SW-846 Update V 6800 - 1 Revision 1 July 2014 METHOD 6800

ELEMENTAL AND MOLECULAR SPECIATED ISOTOPE DILUTION MASS SPECTROMETRY

SW-846 is not intended to be an analytical training manual. Therefore, method procedures are written based on the assumption that they will be performed by analysts who are formally trained in at least the basic principles o f chemical analysis and in the use of the subject technology.

In addition, SW-846 methods, with the exception of required method use for the analysis of method-defined parameters, are intended to be gu idance methods which contain general information on how to perform an analytical procedu re or technique which a laboratory can use as a basic starting point for generating its own de tailed standard operating procedure (SOP), either for its own general use or for a specific pr oject application. The performance data included in this method are for guidance purposes o nly, and are not intended to be and must not be used as absolute quality control (QC) acceptance criteria for purposes of laboratory
accreditation.

1.0 SCOPE AND APPLICATION

For a summary of changes in this version from the p reviously published draft method 6800, February 2007 Rev 0, please see Appendix A at the end of this document.

1.1 This method consists of two approaches: (1) is otope dilution mass spectrometry (IDMS) for the determination of the total concentrations o f elements and molecules and (2) molecular speciated isotope dilution mass spectrometry (SIDMS ) for the determination of elemental and molecular species. IUPAC defines speciation analys is as “analyzing chemical compounds that differ in isotopic composition, conformation, oxida tion or electronic state, or in the nature of their complexed or covalently bound substituents, which c an be regarded as distinct chemical species.” This method is applicable to the determi nation of total elements as well as elemental species and molecular species at parts per billion (ppb), parts per trillion (ppt) and sub-ppt levels in samples of various types; in waters, solids, blo od, foods or in extracts or digests. In general,
elements and molecules that have more than one avai lable stable isotope or combination of enriched isotopes in molecules can be analyzed by I DMS and SIDMS. SIDMS may require more isotopes of an element and/or molecule, depend ing on the number of interconvertible species.SW-846 Update V 6800 - 2 Revision 1 July 2014 The analyses of the following elements are applicab le by this method:

Element CASRN a

Antimony (Sb) 7440-36-0

Boron (B) 7440-42-8

Barium (Ba) 7440-39-3

Cadmium (Cd) 7440-43-9

Calcium (Ca) 7440-70-2

Chromium (Cr) 7440-47-3
Copper (Cu) 7440-50-8

Iron (Fe) 7439-89-6

Lead (Pb) 7439-92-1
Magnesium (Mg) 7439-95-4
Mercury (Hg) 7439-97-6
Molybdenum (Mo) 7439-98-7

Nickel (Ni) 7440-02-0

Potassium (K) 7440-09-7

Selenium (Se) 7782-49-2

Silver (Ag) 7440-22-4

Strontium (Sr) 7440-24-6

Thallium (TI) 7440-28-0

Vanadium (V) 7440-62-2

Zinc (Zn) 7440-66-6

a Chemical Abstract Service Registry Number

Molecules and species may be analyzed by SIDMS if a ppropriate performance is demonstrated for an analyte, in the matrix type and at concentration levels of interest. See examples in the list below (see Sec. 9.0).

 Glyphosate and other pesticides

 Sodium azide and other toxicant and explosives

 Molecular Species, such as oxidized and reduced fo rms of glutathione, and peptides and proteins,

 Toxin and toxicant species conjugated to glutathio ne

 Converting related species such as isoforms and bi omarkers

 Enzymes in tissues and body fluids

 Toxins and toxicants in dietary supplements, food and consumer products

 Toxin and toxicant species in water and foods

 Chemical, Biological, Radiological, Nuclear, Explo sives (CBRNE) for Homeland Defense and Homeland Security

1.2 IDMS is based on the addition of a known amount of enriched isotope to a sample. Equilibration of the spike isotope with the natural element/molecule/species in the sample alters the isotope ratio that is measured. With the known isotopic abundance of both the spike and the analyte in the sample (element/molecule/species ), the amount of the spike added to the known amount of analyte, concentration of the spike added, and the altered isotope ratio, the concentration of the analyte in the sample can be c alculated. Direct calculations independent of
traditional calibration curves are described in sec tion 12.

1.3 IDMS has proven to be a technique of high accur acy for the determination of the total concentration of elements or molecules in var ious matrices (Refs. 1, 9, 10, 33, 34, and 43).SW-846 Update V 6800 - 3 Revision 1 July 2014 IDMS has several advantages over conventional calib ration methodologies. Partial loss of the analyte after equilibration of the spike and the sa mple will not influence the accuracy of the measurement. Fewer physical and chemical interfere nces influence the determination as they have the same effects on each isotope of the same e lement or molecule. The isotope ratio to be measured for quantification by IDMS can be measu red at a high level of precision, typically with a relative standard deviation (RSD) of 0.25% t o 1.0%. Quantification is based on direct mathematical calculation from determined isotopic r atios and known constants, and does not depend on a traditional calibration curve or sample recovery to achieve accuracy.

1.4 SIDMS takes a unique approach to speciation ana lysis that differs from traditional methods. Traditional methods of speciation analysi s attempt to hold each species static during sample collection and preparation and while making the measurement. Unfortunately, interconversion and transformation of species can o ccur, resulting in biased results. SIDMS has been developed to correct for such species conversi ons. In SIDMS, each species is “labeled” with an isotopically enriched spike in the correspo nding species form. Thus, the interconversions that occur after spiking are trace able and can be corrected. While SIDMS maintains the advantages of IDMS, it is capable of correcting for the degradation of the species or the interconversions between the species for mas s spectrometer drift and other errors, such as incomplete extraction of species (Refs. 2, 3, 5 - 8, 11-22, 24-32, and 35). SIDMS is also a diagnostic tool that permits the assessment of spec ies-altering procedures through evaluation and validation of other more traditional speciation analysis methods. SIDMS is to be used in conjunction with other methods when knowledge of sp ecies concentration, conversion and stability are necessary.

1.5 Both IDMS and SIDMS require the equilibration o f the isotopically enriched species spike with the natural isotopes of the species of i nterest present in the sample. For IDMS, the spike and sample can be in different chemical forms , and later grouped as the same species form and only total elemental and or molecular conc entrations will be measured. In general, IDMS equilibration of the spike and sample isotopes occurs during sample decomposition or transformation, which also destroys all species-spe cific information when the isotopes of an
element or molecule are all oxidized or reduced or transformed to the same oxidation state.

For SIDMS, spikes and samples must be in the same s peciated form and be equilibrated in solution (gaseous, liquid, solid), during extrac tion or on a separation or extraction column, as demonstrated with i-Spike cartridges (Refs. 34 and 43). This requires the analyte in spikes to be in the same molecular form as those in the sample. Efforts are taken to keep the species in their original forms after spiking as much as possible. F or solutions or liquid samples, spiking and equilibration procedures can be as simple as mixing a known amount of the sample and the spikes prior to analysis. Aqueous samples such as d rinking water, groundwater, etc. may be directly spiked and analyzed. Solid samples such as soils, sludges, sediments, industrial materials, biological tissues, botanicals, lysed ce lls, foods, including dietary supplements and consumer products, mixed samples, blood, urine and other samples containing solid matrices require spiking before, during or after extraction or prior to analysis to solubilize and equilibrate the species before introduction to the mass spectro meter. Animal and human clinical studies are areas where the higher accuracy and precision provi ded by method 6800 yield more statistically significant data than conventional methods. This m ethod has been and can be used to certify reference materials, for environmental forensic ana lysis, and for detecting chemical and biological agents (specifically referred to as CBRN E, Chemical Biological Radiological Nuclear and Explosives) in homeland defense and homeland se curity applications.

1.6 Sensitivity and optimum ranges of elements may vary with the matrix, separation method, and isotope ratio measurement methods and s pecific ionization source and specific mass spectrometer. Using a liquid chromatography ( LC) in-line with inductively coupled plasmaSW-846 Update V 6800 - 4 Revision 1 July 2014 mass spectrometry (ICP-MS), it is convenient to sep arate elemental or molecular species and to measure their isotope ratios. Using a liquid chrom atography (LC, or Nano-LC in-line with QTOF, triple quadrupole mass spectrometry (QQQ) or other MS instruments, it is appropriate to separate molecular species and to measure their iso tope ratios. Although Method 6800 is not restricted to LC as the separation method of the sp ecies and the ICP-MS as the isotope ratio measurement MS, this document will use these techni ques as examples for the most part in describing the Method’s procedures. Molecular spec ies separation methods such as extraction, precipitation, solid phase chelation, and isotope r atio measurement techniques such as thermal ionization mass spectrometry (TIMS), electrospray i onization mass spectrometry (ESI-MS), matrix assisted laser desorption ionization mass sp ectrometry (MALDI-MS) and other mass spectrometers such as time-of-flight mass spectrome ters (TOF-MS) can also be used. NanoESI-MS –TOF, quadrupole time-of-flight (QTOF), QQQ, GC-MS, GC-QTOF, and GC-QQQ are mass spectrometric techniques that also separate an d measure the species and isotopic ratios of molecular species and therefore are included as mass spectrometry platforms.

1.7 Prior to employing this Method, analysts are a dvised to consult the base method for each type of procedure that may be employed in the overall analysis for additional information on quality control procedures, developm ent of QC acceptance criteria, calculations, and general guidance. Analysts also should consult the disclaimer statement at the front of the manual and the information in SW-846 Chapter Two fo r guidance on the intended flexibility in the choice of methods, apparatus, materials, reagen ts, and supplies, and on the responsibilities of the analyst for demonstrating that the technique s employed are appropriate for the analytes of interest, in the matrix of interest, and at the levels of concern.

In addition, analysts and data users are advised th at, except where explicitly specified in a regulation, the use of SW-846 methods is not mandatory in response to Federal testing requirements. The information contained in this me thod is provided by EPA as guidance to be used by the analyst and the regulated community in making judgments necessary to generate results that meet the data quality objectives for t he intended application.

1.8 This method is restricted to be used by, or und er supervision of, properly experienced and trained personnel. Each analyst mu st demonstrate the ability to generate acceptable results with this method. The scientific literature should be consulted for fundamental training and for updating the fundamental of this m ethod and for additional examples of application.

2.0 SUMMARY OF METHOD

2.1 IDMS method

2.1.1 Samples may require a variety of sample prepa ration procedures, depending on sample matrices and the isotope ratio measurement methods. One primary purpose of sample preparation is to solubilize the analyte of interest and equilibrate the spike isotopes with the isotopes in the sample. So lids, slurries, and suspended material must be subjected to digestion after spiking by usi ng appropriate sample preparation methods (such as Method 3052). Water samples may n ot require digestion when ICP-MS is used as a detection method because ICP can destr oy elemental species and thus many species may become indistinguishable for ICP-MS. M olecular species usually have the molecule-specific isotopes added during extraction to achieve equilibration of the species prior to introduction to ESI-MS, nano-ESI-TOF-MS, G C-MS, MALDI-MS, LC-QQQ, GCTOF, GC-QQQ, LC(IC)-ICP-MS, GC-ICP-MS and other mol ecular mass spectrometric analysis techniques.SW-846 Update V 6800 - 5 Revision 1 July 2014 2.1.2 A measured representative sample is thoroughl y mixed with a measured amount of the isotopic spike. If a digestion proce dure is required, the spiked sample is then digested to equilibrate the spikes and samples . The sample solutions are then measured with mass spectrometry such as ICP-MS to o btain the altered isotope ratios. Method 6020 can be used as a reference method for I CP-MS detection. In addition to Method 6020, dead time correction and mass bias cor rection must be included in the measurement protocol. The equations described in S ec. 12.1 are used to calculate the
concentrations.

Figure 2 shows an example of an IDMS determination of vanadium in crude oil (Ref. 1). IDMS determination of the restricted ele ments Cd, Cr, Pb and Hg in electrical components are demonstrated in Table 11 (Ref. 26).

2.2 SIDMS method

2.2.1 Speciated samples generally require sample pr eparation specific to the sample matrices, species, and the isotope ratio mea surement method. The purpose of sample preparation is to solubilize the species of interest and to equilibrate the natural and spiked species, creating a homogeneous solution. S olids, slurries, and suspended material must be subjected to extraction before or after spiking, using appropriate sample preparation methods (such as Method 3060A for the d etermination of Cr(VI) in soils, and Method 3200 for the determination of mercury specie s in food, blood and tissues, and helix chromatography in the case of protein, peptid e or proteomics). Method 3546 and other sample preparation methods in SW-845 Chapters Three and Four are applicable for
environmental health and other toxicants in the env ironment and human health. Water samples may not need extraction and in many cases m ay be equilibrated and analyzed directly. In contrast to total molecular and eleme nt analysis, efforts must be taken to avoid the destruction of the species in SIDMS. For examp le, in molecular species analysis, reduced glutathione in blood, plasma and/or serum i s transformed to oxidized glutathione (dimer species) by oxygen in the air during blood d raw, storage, shipment or analysis. Sample preparation, spiking, and spike equilibratio n must be carried out in the absence of oxygen prior to LC-ESI-MS, nano-ESI-MS or MALDI-MS analysis. Species of mercuryglutathione complex appear to be more stable to oxy gen but methylation and demethylation of these mercury species can occur du ring sample preparation and analysis.

2.2.2 Although SIDMS is a general method applicable to many elements in various species forms, for example chromium species , Cr(III) and Cr(VI), or proteomic species such as oxidized and reduced glutathione in human health and environmental samples such as water samples or soil extracts bloo d and serum will be considered here for demonstration of molecular species analysis. F or the chromium species two isotopic spikes are prepared and characterized as follows: 50 Cr(III) spike enriched in 50 Cr and

53 Cr(VI) spike enriched in 53 Cr. The dominant natural isotope for Cr is 52 Cr, at 83.79% abundance ( 50 Cr, 4.35%; 53 Cr, 9.50%; 54 Cr, 2.36%). A measured amount of a representative aqueous sample is mixed well with an appropriate amount of 50 Cr(III) and

53 Cr(VI) spike solutions. Cr(III) and Cr(VI) are sep arated using chromatography or another separation method (Figure 3), and four isotope rat ios are measured: 50 Cr(III)/ 52 Cr(III),

53 Cr(III)/ 52 Cr(III), 50 Cr(VI)/ 52 Cr(VI), and 53 Cr(VI)/ 52 Cr(VI). The concentrations of the species are determined from speciated isotope dilution calc ulation in section 12.2.SW-846 Update V 6800 - 6 Revision 1 July 2014 Figures 4 and 5 show examples of SIDMS for the dete rmination of chromium species in an aqueous sample. Any species transfor mation between Cr(VI) and Cr(III) is mathematically corrected, as described in Sec. 12.2

.

2.2.3 SIDMS may be called upon to analyze multiple species of interest that require selection of different isotopically enriche d species and combinations of species. For example, if reduced and oxidized glutathione ar e being determined, the two isotopic forms of reduced glutathione and oxidized glutathio ne are equilibrated prior to shipment of blood samples to correct for oxidation of reduced g lutathione to oxidized glutathione in shipment and during analysis to produce a statistic ally accurate result that is actionable by the practitioners. However, if for example the inor ganic (Hg 2+ ) and methylmercury (CH 3 Hg + ) conjugated forms of glutathione are being quantif ied, then species enriched with C-13, N-15, O-17 or O-18, or Hg-196 through Hg-204 are potential alternative spikes to quantify conjugates of glutathione and mercury spec ies.

Figures 8 A-D show examples of SIDMS for the deter mination of glutathione mercury species of glutathione-Hg dimer and glutath ione-methylmercury monomer. Figure 9 demonstrates nano-ESI-QTOF-MS abundance of reduced and oxidized glutathione for the analysis of Total GSH and GSH/G SSG ratios.

3.0 DEFINITIONS

See the "Glossary" at the end of this document for definitions of applicable terms. Also refer to SW-846 Chapters One and Three, and the man ufacturer’s documentation for definitions that may be relevant to these procedures.

4.0 INTERFERENCES

4.1 Solvents, reagents, glassware, and other sample processing hardware may yield artifacts and/or interferences to sample analysis. All of these materials must be demonstrated to be free from interferences under the conditions of the analysis by analyzing method blanks. Specific selection of reagents and purification of solvents by distillation of all-glass systems may be necessary. Refer to each method to be used for specific guidance on quality control procedures and to SW-846 Chapter Three for general guidance on the cleaning of glassware.

4.2 Sample preparation

4.2.1 Because this Method requires equilibration of the spike isotope(s) with the natural isotopes, solid samples must be digested, d issolved and/or extracted into solution. If the analyte of interest does not completely diss olve, if the spike or sample isotopes are selectively lost before equilibration, or if contam ination occurs in the sample preparation process, the measured isotope ratio will not reflec t the accurate ratio of the added spike atoms to sample atoms for that element or species ( Refs. 1 and 3).

4.2.2 In general, SIDMS incorporates the requiremen t that all the converted species can be found in other species that are bein g monitored. As an example, in the interconversion between Cr(III) and Cr(VI), the los t Cr in one species must be found in the other species. Similarly, for example, in molecula r species, if reduced and oxidized glutathione or inorganic glutathione and methylmerc ury-glutathione are found to transform among them, the isotopic signature from the enriche d species spike are able to correct for these transformations. Thus, efforts should be made to keep all species in solution after equilibration with the enriched isotope species of interest.SW-846 Update V 6800 - 7 Revision 1 July 2014 4.2.3 Preservation of the species is required in SI DMS since interconversion degrades the precision of the measurement. Complet e conversion of the species will prevent the deconvolution of the species concentrat ion. Thus, sample decomposition applied for total element determination is inapprop riate for SIDMS. However, the altered isotope ratios will indicate the conversion that ha s occurred and will not lead to an incorrect answer, but to a situation where the conc entration cannot be determined and the species interconversions has been revealed. As lit tle as 1 to 5% of the species have remained with correction to the original concentrat ion based on the calculations and equations are provided (Refs. 30, 34, and 35). Ap proaches developed to maintain the species are applicable to SIDMS.

4.3 Isotope ratio measurement

4.3.1 Discussions about isobaric interference, doub ly-charged ion interference, and memory interference in Method 6020B are applica ble to this method. The discussion about the physical interference, suggesting the add ition of an internal standard, does not apply. The internal standard is unnecessary becaus e the isotope ratio measurement is free from physical interferences and each measureme nt is a standardized measurement without traditional calibration curves (General con siderations for isotope ratio measurement can be found in Sec. 13.5.1).

4.3.2 Dead time measurement in ICP-MS must be perfo rmed daily. At high count rates, two effects cause pulse counting syste ms to count fewer events than actually occur (Sec. 13.5.2 and 13.5.3). The first is dead time ( τ ), the interval during which the detector and its associated counting electronics ar e unable to resolve successive pulses. If the true rate, n, is much less than 1/ τ , then:



### ) 1( τ 

### m n m

− ≈

where m is the observed rate. The second effect is the lo ss of gain at high rates caused by the inability of the multiplier’s dynode string to supply enough current to maintain constant dynode voltage drops. This effect is indi cated by a sharp increase in apparent dead time at high count rates. Both effects cause the measured isotope ratios to diverge from the true isotope ratios with increasing count rate. While the dead time can be mathematically corrected, the gain loss cannot.

In molecular measurements on a TOF, QTOF or QQQ at low concentrations dead time may not be significant compared to the signal of th e analytes. In these cases dead time may be neglected if it does not significantly affec t the statistical uncertainty of the
measurements.

