“LC-MS/MS Determination of Vitamin D Profiles in serum ... · Vitamin D Essential in bone health ( →rickets, osteoporosis). Most tissues/cells in human body possess vitamin D

“LC-MS/MS Determination of

Vitamin D Profiles in serum

after Derivatization with

Amplifex Reagent“

Dietrich A. Volmer

Institute of Bioanalytical Chemistry

Saarland University

Saarbrücken, Germany

http://www.bioanalytik.uni-saarland.de

@Bio_MassSpec

IBC Institute ofBioanalytical ChemistryIBC Institute ofBioanalytical Chemistry

SCIEX Innovation Day ⋅⋅⋅⋅ 11 May 2016

Vitamin D

� Essential in bone health (→ rickets, osteoporosis).

� Most tissues/cells in human body possess vitamin D receptors.

� Extra-skeletal benefits, reduced risk for: diabetes, cancer,

neurodegenerative, cardiovascular diseases…

� Significant interest in links between vitamin D and disease.

� Reliable and accurate measurement of status required!

D3 D2

Vitamin D vitamers

Holick. Am. J. Clin. Nutr.

2004, 80, 1678S

München

UVB exposureUVB exposure

Vitamin D biosynthesis and metabolism

Stokes, Volmer, Grünhage, Lammert,

Liver Int. 2013, 33, 338.

Common status marker 25(OH)D → long half-life (~21 d)Common status marker 25(OH)D → long half-life (~21 d)

+ C-3 epimer contributions!

3

Double bonds absorb UVBDouble bonds absorb UVB

Sunlight overdoseSunlight overdose

active formactive form

Defining vitamin D levels

ng/mL nmol/L

10

20

30

40

50

0

25

0

50

75

100

125

Severe

Deficient

Insufficient

Serum 25-hydroxyvitamin D

Normal

Insufficient vitamin D

levels in Germany

ca.50% adults

(Nat. Kohorte)

ca. 60% children

(KiGGS)

Institute of Medicine; Endocrine Society

Hintzpeter, Mensink, Thierfelder, Müller, Scheidt-Nave. European Journal of Clinical Nutrition 2007, 1-11.

Richter, Heidemann, Schulze, Roosen, Thiele, Mensink. BMC Pediatrics 2012, 12, 35.4

Other species

Mean 25-hydroxyvitamin D

levels in marmoset versus

human

~400 ng/mL (marmoset)

~50 ng/mL (human)

Ziegler et al., Am. J. Primatol. 2015, 77, 801.

6

Not endorsed for achieving vit D sufficiency…

Laboratory assessment

� No agreement on reference levels for healthy individuals.

� Common status marker is 25(OH)D → long half-life (21 d).

� Various immunoassay common in clinical analyses.

� Today: LC-MS/MS often coined “gold standard method”.

7

Mass spectral inferences from isobars/isomers

� Issues of LC-MS detection sensitivity.

� Many LC-MS/MS assays for vitamin D use loss of H2O from [M+H]+ as

“specific” transition for MRM.

� Serious problem for specificity as HPLC gives co-eluting isobars.

� Isobaric/isomeric interferences expected in MS/MS → systematic errors

for low resolution QqQ (usually in vitamin D labs!)

� Obvious solution � more structure-diagnostic product ions for MS/MS.

� However…

CID

Multiple reaction monitoring (MRM)

m/z 401 m/z 383

Q1 Q2 Q3

8

Problems with limited selectivity

CID spectrum of 25(OH)D illustrating limited structural information at

low/intermediate collision energy → selectivity problem in MRM!

150 200 250 300 350 400 450

50

100255

365

241

269

309283

199227

159145 295185 323

135121337

349

150 200 250 300 350 400

50

100383.32962

365.31927

m/z m/z

Re

l. A

bu

nd

an

ce (

%)

Low energy Medium energy

Volmer, Mendes, Stokes. Mass Spectrom. Rev. 2015, 34, 2-23. 9

MRM chromatogram of 25(OH)D of CLD patient serum

after 96-well micro-extraction; level at 28.2 ng/mL.

