Calculate the mean and standard deviation of the spike calibration mixtures. Unlike ICP-MS measurements, where the spike calibration is normally made on simple mixtures without extensive sample processing, a spike calibration procedure blank must be accounted for and should be

subtracted from the data for the spike calibration.

[CL-1] Page 9 of 13
CL-1 Chloride by Isotope Dilution TIMS

.3 Calculation of Chloride Sample Concentrations

Calculate the chloride concentration (pg/g) in the sannple using the

following equation:

c = fM^KCA^-B.R) 3

1 1 X —

(BR-A) ^ W

where, C is the concentration of chloride in the sample (|jg/g) Ms is the absolute mass of ^^01 spike added (pg) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike R is the corrected ^^CI/^^CI ratio B is the natural abundance of the spike isotope A is the natural abundance of the reference isotope Sb is the absolute mean measured blank (pg) W is the mass of sample aliquot taken (g) An alternative approach, if desired, is to calculate the spike and sample

concentrations in mol/g. In this casfc the equations are slightly modified.

10. Uncertainty Analysis

The expanded uncertainty for the set of samples should be calculated according to ISO guidelines [2] by combining both Type A and Type B

uncertainties. Typical Type A uncertainty components for this measurement

might be the variability of the measured concentrations of chloride in the samples, the measurement of the spike concentration and the variability of the correction for the blank. Typical Type B uncertainty components might be

the uncertainty of the assay of the primary calibrant, weighing measurements, conversion correction for the density of the sample and

instrumental parameters such as drift in the instrument mass discrimination correction and instrument background correction.

For a 95 % confidence level, combine the Type A and Type B components

and calculate the total expanded uncertainty using the following equation: U= k [Si'/dfi + ...+ Sn'/dfn + Bi' +...+ Bn' ]

where, U is the expanded uncertainty k is the coverage factor s is the observed standard deviation of (n) Type A components

[CL-1] Page 10 of 13
CL-1 Chloride by Isotope Dilution TIMS

df is the degrees of freedom associated with each component

B is the Type B component (n components) with infinite degrees

of freedom.

Report the expanded uncertainty in the same units as the sample

concentrations.

11. Data Reporting

Convert data from a mass/mass basis to a mass/volume basis using the measured density of the serum. For clinical use, report data in units of mmol/L. To convert from mmol/L to mg/dL multiply by 3.54527.

12. Performance Statement

12.1 Measurement Repeatability The isotope ratio measurement repeatability is limited by short term

drift in the ^^Cl and ^''Cl signal amplitudes, which tends to be higher in the negative ion mode of operation. Typical measurement repeatability that can be obtained on a NIST TIMS instrument is around 0.1 %.

12.2 Instrument Background Correction and Procedure Blank The instrument background is fairly low and is not a significant factor in the measurement process under normal conditions. Sample-to-sample

carryover may arise from contamination of the ion source or other components, and needs to be minimized by correct sampling

protocols. A typical procedure blank using this method is approximately 1 pg, depending on the purity of the reagents and cleanliness of the sample

containers. This represents approximately a 0.1 % correction to the sample data, using aliquot sizes described in this method.

12.3 Performance Summary A summary of typical method performance parameters expected for use of this method are provided in Table 12-1.

[CL-1] Page 11 of 13
CL-1 Chloride by Isotope Dilution TIMS

Table 12-1: Method Performance Summary

Parameter Typical Value or Range

Isotope ratio measurement repeatability 0.1 %

Applicable concentration range

All ranges found in clinical specimens

Procedure blank = 1 pg

Procedure blank to sample ratio «0.1%

Sample throughput (measurement) 5-6 samples per day

13. Reference Citations

1 . Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline - Third Edition, NCCLS Document C3-A3, NCCLS,

Wayne, Pennsylvania (1997).

2. Guide to the Expression of Uncertainty in Measurement, ISBN 92-67-

10188-9, 1st Ed. ISO, Switzerland, 1993.

[CL-1] Page 12 of 13
CL-1 Chloride by Isotope Dilution TIMS

CL-1 Revision History

Revision Date Author Record of Changes and Comments

1.0 9/2006 SELong Initial draft method

[CL-1] Page 13 Of 13
II
Section 4 - Lithium 4 Lithium

4.1 Clinical Significance

Although lithium is normally present in blood plasma or serum, clinical measurements are usually made in connection with its administration as a

first-line agent in the treatment of affective disorders, such as manic

depressive psychosis (bipolar disorder). In this respect, lithium is highly

effective, the usual pharmaceutical dosage forms being lithium carbonate or

lithium citrate. Lithium is absorbed from the gastro-intestinal tract within approximately eight hours following administration, however, intracellular transport, which is necessary for therapeutic mechanism, is relatively poor, which necessitates the use of high dosages to obtain therapeutic benefit. There is a narrow margin between these therapeutic dosages and the onset

of toxic effects, which therefore mandates a system of intensive patient monitoring for both physical status and serum lithium concentrations. Serum

lithium measurements are normally made approximately twelve hours after a

previous dose has been taken. The therapeutic interval should lie within the range 0.4-1.4 mmol/L. Higher levels than this are considered to be toxic, which may result in a number of effects which include hand tremor, thirst, nausea, vomiting and slurred speech. Some studies which were made in the

late 1970s following FDA approval of lithium for the treatment of manic

depressive disorder and maintenance therapy, presented some evidence for long-term renal damage from extended use. More recent studies have shown

that this is not the case and that the incidence rate among patients taking

lithium medication is typically low.

4.2 Normal Range and Reporting Units The normal therapeutic range for lithium in serum or plasma is about 0.4-1.4 mmol/L (levels > 1.5 mmol/L are considered toxic). Lithium clinical data are

normally reported in mmol/L.

4.3 Routine Clinical Measurement Methods

Classical reference methods for the determination of lithium in serum or plasma involve atomic absorption spectrometry and atomic emission

spectrometry (flame photometry). These methods require transport of the sample to a specialized laboratory and a relatively slow turnaround time for

4-1
Section 4 - Lithium

the transmission of utility data back to the patient. The use of more rapid point-of-care testing methods are advantageous, given the need for the frequent monitoring of lithium levels to obtain the correct therapeutic index in the patient. Thus ion selective electrodes are now extensively used for the measurement of lithium in serum and plasma. A rapid colorimetric system based on the formation of a lithium porphyrin complex, which absorbs at 505

nm, is also used.

4.4 Available Standard Reference Materials Two blood serum SRMs are available with certified values for lithium, which

are SRM 909b {Human Serum), a two-level lyophilized material, and SRM

956b {Electrolytes in Frozen Human Serum), which is a three-level frozen

material intended for standardizing and calibrating ion selective electrode

analyzers. One clinical assay standard may be used for lithium: SRM 924a

{Lithium Carbonate, Clinical Standard) is sold as a clinical primary standard intended for instrument calibration and standardization. Certificate information for these SRMs is summarized in Table 4-1.

Table 4-1, Clinical Standard Reference Materials for Lithium

SRM Description Value

(mmoi/L)

Expanded

Uncertainty

(mmol/L) 909b Level 1 Human Serum 0.6145 0.0050 909b Level 2 Human Serum 2.600 0.023 956b Level 1 Electrolytes in Frozen Human Serum 1.920 0.027 956b Level 2 Electrolytes in Frozen Human Serum 1.207 0.017 956b Level 3 Electrolytes in Frozen Human Serum 0.486 0.007 SRM Clinical Standard

Purity (% mass

fraction)

Expanded

Uncertainty

(%) 924a

Lithium Carbonate (Clinical Standard) 99.867 0.017

4-2
LI-1 Lithium by Isotope Dilution ICP-MS

Version Number 1.0 Approved S E Long

Effective Date Sep 2006

LI-1 LITHIUM BY ISOTOPE DILUTION

INDUCTIVELY COUPLED PLASMA - MASS

SPECTROMETRY

1. Applicability The method is applicable to the determination of lithium at physiological and

therapeutic levels in blood plasma, serum (frozen and lyophilized) and urine.

2. Principle of Measurement

The method is based on isotope dilution mass spectrometry using a quadrupole inductively coupled plasma - mass spectrometer (ICP-MS). A

stable isotopic spike of ^Li is added to the sample, equilibrated by oxidation

with nitric acid in a microwave digestion system, and the '^Li/^Li isotope ratio measured under optimized conditions. The method is extremely accurate and

very selective for lithium.

3. Measurement Strategy and Experimental Design

The determination of lithium in biological fluids by isotope dilution ICP-MS

has not been widely employed in the past because of the difficulties associated with memory effects, high instrument background and relatively large instrument mass discrimination. The lithium isotopes at masses 6 and 7

are free from spectral interferences but, because they are very light isotopes, the mass discrimination is usually high and unstable on a typical ICP-MS

instrument. For these reasons, the determination of lithium in biological fluids at NIST has usually been carried out by TIMS. In this ICP-MS method, the plasma power is slightly reduced to mitigate secondary ionization effects from lithium on the skimmer cone. The instrument background is then fairly low and washout between samples is very rapid. The voltages on the lens system are adjusted specifically to reduce the instrument discrimination to less than 5 %. This does result in some minor ion transmission loss, but the instrument detection limit for lithium is so low that this is of no consequence. Under these conditions, drift in the mass discrimination is low. Oxidation with

nitric acid in a microwave digestion system is necessary to chemically

[LI-1] Page1 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

equilibrate the ^Li isotopic spike with the lithium in the sample. Chemical

separation of the lithium is not necessary. Large dilution factors are employed which reduces the incidence of matrix effects from other components, such as sodium, in the sample.

The amount of spike added to the sample prior to oxidation needs to be

optimized. The error propagation has been calculated and plotted in Figure

3-1 for a typical measurement system employing a \\ spike of 99 % isotopic

purity. The calculated error magnification factor (EM) is at a minimum (EM =

1.06) for a ^UI^Li ratio of approximately 0.35. However, although this is the

ideal ratio, it is considered better to underspike the system to yield an isotope

ratio closer to unity. Under these conditions, the effects of detector dead-time and pulse counting statistics, which are important aspects of ICP-MS

measurement systems, are minimized. As is evident from Figure 3-1 , at a

ratio of approximately unity, the EM factor is about 1.10, which is totally acceptable. The other approach to measurement would be to exactly match

the ratios of the spike calibration mixtures to the sample measurement

mixtures, and run them in the same temporal space. This is not easily achieved and does not circumvent the counting statistics limitation. FIGURE 3-1

Isotope Dilution Error Propagation Plot \i / ^Li

1.40 T

1.00 J , : ^ ^

0.01 0.1 1 10

[LI-1] Page 2 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

4. Safety

Clinical fluids are biohazardous materials which may contain pathogenic

substances. The usual safety precautions should be taken to prevent exposure during handling, including the use of gloves at all times. Although most materials will have been screened for such substances, a clinical monitoring regimen, including screening for HIV 1 and 2 and vaccination against hepatitis B is recommended for personnel handling biological specimens on an extended basis.

5. Required Reagents

5.1 ^Li Isotopic Spike A spike having an isotopic purity >99 % can be obtained through Oak

Ridge National Laboratory (ORNL), or other stable-isotope supplier, in a

form such as ^Li2C03. A stock solution can be prepared by dissolving a

small amount of the spike in 2 % (volume fraction) high-purity nitric acid

to yield a concentration of about 10 pg/g (1 .7 pmol/g) and storing in a

cleaned PTFE bottle. Tracking the tare weight of the bottle is useful for long-term storage and mor itonng the concentration of the spike on a

multiple-use basis.

