deficiencies of deuterium as an internal standard in...
TRANSCRIPT
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Deficiencies of Deuterium
as an Internal Standard in MS
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Deficiencies of Deuterium as an Internal Standard in MS Presentation
Scott Landvatter, Ph.D. (President)
Rich Tyburski (Vice President)
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About IsoSciences
• Formed in 2002 by Scott Landvatter and David Saunders
• Both were isotope chemists at SmithKline Beecham and have >35 years labeling experience
• Custom Synthesis of Labeled Standards
• Catalog of Labeled Internal Standards
• Work with NIST on Vitamin Internal Standards
• Supplier of high purity unlabeled vitamin standards to NIST
• Worked with International Vitamin D Harmonization group
• Collaborator with Diagnostic Labs to develop the next generation of internal standards (e.g. steroids)
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Which 25-OH D3 Internal Standard Do I Choose?
D3 or D6 or 13C3 or
13C5 or 13C3 (Not Yet Available)
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Which Testosterone Internal Standard Do I Choose?
C13
C13
C13
O
OH
O
OH
D
D
D D D
O
OH
D
DD
D5 or D3 or 13C3
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Selecting a Labeled Internal Standard:
What is Typical Now
• ‘Old’ Internal Standards Continued to be Used
• ‘SOP’s Complete
• Validation Complete
• Why Change?
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Selecting a Labeled Internal Standard:
What is Typical Now
• ‘Old’ Standards Continue to be Used
• ‘SOP’s complete
• Validation Complete
• Why Change?
• What Do ‘Old’ Internal Standards Look Like?
• Usually Deuterated
• Usually the Least Expensive
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Selecting a Labeled Internal Standard:
What about New Internal Standards?
• ‘New’ Standards Rapidly Being Developed
• ‘New’ Standards Represent a Shift in Approach
• Minimizing the use of Deuterium
• 13C
• 15N
• D in more stable Positions
• Combination of Labels
• Minimize chance of unlabeled material
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Selecting a Labeled Internal Standard:
What about New Internal Standards?
• ‘New’ Standards Rapidly Being Developed
• ‘New’ Standards Represent a Shift in Approach
• Minimizing the use of Deuterium
• 13C
• 15N
• D in more stable Positions
• Combination of Labels
• Minimize chance of unlabeled material
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Question:
Are There Reasons to Switch
Standards?
Answer:
In Many Cases the Answer is Yes.
But Why?
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Question:
Are There Reasons to Switch
Standards?
Answer:
In Many Cases the Answer is Yes.
But Why?
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To understand why we need
to look at the factors
effecting the synthesis
of the labeled standards.
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Factors in Isotopic Labeling:
Use of Final Compound
Ultimate Use of Labeled Compound
• MS Standard?
• Chemical Stability
• Isotope Stability
• Molecular weight enhancement required- At least M+3 is Standard
- Is Cl present? (Then M+5 Required)
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Factors in Isotopic Labeling:
Use of Final Compound
Ultimate Use of Labeled Compound
• MS Standard?
• Chemical Stability
• Isotope Stability
• Molecular weight enhancement required- M+3 is Standard
- Is Cl present? (Then M+5 Required)
• Biological study?
• Metabolic Stability Required
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Factors in Isotopic Labeling:
Use of Final Compound
Ultimate Use of Labeled Compound
• MS Standard?
• Chemical Stability
• Isotope Stability
• Molecular weight enhancement required- M+3 is Standard
- Is Cl present? (Then M+5 Required)
• Biological study?
• Metabolic Stability Required
• Human study?
