DE20308472U1 - mass spectrometry - Google Patents

Description

Micromass Limited August 25, 2003/kr/tk

mass spectrometry

It is known to measure the isotopic abundance of a particular compound in a mixture by combusting the compound in a furnace (e.g. in oxygen) or pyrolyzing it (e.g. in an inert gas) as it elutes from a gas chromatograph ("GC") or a liquid chromatograph ("LC"). This procedure has the advantage that the chemical properties of the compound do not affect the sample isotope ratio of the products from the furnace.

However, the known procedure has the disadvantage that the background is observed to increase with the increase in the temperature of the chromatograph. This may be due to column bleed of the mixture of interest, which may contain a large number of low level components which elute and are not completely separated. However, in these mixtures, the compounds have a similar isotopic ratio if they have been equilibrated during their course. The compound or compounds to be analyzed are of interest because of their difference from the equilibrated background.

The nature of the background usually makes it difficult to accurately determine the beginning and end of the mass chromatography peaks. Measuring the isotope ratio from the areas of the mass chromatogram

·

peaks of the major isotope and the minor isotope is therefore not accurate under normal experimental conditions.

It is therefore desirable to provide an improved method for mass spectrometry and an improved mass spectrometer.

The present invention provides a method for determining isotope ratios of ions eluting from a chromatograph, which comprises the following steps:

Obtaining a mass chromatogram of a first isotope (major isotope), the mass chromatogram having a first mass chromatogram peak with a first intensity (A) and a first background level (B),

Obtaining a mass chromatogram of a second isotope (minor isotope), the mass chromatogram having a second mass chromatogram peak with a second intensity (a) and a second background level (b),

Determining the ratio R _a between the height (a + b) of the second mass chromatogram peak plus a shift value (bx) and the height (A + B) of the first mass chromatogram peak,

Determining the ratio Rß between the second background level (b) plus the shift value (bx) and the first background level (B),

Determining a specific displacement value (bx) which results in the ratio R _a being substantially equal to

the ratio Rß, and

Determining the ratio between the abundance of the second isotope (minor isotope) and the abundance of the first isotope (major isotope) using either the ratio R _a or the ratio Rp if the shift value (bx) is such that the ratio R _a is substantially equal to the ratio Rp.

The present invention provides a method for determining isotope ratios of ions eluting from a chromatograph, which comprises the following steps:

Obtaining a mass chromatogram of a first isotope (major isotope), the mass chromatogram having a first mass chromatogram peak with a first intensity (A) and a first background level (B),

Obtaining a mass chromatogram of a second isotope (minor isotope), the mass chromatogram having a second mass chromatogram peak with a second intensity (a) and a second background level (b),

Determining the ratio R _a between the height (a + b) of the second mass chromatogram peak and the height (A + B) of the first mass chromatogram peak plus a shift value (bx),

Determining the ratio Rp between the second background level (b) and the first background level (B) with a shift value (bx),

Determining a specific shift value (bx) which results in the ratio R _a being substantially equal to the ratio Rß, and

Determining the ratio between the abundance of the second isotope (minor isotope) and the abundance of the first isotope (major isotope) using either the ratio R _a or the ratio R p if the shift value (bx) is such that the ratio R _a is substantially equal to the ratio R ß.

The first and second isotopes are preferably the combustion or pyrolysis products of a compound. For example, carbon containing ¹³ C and ¹² C can be burned to form carbon dioxide, which is then ionized so that the major isotope, which has the most mass,

12 ■+■

being analyzed is CO2 with a mass-to-charge ratio of 44 and the minor isotope being mass-analyzed is CC>2 ⁺ with a mass-to-charge ratio of 45.

The first and/or second mass chromatogram peaks may not actually appear as a single sharp peak. Instead, the peaks may appear somewhat noisy and there may be a number of so-called "subpeaks" instead of a single sharp peak. The heights of the first and/or second mass chromatogram peaks can be determined by averaging the subpeaks.