5.2 SRM 924a, Lithium Carbonate, Clinical Standard The material should be dried according to the directions on the SRM

certificate, which specify 4 h at 200 °C in a convection oven. Allow to cool. Store in a desiccator at room temperature when not in use. The

purity of the material is 99.867 % with an expanded uncertainty of 0.017 %. The mass fraction of lithium in SRM 924a, adjusted for the purity, is 0.18763.

5.3 High-Purity Nitric Acid

High-purity acid is essential to minimize the extent of lithium contamination from reagent sources. High-purity acid containing less than 20 pg/g lithium may be obtained from a number of commercial

suppliers.

5.4 High-Purity Water

De-ionized or quartz-distilled water, which has been tested and is low in lithium contamination, is used for sample dilution. The water should meet

or exceed the specifications of CAP/NCCLS Type I water [1].

[LI-1] Page 3 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

6. Equipment

6.1 ICP-MS

The method utilizes a standard ICP-MS system employing a quadrupole mass spectrometer. The instrument should be capable of operating in the pulse counting detection mode, and should be fitted with a highefficiency pneumatic nebulizer system, if available. The use of a sample

introduction system composed of an inert material is beneficial in reducing instrument background and improving sample washout.

6.2 Microwave Digestion System A commercial system should be used which is capable of reaching at least 70 bar (7 MPa) vessel pressure and a maximum vessel temperature of at least 200 °C using either PTFE or quartz reaction vessels. The system should be fitted with industry-standard safety

interlocks. A continuous (un-pulsed) variable power delivery system is advantageous in maintaining optimum reaction control.

6.3 Sample Bottles Clean 60 mL polyethlylene sample bottles (or equivalent material) for sample analysis and storage. Note on Sample Bottles: Polyethylene or Teflon® fluoropolymer sample bottles formulated for low trace-metal content should be used. These contain very low levels of electrolyte contaminants. Containers such as polypropylene centrifuge tubes should not be used for this purpose, as they frequently contain appreciable levels of contaminants.

6.4 Micro-Pipette Automatic or manual micro-pipette for dispensing sample, spike and

standard aliquots. A capped plastic syringe fitted with a Teflon®

fluoropolymer uptake tube may also be used for this purpose.

6.5 Analytical Balance

Five-place analytical balance for weighing sample, spike and standard

aliquots. The balance should be calibrated and verified to be functioning

correctly prior to weighing measurements.

7. Sample Handling, Preparation and Quality Control

7.1 Analytical Sample

The analytical sample should be equilibrated to room temperature prior to measurement. Follow supplier instructions for reconstitution of lyophilized material or equilibration from freezer temperature. All sample

[LI-1] Page 4 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

preparation should be carried out in a Class 10 clean environment with the use of protective clothing and gloves.

7.2 Aliquoting and Acidification Approximately 0.5 g of the liquid sample should be weighed accurately, using a five-place analytical balance, into a cleaned microwave digestion vessel manufactured from quartz or Teflon® fluoropolymer and spiked

with an aliquot of ^Li stock (ranging from 0.1 g to 0.4 g depending on the

level in the material) to yield a ^U/^L\ ratio of approximately unity. Five grams of high-purity nitric acid should be added to each vessel, and the vessels then sealed using the appropriate procedure specified by the manufacturer.

7.3 Digestion The prepared solutions should be placed into a high-pressure microwave

digestion system and digested using the manufacturers recommended

conditions, or user-developed digestion protocol. A vessel pressure approaching 70 bar (7 MPa) for at least 30 min will usually ensure

complete oxidation of the sample and conversion of all carbon in the sample to carbon dioxide.

7.4 Dilution The digested samples should be carefully removed from the digestion vessels and diluted with high-purity water to a concentration level appropriate to the ICP-MS instrumentation used for the measurements.

Typically, the working concentration is around 5 ng/g.

7.5 Stability The prepared solutions are stable for several days. However,

determinations should be carried out as soon as possible to avoid the

risk of contamination, losses or stability issues.

7.6 Spike Calibration Mixtures The concentration of the ^Li spike stock solution must be measured by

preparing at least four independent spike calibration mixtures. Prepare two stock calibration solutions of natural lithium from SRM 924a. Prepare two spike calibration mixtures from each of the natural lithium calibration solutions by aliquoting known amounts of the calibration stock and ^Li spike into sample bottles and diluting with 2 % (volume fraction) nitric acid to a concentration suitable for measurement by ICP-MS. The

aliquots should be adjusted to yield ^Li/^Ll isotope ratios as similar as

possible to the analytical samples. A five-place analytical balance should be used for all weighing operations.

[LI-1] Page 5 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

7.7 Procedure Blanks

Prepare a minimum of three procedure blanks by adding approximately

0.05 |jg or less of the ^Li spike to microwave digestion vessels and carry them through the entire preparation procedure.

7.8 Composition Sample

Prepare a composition sample by aliquoting approximately 0.5 g of sample into a microwave digestion vessel and carrying it through the same preparation procedure as the samples. The composition sample is not spiked with ^Li and is used to assess the isotopic composition of the

material being measured. The isotopic composition of lithium has natural

variability and must be measured to assure accuracy of the data. The

measured isotopic composition data are used in the calculations of the sample concentrations. Isotopic composition data for some relevant NIST SRMs are compiled in Appendix A.

7.9 Controls To assess the accuracy of the measurements, prepare at least two

separate SRM control materials having certified values for lithium, preferably of the same matrix type the samples, and carry the controls through the same sample preparation and measurement procedures as

the samples. An additional control material, consisting of an accurately known amount of standard, should be prepared and carried through the whole measurement and calculation process. The gravimetric recovery

of the standard facilitates assessment of the accuracy of the measurement process and veracity of the calculations.

8. Analytical Measurement

8.1 Instrument Preparation

Prior to analytical measurements, the ICP-MS system should be

equilibrated for at least 30 min. The sample introduction system should be cleaned rigorously before use, including the sampler and skimmer

cones, penstaltic pump tubing, plasma torch and spray chamber. These

procedures are designed to mitigate the instrument lithium background

as much as possible. It is recommended that new peristaltic pump tubing be installed prior to measurements. A dilute nitric acid solution (2 %

volume fraction) should be pumped through the new tubing for a while to remove any leachable lithium from the tubing.

[LI-1] Page 6 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

8.2 Instrument Optimization

In order to reduce instrument background, the plasma rf power should be

reduced slightly from the normal setting. For an instrument operating normally at 1 350 W, a setting of 11 00 W would be typical. The

instrument should be optimized for maximum ion transmission at m/z 7 by nebulizing a standard solution of lithium and adjusting ion lens

voltages, plasma gas flows and torch position (relative to the sample

cone). The ion lens(es) should then be adjusted slightly to minimize as

far as possible the mass discrimination. Although this results in some

loss of sensitivity, this should not compromise measurements of lithium

in clinical materials.

8.3 Run Sequence

Software acquisition menus should be prepared for measuring the ^Li and ''LI isotopes. The run order for the samples should be established according to the following protocol:

Analytical procedure blanks should be run at the beginning of the sequence, followed by the composition sample and then the analytical samples (including control materials) in randomized order. Instrument mass discrimination measurement standards (see note) should be run

after every three to four samples depending on the stability of the

instrument. Note on Mass Discrimination Measurement: The measured ^U/^U

ratios should be corrected for the ICP-MS mass discrimination. This can be achieved by periodic measurement of an isotopic calibration standard consisting of natural lithium. Suitable calibration materials are IRMM (Institute for Reference Materials and Measurements, Geel, Belgium), IRMM-016 which has a ^Li / \i ratio of 12.177 (molar mass

6.9401 g/mol) and NIST LSVEC (RM 8545). The LSVEC material is identical to the IRMM material. The natural ratio is very different from

the working sample ratios. A more elegant way to assess discrimination and discrimination drift is to carefully cross-calibrate a solution matched more closely to the sample ratios. A spike calibration solution can

conveniently be used for this purpose.

8.4 Isotope Ratio Acquisition The isotope ratio ^Li/^Li should be acquired in peak jumping mode. A

typical acquisition scheme would be five to seven sets of ratios with a

total acquisition time of 60 s per set, using a dwell-time of 10 ms per

isotope. The isotope ratio data should be downloaded to a spreadsheet program such as Microsoft Excel. Inspect the ratios and remove any

considered to be statistical outliers.

[LI-1] Page 7 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

9. Data Reduction

9.1 Mass Discrimination Corrections

Convert the experimentally measured ''Li/^Li isotope ratios to absolute

ratios using the following equation:

TRUE CORR

MEAS

where, is the dischmination corrected sample ratio

is the true ratio for the isotopic standard

'Li 6,,

is the measured ratio for the isotopic standard

The measured ratios for the isotopic standard can be applied to sample

discrimination corrections in a number of ways, which will depend on the

discrimination drift as a function of time: (1) the ratio can be calculated by

averaging the two standard measurements bracketing the samples of

interest, (2) a more sophisticated approach is to mathematically model

the drift in the standard ratio as a function of time and interpolate the

ratio for each sample. This may require more frequent measurement of the isotopic standard to define the drift function adequately.

.2 Spike Calibration

Calculate the concentration of the ^Li spike in each spike calibration mixture using the following equation:

^SPK - Ms,o(BR - A)

M3 K(A3-B3R)

where, Cspk is the concentration of lithium in the spike in |jg/g

IVIsTD is the mass of lithium added to the spike mix (|jg) B is the natural abundance of the spike isotope (6) R is the corrected ''Li/^Li ratio in the spike mix A is the natural abundance of the reference isotope (7)

[LI-1]Page8of13
LI-1 Lithium by Isotope Dilution ICP-MS

Ms is the mass of ^Li spike aliquot added to the mix (g) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike

Calculate the mean and standard deviation of the spike calibration mixtures. Blank corrections to the spike calibration are not normally necessary because they are simple mixtures prepared in dilute acid.

9.3 Calculation of Lithium Sample Concentrations

Calculate the lithium concentration (|jg/g) in the sample using the

following equation:

c = rM3K(A3-B3R) 1 1 X —

(BR-A) ^ W

where, C is the concentration of lithium in the sample (|jg/g) Ms is the absolute mass of ^Li spike added (|jg) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike R is the corrected ^L\/^L\ ratio B is the natural abundance of the spike isotope A is the natural abundance of the reference isotope Sb is the absolute mean measured blank (|jg) W is the mass of sample aliquot taken (g) An alternative approach, if desired, is to calculate the spike and sample

concentrations in mol/g. In this case the equations are slightly modified.

10. Uncertainty Analysis

The expanded uncertainty for the set of samples should be calculated according to ISO guidelines [2] by combining both Type A and Type B

uncertainties. Typical Type A uncertainty components for this measurement

might be the variability of the measured concentrations of lithium in the samples, the measurement of the spike concentration and the variability of the correction for the blank. Typical Type B uncertainty components might be

the uncertainty of the assay of the primary calibrant, weighing measurements, conversion correction for the density of the sample and

instrumental parameters such as detector dead-time correction, instrument mass discrimination correction and instrument background correction.

[LI-1] Page 9 of 13
LI-1 Lithium by Isotope Dilution I CP-MS

For a 95 % confidence level, combine the Type A and Type B components

and calculate the total expanded uncertainty using the following equation: U= k [Si^/dfi + ...+ Sn'/dfn + Bi^ +...+ Bn^ ] where, U is the expanded uncertainty k is the coverage factor s is the observed standard deviation of (n) Type A components

df is the degrees of freedom associated with each component

B is the Type B component (n components) with infinite degrees

of freedom.

