Advantage of HRAM

1
The world leader in serving science
Manoj Kushwaha
Utilizing Very High Resolution
Fine Isotopic Data To Refine
Elemental Composition Determination

2
Accurate Mass Instruments - Orbitrap
• What they are good at?
• Accurate mass qualitative work – when used as the detector for an ion
trap, they can perform accurate mass MSn.
• Quantitation – Scan-to-scan mass accuracy is typically very good with
a deviation across a peak of 1-3 ppm allowing for narrow selection.
• Mass accuracy stability – Typical performance with once a day
external calibration is <3 ppm.
• Resolution – Can provide very high resolution (up to 480,000 or more)

3
High Resolution MS
What is the information High resolution Mass spectrometer provides
Mass accuracy (ppm) – how accurate is the mass spectrometer at predicting
the mass of an analyte relative to it’s theoretical mass.
Example:
Theoretical mass = 222.1125
Predicted by triple quad mass spectrometer = 222.1
Predicted by Q Exactive = 222.1129
Resolution (R) – how capable is mass spectrometer at identifying two
masses from each other.
Example:
Mass 1 = 222.1125
Mass 2 = 222.1237

4
Using High Resolution: Counting Atoms
• Splitting fine isotopes allows for:
• Confirmation of elemental composition
• Confirmation of a component
• What about isotope ratios?
• Can we split elements and count the atoms?

5
Real World Example – Melamine in Albumin
• Melamine analysis in pharmaceutical excipients was a hot
topic following contamination events in China
NN
N N 2H
N 2H
N2H
NN
N OH
OH
OH
NN
N OH
N 2H
OH
NN
N OH
N 2H
N2H
N=6
Melamine
N=5
Ammeline
N=4
Ammelide
N=3
Cyanuric Acid

6
Melamine Injection – Real Data
RT: 0.00 - 8.36
0 5
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
2.57
3.45
Melamine_FDA_500ul_02 #271 RT: 2.56913 AV: 1 NL: 1.41E7
F: FTMS + p ESI Full ms [85.00-500.00]
126.4 126.6 126.8 127.0 127.2 127.4 127.6 127.8 128.0 128.2 128.4 128.6 128.8
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
127.07287
C3 H7 N6
128.07614
C2
13C H7 N6
Melamine_FDA_500ul_02 #271 RT: 2.56913 AV: 1 NL: 4.69E5
F: FTMS + p ESI Full ms [85.00-500.00]
128.05 128.10
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
128.07614
C2
13C H7 N6
128.07001
C3 H7 N5
15N 128.08173
C4 H9 N4
15N
Scan at Peak Apex
Clear split of 15N and 13C in the A1 isotope
peak of Melamine.
Does the isotopic ratio match?
Can we count the number of N in a structure?
A1

7
Using High Resolution: Counting Nitrogen
• To count nitrogen we need to measure the isotope ratio of
the 15N signal in A1 [A1,N] and the 13C signal in A1 [A1,C]
• As the number of nitrogen atoms (#N) increases the value of
the ratio [A1,N] / [A1,C] increases
• If we know #C, then we can calculate #N
• We can get a reasonable estimate of #C from:
Relative Abundance 13C
A1,C
A0,C
/ = # C (3 for our case)
Note: This formula provides an estimate only, however it breaks down for as MW increases
(>400 MW) and molecules where carbon makes up less than ~65% of the non-hydrogen atoms.

8
• Estimating the value of #N can be performed by recognizing
that the ratio of 15N to 13C in A1 is reflected by:
• Which gives an equation for #N:
• Our observed 15N/13C ratio calculated a value of 6 nitrogen.
A1,N
A1,C
=
#N X Relative Abundance 15N
#C X Relative Abundance 13C
#N =
A1,N
A1,C Relative Abundance 15N
X

9
Using High Resolution: Counting Carbon
Melamine
= 3.3/100* 100/1.08
= 3.05
Relative Abundance 13C
A1,C
A0,C
/ = # C (3 for our case)