• cGMP
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Factors in Isotopic Labeling:
Choice of Isotope(s)
2H (Deuterium)
Pros:
• Easy to incorporate
• Inexpensive (usually)
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Factors in Isotopic Labeling:
Choice of Isotope(s)
2H (Deuterium)
Pros:
• Easy to incorporate
• Inexpensive (usually)
Cons:
• Prone to exchange/loss of label (chemically and in MS)
• Difficult to get a clean molecular ion
• LC/MS co-elution problems: HPLC can sometimes separate deuterated from non-deuterated compound
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Factors in Isotopic Labeling:
Choice of Isotope(s)
13C (Carbon-13)
Pros:
• High isotopic purity/Clean molecular ion
• Chemically stable
• No exchange/loss of label problems in MS
• No LC/MS co-elution problems
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Factors in Isotopic Labeling:
Choice of Isotope(s)
13C (Carbon-13)
Pros:
• High isotopic purity/Clean molecular ion
• Chemically stable
• No exchange/loss of label problems in MS
• No LC/MS co-elution problems
Cons:
• Requires more elaborate syntheses
• More expensive than deuterium (usually)
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Factors in Isotopic Labeling:
Choice of Isotope(s)
15N (Nitrogen-15)
Pros:
• Useful label for compounds containing multiple nitrogens
• No LC/MS co-elution problems
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Factors in Isotopic Labeling:
Choice of Isotope(s)
15N (Nitrogen-15)
Pros:
• Useful label for compounds containing multiple nitrogens
• No LC/MS co-elution problems
Cons:
• Limited choice of expensive starting materials
• Requires total synthesis
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Factors in Isotopic Labeling:
Choice of Isotope(s)
18O (Oxygen-18)
Pros:
• Molecular weight gain of 2 amu per label
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Factors in Isotopic Labeling:
Choice of Isotope(s)
18O (Oxygen-18)
Pros:
• Molecular weight gain of 2 amu per label
Cons:
• Totally exchangeable in easily accessible functional groups (acids, esters, ketones)
• Only useful in ethers (and those are difficult and expensive to prepare)
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Factors in Isotopic Labeling:
Choice of Isotope(s)
Multiple Sources of Label (13C,2H – 13C,13C – 13C,15N)
Pros:
• Lowest possible amount of unlabeled compound
• 1 source of 13C2 at 99% 13C: unlabeled contamination (worst case) = 1%
• 2 sources of 13C at 99% 13C : unlabeled contamination (worst case) = 0.01 x 0.01 =0.0001= 0.01%
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Factors in Isotopic Labeling:
Choice of Isotope(s)
Multiple Sources of Label (13C,2H – 13C,13C – 13C,15N)
Pros:
• Lowest possible amount of unlabeled compound
• 1 source of 13C2 at 99% 13C: unlabeled contamination (worst case) = 1%
• 2 sources of 13C at 99% 13C : unlabeled contamination (worst case) = 0.01 x 0.01 =0.01%
Cons:
• Requires a total synthesis
• More expensive
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
1. Labeling by Exchange
• Only useful for deuterium
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
1. Labeling by Exchange
• Only useful for deuterium
• Compound must contain active carbons
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
1. Labeling by Exchange
• Only useful for deuterium
• Compound must contain active carbons
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
1. Labeling by Exchange
• Only useful for deuterium
• Compound must contain active carbons
Base
MeOD/D2O
Testosterone Testosterone-d5
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
2. Labeling by Deconstruction/Reconstruction
• Take unlabeled final product (or analog)
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
2. Labeling by Deconstruction/Reconstruction
• Take unlabeled final product (or analog)
• Remove part of the molecule
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
2. Labeling by Deconstruction/Reconstruction
• Take unlabeled final product (or analog)
• Remove part of the molecule
• Remake the molecule with label
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
2. Labeling by Deconstruction/Reconstruction
• Take unlabeled final product (or analog)
• Remove part of the molecule
• Remake the molecule with label
Boldenone Testosterone-13C3
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
3. Total Synthesis
• Construct the molecule from basic materials
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
3. Total Synthesis
• Construct the molecule from basic materials
• Tends to be the most expensive
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Factors in Isotopic Labeling:
Route of Synthesis
Three General Routes:
3. Total Synthesis
• Construct the molecule from basic materials
• Tends to be the most expensive
29 Steps (!)
Saxitoxin-[15N4]
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Case Study: Aldosterone
O
O
OH OHO
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Which carbons are activated for deuterium labeling by base-catalyzed exchange?
O
O
OH OHO
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Which carbons are activated for deuterium labeling by base-catalyzed exchange?