Report the expanded uncertainty in the same units as the sample

concentrations.

11. Data Reporting

Convert data from a mass/mass basis to a mass/volume basis using the measured density of the serum. For clinical use, report data in units of mmol/L. To convert from mmol/L to mg/dL multiply by the relative atomic mass of the material and divide by ten. To convert data from pg/mL to mg/dL

divide by ten.

12. Performance Statement

12.1 Measurement Repeatability The isotope ratio measurement repeatability is usually limited by

detector counting statistics and instrument drift. Typical measurement

repeatability on a quadrupole ICP-MS system for a ^Li/^Li ratio approaching unity is 0.2-0.3 %.

12.2 Detection Limit The instrument detection limit is influenced by the instrument background and the operating conditions chosen for the analytical measurements. A typical instrument detection limit (3 sigma) for a quadrupole ICP-MS instrument is 0.5 pg/mL. For a 0.5 g sample

aliquot this corresponds to a detection limit of approximately 0.1 ng/mL

which is far below the lithium concentrations typically found in clinical

fluids.

[LI-1] Page 10 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

12.3 Instrument Background and Procedure Blank The ICP-MS instrument background is average to high for lithium. The

background is typically in the range from 0-1000 counts per second

depending on the cleanliness of the system and history of use. A typical procedure blank using this method is approximately 100 pg depending on the purity of the reagents and storage containers. This is extremely low in relation to the lithium content in clinical fluids. For example, the blank correction for the determination of lithium in blood serum should be < 0.01 %.

12.4 Performance Summary A summary of typical method performance parameters expected for

this method are provided in Table 12-1.

Table 12-1: Method Performance Summary

Parameter Typical Value or Range

Isotope ratio measurement repeatability 0.2 %

Applicable concentration range

All ranges found in clinical specimens

Instrument background / sample ratio «0.1 %

Procedure blank = 100 pg Sample throughput (measurement) 10 samples per hour

13. Reference Citations

1 . Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline - Third Edition, NCCLS Document C3-A3, NCCLS,

Wayne, Pennsylvania (1997).

2. Guide to the Expression of Uncertainty in Measurement, ISBN 92-67-

10188-9, 1st Ed. ISO, Switzerland, 1993.

i

[LI-1] Page 11 of 13
LI-1 Lithium by Isotope Dilution ICP-MS

LI-1 Revision History

Revision Date Author Record of Changes and Comments

1.0 9/2006 SELong Initial draft method

[LI-1] Page 12 of 13
LI-1 Lithium by Isotope Dilution ICR-MS APPENDIX A

Measured Absolute Lithium Isotopic Compositions for Selected NIST Clinical SRMs

SRM 924a, Lithium Carbonate

0.08082 Atom (%) 7.4779

92.5221

Relative Atomic Mass 6.9412

SRM 956a, Serum Level 1,2,3

\\/\\ 0.08200 Atom (%) \\ 7.5790 92.4210

Relative Atomic Mass 6.9402

SRM 956b, Serum Level 1,2,3

\\/\\ 0.080823 Atom (%) \\ 7.5816 92.4184

Relative Atomic Mass 6.9401

SRM 909b, Serum

0.080823 Atom (%) \\ 7.4779 \\ 92.5221

Relative Atomic Mass 6.9412

[LI-1] Page 13 of 13

Sectiion 5 - Magnesium

5 Magnesium

5.1 Clinical Significance

Magnesium is an essential element which plays a critical role in metabolic

processes. Like calcium, the majority of magnesium is localized within the

skeletal system, while approximately 2 % is present in the extra-cellular fluid compartment. Of the magnesium found in plasma, just over half is in the

active ionized form, while one third is bound to protein material. The

remainder is bound to other free ligands such as citrate and phosphate. The

ionized fraction is maintained under homeostatic control by renal excretion

with replenishment through dietary sources. Magnesium is important in many

physiological processes, which include, principally, the storage and use of energy, neuromuscular function, activation of enzyme reactions involving phosphate, and carbohydrate, lipid and protein metabolism. A reduced magnesium level (hypomagnesemia) can result from lowered dietary absorption of magnesium or excessive renal excretion, and often occurs in tandem with hypokalemia and fiypocalcemia, and is associated with a variety of pathological conditions including cardiovascular disease. The condition presents a variety of symptoms which range from muscle weakness to increased reflexes, cramps, tremors and convulsions. Conversely, high levels of magnesium {hypermagnesemia) are predominantly associated with renal

failure, but occasionally with impaired renal function combined with abnormally high intake, such as would result from excessive consumption of magnesium-containing pharmaceuticals such as antacids or laxatives.

5.2 Normal Range and Reporting Units The normal range for total magnesium in serum or plasma is about 1 .8-2.5 mg/dL. Magnesium clinical data are conventionally reported in mg/dL. To convert from mmol/L to mg/dL multiply by 2.43050. To convert from mg/dL to mmol/L multiply by 0.41 144.

5.3 Routine Clinical Measurement Methods

Both flame atomic absorption and atomic emission systems are accurate,

specific and sensitive for the measurement of magnesium in a variety of

clinical media. The use of ion selective electrodes for such measurements

has so far been limited because of the lack of specificity particularly with respect to calcium ions which are almost always present in the sample. An

5-1
Sectiion 5 - Magnesium

important class of routine determinations is spectrophotometry. A variety of reactions have been employed. One of the most widely used is Calmagite which forms a red colored complex with magnesium, absorbing at 530 nm.

Other systems based on chlorophosphonazo III and Magon dye complex

reactions are also employed.

5.4 Available Standard Reference Materials Two blood serum SRMs are available with certified values for magnesium,

which are SRM 909b (Human Serum), a two-level lyophilized material, and SRM 956b {Electrolytes in Frozen Human Serum), which is a three-level frozen material intended for standardizing and calibrating ion selective electrode analyzers. There are also reference values for magnesium in SRM

2670a (Toxic Elements in Freeze-Dried Urine), a two-level, freeze-dried

material. One clinical assay standard may be used for magnesium: SRM

929a (Magnesium Gluconate, Clinical Standard) which is sold as a clinical primary standard intended for instrument calibration and standardization.

Certificate information for these SRMs is summarized in Table 5-1.

Table 5-1. Clinical Standard Reference Materials for Magnesium

SRM Description Value

(mg/dL)

Expanded

Uncertainty

(mg/dL) 909b Level 1 Human Serum 1.855 0.012 909b Level 2 Human Serum 4.661 0.051 956b Level 1 Electrolytes in Frozen Human Serum 3.699 0.048 956b Level 2 Electrolytes in Frozen Human Serum 2.417 0.031 956b Level 3 Electrolytes in Frozen Human Serum 1.113 0.014 2670a Level 1 Toxic Elements in Urine (Freeze-Dried) (2.10) (0.02) 2670a Level 2 Toxic Elements in Urine (Freeze-Dhed) (2.12) (0.02) SRM Clinical Standard

Purity (% mass

fraction )

Expanded

Uncertainty (%) 929a Magnesium Gluconate

(Clinical Standard) 5.362 0.027

(

) Values in Parentheses are Reference Values.

5-2
MG-1 Magnesium by Isotope Dilution ICP-MS

Version Number 1.0 Approved S E Long

Effective Date Sep 2006

MG-1

MAGNESIUM BY ISOTOPE DILUTION

INDUCTIVELY COUPLED PLASMA - MASS

SPECTROMETRY

1. Applicability The method is applicable to the determination of magnesium at physiological

levels in blood plasma, serum (frozen and lyophilized) and urine.

2. Principle of Measurement

The method is based on isotope dilution mass spectrometry using a quadrupole inductively coupled plasma - mass spectrometer (ICP-MS). A

stable isotopic spike of ^^Mg is added to the sample, equilibrated by oxidation with nitric acid in a microwave digestion system, and the ^'*Mg/^^l\/lg isotope

ratio measured under standard conditions. The method is extremely accurate and very selective for magnesium.

3. Measurement Strategy and Experimental Design

The determination of magnesium in biological fluids by isotope dilution ICP- MS is a relatively straightforward application. Oxidation with nitric acid in a microwave digestion system is necessary to avoid potential interferences on

the isotopes ^'^Mg {C2^) and ^^Mg (C2*, C2\-\*) arising from carbon in the sample. The potential for an additional interference on ^"^Mg from "^Ca^* does

exist, but the natural abundance of '*^Ca is so low that the interference is negligible. Two spikes could theoretically be used for the isotope dilution measurement, namely ^^Mg and ^^Mg. These are available in approximately 99% isotopic purity and both inexpensive when purchased from commercial

sources. The isotope ^^Mg is considered to be a better choice for the measurement of magnesium because of its proximity in mass to the reference isotope ^'^Mg. This reduces the effects of mass discrimination in the measurement system relative to ^^Mg. Mass discrimination in this region of the mass scale tends to be high on a quadrupole ICP-MS system.

[MG-1] Page 1 of 12
MG-1 Magnesium by Isotope Dilution ICP-MS

The amount of spike added to the sample prior to oxidation needs to be

optimized. The error propagation has been calculated and plotted in Figure

3-1 for a typical measurement system employing a ^^Mg spike of 99 %

isotopic purity. The calculated error magnification factor (EM) is at a minimum (EM = 1.07) for a ^''Mg/^^Mg ratio of 0.28. However, although this is the ideal ratio, it is considered better to underspike the system to yield an

isotope ratio closer to unity. Under these conditions, the effects of detector dead-time and pulse counting statistics, which are important aspects of ICP- MS measurement systems, are minimized. As is evident from Figure 3-1 , at a

ratio of approximately unity, the error magnification factor is about 1.15, which is totally acceptable. The other approach to measurement would be to exactly match the ratios of the spike calibration mixtures to the sample measurement mixtures, and run them in the same temporal space. This is not easily achieved and does not circumvent the counting statistics limitation. FIGURE 3-1

Isotope Dilution Error Propagation Plot ^^Mg / "wig

1.45

ai 1.05

1.00 J ,

,— .

:— ,

,—

,

1, 1 . 0.01 0.1 1 10

4. Safety

Clinical fluids are biohazardous materials which may contain pathogenic

substances. The usual safety precautions should be taken to prevent

[MG-1] Page 2 of 12
MG-1 Magnesium by Isotope Dilution ICP-MS

exposure during handling, including the use of gloves at all times. Although most materials will have been screened for such substances, a clinical monitoring regimen, including screening for HIV 1 and 2 and vaccination against hepatitis B is recommended for personnel handling biological specimens on an extended basis.

5. Required Reagents

5.1 ^^Mg Isotopic Spike A spike having an isotopic purity >99 %, can be obtained through Oak

Ridge National Laboratory (ORNL) or other stable-isotope supplier in a

form such as ^^MgO. A stock solution can be prepared by dissolving a

small amount of the ^^MgO in 2 % (volume fraction) high-purity nitric acid to yield a concentration of about 7.5 pg/g (0.3 |jmol/g) and storing in a cleaned Teflon® fluoropolymer bottle. Tracking the tare weight of the

bottle is useful for long-term storage and monitoring the concentration of the spike on a multiple-use basis.

5.2 SRM 929a, Magnesium Gluconate, Clinical Standard The material should be dried according to the directions on the certificate for 24 h over magnesium perchlorate in a desiccator. The relative mass fraction of magnesium is 5.362 % with an expanded

uncertainty of 0.027 %.

Alternatively, magnesium standard solution (SRM 3131a), which has a

concentration of 9.99 mg/g ± 0.02 mg/g (0.2 % relative) can be used.