A series of solutions with different concentrations can be prepared from isotopically certified standards for the determination of dead t ime. The concentrations may not be accurate, but the concentrations should spread out evenly, covering the blank to the highest count rate that may be used in measurements . The isotope pairs that are monitored should have large differences between the ir isotopic abundances, since the major isotopes suffer dead time effects much more s eriously than minor isotopes; this makes the dead time correction significant. The sum of the dead-time-corrected counts is used for calculating the isotope ratios after backg round subtraction.SW-846 Update V 6800 - 8 Revision 1 July 2014 _ R m is the dead-time-corrected isotope ratio;

_ Isotope1 

### S sample/standard and Isotope2 

### S sample/standard are the integrated dead-timecorrected counts for the sample or standard of Isot ope1 and Isotope2, respectively;

_ Isotope1 

### S background and Isotope2 

### S background are the integrated dead-time-correctedcounts for the background of Isotope1 and Isotope2, respectively.

As shown in Figure 1, which displays the 50 Cr/ 52 Cr ratios for SRM 979 (Cr(NO 3 ) 3 • 9H 2 O) as a function of the count rate, the isotope rat ios are highly dependent on the number used for dead time correction. When the dead time is set to 43.5 ns, the isotope ratios are approximately constant up to the count rate of 5.8 x 10 5 . At higher count rates, gain loss will occur and cannot be mathemati cally corrected. Therefore, the
solutions must be diluted in the case where the cou nt rate is higher than this value.

NOTE : Dead time correction in ICP-MS is performed befo re mass bias correction, so

the dead-time-corrected isotope ratios may be diffe rent from the certified isotope ratios. Although it is unnecessary to use isotopically certified material for the determination of dead time, the certified m aterial is still required for the measurement of mass bias factors. Thus, it is conv enient to use the same certified material for both dead time and mass bias factor measurement. Dead time correction in Nano-ESI-QTOF is unnecessary due to the small sample size and low demand on detector capacity.

NOTE : It has been observed in ICP-MS that using differ ent isotope pairs for dead time

measurement may obtain different dead times. Thus, it is required to do the dead time measurement for each isotope pair that wi ll be used. The dead time must be determined daily.

4.3.3 Instrumental discrimination/fraction effects are changes induced in the “true” isotope ratios from the ionization process o r from differential transmission/detection by the mass spectrometer. This effect can bias the ratios either positively or negatively. To correct for the mass bias, mass bias factors sho uld be determined with isotopically certified materials.

mass bias factor = R t /R
m

where:

_ R t and R m are the certified isotope ratio and the measured de ad-time-correctedisotope-ratios of the standard material, respective ly.

The dead-time-corrected isotope ratios of the sampl es can be corrected using:

R c = mass bias factor x R
m

where: background 2 Isotope
dard tan s/ sample 2 Isotope background 1 Isotope
dard tan s/ sample 1 Isotope

m 

### S S S S R

−

−

=SW-846 Update V 6800 - 9 Revision 1 July 2014 _ R c and R m are the corrected isotope ratio and the measured d ead-time-correctedisotope-ratios of the sample, respectively.

Mass discrimination is a time-dependent instrumenta l effect, therefore, the mass bias factors must be determined periodically during the measurement of the samples. Samples are run with the assumption that mass bias factors remain constant. In general, the mass bias factors are stable over several hours of ICP-MS operation. However, the measurement interval for determining mass bias fact ors should generally not exceed four
hours.

NOTE : Some previous work observed the following relati onship between the measured

and the true isotope ratios for ICP-MS: R
m =R t (1+ an ), where a is the bias per
mass unit and n is the mass difference between isotopes (12). Thi s enables the calculation of the mass bias factors of other i sotope pairs based on the measurement of one pair of isotopes. However, this must be verified experimentally. Otherwise, the mass bias factor fo r each isotope pair must be determined.

5.0 SAFETY

5.1 This method does not address all safety issues associated with its use. The laboratory is responsible for maintaining a safe wo rk environment and a current awareness file of Occupational Safety and Health Administration (O SHA) regulations regarding the safe handling of the chemicals listed in this method. A reference file of material safety data sheets (MSDSs) should be available to all personnel involv ed in these analyses.

5.2 Refer to Chapter Three for a discussion on safe ty-related references and issues.

5.3 Many chromium, mercury and molecular compounds are highly toxic if swallowed, inhaled, or absorbed through the skin. Extreme car e must be exercised in handling hexavalent chromium, mercury species and many molecular reagen ts. These toxic and toxicant reagents should only be handled by analysts who are knowledg eable about their risks and of safe handling procedures.

6.0 EQUIPMENT AND SUPPLIES

Trade names or commercial products mentioned in th is manual is for illustrative purposes only, and does not constitute an EPA endorsement or exclusive recommendation for use. The products and instrument settings cited in SW-846 me thods represent those products and settings used during method development or subseque ntly evaluated by the Agency. Glassware, reagents, supplies, equipment, and setti ngs other than those listed in this manual may be employed provided that method performance ap propriate for the intended application
has been demonstrated and documented.SW-846 Update V 6800 - 10 Revision 1 July 2014 6.1 ICP-MS or other mass spectrometers capable of b aseline resolution (at least one unified atomic mass unit, u) are required. For exam ple, mass spectrometers such as TOF may have 20,000 to 60,000 u resolution. Other mass spec trometers of even higher resolution are available. The data system should allow for correct ions of isobaric interferences, dead time and mass bias or the equivalent, or the raw data may be exported to a computer for further processing. For quadrupole mass spectrometers, the dwell time should be adjustable since proper settings of dwell time can significantly imp rove the precision of the isotope ratio measurement in mass scanning instruments. Both sca n mode and peak jump mode and simultaneous (i.e., TOF) systems can be used, depen ding on the instrumentation. The use of a mass-flow controller for the nebulizer argon and a peristaltic pump for the sample solution are recommended. When chromatography is coupled to ICP -MS for on-line detection, the ICP-MS data system must be capable of correcting interfere nces, dead time and mass bias, and calculating the isotope ratios in time resolved ana lysis mode (TRA), or the raw data can be exported for off-line processing. Other mass spect rometers may also be used, providing a precision of 0.5% or better can be obtained for the isotope ratio measurement. Examples of other ionization and mass spectrometers would be ES I, nanoESI and/or MALDI and/or microwave induced plasma (MIP) ionization coupled t o quadrupole, TOF and/or ion-trap and/or QQQ mass spectrometers. Appropriate correction pro tocols for these mass spectrometers should be applied based on the performance specific ations of the mass spectrometer.

6.2 Chromatography or other separation methods are used to isolate species prior to isotope ratio measurement. Chromatography, such as ion exchange chromatography may be used to separate the species on-line in SIDMS (Figu re 3). Chromatography components should be chemically inert based on the specific reagents and analytes. The eluent components and the flow rate of the chromatography system must be compatible with ICP-MS. An interface between the chromatography and ICP-MS may be requir ed for compatibility reasons. Alternatively, any appropriate separation methods, including extraction, solid phase extraction, chelation, reverse phase, normal phase and other ch romatography, precipitation, affinity -based separations and combinations of these and others ca n be used after validation.

7.0 REAGENTS AND STANDARDS

7.1 All reagents should be of appropriate purity to minimize the blank levels due to contamination. Whenever possible, acids should be sub-boiling distilled. All references to water in the method refer to high purity reagent wa ter. Other reagent grades may be used if it is first ascertained that the reagent is of sufficient purity to permit its use without lessening the accuracy of the determination. If the purity of a reagent is questionable, analyze the reagent to determine the level of its impurity.

7.2 For higher precision, solutions may be prepared by weight. For IDMS, standard stock solutions with natural isotopic abundance may be purchased or prepared from ultra-high purity grade chemicals or metals. As an example, s ee Method 6020B for instructions on preparing standard solutions from solids. Generall y, the same procedures are applicable to isotope-enriched materials. However, when a limite d amount of the isotope-enriched material is used (usually due to cost considerations) to prepar e the stock solutions, the solutions require calibration with inverse isotope dilution (see Sec. 7.4.1). Isotope-enriched materials with known enrichment can be purchased from several suppliers, such as the Oak Ridge National Laboratory Electromagnetic Isotope Enrichment Facil ity (ORNL-EMIEF). Other examples are provided below.SW-846 Update V 6800 - 11 Revision 1 July 2014 7.3 Currently, standard stock solutions made for sp eciation analysis are commercially available. Thus, in addition to the dissolution of the standard solid, the chemical conversion of the element and molecule into the desired species i s usually required for SIDMS. The preparation of Cr(VI) and Cr(III) stock standards f or SIDMS will be illustrated as an example and the glutathione reduced and oxidized are also illus trated for molecular examples. These two example also are specifically illustrated in the eq uations that follow with chromium species illustrating the elemental and glutathione species illustrating the molecular species. For other elements and molecular species, procedures must be specifically developed, obtained from the literature or obtained by the manufacture and devel opers. One supplier of stable isotope standards for SIDMS and IDMS is Applied Isotope Tec hnologies (AIT) Inc. These standards are and may be covered by patents. AIT is located at 2 403 Sidney St. Suite 280, Pittsburgh PA 15203, info@sidms.com, http://www.sidms.com.

7.3.1 In this example of inorganic elemental specia tion of chromium species, there are four standards to be prepared for the simultaneous analy sis of Cr(VI) and Cr(III): nat Cr(VI) and

nat Cr(III) with natural abundance, 53 Cr(VI) enriched in 53 Cr, 50 Cr(III) enriched in 50 Cr, and isotopicabundance-calibrated Cr standard solution. In the m olecular example of reduced and oxidized glutathione these species of natural and enriched s pecies have exact analogues and corresponding reagents. For example reduced natura l glutathione (GSH) having C-12, N-14 and O-16 are the natural forms of GSH. There are i sotopically enriched analogues where C-13, N-15, and/or O-17, O-18 are enriched. Oxidized glu tathione (GSSG) has similar natural and isotopically enriched forms. The equations are simi lar to those applied to the Cr natural and enriched species with the Cr(III) corresponding to the GSH both natural and enriched and the Cr(VI) GSSG both natural and enriched. One additio nal factor in molecular SIDMS is that there are small percentages of natural C-13, N-15 and O-1 7 and O-18 that account for a 1% naturally occurring signal that can be corrected as described (Refs. 34, 35, and 36). The calculations are essentially identical using both of the iterative a nd determinative SIDMS equations with the exception of adding the correction for the naturall y occurring isotopes of C-13, N-15, and O-17 and O18 that effect the accuracy within the range o f 1 to 2% in most cases. See the referenced literature and other SIDMS literature for further d etails on these modifications for molecular SIDMS.

7.3.2 1 mg/mL Cr(VI) and Cr(III) standards are comm ercially available. nat Cr(VI)
and nat Cr(III) can also be prepared from K 2 Cr 2 O 7 and Cr metal, respectively.

7.3.2.1 nat Cr(VI) standard solution, stock, 1 g = 1 mg Cr: Di ssolve 0.2829 g of K 2 Cr 2 O 7 in about 80 mL of reagent water and dilute to 100 g with reagent water.

7.3.2.2 nat Cr(III) standard solution, stock, 1 g = 1 mg Cr: D issolve 0.1 g of Cr metal (accurately to at least 4 significant figures) in a minimum amount of 6M HCl and dilute the solution with 1% HNO 3 to 100 g.

7.3.3 53 Cr(VI) standard solution, 1 g ≈ 10 μ g Cr: The following procedure
describes chromium oxide as the source material. A 150-mL glass or quartz beaker is used for the dissolution. Weigh 5.8 mg of 53 Cr-enriched oxide (the exact amount should be calculated based on the content of Cr in the mat erial) into the beaker and add 8 mL of concentrated HClO 4 . Slowly heat the beaker on a hot plate until bubb les form on the bottom; the solution should not boil. Keep heating the solution for up to 6 hrs until all solids are dissolved and only 1 to 2 mL of the solu tion remains. Turn off the hot plate and
wait until the beaker cools down. Rinse the beaker and watch glass with 10 mL of reagent water; the solution should turn intense yellow. A dd 50 μ L of 30% H 2 O 2 and 4.5 mL of
concentrated NH 4 OH. Slowly heat the vessel until the solution gent ly boils to oxidize all CrSW-846 Update V 6800 - 12 Revision 1 July 2014 to Cr(VI). Allow the solution to boil for at least 15 minutes to remove the excessive H 2 O 2

. Transfer the solution to a 500-mL polymeric (e.g., polytetrafluoroethylene [PTFE], polyethylene, polypropylene, etc.) bottle and dilut e the solution to 400 g. The exact concentration of the 53 Cr(VI) spike must be calibrated with nat Cr(VI) standard as described in Sec. 7.4.

NOTE : The procedure may be simpler when the isotope-en riched materials are

available in other molecular forms. For example, w hen K 2 Cr 2 O 7 enriched in

53 Cr is available, the solid can be dissolved in reag ent water without further conversion; when Cr metal is available, the metal c an be dissolved in 6M HCl as described in Sec. 7.3.2.2, followed by the addit ion of H 2 O 2 and NH 4 OH to oxidize Cr(III) to Cr(VI) as described above.

NOTE: The molecular forms of GSH and GSSG have simila r analogues. To prevent

the oxidation of GSH to GSSG in the published appli cation, N-ethylmaleimide (NEM) is added to prevent oxidation of GSH (Ref. 35 ). NEM blocks the cysteine fictional group and slows down oxidation. However addition of NEM is not completely effective and therefore correction b y SIDMS might be required in some cases (Ref. 35)

WARNING : Concentrated HClO 4 is a very strong oxidizer. Safety protocols requi re this

reagent only be used in a perchloric acid hood or e quivalent solution and vapor handling system.

7.3.4 50 Cr(III) standard solution, 1 g ≈ 10 μ g Cr: The following procedure
describes chromium metal as the source material. W eigh 4 mg of the metal into a 30-mL PTFE vessel. Add 4 mL of 6M HCl and gently heat th e solution but do not boil it until the solid is dissolved. Continue to heat the solution u ntil only 1 to 2 mL of the solution remains. The solution is then cooled and transferred to a 50 0-mL polymeric bottle. Dilute the solution with 1% HNO 3 to 400 grams. The exact concentration of the 50 Cr(III) spike must
be calibrated with nat Cr standard as described in Sec. 7.4.

NOTE : The procedure depends on the form of the materia l. For example, when

K 2 Cr 2 O 7 enriched in 50 Cr is available, the solid can be dissolved in 1% H NO 3 , followed by the addition of H 2 O 2 to reduce Cr(VI) to Cr(III). The excessive H 2 O 2 can be removed by boiling the solution.

7.3.5 Isotopic-abundance-certified standard solutio n, stock, 1 g ≈ 10 μ g Cr: Weigh 31 mg of Cr(NO 3 ) 3 9H 2 O (SRM 979) into a 500-mL polymeric container. Dis solve the solid and dilute it with 1% HNO 3 to 400 g.

7.4 The isotope-enriched spikes require characteriz ation since a limited amount of material is usually weighed, complex treatment is i nvolved, or the purity of the source material is limited (usually <99% ). For the SIDMS method, in addition to the total concentration of the standard, the distribution of the species must be d etermined before it can be used (see Section 12.2.2, 2 nd NOTE). Inverse IDMS and inverse SIDMS measurement is used to calibrate the isotope-enriched spike and to determine the species distribution. The characterization of

53 Cr(VI) spike solution will be illustrated as an exa mple.

7.4.1 Calibration of total concentration of spike s olution with natural material: Weigh the proper amount (W X ) of 10 μ g/g (C Standard ) nat Cr standard and the proper amount (W S ) of the 53 Cr(VI) spike (nominal concentration is 10 μ g/g) into a polymeric container, and dilute the mixture with 1% HNO 3 to a concentration suitable for isotope ratioSW-846 Update V 6800 - 13 Revision 1 July 2014 measurement. Use direct aspiration mode to determi ne the isotope ratio of 53 Cr/ 52 Cr (R

53/52 ). The concentration of the spike, C Spike , can be calculated using the following equations:

S S Spike 

### M C C =



      

− −

=

S 53
S 52
52 / 53 X

52
52 / 53 X 53

S X X
S 

### A

A R A R A

W W C C

X dard tan S X 

### M

/ C C =

where C S and C X are the concentrations of the isotope-enriched spik e and the standard with natural isotopic abundance in μ mole/g, respectively. M S and M X are the average atomic weights of the spike and the standard in g/m ole, respectively. 53 A S and 53 A X are the atomic fractions of 53 Cr for the spike and standard, respectively. 52 A S and 52 A X are the atomic fractions of 52 Cr for the spike and standard, respectively.

NOTE : The same procedure is applicable to the calibrat ion of the isotope-enriched

spike solutions in IDMS. The same procedure is als o applicable to the calibration of 50 Cr(III) by changing isotope 53 Cr to 50 Cr.

NOTE : Average atomic weight = Σ (atomic weight of the isotope x atomic fraction)

7.4.2 Calibration of the concentration of the Cr(VI ) in the 53 Cr(VI) spike with

nat Cr(VI): Weigh the proper amount (W X ) of 10 μ g/g (C standard VI ) nat Cr(VI) standard and the proper amount (W S ) of the 53 Cr(VI) spike (nominal concentration is 10 μ g/g ) into a polymeric container, and dilute the mixture with re agent water to a concentration suitable for measurement. Acidify the solution to pH 1.7 - 2.0 with concentrated HNO 3 . Separate the Cr(VI) with chromatography or other separation methods and measure the isotope

ratio of 53 Cr/ 52 Cr in Cr(VI) species ( VI
53/52 

### R ). The concentration of Cr(VI) in the spike, C Spike VI

, can be calculated using the following equations:

S VI
S VI Spike 

### M C C =

       

− −

=

S 53
S 52 VI
52 / 53 X

52 VI
52 / 53 X 53

S X VI

X VI
S 

### A

A R A R A

W W C C

X VI
dard tan S VI

X 

### M

/ C C =

where C S

VI and C X

VI are the concentrations of Cr(VI) in the isotope-en riched spike and standard with natural isotopic abundance in μ mole/g, respectively. M S and M X are the average atomic weights of the spike and the standar d in g/mole, respectively. 53 A S and

53 A X are the atomic fractions of 53 Cr for the spike and standard, respectively. 52 A S and 52 A X are the atomic fractions of 52 Cr for the spike and standard, respectively.

NOTE : This set of equations is similar to those used i n the determination of total Cr in

53 Cr(VI) standard (Sec. 7.4.1). The general equation s for inverse SIDMS are not so simple. However, for speciation of Cr(VI) a nd Cr(III) in standardSW-846 Update V 6800 - 14 Revision 1 July 2014 solutions, because the matrix is so simplified, onl y the reduction of Cr(VI) to Cr(III) is observed at low pH. Thus, the existence of Cr(III) species will not influence the isotope ratio of Cr(VI), and the comp lex equations can be simplified to the equations shown above (Ref. 3).

7.4.3 The distribution of Cr(III) and Cr(VI) in 53 Cr(VI) spike can be calculated as:

percentage of Cr(VI) = VI Spike

Spike 

### C x 100% C

percentage of Cr(III) = VI Spike

Spike 

### C 1 - x 100% C      

NOTE : No determination of the species distribution in 50 Cr(III) spike is required

because only Cr(III) is present in this solution.

7.5 Blank samples -- Three types of blank samples a re required for the analysis; namely, a background blank for subtracting backgrou nd in isotope ratio measurement, a preparation blank for monitoring possible contamina tion resulting from the sample preparation procedures, and a rinse blank for flushing the syst em between all samples and standards.

7.5.1 The background blank consists of the same con centration(s) of the acid(s) used to prepare the final dilution of the s ample solution (often 1% HNO 3 (v/v) in reagent water).

7.5.2 The preparation (or reagent) blank must be ca rried through the complete preparation procedure and contain the same volumes of reagents as the sample solutions.

