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

• Fat-soluble secosteroidal compounds

• Biological regulators of calcium homeostasis

• Possible biomarkers of diseases

• Sources

– Sunlight

– Fungi

– Supplements

– Diet

Vitamin D

2

Metabolism

3

Population at risk of deficiency

4

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

5

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

6

Analytical interferences: Stereoisomers

7

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

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

8

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

10

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

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

11

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

12

=

10 µl 10 µl

6 mm punch 6 mm punch

A. B.

Aliquot variability with traditional

DBS

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

14

Materials and methods

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

16

Sample Collection

17

Traditional DBS spotting

Mitra microsampling

Sample Preparation

18

Traditional DBS workflow

Mitra microsampling workflow

Sample

drying

Derivatisation

19

• Introduces physico-chemical characteristics by addition

of a chemical moeity

• Promotes ionisation

• Increases sensitivity and selectivity

• Enables very low LOD and LOQ

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

Results

22

Figure 1: Chromatogram of PTAD derivatised serum sample

Inte

nsit

y, c

ps

23

Figure 2: PTAD stripped-serum-derivatives calibration curve from 1 to 100 ng/mL

An

aly

te A

rea/I

S A

rea

24

Inte

ns

ity,

cp

s

Figure 3: Chromatogram of Amplifex diene derivatised serum sample

25

Figure 4: Amplifex diene stripped serum-derivatives calibration curve from 1 to

100 ng/mL

An

aly

te A

rea/I

S A

rea

26

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

27

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

Conclusion

• 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

29

For Mitra microsampling device

Acknowledgements

For funding and instrumentationFor ELISA sample analysis

For sponsorship