“lc-ms/ms determination of vitamin d profiles in serum ... · vitamin d essential in bone health...
TRANSCRIPT
“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)
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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