7.5.3 The rinse blank consists of 1 to 2 % HNO 3 (v/v) in reagent water. Prepare a sufficient quantity to flush the system between s tandards and samples. Refer to Method 6020 regarding the interference check solution.

7.6 Refer to Method 6020 for preparing mass spectro meter tuning solution.

8.0 SAMPLE COLLECTION, PRESERVATION, AND STORAGE

Sample collection, preservation and storage require ments may vary by EPA program and may be specified in a regulation or project plannin g document that requires compliance monitoring for a given contaminant. Where such req uirements are specified in the regulation, follow those requirements. In the absence of speci fic regulatory requirements, use the following information as guidance in determining the sample c ollection, preservation and storage requirements.

8.1 See the introductory material to SW-846 Chapter Three for inorganic analytes and Chapter Four for organic analytes.

8.2 Due to the possible degradation or interconvers ion of the species, samples collected for speciation analysis must be isotopica lly spiked as soon as possible. The measurement, however, can be carried out later prov ided that less than 95% degradation orSW-846 Update V 6800 - 15 Revision 1 July 2014 interconversion occurs with validation of specific species groups. The holding time prior to measurement depends on the preservation of the spec ies.

8.3 Proper methods to retard the chemical activity of the species are applicable to SIDMS.

8.4 All sample containers must be prewashed with de tergents, acids, and water. Polymeric containers can be used and containers are chosen for inertness and compatibility of the matrix, molecules and species to be analyzed. See SW-846 Chapters Three and Four for further information on clean chemistry procedures t o reduce blank effects in these
measurements.

9.0 QUALITY CONTROL

9.1 Refer to SW-846 Chapter One for guidance on qua lity assurance (QA) and QC protocols. When inconsistencies exist between QC g uidelines, method-specific QC criteria take precedence over both technique-specific criteria an d those criteria given in Chapter One, and technique-specific QC criteria take precedence over the criteria in SW-846 Chapter One. Any effort involving the collection of analytical data should include development of a structured and systematic planning document, such as a Quality Ass urance Project Plan (QAPP) or a Sampling and Analysis Plan (SAP), which translates project o bjectives and specifications into directions for those that will implement the project and asses s the results. Each laboratory should maintain a formal quality assurance program. The l aboratory should also maintain records to document the quality of the data generated. All da ta sheets and quality control data should be maintained for reference or inspection.

9.2 Initial demonstration of proficiency

Each laboratory must demonstrate initial proficienc y with the appropriate sample preparation and the isotope ratio measurement metho ds by generating data of acceptable bias and precision for target analytes in a clean matrix . Method 8000 provides information on how to accomplish a demonstration of proficiency for stand ard methods. This method, using SIDMS, has unique proficiency requirements as concentratio ns are determined by direct calculation based on isotopic properties of the target analyte and the enriched isotopic species spike without the use of calibration curves as described in Method 8000. During SIDMS analysis by Method 6800, analyte transformations may occur to s ome extent in some matrices and can be corrected by using its protocol. Proficiency is es tablished for each species such as Cr(VI), methylmercury, tributyltin, and any polyisotopic sp ecies and each matrix such as water, soil, tissue and others. These proficiencies are to be d etermined using appropriate standards prepared to test proficiency of spiking, extraction , species separation, mass spectrometric operation and calculation which together constitute this method’s SIDMS protocol. A suite of proficiency testing materials, as well as guideline s, for the implementation of the proficiency testing is available from Applied Isotope Technolog ies (info@sidms.com). Determination of total element or molecule using this method’s IDMS protoc ol is similar to more traditional methods that are spiked with elements as the matrix spike o r molecules in organic analytes. However, in this method, concentration of the species of intere st is calculated directly from the isotopic element/molecule spike and not from calibration cur ves.

9.3 Sample quality control for preparation and ana lysis

The laboratory must also have procedures for docum enting the effect of the matrix on method performance (precision, accuracy, method sen sitivity, species conversion). At aSW-846 Update V 6800 - 16 Revision 1 July 2014 minimum, this should include the analysis of QC sam ples including background blank, preparation blank, rinse blank, matrix spike, dupli cate, and laboratory control sample (LCS) and/or proficiency samples if available and were ap plicable for each analytical batch. A sample of known concentration, such as Standard Reference Material, an appropriate reference material or a suitable set of proficiency materials can be used as LCS. Any blank, matrix spike samples, and replicate samples should be subjected to the same analytical procedures (Sec. 11.0) as those used on actual samples.

9.3.1 Duplicate samples should be processed on a ro utine basis. A duplicate sample is a sample processed through the entire sam ple preparation and analytical procedure. A duplicate sample should be processed with each analytical batch or every 20 samples, whichever is the greater number. A dup licate sample should be prepared for each matrix type (i.e., soil, sludge, water, tissue , etc.). If statistical compilations are desired, a sufficient number of duplicate samples s hould be analyzed to produce the desired statistical results. Consult Method 8000 f or information on developing acceptance criteria for the duplicate.

9.3.2 Spiked samples and/or standard reference mate rials and/or reference materials or proficiency materials (if available) s hould be included with each group of samples processed or every 20 samples, whichever is the greater number. A spiked sample should also be included whenever a new sampl e matrix is being analyzed and proficiency should be established for each sample m atrix. For SIDMS, because the species may degrade or convert to other species whe n they are spiked into samples, low recovery may be observed. Thus, the low recovery d oes not immediately invalidate this method. For example, if Cr(III) is spiked into a b asic solution, due to the hydrolysis of Cr(III) and the limited solubility of chromium hydr oxide, low recovery of Cr(III) may be obtained. Low recovery may indicate an unfavorable matrix for preserving the corresponding species (Ref. 4). The conversion of one species to another should be calculated and recorded with the final corrected co ncentration. If equilibration is reached prior to species-loss, an isotopic ratio may still be measurable and will result in an acceptable species measurement. In most cases, con version can be quantified and corrected. If a sample, isotopic spike of one spec ies is completely converted into another species, then the matrix and/or the sample preparat ion procedure may be responsible for species conversion. This will indicate that this s pecific matrix can support the stability of only select species. The results of concentration and conversion should be recorded as an acceptable set of data. Species concentration a nd species conversion should be measured and recorded in the laboratory report. Sp eciation under this method is a relatively new field and additional equations and o ther QC, proficiency support, and reference materials are updated through Ref. 27.

9.3.3 Blank samples should be prepared using the sa me reagents and quantities used in sample preparation, placed in ve ssels of the same type, and processed with the samples. Also see Sec. 7.5 for more infor mation regarding blank samples.

9.4 Lower Limit of Quantitation (LLOQ) check standa rd

9.4.1 The laboratory should establish the LLOQ as the lowest point of quantitation which, in most cases, is the lowest concentration in the c alibration curve or direct calculation, such as Method 6800. The LLOQ is initially verified by the analysis of at least 7 replicate samples, spiked at the LLOQ and processed through all prepar ation and analysis steps of the method. The mean recovery and relative standard deviation o f these samples provide an initial statement of precision and accuracy at the LLOQ. In most case s the mean recovery should be +/- 35% of the true value and RSD should be < 20%. In-house li mits may be calculated when sufficientSW-846 Update V 6800 - 17 Revision 1 July 2014 data points exist. Monitoring recovery of LLOQ over time is useful for assessing precision and bias. Refer to a scientifically valid and published method such as Chapter 9 of Quality Assurance of Chemical Measurements (Taylor 1987) or the Report of the Federal Advisory Committee on Detection and Quantitation Approaches and Uses in Clean Water Act Programs (http://water.epa.gov/scitech/methods/cwa/det/index .cfm) for calculating precision and bias for LLOQ.

9.4.2 Ongoing LLOQ verification, at a minimum, is on a quarterly basis to validate quantitation capability at low analyte concentratio n levels. This verification may be accomplished either with clean control material (e. g., reagent water, method blanks, Ottawa sand, diatomaceous earth, etc.) or a representative sample matrix (free of target compounds). Optimally, the LLOQ should be less than or equal to the desired regulatory action levels based on the stated project-specific requirements.

10.0 CALIBRATION AND STANDARDIZATION

10.1 IDMS calibration

10.1.1 Follow the appropriate sections in Method 60 20B to set up and tune the ICP-MS. Sample introduction is performed in direct aspiration mode. The following procedure is illustrated with the measurement of 50 Cr/ 52 Cr and 53 Cr/ 52 Cr isotope ratios.

10.1.2 Determine the dead time (Sec. 4.3.2). Solut ions prepared from reference material SRM 979 (Cr(NO 3 ) 3 • 9H 2 O) are used in this determination. A range of solu tions of different concentrations should be prepared, but th eir concentrations do not need to be accurately known. Masses 50, 52 and 53, as well as masses which could affect data quality, should be monitored. The raw count rates for each solution are measured and integrated. Assume a dead time and use the equatio n described in Sec. 4.3.2 to correct the integrated counts. The dead-time-corrected cou nts are then used for calculating the isotope ratios after background subtraction. By tr ial and error, the dead time is determined to bring the isotope ratios obtained from solutions of different concentrations to a constant (the relative standard deviation of the isotope rat ios reaches the minimum). The isotope ratios obtained from high counts may be excluded as gain loss may occur.

NOTE : The concentration range of the solutions may be adjusted depending on the

sensitivity and dynamic range of the instrument.

NOTE : For direct aspiration mode, the dead time correc tion can be done either before

or after the integration of the raw data. However, it is simpler to do the dead time correction after the integration.

10.1.3 Determine the mass bias factor (Sec. 4.3.3). The mean of isotope ratios obtained in Sec. 10.1.2 is used for calculating the mass bias factor. The equation is provided in Sec. 4.3.3. The measurement of the mas s bias factor must be done periodically between sample measurements. The inte rval between these measurements depends on the mass bias stability of the instrumen t. The relative difference between two consecutive mass bias factors should not exceed 1%.SW-846 Update V 6800 - 18 Revision 1 July 2014 10.2 SIDMS calibration

10.2.1 Follow the appropriate sections in Method 60 20B to set up and tune the ICP-MS. Follow Sec. 10.1.2 to measure the dead tim e. If the calibration of the isotopeenriched spikes is required, the mass bias factors for direct aspiration mode and the altered isotope ratios for the spiked standards are measured at this step. The measured isotope ratios obtained at this step are used in th e calibration of total concentrations.

10.2.2 Determine the mass bias factor (Sec. 4.3.3). Connect the outlet of the chromatographic column to the nebulizer of the ICP- MS. Background blank and an isotopic abundance certified standard are used for the measurement of the mass bias factors in time resolved analysis (TRA) mode. The raw data at each point are corrected for dead time using the equation described in Sec. 4.3.2 and then integrated by summing the data across each peak. The intervals between t wo consecutive injections must be long enough for the signal to return to baseline. The integrated counts are then used to calculate the isotope ratios with the equation show n in Sec. 4.3.2. Apply the equation in Sec. 4.3.3 to calculate the mass bias factors for e ach isotope pair by comparing the measured isotope ratios to the certified isotope ra tios.

NOTE : For the TRA mode, the dead time correction must be done at each data point

before the data integration.

NOTE : For other ionization and mass spectrometer types such as ESI and/or MALDI

and/or MIP ionization coupled to quadrupole, TOF an d/or ion-trap mass spectrometers, appropriate correction protocols sho uld be utilized.

11.0 PROCEDURE

11.1 IDMS

11.1.1 Closed-vessel microwave digestion is used as an example method to decompose samples and to solubilize, equilibrate an d stabilize the elements of interest. The following procedure is applicable to samples de composed using Method 3052. Refer to Method 3052 for specifications of the microwave apparatus. Method 3546 is a microwave extraction method for organic analytes an d is suited for IDMS and SIDMS of molecular species. Other extraction methods are ava ilable in the literature for additional species and matrices. Separation may be directly a pplied to molecular and elemental species that are amenable to such measurements such as water, blood, serum, plasma, drinks, and other samples.

11.1.2 Prepare or purchase the isotope-enriched spi ke and calibrate it with the inverse isotope dilution mass spectrometry procedur e described in Secs. 7.3 and 7.4.1. Isotope enriched spikes are available as commercial products for both elemental and molecular species from Applied Isotope Technologies , Inc. (e-mail: info@sidms.com; http://www.sidms.com).

11.1.3 Weigh a representative sample to the nearest 0.001 g into an appropriate microwave digestion vessel equipped with a pressure relief mechanism. Spike the sample with the calibrated isotope-enriched spike. The co ncentration of the spike should be high enough so that only a small volume of the solution is used. Weigh the mass of the spike to at least three significant figures.SW-846 Update V 6800 - 19 Revision 1 July 2014 11.1.4 Digest the sample according to the procedure described in Method 3052 and/or extract the species from the sample matrix w ith an extraction method such as 3546, or separate the analytes by chromatography (see SW- 846 Chapters Three and Four and the literature for additional sample preparation me thods).

NOTE : For filtered and acidified aqueous samples, dige stion may not be required.

Sample solutions can be directly analyzed with ICP- MS, TOF, QTOF, QQQ or and/or other appropriate mass spectrometers after s piking and equilibration.

11.1.5 Measurement of the isotope ratios can be car ried out using ICP-MS or ESI-TOF, QTOF, QQQ and/or other appropriate mass sp ectrometers.

11.1.5.1 Determine the mass bias factor periodicall y as described in Sec. 10.1.3.

11.1.5.2 Measure the isotope ratio of each sample. Flush the system with the rinse blank. In many cases the ideal isot ope ratio is 1:1. Isotope ratios are ideally within the range from 0.1:1 to 10:1, ex cept for blanks and samples with extremely low concentrations. Samples may be re-sp iked to achieve an isotope ratio close to 1:1. If too high of a count rate is observed, samples must be diluted to avoid gain loss of the detector. Some isotope rati os can be quantified at 1:100 and 100:1 in cases of instrument sensitivity and stabil ity. Error Propagation Factors (EPF) can be calculated to optimize the ratios for quantification.

NOTE : Unspiked solutions are used to measure the isotop ic abundance of

elements and molecules such as lithium, lead, and u ranium, and molecular enriched ions whose isotopic abundances c an vary in nature.

11.2 SIDMS

11.2.1 SIDMS is applicable to the quantification of elemental and molecular species in various sample matrices such as water, s oil, sludge, tissue, food, blood, plasma, serum, drugs, dietary supplements, etc. Sol id samples require or benefit from isolation/solubilization and separation of the elem ental species before analysis. Procedures for such extraction of the species from different matrices must be specifically designed to minimize and measure species conversion . Extraction procedures for tissue, hair and bacteria are found in references (Refs. 20 through 22, respectively). The following procedure is an illustration of the simul taneous determination of Cr(III) and Cr(VI) in water samples or in soil or sediment extracts. C r(VI) is extracted from the solid using Method 3060A, mercury species by Method 3200, and o rganic molecules by Method 3546 and other appropriate methods (See SW-846 Chapters Three and Four).

11.2.2 There are two ways of obtaining the isotopic ally enriched standards used in IDMS and SIDMS. Prepare the isotope-enriched sp ikes of each target species and calibrate them with inverse isotope dilution mass s pectrometry as described in Sec. 7.4. Species-specific isotope-enriched spikes are availa ble as commercial products from Applied Isotope Technologies (e-mail: info@sidms.co m; http://www.sidms.com).

11.2.3 Weigh an appropriate aliquot of the water, s olid or semi-solid sample, or extract to the nearest 0.0001 g into a polymeric co ntainer. Spike the sample with 10 μ g/g
of 53 Cr(VI) spike to a concentration so that the isotope ratio of 53 Cr/ 52 Cr in Cr(VI) will be approximately 1:1. Spike the sample with 50 Cr(III) spike to a concentration so that the isotope ratios of 50 Cr/ 52 Cr in Cr(III) will also be approximately 1:1 and th e speciesSW-846 Update V 6800 - 20 Revision 1 July 2014 concentrations are suitable for measurement. Thoro ughly mix the spike and the sample. The isotope ratios 53 Cr/ 52 Cr for samples must be within the range of 0.1:1 to 10:1, except for blanks or samples with extremely low concentrat ions. If isotope ratios are found to be outside these ranges, samples should be re-spiked a nd re-analyzed.

NOTE : If only the Cr(VI) is of interest, the sample ca n be spiked only with 53 Cr(VI).

However, this is based on the assumption that only unidirectional conversion can occur after spiking, i.e. reduction of Cr(VI) t o Cr(III). This is usually true if the sample is acidified to a low pH after spiking, especially for matrices containing reducing agents. Molecular SIDMS uses GS H as the example of molecular species that have unidirectional species oxidation to GSSG.

11.2.4 Extract the species from the sample matrix ( see Section 11.2.1)

NOTE : For aqueous samples, extraction may not be requi red. Sample solutions can

be directly analyzed after spiking and equilibratio n. Highly concentrated samples may need to be adjusted in spike to sample ratio and may be adjusted to a lower spike ratio to conserve spike or sub-sam pled. Chromium ore processing residues (COPR) samples are a good examp le of high chromium concentration where this can be applied.

11.2.5 If the solution is strongly basic, neutraliz e the sample with concentrated HNO 3 to avoid the hydrolysis of Cr(III). Sample prepar ation to preserve the molecular species is specific to the chemistry of the species and is performance based and depends on the analyses being conducted. The example of ch romium species and glutathione are used to illustrate elemental and molecular species respectively. The sample preparation is dependent on the species chemistry, stoichiometry a nd equilibrium relationships of the species.

11.2.6 Acidify the spiked samples to pH 1.7 to 2.0; under these conditions Cr is usually retained in solution, although there might be interconversion between Cr(III) and Cr(VI). The spiked samples can be stored at ≤6 ° C to retard interconversion of the Cr species. Other methods that can slow down the tran sformation of the species can be applied as long as no interference with the isotope ratio measurement is introduced. For example, some soil extracts contain large concentra tions of reducing agents that reduce Cr(VI) rapidly after acidification. To slow down t he reduction, stoichiometric amounts of KMnO 4 can be added to the sample; KMnO 4 easily oxidizes the reducing matrices.

NOTE : Studies have shown that the lower the interconve rsion between the species,

the more precise the determination is (Ref. 3). Th us, efforts should be made to prevent or retard species interconversions.

11.2.7 The measurement of the isotope ratios in eac h species can be carried out using ICP-MS or other equivalent mass spectrometers following the separation of the species using chromatography or other separation me thods. The use of ion-exchange chromatograph coupled with ICP-MS will be illustrat ed as an example in the measurement of 50 Cr/ 52 Cr and 53 Cr/ 52 Cr isotope ratios in samples containing both Cr(III ) and Cr(VI) species.

11.2.7.1 Determine the mass bias factors periodical ly as described in Sec. 10.2.2.SW-846 Update V 6800 - 21 Revision 1 July 2014 11.2.7.2 Measure the isotope ratios of each sample. Flush the system with the eluent until the signal returns to the bas eline. The ideal isotope ratios for

50 Cr/ 52 Cr in Cr(III) and 53 Cr/ 52 Cr in Cr(VI) are 1:1. Ratios between 0.1:1 and 10: 1 are also appropriate. Samples may be re-spiked to achieve an isotope ratio close to 1:1. If excessively high count rates are observ ed, samples must be diluted to avoid gain loss of the detector.

NOTE : Unspiked solutions are used to measure the isotop ic abundance of

elements such as lithium, lead, and uranium, whose isotopic abundances can vary in nature. NOTE : A different set of equations and examples are re quired for the

simultaneous determination of three species with co rrection following the SIDMS protocol,. One example for three mercury spe cies can be found in Ref. 24 along with a version of the equations de veloped for three species. Equations for the determination of two an d three species are included in this reference and the formats are cons tructed to simplify these calculations. New explicit and determinative equations will be published in the literature and updated references and technical support will be made available through the www.SIDMS.com we bsite (Ref. 27).