The chromatograph may comprise a gas chromatograph,

which is preferably connected to an ion source such as an electron impact ion source ("EI ion source") via a combustion or pyrolysis chamber or stage. Alternatively, the chromatograph may comprise a liquid chromatograph which is preferably connected to an ion source such as an electron impact ion source ("EI ion source") via a combustion or pyrolysis chamber or stage and a preceding or upstream solvent removal stage.

The present invention provides a method for mass spectrometry in which the sample isotope ratio between a second isotope (minor isotope) and a first isotope (major isotope) is determined by the following steps:

Adding a shift value to the data relating to the second isotope (minor isotope) or to the data relating to the first isotope (major isotope),

Determining a background isotope ratio and

Change the shift value until the average sample isotope ratio summed over the time period during which the first and second isotopes are eluting is substantially equal to the background isotope ratio.

According to another aspect of the present invention there is provided a mass spectrometer comprising:

an ion source connected to a chromatography source

is coupled,

a magnetic sector mass analyzer,

a first detector for detecting ions having a first mass-to-charge ratio and a second different detector for detecting ions having a second different mass-to-charge ratio and

a data processing facility that:

(a) obtaining a mass chromatogram of a first isotope (major isotope), the mass chromatogram having a first mass chromatogram peak with a first intensity (A) and a first background level (B),

(b) obtaining a mass chromatogram of a second isotope (minor isotope), the mass chromatogram having a second mass chromatogram peak with a second intensity (a) and a second background level (b),

(c) the ratio R _a between the height (a + b) of the second mass chromatogram peak plus a shift value (bx) and the height (A + B) of the first mass chromatogram peak is determined,

(d) the ratio Rß of the second background level (b) plus the shift value (bx) and the first background level (B) is determined,

(e) determines a specific displacement value (bx) which results in the ratio R _a substantially corresponding to the

· t

~&eegr; · I · «· I I It I

Ratio Rp equals, and

(f) the ratio between the abundance of the second isotope (minor isotope) and the abundance of the first isotope (major isotope) is determined using either the ratio R _a or the ratio Rß, if the shift value (bx) is such that the ratio R _a is substantially equal to the ratio R p.

According to another aspect of the present invention there is provided a mass spectrometer comprising:

an ion source coupled to a chromatography source,

a magnetic sector mass analyzer,

a first detector for detecting ions having a first mass-to-charge ratio and a second different detector for detecting ions having a second different mass-to-charge ratio and

a data processing facility that:

(a) obtaining a mass chromatogram of a first isotope (major isotope), the mass chromatogram having a first mass chromatogram peak with a first intensity (A) and a first background level (B),

(b) obtaining a mass chromatogram of a second isotope (minor isotope), the mass chromatogram having a second mass chromatogram peak with a second intensity (a)

and a second background level (b),

(c) the ratio R _a between the height (a + b) of the second mass chromatogram peak and the height (A + B) of the first mass chromatogram peak plus a shift value (bx) is determined,

(d) the ratio Rp between the second background level (b) and the first background level (B) is determined with a shift value (bx),

(e) determining a specific displacement value (bx) which results in the ratio R _a being substantially equal to the ratio Rp, and

(f) the ratio between the abundance of the second isotope (minor isotope) and the abundance of the first

isotope (major isotope) is determined either by the ratio R _a or the ratio Rp if the shift value (bx) is such that the ratio R _a is substantially equal to the ratio Rp.

The problem addressed by the preferred embodiment is the determination of accurate isotope ratios from gas or liquid chromatograph peaks in the presence of an uncertain and potentially changing background and in the absence of any knowledge of the exact location of the start and end points of an observed mass chromatogram peak.

According to the preferred embodiment, the experimental data are manipulated in a manner such that the beginning and end of a mass chromatogram peak do not have a strong effect on the isotope enrichment calculation. A shift value is added to the minor isotope (or alternatively the major isotope) data such that the determined background isotope ratio between the minor isotope and the major isotope equals the determined sample isotope ratio between the minor isotope and the major isotope.

