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De Deficienc ficiencies ies of of Deu Deute teriu rium m as an as an Inte Interna nal l Sta Stand ndar ard d in in MS MS Deficiencies of Deuterium as an Internal Standard in MS Presentation Scott Landvatter, Ph.D. (President)

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SLIDE 1

De Deficienc ficiencies ies of

  • f Deu

Deute teriu rium m as as an an Inte Interna nal l Sta Stand ndar ard d in in MS MS

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SLIDE 2

Deficiencies of Deuterium as an Internal Standard in MS Presentation

Scott Landvatter, Ph.D. (President) Rich Tyburski (Vice President)

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SLIDE 3

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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SLIDE 4

Which 25-OH D3 Internal Standard Do I Choose?

D3

  • r D6
  • r 13C3 or

13C5

  • r 13C3 (Not Yet Available)
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SLIDE 5

Which Testosterone Internal Standard Do I Choose?

C

13

C

13

C

13

O OH O OH D D D D D O OH D D D

D5

  • r D3
  • r 13C3
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SLIDE 6

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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SLIDE 7

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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SLIDE 8

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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SLIDE 9

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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SLIDE 10

Question: Are There Reasons to Switch Standards? Answer: In Many Cases the Answer is Yes. But Why?

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SLIDE 11

Question: Are There Reasons to Switch Standards? Answer: In Many Cases the Answer is Yes. But Why?

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SLIDE 12

To understand why we need to look at the factors effecting the synthesis

  • f the labeled standards.
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SLIDE 13

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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SLIDE 14

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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SLIDE 15

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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SLIDE 16

Factors in Isotopic Labeling: Choice of Isotope(s)

2H (Deuterium)

Pros:

  • Easy to incorporate
  • Inexpensive (usually)
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SLIDE 17

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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SLIDE 18

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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SLIDE 19

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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SLIDE 20

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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SLIDE 21

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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SLIDE 22

Factors in Isotopic Labeling: Choice of Isotope(s)

18O (Oxygen-18)

Pros:

  • Molecular weight gain of 2 amu per label
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SLIDE 23

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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SLIDE 24

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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SLIDE 25

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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SLIDE 26

Factors in Isotopic Labeling: Route of Synthesis

Three General Routes:

  • 1. Labeling by Exchange
  • Only useful for deuterium
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SLIDE 27

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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SLIDE 28

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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SLIDE 29

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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SLIDE 30

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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SLIDE 31

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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SLIDE 32

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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SLIDE 33

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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SLIDE 34

Factors in Isotopic Labeling: Route of Synthesis

Three General Routes:

  • 3. Total Synthesis
  • Construct the molecule from basic materials
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SLIDE 35

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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SLIDE 36

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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SLIDE 37

Factors in Isotopic Labeling: Label Stability (Deuterium)

Case Study: Aldosterone

O O OH OH O

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SLIDE 38

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone – Which carbons are activated for deuterium labeling by base-catalyzed exchange?

O O OH OH O

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SLIDE 39

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

OH O

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SLIDE 40

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

OH O D D D D D D D D(H)

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SLIDE 41

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone Standard

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SLIDE 42

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 isotopomers d8 d7 d6 d5

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SLIDE 43

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone-d7 Aldosterone Standard

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SLIDE 44

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone – Label Stability Problems

O O OH OH O D D D D D D D D(H) Base or Mass Spec

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SLIDE 45

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone – Label Stability Problems

O O OH OH O D D D D D D D D(H) O O OH OH O

H

D H D

H H H

D(H) Base or Mass Spec

Easy to Incorporate Deuterium Means Easy to Lose Deuterium!

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SLIDE 46

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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SLIDE 47

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone – Solving the Label Stability Problem

O O OH OH O D D D D

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SLIDE 48

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone – Solving the Label Stability Problem

O O OH OH O D D D D

  • Deuterium from 2 sources
  • Deuterium in non-exchangeable positions.
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SLIDE 49

Factors in Isotopic Labeling: Label Stability (Deuterium)

Aldosterone-d4 96.9% d4 3.1% d3 d4 d3

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SLIDE 50

Factors in Isotopic Labeling: Label Stability (Deuterium)

Note: All 3 are at exactly the same concentration

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SLIDE 51

Quality Controls Necessitated by Deuterium

Unlabeled standard at m/z 343

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SLIDE 52

Quality Controls Necessitated by Deuterium

D4 label at m/z 347

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SLIDE 53

Quality Controls Necessitated by Deuterium

Select MRMs

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SLIDE 54

Quality Controls Necessitated by Deuterium

Carry Out Analysis with selected MRM

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SLIDE 55

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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SLIDE 56

Quality Controls Necessitated by Deuterium

T=0

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SLIDE 57

Quality Controls Necessitated by Deuterium

T=n hrs

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SLIDE 58

Quality Controls Necessitated by Deuterium

T=n hrs Deuterium has exchanged and the molecular ion intensity has decreased

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SLIDE 59

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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SLIDE 60

How Much of a Potential Problem is There?

