**SW-846 Update V 6800 - 1 Revision 1 October 2012**  **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**  **October 2012 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 October 2012**  **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 plasma** **SW-846 Update V 6800 - 4 Revision 1 October 2012**  **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. Nano**  **ESI-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, GC**  **TOF, 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**  **October 2012 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 mercury**  **glutathione 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 October 2012**  **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 October 2012**  **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 October 2012**  _ **R m**  **is the dead-time-corrected isotope ratio;**

_ **Isotope1** 

### S **sample/standard and**  **Isotope2** 

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

_ **Isotope1** 

### S **background and**  **Isotope2** 

### S **background are the integrated dead-time-corrected**  **counts 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-corrected**  **isotope-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 October 2012**  _ **R c**  **and R m**  **are the corrected isotope ratio and the measured d ead-time-corrected**  **isotope-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**  **October 2012 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**  **October 2012 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 isotopic**  **abundance-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 Cr** **SW-846 Update V 6800 - 12 Revision 1 October 2012**  **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 ratio** **SW-846 Update V 6800 - 13 Revision 1 October 2012**  **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 standard SW-846 Update V 6800 - 14 Revision 1**  **October 2012 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 or** **SW-846 Update V 6800 - 15 Revision 1 October 2012**  **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 a** **SW-846 Update V 6800 - 16 Revision 1 October 2012**  **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 sufficient** **SW-846 Update V 6800 - 17 Revision 1 October 2012**  **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**  **October 2012 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 isotope**  **enriched 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**  **October 2012 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 species SW-846 Update V 6800 - 20 Revision 1**  **October 2012 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**  **October 2012 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**  **October 2012 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 R** **SW-846 Update V 6800 - 23 Revision 1 October 2012**  **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 M**SW-846 Update V 6800 - 24 Revision 1 October 2012**  **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**  **October 2012 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 and** **SW-846 Update V 6800 - 26 Revision 1 October 2012**  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 October 2012**  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 B**SW-846 Update V 6800 - 28 Revision 1 October 2012**  **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 pre**  **equilibrating 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 species**  **shifts 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 to** **SW-846 Update V 6800 - 29 Revision 1 October 2012**  **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 of** **SW-846 Update V 6800 - 30 Revision 1 October 2012**  **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 October 2012**  **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 MALDI**  **QTOF-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 these** **SW-846 Update V 6800 - 32 Revision 1 October 2012**  **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**  **October 2012 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**  **October 2012 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-ICP**  **MS,”** 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 cost**  **effective environmental remediation solutions using acid mine drainage,”**  J. Environ.
Monit. **, 2005, 7, 899-905. SW-846 Update V 6800 - 35 Revision 1**  **October 2012 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 Double**  **Spike 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 October 2012**  **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 “International** **SW-846 Update V 6800 - 37 Revision 1 October 2012**  **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-of**  **Flight 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 October 2012**  **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**  **October 2012 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 October 2012**  **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 filed** **SW-846 Update V 6800 - 41 Revision 1 October 2012**  **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 applicable SW-846 Update V 6800 - 42 Revision 1**  **October 2012 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 October 2012**  **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 October 2012**  **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 ~ 100 Material-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 October 2012**  **TABLE 9**

**SIDMS ANALYSIS OF FISH TISSUE (FISH REFERENCE MATER IALS) (ERM-CE464, DOLT**  **3, 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 October 2012**  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 October 2012**  **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) Tot al 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 October 2012**  **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 October 2012**  **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 parentheses** **SW-846 Update V 6800 - 50 Revision 1 October 2012**  **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**  **October 2012 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 Con**  **centration 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 October 2012**  **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% CL SW-846 Update V 6800 - 53 Revision 1**  **October 2012 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 October 2012**  **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 October 2012**  **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**  **October 2012 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 combo** μ

**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**  **October 2012 FIGURE 7** 

**ALPHA-GLACTOSIDASE A (GLA) ENZYME MEASURED BY 6800 IDMS IN MOUSE BY NANO**  **ESI-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**  **October 2012 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 October 2012**  **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 October 2012**  **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 October 2012**  **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 October 2012**  **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 ESI ID-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**  **October 2012 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 October 2012**  **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.**

**Stop SW-846 Update V 6800 - 65 Revision 1**  **October 2012 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. Stop** **SW-846 Update V 6800 - 66 Revision 1 October 2012**  **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 October 2012**  **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**  **October 2012 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.**  **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.**