5.3 High-Purity Nitric Acid

High-purity acid is essential to minimize the extent of magnesium

contamination from reagent sources. High-purity acid containing less than 100 pg/g magnesium may be obtained from a number of commercial suppliers.

5.4 High-Purity Water

De-ionized or quartz-distilled water, which has been tested and is low in magnesium contamination, is used for sample dilution. The water should meet or exceed the specifications of CAP/NCCLS Type I water [1].

6. Equipment

6.1 ICP-MS

The method utilizes a standard ICP-MS system employing a quadrupole mass spectrometer. The instrument should be capable of operating in

[MG-1] Page 3 of 12
MG-1 Magnesium by Isotope Dilution I CP-MS

the pulse counting detection mode, and should be fitted with a highefficiency pneumatic nebulizer system, if available. The use of a sample

introduction system composed of an inert material is beneficial in reducing instrument background.

6.2 Microwave Digestion System A commercial system should be used which is capable of reaching at least 70 bar (7 MPa) vessel pressure and a maximum vessel temperature of at least 200 °C using either PTFE or quartz reaction vessels. The system should be fitted with industry-standard safety

interlocks. A continuous (un-pulsed) variable power delivery system is advantageous in maintaining optimum reaction control.

6.3 Sample Bottles Clean 60 mL polyethylene (Nalgene) sample bottles (or equivalent

material) for sample storage and analysis. Note on Sample Bottles: Polyethylene or Teflon® fluoropolymer sample bottles formulated for low trace-metal content should be used. These contain very low levels of electrolyte contaminants. Containers such as polypropylene centrifuge tubes should no. be used for this purpose, as they frequently contain appreciable levels of contaminants.

6.4 Micro-Pipette Automatic or manual micro-pipette for dispensing sample, spike and

standard aliquots. A capped plastic syringe fitted with a Teflon®

fluoropolymer uptake tube may also be used for this purpose.

6.5 Analytical Balance

Five-place analytical balance for weighing sample, spike and standard

aliquots. The balance should be calibrated and verified to be functioning

correctly prior to weighing measurements.

7. Sample Handling, Preparation and Quality Control

7.1 Analytical Sample

The analytical sample should be equilibrated to room temperature prior to measurement. Follow supplier instructions for reconstitution of

lyophilized material or equilibration from freezer temperature. All sample

preparation should be carried out in a Class 10 clean environment with the use of protective clothing and gloves.

[MG-1] Page 4 of 12
MG-1 Magnesium by Isotope Dilution ICP-MS

7.2 Aliquoting and Acidification Approximately 0.5 g of the liquid sample should be weighed accurately, using a five-place analytical balance, into a cleaned microwave digestion vessel manufactured from quartz or Teflon® fluoropolymer and spiked

with an aliquot of ^^Mg stock to yield a ^''Mg/^^Mg ratio of approximately

unity. Five grams of high-purity nitric acid should be added to each

vessel and the vessels then sealed using the appropriate procedure

specified by the manufacturer.

7.3 Digestion The prepared solutions should be placed into a high-pressure microwave

digestion system and digested using the manufacturers recommended

conditions or user-developed digestion protocol. A vessel pressure approaching 70 bar (7 MPa) for at least 30 min will usually ensure complete oxidation of the sample and conversion of all carbon in the sample to carbon dioxide.

7.4 Dilution The digested samples should be carefully removed from the digestion vessels and diluted with high-purity water to a concentration level appropriate to the ICP-MS instrumentation used for the measurements.

7.5 Stability The prepared solutions are stable for several days. However,

determinations should be carried out as soon as possible to avoid the

risk of contamination, losses or stability issues.

7.6 Spike Calibration Mixtures The concentration of the ^^Mg spike stock solution must be measured by

preparing at least four independent spike calibration mixtures. Prepare two stock calibration solutions of natural magnesium from SRM 929a or SRM 3131a. Prepare two spike calibration mixtures from each of the

natural magnesium calibration solutions by aliquoting known amounts of the calibration stock and ^^Mg spike into sample bottles and diluting with 2 % (volume fraction) nitric acid to a concentration suitable for measurement by ICP-MS. The aliquots should be adjusted to yield ^'*Mg/^^Mg isotope ratios as similar as possible to the analytical samples. A five-place analytical balance should be used for all weighing

operations.

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MG-1 Magnesium by Isotope Dilution ICR-MS

7.7 Procedure Blanks

Prepare a minimum of three procedure blanks by adding approximately

0.5 |jg or less of the ^^Mg spike to microwave digestion vessels and

carrying them through the entire preparation procedure.

7.8 Composition Sample

Prepare a composition sample by aliquoting approximately 0.5 g of sample into a microwave digestion vessel and carrying it through the same preparation procedure as the samples. The composition sample is not spiked with ^^Mg and is used to assess the presence or absence of spectral interferences in the ICP-MS measurement process.

7.9 Controls To assess the accuracy of the measurements, prepare at least two

different SRM control materials (preferably of the same mathx as the samples) having certified values for magnesium, and carry them through

the same sample preparation and measurement procedures as the samples.

8. Analytical Measurement

8.1 instrument Preparation

Prior to analytical measurements, the ICP-MS system should be

equilibrated for 30 min. The sample introduction system should be

rigorously cleaned before use, including the sampler and skimmer cones,

peristaltic pump tubing, plasma torch and spray chamber. Magnesium

background from the instrument is usually quite high and these procedures are designed to mitigate the background as much as

possible. It is recommended that new pehstaltic pump tubing be installed prior to measurements. A dilute nitric acid solution (2 % volume fraction) should be pumped through the tubing for a while to remove any

leachable magnesium from the tubing.

8.2 Instrument Optimization The instrument should be optimized for maximum ion transmission at m/z 24 by nebulizing a standard solution of magnesium and adjusting ion lens voltages, plasma gas flows, torch position (relative to the sample

cones) and rf forward power.

[MG-1] Pages of 12
MG-1 Magnesium by Isotope Dilution ICP-MS

8.3 Run Sequence

Software acquisition menus sliould be prepared for measuring the ^"^Mg and ^^Mg isotopes. The run order for the samples should be established according to the following protocol:

Analytical procedure blanks should be run at the beginning of the sequence, followed by the composition sample and then the analytical samples (including control matehals) in randomized order. Instrument mass discrimination measurement standards (see note) should be run

after every three to four samples depending on the stability of the

instrument. Note on Mass Discrimination Measurement: The measured

^"^Mg/^^Mg ratios should be corrected for the ICP-MS mass

discrimination. This can be achieved by periodic measurement of a

calibration standard consisting of natural magnesium. The natural ratio of ^"Mg/^^Mg is approximately 7.9, which is very different from the sample ratios. A more elegant way to assess discrimination is to

carefully calibrate a solution having a ratio closer to that of the samples. A spike calibration solution can conveniently be used for this purpose.

8.4 Isotope Ratio Acquisition The isotope ratio ^'^Mg/^^Mg should be acquired in peak jumping mode. A

typical acquisition scheme would be five to seven sets of ratios with a

total acquisition time of 60 s per set, using a dwell-time of 10 ms per

isotope. The isotope ratio data should be downloaded to a spreadsheet program such as Microsoft Excel. Inspect the ratios and remove any

considered to be statistical outliers.

9. Data Reduction

9.1 Mass Discrimination Corrections

Convert the experimentally measured ^''Mg/^^Mg isotope ratios to absolute ratios using the following equation:

24,

24,JWg

'Mg

25, Utrue

25, 24,

'JcORR 25,Mg MEAS Where, r24„g" is the discrimination corrected sample ratio

CORR

[MG-1] Page 7 of 12
MG-1 Magnesium by Isotope Dilution ICP-MS

'Mg

*Mg

is the true ratio for the isotopic standard

is the measured ratio for the isotopic standard

The measured ratios for the isotopic standard can be applied to sample

discrimination corrections in a number of ways, which will depend on the

discrimination drift as a function of time: (1) the ratio can be calculated by

averaging the two standard measurements bracketing the samples of

interest, (2) a more sophisticated approach is to mathematically model

the drift in the standard ratio as a function of time and interpolate the

ratio for each sample. This may require more frequent measurement of the isotopic standard to define the drift function adequately.

9.2 Spike Calibration

Calculate the concentration of the ^"^Mg spike in each spike calibration mixture using the following equation:

^SPK -

Ms,, (BR - A)

M, K(A,-B,R)

where, Cspk is the concentration of magnesium in the spike in pg/g

IVIsTD is the mass of magnesium added to the spike mix (|jg) B is the natural abundance of the spike isotope R is the corrected ^'^Mg/^^Mg ratio in the spike mix A is the natural abundance of the reference isotope Ms is the mass of ^^Mg spike aliquot added to the mix (g) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike

Calculate the mean and standard deviation of the spike calibration mixtures. Blank corrections to the spike calibration are not normally necessary because they are simple mixtures prepared in dilute acid.

.3 Calculation of Magnesium Sample Concentrations

Calculate the magnesium concentration (fjg/g) in the sample using the

following equation:

[MG-1] Page 8 of 12
MG-1 Magnesium by Isotope Dilution I CP-MS

c = rM3K(A3-B3R) 1 1 X —

[ (BR-A) "J W

where, C is the concentration of magnesium in the sample (|jg/g) Ms is the absolute mass of ^^Mg spike added (pg) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike R is the corrected ^"^Mg/^^Mg ratio B is the natural abundance of the spike isotope A is the natural abundance of the reference isotope Sb is the absolute mean measured blank (pg) W is the mass of sample aliquot taken (g) An alternative approach, if desired, is to calculate the spike and sample

concentrations in mol/g. In this case the equations are slightly modified and the relative atomic masses of natural magnesium and the spike are

not required for the calculations.

10. Uncertainty Analysis

The expanded uncertainty for the set of samples should be calculated according to ISO guidelines [2] by combining both Type A and Type B

uncertainties. Typical Type A uncertainty components for this measurement

might be the variability of the measured concentrations of magnesium in the samples, the measurement of the spike concentration and the variability of the correction for the blank. Typical Type B uncertainty components might be

the uncertainty of the assay of the primary calibrant, weighing measurements, conversion correction for the density of the sample and

instrumental parameters such as detector dead-time correction, instrument mass discrimination correction and instrument background correction.

For a 95 % confidence level, combine the Type A and Type B components

and calculate the total expanded uncertainty using the following equation: U= k [si'/dfi + . . . + Sn'/dfn + Bi^ +. . . + Bn^ ]

^-"^

where, U is the expanded uncertainty k is the coverage factor s is the observed standard deviation of (n) Type A components

df is the degrees of freedom associated with each component

[MG-1] Page 9 of 12
MG-1 Magnesium by Isotope Dilution ICP-MS

B is the Type B component (n components) with infinite degrees

of freedom.

Report the expanded uncertainty in the same units as the sample

concentrations.

11. Data Reporting

Convert data from a mass/mass basis to a mass/volume basis using the measured density of the serum. For clinical use, report data in units of mg/dL. To convert data from pg/mL to mg/dL divide by 10. To convert from mg/dL to mmol/L multiply by 0.41 144.

12. Performance Statement

12.1 IVIeasurement Repeatability The isotope ratio measurement repeatability is usually limited by

detector counting statistics. Typical measurement repeatability on a quadrupole ICP-MS system for a ^'*Mg/^^Mg ratio approaching unity is 0.1-0.2 %.

12.2 Detection Limit The instrument detection limit is influenced by the instrument background. A typical instrument detection limit for a well-optimized quadrupole ICP-MS instrument is 5 pg/mL. For a 0.5 g sample aliquot

this equates to a detection limit of approximately 1 ng/mL which is far below the magnesium concentrations typically found in clinical fluids.