The sample isotope ratio may be determined initially from the raw data so that the zero value is ignored. If the background is low, the sample isotope ratio will appear noisy. A value may be added to both the major and minor isotope data to reduce the effects of noise. However, this is not essential to the present invention.

The data are preferentially analyzed and mass chromatogram peaks are identified in the major isotope track. Non-peak points on the major isotope track and the minor isotope track are selected and the ratio between the minor isotope background and the major isotope background is calculated from the data. Background isotope ratio values are interpolated so that a background isotope ratio can be assigned to each of the sample isotope peaks measured. The start and end point of each mass chromatogram peak is determined in the first data pass and the total peak area of the major and minor isotopes and the

Sample isotope ratio determined.

The background isotope ratio and the sample isotope ratio are therefore calculated using non-zero corrected values. By adding a shift value to the minor (or major) isotope data, the isotope ratio can be changed until the background isotope ratio is substantially equal to the sample isotope ratio. This then allows the desired sample isotope ratio to be determined accordingly.

The preferred method is not particularly sensitive to the beginning and end of mass chromatogram peaks because there is little difference in isotope ratio at the flanks of a mass chromatogram peak and the flanks do not contribute significantly to the determination of the sample isotope ratio.

Various embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

Figure 1 shows a simplified mass chromatogram peak of the minor isotope and a simplified mass chromatogram peak of the major isotope,

Figure 2 shows a portion of a mass chromatogram of a major isotope formed by burning a substance eluted from a GC mass chromatograph and the sample isotope and background ratio between the minor isotope and the major isotope, with a first shift value added to the minor isotope data,

Figure 3 shows a portion of a mass chromatogram of a major isotope formed by burning a substance eluted from a GC mass chromatograph and the sample isotope and background ratio between the minor isotope and the major isotope, with a second shift value added to the minor isotope data,

Figure 4 shows a portion of a mass chromatogram of a major isotope formed by burning a substance eluted from a GC mass chromatograph and the sample isotope and background ratio between the minor isotope and the major isotope, with a third shift value added to the minor isotope data,

Figure 5A shows a mass chromatogram of a major isotope and Figure 5B shows a corresponding mass chromatogram of a minor isotope and

Figure 6 shows a graph of compensating displacement value as a function of ratio difference for tip 4 shown in Figures 5A and 5B.

A preferred embodiment of the present invention will now be described with reference to Figure 1. As can be seen from this figure, the intensity of the mass chromatogram peak corresponding to a major isotope (i.e., the abundance of the major isotope) can be considered as "A" and the corresponding background intensity with an arbitrarily defined baseline can be considered as "B". Similarly, the intensity of the mass chromatogram peak corresponding to a minor isotope (i.e., the abundance of the minor isotope) can be considered as "a",

&iacgr; ·

and the corresponding background intensity with an arbitrarily specified baseline can be considered as "b". From these experimental data, the values of B, A + B, b and a + b can be determined, but it is not trivial to determine a and A precisely because it is difficult to determine exactly where the mass chromatogram peaks begin and end. Because the background continues to change with time, the background at the time the major or minor isotope elutes is not precisely known.

Two ratios can be calculated directly from the experimental data. First, the ratio of the peak heights (or areas) Rq can be determined:

a + b

_R0 =

^V A + B

Secondly, the ratio of the backgrounds Rb can be determined:

However, the ratio to be determined is the ratio R of the isotopes above their corresponding backgrounds:

According to the preferred embodiment, the values of the background b of the mass chromatogram peak of the secondary

isotope a compensating shift value bx is added so that the background of the mass chromatogram peak of the minor isotope then becomes b + bx and the height of the mass chromatogram peak of the minor isotope becomes a + b + bx.

The new ratio R _a of the peak heights ("sample isotope ratio") is given by

a + b + bx

^R a =

A+B

given.

