Method of speciated isotope dilution mass spectrometry

The method of determining the concentration of a specie in a sample includes providing at least one predetermined, enriched isotope in the same speciated form as the species to be measured, spiking the sample containing the species to be measured, equilibrating the spiked species with the species to be measured, separating the species from the sample and subsequently determining the concentration of the species to be measured by employing isotopic element specie ratios. In one embodiment, a single speciated isotope spike is employed and, in others, two or more such spikes may be employed. In a preferred embodiment, time resolution chromatography is used to effect separation of these species from the sample and mass spectrometer is employed in determining isotopic elemental ratios. It is also preferred that a method be employed to determine if there has been conversion from one species to another. In another embodiment, spiking of the several different isotopically enriched analogs of the same specie are added at various steps in the sampling procedure and the stability and integrity of the specie with respect to these processes is evaluated by mass spectrometric measurements of the various isotopic ratios. Chemical processes, extraction methods, dissolution procedures and storage procedures are evaluated. In another embodiment, speciated isotope dilution is used to determine the effect on species of various sample preparation methods and portions of sample preparation techniques. Extraction and separation procedures employ the technique to provide definitive evidence of accurate specie manipulation and provide for performance based measurement.

BACKGROUND OF THE INVENTION 
1. Field Of the Invention 
The present invention relates to a method of measurement of species which 
employs enriched isotope spikes in the same speciated form as the species 
to be measured, equilibration, separation and subsequent determining of 
concentration by employing isotopic element specie ratios. 
2. Description Of the Prior Art 
The need for making quantitative determinations of a specie of interest 
occurs in many environments including environmental, biological, 
pharmaceutical and industrial samples. For example, certain forms of an 
element or molecular species may exhibit different toxicities or chemical 
behaviors from others. Existing techniques, with the exception of 
electrochemical methods, rely predominately on physical separation in 
time. They are incapable of determining either cross-over (transformation 
of one specie form into another), are lost, are altered or are completely 
recovered. Such techniques cannot be used to determine transformation of 
one species into another during storage, manipulation and sample 
preparation or during the measurement process. 
An example of the criticality of such measurements would be to consider 
chromium. While Cr(III) is a trace dement essential for human health, 
Cr(VI) is poisonous to humans and most other animals and is also a 
carcinogen. As a result, the difference between these two species, which 
resides in the oxidation state of the dement, may be of critical 
importance. While chromatography can be used to separate Cr(III) in time 
resolution from Cr(VI), as each specie can react with its surroundings and 
even with separating agencies, the chromatographic separation is only a 
snapshot in time recording the state of affairs at the end of the 
manipulation. Each specie may have reacted with many other reagents and 
transformed during the analysis. There is, therefore, with time 
resolution, no way of determining how much chromium was actually in each 
specie when the experiment began or when the sample was actually taken. 
Specific species are often required for a particular process. For example, 
barium is toxic in some compound forms, but is prescribed for medical 
diagnostic x-ray tests, usually as barium sulfate in liquid slurry form. 
The conformation and evaluation of a body's processing of barium into 
another specie can be accomplished with isotopically labeled barium 
sulfate. These studies have been done, but the use of speciated isotope 
dilution measurements has not been used for analysis. 
Some isotopic tracking has been done for lead due to terrestrially unique 
and naturally occurring isotopic compositions of this dement. The isotopic 
ratios can be matched with a particular source to determine the origin of 
the lead. These measurements are not speciated measurements, but depend on 
the isotopic ratio differences of the natural material to be detected. 
This technique has also been used for lead pottery glazes to determine the 
origin of art objects, however in this case also, naturally occurring 
isotopic ratios were determined. Lead is a uniquely feasible 
non-radioactive dement to be evaluated (as to origin) by the isotopic 
ratio method, as its isotopic ratios change with the amount of uranium 
mixed with the lead in the original ore deposits. The decay of uranium 
into different lead isotopes creates unique isotopic ratios for different 
lead deposits. Uranium also permits this origin-specific ratio 
identification method. 
All elements, with the exception of Pb, U, and Pu, have constant isotopic 
ratios throughout the earth's crust. Hinners, T. A.; Heithmar, E. M.; 
Spittier, T. M.; Henshaw, J. M., "Inductively Coupled Plasma Mass 
Spectrometric Determination of Lead Isotopes" Analytical Chemistry, 59, 
2658-2662, 1987. It is this constant elemental isotopic ratio that 
provides the basis of isotope dilution analysis. When this ratio for an 
element is artificially altered with an enriched isotope of that element, 
and bulk analysis of an element is measured from the isotopic ratio, the 
method is referred to as isotope dilution analysis. The measurement 
employs a mass spectrometer to determine the isotopic ratio, but only the 
total elemental concentration is determined. Examples of isotope dilution 
analysis for total elemental concentration are described for standard 
reference materials and for general analysis using thermal isotope 
dilution mass spectrometry. See Moore, Larry J.; Kingston, Howard M.; 
Murphy, Thomas J.; and Paulsen, Paul J., "The Use of Isotope Dilution Mass 
Spectrometry for the Certification of Standard Reference Materials", 
Environment International, 10, 169-173, 1984; Fassett, Jack D. and 
Paulsen, Paul J., "Isotope Dilution Mass Spectrometry for Accurate 
Elemental Analysis", Analytical Chemistry, 61, 386-390, 1989. Specific 
analysis of total chromium and selenium and other metals by this method of 
isotope dilution mass spectrometry using a gas chromatography mass 
spectrometer (GC-MS) are described in other references. Reamer, Donald C. 
and Veillon, Claude, "A Double Isotope Dilution Method for Using Stable 
Selenium Isotopes in Metabolic Tracer Studies: Analysis by Gas 
Chromatography/Mass Spectrometry (GC/MS)", Journal of Nutrition, 113, 
786-792, 1983. Reamer, Donald C. and Veillon, Claude, "Determination of 
Selenium in Biological Materials by Stable Isotope Dilution Gas 
Chromatography-Mass Spectrometry", Analytical Chemistry, 53, 2166-2169, 
1981. Veillon, Claude; Wolf, Wayne, and Guthrie, Barbara, "Determination 
of Chromium in Biological Materials by Stable Isotope Dilution", 
Analytical Chemistry, 51, 1022-1024, 1979. 