C
C C
C
O
O
OH
C
OHO
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Deuterated Aldosterone – Prepared by H/D exchange under basic conditions.
C
C C
C
O
O
OH
C
OHO
D
DD
D D
DD
D(H)
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone Standard
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Note that a 1:1 mass mix of standard and labeled
standard will not a give a 1:1 molecular ion intensity
Aldosterone-d7
showing mix of
isotopomersd8
d7
d6
d5
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone-d7
Aldosterone Standard
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Label Stability Problems
O
O
OH OHO
D
DD
D D
DD
D(H)
Base or Mass Spec
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Label Stability Problems
O
O
OH OHO
D
DD
D D
DD
D(H)
O
O
OH OHO
H
DH
D H
HH
D(H)
Base or Mass Spec
Easy to Incorporate Deuterium Means Easy to Lose Deuterium!
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Label Stability Problems
H2O
Base or Mass Spec
This exchange process is always occurring, but you only
detect it when deuterium is present.
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Solving the Label Stability Problem
O
O
OH OHO
D
D
DD
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone – Solving the Label Stability Problem
O
O
OH OHO
D
D
DD
•Deuterium from 2 sources
•Deuterium in non-exchangeable positions.
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Aldosterone-d4
96.9% d4
3.1% d3
d4
d3
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Factors in Isotopic Labeling:
Label Stability (Deuterium)
Note: All 3 are at exactly the same concentration
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Quality Controls Necessitated by
Deuterium
Unlabeled standard at m/z 343
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Quality Controls Necessitated by
Deuterium
D4 label at m/z 347
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Quality Controls Necessitated by
Deuterium
Select MRMs
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Quality Controls Necessitated by
Deuterium
Carry Out Analysis with
selected MRM
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Quality Controls Necessitated by
Deuterium
Carry Out Analysis with
selected MRM
BUT…How do you know your
internal standard is still
accurate?
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Quality Controls Necessitated by
Deuterium
T=0
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Quality Controls Necessitated by
Deuterium
T=n hrs
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Quality Controls Necessitated by
Deuterium
T=n hrs
Deuterium has exchanged
and the molecular ion
intensity has decreased
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Quantification Complications
• Blank + Internal Standard sample only catches the issue if
there is a complete loss of isotope. If a [d4] internal
standard begins to lose deuterium and could be detectable
at [d3], [d2] and [d1] then the [d4] mass spec internal
standard signal is decreased. There is no red flag from the
Blank + Internal Standard sample unless it loses all of the
deuterium.
• Since the internal standard ratio is used to quantify, if the
internal standard signal drifts because of loss of deuterium,
then the analyte signal is normalized to the higher internal
standards signal, giving erroneously higher diagnostic
results.
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How Much of a Potential Problem is There?
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Examples: Androstenedione
Androstenedione- [2H7] Androstenedione-[13C3]
• Old Standard Used
• D is Exchangeable
• New Standard
• 13C Labeled
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Examples: 17a-Hydroxyprogesterone
17a-Hydroxyprogesterone-[2H8] 17a-Hydroxyprogesterone-[13C3]
• Old Standard
•D is Exchangeable
• New Standard
• 13C Labeled
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Examples: Dehydroepiandrosterone
Dehydroepiandrosterone-[2H2] Dehydroepiandrosterone-[2H6]
• Old Standard
• D is Exchangeable
• Only M+2
• New Standard
• D is non-exchangeable
•13C3 is also available
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Examples: Dihydrotestosterone
Dihydrotestosterone-[2H4] Dihydrotestosterone-[13C3]
• Old Standard Used
• D is Exchangeable
• New Standard
• 13C Labeled
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Examples: 17b-Estradiol
17b-Estradiol-[2H5] 17b-Estradiol-[13C3]
• Old Standard Used
• 2 of the D are
Exchangeable
• New Standard
• 13C Labeled
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Examples: Vitamin B3
(Niacin; Nicotinic Acid)
Nicotinic Acid- [2H3] Nicotinic Acid-[13C3,15N ]
N
O
OH
D D
D
C13
N15
C13
C13
O
OH
OR
• Old Standard Used
• D is Exchangeable
• New Standard
• 13C,15N Labeled
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Examples: Vitamin B9 (Folic Acid)
Folic Acid-[2H4] Folic Acid-[13C5]
NH
C13
C13
C13
C13
C13 OHO
O
OHNH
N
N
NH
NNH2
O
O
NH
OHO
O
OHNH
N
N
NH
NNH2
O
O
D
D
D
D
OR
• Old Standard Used
• Potentially Labile D
• New Standard
• 13C Labeled
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Examples: Vitamin B12
Old Standard
None
New Standard
Vitamin B12-[13C7] (!!!)