10
Melamine
#N =
A1,N
A1,C Relative Abundance 15N
X
= 2.3/3.3*3*1.08/0.369
= 6.1

11
F: FTMS + p ESI Full lock ms [140.00-2000.00]
690 700 710 720 730 740 750 760 770 780 790 800 810 820 830 840
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
792.5536
776.5591
748.5280
752.5594
768.5543 780.5906
764.5229
796.5852
718.5383702.5433 814.5358728.5592 744.5544
692.5229 835.6172706.5406
C 45
H 79
O 8
N P
-0.21 ppm
HRAM provides Elemental Composition
Using
Spectral Distance

12
Table of Relative Abundance for Common Elements
Name Mass % Abundance
Rel.
Abundance
Δ Most
abundant Name Mass % Abundance
Rel.
Abundance
Δ Most
abundant
1H 1.007825 99.9558 100.0% - 32S 31.972071 94.93 100.0% -
2H 2.014102 0.115 0.12% 1.006277 33S 32.971458 0.76 0.80% 0.999387
3H 3.016049 - 2.008224 34S 33.967867 4.29 4.52% 1.995796
12C 12 98.93 100.0% - 36S 35.967081 0.02 0.02% 3.995010
13C 13.003355 1.07 1.08% 1.003355 35Cl 34.968853 75.78 100.0% -
14C 14.003242 - 2.003242 37Cl 36.965903 24.22 31.96% 1.997050
14N 14.003074 99.632 100.0% - 79Br 78.918338 50.69 100.0% -
15N 15.000109 0.368 0.37% 0.997035 81Br 80.916291 49.31 97.28% 1.997953
16O 15.994915 99.757 100.0% -
17O 16.999132 0.038 0.04% 1.004217
18O 17.999160 0.205 0.21% 2.004245
• A good reference can be found at:
http://www.chem.ualberta.ca/~massspec/atomic_mass_abund.pdf

13
Isotopic Spectral Match – Theoretical vs Experimental
True Identification with High Resolution only!

14
Exactive – Isotope Fidelity
• Early in the peak – Low signal
(1.6e5)
C14H11Cl2NO2 +H: C14 H12 Cl2 N1O2 p(gss, s/p:40) Ch...
296.0 296.5 297.0 297.5 298.0 298.5 299.0 299.5 300.0
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
296.02
298.02
297.03
300.02299.02
RT: 3.40 - 4.01
3.4 3.5 3.6 3.7 3.8 3.9 4.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
3.72
Theoretical
C14H12Cl2N1O2
296 297 298 299 300
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
296.0242
R=23300
298.0212
R=23600
296.2200
R=22200 297.0274
R=21700 300.0181
R=22300
299.0247
R=20600297.2228
R=19700
65%
16%
10%10%
+10%
-1%
+/-0%+/-0%
Observed

15
• Center of the peak – Signal
(9.8e5)
C14H11Cl2NO2 +H: C14 H12 Cl2 N1O2 p(gss,s/p:40) Ch...
296.0 296.5 297.0 297.5 298.0 298.5 299.0 299.5 300.0
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
296.02
298.02
297.03
300.02299.02
RT: 3.40 - 4.01
3.4 3.5 3.6 3.7 3.8 3.9 4.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
3.72
296 297 298 299 300
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
R=23500
298.0211
R=23800
297.0276
R=23200 299.0247
R=23600
300.0182
R=23500296.2202
R=19800
297.2407
R=18500
Theoretical
C14H12Cl2N1O2
65%
16%
10%10%
+1%
-3%
+/-0%+/-0%
Observed

16
• Tail of the Peak – 5.3e4
C14H11Cl2NO2 +H: C14 H12 Cl2 N1O2 p(gss, s/p:40) Ch...
296.0 296.5 297.0 297.5 298.0 298.5 299.0 299.5 300.0
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
296.02
298.02
297.03
300.02299.02
RT: 3.40 - 4.01
3.4 3.5 3.6 3.7 3.8 3.9 4.0
Time (min)
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
3.72
Theoretical
C14H12Cl2N1O2
296 297 298 299 300
m/z
0
10
20
30
40
50
60
70
80
90
100
RelativeAbundance
R=22800
298.0214
R=22500
299.1621
R=20300
296.2196
R=20000
297.0272
R=16100
299.0255
R=18400
300.0186
R=18000
65%
16%
10%10%
-8%
-3%
+/-0%+/-0%
Observed

17
Calculating Elemental Composition from A0
• For any given accurate mass value, only a finite number of
elemental compositions can be predicted.
• If all elements on the periodic chart and any number of each
were allowed, the possible compositions would be a function
of the observed m/z however it would be a very large
number.
• If we constrain the elements and their allowed ranges we can
begin to determine a reasonable list of possible elemental
compositions.