12.3 Instrument Background and Procedure Blank The ICP-MS instrument background tends to be fairly high for magnesium. The background can vary from a few hundred counts per second to several thousand counts per second depending on the

cleanliness of the system and history of use. A typical procedure blank using this method is approximately 5 ng, depending on the purity of the reagents and storage containers. This is low in relation to the magnesium content in clinical fluids. For example,

the blank correction for the determination of magnesium in blood serum should be < 0.1 %.

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MG-1 Magnesium by Isotope Dilution I CP-MS

12.4 Performance Summary A summary of typical method performance parameters expected for this method are provided in Table 12-1.

Table 12-1: Method Performance Summary

Parameter Typical Value or Range

Isotope ratio measurement repeatability 0.1-0.2 %

Applicable concentration range

All ranges found In clinical specimens

Instrument background / sample ratio = 0.2 %

Procedure blank = 5 ng Sample throughput (measurement) 10 samples per hour

13. Reference Citations

1 . Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline - Third Edition, NCCLS Document C3-A3, NCCLS,

Wayne, Pennsylvania (1997).

2. Guide to the Expression of Uncertainty In Measurement, ISBN 92-67-

101 88-9, 1 St Ed. ISO, Switzerland, 1 993.

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MG-1 Magnesium by Isotope Dilution ICP-MS

MG-1 Revision History

Revision Date Author Record of Changes and Comments

1.0 9/2006 SELong Initial draft method

[MG-1] Page 12 of 12
Section 6 - Potassium

Potassium

6.1 Clinical Significance

Potassium is tlie most abundant cation in intra-cellular fluid. It has a major

role in maintaining the resting membrane potential of cells, which is dependent on the concentration gradient of potassium inside and outside the

cell. Cell membranes are highly permeable to potassium ions, so that there is free exchange of potassium ions across the membrane. The majority of the potassium in the body is in the intra-cellular fluid, with approximately 2 % of the total in the extra-cellular fluid. The physiological action of potassium is intimately associated with that of sodium and, like sodium, is regulated by

aldosterone and other hormones. Potassium homeostasis is maintained by

dietary intake and renal excretion. A lowered potassium level (hypokalemia)

results when there is excessive loss of potassium, such as renal losses

during diuretic treatment and in both chronic and acute metabolic alkalosis. Losses can also occur by dehydration resulting from fluid and electrolyte losses from burns and hemorrhages, and from gastrointestinal losses associated with excessive vomiting and diarrhea. In some cases, extreme

losses of potassium can be life threatening. Elevated potassium levels {hyperl<alemia) can occur during renal failure, from reduced aldosterone

output associated with Addison's disease, dehydration and from movement

of potassium out of cells as a result of large muscle injury or tissue damage.

6.2 Normal Range and Reporting Units The normal range for potassium in serum or plasma is 3.5-5.3 mmol/L. Potassium clinical data are conventionally reported in mmol/L. To convert from mmol/L to mg/dL multiply by 3.90983. To convert from mg/dL to mmol/L multiply by 0.25577.

6.3 Routine Clinical Measurement Methods

Blood and urine are the most commonly used media for assessing patient health status. Flame photometry and flame atomic absorption spectrometry

are reference methods used for the measurement of potassium, providing accuracy and specificity. For routine use, especially in point-of-care environments, ion selective electrodes are now well established. These

systems, which typically measure potassium and sodium in tandem, are capable of providing accurate and consistent data in a very short

6-1
Section 6 - Potassium

measurement time frame and are therefore extremely useful In environments where rapid measurements are Important such as Intensive care units and

operating theaters.

6.4 Available Standard Reference Materials Two blood serum SRMs are available with certified values for potassium, which are SRM 909b {Human Serum), a two-level lyophilized material, and SRM 956b (Electrolytes in Frozen Human Serum), which is a three-level frozen material intended for standardizing and calibrating ion selective electrode analyzers. There are also reference values for potassium in SRM

2670a (Toxic Elements in Freeze-Dried Urine), a two-level, freeze-drled

material. One clinical assay standard may be used for potassium: SRM 918a (Potassium Chloride, Clinical Standard) which is sold as a clinical phmary

standard Intended for instrument calibration and standardization. Certificate information for these SRMs is summarized in Table 6-1

.

Table 6-1. Clinical Standard Reference Materials for Potassium

SRM Description Value

(mmol/L)

Expanded

Uncertainty

(mmoi/L) 909b Level 1 Human Serum 3.424 0.025 909b Level 2 Human Serum 6.278 0.052 956b Level 1 Electrolytes in Frozen Human Serum 5.973 0.045 956b Level 2 Electrolytes in Frozen Human Serum 3.983 0.029 956b Level 3 Electrolytes in Frozen Human Serum 1.987 0.014 2670a Level 1 Toxic Elements in Urine (Freeze-Dried) (10.49) 0.26 2670a Level 2 Toxic Elements in Urine (Freeze-Dried) (10.61) 0.26 SRM Clinical Standard

Purity (% mass

fraction)

Expanded

Uncertainty

(%) 918a Potassium Chloride (Clinical Standard) 99.9817 0.0084

(

) Values In Parentheses are Reference Values.

6-2
K-1 Potassium by ICR-MS

Version Number 1.0 Approved S E Long

Effective Date Sep 2006

K-1

POTASSIUM BY ISOTOPE DILUTION

INDUCTIVELY COUPLED PLASMA - MASS

SPECTROMETRY

1. Applicability The method is applicable to the determination of potassium at physiological

levels in blood plasma, serum (frozen and lyophilized) and urine.

2. Principle of Measurement

The method is based on isotope dilution mass spectrometry using a quadrupole inductively coupled plasma - mass spectrometer (ICP-MS)

operated in "cool plasma" (shielded) mode [1]. A stable isotopic spike of

is added to the sample, equilibrated by oxidation with nitric acid in a microwave digestion system, and the ^^K/'V isotope ratio measured using low power plasma conditions. The method is extremely accurate and

selective for potassium.

3. Measurement Strategy and Experimental Design

The determination of potassium in biological fluids by isotope dilution ICP-MS

is complicated by the presence of potential spectral interferences from ^^Ar^H which interferes with '^K, and '*°Ar'H, ''Na''(H20) and ''Mg^^O which

interfere with "^V. The use of "cool" or shielded plasma conditions effectively attenuates the Ar based spectral background interferences. The other

interferences are minor, except for the ^^Na^^(H20) interference which is significant because of the relatively high level of sodium in biological fluids. For this reason, a chemical separation of potassium and sodium is necessary. For convenience, potassium and calcium (which is similarly

afflicted) can be separated, re-combined and determined together in the same measurement system if desired. The isobaric interference from ''°K on

'*°Ca is negligible for the potassium levels normally found in clinical fluids. Treatment of the sample with nitric acid in a microwave digestion system is

[K-1] Page 1 of 13
K-1 Potassium by ICP-MS

necessary to digest the sample matrix. Normally, is used as the spike, which is available in approximately 99 % isotopic purity.

The amount of spike added to the sample prior to digestion needs to be

optimized. The error propagation has been calculated and plotted in Figure

3-1 for a typical measurement system employing a '^V spike of 99 % purity. The calculated error magnification factor (EM) is at a minimum (EM = 1 .06)

for a ^^K/^V ratio of 0.37. However, although this is the ideal ratio, it is considered better to underspike the system to yield an isotope ratio closer to

unity. Under these conditions, the effects of detector dead-time and pulse counting statistics, which are important aspects of ICP-MS measurement

systems, are minimized. As is evident from Figure 3-1, at a ratio of approximately unity, the EM factor is about 1.09, which is acceptable. An

additional approach to measurement is to closely match the ratios of the spike calibration mixtures to the sample measurement mixtures, and run them in the same temporal measurement space. This requires a fairly accurate knowledge of the concentration of potassium in the unknown

sample prior to spiking. FIGURE 3-1

Isotope Dilution Error Propagation Plot ^^K / ^^K

1.60

[K-1] Page 2 of 13
K-1 Potassium by ICP-MS

4. Safety

Clinical fluids are biohazardous materials which may contain pathogenic

substances. The usual safety precautions should be taken to prevent exposure during handling, including the use of gloves at all times. Although most materials will have been screened for such substances, a clinical monitoring regimen, including screening for HIV 1 and 2 and vaccination against hepatitis B is recommended for personnel handling biological specimens on an extended basis.

5. Required Reagents

5.1 Isotopic Spike A spike having an isotopic purity > 99 %, can be obtained through Oak

Ridge National Laboratory (ORNL) or other stable-isotope supplier in a form such as "^^KCI. A stock solution can be prepared by dissolving a

small amount of the ''Vci in 1 % (volume fraction) high-purity nitric acid

to yield a concentration of about 275 pg/g (6.7 pmol/g) and storing in a

cleaned PTFE bottle. Tracking the tare weight of the bottle is useful for long-term storage and monitoring the concentration of the spike on a

multiple-use basis.

5.2 SRM 918a, Potassium Chloride, Clinical Standard The material should be dried according to the directions on the certificate

in a laboratory oven at 11 0-1 20 °C for 2 h. The certified purity of the material is 99.9917 % (mass fraction) with an expanded uncertainty of 0.0084 %. The relative mass fraction of potassium in SRM 918a, adjusted for the purity, is 0.52435.

Alternatively, potassium standard solution (SRM 3141a), which has a

concentration of 10.295 mg/g ± 0.023 mg/g (0.2 % relative) can be used.

5.3 High-Purity Nitric Acid

High-purity acid is essential to minimize the extent of potassium

contamination from reagent sources. High-purity acid containing less than 100 pg/g potassium may be obtained from a number of commercial

suppliers.

5.4 High-Purity Hydrochloric Acid

For chemical separation of potassium from sample matrix. High-purity acid is essential to minimize the extent of potassium contamination from

reagent sources. High-purity acid containing less than 100 pg/g potassium may be obtained from a number of commercial suppliers.

[K-1] Page 3 of 13
K-1 Potassium by ICP-MS

5.5 High-Purity Water

De-ionized or quartz-distilled water, which has been tested and is low in potassium contamination, is used for sample dilution. At a minimum the water should meet or exceed the specifications of CAP/NCCLS Type I water [2].

5.6 Ion-Exchange Resin AG 50-X8, 100-200 mesh (75-150 pm) cation-exchange resin, or equivalent for chemical separation of potassium from the sample matrix. The resin should be bulk cleaned, packed into columns and batch cleaned twice by alternating between 25 mL of 5 mol/L hydrochloric acid and sufficient high-purity de-ionized water to achieve neutral pH.

6. Equipment

6.1 ICR-MS System

The method utilizes a standard ICP-MS system employing a quadrupole mass spectrometer and an automated shielded torch system operating at low RF forward power ("cool-plasma") for reducing argon-based spectral

interferences. The instrument should be capable of operating in the pulse

counting detection mode, should be fitted with a low dead-volume,

cooled spray chamber, and a high-efficiency pneumatic nebulizer system, if available.

6.2 Microwave Digestion System A commercial system should be used which is capable of reaching at least 70 bar (7 MPa) vessel pressure and a maximum vessel temperature of at least 200 °C using either PTFE or quartz reaction vessels. The system should be fitted with industry-standard safety

interlocks. A continuous (un-pulsed) variable power delivery system is advantageous in maintaining optimum reaction control.

6.3 Teflon® Fluoropolymer Beakers

Clean 50 mL Teflon® fluoropolymer beakers for solution evaporation

prior to column separations.