12.0 DATA ANALYSIS AND CALCULATIONS

12.1 IDMS calculations

The quantitative values must be reported in appropr iate units, such as micrograms per liter ( μ g/L) for aqueous samples and milligrams per kilogra m (mg/kg) for solid samples. If dilutions are performed, the appropriate corrections must be applied to the sample values.

12.1.1 Calculate the isotope ratios. Calculations should include appropriate interference corrections (see Sec. 4.3 for data int egration, dead time correction, and mass bias correction).

12.1.2 The following equations are applied to the c alculation of the concentration of the element, C Sample ( μ g/g), in the final sample solutions:

x X Sample 

### M C C =

S

Spike S 

### M / C C =



      

− −

=

X 53

X 52
52 / 53 S

52
52 / 53 S 53

X S S
X 

### A

A R A R A

W W C C

where, C S and C X are the respective concentrations of the isotope-en riched spike and the analyte in the sample, in μ mole/g. M S and M X are the average atomic weights of the isotope-enriched spike and the sample in g/mole, re spectively. 53 A S and 53 A X are the
atomic fractions of 53 Cr for the isotope-enriched spike and sample, respe ctively. 52 A S and

52 A X are the atomic fractions of 52 Cr for the isotope-enriched spike and sample, respectively. C spike is the concentration of the isotope-enriched spike in μ g/g. A general guideline for IDMS calculations can be found in the documents of Section 13.5.4.SW-846 Update V 6800 - 22 Revision 1 July 2014 NOTE : When isotope 50 Cr is used, 53 Cr is substituted with 50 Cr in the above equations.

12.1.3 If appropriate or required, calculate result s for solids on a dry-weight basis as follows:

(1) A separate determination of percent solids must be performed. (2) The concentrations determined in the digest are to be reported on the basis of the dry weight of the sample as follows:

Concentration (dry weight)(mg/kg) = Sample 

### C

S

where, C Sample = Concentration based on the wet sample ( μ g/g)

12.2 SIDMS calculations

The quantitative values must be reported in appropr iate units, such as μ g/L for aqueous samples and mg/kg for solid samples. If dilutions are performed, the appropriate corrections must be applied to the sample values.

12.2.1 Calculate the isotope ratios. Calculations should include appropriate interference corrections, dead time correction, and mass bias correction (Sec. 4.3).

12.2.2 The following equations are used to deconvol ute the concentrations of the species at the time of spiking, as well as the conv ersion of the species after spiking:

where,
R III

50/52 is the measured isotope ratio of 50 Cr to 52 Cr of Cr(III) in the spiked sample

50 A X is the atomic fraction of 50 Cr in the sample (usually a constant in nature) 

### % Solids S = 100



# ( ) ( ) 

# ( ) ( ) ( ) 

# ( ) β α β α

VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X III 

### W C A W C A W C A W C A W C A W C A W C A W C A R 52 52 52 52 50 50 50 50

52 / 50 

### 1 1

+ +

− + + +

− +

= 

# ( ) ( ) 

# ( ) ( ) ( ) 

# ( ) β α β α

VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X III 

### W C A W C A W C A W C A W C A W C A W C A W C A R 52 52 52 52 53 53 53 53

52 / 53 

### 1 1



### + +

− + + +

− +

= 

# ( ) ( ) ( ) 

# ( ) ( ) ( ) β α β α



### −

+ + + − + + +

= 1 1

52 52 52 52 50 50 50 50

52 / 50 VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X VI 

### W C A W C A W C A W C A W C A W C A W C A W C A R 

# ( ) ( ) ( ) 

# ( ) ( ) ( ) β α β α



### −

+ + + − + + +

= 1 1

52 52 52 52 53 53 53 53

52 / 53 VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X VI
S VI
S VI
S X VI

X X III
S III
S III
S X III

X X VI 

### W C A W C A W C A W C A W C A W C A W C A W C A RSW-846 Update V 6800 - 23 Revision 1 July 2014 C x III is the concentration of Cr(III) in the sample ( μ mole/g, unknown)

W x is the weight of the sample (g)

50 A s III is the atomic fraction of 50 Cr in the 50 Cr(III) spike

C s III is the concentration of Cr(III) in the 50 Cr(III) spike ( μ mole/g)

W s III is the weight of the 50 Cr(III) spike (g)

C x VI is the concentration of Cr(VI) in the sample ( μ mole/g, unknown)

α is the percentage of Cr(III) oxidized to Cr(VI) af ter spiking (unknown)

β is the percentage of Cr(VI) reduced to Cr(III) aft er spiking (unknown)

NOTE : The unit of the concentrations shown above is μ mole/g. The conversion factor

from μ mole/g to μ g/g is: M, where M is the average atomic weight of the
element in μ g/ μ mole (Sec. 7.4.1). The following equation can be u sed to convert the unit of the concentration. Be aware th at samples with different isotopic abundance have different average atomic we ights.

Concentration ( μ mole/g) x M = Concentration ( μ g/g)

NOTE : Although the species distribution of the isotopi c spike is determined (Sec. 7.4),

the above equations assume that each isotope-enrich ed spike is only in one species form to simplify the equations. This has b een validated for 50 Cr(III) and

53 Cr(VI) spikes prepared using the procedures describ ed in Sec. 7.3. For other speciation analysis, this assumption must be verifi ed experimentally, or the distribution of the species in the isotope-enriched spikes must be taken into
account.

NOTE : For the quantification of the single-spiked samp les, the following equations are

used:



### C X VI



### = C S VI 

### W S 

### W X 53 

### A S

VI



### − R 53/52 VI 52 

### A S

VI



### R 53/52 VI 52 

### A X 

### − 53 

### A X 

### 
  

  VI VI X Sample X 

### = C C M

VI VI VI

S S Spike 

### = / C C MSW-846 Update V 6800 - 24 Revision 1 July 2014 where C s

VI and C x

VI are the respective concentrations of the isotope-en riched spike and the
analyte in the sample, in μ mole/g. M s VI and M X are the average atomic weight of the
isotope-enriched spike and the sample in g/mole, re spectively. 53 A s VI and 53 A X are the

atomic fraction of 53 Cr for the isotope-enriched spike and sample, respe ctively. 52 A s VI and

52 A X are the atomic fractions of 52 Cr for the isotope-enriched spike and sample, respectively. C spike VI is the concentration of the isotope-enriched spike in μ g/g.

NOTE : When isotope 50 Cr is used, 53 Cr is substituted with 50 Cr in the above equations.

12.2.3 A computer program such as a spreadsheet can be developed to solve this set of second power, four variable equations. Solutions for the four unknowns, C X III

, C X VI

, α and β are required, and can be found from various mathem atical approaches. An iterative approach was originally used to solve for the four unknowns (Refs. 11, 12, and 28), but an explicit, deterministic solution has si nce been developed. The explicit, determinative solution method eliminates the need f or initialization, and recognizes the conditions under which a unique solution will be ob tained.

To implement the explicit, deterministic solution m ethod, it is helpful to first express the four equations in simplified notation:

III VI

1 x 1 1 x 2
1 III VI

2 x 3 2 x 4 

### ( C )(1 ) ( C ) ( C )(1 ) ( C ) 

### A B A B R A B A B

α β α β 

### + − + +

= + − + +

III VI
3 x 5 3 x 6
2 III VI

2 x 3 2 x 4 

### ( C )(1 ) ( C )
( C )(1 ) ( C ) 

### A B A B R A B A B

α β α β 

### + − + +

= + − + +

III VI

1 x 1 1 x 2
3 III VI

2 x 3 2 x 4 

### ( C ) ( C )(1 ) ( C ) ( C )(1 ) 

### A B A B R A B A B

α β α β 

### + + + −

= + + + −

III VI
3 x 5 3 x 6
4 III VI

2 x 3 2 x 4 

### ( C ) ( C )(1 )
( C ) ( C )(1 ) 

### A B A B R A B A B

α β α β 

### + + + −

= + + + −

These are the exact equations presented in 12.2.2, expressed in terms of thirteen known quantities:

• Four ion intensity ratios, corrected for dead-time and mass-bias: 

## { } 

# { } III III VI VI
1 2 3 4 50/52 53/52 50/52 53/52 

### , , , R ,R ,R ,R R R R R =

.

• The three sample weights of each chromium isotope, using naturally occurring isotope
proportions

x



### W : 

## { } 

# { } 50 52 53
1 2 3 x x x x x x 

### , , A W , A W , A W A A A =

.

• The amounts of 50 Cr(III), 52 Cr(III) and 53 Cr(III) in the Cr(III) spike: 

## { } 

# { }

50 III III 52 III III 53 III III
1 3 5 s s s s s s 

### , , A N , A N , A N B B B = , as well as the amounts of 50 Cr(VI), 52 Cr(VI)

and 53 Cr(VI) in the Cr(VI) spike: 

## { } 

# { } 50 VI VI 52 VI VI 53 VI VI
2 4 6 s s s s s s 

### , , A N , A N , A N B B B = . If III

s 

### W is

the weight of the Cr(III) spike, and III

s 

### C is the concentration of Cr(III) in that spike, the n

III III III

s s s 

### N =W C

.SW-846 Update V 6800 - 25 Revision 1 July 2014 Solving equations (1)–(4) for the unknowns 

### α

, 

### β

, III

x 

### C , and VI

x 

### C , gives the following explicit

solutions:

2 4 2 2 3 3 4 3 6 3 2 6 1 4 4 1 1 3 1 2 2 1 3 3

5 1 5 1 2 2 3 1 1 4 3 2 3 2 2 2 2 4 1 6 1 6 1 2 

### ( )
) ( 

### R A B B R R

B B R R B B R A B A B R A A B A B A B R A B A

A R A B A B A A R A B A A B K R B A R B α 

### + − + − −

− + + − + − =

+ − +

− −

1 2 2 1 3 1 3 3 5 1 5 1 2 2 3 1 1 3 3 3 3 1 4 2

4 3 1 5 3 2 5 1 2 4 2 2 3 3 4 3 6 3 2 6 1 4 4 1 

### ( )(
) 

### R A A B A A R A B A B A B A R A

B A R A A R A A B A R A A B A B B R B B R R B

R B B B B R B B R K β 

### − + + − − − −

+ + − − + − + − + =

3 5 1 4 3 1 5 2 1 4 1 3 2 3

3 2 2 3 3 1 3 1 4 1 2 4 2 1 III

x 

### C R R R R

R R R A R B R B B B R B R R B

R A A A R R A R A + − +

= − + + − − −

−

3 1 4 2 3 2

3 2 2 3 3 1 3 1 4 1 2 4 VI 6 4 4 2 2

x
1 1 4

2 6 

### C R R R R

R B R B B B R B R R B

R A A A R R R

R A A A R R + − +

= − + + − − −

− .

The common value of 

### K in the denominators of the solutions for 

### α and 

### β is given by:

1 1 1 2 3 2 2 1 3 2 3 4 2 

### ( )( )( )( ) 

### A R A A R A A K d R A A R A

− − − − = ,

where

1 11 21 42 32 22 12 31 41 

### ( )( ) ( )( ) 

### K K K K K K K d K = − − − − −

and

1 1 3 2 1 4
11 12

1 1 2 1 1 2 

### B R B B R B K K A R A A R A − −

= =

− −

5 2 3 6 2 4
21 22

3 2 2 3 2 2 

### B R B B R B K K A R A A R A − −

= =

− −

1 3 3 2 3 4
31 32

1 3 2 1 3 2 

### B R B B R B K K A R A A R A − −

= =

− −

5 4 3 6 4 4
41 42

3 4 2 3 4 2 

### B R B B R B K K A R A A R A − −

= =

− − .

If the coefficients ij



### K

, 

### 1 4 i ≤ ≤ and 

### 1 2

j ≤ ≤ , exist and 1 

### 0



### d ≠ , then the crossover proportions
and concentrations obtained from equations (5)–(8) exist and are unique

.

NOTE : This method does not require the use of calibrati on curves, and that the set of explicit solutions for three (or more) species is analogous to the above (albeit more lengthy).

The following mathematics is a way to solve the equ ations iteratively. This method was the original iterative method and can now be co nfirmed with the previously introduced explicit approach. To assist the analyst, addition al information, technical support andSW-846 Update V 6800 - 26 Revision 1 July 2014 1 1

2 2

1 1

2 2 

### C A

C A

= A B

A B 

### β and analyst training have been made available for both iterative and explicit solutions (Refs. 11, 27, and 28).

To make the expression simpler, assume

VI
S VI
S VI
S III
S III
S III
S VI

X X VI

X III

X X III

X 

### N W C

, N W C

, N W C

, N W C = = = =

At the beginning of the iteration, arbitrary values can be assigned to N X VI and α . For example, both of them are assigned as 0s. Now we n eed to know the expression of N X III

and β . After careful derivation, we can get the followi ng equations:


## ( ) 

# ( ) ( ) ( )



# [ ] ( ) ( ) α β α

−

+

− = +

− + +

− −

1

N A A R N A N A N A N A R N A A R 1

III
S III
S 50 III
S 52 III
52 / 50 VI
S VI
S 50 VI

X X 50 VI
S VI
S 52 VI

X X 52 III
52 / 50 III

X X 50

X 52 III
52 / 50



## ( ) 

# ( ) ( ) ( )



# [ ] ( ) ( ) α β α

−

+

− = +

− + +

− −

1

N A A R N A N A N A N A R N A A R 1

III
S III
S 53 III
S 52 III
52 / 53 VI
S VI
S 53 VI

X X 53 VI
S VI
S 52 VI

X X 52 III
52 / 53 III

X X 53

X 52 III
52 / 53

These equations can be rewritten as:

The solutions are:

Use these two values in the following equations to solve N X VI and α



## ( ) 

# ( ) ( ) ( )



# [ ] ( ) ( ) β α β

−

+

− = +

− + +

− −

1

N A A R N A N A N A N A R N A A R 1

VI
S VI
S 50 VI
S 52 VI
52 / 50 III
S III
S 50 III

X X 50 III
S III
S 52 III

X X 52 VI
52 / 50 VI

X X 50

X 52 VI
52 / 50



### 1

− β 

## ( ) R 53/52

VI 52 

### A X 

### − 53 

### A X 

# ( ) N X VI 

### + R 53/52

VI 52 

### A X 

### N

X III 

### + 52 

### A S

III 

### N S III 

# ( )

− 53 

### A X 

### N X III 

### + 53 

### A S

III 

### N S III 

# ( )     α



### = − R 53/52

VI 52 

### A S

VI 

### + 53 

### A S

VI 

# ( ) N S VI 

### 1

− β 

## ( )

Rewrite the equation as: III 1 1 X 1

III 2 2 X 2 

### + = N C A B

+ = N C A B 

### β

β   

1

1

2 2 III X

1 1

2 2 

### C B

C B

= N A B

A B

VI 3 3 X 3

VI 4 4 X 4 

### + = N C A B

+ = N C A B 

### α

α   SW-846 Update V 6800 - 27 Revision 1 July 2014 3 3

4 4

3 3

4 4 

### C A

C A

= A B

A B 

### α and again:

Repeating the calculation, the variables N x III , N x VI

, α and β will converge to constant
values, and these values are the solution of the eq uations.

12.2.4 If conversion is complete from one species t o another, then the starting samples may be re-spiked with the isotope-enriched spikes and analyzed, with a particular attention to retard the conversion of the species, if possible. As long as the isotope ratio can be determined, the SIDMS protocol will correct for the conversion even if extensive conversion has occurred. If 100% conversion occurs , this is the result of an active matrix. This indicates that the species converted is highly unstable in that matrix under the extraction procedure chosen.

12.2.5 If appropriate or required, calculate result s for solids on a dry-weight basis as described in Sec. 12.1.3.

12.3 Examples, calculation aids, and help documents for SIDMS are available as referenced in the web (Refs. 11, 27, and 28).

12.4 Results must be reported in units commensurate with their intended use and all dilutions must be taken into account when computing final results.

13.0 METHOD PERFORMANCE

13.1 Performance data and related information are p rovided in SW-846 methods only as examples and guidance. The data do not represent r equired performance criteria for users of the methods. Instead, performance criteria should be developed on a project-specific basis, and the laboratory should establish in-house QC per formance criteria for the application of this method. These performance data are not intended to be and must not be used as absolute QC acceptance criteria for purposes of laboratory accr editation.

13.2 Performance and use of IDMS as a definitive me thod in standard reference material certification has been well established in practice and in the literature. Review and discussion articles are referenced for performance criteria of this highly accurate method (Refs. 1, 9, and 10). IDMS has also been applied to analyze nutriti onal supplements where toxic materials were reported to contaminate zinc dietary supplements wi th antimony in 2010. Thirteen batches of the products and the raw material used as ingredien ts and the contaminated bottles were tested by Method 6800. Testing revealed that four out of t he thirteen batches were not contaminated. The results were independently tested and confirmed by the manufacturer. Quantitative measurements of incoming raw material and final pro ducts tested with Method 6800 IDMS were found to be superior to less accurate results perfo rmed by other methods which were replaced by the manufacturer with Method 6800 using ICP-MS ( Ref. 33). Table 16a and 16b show IDMS concentrations of tested ingredients in batches of recalled dietary supplement prescribed and distributed to hospitals. 3 3

4 4 VI X

3 3

4 4 

### C B

C B

= N A B

A BSW-846 Update V 6800 - 28 Revision 1 July 2014 IDMS by Method 6800 has been shown to be suitable f or the analysis of highly toxic materials. Glyphosate is the active ingredient of a popular he rbicide and is also being used as surrogate for VX nerve agent. I-Spike is a novel solid phase extraction (SPE) technique in which the isotopically enriched spike is preloaded onto a SPE column prior to the introduction of the sample. I-Spike solid-phase sorbent cartridges were tested to provide the placement of a molecular toxic analyte directly on the SPE column (pre-loaded enriched spikes) for safety and quality control purposes. Table 17 demonstrates th e equivalent results comparing preequilibrating the spike with the sample prior to lo ading on the SPE column (liquid phase) or loading the spike and sample individually on the SP E column (solid phase) for added safety of the analyst (Ref. 34).

13.3 Accuracy, precision, and use of Method 6800 SI DMS in quantifying and correcting for species inter-transformations are shown in Tabl es 1 through 15, 18. Table 2(A) and Table 2(B) compare data between Method 6800 and Method 71 96 for Cr(VI) in chromium ore process residues (COPR) and soil extracts. Table 1 demonst rates the ability of this method to correct for transformations of both Cr(VI) and Cr(III) in a queous samples. Table 1 also displays the magnitude of errors that may be expected when using other methods unable to track and determine the conversion of these species. Table 2 (A) indicates a sample type where double spikes were added into the same extract (spiked aft er extraction with Method 3060A) used for determination of Cr(VI) with Method 7196 and then a nalyzed with Method 6800. In this case, both the traditional 3060A/7196 methods and 3060A/6 800 methods produced statistically similar data indicating that transformation had already occ urred and confirmation between these two analytical methods was observed (Refs. 8 and 12). Whereas Table 2(B) indicates a situation where one set of samples were extracted with Method 3060A and analyzed with Method 7196 and another set of sub-samples of the same samples were double spiked before extraction with Method 3060A and then analyzed with Method 6800. I n this case, statistically significant different results were obtained from both the tradi tional 3060A/7196 and the 3060A/6800 methods (Refs. 3 and 12). Method 6800 provided the ability to correct for the transformation of Cr(III) to Cr(VI) using the mathematical protocol d escribed in Sec.12.2.3. This is also an example of how Method 6800 can be used as a diagnos tic tool to isolate the origin of speciesshifts in sample handling, and in analytical prepar ation steps of methods. Recently, NIST has released a standard reference material (SRM) where, according to the SRM’s certificate of analysis, the only true value for the species of Cr (VI) was obtained by EPA Method 6800 and that the measurements made by EPA Methods 7199 and 7196A were in error by as much as 40% (Refs. 37 and 38). The European Committee for Standardization has made a similar declaration (signed by all EU member experts) accep ting Method 6800 as the preferred legally defensible method for Cr(VI) measurement (Ref. 39).