18
• Example (If you recognize the m/z don’t shout it out…)
385 386 387 388 389 390
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
386.2553
387.2522
388.2533
It’s a small pharma molecule so we limit
our elements and set reasonable max
values
Element Max
C 25
H 40
O 6
N 6
S 2
Cl 1
Br 1
F 3

19
• Multiple formulas calculated from A0
385 386 387 388 389 390
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
386.2553
387.2522
388.2533
We also apply a limit of 5 ppm tolerance
for any formula we calculate.
Formula
C21 H32 O2 N5
C15 H38 O5 N4 S
C21 H40 O N S2
C16 H35 N5 F3 S
C16 H39 O N4 F S2
C21 H36 N2 F2 S
C18 H33 O3 N5 F
C23 H34 O3 N2
C21 H33 O N2 F3
C20 H36 O6 N
C15 H35 O6 N4 F
C18 H38 O3 N F2 S
A0
A1
A2

20
• Consider the actual mass of natural isotopes.
385 386 387 388 389 390
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
386.2553
387.2522
388.2533
∆15N 13C = 0.00632
387.24 387.26
m/z
0
20
40
60
80
100
RelativeAbundance
387.2522
387.2466
N
Requires Resolution
>50,000

21
• Consider the actual mass of natural isotopes.
385 386 387 388 389 390
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
386.2553
387.2522
388.2533
∆34S and 2X 13C = 0.01914
389.25 389.30
m/z
0
20
40
60
80
100RelativeAbundance
389.2587
2 13C
No
S

22
• How do we further refine our results?
385 386 387 388 389 390
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
386.2553
387.2522
388.2533
Formula
C21 H32 O2 N5
C15 H38 O5 N4 S
C21 H40 O N S2
C16 H35 N5 F3 S
C16 H39 O N4 F S2
C21 H36 N2 F2 S
C18 H33 O3 N5 F
C23 H34 O3 N2
C21 H33 O N2 F3
C20 H36 O6 N
C15 H35 O6 N4 F
C18 H38 O3 N F2 S

23
• How do we further refine our results?
385 386 387 388 389 390
m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
RelativeAbundance
386.2553
387.2522
388.2533
Formula
C21 H32 O2 N5
C18 H33 O3 N5 F
C23 H34 O3 N2
C21 H33 O N2 F3
C20 H36 O6 N
C15 H35 O6 N4 F
We can further limit the elements (especially
fluorine).
As m/z is odd by nitrogen rule
C23 H34 O3 N2 ruled out
Formula
C21 H32 O2 N5
C23 H34 O3 N2
C20 H36 O6 N

24
If Sulfur is present -Elemental Composition with
Isotope Pattern
• Example – The applied “spectral fit” for a small sulfur
containing molecule require very high resolution.
34S
2 13C
Resolution >50000

25
CoA at - 60,000 Resolution m/z 768
34S and 13C2 not resolved

26
CoA at Different Resolutions, m/z 770
60 K @ m/z 400 100 K @ m/z 400 240 K @ m/z 400

27
311.0 311.5 312.0 312.5 313.0 313.5
m/z
311.1689
312.1715
313.1641
Why Use Ultra High resolution
313.14 313.16 313.18 313.20
m/z
313.1641
313.1741
34S
13C2
Calculated
35,000 Resolution
C17H27O3S
313.10 313.15 313.20 313.25
m/z
313.1669

