quantification of vitamin d metabolites in limited volume ...€¦ · mitra™ microsampling device...
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Quantification of vitamin D metabolites in limited volume whole blood collected using an innovative microsampling device and dry blood spot matrix using ionisation enhancing derivatisation reagent by LC-MS/MSRapholo, A.F.1; Cardeano, J.1; Usher, A.1; Van Tonder J.J.2; Cromarty, A.D.1
1Dept. of Pharmacology, University of Pretoria2Triclinium, 121 Amkor Road, Lyttelton Manor
14 September 2016
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• Fat-soluble secosteroidal compounds
• Biological regulators of calcium homeostasis
• Possible biomarkers of diseases
• Sources
– Sunlight
– Fungi
– Supplements
– Diet
Vitamin D
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Metabolism
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Population at risk of deficiency
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Chromatography and vitamin D
analysis
• > 50 years of continuous tremendous growth in
research worldwide
• LC-MS/MS is the “gold standard”
– Highly sensitive and specific analytical technique
– Accurate quantitation
– Targeted analysis
• Separation of D2 from D3
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Analytical challenges with vitamin D
analysis
• Issues of sensitivity– Biologically active metabolite available at [pg/mL]
– Protein binding
• Cross-reactivity in immunoassays – Overestimation
• Misclassification of vitamin D status
• Ion suppression– Mass spectral isobaric interferences in MS/MS
– C-3 epimer contributions
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Analytical interferences: Stereoisomers
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25-Hydroxyvitamin D3 3-epi-25-Hydroxyvitamin D3 1-α--Hydroxyvitamin D3
Monoisotopic Mass: 400.3341 Da Monoisotopic Mass: 400.3341 Da Monoisotopic Mass: 400.3341 Da
Molecular formula: C27H44O2 Molecular formula: C27H44O2 Molecular formula: C27H44O2
• Characteristic display of similar fragmentation ions spectra
(MRM transitions) and chromatography upon ionisation
• Co-elution isobaric interferences
• Overestimation
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Study Design
• Pilot study
• Not powered to assess group differences (age,
sex, ethnicity)
• ELISA method evaluation
– To quantify vitamin D metabolites from samples of 30
healthy native South African volunteers
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Study Population
Table 1: Participants characteristics
Participants (n=30)
Age (years) 19 – 68
Height (m) 1.53 – 1.8
Weight (kg) 43.2 - 96
BMI (kg/m2)a 18.2 – 38
SPIb 28 - 35
Sunscreen use 4
Sunlight exposure
(hours/day)0.5 – 8
9 aBMI – was calculated as kg/m2
bSPI – Felix van Luschen chart
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Table 2: Participants ELISA vitamin D status levels
Vitamin D status
Interpretation
Participants
(n=30)
Severe deficiency
(5 – 10 ng/mL)0
Deficiency
(10 – 20 ng/mL)7
Suboptimal provision
(20 – 30 ng/mL)8
Optimal level
(30 – 50 ng/mL)15
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Study Aim
To quantify the vitamin D metabolites, 25-(OH)-D2 and
D3 in limited volumes of whole blood collected using an
innovative microsampling device and dry blood spot
(DBS) using LC-MS/MS methodology after derivatisation
with an ionisation enhancing reagent
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DBS Sampling
Pros:
• Ease of sampling/home
sampling
• Minimally invasive
• Small blood volumes
• Representative matrix
(blood)
• Reduced biohazard
• Convenient & cost-effective
transport and storage
• Automatable
Cons:
• Incorrect sampling
– Aliquot variability
• Contamination risk
– Interstitial fluid
• Sensitive analysis required
• Extensive validation required
– Site of card punching
– Blood volume based haematocrit
effect
• Blood viscosity
• Extraction efficiency (recovery)