The SIDMS analysis technique, applied to difficult environmental sample matrices, demonstrates the advantages of having the capabilit y to identify and understand the transformations and method perturbations of dynamic species (Refs. 13 and 14). The study
described in Table 3 demonstrates how Method 6800 c an be used to field-spike reactive samples that would have degraded by the time they r eached the analytical laboratory. Table 3 analytically evaluates and validates the field appl ication of chemical spiking at the sampling site and permits evaluation of the process involving fer rous iron and chromate in the treatment or remediation of hexavalent chromium runoff by using another waste by-product, acid mine drainage (AMD) (Ref. 14). Figure 6 demonstrates th e analysis of the isolated fly ash runoff and
the waste stream mixed with the second waste stream of AMD. These data in Table 3 and Figure 6 as cited in the literature (Ref. 14) demon strate the accuracy and precision of both a stable and a less stable set of samples. It was not ed in Ref. 14 that applying Method 6800 at the time of sampling the small amount of residual Cr(VI ) left in the sample for equilibration was enough to make quantitative measurements of Cr(VI). This can be an effective solution toSW-846 Update V 6800 - 29 Revision 1 July 2014 circumventing holding time issues. When a sample is highly reactive, implementing Method 6800 in the field to establish the species content at the time of sampling is an effective way of preserving the quantitative and qualitative species content.

Tables 4(A) and 4(B) demonstrate how Method 6800 ca n be used as a diagnostic tool to observe whether chromium species (and other species ) transformation occurs during or after extraction steps. It is reported in the literature that there is a possibility of precipitation of Pb as PbCrO 4 during acidification. In order to determine if th ere is a bias introduced for Cr(VI) during acidification, the extracts were analyzed directly after acidification, without filtration and after filtration. From Table 4(A), it is observed that the results obtained for the same extract aliquots after performing filtration completely agree with t hose obtained from direct analysis of unfiltered samples after neutralization (Ref. 13). In Method 6800, loss of some portion of an already equilibrated sample does not cause an error, as mat hematical basis of quantitation does not depend on recovery of the analyte but only on the i sotopic ratio (Refs. 13 and 18). Standard calibration is adversely affected by the loss of an alyte. The SIDMS, on the other hand, is unaffected by loss of analyte after isotopic specie s equilibration. From Table 4(B), it is observed, following extraction, that the determinat ion of Cr(VI) is the same for three different instrumental calibrations. In most cases, similar results were obtained by analyzing the same extract using external calibration, IDMS (single sp ike after extraction) or SIDMS (double spike after extraction). This is because the equilibrati on chemistry, including species transformation, usually takes place during extraction of the sample . It can be observed from Table 4(A) and 4(B) that substantial transformation of Cr(III) to Cr(VI) can take place during extraction of the sample, and transformations generally occur nominal ly during post-extraction manipulation.

Table 5 demonstrates the correction necessary in so me soil samples where the sample matrix causes a bias in more traditional methods. These bias corrections demonstrate the ability of Method 6800 to identify and correct for the degradation and transformation of species during the measurement process. Traditional methods such as Methods 7196, 7196A and 7199 are not able to correct for Cr(VI) transformations which may increase or decrease during Method 3060A extraction. Transformations occurring in transportation, storage, extraction, and sample preparation that have changed the species ar e not accounted for and all errors add to
create biases that are undetectable. Method 6800 c orrects for transformations occurring during extraction and other targeted steps when the enrich ed isotopic spike(s) are added to include those step including in the field at collection.

The SIDMS approach in Method 6800 was applied in th e simultaneous determination of chromium species in food supplement, in this case b iological samples, by an independent laboratory (Ref. 22). The Institute of National Me asurement Standards, National Research Council of Canada has independently validated Metho d 6800 on brewers’ yeast where they found Cr(VI) converting to Cr(III) and vise versa, and concluded that the SIDMS method enabled the accurate determination of both Cr(VI) a nd Cr(III) under these conditions. This work
is summarized in Table 6 which was abstracted with permission from the author in Ref. 22. This study concluded that despite the presence of signif icant bi-directional transformation of chromium species during sample preparation, an accu rate simultaneous determination of both species in yeast was made possible through the appl ication of the SIDMS method (Table 6).

13.4 The SIDMS analysis technique has been successf ully been applied for the determination of other species. For example, methy lmercury and inorganic mercury have been successfully determined in biological, human and ot her tissues, and in soils. Table 7 demonstrates concentration measurements in soil sam ples (Refs. 15 and 16). Method 6800 has also been applied as a diagnostic tool to deter mine the concentration and to identify analytical biases caused by species transformations using the Microwave-Assisted Extraction methods such as Method 3200, used in mercury specie s extraction. The SIDMS protocol ofSW-846 Update V 6800 - 30 Revision 1 July 2014 Method 6800 provides the ability to measure the con centration of species and to evaluate if species transformations have occurred. One set of s amples was double-spiked before extraction and another aliquot of the same sample w as double-spiked after extraction. It was
observed that this extraction method did not alter the species or permit them to transform in this matrix, using this extraction protocol. The deconvo luted concentrations for each species (Table 7) obtained from both sets of extractions overlap a t the 95% CI and are statistically indistinguishable (Ref. 15).

In a separate study, the SIDMS protocol was used as a diagnostic tool for the evaluation of different mercury speciation methods published i n the literature and in Method 3200 (Refs. 16 and 17). From this study, it was observed that two of the five literature methods were highly prone to form inorganic mercury from methylmercury during extraction. These two published methods converted approximately ~100% of the methyl mercury to inorganic mercury, including the spiked isotope enriched methylmercury. In anot her literature method, as much as 45% methylmercury was converted into inorganic mercury during extraction. Method 3200 and a sonication-based extraction method induced very lit tle or no conversion of methylmercury to inorganic mercury (Table 8) (Ref. 17). Method 6800 has also been used to validate other speciation method. For example, if used in conjunc tion with Method 3200, Method 6800 corrects for matrix-induced and method-induced tran sformations of species, thus permitting the evaluation of Method 3200 and other speciation meth ods (Refs. 15, 17, 20, and 21).

The SIDMS protocol of Method 6800 along with Method 3200 was applied for the determination of mercury species in tissue samples (Ref. 20) (Table 9). After analyzing several fish tissue reference materials (ERM-CE464, DOLT-3, NIST-1946) with better than 20% precision, approximately 20% of the inorganic mercu ry was converted to methylmercury during analysis and methylmercury converted to inorganic m ercury species. Here, the creation of the species of interest from inorganic mercury would be undetected without the ability of Method 6800 to evaluate and correct for the species-conver sions in these speciated measurements.

The SIDMS method was applied as a diagnostic tool t o evaluate eight published literature methods for mercury speciation in human hair (IAEA- 085) (Ref. 21). From this study, it was observed that most of the methods induced minor or no statistically significant species transformation during extraction. However, two of t he eight methods induced larger amounts of species transformation from methylmercury to inorga nic mercury. One method transformed approximately 90% and the other transformed almost 20% of methylmercury to inorganic mercury during extraction (Table 10). Because an I AEA reference material was used in this study, the reference value was available for valida tion of all methods. The inorganic and methylmercury concentrations were obtained by the S IDMS protocol of Method 6800 as were the amount of inorganic mercury converted into meth ylmercury. The correction for methylmercury and inorganic mercury conversion was also accomplished. Total mercury was determined mathematically by adding inorganic and m ethylmercury. Without conversion correction, the accurate concentration measurement cannot be assured.

In Ref. 30, three mercury species were extracted us ing Method 3200 and measured individually while others were grouped as discussed in Method 3200. After the removal of the extractable mercury species, the remaining mercury species were spiked collectively and measured as a group. Ref. 30 demonstrates the abili ty of Method 6800 to correct for species transformations as much as 80% and obtain mass bala nce, after analysis. Mass balance simply requires addition of all species forms in a sample to equal to the sum of all related species. In this case, mass balance was achieved when all the m ercury species that are collectively or individually present in crude oil were accounted fo r.SW-846 Update V 6800 - 31 Revision 1 July 2014 Ref. 31 and Table 14 show the application of SIDMS to human blood SRM. Here, inorganic, methyl and ethyl mercury species from ai r, food and water contamination are measured. Interspecies conversions of each of thes e species are calculated and corrected for. Through the analysis of an analytical reference mat erial (ERMOCE464, Tuna Fish), Ref. 32 demonstrates that once the equilibration is achieve d in the extraction step, incomplete extraction of the species does not affect accuracy. Ref. 30 describes the way existing methods in SW-846 Chapters Three and Four, and the literatu re may be used to implement 6800, along with other methods by adding the enriched species d uring sample preparation to overcome errors such as recovery.

The SIDMS protocol can also be used successfully t o determine the concentration of one or more analytes in the sample where species conver sion, degradation and/or incomplete separation may have taken place before or during an alysis (Refs. 2, 3, 5-8, 11-32, and 34-40). Additional equations and mathematical manipulations required for the determination of three simultaneous species have been developed and publis hed. The first mathematical solutions for SIDMS were iterative. In addition to iterative equ ations, new explicit and deterministic SIMDS solution for three species analysis has been develo ped, published and is available in 6800 and Ref. 28. The explicit and determinative equations w ere developed for mission-critical applications, such as homeland defense and homeland security, where the detection method is required to perform predictable precision and accur acy to obtain actionable data in the field under safety scenarios. For mathematical solutions using three simultaneous species equations, tutorials and calculation aids, you are referred to the references, web sites and related publications cited in Refs. 24, 27, 32, and 35-40.

SIDMS is a general application for the detection o f active species that may transform during sample collection, storage, shipping, prepar ation, analysis and other manipulations. In Figures 8 and 9, and Table 18 (Refs. 21, 34, 35, 36 , 39, 40, and 41), glutathione, one of the most ubiquitous anti-toxin species in the human bod y is quantified in the oxidized and reduced form, together with the conjugated toxicant species such as inorganic and methyl mercury using ESI-QTOF-MS. This type of combined biological and i norganic quantitation is now being applied to autism and other medical research. In Ref. 35, t he application of Method 6800 with MALDIQTOF-MS demonstrates that glutathione and conjugate d mercury species are quantifiable by applying the SIDMS equations to data generated with either of the commonly used ionization sources (ESI and MALDI)(Ref. 35, 42). Ref. 41 demon strates that ESI-ion trap instruments can make the same qualitative measurements of glutathio ne with toxicants. However, quantitation is not possible without direct isotopic ratios as a ca libration curve of glutathione with toxicants is not feasible. Figure 10 and Refs. 40 and 42 demonst rate the quantitative SIDMS analysis of sodium azide which is a toxicant and an explosive t hat is included with the air bags in automobiles. Lead azide is a primary component of primers in munitions and a toxicant in water. It is difficult to analyze sodium azide by G C-MS as it is not readily ionized. Method 6800 demonstrates the quantitative measurement on a TOF- MS directly without calibration curves using a mathematical determination. False positives do not occur in Method 6800 since these quantitative isotopic ratios are not achievable unl ess both the natural and isotopic forms of the analyte are present. Here, incomplete extraction di d not affect accuracy with Method 6800. Also adduct formation due to sodium or other compounds i n the sample matrix do not require matrix matching as all adduct ratios provided the same num erical concentration of the solution indicating that equilibration with the sample was a chieved. Sodium azide (Figure 10 and Refs. 42 and 40), potassium cyanide, toxicants and other difficult analytes in molecular and ionic forms were tested for homeland security and environ mental health assessment purposes. All requested priority toxins (including those that cou ld not be assessed by conventional field methods) and explosives were successfully quantifie d in a similar manner. Analytical results of these analytes (Ref. 42) were selected from a group of analytes generally classified as Chemical, Biological, Radioactive, Nuclear, and Exp losive (CBRNE) agents. Details of theseSW-846 Update V 6800 - 32 Revision 1 July 2014 tests included in this document exist as “task repo rts” provided to US Air Force. Portions of these reports may be obtained from info@sidms.com ( www.sidms.com), with prior request and approval.

The IDMS protocol can be used to determine simulta neously elemental concentrations as total elemental concentrations using sample decompo sition methods such as Method 3052. An example of such IDMS analysis is provided for groun ded and pulverized electronic components and circuit boards. The European environmental reg ulations referred to as Reduction of Hazardous Substances (RoHS), Waste, Electrical and Electronic Equipment (WEEE) and Registration; Evaluation, Authorization and Restric tion of Chemicals (REACH) require total and speciated analyses. Table 11 demonstrates total el emental analysis by IDMS using Method 3052 microwave-enhanced sample decomposition where Cd-112, Cr-50, Pb-206 and Hg-199 isotopes were added into the sample. IDMS analysis was accomplished using a commercially available ICP-MS and total elemental content is rep orted in Table 11 (Ref. 26). Chromium species can be determined as described in Sec. 13.3 to complete the analysis requirements under the European RoHS regulations. The concentra tion of Cr(VI) was negligible in these specific samples as measured by Method 6800.

Molecular SIDMS protocols are demonstrated as exam ples by Refs. 34, 35, and 36 where molecular species such as reduced and oxidized glut athione are the molecular examples used to illustrate the SIDMS deconvolution of transforma tions of molecular species such as peptides and proteins in the immune system. These molecular species measure the red blood cell concentration of reduced and oxidized glutathione ( GSH and GSSG, respectively) and the ratio of these species that is an indication of the funct ion of the immune system and their ability to detoxify toxicants. Like Cr(VI) the GSH and GSSG mu st be spiked during the sampling of the blood and before shipment to the laboratory to make possible the accurate quantification of GSH and GSSG species in the blood sample. These SI DMS molecular measurements use specific sample preparation and separation specific to these species. The above equations have an almost exact adaptation of the algorithms f or the Cr(III) and Cr(VI) calculated examples in sections 7.3.1 and in example calculations secti on 12.2.

13.5 The following documents may provide additional guidance and insight on the elemental and molecular isotope ratio measurement u sing ICP-MS, ESI-QTOF and ESI-QQQ, respectively:

13.5.1 K. E. Jarvis, A. L. Gray and R. S. Houk, Handbook of Inductively Coupled Plasma Mass Spectrometry ; Blackie Academic & Professional: London, U.K. 199 2, p-315.

13.5.2 G. P. Russ III, J. M. Bazan, Spectrochim. Acta, Part B 1987, 42B, 49- 62.

13.5.3 H. E. Taylor, Inductively Coupled Plasma Mass Spectrometry: Pract ice and Techniques , Academic Press: San Diego, CA, USA, 2001, p-48.

13.5.4 M. Sargent, R. Harte and C. Harrington, Guidelines for Achieving High Accuracy in Isotope Dilution Mass Spectrometry , The Royal Society of Chemistry: Cambridge, UK, 2002.

13.5.5 “Characterization of waste – State of the art document – Chromium VI specification in solid matrices” PD CEN/TR 14589:20 03, British Standards, EU publication “International Standards Correspondence Index, Technical Report CEN Technical Committee CEN/TC 292, European Committee for Standardization,
2003.SW-846 Update V 6800 - 33 Revision 1 July 2014 13.5.6 The web sites: http://www.epa.gov/epaoswer/hazwaste/test/new-meth. htm contains Method 6800 and www.sampleprep.duq.edu and www.sidms.com, conta in SIDMS/Method 6800 analysis aids, tutorials, update information and ap plications (Refs. 27, 28, 24, 35, 36, and 42).

Acknowledgement and Disclaimer: Some of the work and data in this document associat ed with homeland security and homeland defense were supported by the Headquarters of the U S Air Force Surgeon General under awards No.FA7014-07-0014 and No.FA7014-08-C-0029. T he opinions interpretations, conclusions and recommendations are those of the au thor and not necessarily endorsed by the US Air Force.

14.0 POLLUTION PREVENTION

14.1 Pollution prevention encompasses any technique that reduces or eliminates the quantity and/or toxicity of waste at the point of g eneration. Numerous opportunities for pollution prevention exist in laboratory operations. The EPA has established a preferred hierarchy of environmental management techniques that places pol lution prevention as the management option of first choice. Whenever feasible, laborat ory personnel should use pollution prevention techniques to address their waste generation. When wastes cannot be feasibly reduced at the source, the Agency recommends recycling as the next best option.

14.2 For information about pollution prevention tha t may be applicable to laboratories and research institutions consult Less is Better: Laboratory Chemical Management for Waste
Reduction , a free publication available from the American Ch emical Society (ACS), Committee on Chemical Safety, http://portal.acs.org/portal/fileFetch/C/WPCP_01229 0/pdf/WPCP_012290.pdf.

15.0 WASTE MANAGEMENT

The Environmental Protection Agency requires that l aboratory waste management practices be conducted consistent with all applicab le rules and regulations. The Agency urges laboratories to protect the air, water, and land by minimizing and controlling all releases from hoods and bench operations, complying with the lett er and spirit of any sewer discharge permits and regulations, and by complying with all solid an d hazardous waste regulations, particularly the hazardous waste identification rules and land d isposal restrictions. For further information on waste management, consult The Waste Management Manual for Laboratory Personne l available from the American Chemical Society at the address listed in Sec. 14.2.

16.0 REFERENCES

1. J. D. Fasset and P. J. Paulsen, "Isotope Dilutio n Mass Spectrometry," Anal. Chem . 1989, 61(10), 643A-649A.

2. H. M. Kingston, "Method of Speciated Isotope Dil ution Mass Spectrometry," US Patent Number: 5,414,259, 1995.SW-846 Update V 6800 - 34 Revision 1 July 2014 3. D. Huo and H. M. Kingston, "Correction of Specie s Transformations in the Analysis of Cr(VI) in Solid Environmental Samples Using Speciat ed Isotope Dilution Mass Spectrometry," Anal. Chem. 2000, 72(20), 5047-5054

.

4. B. R. James, J. C. Petura, R. J. Vitale and G. R . Mussoline, "Hexavalent Chromium Extraction from Soils: A Comparison of Five Method s," Environ. Sci. Tech. 1995, 29(9)

, 2377-2381.

5. H. M. Kingston, D. Huo, Y. Lu and S. Chalk, "Acc uracy in Species Analysis. Speciated Isotope Dilution Mass Spectrometry (SIDMS) Exemplif ied by the Evaluation of Chromium Species," Spectrochim. Acta, Part B: Atomic Spectroscopy , 1998, 53B(2), 299-309.

6. D. Huo, H. M. Kingston and B. Larget, “Applicati on of Isotope Dilution in Elemental Speciation: Speciated Isotope Dilution Mass Spectro metry (SIDMS),” In: Comprehensive Analytical Chemistry, J. A. Caruso, K. L. Sutton and K. L. Ackley (eds.), Vol. 33 (Elemental Speciation: New Approaches for Trace Element Analys is), Elsevier, New York, USA, 2000, p-277-313.

7. H. M. Kingston, D. Huo, S. Chalk and P. Walter, "The Accurate Determination of Species by Speciated Isotope Dilution Mass Spectrometry: Ex emplified by the Evaluation of Chromium (VI) in Soil," The Twelfth Annual Waste Te sting & Quality Assurance Symposium; Washington, DC, July 23-26, 1996, 112-11 9.