Similarly, the new ratio Rß of the background heights ("background isotope ratio") is given by

b + bx

^R ß = —&tgr;—

given.

According to the preferred embodiment, the value of bx is then adjusted until

R _a = R _ß = R*

applies. Accordingly:

b + bx = R*. B

^: &Sgr; * &idigr;&Sgr;&idigr;

a + £> + jbx = #*. A + R*. B

By inserting this we get:
a + (b + bx) = a + (r*. b) = R*. A+R*. b
and thus:

a = R*. A

Therefore, we have:

R* =1 = R

Therefore, the required isotope ratio R is equal to R _a or Rp if R _a = Rp.

Figures 2-4 show typical experimental data and illustrate the effect of adding various shift parameters bx to the background of the minor isotope. A portion of the mass chromatogram of the major isotope is shown in Figure 2. A first shift value bx was added to the background of the minor isotope. The mass chromatogram peak of the minor isotope (not shown) is similar to that of the major isotope shown in Figure 2.

Figure 2 also shows the ratio between the ions of the minor isotope (with a mass-to-charge ratio of 45) and the ions of the major isotope (with a mass-to-charge ratio of 44) as a function of time, where

a first shift value is added to the data of the minor isotope.

The mass chromatogram peak of the major isotope shows how the background increases progressively over time in this particular case. As shown, the background isotope ratio and the sample isotope ratio increase in the same way as the major isotope trace. There may be cases where the background decreases over time.

A second different shift value bx was then added to the minor isotope data and the resulting curves are shown in Figure 3. It can be seen that by changing the shift value bx added to the minor isotope data, both the sample isotope ratio and the background isotope ratio are changed. It can be seen in this case that the peaks of the sample isotope ratio with respect to the background isotope ratio are negative for the second shift value, whereas the peaks of the sample isotope ratio with respect to the background isotope ratio are positive for the first shift value used in the example shown in Figure 2. The slope of the background isotope ratio has also changed from positive in Figure 2 to negative in Figure 3.

According to the preferred embodiment, a shift value can be chosen so that the background isotope ratio is equal to the sample isotope ratio. This is shown in Figure 4. The background isotope ratio shown in Figure 8 is about -8, and the

The average sample isotope ratio summed over the period during which the first and second isotopes elute is also essentially -8.

For very precise measurement of isotope ratios, where areas under the mass chromatogram peaks of the major and minor isotopes are measured, the start and end points normally chosen can have a significant effect on the determined sample isotope ratio, even in cases where single pure mass chromatogram peaks are measured. In the examples shown in Figures 2-4, it can be seen that the start and end points of the mass chromatograph peaks are obscured by the background. However, in the preferred embodiment, the values on the flanks of each peak are placed close to the ratio of the peak itself, and therefore the start and end points are not critical.

In the examples shown in Figures 2-4, the minor isotope with a mass-to-charge ratio of 45 and the major isotope with a mass-to-charge ratio of 44 do not elute together. This is evident from Figure 4, where the sample isotope ratio looks like a derivative curve. The minor isotope with a mass-to-charge ratio of 45 elutes a few milliseconds before the major isotope with a mass-to-charge ratio of 44, and therefore the sample isotope ratio of the minor isotope with respect to the major isotope shown in Figure 4 shows an increase at the beginning of the peak of the sample isotope ratio.

The mass chromatogram for the main isotope cannot

_ 17 -..

can be easily aligned with the mass chromatogram for the minor isotope. However, because the background isotope ratio has been set equal to the sample isotope ratio in the preferred embodiment, the effect of the flanks of the peaks is very small.