Examples of typical prior art are those that use ICP-MS detection with some 
form of chromatography (sometimes called "flow injection analysis") to 
separate the species and then uses the instrument as a total elemental 
detector as described in Thompson, J. J.; Houk, R. S., "Inductively 
Coupled Plasma Mass Spectrometric Detection for Multielement Flow 
Injection Analysis and Elemental Speciation by Reversed-Phase Liquid 
Chromatography" Analytical Chemistry, 58, 2541-2548, 1986. As previously 
stated herein, in general, when isotope dilution has been used with 
inductively coupled plasma for environmental analysis, the applications 
have focused on total analysis and although it is an excellent method for 
total elemental composition, it has not been effectively applied to 
species. Examples of ICP-MS isotope dilution analysis for environmental 
natural water and geological samples are given in Garbarino, H. R.; 
Taylor, H. E., "Stable Isotope Dilution Analysis of Hydrologic Samples by 
Inductively Coupled Plasma Mass Spectrometry" Analytical Chemistry, 59, 
1568-1575, 1987; McLaren, J. W.; Beauchemin, D.; Berman, S. S., 
"Application of Isotope Dilution Inductively Coupled Plasma Mass 
Spectrometry to the Analysis of Marine Sediments" Analytical Chemistry, 
59, 610-613, 1987, respectively. 
Studies to measure metabolic transformation of elements in humans have been 
performed where stable isotopes, such as Se-74, have been metabolized in 
the body and transformed into various species. An enriched isotope is 
ingested as an inorganic salt and the different forms of the isotope are 
made by the body. By feeding Se-74 to patients, allowing metabolism, and 
then removing and storing the blood, previously metabolized Se could be 
stored for each individual. After the body was free of the labeled Se-74, 
11 months later, the blood was reintroduced into the subject and the exact 
excretion method was studied without the normal body burden of Se 
confusing the excretion in body fluids. This experiment used stable 
isotope tracers to studied blood excretion mechanisms and definitively 
established the dominant pathway to be the urinary path. The studies 
observed whether body pools could be labeled and how the element is 
excreted. The metabolic study was specifically for selenium and no 
specific speciated isotopes or specific species were used to spike in the 
measurement process. The species were destroyed in the measurement process 
and total selenium was reported. Following repeated ingestion of an 
enriched stable isotope of selenium, their blood plasma became labeled 
with the element in all of the natural, biologically relevant chemical 
forms. Veillon, Claude; Patterson, Kristine; Button, Lawrence; and 
Sytkowski, Arthur, "Selenium Utilization in Humans: a Long-Term, 
Self-Labeling Experiment with Stable Isotopes", American Journal of 
Clinical Nutrition 52, 155-158, 1990. By spreading the enriched isotope 
over all species the identification of any specific species becomes 
impossible by speciated isotope dilution mass spectrometry. "Selenium 
Utilization in Humans--A Long-Term, Self-Labeling experiment with stable 
Isotopes", Veillon et at., Am. J. Clin, Nutr.; 52:155-8 (1990) describes 
this study. Reamer, Donald C. and Veillon, Claude, "A Double Isotope 
Dilution Method for Using Stable Selenium Isotopes in Metabolic Tracer 
Studies: Analysis by Gas Chromatography/Mass Spectrometry (GC/MS)", 
Journal of Nutrition, 113, 786-792, 1983 describes the measurement method 
of isotope dilution using GC-MS. This method is identical to conventional 
isotope dilution mass spectrometry as described in Moore, Larry J.; 
Kingston, Howard M.; Murphy, Thomas J.; and Paulsen, Paul J., "The Use of 
Isotope Dilution Mass Spectrometry for the Certification of Standard 
Reference Materials", Environment International, 10, 169-173, 1984; and 
Fassett, Jack D. and Paulsen, Paul J., "Isotope Dilution Mass Spectrometry 
for Accurate Elemental Analysis", Analytical Chemistry, 61, 386-390, 1989, 
except for the type of mass spectrometry equipment used. 
Since different species have different reactivates and can react with 
reagents, container material, oxygen from the air, or other portions of 
the sample itself, only a final indication of the species separated and 
detected in time has been employed. The original condition, or the ratio 
and type of species at the time of sampling or in the sample prior to 
sampling, cannot be assessed with certainty because transformations or 
exchanges of the species during storage, chemical manipulation, or the 
measurement process cannot be measured by conventional species measurement 
techniques. 
Traditional speciation methods rely on separation of the species using 
physical and/or chemical separation methods. These methods have been 
summarized in two books. Trace Element Speciation: Analytical Methods and 
Problems, Ed. Graeme E. Batley, CRC Press, Boca Raton, Fla., 1989 (ISBN 
0-8493-4712-2); and Metal Speciation: Theory, Analysis and Application, 
Eds. James R. Kramer and Herbert E. Allen, Lewis Publishers, Chelsea, 
Mich., 1991 (ISBN 0-87371-140-8). Neither reference mentions the word 
"isotope" and no mention of isotopic differentiation, isotopic spike 
equilibration, or measurement or monitoring using isotopes is mentioned in 
the speciation literature. Currently, chromatography and other methods of 
chemical and physical separation are used to perform speciation. Examples 
of these specific methods are compiled in the Batley and Kramer books, and 
additional examples are provided. 