6.4 Ion-Exchange Columns

Clean plastic or glass ion-exchange columns containing a cation- exchange resin bed (see Reagents) of approximate dimensions 10 cm

long X 0.7 cm wide. This bed should contain sufficient capacity and

column resolution for most clinical samples.

[K-1] Page 4 of 13
K-1 Potassium by I CP-MS

6.5 Sample Bottles Clean 30 mL polyethylene (Nalgene) sample bottles (or equivalent

material) for sample storage and analysis. Note on Sample Bottles: Polyethylene or Teflon® fluoropolymer sample bottles formulated for low trace-metal content should be used. These contain very low levels of electrolyte contaminants. Containers such as polypropylene centrifuge tubes should not be used for this purpose as they frequently contain appreciable levels of contaminants. Clean the sample bottles prior to use by soaking in dilute nitric acid for a minimum of 24 h.

6.6 Micro-Pipette Automatic or manual micro-pipette for dispensing sample, spike and

standard aliquots. A capped plastic syringe fitted with a Teflon®

fluoropolymer uptake tube may also be used for this purpose, depending

on the weighing method employed.

6.7 Analytical Balance

Five-place analytical balance for weighing sample, spike and standard

aliquots. The balance should be calibrated and verified to be functioning

correctly prior to weighing measurements.

7. Sample Handling, Preparation and Quality Control

7.1 Analytical Sample

The analytical sample should be equilibrated to room temperature prior

to measurement. Follow supplier instructions for reconstitution of lyophilized material or equilibration from freezer temperature. All sample

preparation should be carried out in a Class 10 clean environment with the use of protective clothing and gloves.

7.2 Aliquoting and Acidification Approximately 0.7 g of the liquid sample should be weighed accurately, using a five-place analytical balance, into a cleaned microwave digestion vessel manufactured from quartz or Teflon® fluoropolymer and spiked

with an aliquot of stock (approximately 0.4-0.5 g for normal blood serum) to yield a ^^K/'V ratio of approximately unity. Six grams of highpurity nitric acid should be added to each vessel and the vessels then sealed using the appropriate procedure specified by the manufacturer.

[K-1] Page 5 of 13
K-1 Potassium by ICP-MS

7.3 Digestion The prepared solutions should be placed into a high-pressure microwave

digestion system and digested using the manufacturers recommended

conditions or user-developed digestion protocol. A vessel pressure approaching 70 bar (7 MPa) for at least 30 min will usually ensure complete oxidation of the sample and conversion of all carbon in the sample to carbon dioxide.

7.4 Evaporation and Chemical Separations The digested samples should be evaporated to near dryness using the microwave digestion system (if this is a feature), or alternatively by

heating on a hot-plate in 50 mL Teflon® fluoropolymer beakers. Redissolve in a small quantity of dilute 2 % (volume fraction) nitric acid. Transfer to cation-exchange columns. Elute the sodium using approximately 40 mL of 0.3 mol/L hydrochloric acid. Elute the potassium

with approximately 20 mL of 0.5 mol/L hydrochloric acid. Evaporate the potassium fraction to dryness and re-dissolve in 2 % (volume fraction)

nitric acid to yield a concentration suitable for ICP-MS measurement.

Store in clean 30 mL polyethylene bottles.

7.5 Stability The prepared solutions are stable for several days. However,

determinations should be carried out as soon as possible to avoid the

risk of contamination, losses or stability issues.

7.6 Spike Calibration Mixtures The concentration of the '*V spike stock solution must be measured by

preparing at least four independent spike calibration mixtures. Prepare two different stock calibration solutions of natural potassium from SRM

918a or SRM 3141a. Prepare two spike calibration mixtures from each of the natural potassium calibration solutions by aliquoting known amounts

of the calibration stock and '^^K spike into sample bottles and diluting with 2 % (volume fraction) nitric acid to a concentration suitable for measurement by ICP-MS. The aliquots should be adjusted to yield ^^K/*V isotope ratios as similar as possible to the analytical samples. A

five-place analytical balance should be used for all weighing operations.

7.7 Procedure Blanks

Prepare a minimum of three procedure blanks by adding approximately 100 ng of the '*^K spike to microwave digestion vessels and carrying them through the entire preparation and analysis procedure.

[K-1] Page 6 of 13
K-1 Potassium by ICP-MS

7.8 Composition Sample

Prepare a composition sample by aliquoting approximately 0.7 g of sample into a microwave digestion vessel and carrying it through the same preparation procedure as the samples. The composition sample is not spiked with "^^K and is used to assess the presence or absence of

spectral interferences in the ICP-MS measurement process.

7.9 Controls To assess the accuracy of the measurements prepare at least two

different SRM control materials (preferably of the same matrix type as

the samples) having certified values for potassium, and carry the controls through the same sample preparation and measurement procedures as

the samples.

8. Analytical Measurement

8.1 Instrument Preparation

Prior to analytical measurements, the ICP-MS system should be

equilibrated for 30 min. The sample introduction system should be

rigorously cleaned before use, including the sampler and skimmer cones,

peristaltic pump tubing, plasma torch and spray chamber. These

procedures are designed to mitigate the background from potassium as much as possible. It is recommended that new peristaltic pump tubing be

installed prior to measurements. A dilute nitric acid solution (2 % volume

fraction) should be pumped through the tubing for a while to remove any

leachable potassium from the tubing.

8.2 Instrument Optimization The instrument should be optimized for maximum ion transmission and minimum background at m/z 39 and 41 (maximum signal to background

ratio) using a suitable potassium standard, and adjusting ion lens voltages, plasma gas flows, torch position (relative to the sample cones) and rf forward power as necessary.

8.3 Run Sequence

Software acquisition menus should be prepared for measuring the ^^K and isotopes. The run order for the samples should be established according to the following protocol:

Analytical procedure blanks should be run at the beginning of the sequence, followed by the composition sample and then the analytical samples (including control materials) in randomized order. Instrument

[K-1] Page 7 of 13
K-1 Potassium by ICP-MS

mass discrimination measurement standards (see note) slnouid be run

after every three to four samples depending on tine stability of the

instrument.

Note on Mass Discrimination Measurement: The measured ^^K/^V

ratios should be corrected for the ICP-MS mass discrimination. This can be achieved by measurement of a calibration standard (SRM

918a) consisting of natural potassium. However, the natural ratio of ^^K / is approximately 14, which is very large and different from

the measured sample ratios. A better way to assess discrimination and measurement run discrimination drift is to carefully calibrate a

solution having a ratio matched more closely to the samples. A spike

calibration solution can conveniently be used for this purpose.

8.4 Isotope Ratio Acquisition The isotope ratio ^^K/'V should be acquired in peak jumping mode. A

typical acquisition scheme would be five to seven sets of ratios with a

total acquisition time of 60 s per set, using a dwell-time of 10 ms per

isotope. The isotope ratio data should be dead-time corrected and downloaded to a spreadsheet program such as Microsoft Excel. Inspect the ratios and remove any considered to be statistical outliers.

9. Data Reduction

9.1 Mass Discrimination Corrections

Convert the experimentally measured ^^K/*V isotope ratios to absolute

ratios using the following equation:

39,

' JcORR 39k

'Jtrue

MEAS where, [39^ is the discrimination corrected sample ratio

CORK

is the true ratio for the isotopic standard

TRUE

is the measured ratio for the isotopic standard

[K-1] Page 8 of 13
K-1 Potassium by I CP-MS

The measured ratios for the isotopic standard can be applied to sample

discrimination corrections in a number of ways, which will depend on the

discrimination drift as a function of time; (1) the ratio can be calculated by averaging the two standard measurements bracketing the samples of

interest, (2) a more sophisticated approach is to mathematically model

the drift in the standard ratio as a function of time and interpolate the

ratio for each sample. This may require more frequent measurement of the isotopic standard to define the drift function adequately.

9.2 Spike Calibration

Calculate the concentration of the spike in each spike calibration mixture using the following equation:

Mstd(BR -A)

where, Cspk is the concentration of potassium in the spike in |jg/g MsTD is the mass of potassium added to the spike mix (|jg) B is the natural abundance of the spike isotope R is the corrected ^^K/*V ratio in the spike mix A is the natural abundance of the reference isotope

H/ls is the mass of ''^ K spike aliquot added to the mix (g) K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike

Calculate the mean and standard deviation of the spike calibration mixtures. Blank corrections to the spike calibration are not normally necessary because they are simple mixtures prepared in dilute acid with minimal sample processing.

9.3 Calculation of Potassium Sample Concentrations

Calculate the potassium concentration (pg/g) in the sample using the

following equation:

c = rM3K(A3-B3R) 3 1 1 X —

(BR-A) ^ W

where, C is the concentration of potassium in the sample (|jg/g) Ms is the absolute mass of

'^^ K spike added (|jg)

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K-1 Potassium by ICP-MS

K is the natural to spike relative atomic mass ratio As is the abundance of the reference isotope in the spike Bs is the abundance of the spike isotope in the spike R is the corrected ^^K/*^ K ratio B is the natural abundance of the spike isotope A is the natural abundance of the reference isotope Sb is the absolute mean measured blank (pg) W is the mass of sample aliquot taken (g) An alternative approach, if desired, is to calculate the spike and sample

concentrations in mol/g. In this case the equations are slightly modified and the relative atomic masses of natural potassium and the spike are

not required for the calculations.

10. Uncertainty Analysis

The expanded uncertainty for the set of samples should be calculated according to ISO guidelines [3] by combining both Type A and Type B

uncertainties. Typical Type A uncertainty components for this measurement

might be the variability of the measured concentrations of potassium in the samples, the measurement of the spike concentration and the variability of the correction for the blank. Typical Type B uncertainty components might be

the uncertainty of the assay of the primary calibrant, weighing measurements, conversion correction for the density of the sample and

instrumental parameters such as detector dead-time correction, instrument mass discrimination correction and instrument background correction.

For a 95 % confidence level, combine the Type A and Type B components

and calculate the total expanded uncertainty using the following equation: U= k [Si'/dfi + ...+ Sn'/dfn + Bi^ +...+ Bn' ]

where, U is the expanded uncertainty k is the coverage factor s is the observed standard deviation of (n) Type A components

df is the degrees of freedom associated with each component B is the Type B component (n components) with infinite degrees

of freedom.

Report the expanded uncertainty in the same units as the sample

concentrations.

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K-1 Potassium by ICP-MS

11. Data Reporting

Convert data from a mass/mass basis to a mass/volume basis using the measured density of tlie serum. For clinical use, report potassium data in units of mmol/L. To convert data from |jg/mL to mg/dL divide by 10. To

convert from mmol/L to mg/dL multiply by 3.90983.

12. Performance Statement

12.1 IVIeasurement Repeatability The isotope ratio measurement repeatability is usually limited by

detector counting statistics. Typical measurement repeatability on a

quadrupole ICP-MS system for a ^^K/'V ratio approaching unity is 0.1- 0.2 %.

12.2 Detection Limit The instrument detection limit is influenced by the instrument background. A typical instrument detection limit for a well-optimized quadrupole ICP-MS instrument is 15 pg/mL. For a 0.7 g sample aliquot

this equates to a matrix detection limit of approximately 0.1 ng/mL, which is far below the potassium concentrations typically found in clinical fluids.

12.3 Instrument Background and Procedure Blank The ICP-MS instrument background tends to be very high for potassium owing to the direct isobaric interference of argon species. This can be very effectively reduced by means of "cool plasma"

technology. It should be possible to reduce the background for to around 1000 counts per second or lower by judicious choice of operating conditions. A typical procedure blank using this method is of the order of 50-100

ng (1-2 nmol), depending on the purity of the reagents, cleanliness of the containers and processing conditions. This is extremely low in relation to the normal potassium content in clinical fluids. For example,

the blank correction for the determination of potassium in blood serum

should be < 0.1 %.