N
NN
N
NH
OP
O
OOH
NC
13
C13
C13
C13
C13
C13N
C13
CH3
CH3OH
O O
O
Co+
CONH2
NH2CO
NH2CO
CONH2
NH2CO
CONH2
CN
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Which Do I Choose?
C13
C13
C13
O
OH
O
OH
D
D
D D D
O
OH
D
DD
•Totally Exchangeable
Deuterium
•Significant Amount of
m0 detected
•Non-Exchangeable
Deuterium, But
•m0 still detected
under MS Conditions;
MS itself induces
some loss of D (up to
all 3 D’s)
•No Exchange
•No loss of label in MS
•No m0
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Which Do I Choose?
OH
OH
D
D
D
OH
OH
CD3
CD3
C13
C13
C13
OH
OH
C13
C13
C13
C13
C13
OH
OH OH
C13
C13
C13
OH
There Can Be
No Exchange
or Loss of
Isotope In MS
There Can Be
No Exchange
or Loss of
Isotope In MS
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OH
D
DD
OH
OH
CD3
CD3
OH
N
NN
O
O
RN
NN
O
O
R
Which Do I Choose?
If PTAD Derivitization is Used:
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D3 D0
Label Retained Label Lost
Which Do I Choose?
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Factors in Isotopic Labeling:
Chromatographic Separation of Isotopes
EPA Ester and EPA-d5 Ester Co-injection
d5 d0
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Factors in Isotopic Labeling:
Chromatographic Separation of Isotopes
Analytical Chemistry (1988), 60(19) 2131
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Factors in Isotopic Labeling:
Chromatographic Separation of Isotopes
• Deuterium elutes before hydrogen
• We’ve seen it with 24(R),25-Dihydroxyvitamin D3-d6
• Many other examples in the literature:
• Benzazepines
• Dopamines
• Aminoaromatics
• Carotenes
• Fatty Acids
• Issue Will Become More Apparent with Improvements
in HPLC
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What Does Percent Isotope Incorporation
Mean?
Example: Estriol-d3 (98% D incorporation):
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What Does Percent Isotope Incorporation
Mean?
Example: Estriol-d3 (98% D incorporation):
What does 98% mean?
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What Does Percent Isotope Incorporation
Mean?
Isotope Incorporation (technical definition):
“Sum of the isotopic content of all possible isotopomers divided by the theoretical isotope content.”
For Estriol-d3 , it would be:
d0+d1+d2+d3 / 3
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What Does Percent Isotope Incorporation
Mean?
Incorp. #D's Total D's
2.00% 0 0.00
0.00% 1 0.00
0.00% 2 0.00
98.00% 3 2.94
100.00% 2.94
98.00%
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What Does Percent Isotope Incorporation
Mean?
Incorp. #D's Total D's Incorp. #D's Total D's
2.00% 0 0.00 1.50% 0 0.00
0.00% 1 0.00 0.00% 1 0.00
0.00% 2 0.00 1.50% 2 0.03
98.00% 3 2.94 97.00% 3 2.91
100.00% 2.94 100.00% 2.94
98.00% 98.00%
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What Does Percent Isotope Incorporation
Mean?