Aspects of elemental speciation for biological materials are reviewed in 
Behne, Dietrich, "Speciation of Trace Elements in Biological Materials: 
Trends and Problems", Analyst, 117, 555-557, 1992. The scope of speciation 
as related to high performance liquid chromatography can be understood 
from Cappon, C. J. "HPLC Speciation of Selected Trace Elements" LC-GC, 6, 
584-599. 1988. 
Early work in chromium speciation prior to spectroscopic equipment is 
described in Jones, D. R. and Manahan, S. E., "Atomic Absorption Detector 
for Chromium Organometallic Compounds Separated by High Speed Liquid 
Chromatography", Analytical Letters, 8, 569-574, 1975. Two current methods 
for determining chromium species in water, including one commercial 
version, are described in Lan, Chi-Ren; Tseng, Chia-Liang; Yang, 
Mo-Hsiung; and Alfassi, Zeer B., "Two-Step Coprecipitation Method for 
Differentiating Chromium Species in Water Followed by Determination of 
Chromium by Neutron Activation Analysis", Analyst, 116, 1991; and 
Determination of Chromium, Application Note 26, Dionex Corporation, May 
1986. Energy based separation using microwave extraction is described in 
Ganzler, K.; Szinai, I.; and Salgo, A., "Effective Sample Preparation 
Method for Extracting Biologically Active Compounds From Different 
Matrices by a Microwave Technique", Journal of Chromatography, 520, 
257-262, 1990. Most of these methods could be converted to a definitive 
speciation technique with the addition of spiking with a stable separated 
isotope specie and use of mass detection following the particular 
separation method described. 
Currently, no definitive method of species measurement is available, 
although research is needed on many important species of almost every 
element. In addition to more than 100 elements, literally thousands of 
species of these elements need to be studied and evaluated. Organic 
molecules also need investigation. To evaluate the significance and 
reactions of each, a definitive measurement method is proposed that will 
work for all elements having more than one isotope. Elements that have 
radioactive isotopes can also be evaluated. For the examples previously 
given, chromium has four stable isotopes at constant ratio in nature 
(Cr-50 at 4.35%, Cr-52 at 83.79%, Cr-53 at 9.50%, and Cr-54 at 2.36%). 
Within the limits of current isotopic measurement, these ratios are 
constant throughout the entire earth's crust. 
There remains, therefore, a very real and substantial need to provide a 
method for measuring quantitatively and accurately one or more species 
within a sample employing speciated isotope dilution measurement and 
sampling techniques. 
SUMMARY OF THE INVENTION 
The method of the present invention employs speciated isotope dilution in 
measuring or preparing a sample. It includes providing at least one 
speciated enriched isotope spike in the same speciated form as the species 
to be measured, spiking the sample containing the species to be measured 
therewith, and equilibrating said spiked species with said species to be 
measured, separation of the species to be measured and the enriched 
isotope spike from the remainder of the sample is then effected. 
Subsequently a determination of the concentration of the species to be 
measured is made by employing isotopic element specie ratio. 
In preferred embodiments of the present invention, time related 
chromatographic means may be employed to effect the separation and a mass 
spectrometer may be employed in the isotope dilution measurements. 
In a preferred process of the method, a determination as to whether 
isotopic conversion has occurred is made. 
It is an object of the present invention to provide a method of measurement 
of elemental, ionic, molecular or complex species using isotope spiking of 
species, separation and isotope dilution fraction measurement to provide a 
determination of the quantity of the species in the example. 
It is an object of the present invention to effect such determination by 
chemical means. 
It is an object of the present invention to effect such determination by a 
combination of energy coupling and chemical means such as microwave or 
infrared energy. 
It is a further object of the invention to employ such method in both 
measurement and sample preparation. 
It is yet another object of the present invention to accomplish this 
objective by determining the oxidation state of the elemental species, 
ionic species, molecular species or complex species, such as 
organometallic species. 
It is a further object of this invention to provide such determinations in 
an accurate, economical and reliable fashion. 
Other objects of the invention will be more fully understood from the 
following description of the invention on reference to the illustration 
appended hereto.

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
As used herein, "specie" as employed in respect of the sample containing 
the specie which is to be analyzed quantitatively, shall refer to 
elemental species, ionic species, molecular species, complex species such 
as organometallic species and other species which are adapted to chemical 
quantitative speciated analysis of the present invention. 
As used herein, the term "isotopic element specie ratio" shall refer to an 
isotopic ratio of a "specie." 
The pure isotopic spikes (99.99+ % single separated isotopes) which may 
advantageously be employed in the present invention as the speciated 
isotope spike may be obtained from suitable sources known to those skilled 
in the art such as pure isotopic spikes which may be obtained from Los 
Alamos National Laboratories or enriched, separated isotopes spikes which 
are available from many sources including Los Alamos National Laboratories 
and Oak Ridge National Laboratories. These latter spikes are generally 
more than 90% enriched in the minor isotopes of interest. 