8. D. Huo, Y. Lu and H. M. Kingston, "Determination and Correction of Analytical Biases and Study on Chemical Mechanisms in the Analysis of Cr( VI) in Soil Samples Using EPA Protocols," Environ. Sci. Tech. , 1998, 32(21), 3418-3423.

9. G. N. Bowers, Jr., J. D. Fassett and E. V. White , "IDMS and the National Reference System: Accuracy Transfer in Clinical Chemistry," Anal. Chem. , 1993, 65(12), 475 - 479.

10. L. J. Moore, H. M. Kingston and T. J. Murphy, " The Use of Isotope Dilution Mass Spectrometry for the Certification of Standard Refe rence Materials," Environ. Intern. , 1984, 10(2), 169-173.

11. H. M. ‘Skip’ Kingston, D. Huo, G. M. M. Rahman, and P. Walter, SamplePrep Web [Homepage of SamplePrep Web], Available: http://www .sampleprep.duq.edu/, referenced
2011.

12. D. Huo, “The Development of Methods in Speciate d Isotope Dilution Mass Spectrometry (SIDMS) for Elemental Speciation of Chromium in Env ironmental Samples Using LC-ICPMS,” Doctoral Dissertation , Duquesne University, July 30, 1999.

13. G. M. M. Rahman, H. M. Skip Kingston, T. G. Tow ns, R. J. Vitale and K. R. Clay, “Determination of hexavalent chromium by using spec iated isotope-dilution mass spectrometry after microwave speciated extraction o f environmental and other solid materials,” Anal. Bioanal. Chem ., 2005, 382, 1111-1120.

14. H. M. “Skip” Kingston, R. Cain, D. Huo and G. M . M. Rahman, “Determination and evaluation of hexavalent chromium in power plant co al combustion by-products and costeffective environmental remediation solutions using acid mine drainage,” J. Environ. Monit. , 2005, 7, 899-905.SW-846 Update V 6800 - 35 Revision 1 July 2014 15. G. M. M. Rahman and H. M. ‘Skip’ Kingston, “Dev elopment of a microwave-assisted extraction method and isotopic validation of mercur y species in soils and sediments,” J. Anal. At. Spectrom. , 2005, 20, 183-191.

16. G. M. M. Rahman, H. M. ‘Skip’ Kingston, John Ke rn, Sarah W. Hartwell, Ramond F. Anderson and Shen-yi Yang, “Inter-laboratory Valida tion of EPA Method 3200 for mercury species analysis using prepared soil reference mate rials,” J. Applied Organometallic Chem. , 2005, 19, 301-307.

17. G. M. M. Rahman and H. M. ‘Skip’ Kingston, “App lication of Speciated Isotope Dilution Mass Spectrometry to Evaluate Extraction Methods fo r Determining Mercury Speciation in Soils and Sediments,” Anal. Chem ., 2004, 76, 3548-3555.

18. H. M. Kingston, “Speciated isotope dilution mas s spectrometry of reactive species and related methods,” US Patent Number 6 790 673 B1, 20 04 and PCT Int. Appl . January 28, 1999, Patent No. WO 9939198.

19. H. M. Skip Kingston, U.S. Patent Number 6,974,9 51 B1, “Automated In-Process Ratio Mass Spectrometry” Dec 13, 2005.

20. L. Hinojosa Reyes, G. M. M. Rahman, H. M. Skip Kingston, Robust microwave-assisted extraction protocol for determination of total merc ury and methylmercury in fish tissues, Analytica Chemica Acta, 631, 121-128, 2009.

21. G. M. M. Rahman, T. Fahrenholz and H. M. Skip Kingston, “Application of Speciated Isotope Dilution Mass Spectrometry to Evaluate Meth ods for Efficiencies, Recoveries, and Quantification of Mercury Speciation Transformatio ns in Human Hair” Journal of Analytical Atomic Spectrometry, 24, 83-92, 2009.

22. Lu. Yang, Elena Ciceri, Zoltan Mester and Ralp h E. Sturgeon, “Application of DoubleSpike Isotope Dilution for the Accurate Determinati on of Cr(III), Cr(VI) and Total Cr in Yeast”, Anal. Bioanal. Chem ., 2006, 386: 1673-1680

23. H. M. Skip Kingston, Robert McDonald, Ye Han, J ason Wang, June Wang, Michael West, Larry Stewart, Bob Ormond, and Rudy Mui, “Automated , On-Line, Trace Contamination and Chemical Species Analysis For the Semiconductor Industry,” American Institute of Physics (AIP), Conference Proceedings, Proceedings form the 2003 Characterization and Metrology for ULSI Technology Conference, Volume 68 3, Melville, NY, 2003.

24. G. M. Mizanur Rahman, “Development, Research a nd Validation of Environmental Speciation Methods: Evaluation by Speciated Isotope Dilution Mass Spectrometry in Mercury and Chromium Speciation Analysis.” Doctoral Dissertation, Duquesne University, Pittsburgh, PA USA, 2004.

25. Juris Meija, Lu Yang, Joseph A. Caruso, Zoltan Mester, “Calculations of Double Spike Isotope Dilution Results Revisited.” J. Analytical Atomic Spectrometry, 2006 21, 1294- 1297,

26. Gonzalo Manchego, G. M. Mizanur Rahman, Laura Reyes, Panxi Zhao, H. M. Skip Kingston, “Testing is an Essential Part of Internat ional Environmental Regulation and Commerce Compliance”, Washington DC, NEMC Presentat ion 2008.SW-846 Update V 6800 - 36 Revision 1 July 2014 27. 

### http://www.epa.gov/epaoswer/hazwaste/test/new-meth. htm and www.sampleprep.duq.edu and www.sidms.com.

28. John Kern, H. M. Skip Kingston, G. M. M. Rahma n, J. Sun, L. H. Reyes, Accurate, Explicit Determinations in Speciated Isotope Dilution Mass S pectrometry without Calibration Curves, in preparation 2012.

29. C. Toews, J. Kern, H. M. Kingston and M. Rahma n, “Sensitivity Analysis for Speciated Isotope Dilution Mass Spectrometry” Journal of Anal ytical and Atomic Spectrometry, 25, 196-200, 2010.

30. G. M. Mizanur Rahman, Timothy M. Fahrenholz, H. M. Skip Kingston, Matt Pamuku, J. David Hwang, and Lyman A. Young, “Speciation of Mer cury in Crude Oil Using Speciated Isotope Dilution Mass Spectrometry” Spectroscopy, 2 5,1, 36-45, 2010.

31. G. M. Mizanur Rahman, Timothy M. Fahrenholz, Ma tt Pamuku, Panxi Zhao, Laura Reyes, Scott Faber, H. M. Skip Kingston, “Speciation of Me rcury in whole human blood by SIDMS SPME-GC-ICP-MS with Microwave Assisted Enhanced Equ ilibration and Extraction” (in preparation 2012)

32. Laura H. Reyes, G. M. Mizanur Rahman, Timothy Fahrenholz, H. M. Skip Kingston, “Comparison of methods with respect to efficiencies , recoveries, and quantitation of mercury species interconversions in food demonstrat ed using tuna fish” Analytical Bioanalytical Chemistry, 390, 2123-2132, 2008.

33. Gregory M. Zinn, G. M. Mizanur Rahman, Curtis Frantz, Larry Newman, Scott Faber, Matt Pamuku, H. M. Skip Kingston, “Dietary Supplement Co ntamination with metals, (in preparation 2012)

34. Rebecca L. Wagner, Stephanie J. Wetzel, John Ke rn, Mizanur Rahman, Matt Pamuku, H. M. Skip Kingston, “Improved sample preparation of g lyphosate and methylphosphonic acid by EPA Method 6800 and time-of-flight mass spectrom etry using a novel solid phase extraction,” ( Accepted : Journal of Mass Spectrometry, 2012)

.

35. Timothy Fahrenholz, H. M. Skip Kingston, John K ern, Matt Pamuku, Hemasudha Chatragadda, “Quantification of Protein Biomarkers of Glutathione Reduced and Oxidized ” (in preparation 2012)

36. Michael Passineau, Timothy Fahrenholz, Laurie M achen, Zourelias Lee, Katherine Nega, Rachel Paul, Mary MacDougall, Olga Mamaeva, Richard Steet, Jarrod Barnes, H. M. Skip Kingston, Raymond Benza. “α-Glactosidase A Expresse d in the Salivary Glands Partially Corrects Organ Biochemical Deficits in the Fabry Mo use Through Endocrine Trafficking”Human Gene Therapy, 22:1-9 (March 2011)

.

37. NIST Certificate of Analysis SRM 2701 “Hexavale nt Chromium in Contaminated Soil (High Level)” (2009),

38. Stuart Nagourney, Stephen A. Wilson, Brian Buck ley, H. M. Skip Kingston, Shen-Yi Yang and Stephen E. Long. Journal of Analytical Atomic S pectroscopy, 23, 1550-1554, 2008.

39. “Characterization of waste – Stat of the art do cument – Chromium VI specification in solid matrices” PD CEN/TR 14589:2003, British Standards, EU publication “InternationalSW-846 Update V 6800 - 37 Revision 1 July 2014 Standards Correspondence Index, Technical Report CE N Technical Committee CEN/TC 292, European Committee for Standardization, 2003.

40. Gregory M. Zinn, G. M. Mizanur Rahman, Sara Kal lop, H. M. Skip Kingston, “Sodium Azide and Potassium Cyanide Quantification for homeland d efense by isotope dilution mass spectrometry EPA Method 6800 (in preparation 2012).

41. Eva Krupp, Bruce Milne, Adrin Mestrot, Andrew Meharg and Jorg Feldmann, “Investigation into mercury bound to biothiols: structural identif ication using ESI-ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS.” Analytical Bioanalytical Chemistry, 3 90, 1753-1764, 2008.

42. US AIR FORCE Task Reports for the Homeland Secu rity and Homeland Defense: extension of EPA Method 6800 conducted between 2007 and 2010 with funding from the Headquarters of the US Air Force Surgeon General un der the awards, No.FA7014-07-0014 and No.FA7014-08-C-0029. Report authors: H. M. Skip Kingston, M. Pomuku, M. Rahman, G. M. Zinn, R. L. Wagner. This report can be obtai ned from the US Air Force (with their permission) as some “sensitive” discussions are con tained in this report.

43. Timothy Fahrenholz, Scott Faber, H. M. Skip Ki ngston, John Kern, Matt Pamuku, Hemasudha Chatragadda, Becky Pekar, “Quantification Glutathione Biomarkers in Autism” (in preparation 2012).

44. Rebecca L. Wagner, Stephanie Wetzel, H. M. Ski p Kingston“ Direct Speciated Isotope Dilution Mass Spectrometry of Analytical Methodolog y of Illicit Drug in Urine Using Time-ofFlight Mass Spectrometry” (in review 2012).

17.0 TABLES, DIAGRAMS, FLOW CHARTS AND VALIDATION D ATA

The following pages contain the tables and figures referenced by this method. The pages also contain two work-flow diagrams (one each for I DMS and SIDMS). A glossary follows these
materials.

The data below are provided for guidance purposes o nly.SW-846 Update V 6800 - 38 Revision 1 July 2014 TABLE 1

ANALYSIS OF AN ARTIFICIALLY SYNTHESIZED WATER SAMP LE (Refs. 6 and 7)

Concentration (ng/g) Conversion (%)

Aliquot Days after

spiking Cr(III) Cr(VI) Cr(III) to Cr(VI) Cr(VI) to

Cr(III)

1 69.8 ± 0.3 68.8 ± 0.3 4.87 ± 0.22 3.57 ± 0.03

1 4 69.2 ± 0.6 69.4 ± 0.3 3.47 ± 0.11 11.9 ± 0.5

13 70.5 ± 0.9 68.5 ± 0.4 2.80 ± 0.13 22.4 ± 0.2

1 69.6 ± 0.2 68.8 ± 0.4 17.6 ± 0.1 2.95 ± 0.02

2 4 69.3 ± 0.7 69.6 ± 0.6 14.6 ± 1.3 11.4 ± 0.7

13 70.7 ± 0.4 68.8 ± 0.3 12.8 ± 0.1 22.1 ± 0.3

1 69.8 ± 0.6 69.0 ± 0.2 23.8 ± 0.3 2.76 ± 0.08

3 4 69.0 ± 0.8 69.6 ± 0.3 21.6 ± 0.2 10.2 ± 0.1

13 70.4 ± 0.5 68.9 ± 0.8 17.6 ± 0.3 22.1 ± 0.1

True 69.67 68.63

mean ± 95% confidence interval

Aliquots 1, 2 and 3 were from the same isotopically -spiked synthesized sample. These aliquots were treated in different ways to permit different degrees of interconversion between Cr(III) and Cr(VI). Measurements were done on different days t o check the stability of the species during storage. Despite the different degrees of intercon version, the deconvoluted concentrations for both Cr(III) and Cr(VI) were always corrected to th e true concentrations within experimental

error.SW-846 Update V 6800 - 39 Revision 1 July 2014 TABLE 2 (A)

CONCENTRATIONS OF Cr(VI) IN COPR SAMPLES DETERMINED WITH METHOD 7196

AND SIDMS (SPIKED AFTER EXTRACTION)

(Refs. 8 and 12)

Method 7196 SIDMS
Sample Conc. of Cr(VI)

( μ g/g) Average

(mean ± std) Conc. of Cr(VI) ( μ g/g) Average

(mean ± std)

COPR1 1330 1410 ± 85 1373 1445 ± 70

1410 1449

1500 1512

COPR3 91.2 85.3 ± 5.2 93.9 88.8 ± 6.1

81.5 82.1

83.1 90.4

COPR4 408.9 407.8 ± 7.2 419.8 418.0 ± 9.2

414.4 426.1

400.2 408.0

COPR: chromite ore processing residue. Method 3060A was used for Cr(VI) extraction. Results obtained from SIDMS and Method 7196 are com parable for COPR samples.SW-846 Update V 6800 - 40 Revision 1 July 2014 TABLE 2(B)

OXIDATION OF Cr(III) TO Cr(VI) DURING EXTRACTION WI TH METHOD 3060A FROM THREE COPR SAMPLES AND DETERMINED WITH METHOD 7196 AND SIDMS (SPIKED BEFORE EXTRACTION)

(Refs. 3 and 12)

Total Cr Cr(VI) ( μ g g - 1 ) RE e

(mg g - 1 ) SIDMS

Corrected for Cr(III) to

Cr(VI) conversion Method 7196

Uncorrected for Cr(III) to

Cr(VI) conversion (%)

COPR 1 10.4 a 2573 ± 35 d 2671 ± 17 3.8

COPR 3 1.97 a 161 ± 6 351 ± 8 118

COPR 4 4.60 a 614 ± 13 877 ± 21 43

Fresh Fly Ash 0.0475 b 8.3 ± 0.3 10.0 ± 0.4 21

Fly Ash (at 41 ft.) 0.0582 b 3.3 ± 0.3 4.1 ± 0.1 24

SRM 1645 (River

sediment) 29.6 c 1045 ± 46 2753 ± 31 163

a provided by Environmental Standards, Inc.

b determined with EPA method 3052

c certified values

d mean ± 95% confidence interval

e Relative error

TABLE 3

ANALYSIS OF Cr(VI) IN LEACHATE FROM COAL FLYASH AND ACID MINE DRAINAGE (AMD) COMBINATION SAMPLES

(Ref. 14)

Sample mixture Aliquot Cr(VI) calculated/

ng g -1 b Cr(VI) measured/

ng g -1
1:1 F Aliquot 1 704 46.6 ± 8.6

1:1 L Aliquot 2 704 17.5 ± 4.2 5:1 F Aliquot 1 1172 963 ± 53 5:1 L Aliquot 2 1172 25.5 ± 3.5

a Uncertainties are at 95% confidence interval, n = 3

b Only dilution of the leachate is considered L – spiked in laboratory F – spiked in filedSW-846 Update V 6800 - 41 Revision 1 July 2014 TABLE 4(A)

HEXAVALENT CHROMIUM RESULTS OBTAINED FROM INDUSTRIA L MATERIALS EXTRACTED WITH METHOD 3060A AND ANALYZED WITH METHO D 6800 (DOUBLE SPIKED BEFORE EXTRACTION)

(Ref. 13)

Sample

Matrix Sample ID EPA Method 6800 (diagnostic SIDMS)

Cr 6+ before

filtration Cr 6+ after

filtration Cr 3+ to Cr 6+

before filtration Cr 3+ to Cr 6+ after

filtration

( μ g g - 1 ) ( μ g g - 1 ) (%) (%)

SRM SRM 2704 7 ± 3 8 ± 1 50 ± 2 45 ± 4

SRM 2711 N/A N/A N/A N/A

Sediments Sediment-1 ND N/A N/A N/A

Sediment-2 1.77 ± 0.34 N/A N/A N/A

Aggregate

Material Aggregate-1 308 ± 44 314 ± 28 56 ± 3 57 ± 2

Aggregate-2 341 ± 29 334 ± 36 46 ± 1 47 ± 1

Aggregate-3 206 ± 9 205 ± 16 48 ± 6 49 ± 7

Aggregate-4 141 ± 6 141 ± 14 33 ± 3 33 ± 2

Aggregate-5 223 ± 28 216 ± 17 41 ± 2 41 ± 1

Aggregate-6 289 ± 27 291 ± 22 19 ± 3 19 ± 3

Aggregate-7 278 ± 32 306 ± 29 20 ± 1 20 ± 1

Uncertainties are at 95% CI, n = 3

ND = not detectable (lowest measurable chromium = 0 .5 ng mL -1 and this corresponds to 12.5 ng g -1 in

soil or sediment sample)

NA = not applicableSW-846 Update V 6800 - 42 Revision 1 July 2014 TABLE 4(B) HEXAVALENT CHROMIUM RESULTS OBTAINED FROM INDUSTRIA L MATERIALS EXTRACTED WITH METHOD 3060A AND ANALYZED WITH IC-IC P-MS (EXTERNAL CALIBRATION) AND METHOD 6800 (IDMS & SIDMS), SINGLE SPIKED & DOUBLE SPIKED AFTER EXTRACTION

(Ref. 13)

Sample External

calibration IDMS a EPA method 6800 b

( μ g g - 1 ) ( μ g g - 1 ) ( μ g g - 1 ) Cr 3+ converted to Cr 6+ (%)

SRM 2704 7 ± 2 12 ± 1 14 ± 2 10 ± 1

SRM 2711 7 ± 1 1.5 ± 0.1 12 ± 4 9 ± 4

Aggregate-1 342 ± 19 367 ± 25 342 ± 26 12 ± 3

Aggregate-2 440 ± 6 448 ± 17 409 ± 11 5 ± 2

Aggregate-3 245 ± 17 259 ± 12 242 ± 16 4 ± 1

Aggregate-4 164 ± 8 171 ± 7 184 ± 11 10 ± 2

Aggregate-5 199 ± 22 215 ± 17 214 ± 23 9 ± 2

Aggregate-6 277 ± 22 295 ± 15 319 ± 23 10 ± 2

Aggregate-7 291 ± 22 298 ± 18 328 ± 24 17 ± 4

Uncertainties are at 95% CI, n = 3

a Extracts were spiked with 53 Cr 6+ spike after extraction

b Extracts were double spiked with iso-50 Cr 3+ and 53 Cr 6+ after extraction

TABLE 5

RECOVERY OF Cr(VI) SPIKED INTO SOIL EXTRACTS (Ref. 8)

Sample Mass of Soil (g) Spiked nat Cr(VI) Recovery (%)

( μ g/g) Method 7196 SIDMS

1 0 2.997 101 ± 0.4 100 ± 1.3

2 1.53 3.033 91.8 ± 1.7 100 ± 0.3

3 3.06 2.993 81.9 ± 1.1 101 ± 0.3

4 3.12 1.587 71.6 ± 2.5 99.3 ± 0.3

Results obtained from SIDMS and Method 7196 are inc omparable for soil extracts due to the serious matrix effects resulting from the coexistin g reducing agents in soil. Method 7196 is incapable of correcting conversion of Cr(VI) leadin g to low recoveries. Results are based on N = 3 with uncertainties expressed in standard deviatio n.SW-846 Update V 6800 - 43 Revision 1 July 2014 TABLE 6

RESULTS FOR SPECIATION OF Cr IN YEAST

(Ref. 22)

Sample Nat Cr(III)

added, (mg/kg) Nat Cr(VI)

added, (mg/kg) Measured

Cr(III), (mg/kg) Measured

Cr(VI), (mg/kg) Nat Cr(III)

Recovery (%) Nat Cr(VI)

Recovery (%) Measured

Cr(III) + Cr(VI) (mg/kg) Measured

Total Cr (mg/kg)

Yeast 0 0 1952 ± 103 76 ± 48 NA NA 2028 ± 57 2014 ± 16 Spiked
Yeast 1784 2398 3749 ± 43 2466 ± 40 101 ± 2 100 ± 2 NA NA

NA – not applicable Uncertainties are at 95% confidence interval, n = 3

.

