Analysis of 1,25 DHVD3 and 1,25 DHVD2 Using … · RUO-MKT-11-3442-A For Research Use Only. Not for use on diagnostic procedures. Analysis of 1,25 DHVD 3 and 1,25 DHVD 2 Using Amplifex™

RUO-MKT-11-3442-A For Research Use Only. Not for use on diagnostic procedures.

Analysis of 1,25 DHVD3 and 1,25 DHVD2 Using

Amplifex™ Diene Reagent

2 © 2016 AB Sciex RUO-MKT-11-3442-A For Research Use Only. Not for use on diagnostic procedures.

7-Dehydrocholesterol

Cholecalciferol

(Vitamin D3) Vitamin D3 (fish, meat)

Vitamin D2 (supplements)

25-Hydroxy Vitamin D3

1a, 25-Dihydroxy Vitamin D3

HO

OH

13

25

HO

OH

OH13

25

HO 13

25

Skin

Dietary intake

Liver

Kidney

Maintains

Calcium

balance in

the body

Deficiency: Less than 20 ng/mL

Insufficiency: 20-29 ng/mL

Optimum Level: 30-80 ng/mL

• 1a, 25-di-OH-Vitamin-D3 (DHVD3,

Calcitriol) is present in the plasma in

low pg/mL ranges.

• Recently Calcitriol found to play an

important role in breast cancer a. Vitamin D and breast cancer: Inhibition of estrogen synthesis

and signaling. Krishnan, Aruna V.; Swami, Srilatha; Feldman,

David Journal of Steroid Biochemistry and Molecular

Biology (2010), 121(1-2), 343-348.

b. 1α,25-Dihydroxyvitamin D3 inhibits transcriptional potential of

nuclear factor kappa B in breast cancer cells . Tse, Anfernee

Kai-Wing; Zhu, Guo-Yuan; Wan, Chi-Keung; Shen, Xiao-Ling; Yu,

Zhi-Ling; Fong, Wang-Fun Molecular

Immunology (2010), 47(9), 1728-1738.

c. Vitamin D deficiency promotes human breast cancer growth in

a murine model of bone metastasis. Ooi, Li Laine; Zhou, Hong;

Kalak, Robert; Zheng, Yu; Conigrave, Arthur D.; Seibel, Markus J.;

Dunstan, Colin R. Cancer Research (2010), 70(5), 1835-1844.

d. CCAAT enhancer-binding protein α is a molecular target of

1,25-dihydroxyvitamin D3 in MCF-7 breast cancer cells

Dhawan, Puneet; Weider, Robert; Christakos, Sylvia Journal of

Biological Chemistry (2009), 284(5), 3086-3095.

Introduction and Overview


Analysis of 1,25DHVD3: Challenges

•Low abundance (<100 pg/mL)

•Neutral, difficult to ionize molecule (in

MS based methods)

•Dehydration prone, thermally and

photochemically labile

•Presence of multiple isomeric

(structurally similar) and /or isobaric

(same molecular weight) metabolites in

biological matrix can cause interferences

to mass spectrometry and immuno

assays

Improved ionization efficiency

and stability of these compounds

via derivatization during MS

analysis is critical for a highly

selective and reliable analysis

~1000 fold less abundant

than 25-HO-VD3

It is absolutely necessary to separate the

isobaric isomers of 1,25DHVD3, for example

24(R), 25-DHVD3,

24(S), 25-DHVD3, and

23(R), 25-DHVD3,

to avoid interferences during MS analysis

1. Pelc, B.; Marshall, D. H. Steroids. 1978, 31, 23-29.

2. Grady, L. T.; Thakker, K. D. J. Pharmaceutical Sciences.

1980, 69, 1099-1102.

3. Holmberg, B. I.; Kristiansen, T.; Pedersen, J. I. Clinical

Chem. 1979, 25, 584-588.

4. Isolation and Identification of Vitamin D Metabolites.

Napoli, J. L.; Koszewski, N. J.; Horst, R. L. Methods in

Enzymology, 1986, 123, 127-140.

5. C-3 Epimers Can Account for a Significant Proportion

of Total Circulating 25-Hydroxyvitamin D in Infants,

Complicating Accurate Measurement and Interpretation of

Vitamin D Status. Singh, R. J.; Taylor, R. L.; Reddy, G. S.;

Grebe S. K. G. The Journal of Clinical Endocrinology &

Metabolism, 2006, 91, 3055-3061.