In the method of the present invention (speciated isotope dilution), one or 
more spikes (speciated enriched isotope) will be prepared from an enriched 
isotope (usually a stable isotope), and the specie form of the spike will 
be altered to match that of the specie of interest of a particular element 
or molecule. More than one specie can be prepared from different enriched 
isotopes, depending on the number of available separated isotopes and 
depending on the number of isotopes the element of interest has. The spike 
will be added to the material that is to have the species determined. The 
specie's isotopic spike and the sample's natural specie will be 
equilibrated and extracted and/or separated physically by some means. This 
can be done in either a batch or on-line continuing mode. The isotopic 
specie spike and natural specie from the sample are both separated from 
the sample as they now have the same chemical reactivity and are 
chemically indistinguishable, with the exception of mass. Both are 
separated from other species in time and space and both have the same 
reactivity as that particular specie. As in the measurement method of 
isotope dilution mass spectrometry (IDMS), the measurement is made on the 
isotopic spike ratio. This leads to the evaluation of different isotopic 
ratios to determine the concentration of the species. In IDMS, the total 
concentration is calculated from the isotopic ratio. In this case, only 
the specie of interest will be present and only the specie concentration 
will be calculated. Two or more species can be measured simultaneously by 
using multiple spikes. The absence of a separated isotopic spike of one 
form in the other definitively demonstrates the absence of conversion of 
one specie to another. Any conversion of the specie of interest to another 
form will alter the isotopic ratio of that other specie form by the amount 
of spike and natural specie that was transformed in the measurement 
process. 
The method of the present invention relies on several procedures that are 
summarized as follows: 
The sample is defined and selected by the analyst and may be one of a 
variety of sample types previously described herein. 
1. Tag the specie(s) in the sample with an isotopic tag in the same 
speciated form as the specie(s) of interest in the sample; 
2. Equilibrate the spiked specie(s) with the natural sample specie(s) to 
thereby facilitate all chemical reactions occurring in equal probability 
to both the tagged specie and the natural specie. 
3. Separate the specie(s) of interest from one another by some method, 
either chemical or physical, including but not limited to chromatography, 
digestion, extraction, centrifugation; 
4. Determine each isotopic dement specie ratio, such as by using a mass 
spectrometer, for example. The total concentration of the fraction 
containing the specie(s) is also determined by standard methods for 
comparison; 
5. Deconvolute the movement between species from the time of spiking, using 
isotopic ratios and other mathematical methods. The shifting of the ratio 
of any other specie from natural provides the cross over to that specie 
from the specie of interest under study. The absence of any ratio 
alteration from the speciated isotope, except for the specie of interest, 
definitively proves no crossover occurred; 
6. Calculate the concentration of the specie(s) in the sample. While both 
conventional and the speciated isotope dilution can be employed 
simultaneously, only the speciated method can definitively prove whether 
all the specie of interest is in the original form as spiked; 
7. Spiking can be just prior to measurement or at the time of sampling to 
determine the original concentration and stability of the species in the 
entire measurement process. The speciated isotope dilution method can be 
used to determine only measurement parameters, or can include sampling 
stability and chemical manipulation procedures, depending on the time and 
conditions of spiking. Lack of isotopic alteration of other species is 
definitive evidence of a lack of interchange between species. 
These steps remain essentially the same if more than one specie is spiked, 
but the mathematical deconvolution is different, depending on the number 
of spiked species and the extent to which the species interact. 
The calculations can be done by many different methods. The classical IDMS 
equations which are known to those skilled in the art may be employed 
where there is no crossover and may be employed with modification where 
crossover has occurred. 
In the method disclosed herein, the traditional IDMS equation methods of 
calculating the concentration of an isotopically spiked portion of a 
sample are used in a different manner. The normal equilibration with spike 
and destruction of different species limit the information to bulk total 
elemental analysis. In this approach, the speciated information is 
retained and treated in an entirely different manner, preserving the 
species and separating them in time and space to permit quantitative and 
qualitative specie determinations. The traditional application of IDMS was 
developed as a measurement technique for the nuclear industry and was 
later applied to the geological community where isotopic composition is 
the main objective and speciation is ignored. The traditional use of the 
bulk element (total element), high accuracy analysis technique of IDMS is 
described in Moore, Larry J.; Kingston, Howard M.; Murphy, Thomas J.; and 
Paulsen, Paul J., "The Use of Isotope Dilution Mass Spectrometry for the 
Certification of Standard Reference Materials", Environment International, 
10, 169-173, 1984; Fassett, Jack D. and Paulsen, Paul J., "Isotope 
Dilution Mass Spectrometry for Accurate Elemental Analysis", Analytical 
Chemistry, 61, 386-390, 1989; and Kingston, H. M. and Fassett, J. D., 
"Report of Analysis: Determination of Vanadium in Oil"; National Bureau of 
Standards, Gaithersburg, Md., Jul. 17, 1984. 
Speciated measurements are based on transforming the enriched speciated 
isotope ("spike") into the same speciated form as the specie of interest. 
This is then added to a sample. The sample is then equilibrated. 
Equilibration is achieved by various methods depending on the medium in 
which the specie is found. For aqueous or other solutions, mixing prior to 
any manipulation is all that is necessary. Mixing the spike with the 
sample can occur at sampling or at the time of analysis. The ramification 
of one is the inclusion of storage, and the other is only from the 
analysis point. Solids such as tissues or soils prior to decomposition or 
extraction and the spike and specie are extracted or the material around 
them is decomposed at the same time to provide a homogeneous solution 
which is mixed at the molecular level and any chemical process happening 
to the one happens to the other with equal probability as they are 
indistinguishable chemically. 