TABLE 7

THE DECONVOLUTED CONCENTRATION AND TRASFORMATION OF MERCURY SPECIES

IN REFERENCE SOIL (MATERIAL-1) USING SIDMS CALCULAT IONS. (Ref. 15)

Deconvoluted Concentration Interconversion

Hg 2+

( μ g/g) CH 3 Hg +

( μ g/g) Hg 2+ to CH 3 Hg +

(%) CH 3 Hg + to Hg 2+

(%)

DSBE 3.05 ± 0.12 2.69 ± 0.10 1.3 ± 1.5 0.1 ± 1.4

DSAE 2.94 ± 0.07 2.62 ± 0.09 0.8 ± 1.5 0.7 ± 0.6

DSBE = double spiked before extraction; DSAE = doub le spiked after extraction.

Uncertainties are expressed at 95% CL with n = 3.

Material-1: 100% processed topsoil containing both inorganic mercury and methylmercury.SW-846 Update V 6800 - 44 Revision 1 July 2014 TABLE 8 EVALUATION OF DIFFERENT LITERATURE METHODS FOR MERC URY SPECIATION IN

SOILS AND SEDIMENTS USING METHOD 6800.

(Ref. 16)Sample Extraction

Method Deconvoluted Concentration

( μ g/g) % Recovery Interconversion (%)

Hg 2+ CH 3 Hg + Hg 2+ CH 3 Hg + Hg 2+ to

CH 3 Hg + CH 3 Hg + to

Hg 2+Material-1 M-3200 2.68 ± 0.34 2.20 ± 0.29 89 ± 12 73 ± 10 0 ± 3 0 ± 9

SONI-1 2.49 ± 0.16 1.83 ± 0.13 83 ± 5 61 ± 4 5 ± 1 45 ± 4

SONI-2 1.88 ± 0.20 1.96 ± 0.24 63 ± 7 65 ± 8 2 ± 3 10 ± 5

MAE 1.99 ± 0.14 2.01 ± 0.16 66 ± 8 67 ± 5 0 ± 3 7 ± 3

CAE NA NA NA NA NA ~ 100Material-2 M-3200 4.09 ± 0.93 2.79 ± 0.29 91 ± 21 62 ± 6 2 ± 2 6 ± 5

SONI-1 3.67 ± 0.16 2.65 ± 0.09 81 ± 3 59 ± 2 2 ± 1 44 ± 4

SONI-2 3.09 ± 0.23 2.29 ± 0.20 67 ± 5 51 ± 5 1 ± 1 2 ± 2

MAE 3.09 ± 0.24 2.26 ± 0.13 69 ± 5 50 ± 3 2 ± 1 4 ± 3

CAE NA NA NA NA NA ~ 100

Uncertainties are expressed at 95% CL, n = 9.

NA – analyzed but could not perform SIDMS calculati ons.SW-846 Update V 6800 - 45 Revision 1 July 2014 TABLE 9

SIDMS ANALYSIS OF FISH TISSUE (FISH REFERENCE MATER IALS) (ERM-CE464, DOLT3, NIST-1946) AFTER OPTIMIZED MICROWAVE-ASSISTED EX TRACTION (Ref. 20)

Reference
material ICP-MS LC-ICP-MS

Total Hg (mg kg -1 ) Hg 2+ as Hg

(mg kg -1 ) CH 3 Hg + as Hg

(mg kg -1 ) Sum of species a as

Hg (mg kg -1 ) Mean conversion ± 95% C.L. (%) Hg 2+ to CH 3 Hg + CH 3 Hg + to Hg 2+

ERM-CE464

Certified Values 5.24 ± 0.10 5.12 ± 0.16

EC 5.29 ± 0.24 0.17 ± 0.01 5.14 ± 0.10 5.31 ± 0.10 IDMS (SSAE) 5.30 ± 0.02 SIDMS (DSAE) 0.14 ± 0.01 5.09 ± 0.07 5.23 ±0.08 3. 80 ± 0.30 0.53 ± 0.26 SIDMS (DSBE) 0.14 ± 0.02 5.16 ± 0.04 5.30 ±0.04 7. 05 ± 0.78 0.10 ± 0.05

DOLT-3

Certified Values 3.37 ± 0.14 1.72 b

EC 3.32 ± 0.09 1.63 ± 0.06 1.59 ± 0.07 3.22 ± 0.08 IDMS (SSAE) 3.24 ± 0.06 SIDMS (DSAE) 1.37 ± 0.02 1.78 ± 0.05 3.15 ± 0.06 1 .99 ± 0.42 0.62 ± 0.22 SIDMS (DSBE) 1.52 ± 0.03 1.68 ± 0.03 3.20 ± 0.05 4 .39 ± 0.40 1.57 ± 0.36

NIST 1946

Certified Values 0.43 ± 0.01 0.40 ± 0.01

EC 0.43 ± 0.02 0.016 ± 0.003 0.42 ± 0.02 0.44 ± 0.0 1 IDMS (SSAE) 0.42 ± 0.01 SIDMS (DSAE) 0.012 ± 0.005 0.412 ± 0.005 0.42 ± 0. 01 6.76 ± 1.82 0.41 ± 0.13 SIDMS (DSBE) 0.010 ± 0.002 0.41 ± 0.01 0.42 ± 0.01 7.91 ± 1.20 0.48 ± 0.09

a Sum of species = Hg 2+ + CH 3 Hg + (HPLC-ICP-MS). b Indicative value.SW-846 Update V 6800 - 46 Revision 1 July 2014 Reference
material ICP-MS LC-ICP-MS

Total Hg (mg kg -1 ) Hg 2+ as Hg

(mg kg -1 ) CH 3 Hg + as Hg

(mg kg -1 ) Sum of species a as

Hg (mg kg -1 ) Mean conversion ± 95% C.L. (%) Hg 2+ to CH 3 Hg + CH 3 Hg + to Hg 2+

ERM-CE464

Certified Values 5.24 ± 0.10 5.12 ± 0.16

EC 5.29 ± 0.24 0.17 ± 0.01 5.14 ± 0.10 5.31 ± 0.10 IDMS (SSAE) 5.30 ± 0.02 SIDMS (DSAE) 0.14 ± 0.01 5.09 ± 0.07 5.23 ±0.08 3. 80 ± 0.30 0.53 ± 0.26 SIDMS (DSBE) 0.14 ± 0.02 5.16 ± 0.04 5.30 ±0.04 7. 05 ± 0.78 0.10 ± 0.05

DOLT-3

Certified Values 3.37 ± 0.14 1.72 b

EC 3.32 ± 0.09 1.63 ± 0.06 1.59 ± 0.07 3.22 ± 0.08 IDMS (SSAE) 3.24 ± 0.06 SIDMS (DSAE) 1.37 ± 0.02 1.78 ± 0.05 3.15 ± 0.06 1 .99 ± 0.42 0.62 ± 0.22 SIDMS (DSBE) 1.52 ± 0.03 1.68 ± 0.03 3.20 ± 0.05 4 .39 ± 0.40 1.57 ± 0.36

NIST 1946

Certified Values 0.43 ± 0.01 0.40 ± 0.01

EC 0.43 ± 0.02 0.016 ± 0.003 0.42 ± 0.02 0.44 ± 0.0 1 IDMS (SSAE) 0.42 ± 0.01 SIDMS (DSAE) 0.012 ± 0.005 0.412 ± 0.005 0.42 ± 0. 01 6.76 ± 1.82 0.41 ± 0.13 SIDMS (DSBE) 0.010 ± 0.002 0.41 ± 0.01 0.42 ± 0.01 7.91 ± 1.20 0.48 ± 0.09

a Sum of species = Hg 2+ + CH 3 Hg + (HPLC-ICP-MS). b Indicative value.SW-846 Update V 6800 - 47 Revision 1 July 2014 TABLE 10

EVALUATION OF DIFFERENT LITERATURE METHODS FOR MERC URY SPECIATION IN

HUMAN HAIR (IAEA-085) USING METHOD 6800

(Ref. 21)

Extraction Method Hg 2+

(μg/g) MeHg +

(μg/g) Total Mercury

(μg/g) Hg 2+ to

MeHg + (%) MeHg + to

Hg 2+ (%)

Certified value 0.3 22.9 ± 1.4 23.2 ± 0.8 ------ -- ---- Akagi et. al. 1.96 ± 0.59 23.81 ± 0.67 25.77 ± 0.89 < 0 < 0

Diez et. al

. 1.36 ± 0.39 24.82 ± 1.59 26.18 ± 1.64 < 0 4 ± 1

Bermejo et. al. 1.32 ± 0.16 22.39 ± 0.49 23.71 ± 0.52 4 ± 1 2 ± 1

Feng et. al. 1.15 ± 0.40 23.49 ± 0.49 24.64 ± 0.63 4 ± 2 3 ± 1

Chai et. al. 1.00 ± 0.56 23.62 ± 0.57 24.62 ± 0.80 2 ± 2 5 ± 2

Montuori et. al. 1.15 ± 0.42 25.17 ± 0.96 26.32 ± 1.05 14 ± 2 22 ± 1

Morton et. al. 1.09 ± 0.23 23.50 ± 1.21 24.59 ± 1.23 < 0 90 ± 5

Method 3200

(MAE) 0.59 ± 0.22 23.65 ± 1.42 24.24 ± 1.44 4 ± 2 6 ± 1

Method 3200 (UAE) 1.13 ± 0.25 19.80 ± 1.25 20.93 ± 1.28 9 ± 3 0 ± 1

Uncertainties are at 95% CL, n = 12.

MAE – Microwave-assisted extraction; UAE – Ultrasou nd-assisted extraction.SW-846 Update V 6800 - 48 Revision 1 July 2014 TABLE 11

ANALYSIS OF RoHS GROUND AND PULVERIZED CIRCUIT BOAR DS FOR ALL

REGULATED METALS SIMULTANEOUSLY USING IDMS (Ref. 26)

Sample ID Cadmium (μg/g) Chromium

(μg/g) Lead

(μg/g) Mercury

(μg/g) 2811 1.916 ± 0.054 23.829 ± 0.866 19,806 ± 973 4.13 4 ± 1.059 Compaq #1 2.087 ± 0.107 13.462 ± 1.252 15,257 ± 917 6.316 ± 1.709 Dell Optiplex #5 2.081 ± 0.049 20.624 ± 1.010 23,49 6 ± 1,354 3.838 ± 0.871 Dell Optiplex #3 2.013 ± 0.086 38.512 ± 3.113 21,63 4 ± 972 N/A AVVA 2.017 ± 0.115 35.399 ± 1.045 23,883 ± 1,243 N/ A Uncertainties are at 95% CI with n = 3, Cr(VI) was less than 1% by 6800. N/A – not analyzed



### .

TABLE 12

SIDMS BY HPLC-ICP-MS ANALYSIS OF THREE EXTRACTABLE MERCURY SPECIES AND NON-EXTRACTABLE MERCURY FROM CRUDE OIL AFTER EPA ME THOD 3200 EXTRACTION AND 6800 QUANTIFICATION WITH MASS BALANC E. (Ref. 30)

Sample Name Extractable Mercury Species, μg/kg Non-Extractable Mercury Species, μg/kg Total Mercury*, μg/kg

Hg 2+ MeHg + EtHg +

Serial # 871833 17.3 ± 3.6 0.8 ± 0.1 10.7 ± 1.1 342.5 ± 46.8 370.2 ± 47.0 (414.1 ± 24.7) Serial # 870987 23.6 ± 3.7 0.5 ± 0.1 7.4 ± 2.2 323.3 ± 30.7 354.8 ± 31.0 (437.8 ± 25.4) Uncertainties are at 95% CL, n = 12 (3 x 4). *values in parentheses are total mercury determined by isotope dilution mass spectrometry (IDMS) of th e crude oil after Method 3052 digestion.SW-846 Update V 6800 - 49 Revision 1 July 2014 TABLE 13

INTERCONVERSION OF EXTRACTABLE SPECIES USING METHOD 3200 DEMONSTRATING THAT GREATER THAN 80% CONVERSIONS ARE QUANTIFIABLE BY METHOD 6800. THESE DATA ARE PART OF THE OVERALL DA TA IN TABLE 12 AND ARE DETAILED IN THE REFERENCE.

(Ref. 30)

Sample
Name Deconvoluted

Concentration, μg/kg Interconversion (%)

Hg 2+ MeHg + EtHg + Hg 2+ to MeHg + MeHg + to

Hg 2+ MeHg + to

EtHg + EtHg + to

MeHg + EtHg + to

Hg 2+ Hg 2+ to

EtHg +

Serial #
871833 17.3 ±

3.6 0.8 ±

0.1 10.7 ±

1.1 9.6 ± 0.9 2.9 ± 0.8 0.8 ± 0.1 6.1 ± 0.6 55.1 ±
2.2 2.1 ± 1.0

Serial #
870987 23.6 ±

3.7 0.5 ±

0.1 7.4 ±

2.2 7.4 ± 1.0 6.2 ± 1.0 0.9 ± 0.1 7.9 ± 0.8 86.9 ±
1.5 1.3 ± 0.1

Uncertainties are at 95% CL, n = 12 (3 x 4).

TABLE 14

MERCURY SPECIES IN NIST HUMAN BLOOD SRM-966 BY SIDM S

(Refs. 31 and 42)

Sample Total Mercury (ng/g) Deconvoluted Concentration (ng/g) Hg2+ MeHg+ EtHg+

SRM-966 (Level 2) 31.9 ± 2.1 (29.8 ± 1.6) a 16.7 ± 1.2

(14.1 ± 0.9) b 15.3 ± 1.5

(15.6 ± 1.3) b NA

HB-1 ------- 1.54 ± 0.12 3.72 ± 0.48 0.09 ± 0.02

HB-2 30.63 ± 3.19

(23) b 5.43 ± 1.52 25.2 ± 2.8 NA

HB-3 23.38 ± 2.25

(17) b 4.28 ± 2.29 19.1 ± 2.3 NA

Uncertainties are at 95% CL, n = 4

a Certified values are in parentheses.

b Reference values are in parenthesesSW-846 Update V 6800 - 50 Revision 1 July 2014 TABLE 15 CONCENTRATIONS OF MERCURY SPECIES (IN mg/Kg of Hg s pecies) DETERMINED IN TUNA FISH TISSUE SRM (ERM0CE464) BY EXTERNAL CALIBR ATION AND METHOD 6800 (SIDMS) DEMONSTRATING THAT EFFICIENCY OF EXTRACTION DOES NOT AFFECT

ACCURACY AFTER EQUILIBRATION HAS BEEN REACHED IN ME THOD 6800



### ( Ref. 

### 32)

External calibration EPA Method 6800 (SIDMS)

Extraction procedure Hg 2+ CH 3 Hg + sum of

species Hg 2+ CH 3 Hg + sum of species

Certified

value 0.12 a 5.12 ± 0.16 5.24 ± 0.10 0.12 a 5.12 ± 0.16 5.24 ± 0.10

A 0.06 ± 0.02 5.05 ± 0.13 5.11 ± 0.13 0.07 ± 0.02 5.22 ±0.31 5.29 ± 0.31

B 0.12 ± 0.03 5.05 ± 0.18 5.17 ± 0.18 0.07 ± 0.03 5.20 ± 0.18 5.27 ±0.18

C 0.18 ± 0.05 4.88 ± 0.17 5.06 ± 0.18 0.30 ± 0.07 5.18 ± 0.13 5.48 ± 0.15

D 0.07 ± 0.02 4.29 ± 0.39 4.36 ± 0.39 0.13 ± 0.05 5.11 ± 0.38 5.24 ± 0.38

E 0.06 ± 0.04 3.94 ± 0.12 4.00 ± 0.13 0.11 ± 0.07 5.60 ± 0.33 5.71 ± 0.34

F 0.35 ± 0.08 3.29 ± 0.14 3.64 ± 0.16 0.27 ± 0.12 5.12 ± 0.19 5.39 ± 0.22

G 0.45 ± 0.10 4.87 ± 0.20 5.32 ± 0.22 1.05 ± 0.14 5.08 ± 0.25 6.13 ± 0.29

H 0.16 ± 0.07 4.42 ± 0.14 4.58 ± 0.16 0.07 ± 0.02 5.22 ±0.31 5.29 ± 0.31

a Inorganic mercury concentration was calculated as t he difference between certified total mercury and methylmercury concentrations. The values are means ± 95% CL (n = 3).SW-846 Update V 6800 - 51 Revision 1 July 2014 TABLE 16a ANTIMONY TOXICANT CONTAMINATION IN Zn DIETARY SUPPL EMENT IN ug/g BY IDMS METHOD 6800

( Ref. 33)

Samples Directly From Manufacturer Commercially Ava ilable Samples Lot Concentration of Antimony (ppb) Lot Concentrati on of Antimony (ppb)
1 17,650 ± 145.9 3-1 14,430 ± 479.8 2 15,770 ± 246.1 3-2 15,720 ± 47.07 3 17,000 ± 67.03 3-3 15,370 ± 31.41 4 27,310 ± 276.2 11-1 96.15 ± 12.79 5 16,830 ± 236.5 11-2 79.55 ± 5.289 6 18,060 ± 84.62 11-3 65.00 ± 1.128 7 15,590 ± 88.14 14-1 0.4486 ± 0.1586 8 24,450 ± 159.0 14-2 0.3350 ± 0.05088 9 16,120 ± 57.58 14-3 0.3040 ± 0.03636
10 218.4 ± 24.50 15 0.6307 ± 0.09681
11 78.84 ± 1.337

12 454.1 ± 12.16

13 380.9 ± 29.22 Stevia 31,620 ± 298.5 n = 12, at the 95% Confidence Interval.

TABLE 16b ZINC CONCENTRATION IN Zn DIETARY SUPPLEMENT IN ug/g BY IDMS METHOD 6800.