12.4 Performance Summary A summary of typical method performance parameters expected for

this method are provided in Table 12-1.

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K-1 Potassium by ICP-MS

Table 12-1: Method Performance Summary

Parameter Typical Value or Range

Isotope ratio measurement repeatability 0.1-0.2%

Applicable concentration range

All ranges found in clinical specimens

Instrument background / sample ratio = 0.1 %

Procedure blank * 50 - 100 ng Sample throughput (measurement) 10 samples per hour

13. Reference Citations

1. Murphy K.E., Long S.E., Rearick M.S. and Ertas O.S., "The Accurate

Determination of Potassium and Calcium using Isotope Dilution Inductively Coupled "Cold" Plasma Mass Sectrometry."

J. Anal. At. Spectrom. 17, pp. 469-477 (2002).

2. Preparation and Testing of Reagent Water in the Clinical Laboratory; Approved Guideline - Third Edition, NCCLS Document C3-A3, NCCLS,

Wayne, Pennsylvania (1997).

3. Guide to the Expression of Uncertainty in Measurement, ISBN 92-67- 10188-9, 1st Ed. ISO, Switzerland, 1993.

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K-1 Potassium by I CP-MS

K-1 Revision History

Revision Date Author Record of Changes and Comments

1.0 9/2006 S E Long Initial draft method

[K-1] Page 13 of 13

Section 7 - Sodium Sodium

7.1 Clinical Significance Sodium is the predominant cation in serum or plasma. The majority of the sodium, approximately 140 mmol/L, is distributed in the extra-cellular fluid, while the intra-cellular fluid contains less than 10 mmol/L. The gradient between the two is closely associated with potassium, the major intra-cellular cation, through a sodium-potassium ion pump. Sodium controls the osmotic

pressure in the extra-cellular fluid and therefore influences the amount of water retained in the extra-cellular fluid. Sodium plays a critical role in the

function of the renal, circulatory and neuro-muscular systems of the body. The concentration of sodium in blood is maintained through dietary intake, balanced by renal excretion, which is modified by the hormone aldosterone. Elevated concentrations (>145 mmol/L) of sodium {hypernatremia) can result from excessive loss of water from the body through dehydration or excess

ingestion. Equally, depressed concentrations (<130 mmol/L) of sodium

{hyponatremia) may result from excessive loss through sweating, insufficient dietary intake, renal tubular acidosis (such as that resulting from burns), renal impairment or disorders of the gastro-intestinal system.

7.2 Normal Range and Reporting Units The normal range for sodium in serum or plasma is 130-145 mmol/L. Sodium clinical data are conventionally reported in mmol/L. Occasionally mEq/L is also used. To convert from mmol/L to mg/dL multiply by 2.29898. To convert from mg/dL to mmol/L multiply by 0.43498.

7.3 Routine Clinical Measurement Methods

In the clinical laboratory, the classical method for measuring sodium is flame atomic emission (flame photometry). Sodium has a very intense emission

doublet consisting of the D lines at 589.00 nm and 589.59 nm. This is a very

selective method but is not as easily automated or as rapid as some of the

potentiometric analyzers. Ion selective electrodes are now widely employed

for electrolyte measurements. These systems can provide analytical data in minutes with accuracy which is competitive with flame photometry. The

7-1
Section 7 - Sodium

compact size of some of these systems makes them amenable to portable use in a variety of settings including emergency care.

7.4 Available Standard Reference Materials Two blood serum SRMs are available with certified values for sodium, which

are SRM 909b {Human Serum), a two-level lyophilized material, and SRM

956b {Electrolytes in Frozen Human Serum), which is a three-level frozen

material intended for standardizing and calibrating ion selective electrode

analyzers. There are also reference values for sodium in SRM 2670a {Toxic Elements in Freeze-Dried Urine), a two-level, freeze-dried material SRM

919a {Sodium Chloride, Clinical Standard) is sold as a clinical primary standard which is intended for the purpose of instrument calibration and

standardization. Certificate information for these SRMs is summarized in Table 7-1

.

Table 7-1. Clinical Standard Reference Materials for Sodium

SRM Description Value

(mmol/L)

Expanded

Uncertainty

(mmol/L) 909b Level 1 Human Serum 120.76 0.92 909b Level 2 Human Serum 141.0 1.3 956b Level 1 Electrolytes in Frozen Human Serum 120.1 1.4 956b Level 2 Electrolytes in Frozen Human Serum 141.0 1.6 956b Level 3 Electrolytes in Frozen Human Serum 160.7 1.8 2670a Level 1 Toxic Elements in Urine (Freeze-Dried) (37.2) (0.7) 2670a Level 2 Toxic Elements in Urine (Freeze-Dried) (41.0) (0.9) SRM Clinical Standard

Purity (% mass

fraction)

Expanded

Uncertainty

(%) 919a Sodium Chloride (Clinical Standard) 99.89 0.03

( ) Values in Parentheses are Reference Values

7-2
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

Version Number 1.0 Approved S E Long

Effective Date Sep 2006

NA-1

SODIUM BY STABLE-ISOTOPE INTERNAL STANDARD

INDUCTIVELY COUPLED PLASMA - MASS

SPECTROMETRY

1. Applicability The method is applicable to the determination of sodium at physiological

levels in blood serum (frozen and lyophillzed), sweat and urine.

2. Principle of Measurement

The method is based on measurement of the sodium ion at m/z 23 using an

inductively coupled plasma - quadrupole mass spectrometer (ICP-MS). The

instrument is operated in analog detection mode, which reduces the

contribution from the instrument background which would otherwise be a

significant portion of the measured ion signal. As the ions are separated on

the basis of their mass to charge ratio, the method is extremely selective for sodium. There are no spectral interferences at this mass from species

normally found in clinical fluids. A separated stable-isotope of ^^Mg is added

to the sample as an internal standard.

3. Measurement Strategy and Experimental Design

Sodium has not commonly been measured by pulse counting detection ICP- MS because of high residual background contamination, which comprises a

substantial fraction of the total instrument dynamic range. Because of the

relatively high concentration of sodium in clinical materials, however, it is possible to measure sodium using analog ion detection. This approach has a much more effective dynamic range as well as immunity to detector deadtime effects and the limitations of counting statistics. Since sodium is monoisotopic, it is not possible to use isotope dilution. However, a ^^Mg

enriched isotope can be used as an internal standard with measurement of the ^^Na/^^Mg isotope ratio. ^^Mg is used for the internal standard because it is close in mass to sodium and similar in ionization behavior. The ^^Mg

isotope is used because clinical materials also contain a small amount of

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NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

magnesium and the background contribution from this is minimized by the use of ^^Mg as the internal standard rather than natural magnesium, which is predominantly ^'^Mg. All of the ^^Na/^^Mg ratios are internally corrected by concurrent measurement of the ^''Mg/^^Mg ratio and subtracting out the small

contribution from the ^^Mg component indigenous to the sample.

4. Safety

Clinical fluids are biohazardous materials which may contain pathogenic

substances. The usual safety precautions should be taken to prevent exposure during handling, including the use of gloves at all times. Although most materials will have been screened for such substances, a clinical monitoring regimen, including screening for HIV 1 and 2 and vaccination against hepatitis B is recommended for personnel handling clinical specimens on an extended basis.

5. Required Reagents

5.1 ^^Mg Isotopic Spike A spike having an isotopic purity >99 % can be obtained through Oak

Ridge National Laboratory (ORNL) or other stable-isotope supplier in the form of ^^MgO. A stock solution can be prepared by dissolving 100 mg of the ^^MgO in 20 mL of 2 % (volume fraction) high-purity nitric acid and

storing in a cleaned Teflon® fluoropolymer bottle.

5.2 SRM 919a, Sodium Chloride Clinical Standard

Dry for 3 h in a convection oven at 110 °C. Store in a desiccator when

not in use. The certified purity of the material is 99.89 % (mass fraction) with an expanded uncertainty of 0.03 %. The relative mass fraction of sodium in SRM 919a adjusted for the purity is 0.39294.

5.3 High-Purity Nitric acid

High-purity acid is essential to minimize the extent of sodium

contamination from reagent sources. High-purity acid may be obtained from a number of commercial suppliers.

5.4 High-Purity Water

De-ionized or quartz-distilled water which has been tested and is low in sodium contamination is used for sample dilution. The water should meet

or exceed the specifications of CAP/NCCLS Type I water [1].

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NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

6. Equipment

6.1 ICP-MS

The method utilizes a standard ICP-MS system employing a quadrupole mass spectrometer. The instrument should be capable of operating in the analog detection mode using a high-efficiency pneumatic nebulizer system. The use of a sample introduction system using an inert material

is beneficial in reducing instrument background.

6.2 Polyethylene Bottles Clean 30 mL polyethlylene sample bottles for sample preparation and

analysis. Note on Sample Bottles: Polyethylene sample bottles formulated for low

trace-metal content should be used. These contain very low levels of

electrolyte contaminants. Containers such as polypropylene centrifuge tubes should not be used for this purpose as they frequently contain appreciable levels of contaminants.

6.3 Micro-Pipette A 1 mL capacity automatic or manual micro-pipette for dispensing sample and standard aliquots. A capped plastic syringe fitted with a Teflon® fluoropolymer uptake tube may also be used for this purpose.

6.4 Analytical Balance

Five-place analytical balance for weighing sample and standard aliquots. The balance should be calibrated and verified to be functioning correctly

prior to weighing measurements.

7. Sample Handling and Preparation

7.1 Analytical Sample

The analytical sample should be equilibrated to room temperature prior to measurement. Follow provider instructions for reconstitution of lyophilized material or equilibration from freezer temperature. All sample

preparation should be carried out in a Class 10 clean environment with the use of protective clothing and gloves.

7.2 Aliquoting and Dilution Approximately 0.25 g of the liquid sample should be weighed accurately using a five-place analytical balance into a 30 mL polyethylene bottle by means of a transfer pipette or syringe. The sample is diluted with highpurity water to approximately 25 mL. The exact volume is not critical. An

[NA-1] Page 3 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

accurately weighed aliquot of Mg spike (5000 pg/g as MgO) is then added to the solution. The aliquot volume is typically 0.25 g but the optimum volume will vary depending on the characteristics of the ICP- MS system and the concentration of sodium in the matrix. The spike should not be added to the sample until dilution has taken place because

the acid in the spike solution could otherwise precipitate proteins in the sample.

7.3 Acidification The solution should be acidified to 0.25 % (volume fraction) nitric acid by adding approximately 60 pL of high-purity nithc acid and mixing

thoroughly. On no account should the acid concentration be greater than

this as there is a risk of precipitating proteins from the sample. Although

the sample could still be analyzed, it is better to avoid doing this. Addition of a small amount of acid is necessary to improve sample washout during the measurement process.

7.4 Stability The prepared solutions should be stable for several days. However,

determinations should be carried out as soon as possible to avoid the

risk of contamination, losses or stability issues.

7.5 Calibration Solution Prepare an instrument calibration solution from SRM 919a which falls within the expected range of concentrations of the samples, and spike

this solution as for the samples from the same stock of ^^Mg isotope.

7.6 Procedure Blanks

Prepare a minimum of three procedure blanks by adding approximately

0.25 g of the ^^Mg spike to a 30 ml polyethylene bottle and treating in an

identical manner to the samples.