After equilibrating, the altered speciated spike isotope with the natural 
element species in the sample, physical separation of the individual 
species is performed. Isotopic element specie ratio analysis, preferably 
by mass spectrometry, is performed on each separate component (specie) and 
is used to measure the altered isotopic element specie ratio(s). The 
measured isotope ratio of isotope A to isotope B (Rm) can be calculated as 
follows: 
##EQU1## 
Where: R.sub.m is the measured isotope ratio of isotope A to isotope B 
A.sub.x is atom fraction of isotope A in the sample (usually a constant 
ratio in nature) 
C.sub.x is concentration of speciated element in the sample 
W.sub.x is the weight of sample 
A.sub.s is atom fraction of speciated isotope A in the spike (an enriched 
separated isotope) 
C.sub.s is concentration of speciated elemental in the spike 
W.sub.s is the weight of the isotopic spike 
B.sub.x is atom fraction of isotope B in the sample (usually a constant 
ratio in nature) 
B.sub.s is atom fraction of speciated isotope B in the spike (an enriched 
separated isotope) 
See generally Fassett et al., "Isotope Dilution Mass Spectrometry for 
Accurate Elemental Analysis," Analytical Chemistry, Vol. 61, No. 10 
(1989). 
With a speciated spike, preservation and separation of the species, and 
analysis of individual isotopically spiked species, the concentration of 
the speciated element in the sample is calculated as distinguished from 
the total elemental concentration. The concentration of the specie without 
crossover is calculated as follows: 
##EQU2## 
The method can best be demonstrated through examples. The combination of 
isotope transformation into a speciated form, species, equilibration, 
spike and natural species separation, and determination of isotopic 
element specie ratios for individualized species can be illustrated for 
chromium (III) and chromium (VI) with reference to the figure. The FIGURE 
illustrates the chromatographic separation of Cr(VI) and Cr(III). The 
FIGURE includes the separated isotope ratio to be measured by using Cr-50 
separated isotopes equilibrated in the separate speciated forms of 
Cr(VI)-50 in the sample spiked and equilibrated with the enriched 
separated isotope. 
A general overview of the preferred process of the present invention may be 
summarized as follows. The method being disclosed is generally feasible 
for most elements with multiple isotopes, multiple species (either ionic, 
molecular, metallic or complexed), and a method of physically separating 
the species. The technique being described uses separation methods that 
separate the elemental species physically and, in time from one another to 
permit the isotopic dement specie ratios of each specie to be determined 
independently. 
The general concept is applicable to single or double spikes of ionic, 
molecular, or combination species (including metallic species). The first 
example considered herein uses the two oxidative elemental species Cr(III) 
and Cr(VI). This example will determine the concentration of Cr(VI) and 
indicate if any of it was transformed into Cr(III) during sample storage, 
separation, or manipulation. 
EXAMPLE I 
This example is shown graphically in the FIGURE. 
Step 1. Spike Preparation 
A separated (enriched) isotope spike for a single or for each of two 
species is prepared for Cr(VI) employing the single enriched specie spike 
of Cr-50 for Cr(VI). The breakdown is as follows: 
______________________________________ 
Natural Isotopic Abundance 
Separated Isotopic Spike 
for Chromium (VI) 
______________________________________ 
50- Cr(VI) Spike 50- 4.35% 
50- &gt;99.99% 52- 83.79% 
52- &lt;0.01% 53- 9.50% 
53- &lt;0.01% 54- 2.36% 
54- &lt;0.01% 50- 4.35% 
______________________________________ 
Know to 3+ significant figures .+-.0.02% 
Step 2. Sample Collection and Spiking 
Water is collected from a natural water aquifer. Using the single enriched 
specie spike of Cr-50 for Cr(VI), the sample is spiked with a known 
quantity of the spiked (Cr-50, Cr(VI)) species. 
The optimal ratio would be Cr(VI)-50 in a concentration that approaches 
approximately a 1:1 ratio with the natural Cr(IV)-52. This permits the 
highest accuracy during the measurement. 
Step 3. Sample Specie and Spike Specie Equilibration 
Equilibrate the sample (natural) Cr(VI) isotope 52 (83.79%) and the species 
Cr(VI) spike isotope Cr-50. For this example, mixing both the natural and 
enriched material in aqueous form is accomplished. At this point the total 
Cr(VI) has a concentration from total material and a ratio Cr(VI)-50 
(concentration established) to: Cr(VI)-52 in the sample of approximately 
1:1 depending on the amount spiked, the purity of the isotope and the 
amount of Cr(VI) in the original sample. 
Step 4. Resolve the Species Temporally or Spatially 
For this example, separate the species using chromatography. (The preferred 
method of separation will be determined by the chemistry of the species 
and will be known to those skilled in the art.) Separation in time on-line 
to an inductively coupled plasma mass spectrometer (ICP-MS) creates 
physical resolution of Cr(III) fraction and Cr(VI) fraction in different 
portions of a chromatogram. Cr(III) and Cr(VI) are separated from each 
other and all isotopes of chromium in each speciated form are separated as 
one of these Cr(III) or Cr(VI) speciated forms and are separated together 
in narrow chromatographic bands. 
Step 5. The Isotope Ratio of Each Speciated and Resolved Component is 
Measured 
Isotope ratio measurement of each individual species (isotope resolved 
component is made separately for Cr(III) and for Cr(VI). 
The concentration of the species is determined from isotope dilution 
calculations. A total concentration for both isotopes can also be 
performed as a check. 
Step 6. Determination Of Specie Conversion 
Deconvolute each species in the presence of each other using isotopic 
element specie ratios. If, for example, Cr(VI) was converted to Cr(III), 
the natural ratio will show a shift in the Cr(III)-50 isotopic ratio. If 
no Cr(VI) was converted to Cr(III), then the isotopic ratio of Cr((III) 
will be that of natural Cr. A natural isotopic ratio for Cr(III) 
definitely proves no conversion of Cr(VI) to Cr(III). This definitively 
proves the maximum concentration of Cr(VI) in the water. The FIGURE 
illustrates this example. 