Some typical experimental data are shown in Figures 5A and 5B, which show four mass chromatogram peaks of the major isotope and the minor isotope, respectively, from four separate experimental runs. Mass chromatogram peak 4, corresponding to the fourth experimental run, was analyzed. The data shown in Figure 5A relating to the intensity of the major isotope were analyzed using conventional peak detection algorithms. The prepeak background onset time was determined to be 22117, and the prepeak background end time was determined to be 22317. The peak onset time was 22359, the peak peak time was 22397, and the peak end time was 22443. The postpeak background onset time was determined to be 22485, and the postpeak background end time was determined to be 22685.

The following table shows how the various ratios calculated according to the preferred embodiment change as a function of a compensating displacement value bx.

-600 CO CO CO CO N 0.001466 -400 -H ■&EEgr; I-H -H G 0.001190 -200 G C -P G G &phgr; 0.000914 U 0 -P -P U -P -P M 0.000638 &phgr; 200 ■&EEgr; φ M rH Φ 0.000362 400 I :(&Tgr;3 :&iacgr;&Tgr;5 4-t ^^ 0.000086 CO 600 G&Lgr; C &ggr;"
(]) ^~* -P ,G &khgr;; &EEacgr; ca -0.000190 Φ M co M ■&EEgr; CC NCD '— Xi Φ; £S? TJ -P > OQ ■y > PQ &ugr; > "2, G
Φ COI
■&EEgr; Φ β -H TJ \
Qj G -Q "2, TJ \ :&Ogr; TJ &igr;&Ugr;[ N 5P? -H CO 3 —
MM &igr; d
G M ■&Rgr; r—) &ugr; O t7> φtn -H :ro co N M p _t .G M Φ ^ φ -P Φ cn Φ ■&Rgr; -H -P Φ G α G -H -H tn -&eegr; 0.010410 PC 0.010677 0.008909 0.008981 0.008944 0.010944 0.009459 0.009517 0.009487 0.011211 0.010008 0.010053 0.010030 0.011478 0.010558 0.010588 0.010573 0.011745 0.011107 0.011124 0.011116 0.012012 0.011657 0.011660 0.011658 0.012206 0.012196 0.012201

Figure 6 shows a graph showing how the compensating shift value bx changes with the ratio difference (R _a - Rp). The value of the compensating shift value bx required to achieve a zero ratio difference was calculated to be 462.634055. At this compensating shift value bx, the pre-peak background ratio was 0.011829 and the post-peak background ratio was 0.011828. The minor isotope/major isotope peak to peak background ratio was 0.011828, the peak ratio was 0.011828, and the ratio difference was 0.000000. Accordingly, the required sample isotope ratio equal to R _a or Rp is 0.011828. Without a compensating shift value, the sample isotope ratio R _a would have to be determined as 0.011211, which would result in a difference of 0.0006 or approximately

• t ··

6%. This shows that a significant improvement in accuracy can be achieved according to the preferred embodiment.

Although the preferred embodiment requires a reasonable estimate of the peak start points and the peak end points, the peak start points and the peak end points do not need to be determined with the same accuracy as in the conventional ratio determination methods. Accordingly, conventional peak detection algorithms (usually based on the first and second derivatives) are entirely adequate.

Similarly, the location of the background regions before and after the peak is not critical. Suitable criteria are that 200 data points are used for each background region. The end of the pre-peak background region should preferably be approximately half a peak width before the peak start position, and the beginning of the post-peak background region should preferably be approximately half the peak width after the peak end position. The location of the background regions can be adjusted algorithmically in the event that other peaks are detected within the region.

The value of the prepeak background ratio can be defined as follows:

where i is the beginning of the prepeak background, j is the end of the prepeak background, bi is the intensity value of the minor isotope track, Bi is the intensity value of the major isotope track, and &khgr; is the postulated compensating shift value.

Similarly, the value of the post-peak background ratio can be defined as:

where k is the beginning of the postpeak background, 1 is the end of the postpeak background, b2 is the intensity value of the minor isotope trace, B ₂ is the intensity value of the major isotope trace and &khgr; is the postulated compensating shift value.

The value of the peak-to-peak background ratio is preferably calculated by interpolation between the pre-peak and post-peak background ratios.