Problems hidden in MRM analyses

m/z 401 → 383

Geib, Meier, Schorr, Lammert, Stokes, Volmer. J. Mass Spectrom. 2015, 50, 275-279.10

0 1 2 3 4 5 6 7 8 9 10

Time [min]

1007.12

7.32

epi-25(OH)D

25(OH)D

% R

A

The isobaric space of 25(OH)D in human serum

m/z 401 → 383

a b cd e

f gh

ij k

401.15 401.20 401.25 401.30 401.35 401.40m/z

a No LC

b LC fraction

c Reconstructed “neutral loss” (H2O) spectrum

FTICR-MS → serum isobars of m/z 401 FTICR-MS → serum isobars of m/z 401

Very similar for different sample preparation techniques (PP → SPE, LLE, SLE)

11

LC-MS/MS separation of 25(OH)D and pentaerythritol oleate (PEO). SRM traces

correspond to m/z 401→383 transition.

True identity of ‘peak g’: pentaerythritol oleate

C23H44O5

2 3 41 5 6 7 8 9

25(OH)D

PEO

LC-MS/MS

SRM

m/z 401→383100

50

Retention time (min)

PEO

O

O

OH

OH

OH

g C23H44O5 401.32617 401.32615 0.05

h C27H44O2 (=25(OH)D) 401.34138 401.34141 -0.07

no. Proposed formula

(neutral compound)

Measured

(m/z)

Calculated

(m/z)

Error

(ppm)

1,2-didecanoyl-sn-glycerol

DAG ?

C23H44O5

Qi, Geib, Schorr, Meier, Volmer. Rapid Commun. Mass Spectrom. 2015, 25, 1-9.

...

12

13Qi, Geib, Schorr, Meier, Volmer. Rapid Commun. Mass Spectrom. 2015, 25, 1.

Mimicking endogenous background

25(OH)D + 8 isobaric compounds SelexIon DMS

Volmer.

GIT 2012, 12, 855

Removing interferences with DMS-MS/MS

14

Low versus high resolution for quantification

TOF-MS TOF-MS/MS

Low versus high resolution for quantification

Passing-Bablok regression and Bland-Altman analysis of 97 serum samples from

CLD patients receiving vitamin D substitution

15

QTRAP 5500 vs TrileTOF 5600

Vit D metabolic fingerprints (“chemotypes”)

MRM chromatograms from human

plasma after PP/SPE/PTAD:

Ding, Schoenmakers, Jones, Prentice, Volmer. Anal. Bioanal. Chem. 2010, 398, 779

Overcoming issues of detection sensitivity

→ chemical derivatization:

Cookson-type reagents (e.g. 4-phenyl-

1,2,4-triazoline-3,5-dione, PTAD)

25-OH D3

(53.2 nmol/L)

25-OH D2

(0.9 nmol/L)

24,25-(OH)2 D3

(3.5 nmol/L)

1.0 2.0 3.0

Re

lati

ve I

nte

nsi

ty(%

)

1.0 2.0 3.0 4.0 5.00

1,25-(OH)2 D3

(78 pmol/L)

Time (min)

50

100

Time (min)

Re

lati

ve I

nte

nsi

ty(%

)

50

100

16

Amplifex reagent and serum extraction

17

Defining pathobiological significance of vitamin D and its

metabolites and deciphering relevance for progression /

outcome of chronic diseases

Vit D metabolic fingerprints (“chemotypes”)

18Müller, Stokes, Lammert, Volmer. Sci. Rep. 2016, 21080.

Vit D metabolic fingerprints (“chemotypes”)

19Müller, Stokes, Lammert, Volmer. Sci. Rep. 2016, 21080.

Acknowledgements

Saarbrücken groupCaroline ByldaElizabeth CrawfordDr Stella de BortoliVerena KellerMiriam MüllerDavid AuerbachMathias Baltes

Mathias BaltesTobias DierSebastian FreyTobias GeibDr Klaus HollemeyerFlorian MeierDr Yulin QiIgnacy Rzagaliński

Pascal SchorrRainer Wintringer

UdS Med SchoolDr Caroline StokesProf Frank Lammert