If Cr(III) was converted to Cr(VI), the Cr(VI) concentration will be 
increased and show a larger concentration. This experiment will only 
provide a definitive maximum concentration for Cr(VI). 
To definitively determine both Cr(III) and Cr(VI), Step 1 would be altered 
for a double speciated isotopic spike. This is described in connection 
with Example II. 
With reference to the FIGURE, a specific example of the general concept to 
be applied with a single specie spike of ionic Cr(VI) is illustrated. 
______________________________________ 
Natural Isotopic Abundance 
for Cr Spike Separated Isotopic Spike 
______________________________________ 
50- 4.35% 50- Cr(VI) 
52- 83.79% 50- &gt;99.99% 
53- 9.50% 52- &lt;0.01% 
54- 2.36% 53- &lt;0.01% 
50- 4.35% 54- &lt;0.01% 
______________________________________ 
Cr(III) Cr(VI) 
______________________________________ 
B. Spike Resolution 
Nat. Cr(III) & (VI) 
Nat. Cr(III) 
Nat. Cr(VI) 
52- 83.79% 52- 83.79% 52- 83.79% 
53- 9.50% 53- 9.50% 53- 9.50% 
54- 2.36% 54- 2.36% 54- 2.36% 
50- 4.35% 50- 4.35% 50- 4.35% 
50- Cr(VI) Spike 
50- &gt;99.99% % 
52- &lt;0.01% 
53- &lt;0.01% 
______________________________________ 
The ratios are as follows: 
##EQU3## 
Where "x" is the sample specie, "sp" is the spike specie, and "xsp" is the 
final combination of sample and spike for a single specie. This process 
provides the ratio measurement of 52:50 for Cr(VI) and for Cr(III) that 
establishes the stability or lack of stability of the species during 
storage, sample preparation, processing and analyzing. It also provides 
for quantification of Cr(VI) if no conversion occurred. 
EXAMPLE II 
This example provides a definitive determination of both Cr(III) and 
Cr(VI). 
If Cr(III) was converted to Cr(VI), the Cr(VI) concentration will be 
increased and show a larger concentration. This experiment will only 
provide a definitive maximum concentration for Cr(VI). 
To definitively determine both Cr(III) and Cr(VI), Step 1 of Example I 
would be altered for a double speciated isotopic spike. 
Example II is shown graphically in the FIGURE. Only steps 1, 5 and 6 of 
Example I are altered in Example II. 
Step 1. Spike Preparation 
Separated (enriched) isotope spikes for both Cr(III) and Cr(VI) species are 
prepared. Use the isotopically enriched specie spike of Cr-50 for Cr(VI), 
as in Example I, and the isotopically enriched specie spike of Cr-53 for 
Cr(III): 
__________________________________________________________________________ 
Natural Isotopic Abundance 
Enr. Isotope Cr(VI)-50 
Enr. Iso. Cr(III)-53 
for Cr (III) & (VI) 
Separated Isotopic Spike 
Sep. Isotopic Spike 
__________________________________________________________________________ 
50- 4.35% 50- Cr(Vl) Spike 
53- Cr(III) Spike 
52- 83.79% 50- &gt;99.99% 50- &lt;0.01% 
53- 9.50% 52- &lt;0.01% 52- &lt;0.01% 
54- 2.36% 53- &lt;0.01% 53- &gt;99.99% 
50- 4.35% 54- &lt;0.01% 54- &lt;0.01% 
__________________________________________________________________________ 
Know to 3+ significant figures .+-.0.02% 
Steps 2 through 5 are essentially the same as in Example I. 
Step 2. Sample Collection and Spiking 
Step 3. Sample Specie and Spike Specie Equilibration 
Step 4. Resolve the Species Temporally or Spatially 
Step 5. Isotope Ratio of Each Speciated and Resolved Component 
Step 6. Determination of Specie Conversion and Concentration 
Deconvolute each specie in the presence of the other using isotopic element 
specie ratios. If Cr(VI) was converted to Cr(III), the natural ratio will 
show a shift in the Cr(III)-50 isotopic ratio Cr52/50. If no Cr(VI) was 
converted to Cr(III), then the isotopic ratio of Cr(III) will be that of 
natural Cr52/50. If no Cr(III) was converted to Cr(VI), then the isotopic 
ratio of Cr(VI) will have a normal Cr52/53 ratio (to within 0.04%, or the 
ability of the instrument to determine the ratio). The absence of other 
than a natural isotopic ratio for Cr(iii) Cr52/50 precludes the conversion 
of Cr(VI) to Cr(III). The equation as described can be used to 
definitively calculate the concentration of Cr(III) [Cr(III) 52/53] 
species and Cr(VI) [Cr(VI) 52/50] species in the water. 
One may determine the extent of conversion of each specie to another, and 
one or both may need to be corrected for conversion in different examples. 
The relative concentration and quantity of each isotope converted to the 
other can be calculated using mathematical relationships established for 
the specific isotopes, enrichment factors, and resolutions of the 
analyzing instrument. 
With reference to Example I and the FIGURE, a specific example of the 
general concept to be applied with double spike of ionic chromium (III) 
and (VI) species will be considered. The data is as follows: 
______________________________________ 
A. Time Resolution 
Cr(III) Cr(VI) 
B. Spike Resolution 
Nat. Cr(III) & (VI) 
Nat. Cr(III) Nat. Cr(VI) 
50- 4.35% 50- 4.35% 50- 4.35% 
52-83.79% 52- 83.79% 52- 83.79% 
53- 9.50% 53- 9.50% 53- 9.50% 
Spike 53- Cr(III) Spike 
54- Cr(VI) Spike 
50- &lt;0.00% 50- &gt;99.99% 
52- &lt;0.00% 52- &lt;0.00% 
53- &gt;99.99% 53- &lt;0.00% 
##STR1## 
______________________________________ 
Where "x" is the sample specie, "sp" is the spike specie, and "xsp" is the 
final combination of sample and spike for a single specie. 