The value of the chromatograph mass peak ratio can be defined as follows:

&Sgr;*

* * t9

where m is the beginning of the peak of interest, η is the end of the peak of interest, a is the intensity value of the minor isotope trace, A is the intensity value of the major isotope trace, and &khgr; is the postulated compensating shift value.

The range of values used for the compensating shift value can be arbitrary initially, but can later be adjusted algorithmically to ensure bracketing of the value resulting in a ratio difference of zero.

Other minor improvements can be made to the data. The mass chromatogram peak of the minor isotope can be moved so that it is directly below the mass chromatogram peak of the major isotope. The peak shape can also be mathematically improved to improve the measurement of mass chromatogram peaks that are adjacent or that overlap.

While the present invention has been described with reference to preferred embodiments, it will be understood by those skilled in the art that various changes in form and details may be made without departing from the scope of the invention as set forth in the appended claims.

Claims (7)

1. Mass spectrometer which has:
a ton source coupled to a chromatography source,
a magnetic sector mass analyzer,
a first detector for detecting ions having a first mass-to-charge ratio and a second different detector for detecting ions having a second different mass-to-charge ratio and
a data processing facility that: a) obtaining a mass chromatogram of a first isotope (major isotope), the mass chromatogram having a first mass chromatogram peak with a first intensity (A) and a first background level (B), b) obtaining a mass chromatogram of a second isotope (minor isotope), the mass chromatogram having a second mass chromatogram peak with a second intensity (a) and a second background level (b), c) the ratio R _α between the height (a + b) of the second mass chromatogram peak plus a shift value (bx) and the height (A + B) of the first mass chromatogram peak is determined, d) the ratio R _β of the second background level (b) plus the shift value (bx) and the first background level (B) is determined, e) determining a specific displacement value (bx) which results in the ratio R _α being substantially equal to the ratio R _β , and f) the ratio between the abundance of the second isotope (minor isotope) and the abundance of the first isotope (major isotope) is determined using either the ratio R _α or the ratio R _β if the shift value (bx) is such that the ratio R _α is substantially equal to the ratio R _β . 2. Mass spectrometer, which has:
a ton source coupled to a chromatography source,
a magnetic sector mass analyzer,
a first detector for detecting ions having a first mass-to-charge ratio and a second different detector for detecting ions having a second different mass-to-charge ratio and
a data processing facility that: a) obtaining a mass chromatogram of a first isotope (major isotope), the mass chromatogram having a first mass chromatogram peak with a first intensity (A) and a first background level (B), b) obtaining a mass chromatogram of a second isotope (minor isotope), the mass chromatogram having a second mass chromatogram peak with a second intensity (a) and a second background level (b), c) the ratio R _α between the height (a + b) of the second mass chromatogram peak and the height (A + B) of the first mass chromatogram peak plus a shift value (bx) is determined, d) the ratio R _β between the second background level (b) and the first background level (B) is determined with a shift value (bx), e) determining a specific displacement value (bx) which results in the ratio R _α being substantially equal to the ratio R _β , and f) the ratio between the abundance of the second isotope (minor isotope) and the abundance of the first isotope (major isotope) is determined using either the ratio R _α or the ratio R _β if the shift value (bx) is such that the ratio R _α is substantially equal to the ratio R _β . 3. A mass spectrometer according to claim 1 or 2, wherein the chromatograph comprises a gas chromatograph. 4. A mass spectrometer according to claim 3, wherein the gas chromatograph is connected to an ion source via a combustion or pyrolysis stage. 5. A mass spectrometer according to any one of claims 1 or 2, wherein the chromatograph comprises a liquid chromatograph. 6. A mass spectrometer according to claim 5, wherein the liquid chromatograph is connected to a ion source via a solvent removal stage and a combustion or pyrolysis stage. 7. A mass spectrometer according to claim 4 or 6, wherein the ion source comprises an electron impact ion source ("EI ion source").