(Ref. 33)



### Samples Directly From Manufacturer Commercially Available Samples
Lot Concentration of Zinc (ppm) Lot Concentration of Zinc (ppm) 1 4,332 ± 84 3-1 4,150 ± 178 2 4,223 ± 96 3-2 4,034 ± 207 3 4,307 ± 94 3-3 4,105 ± 77 4 4,128 ± 77 11-1 4,121 ± 55 5 4,173 ± 117 11-2 4,146 ± 83 6 4,340 ± 126 11-3 4,041 ± 129 7 3,930 ± 81 14-1 3,935 ± 110 8 4,232 ± 134 14-2 4,142 ± 72 9 4,203 ± 93 14-3 4,131 ± 62 10 4,198 ± 100 15 4,656 ± 65
11 4,115 ± 89 12 4,174 ± 95 13 4,170 ± 77
Stevia 0.9347 ± 0.1085 Concentrations are in ppm, ug/g of dietary suppleme nt n = 12, at the 95% Confidence Interval.SW-846 Update V 6800 - 52 Revision 1 July 2014 TABLE 17

ANALYSIS OF THE PESTICIDE GLYPHOSATE (ANALOGUE FOR VX NERVE GAS) BY IDMS SOLID PHASE EXTRACTION AND i-SPIKE FROM DRINKING WA TER WHERE THE ENRICHED ISOTOPES ARE ADDED FIRST TO THE EXTRACTION MEDIA FOR AUTOMATION AND FOR PERSONNEL SAFETY IN EXTREMELY TO XIC ANALYSES

(Refs. 34 and 42)

Normalized Actual Concentration (ppm) Normalized Measured Concentration (ppm) 95% Confidence Interval % RSD

SPE-IDMS
100 99.54 ± 2.17 2.10%
50 53.05 ± 0.90 1.55% i-Spike
100 99.54 ± 1.90 1.83%
50 53.77 ± 1.16 1.96%

TABLE 18

GLUTATHIONE CONGIGATE MERCURY SPECIES QUANTIFIED BY 6800 USING ESI-TOF AND MALDI-TOF (TRUE VALUE 480.3 ug/g)

(Refs. 35 and 41)

Glutathione mercury (Glutathione 2 -Hg 2+ in sample by nanoESI-TOF IDMS 485 ± 18 ppm Glutathione mercury (Glutathione 2 -Hg 2+ in sample by MALDI-TOF IDMS 474 ±73 ppm Actual Concentration of Glutathione mercury dimer 480.3 ppm Quantification by EPA Method 6800 using both nanoES I-TOF-MS and MALDI-TOF- MS

n-4, uncertainty is 95% CLSW-846 Update V 6800 - 53 Revision 1 July 2014 FIGURE 1

THE INFLUENCE OF THE DEAD TIME CORRECTION ON THE IS OTOPE RATIOS MEASURED WITH ICP-MS EQUIPPED WITH A CONTINUOUS DYN ODE MULTIPLIER (Ref. 12)

Gain loss occurs when the count rate exceeds 5.8x10 5

.

FIGURE 2

IDMS DETERMINATION OF VANADIUM IN CRUDE OIL. NUMBE RS SHOWN ABOVE THE BARS ARE THE ATOMIC FRACTION (Revised from Ref. 1)SW-846 Update V 6800 - 54 Revision 1 July 2014 FIGURE 3

SEPARATION AND DETECTION OF Cr(III) AND Cr(VI) WITH ION-EXCHANGE

CHROMATOGRAPHY COUPLED WITH AN ICP-MS (Ref. 5)

FIGURE 4

SEPARATION OF THE UNSPIKED SAMPLE AND ISOTOPICALLY SPIKED SAMPLE (Ref. 6)

(a): Chromatograms of a solution containing Cr(III) and Cr(VI) with natural isotopic abundance.

(b): Chromatograms of the same solution spiked with isotope-enriched spikes 50 Cr(III) and 53 Cr(VI)

.SW-846 Update V 6800 - 55 Revision 1 July 2014 FIGURE 5

GRAPHIC CALCULATED ILLUSTRATION OF THE APPLICATION OF SIDMS TO THE

SIMULTANEOUS DETERMINATION OF Cr(III) AND Cr(VI) (R ef. 6)

(a) and (b) show the initial natural isotopic abund ance of species Cr(III) and Cr(VI) in a 50

_ l 200 ppb Cr solution in which the concentrations of both Cr(III) and Cr(VI) are 100 ppb. In (c) and (d), the sample is spiked with 100 ppb 50 Cr(III) (in which 50 Cr is enriched) and 100 ppb 53 Cr(VI) (in
which 53 Cr is enriched), there is no interconversion betwee n Cr(III) and Cr(VI). In (e) and (f), 20% of Cr(III) is converted to Cr(VI), and 20% of C r(VI) is converted to Cr(III). Different degrees of interconversion results in different isotopic ab undances, so the change of the relative isotopic abundance can be applied to the determination of th e species and the degree of the
interconversion.SW-846 Update V 6800 - 56 Revision 1 July 2014 FIGURE 6

HEXAVALENT CHROMIUM CONCENTRATIONS IN FLY ASH LEACH ATE OBTAINED FROM HISTORICAL DATA (HD), STUDY WITH SIDMS (DU); AND BE FORE AND AFTER

COMBINATION WITH ACID MINE DRAINAGE (AMD) (Ref. 14)

0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6

HD-1 HD-2 HD-3 HD-4 HD-5 HD-6 HD-7 DU-1 DU-2 DU-3 1:1 combo 5: 1 comboμ

Cr(VI) concentration in leachate before and after c ombination with AMD, which illustrate the dramatic and statistically significant decrease in Cr(VI) concentrations in the fly ash leachate as a result of treating with AMD. Observations from HD- 1 to HD-7 (dark gray bars) were historical data obtained from the power plant. Observations f rom DU-1 to DU-3 (light gray bars) were obtained from the SIDMS analysis at Duquesne Univer sity during current study, and observations ‘1:1 combo’ and ‘5:1 combo’ were obtai ned by mixing leachate and AMD with the ratios of 1:1 and 5:1 (leachate to AMD), respective ly. Error bars are shown at 95% confidence
intervals with n = 3.SW-846 Update V 6800 - 57 Revision 1 July 2014 FIGURE 7

ALPHA-GLACTOSIDASE A (GLA) ENZYME MEASURED BY 6800 IDMS IN MOUSE BY NANOESI-QTOF MASS SPECTROMETRY AGAINST THE STANDARD FLU ORESCENCE METHODS AND FOUND TO BE SUPERIOR FOR DETECTING LOWE R DOSE CONCENTRATIONS IN LIVER, KIDNEY AND BRAIN (PLASMA W AS NOT DONE BY IDMS IN THIS DIAGRAM).

(Ref. 36)SW-846 Update V 6800 - 58 Revision 1 July 2014 FIGURE 8

NANO-ESI-TOF-MS ANALYSIS OF GLUTATHIONE CONJUGATES WITH CH 3 Hg + and Hg 2+

. THE TWO NATURAL ISOTOPE SPECIES OF INORGANIC MERCUR Y AND METHYLMERCURY BOUND TO GLUTATHIONE IN SPECTRA 8A AN D 8C. THE TWO ISOTOPICALLY ENRICHED MERCURY IN SPECIATED FORMS WI TH THE ENRICHED ISOTOPE EVIDENT IN THE MERCURY GLUTATHIONE SPECTRA OF 8B AND 8D.

(Refs. 35 and 42)

Figure 8A. Natural abundant CH 3 Hg + - Glutathione conjugate (522 m/z peak mostly from 200 Hg – Glutathione conjugate, 524 m/z peak mostly from 202 Hg – Glutathione conjugate)

Figure 8B. Natural abundant CH 3 Hg + spiked with CH 3 200 Hg + - Glutathione conjugate (notice the 522 m/z peak in this figure compared to the one abo ve)SW-846 Update V 6800 - 59 Revision 1 July 2014 Figure 8C. Natural abundant Hg 2+ - Glutathione conjugate with two Glutathione molec ules bound to Hg 2+ (peak at 812 m/z mostly due to 199 Hg 2+ - Glutathione conjugate, peak at 815 m/z mostly due to 202 Hg – Glutathione conjugate).

Figure 8D. Natural abundant Hg 2+ spiked with enriched 199 Hg 2+ (notice the 812 m/z peak in this figure compared to the one above)SW-846 Update V 6800 - 60 Revision 1 July 2014 FIGURE 9

NATURAL ABUNDANT REDUCED (9A) AND OXIDIZED (9B) GLU TATHIONE BIOMARKERS ANALYZED BY NANO-ESI-QTOF-MS.



### (Refs. 35 and 42)

FIGURE 9A. Natural abundant Reduced Glutathione (GS H)

FIGURE 9B. Natural abundant oxidized Glutathione (G SSG)SW-846 Update V 6800 - 61 Revision 1 July 2014 FIGURE 10

SODIUM AZIDE, TOXICANT AND EXPLOSIVE, ANALYZED BY E SI-TOF-MS IN BOTH POSITIVE AND NEGATIVE MODES. EACH MODE GIVES DIFFE RENT SPECIES ADDUCT FORMATION PATTERNS AND RATIOS THAT ARE STOICHIOMETR ICALLY RELATED TO THE DIRECT EQUATIONS AND MATHEMATICAL SOLUTIONS. Q UANTIFICATION ANALYSES WERE WITHIN 5% OF TRUE VALUE BY METHOD 680 0 FOR ALL ANALYSES OF THIS TYPE.

(Refs. 40 and 42)

Figure 10A: An example of the unique spectra obtai ned from natural sodium azide, enriched sodium azide, and a mixed solution containing both natural sodium azide and enriched sodium azide in negative mode. The graphs are offset for clarity. In the mixed solution, a 1:2:1 ratio is observed as expected. Typical sample accuracy is wi thin ± 5% of true concentration.SW-846 Update V 6800 - 62 Revision 1 July 2014 FIGURE 10B. Spectrum of 20 ppm NaN 3 and 20 ppm Na 15 NNN in water in positive mode and enlargement of one of the specific fragmentation pa tters. Each fragmentation pattern gives the same direct mathematical concentration. In positive mode, these two peaks form a mathematical pattern of one natural (left) and one enriched (right) providing quantitative data within 1% of the true value (in this particular cas e). In addition, these positive azide analyses in water are examples of solid phase extraction (SPE) elution solution spectrum for an i-Spike ESIID-TOF-MS of sodium azide in water using 1:1:1 Opti ma ACN:Optima MeOH:Optima H 2 O with 5% 0.5 M NaOH in water. The i-Spike method loads t he enriched isotopic molecule first and then loads the sample where the analyte is extracte d and the two are equilibrated on the solid phase media and then eluted in the final isotopic a nd molecular form after which any portion of the sample analyte gives the same correct quantitat ive solution.SW-846 Update V 6800 - 63 Revision 1 July 2014 FIGURE 10C. Spectrum of 20 ppm NaN 3 and 20 ppm Na 15 NNN in water in negative mode. Here a different fractionation pattern is a 1:2:1 ( similar to the example in Figure 10A) and provides quantification accuracy within 5%. Note t hat the negative and positive ionization patterns confirm each other using the opposite char ged fragmentation pattern.SW-846 Update V 6800 - 64 Revision 1 July 2014 METHOD 6800

ELEMENTAL AND MOLECULAR SPECIATED ISOTOPE DILUTION MASS SPECTROMETRY : ISOTOPE DILUTION MASS SPECTROMETRY (IDMS)

Start

Type of analysis?

11.1 Total Element 11.2 Speciated Isotope Dilution Mass Spectrometry (SIDMS) 11.2 Speciation

11.1.2 Are the isotope
enriched standards ready? 7.2 Prepare isotope enriched
standards No

7.4 Spike the isotope
enriched standards with
the standard solutions
that have natural isotopic abundance. 11.1.3 Spike sample with the isotope
enriched standards.

11.1.4 Is the sample acidified and prefiltered water? 11.1.4 Digest the spiked samples. No Yes

Yes

11.1.5 Is the instrument ready for measuring isotopic ratios?

10.1.1 Set-up and
tune instrument according to Method 6020.

10.1.2 Determine the
dead time. No 11.1.5 Measure the isotope ratios of samples.
Measure mass bias

Yes 11.1.5 Are the isotopic ratios outside 0.1 to 10 for samples containing significant
amounts of Yes

11.1.5.2

Is the response so high that the gain depression might occur? No 11.1.5.2 Dilute

the spiked samples

Yes

12.1.1 Integrate the counts, perform dead
time correction and
mass bias correction.

No

12.1.1 Calculate the isotope ratios.

12.1.1 Calculate the concentrations using the measure isotope ratios.

12.1.3 If required calculate results for dry-weight basis.

StopSW-846 Update V 6800 - 65 Revision 1 July 2014 METHOD 6800 ELEMENTAL AND MOLECULAR SPECIATED ISOTOPE DILUTION MASS SPECTROMETRY : SPECIATED ISOTOPE DILUTION MASS SPECTROMETRY (SIDM S)

Start

Type of analysis 11.1 Total Element 11.1 Isotope
Dilution Mass Spectrometry (IDMS)

11.2 Speciation

11.2.2 Are the isotope
enriched standards

50 Cr(III) & 53 Cr(VI) ready? 7.3 Prepare isotope
enriched standards

50 Cr(III) & 53 Cr(VI) No

7.4 Spike the isotope
enriched standards with
the standard solutions that have natural isotopic
abundance. 11.2.3 Spike sample
with 53 Cr(VI)

11.2.4 Is the sample

an aqueous solution? 11.2.4 Extract the species from the sample No

11.2.5 Neutralize the sample, if

necessary. Yes

Yes

11.2.6 Acidify the solution to pH 2.

11.2.7
Is the instrument ready for measuring isotopic ratios? 10.1.1 Set-up and tune instrument according to
Method 6020

10.1.2 Measure the
dead time.

10.2.2 Connect the chromatograph to ICP-MS No 11.2.7 Measure the isotope ratios of samples. Measure mass bias factors.

Yes 11.2.7.2 Are the isotopic ratios outside 0.1 to 10 for samples containing significant amounts of analyte? Yes

11.2.7.2

Is the response so high that the gain depression might occur? No 11.2.7.2 Dilute

the spiked samples

Yes

12.2.1 Perform dead
time correction at each point across peaks.

No

12.2.1 Integrate the peaks by summing up the counts across peaks.

12.2.1 Calculate the isotope ratios and perform mass bias
correction.

12.2.2 Deconvolute the concentration of each species and the conversion of the species.

12.2.5 If required calculate results for dry-weight basis. StopSW-846 Update V 6800 - 66 Revision 1 July 2014 GLOSSARY

Atomic mass unit (symbol: u or amu) or dalton (symb ol: Da ) – the unit used to indicate mass on an atomic or molecular scale. One amu or u or Da i s defined as the mass of one twelfth of the isotope of carbon-12.

Mass-to-charge ratio (m/z) – is the mass of the ion divided by the charge of the ion and is the functional unit measured in a mass spectrometer.

Isotope dilution mass spectrometry (IDMS) – A quantitative method for total concentration determination of an analyte based on the measuremen t of the isotope ratio of a nuclide using mass spectrometry after isotope dilution, both mole cular and ionic.

Isotope dilution – Mixing of a given nuclide with one or more of it s isotopes. The isotope usually has an enriched isotopic abundance different from t hat occurring naturally.

Speciation analysis – IUPAC defines speciation analysis as analyzing: “Chemical compounds that differ in isotopic composition, conformation, oxidation or electronic state, or in the nature of their complexed or covalently bound substituent, ca n be regarded as distinct chemical species”

Speciated isotope dilution mass spectrometry (SIDMS ) – A quantitative method for determining molecular or ionic or elemental species and interco nversions between them based on the measurement of isotope ratio(s) in each species of a nuclide using mass spectrometry after speciated isotope dilution. Samples are mixed with one or more isotopic spikes which have different isotopic abundances and are artificially converted to chemical forms corresponding to the species to be analyzed. The spiked samples are then subjected to the separation of the species, followed by the measurement of the altered isotope ratios in each species. Both species concentrations and the extent of species co nversions can be mathematically
deconvoluted.

Isotopic abundance – The relative number of atoms of a particular iso tope in a mixture of the isotopes of an element or molecule, expressed as a fraction of all the atoms of the element.

Isotopes – Nuclides having the same atomic number but diffe rent mass numbers.

Species – Chemical forms in which molecule and or an eleme nt exists.

Natural isotopic abundance –Isotopic abundance of elements from natural sourc es. Most elements (except lithium, lead and uranium) found i n nature have a constant isotope abundance.

Isotope ratio – Ratio of the isotopic abundances of two isotopei c forms of molecular ions.

Speciation (or speciated) analysis – Quantification of elements in specific chemical forms.

Isotope-enriched material – Material containing elements artificially enrich ed in minor isotopes.

Isotopic spike (Isotope-enriched spike) – Standards prepared from isotope-enriched materia ls.

i-Spike – Method of isotopic species equilibration that ta kes place rapidly by preloading isotopically enriched spike onto a sorbent-packed c olumn prior to the introduction of the sample in both IDMS and SIDMS.SW-846 Update V 6800 - 67 Revision 1 July 2014 Dead time – The interval during which the detector and its a ssociated counting electronics are unable to resolve successive pulses. The measured counts are lower than the true counts if no correction is performed.

Gain loss – The loss of gain in detector caused by the inabi lity of the multiplier’s dynode string to supply enough current to maintain constant dynod e voltage drops. The measured counts are lower than the true counts, and cannot be mathemati cally corrected if gain loss occurs.

Mass bias – The deviation of the measured isotope ratio from the true value caused by the differential sensitivity of the instrument to mass. This effect may occur in the ionization process or from differential transmission/detection by the mass spectrometer.

Mass bias factor

– A number used to correct for the mass bias of th e measured isotope ratios. Mass bias factor is measured by employing an isotop ically certified standard.

Isotopic-abundance-certified standard (Isotopically certified standard) – Standard material with certified isotopic abundance.

Inverse isotope dilution – Analysis method to determine the concentrations of isotopic spikes. A known quantity and isotopic abundance of an isotopi c spike is mixed with a known amount and isotopic abundance (usually tabulated natural isoto pic abundance or certified isotopic abundance) of standard(s) and the altered isotope r atio(s) is (are) measured and used in the calculation to find the concentration of the isotop ic spike. Usually, a high purity source of the
natural material is used to calibrate and determine the concentration of the separated isotopic spike solution using this method. Only in the case of such elements as uranium, lead, and lithium are the natural isotopic abundances not con stant in terrestrial materials. Molecules have natural isotopes also such as C-13, O-18, O18, N-15 that can alter the purity of the natural molecule and can be mathematically corrected.

Spiking – Addition of the enriched isotopic species (spike ) to the extract or appropriate sample.

Single spiking – Addition of one enriched isotopic spike to the s ample.

Double spiking – Addition of two isotopic spikes to the sample. The two isotopic spikes are enriched in different isotopes, and are prepared in different chemical forms, each of which corresponds to a species form.

Unidirectional conversion – One directional transformation occurring between two species. One species can convert to the other; the reverse trans formation does not occur.

Interconversion – Uni- or bi-directional transformation occurring between two species. Some species convert back and forth between two or more chemical forms.

Time resolved analysis (TRA) – A data collection mode in which the data can be acquired at designated intervals for a continuously aspirated s ample, over a user-defined period of time.SW-846 Update V 6800 - 68 Revision 1 July 2014 APPENDIX A SUMMARY OF REVISIONS TO METHOD 6800 (AS COMPARED TO PREVIOUS REVISION 0, FEBRUARY 2007)

1. Update for molecular SIDMS capability as well as examples of Homeland Defense and Homeland Security and Human Health examples. 2. Improved overall method formatting for consisten cy with new SW-846 methods style guidance. 3. Inserted text describing use and application of LLOQ (Sec. 9.4).
4. Minor editorial and technical revisions were mad e throughout to improve method clarity. The revision number was changed to one and the date published was changed to July 2014. 5. New explicit and determinative SIDMS equations w ere added to the iterative equations previously described. 6. Minor corrections to equations were made for acc uracy. 7. Examples of applications and data validating the se applications were included. 8. Literature examples of more molecular applicatio ns of the method were expanded.