The use of more than one speciated isotope provides the ability to 
calculate the contribution and conversion of one specie to another. This 
is very different from current methods wherein only the total in the final 
form can be determined and conversion is undesirable and unmeasurable. 
If Example II were repeated but with isotopes of lesser purity, the same 
result could be achieved but the reduced purity of the isotopes would have 
to be considered in the calculations. 
It will be appreciated that the present invention not only provides a basis 
for effective quantitative determination of the presence of a specie such 
as in a real, natural sample, but it facilitates use of the process in a 
batch or a continuous process as desired by the user. In addition, it 
provides a means for determining if there has been undesired conversion 
from one isotope or specie form to another during storage, as a result of 
interaction with reagent materials or during measurement. The process of 
spiking with one or more speciated isotopes of know concentration, 
quantity, and specie permits evaluation of various segments of specie 
analysis process. It also provides quality control measurement capability 
for evaluation of the validity of speciated procedures and standard 
methods. The ability to spike the sample at different stages provides 
evaluation beyond the ability to just measure the final specie 
concentration. 
Spiked at a critical stage in a chemical procedure the conversion of 
various species with respect to that particular process can be evaluated 
and all subsequent procedures until mass ratio measurements are performed. 
Spiking during sampling provides a mechanism of evaluating the particular 
specie spiked from sample collection to final ratio measurement and 
provides a method of measuring the conversion for the entire protocol 
including storage, manipulation and the measurement process. 
The ability to extract and separate the species from the bulk sample and 
material can be evaluated by spiking the sample and then extracting using 
such methods a microwave extraction, decomposition, solvent extraction. 
The conversion or interchange of the species with various procedures or 
for a single procedure can be measured. Correction for the process may be 
necessary to may procedures and this is the only method of providing a 
tracking procedure. This is crucial to the conversion from prescription 
based environmental and medical procedures to performance based methods. 
Performance based procedures require demonstration of the ability to 
achieve the precise speciated measurement required and precludes bias 
caused by conversion of other species. 
It is desirable, where practical, to measure several species simultaneously 
on a qualitative and quantitative basis. To do this multiple isotope 
specie spikes are the most informative. 
In order to employ the present invention in determining whether conversion 
has occurred, one should employ the double speciated isotopic spike such 
as is described in Example II. 
Deconvolution of these species in the presence of each other may be 
determined using isotopic ratios such as, for example, CR(III) and CR(VI) 
species crossover by a species III to IV and vice versa shift will result 
in the isotope dement specie ratios being altered for each specie. 
Determination of the amount of crossover of one specie to another by using 
isotopic ratios to calculate the specie interchange becomes possible, 
employing the present invention. 
Even where two isotopic spikes are not available, one spike can be used 
with calculation of crossover by ratio shift in one specie. An alternative 
is to perform the same speciated spiking experiment repeatedly but 
transform the isotopic spike into different species for different 
experiments. By collecting multiple measurements and doing a mass balance 
each time the entire speciation behavior mechanism can be evaluated. 
It will be appreciated that the process is directed primarily toward 
elements, molecules, ions or complexes which have more than one isotope. 
The invention may be employed in a wide range of applications including, 
but not limited to, medical, biological, environmental and industrial 
uses. 
The invention enables measurement of elemental, ionic, and molecular 
species as well as complexes using isotope spiking of species, separation 
and isotope dilution fraction measurement to provide a combined technique 
enabling the reliable and definitive determination of the quantity of 
species in a real, natural sample. 
The process of measurement may include the following features: 
a. The preparation of species in elemental, ionic, molecular, organic 
metallic, complexed, and metallic states used in the speciated isotopic 
alteration of natural material containing natural species. 
b. Equilibration of the species spike will be performed. In contrast to 
equilibration in classical IDMS, the elemental, ionic, molecular or 
complex species will be preserved. The isotopic spike will react in a 
manner similar to the natural isotopic species components. Equilibration 
in an aqueous sample will include mixing; in solid samples it may be 
extracted with the analyte of interest in the preparation methodology. 
c. The physical and chemical separation of species containing isotopically 
altered species is performed to provide a time resolved component for each 
specie. 
d. Extraction and physical separation may include, but not be limited to, 
extraction by microwave-assisted extraction, soxhilate extraction, solvent 
dissolution, acid dissolution, acid or base hydrolysis distillation, 
centrifugation, solvent extraction using a separatory funnel, and other 
chemical and physical separation methods. 
e. Chromatographic separation may be used to separate the species in time 
and capture each fraction for separate evaluation by a mass spectrometer 
or in an on-line mode. 
f. High performance liquid chromatography (HPLC) or flow injection analysis 
(FIA) may be used to separate the species is real time with detection of 
each fraction by a mass spectrometer. 
g. Mass spectrometric measurements will be used to evaluate the isotopic 
element specie ratios of the specific specie fractions. This will produce 
an isotopic resolved component for evaluation. Isotope dilution 
calculations will be applied to each fraction to determine the 
quantitative concentration of the species present. 
h. A shift in the isotopic ratios of other species not spiked with this 
specific isotope will be used to quantitatively determine the 
transformation of one species to another from the time of the spiking 
event. The original composition of the species composition will be 
calculated from these isotopic shifts. 
i. Other algorithm calculations are possible from the two components of 
time resolution of individual species and mass isotopic element specie 
ratio alterations. 
j. The method of the present invention makes it possible to measure the 
stability of species in specific reactions, separating reagents, 
chromatography procedures, extractions, at specific temperatures, and in 
various storage environments. Others are possible if spiked with speciated 
isotopes prior to the reaction occurring. Reactions affecting the natural 
material species will also affect the speciated isotopes. Ratio shifts in 
other natural species caused by the incorporation of the enriched isotopes 
into new molecular forms identifies the origin as the isotopically 
enriched specie. 
k. These measurements may be made in active systems, such as environmental 
or biological systems, where spiking of the system with a specific species 
will be followed by isotopic shifts in species produced from the system. 