28
287 288
m/z
0
100
RelativeAbundance
Using Very High Resolution Accurate Mass
HRAMVHRAM
• Accurate mass and
fragmentation are not the
only tools available to us
• Accurate mass gives us
access to elemental
composition
• Very high resolutions bring
even more power to our
ability to determine correct
elemental composition
287.20287.20 287.22287.22
00
100100
RelativeAbundanceRelativeAbundance
287.2033287.2033
287.1970287.1970
287.2063287.2063
287.20 287.22
0
100
RelativeAbundance
287.2033
287.1970
287.2063
C11H24O2N7
C15H28O4N
or
286.1999
287.2028
288.2051
287.2033
286.1999
288.2067
15N
13C

29
Full Scan Data
Isotopic Pattern + VHRAM = Powerful
C10H12O3N3S (0.05 ppm)
254.0 254.5 255.0 255.5 256.0
m/z
0
100
RelativeAbundance
254.0594
255.0627
R=249602
256.0551
R=244902
255.2318
R=200000
254.9491
R=172100
C13H15FS2 (0.11 ppm)
or
255.06 255.07m/z
0
100
RelativeAbundance
1.0063
Theoretical
R=254207
1.0034
0.9970
0.9994
13C
15N 33S
255.055 255.060 255.065
0
100
RelativeAbundance
R=249602
R=248000
R=249300
1.0033
0.9970
0.9993

30
2 AMU
Isolation Offset and Isotopic Pattern
“Normal”isolationissymmetrical“Normal”isolationissymmetrical——“Upanddown”“Upanddown”
Realdataisn’tRealdataisn’t——PatternsusuallyonlygoupfromAPatternsusuallyonlygoupfromA00
m/z
254.0593
253.0107
255.0626
256.0551251.1852 252.2169 257.9676253.2161
251 252 253 254 255 256 257 258
m/z
0
100
RelativeAbundance
250

31
TogettheisotopicpatterninaSIMorafragmentscanaverywideTogettheisotopicpatterninaSIMorafragmentscanaverywide
isolationisrequired.isolationisrequired.
6 AMU
m/z
254.0593
253.0107
255.0626
256.0551251.1852 252.2169 257.9676253.2161
Nothing “good” is here…
We’ve contaminated
our fragmentation!
251 252 253 254 255 256 257 258
m/z
0
100
RelativeAbundance
250

32
251 252 253 254 255 256 257 258
m/z
0
100
RelativeAbundance
250
4 AMU2 AMU
IsolationOffsetontheOrbitrap massspectrometer:Shiftthecenterofisolation,focusIsolationOffsetontheOrbitrap massspectrometer:Shiftthecenterofisolation,focus
onwhatmatters.onwhatmatters.
m/z
254.0593
253.0107
255.0626
256.0551251.1852 252.2169 257.9676253.2161
Offset = 1

33
Isotopic Pattern + VHRAM + Isolation Offset = Unique
No isotope pattern.
Isolation too narrow.
VHRAMHCDMS2
Isolation2
156.0 156.5 157.0 157.5 158.0 158.5
m/z
0
100
RelativeAbundance
156.0123
R=80300S
NH
N O
N2H
O
O
60 80 100 120 140 160 180 200 220 240 260
m/z
0
100
RelativeAbundance
156.0123
R=80300
108.0451
R=9540092.0501
R=103005
99.0559
R=98300
160.0878
R=78200147.0800
R=79200
173.5372
R=70300
188.0829
R=64000
110.0607
R=87000
68.0500
R=114800
254.0606
R=64700

34 COMPANY CONFIDENTIAL
Refining Prediction- Isotopic Pattern + VHRAM + Isolation Offset
Purpose:
To determine the capability of fine isotopic information to refine the minimum and
maximum values of elements in prediction of the elemental composition from very
high resolution data.
Methods:
Mass spectral information was acquired at very high resolutions (>240,000 FWHM
@ m/z 200) for both full scan and fragmentation (MS2) for known components.
The elemental composition was calculated for the full scan observed isotopic pattern
using a two different elemental composition sets, a limited “pre-known” set and a
more relevant “open” set.
Sample Preparation:
Standard samples of 5 compounds were prepared in a solution of 50:50 Water:
Acetonitrile with 0.1% formic acid.