• Matrix effects
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=
10 µl 10 µl
6 mm punch 6 mm punch
A. B.
Aliquot variability with traditional
DBS
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Mitra™ microsampling device
Pros:
• Volumetric absorptive microsampling– Collects consistent blood
volumes across a range of haematocrits and sampling conditions
– Homogeneous 10 or 20 µL sample
• Simple workflow– Direct sampling from host
– Drying and extraction
– No sample manipulation before extraction
Cons:
• Poor sampling technique
may result in less analyte
recovery
• Cost
– More expensive than
traditional DBS
• Extensive validation
required
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Materials and methods
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Table 3: Analytical method
Samples
25-(OH)-D2, 25-(OH)-D3, D3-25-(OH)-
D3 (IS), 3-epi-25-(OH)-D3 and 1α-
(OH)-D3 (50 ng/mL)
Column Phenomenex Kinetex Biphenyl
Dimensions 100 x 2.1 mm ID, 2.6 µm
Mobile phaseA: 0.1% Formic acid
B: MeOH:ACN (50:50,v/v)
HPLC Agilent 1100 Series
Flow rate 300 µL/min
Injection volume 10 µL
Detection AB Sciex 4000 QTRAP ESI, positive
mode
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Sample Collection
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Traditional DBS spotting
Mitra microsampling
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Sample Preparation
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Traditional DBS workflow
Mitra microsampling workflow
Sample
drying
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Derivatisation
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• Introduces physico-chemical characteristics by addition
of a chemical moeity
• Promotes ionisation
• Increases sensitivity and selectivity
• Enables very low LOD and LOQ
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Amplifex diene • Improves sensitivity/ionisation efficiency
• Permanently + charged moiety
• Reacts specifically with cis-diene configuration under
strictly anhydrous conditions
• 50 µL of 1 mg/mL to derivatise
• Time: 30 minutes at ambient temperature
20 25-(OH)-D3 + Amplifex diene Amplifex-derivative
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Results
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Figure 1: Chromatogram of PTAD derivatised serum sample
Inte
nsit
y, c
ps
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Figure 2: PTAD stripped-serum-derivatives calibration curve from 1 to 100 ng/mL
An
aly
te A
rea/I
S A
rea
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Inte
ns
ity,
cp
s
Figure 3: Chromatogram of Amplifex diene derivatised serum sample
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Figure 4: Amplifex diene stripped serum-derivatives calibration curve from 1 to
100 ng/mL
An
aly
te A
rea/I
S A
rea
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XIC of +MRM (3 pairs): 732.800/673.500 Da ID: AMP-25-OH-D3_1 from Sample 99 (Solvent_MeOH) of D... Max. 3.7e5 cps.
2 4 6 8 10 12Time, min
0.0
5.0e4
1.0e5
1.5e5
2.0e5
2.5e5
3.0e5
3.5e5
4.0e5
4.5e5
5.0e5
5.5e5
5.9e5In
ten
sit
y,
cp
s
11.28
9.33
8.88
9.97
Figure 5: Amplifex-derivatised clinical DBS sample chromatogram
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XIC of +MRM (3 pairs): 732.800/673.500 Da ID: AMP-25-OH-D3_1 from Sample 100 (Solvent_Cyclohex) ... Max. 6.5e4 cps.
2 4 6 8 10 12Time, min
0.0
1.0e4
2.0e4
3.0e4
4.0e4
5.0e4
6.0e4
7.0e4
8.0e4
8.7e4
Inte
ns
ity
, c
ps
9.44
8.98
Figure 6: Amplifex-derivatised clinical Mitra sample chromatogram
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Conclusion
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• Derivatisation of DBS samples allowed simultaneous
25-(OH)-D2 and D3, its corresponding epimer and
isobar quantitation and separation using LC-MS/MS
technology, reducing the overestimation of the 25-(OH)-
D status levels
• Kinetex Biphenyl column provided unique retention
mechanism, separation and resolution of the analytes
– First time using Biphenyl column for vitamin D metabolite
analysis
• High sensitivity in low sample volume was achieved, but
interferences playing a big role in analysis needs to be
addressed
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For Mitra microsampling device
Acknowledgements
For funding and instrumentationFor ELISA sample analysis
For sponsorship