Examples may be the stable isotopic alteration of food and the evaluation 
of blood species components, urine, feces, and tissues using the 
equilibration of the natural system to perform the isotopic mixing and 
transformation of species. The same mathematical alterations may be used 
to deconvolve the final species. In addition, second spiking of another 
isotope of that same specie may be used to quantify the species, creating 
a double spike of different origin, one from the active biological or 
environmental system, and the other from the analytical process of 
measurement. 
1. Stability of species may be determined by using isotope altered spikes 
of the species to track the stability and conversion with time, material, 
reagent, and other conditions. 
The method of the present invention is applicable to elements, ions, 
inorganic molecules, complexes, covalent, organic molecules, 
organometallics, gasses, liquids, solids, solutions, and mixtures. 
Separated isotopic spikes of these same speciated forms can be created and 
thereby renders the method of the present invention a general technique to 
evaluate a variety of speciated forms in almost any sample matrix type. As 
stated hereinbefore, if desired, the process may be employed as a batch 
process or a continuous process. 
Also, additional flexibility is provided in respect of what will be 
accomplished on-line as opposed to off-line. For example, on-line 
separation with off-line component evaluation may be employed. In the 
alternative, off-line sample preparation and spiking with on-line mass 
spectrometric analysis may also be employed, if desired. If desired, 
automatic spiking devices may add the speciated spike to the sample 
automatically or in a batch mode prior to analysis. Spiking of the sample 
may be achieved on-line with the isotopic spike being added in an 
automated fashion. 
If desired, multiple isotopic spikes may be used simultaneously to evaluate 
species qualitatively or quantitatively. Isotopic spikes of the same 
specie but made from different isotopes can be added successively to the 
reaction to track reaction points where species alteration occurs at the 
greatest rate or to entirely different species. Different ratios will be 
produced in the related species depending on when the conversion occurred 
in the process. 
It will be appreciated that the invention also contemplates spiking of the 
several different isotopically enriched analogs of the same specie which 
may be added at various steps in the sampling procedure and the stability 
and integrity of the specie with respect to these processes is evaluated 
by mass spectrometric measurements of the various isotopic ratios. 
Chemical processes, extraction methods, dissolution procedures and storage 
procedures may be evaluated. 
Spiking active, biological and environmental systems with isotopic labelled 
species can be used to identify transformation of the species that permit 
fate and transport to be determined for species as well as providing the 
measurement method. The method may also be employed to evaluate the effect 
of precise energy inputs during extraction when employing energy inputting 
devices such as microwave energy controlled systems, super critical fluid 
extraction, or other solvent extraction systems to separate the species of 
interest from a matrix. 
In connection with evaluation, the methods of the present invention may be 
employed, for example, in extraction chemistry, dissolution chemistry, 
chemical manipulation influences and storage chemistry. 
The spiking of the sample in specific species separation devices such as 
microwave assisted chemical abstraction system permits the evaluation of 
the stability of the species extraction. Different temperatures and 
pressures conditions may shift the equilibrium of various species in the 
sample preparation of the sample. A previous patent application describes 
a method that permits the control of pressure at various temperatures in 
chemical reactions and in different phases in microwave chemical 
processing. See Kingston U.S. Ser. No. 08/127,263 filed on Sep. 24, 1993. 
The spike of samples in specific species separation devices, such as 
chromatographic instrumentation, permits the evaluation of the stability 
of the species extraction and transforms the separation into a 
quantifiable technique. These devices may spike the sample automatically 
or in a batch mode. The speciated isotope dilution method may be used to 
evaluate the stability of the species extraction or to quantify the 
original species concentrations and method dependent devices. 
The speciated enriched species may be added in a gas, liquid, solid or 
chemically or physically bound form. 
It will be appreciated that a wide variety of mass spectrometric 
instruments can be used in the practice of the present invention. These 
include, but are not limited to, inductively coupled plasma mass 
spectrometry (ICP-MS), microwave induced plasma mass spectrometry 
(MIP-MS), thermal ionization mass spectrometry (TIMS), spark source mass 
spectrometry, liquid chromatography mass spectrometry (LC-MS), gas 
chromatography mass spectrometry (GC-MS), and all hyphenated and 
non-hyphenated mass spectrometric measurement techniques. Measurement by 
any mass discriminating device capable of isotopic measurement, including 
neutron activation analysis, can be used to make these measurements. 
In the event that a suitable stable isotope were not available, a 
radiogenic isotope may be employed in lieu thereof and treated as a stable 
isotope for mass ratioing and such use shall be deemed to be a "stable 
isotope" for purposes of the disclosure and claims hereof. 
The present method not only provides a capability not present in prior art 
techniques and one which is very much needed, but also one which can, 
depending upon instrument and method controls, provide the accuracy of 
highly accurate measurements. 
In summary, the present invention provides an efficient means of obtaining 
accurate chemical specie quantitative evaluations in a manner which will 
have wide application and is not present in existing prior art systems. 
Whereas particular embodiments of the invention have been described above 
for purpose of illustration, it will be evident that those skilled in the 
art that numerous variations of the details may be made without departing 
from the invention as defined in the appended claims.