D-IDMS Technology Explained

Introduction

Direct Isotope Dilution Mass Spectrometry (D-IDMS), and Direct Speciated Isotope Dilution Mass Spectrometry (D-SIDMS) are state-of-the-art measurement techniques that dramatically increase the accuracy, precision and reproducibility of analytical instrumentation used in clinical analysis. These tools have the unique ability to track and measure changes in samples from the moment of collection to the instant of analysis. These techniques are closely related, so for simplicity’s sake we are going to refer to both of these techniques as D-IDMS in this introduction. For more rigorous scientific discourse please refer to the journal articles on our Resources page.

D-IDMS is a scientifically recognized technique that has delivered superior results in the chemical, energy, and environmental fields for many years.  However, D-IDMS has only recently been applied to biological and clinical samples. It is regarded by the National Institute of Standards & Technology (NIST) as a “definitive method,” because it is a method of proven high accuracy. The accuracy is afforded by adding the sample mixture to a known amount of a similar compound labeled with a non-radioactive, or so-called “stable,” chemical isotope to the sample. The sample is equilibrated with the isotope standard so that the isotope-labeled sample and unknown samples are well-mixed. The total amount of the compound of interest in the sample and the isotope ratios are determined by the precise measurement of mass made by mass spectrometry.

Ready to learn more? Here is a glossary of helpful terms:

Isotope – Elements are defined by the number of protons in an atom’s nucleus. For example, an atom with 6 protons must be carbon.  In addition to protons, the atoms of every element (except the simplest form of hydrogen) also contain neutrons. When an element’s atoms have different numbers of neutrons they are said to be isotopes of that element.

Mass Spectrometry – Mass spectrometry (MS) is an analytical technique that ionizes chemical species and sorts the ions based on their mass to charge ratio. In simpler terms, a mass spectrum measures the masses within a sample. Mass spectrometry is used in many different fields and is applied to pure samples as well as complex mixtures.

Direct Isotope Dilution (D-ID) – Use of enriched isotope spike for analysis of a compound to provide concentration values without calibration curves.

Direct Speciated Isotope Dilution (D-SID) – Use of multiple isotopically enriched standards to simultaneously analyze related compounds or species, and provide both measured and bias-corrected results without calibration curves.

Dried Blood Spot Analysis (DBS) – A form of bio-sampling where blood samples, typically from a finger or heel stick, are blotted and dried on filter paper for shipment, analysis, and storage.

The principle of isotope dilution

Now that you have the lingo down, let’s take a deeper dive into the technique. We’ll use the measurement of an important biological compound, glutathione, in our example. Glutathione is one of the most abundant antioxidants in aerobic cells, and is critical for protecting the brain from oxidative stress, acting as a free radical scavenger.1

Glutathione can have two different forms in the body, reduced and oxidized, and these forms act differently.  Glutathione (GSH) is typically in a reduced state in the blood. Like many compounds, it can be oxidized in the presence of air or oxygen. This form we will designate as GSSG.  The ratio of the reduced GSH to oxidized GSSG is an important indicator of cellular health. Think of the difference between reduced iron in steel, and oxidized iron or rust. These two forms of iron have very different properties and it is important to know how much of a bridge is reduced versus oxidized or rust.

Still with us? There’s more.

GSH constitutes up to 98% of total cellular glutathione (TCG) under normal conditions. Since GSSG is only about 2% of TCG in the blood, we need an accurate method to measure GSH, GSSG, and the total glutathione in the body. Here’s the trick – the measurement must be made before exposure to the air oxidizes the sample. The fundamental problem D-IDMS solves is that it can quantitatively track any change in the sample (including oxidation) so that the values determined for the sample are from the time of collection, not the time of analysis. That means that D-IDMS removes the error introduced by air oxidation.

To keep things straight, let’s designate a few abbreviations so we can keep track of our molecules:

GSH – Reduced glutathione (natural)
GSSG – Oxidized glutathione (natural)
GSH* – Reduced isotopically labeled glutathione (standard)
GSSG+ – Oxidized isotopically labeled glutathione (standard)
GSSG* – Oxidized isotopically labeled glutathione (from GSH*)

Collection

We need to measure the amount of GSH and GSSG in a drop of blood. The problem is that some of the GSH can oxidize to GSSG before we can get the sample to the lab for analysis. Our D-IDMS technique eliminates this problem by providing a dried blood spot card that contains known amounts of isotope-labeled reduced glutathione (GSH*) and oxidized (GSSG+), respectively. It’s worth noting that we are using different and unique labels for the GSH* and GSSG+, but let’s come back to this in a minute.  We have no idea of knowing how much GSH or GSSG is in the blood sample at this point, but we do know exactly how much added labeled standards are on the card as shown in Figure 1.  For this example, we have measured and placed 90 units GSH* and 10 units GSSG+ on the DBS spot.