8. Analytical Measurement

8.1 Instrument Preparation

Prior to analytical measurements, the ICP-MS system should be

equilibrated for 30 min. The sample introduction system should be

rigorously cleaned before use, including the sampler and skimmer cones,

peristaltic pump tubing, plasma torch and spray chamber. Sodium

background from the instrument is usually very high and these procedures are designed to mitigate the background as much as

possible. It is recommended that new peristaltic pump tubing be installed

[NA-1] Page 4 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

prior to measurements. A dilute nitric acid solution (2 % volume fraction) should be pumped through the tubing for a while to remove any

leachable sodium from the tubing.

8.2 Instrument Optimization The instrument should be optimized for maximum ion transmission at m/z 23 by nebulizing a standard solution of sodium and adjusting ion lens voltages, plasma gas flows and rf forward power. The voltage on the analog stage of the detector should be adjusted such that the signal to background ratio is maximized.

Note on Optimization of Instrument Conditions: The RF forward power to the plasma should be maintained as high as possible to ensure complete decomposition of protein material in the sample. The use of low RF powers (typically less than 1300 W) will result in significantly degraded measurement precision.

8.3 Run Sequence

Software acquisition menus should be prepared for measuring the ^^Na,

^'^Mg and ^^Mg isotopes. The run order for the samples should be

established according to the following protocol: Analytical procedure blanks should be run at the beginning of the sequence, followed by the composition sample and then the analytical samples (including control materials) in randomized order. Instrument

calibration standards should be run after every three to four samples

depending on the mass discrimination stability of the instrument.

8.4 Isotope Ratio Acquisition The isotope ratios ^^Na/^^Mg and ^'*Mg/^^Mg should be acquired in peak

jumping mode. A typical acquisition scheme would be five to seven sets

of ratios with a total acquisition time of 60 s per set, using a dwell-time of 10 ms per isotope. Dead-time corrections to the data are not necessary as analog detection mode is used. The isotope ratio data should be downloaded to a spreadsheet program such as Microsoft Excel. Inspect the ratios and remove any considered to be statistical outliers.

9. Data Reduction

9.1 Correction Equations for Magnesium in the Sample

Convert the measured isotope ratio ^^Na/^^Mgmeas to the true ratio ^^Na/^^Mg,rue which is corrected for the magnesium sample contribution using the following equations:

[NA-1] Page 5 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

26| _ 26| . f> 24| iA\ meas ~ 'true ^ '-'nat • iserum \

I f where, ^^Leas is the measured signal intensity at m/z 26

^^Itrue is the true signal intensity for Mg spike

^'*lseruni is the Signal intensity for serum Mg

Cnat is the correction for the natural composition ^^Mg/^^Mg

24, _ 24, p 26, ,<5v 'serum ~ 'meas " '-'spk • 'true \^) where, ^'^Leas is the measured signal intensity at m/z 24

Cspk is the ratio correction for the spike composition ^''Mg/^^Mg

'meas ~ 'true ^ Cnat ( 'meas " Cspk • '"'true) (3)

^^'meas ~ (

"I " Cnat Cgpk) ^^'true "•" Cnat • ^*'meas (4)

^^'true - (^^'meas~ Cnat ^'*'meas) ("I Cnat • Cgpk) ^ (5) The magnitude of this correction process Is small, typically amounting to < 0.1 % for a sample sodium concentration In the region of 140 mmol/L.

.2 Calculation of Sodium Concentration

Calculate the sodium concentration (pg/g) in the sample using the

following equation:

Csmp - { (Rsmp/ RstdX IVIstd X IWIiSsmp' M|Sstd ) " Bavg } X (IVIsmp)

^

where, Csmp is the concentration of sodium in the sample In pg/g Rsmp is the corrected ^^Na/^^Mg ratio in the sample

Rstd is the ^^Na/^^Mg ratio in the standard

Mstd is the absolute mass of Na in the prepared standard (pg) Mis smp is the mass of ^^Mg spike added to the sample (g) Mis std is the mass of ^^Mg spike added to the standard (g) Bavg is the mean measured procedure blank (pg) Msmp is the mass of sample taken (g)

10. Uncertainty Analysis

The expanded uncertainty for the set of samples should be calculated according to ISO guidelines [3] by combining both Type A and Type B

uncertainties. Typical Type A uncertainty components for this measurement

might be the variability of the measured concentrations of sodium in the samples, the instrument calibration process and the variability of the

contribution of the procedure blank correction. Typical Type B uncertainty components might be the uncertainty of the assay of the SRM 919a calibrant, the weighing measurements, the calibration standard response

[NA-1] Page 6 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

(discrimination) drift, ttie ICP-MS instrument background subtraction, the

correction for the magnesium present in the sample and the conversion

correction for the density of the sample.

For a 95 % confidence level, combine the Type A and Type B components

and calculate the total expanded uncertainty using the following equation: U= /f[Si2/dfi+ ...+ Sn^/dfn + Bi2+...+ Bn']'''

where, U is the expanded uncertainty k is the coverage factor s is the observed standard deviation of (n) Type A components

df is the degrees of freedom associated with each component B is the Type B component (n components) with infinite degrees

of freedom.

Report the expanded uncertainty in the same units as the sample

concentrations.

11. Data Reporting

Convert data from a mass/mass basis to a mass/volume basis using the measured density of the serum. Report data in units of mmol/L. To convert data from pg/mL to mmol/L, multiply by 0.043498.

12. Performance Statement

12.1 Measurement Reproducibility The isotope ratio measurement reproducibility is not limited by

counting statistics. Typical measurement repeatability for both of the

ratios, '''Mg/'^Mg and '^Na/'^Mg is 0.1-0.2 % [2].

12.2 Linearity Analog mode detection is subject to non-linearity at high concentrations. The linearity of the system has been tested [2] using

four sodium calibration standards covering the range 20-75 pg/mL

which were spiked with a constant amount of natural Mg and the ratio ^^Na/^'^Mg measured as a function of the sodium concentration. The

resulting calibration curve was fitted through zero and clearly indicated

that the measurement system is linear (r = 0.999995) at least to this

limit.

[NA-1] Page 7 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

12.3 Instrument Background and Procedure Blank The use of analog mode ion detection dramatically reduces the

effective measured instrument background. By careful optimization, the magnitude of background corrections to the measurement data can be

limited to approximately 0.1 % or less. The instrument background

under these conditions is very stable. A typical procedure blank [2] is 400 ng (n=7, 1s = 69 ng), which represents a very small source of

uncertainty.

12.4 Method Validation Data using NIST SRM 909b The method has been tested using SRM 909b [2]. Nine statistically selected bottles of SRM 909b Level 1 were tested using the method. Four separate bottles were tested using a primary gravimetric method.

In addition, six bottles of the Level 2 material were tested by the ICP- MS method and five separate bottles were tested by gravimetry. The

analytical data obtained by both methods are compiled in Appendix A, Table A-1 , together with the expanded uncertainties and the certified values listed in the SRM 909b Certificate of Analysis. Excellent agreement between the methods was obtained. For Level 1, the

relative difference between the arithmetic means of the methods was

0.14 %, and for Level 2 the relative difference was 0.07 %. In both cases the means of the two methods were within the calculated expanded uncertainties of each method.

12.5 Method Validation Data using NIST SRM 956a The method has been tested using SRM 956a [2]. Five measurements

from separate ampoules were made for each of the three concentration levels. Gravimetric measurements were used to certify the sodium concentrations and were made independently on six ampoules each of Levels 1 and 2 and five ampoules of Level 3. Analytical data obtained by both methods are summarized in Appendix

A, Table A-2, together with the expanded uncertainties and the

certified values in the SRM Certificate of Analysis. Again excellent agreement was obtained between the two methods for all of the concentration levels. The difference between the two methods was

0.16 %, 0.05 % and 0.13 % respectively for Levels 1, 2 and 3, and the means of the two methods were within the calculated expanded

uncertainties of each method.

12.6 Performance Summary A summary of typical method performance parameters expected for

this method are provided in Table 12-1.

[NA-1] Page 8 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

Table 12-1: Method Performance Summary

Parameter Typical Value or Range

Isotope ratio measurement precision 0.1-0.2%

Applicable concentration range 120-160 mmol/L

Instrument background / sample ratio <0.1 %

Procedure blank < 400 ng Sample throughput (measurement) 10 samples per hour

13. Reference Citations

1 . Preparation and Testing of Reagent Water in ttie Clinical Laboratory; Approved Guideline - Third Edition, NCCLS Document C3-A3, NCCLS,

Wayne, Pennsylvania (1997).

2. Long S.E. and Vetter T.W., "Determination of Sodium in Blood Serum by

Inductively Coupled Plasma Mass Spectrometry.

J. Anal. Atom.Spectrom., 17 (12), pp.1589-1594 (2002).

3. Guide to the Expression of Uncertainty in Measurement, ISBN 92-67-

10188-9, 1st Ed. ISO, Switzerland, 1993.

[NA-1] Page 9 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

NA-1 Revision History

Revision Date Author Record of Changes and Comments

1.0 9/2006 S E Long Initial draft method

[NA-1] Page 10 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

Appendix A

Table A-1. ICP-MS and Gravimetric Data for Sodium in SRM 909b

Level 1 , mmol/L Level 2, mmol/L

Sample ICP-MS Gravimetry ICP-MS i Gravimetry

1 120.73 120.75 141.13 141.21 2 121.31 120.90 140.92 140.95

3 120.94 120.53 141.61 141.02 4 120.97 120.83 140.41 140.94

5 120.74 141.23 141.08 6 120.76 141.55

7 121.27

8 120.84

9 120.73 Mean 120.92 120.75 141.14 141.04

Standard Deviation 0.23 0.16 0.44 0.11 RSD (%) 0.19 0.13 0.31 0.08 Expanded Uncertainty^ 0.45 0.31 0.62 0.26

Certified Value' 120.76 ±0.92 141. 0± 1.3

^ Expanded uncertainty calculated at a 95 % confidence interval. "NIST certificate value and expanded uncertainty for a 95 % statistical tolerance interval reflecting the effects of measurement uncertainty and variability in the mass of dry serum

fill mass.

[NA-1] Page 11 of 12
NA-1 Sodium by Stable Isotope Internal Standard ICP-MS

Table A-2. ICP-MS and Gravimetric Data for Sodium In SRM 956a

Level 1, mmol/L Level 2, mmol/L Level 3, mmol/L

Sample ICP-MS Gravimetry ICP-MS Gravimetry ICP-MS Gravimetry

1 191 cil 191 4^; 1 ^ 1 .to A An i^i I*tU.O I 1 H 1 . UU iRn 07 1 1 n 2 120 64 121 59 141 24 141 nc; iRn no iRn R'k

"

3

120 95 12l'^46 141.08 140"91 161.40 160.88""" 4 121.42 121.40 140.61 141.02 161.12 160.89

cO 121.59 121.35 141.41 141.36 160.73 160.75 6 121.22 140.89 Mean 121.22 121.41 140.97 141.04 160.68 160.89 Standard

Deviation

0.41 0.12 0.39 0.17 0.60 0.13 RSD (%) 0.34 0.10 0.28 0.12 0.37 0.08 Expanded

Uncertainty^ 0.59 0.31 0.62 0.29 0.82 0.30

Certified Value'' 121.4 ±0.3 141.0±0.3 160.9 ±0.4

^Expanded uncertainty calculated at a 95 % confidence interval. "NIST certificate value and expanded uncertainty for a 95 % statistical confidence interval.

[NA-1] Page 12 of 12