Incorp. #D's Total D's Incorp. #D's Total D's Incorp. #D's Total D's
2.00% 0 0.00 1.50% 0 0.00 0.00% 0 0.00
0.00% 1 0.00 0.00% 1 0.00 1.99% 1 0.02
0.00% 2 0.00 1.50% 2 0.03 2.01% 2 0.04
98.00% 3 2.94 97.00% 3 2.91 96.00% 3 2.88
100.00% 2.94 100.00% 2.94 100.00% 2.94
98.00% 98.00% 98.00%
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What Does Percent Isotope Incorporation
Mean?
Incorp. #D's Total D's Incorp. #D's Total D's Incorp. #D's Total D's
2.00% 0 0.00 1.50% 0 0.00 0.00% 0 0.00
0.00% 1 0.00 0.00% 1 0.00 1.99% 1 0.02
0.00% 2 0.00 1.50% 2 0.03 2.01% 2 0.04
98.00% 3 2.94 97.00% 3 2.91 96.00% 3 2.88
100.00% 2.94 100.00% 2.94 100.00% 2.94
98.00% 98.00% 98.00%
All are 98% D, but only one is free of d0!
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What Does Percent Isotope Incorporation
Mean?
Deuterated aldosterone:
O
O
OH OHO
D
DD
D D
DD
D(H)
7.50 D/molecule
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What Does Percent Isotope Incorporation
Mean?
Deuterated aldosterone:
O
O
OH OHO
D
DD
D D
DD
D(H)
7.50 D/molecule Is this Aldosterone-d7 or d8?
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What Does Percent Isotope Incorporation
Mean?
Deuterated aldosterone:
O
O
OH OHO
D
DD
D D
DD
D(H)
7.50 D/molecule Is this Aldosterone-d7 or d8?Is this 93.8% D (for d8) or 107.1% D (for d7)?
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Issues if Deuterium is Used
• How Chemically Stable is the Deuterium?
• Is it stable in your matrix?
• How long is it stable in your matrix?
• Is it stable in mobile phase?
• Stable at t=0 does not mean stable at t=60 min or…
• Has this validation been run?
• These answers are unique to each user
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Issues if Deuterium is Used
• How Stable is the Deuterium to MS ?
• Is it stable under your MS conditions?
• As MS becomes more sensitive this will become
more of an issue
• These answers are unique to each instrument
• So There May be No Issue at all with Deuterium
• But all these Quality Controls Should be Run
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Carbon-13 and/or Nitrogen-15 have none of these problems!
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Can Deuterium Still be Used if There are No Alternatives?
Deuterium1.56%
• Deuterium is in a non-exchangeable position
• Deuterium MS stability is verified
•More of a problem with increasing MS sensitivity
• Deuterium in Conjunction with 13C and/or 15N
• Deuterium from more than one synthetic source
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Summary
• Deuterium has inherent limitations
–Instability/ Loss of label / False Positives / Co-elution Issues
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Summary
• Deuterium has inherent limitations
–Instability/ Loss of label / False Positives / Co-elution Issues
• Next generation of standards must:
–Incorporate 13C or
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Summary
• Deuterium has inherent limitations
–Instability/ Loss of label / False Positives / Co-elution Issues
• Next generation of standards must:
–Incorporate 13C or
–Incorporate multiple isotopes 13C,2H ; 15N,2H or
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Summary
• Deuterium has inherent limitations
–Instability/ Loss of label / False Positives / Co-elution Issues
• Next generation of standards must:
–Incorporate 13C or
–Incorporate multiple isotopes 13C,2H ; 15N,2H or
–If only deuterium:
• Synthesized in stable, non-exchangeable positions
• Synthesized from multiple deuterium sources to minimize unlabeled contaminants
• Stability Validated
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Summary
• The ultimate question is what do you require for the best and most unambiguous results?
–Interaction with manufacturer in the design phase
–Selecting the isotope(s)
–Selecting the number of labels
–Selecting labeling sites
–Balancing costs
–Feedback on performance
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Summary
• Don’t know if you have standards with exchangeable deuterium?
• Ask us-
–call (610-337-3762),
–Email ([email protected])
• We’ll tell you
–This is what we do.
–We’re synthetic organic isotope chemists, so this is our area of expertise.
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Summary
“Tell Us What You Need and We’ll Make It”
610-337-3762