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SLIDE 61

Examples: Androstenedione

Androstenedione- [2H7] Androstenedione-[13C3]

  • Old Standard Used
  • D is Exchangeable
  • New Standard
  • 13C Labeled
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SLIDE 62

Examples: 17a-Hydroxyprogesterone

17a-Hydroxyprogesterone-[2H8] 17a-Hydroxyprogesterone-[13C3]

  • Old Standard
  • D is Exchangeable
  • New Standard
  • 13C Labeled
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SLIDE 63

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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SLIDE 64

Examples: Dihydrotestosterone

Dihydrotestosterone-[2H4] Dihydrotestosterone-[13C3]

  • Old Standard Used
  • D is Exchangeable
  • New Standard
  • 13C Labeled
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SLIDE 65

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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SLIDE 66

Examples: Vitamin B3 (Niacin; Nicotinic Acid)

Nicotinic Acid- [2H3] Nicotinic Acid-[13C3,15N ]

N O OH D D D C

13

N

15

C

13

C

13

O OH

OR

  • Old Standard Used
  • D is Exchangeable
  • New Standard
  • 13C,15N Labeled
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SLIDE 67

Examples: Vitamin B9 (Folic Acid)

Folic Acid-[2H4] Folic Acid-[13C5]

N H C

13

C

13

C

13

C

13

C

13

OH O O OH N H N N N H N N H2 O O N H OH O O OH N H N N N H N N H2 O O D D D D

OR

  • Old Standard Used
  • Potentially Labile D
  • New Standard
  • 13C Labeled
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SLIDE 68

Examples: Vitamin B12

Old Standard None New Standard Vitamin B12-[13C7] (!!!)

N N N N N H O P O O O H N C

13

C

13

C

13

C

13

C

13

C

13

N C

13

CH3 CH3 OH

O O

O Co

+

CONH2 NH2CO NH2CO CONH2 NH2CO CONH2 CN

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SLIDE 69

Which Do I Choose?

C

13

C

13

C

13

O OH O OH D D D D D O OH D D D

  • 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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SLIDE 70

Which Do I Choose?

OH O H D D D OH O H CD3 CD3 C

13

C

13

C

13

OH O H C

13

C

13

C

13

C

13

C

13

OH O H O H C

13

C

13

C

13

OH

There Can Be No Exchange

  • r Loss of

Isotope In MS There Can Be No Exchange

  • r Loss of

Isotope In MS

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SLIDE 71

O H D D D OH O H CD3 CD3 OH

N N N O O R N N N O O R

Which Do I Choose?

If PTAD Derivitization is Used:

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SLIDE 72

D3 D0

Label Retained Label Lost

Which Do I Choose?

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SLIDE 73

Factors in Isotopic Labeling: Chromatographic Separation of Isotopes

EPA Ester and EPA-d5 Ester Co-injection d5 d0

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SLIDE 74

Factors in Isotopic Labeling: Chromatographic Separation of Isotopes

Analytical Chemistry (1988), 60(19) 2131

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SLIDE 75

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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SLIDE 76

What Does Percent Isotope Incorporation Mean?

Example: Estriol-d3 (98% D incorporation):

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SLIDE 77

What Does Percent Isotope Incorporation Mean?

Example: Estriol-d3 (98% D incorporation): What does 98% mean?

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SLIDE 78

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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SLIDE 79

What Does Percent Isotope Incorporation Mean?

Incorp. #D's Total D's 2.00% 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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SLIDE 80

What Does Percent Isotope Incorporation Mean?

Incorp. #D's Total D's Incorp. #D's Total D's 2.00% 0.00 1.50% 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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SLIDE 81

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% 0.00 1.50% 0.00 0.00% 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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SLIDE 82

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% 0.00 1.50% 0.00 0.00% 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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SLIDE 83

What Does Percent Isotope Incorporation Mean?

Deuterated aldosterone:

O O OH OH O D D D D D D D D(H)

7.50 D/molecule

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SLIDE 84

What Does Percent Isotope Incorporation Mean?

Deuterated aldosterone:

O O OH OH O D D D D D D D D(H)

7.50 D/molecule Is this Aldosterone-d7 or d8?

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SLIDE 85

What Does Percent Isotope Incorporation Mean?

Deuterated aldosterone:

O O OH OH O D D D D D D D 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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SLIDE 86

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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SLIDE 87

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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SLIDE 88

Carbon-13 and/or Nitrogen-15 have none of these problems!

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SLIDE 89

Can Deuterium Still be Used if There are No Alternatives?

Deuterium 1.56%

  • 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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SLIDE 90

Summary

  • Deuterium has inherent limitations

–Instability/ Loss of label / False Positives / Co- elution Issues

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SLIDE 91

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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SLIDE 92

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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SLIDE 93

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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SLIDE 94

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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SLIDE 95

Summary

  • Don’t know if you have standards with exchangeable

deuterium?

  • Ask us-

–call (610-337-3762), –Email (info@isosciences.com)

  • We’ll tell you

–This is what we do. –We’re synthetic organic isotope chemists, so this is our area of expertise.

slide-96
SLIDE 96

Summary

“Tell Us What You Need and We’ll Make It”

info@isosciences.com scott.landvatter@isosciences.com 610-337-3762