Mass Spectrometry:
The analysis was performed on an Orbitrap Fusion Tribrid™ mass spectrometer in
positive mode both Full MS and MS2. Fragmentation data was acquired using HCD
at 25% for Tryptophan, 35% for Ranitidine, 40% for Oxytetracycline, and 60% for
Guanine and Norfloxacin.
The fragmentation was acquired using an isolation offset method. The isolation
width was set to 4 AMU with an offset of 1 AMU for a net isolation of -1 to +3 AMU.
This allowed for fragmentation scans to have isotopes up to A2.

The Elemental Composition Subsets – “Pre-Known” and “Open”
TABLE 1. The Elements and Ranges used for Composition Determination.
Element
Pre- Known Set Open Set
Min Max Min Max
C p/2 p+6 1 60
H p/2 p+6 2 180
O p-2 or 0 p+3 0 20
N p-2 or 0 p+2 0 15
S p-2 or 0 if p>0 then p+1, else 0 0 4
P p-2 or 0 if p>0 then p+1, else 0 0 3
F 0 p 0 3
p = the number of atoms present in the known parent structure. For the “Pre-known” list, the
minimum and maximum values were set as an expansion of the known “right” answer. For the
minimum values, 0 was used when the formula provided a minimum value < 0.

The Elemental Composition Subsets – “Pre-Known” and “Open”
TABLE 3. Elemental Composition Determination – No Fine Isotope Refinement.
Compound
Measured
Accuracy (ppm)
Total Possible Compositions
Pre-Known Open
Ranitidine -0.1 1 50
Tryptophan -0.1 4 7
Oxytetracycline 1.0 2 324
Guanine 0.8 1 6
Norfloxacin 0.5 2 61

Utilizing Fine Isotope Data to Refine Elemental Composition
Full scan data was acquired at resolution 480,000 and MS2 acquired at 240,000;
fine isotopic information available from Full scan and Fragment scan was used to
refine the elements in use.

TABLE 4. Refinement to Open Set by Direct Elemental Observation
Compound
Refinement from Fine Isotopes
Element Min Max Observation
Ranitidine
S
N
1
1
2
3
Fill scan 33S/34S and 13C/34S ratio
MS2 15N and MS 13C/15N A1 ratio
Tryptophan
N
S
1
0
3
0
MS2 15N and 13C/15N A1 ratio
Full scan lack of 34S in A2
Oxytetracycline
O
S
N
3
0
1
Unchanged
0
Unchanged
MS2 18O and 13C ratio
MS2 15N
Guanine
N
S
1
0
Unchanged
0
MS2 15N
Norfloxacin
N
O
S
1
1
0
Unchanged
Unchanged
0
MS2 15N
MS2 18O
Table 4 shows the detected fine isotope refinement possible by direct observation of
isotopes in fragmentation data for the compounds studied and the resulting improvement
on the possible elemental compositions calculated from the data.

Comparison of Performance – Using Fine Isotope Refinement
Elemental compositions were calculated using the fine isotopes observed in MS and MS2
data to refine the minimum and maximum values.
The inclusion of the additional limits significantly reduced the number of possible
compositions that were determined, for example, the total possible compositions
calculated from the HRAM full scan MS data of Oxytetracycline was reduced by almost an
order of magnitude when taking fine isotopic data into consideration.
Compound
Open Set
Pre- Known Set
Original With Fine Isotopes
Ranitidine 50 5 1
Tryptophan 7 1 4
Oxytetracycline 324 84 2
Guanine 6 1 1
Norfloxacin 61 30 2
Table 5. Comparison of the Three Methods for Elemental Composition.

Conclusion
The determination of elemental composition from accurate mass alone is insufficient
unless the elemental subset is constrained with a priori knowledge of the answer.
For real world analyses, this prior knowledge doesn’t exist and a more open
elemental composition set must be used. Here we have demonstrated that inclusion
of refinements to the minimum and maximum number of atoms for isotopic elements
by direct observation of fine isotope pattern improves our capability to determine the
composition.
• Accurate mass, even below 1 ppm, is insufficient for correct elemental composition
determination unless a priori knowledge is used.
• Very high resolution can give us access to direct observation of fine isotopes.
• Direct observation of the fine patter can refine the determination of elemental composition.
• Fine isotopic refinement of the elemental subset can be applied to real world scenarios to
improve elemental composition determination

Thank You

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