Image of blood drop containing GSH and GSSH being added to the DBS card impregnated with GSH* and GSSG+ for D-IDMS analysis.
Figure 1.  Blood sample with natural glutathione, and a dried blood spot containing isotopically labeled glutathione.
Equilibration

The GSH and GSSG mixes with the GSH* and GSSG+ on the card as illustrated in Figure 2. As we dry the card, transport it to the lab, and prepare it for analysis, some of the GSH and GSH* may naturally oxidize. The GSH and GSH* will oxidize at the same rate, since they are the same compound and are exposed to the same air.  The GSH oxidizes to GSSG, and the GSH* oxidizes to GSSG*, which is isotopically different from the GSSG+ we added to the card.

Image of the mixing of GSH and GSSG from the blood sample with the GSH* and GSSG+ impregnated on the DBS card.
Figure 2.  While GSH, GSSG, GSH* and GSSG+ mix on the dried blood spot, natural oxidation of GSH and GSH* may occur.
Analysis

At the lab, we analyze the dried blood spot using a mass spectrometer to detect and identify the natural and labelled glutathione species. The mass spectrometer can easily measure the mass difference between GSH, GSH*, GSSG, GSSG+ and GSSG* on the card. Unfortunately, it is not able to tell us the history of what happened to the GSH or GSSG prior to analysis. Enter Direct Speciated Ion Mass Spectrometry (D-SIDMS).

Conventional lab tests that can measure the GSH and GSSG levels on the card cannot account for oxidation during collection, transportation, or analysis, which returns an incorrect result (Figure 3).   D-SIDMS, however,  first measures the GSH and GSSG, as well as the GSH*, GSSG+ and GSSG* on the card.   With a selective ion monitoring mass spectrometry measurement, we can then compare the GSH* measured value to the known GSH* we put on the card at time of spotting, and the amount of GSSG* to track the natural oxidation. This returns accurate results compared to the error prone conventional lab test.

Illustration of analysis of labeled and unlabeled GSH and GSSG by D-IDMS.

Figure 3.  SIDMS identifies and measures the natural oxidation changes of samples from collection
to analysis, corrects for those changes and reports the actual values from time of collection.

For example:  If the total amount of natural GSH is measured to be 95.00, and the total amount of natural GSSG is measured 5, conventional labs have to stop here and report that amount. Using D-SIDMS we make the additional measurements of GSH* and find a result of 85.15, GSSG+ at 10.00, and GSSG* at 4.85. The original GSH* number was 90.00, but we can see that 4.85% of that oxidized to GSSG* between collection and analysis.  We therefore know that the measured value for GSH is 4.85% low and can report the corrected value of 95.00 (measured) + 4.85 (deviation) which in this example delivers an actual value of 99.85.  This is the true value at the moment of collection.  Another way to look at this is in Table 1.

Table 1. Illustration of Limitations of Conventional MS Analysis vs. D-SIDMS Analysis
D-SIDMS Identifies and Corrects Errors Natural Labeled
GSH GSSG GSH* GSSG+ GSSG*
Collect Body ?? ?? 0.00 0.00 0.00
DBS Card 0.00 0.00 90.00 10.00 0.00
Analyze Measured Value 95.00 5.00 85.15 10.00 4.85
Measured Deviation - - -4.85 0.00 +4.85
Report Conventional Lab Report 95.00 5.00 - - -
Actual Value 99.85 0.15 - - -
Conventional Lab Error 4.85% 4.85% - - -
D-SIDMS Report 99.85 0.15 90.00 10.00 0.00
Actual Value 99.85 0.15 90.00 10.00 0.00
D-SIDMS Lab Error 0.00 0.00 0.00 0.00 0.00
Why is this important?

So if we remember the beginning of the discussion, very small differences in blood chemistry can have significant health implications. The ratio of reduced to oxidized glutathione have been identified in blood from patients with diabetes, autism spectrum disorder, and respiratory distress syndrome, and are currently being investigated as potential clinical biomarkers for these diseases.The small differences and changes that can occur between time of collection and time of analysis has made accurate trace-level measurement of many interesting and significant compounds extremely problematic.  As scientific instruments like mass spectrometers become more sensitive, the need for more accurate techniques and tests that identify and measure sources of error in analyses becomes increasingly important.  The glutathione example is just one of many different compounds where D-IDMS and D-SIDMS can provide new information through highly accurate and precise analysis.  Recent applications of D-SIDMS and D-IDMS have also provided new information on the health effects from toxic metals for child neurological disorders and identifying correlations between environmental organic pollutants with autism spectrum disorders.3,4

References
  1. Methods in Molecular Biology 2010, 648: 269-7. [LINK]
  2. Analytical Chemistry. 2015, 87: 1232-1240. [LINK]
  3. Biomarkers 2009, 14(3): 171-80. [LINK]
  4. Nature Scientific Reports. 2016, 6: 26185. [LINK]