Pure Amplifex™ Diene Reagent can be synthesized in high yield with ionization enhancing group

Reagent is very hydrophilic (improved LC property of the derivative), yet soluble in organic solvent

Reacts with Vitamin Ds within minutes & unreacted reagents elutes very early in reversed phase LC

The reagent also derivatizes Vitamin D3, 25-hydroxy Vitamin D3, Vitamin D2 and its metabolites

Commercially available Cookson type reagents (PTAD, MBOTAD, DMEQTAD and similar

molecules do not have ionization enhancing group

Addressing the Challenges: Ionization Efficiency/Fragmentation Improvement

Ionization

enhancing

group

Labile dine group

is stabilized after

derivatization


Fragmentation Patterns: SCIEX QTRAP® 5500 System

DHVD3-Underivatized

399.6135.3

DHVD3-PTAD

574.5314.2

DHVD3-Amplifex™ Diene

748.6689.5

M+= 748.5

MH+= 417.3

MH+ = 592.4

quantifying

qualifying

qualifying

•A cleaner and defined fragmentation pattern was observed for DHVD3 using Amplifex™ Diene Reagent:

Most ion current is funneled to the quantifying transition

Includes the whole analyte (structure specific)

Practically free of dehydration

•DHVD3-PTAD and its fragment ion:

•Undergoes dehydration

•Quantifying ion carry only a small portion of the analyte, hence iso-structural species (for example DHVD2) will have common Q3 ion

quantifying

CE in all cases was ~35 ev


DHVD3-Amplifex™ Diene

DHVD3-

Underivatized

Signal Intensity Comparisons of DHVD3 and its Amplifex™ Diene Derivative SCIEX QTRAP® 5500

~180 fold

enhancement

Note: Offsetting of LC retention times for

DHVD3-underivatized was used to position the

peak under DHVD3-Amplifex™ Diene analyte

peak for visual demonstration.

In each case concentration of

analyte was 40 pg (on column)

Transitions used (most intense

transitions)

DHVD3-underivatized

399.6135.3

DHVD3- Amplifex™ Diene

748.6689.5


XIC of +MRM (4 pairs): 748.600/689.510 Da ID: d0DHVD3(1,25:24,25,23:25)_NeuL from Sample 1 (1,25DHVD3_3QAOC) of 1,25DHV... Max. 2.3e5 cps.

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.51 49 98 146 195 243 291 340 388 436

Time, min

0.0

2.0e4

4.0e4

6.0e4

8.0e4

1.0e5

1.2e5

1.4e5

1.6e5

1.8e5

2.0e5

2.2e5

2.3e5

In

te

ns

ity

, c

ps

2.17 DHVD3-Amplifex™ Diene

DHVD3-PTAD

~10-11 fold

In each case concentration of

analyte was 40 pg (on column)

Transitions used (most intense

transitions)

DHVD3-PTAD

574.4314.2

DHVD3- Amplifex™ Diene

748.6689.5

Signal Intensity Comparisons of Amplifex™ Diene and PTAD Derivatives of DHVD3

SCIEX QTRAP® 5500


Interference study


d0-DHVD3-Amplifex™ Diene




1,25 DHVD3-Amplifex™ Diene and DHVD2-Amplifex™ Diene with Internal Standards

Window of elution of 1,25 DHVD3-Amplifex™ Diene


24(R), 25 DHVD3-Amplifex™ Diene

d0-24(R), 25-DHVD3-Amplifex™ Diene

Origin: Possible enantiomeric impurity

in synthetic 24(R), 25 DHVD3



d0-24(S), 25-DHVD3-Amplifex™ Diene


Origin: Possible enantiomeric impurity

in synthetic 24(S), 25 DHVD3

24(S),25 DHVD3-Amplifex™ Diene


Structures (Underivatized) of 1,25 DHVD3 Isobars and Internal Std.


XIC of +MRM (6 pairs): 748.600/689.510 Da ID: d0DHVD3(1,25:24,25,23:25)_NeuL from Sample 11 (d0-1,25DHVD3) of Data_5uL.wiff (... Max. 8.8e5 cps.

1.7 1.8 1.9 2.0 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 3.0Time, 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

6.0e5

6.5e5

7.0e5

7.5e5

8.0e5

8.5e5In

ten

sit

y,

cp

s2.45

d0-1,25DHVD3

d6-3epi-1,25DHVD3

d0-3epi-1,25DHVD3

d0-23(R),25DHVD3

d0-24(S),25DHVD3

d0-24(R),25DHVD3

d6-1,25DHVD3

d0-1,25DHVD2

d6-1,25DHVD2

d6-24(R),25DHVD3

d6-24(S),25DHVD3

Overlaid Chromatograms


Sample Preparation, Derivatization and Analysis

Spike internal standard in

200 mL of human serum

Dry

Label with

Amplifex™ Diene

Reagent

LC-MRM SPE

35 min 30 min 30 min,

ambient

temp

5 min

Simple one step SPE extraction using two inexpensive cartridges and solvents

One Step derivatization process

5 minute LC-MRM analysis─ 20 mL injection, standard water/acetonitrile

(0.1% Formic acid) gradient, 0.7 mL/min

A QTRAP ® 5500 LC-MS/MS system with electrospray ion source in Multiple Reaction

Monitoring (MRM) mode was used for detection

Simple, Fast and Affordable …


Calibration Curves Using Metabolite Depleted Serum

Linear response observed for d0/d6-DHVD3 and d0/d6-DHVD2 in metabolite depleted

serum (0-250 pg/mL range shown. Dynamic range verified up to 1000 pg/mL)

DHVD3

DHVD2


0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

40.0

0.0 10.0 20.0 30.0 40.0 50.0

%C

V (

Ca

lcu

late

d C

on

ce

ntr

ati

on

; p

g/m

L)

[DHVD3] pg/mL

%CV vs. Analyte Conc. (DHVD3)

0.0

5.0

10.0

15.0

20.0

25.0

30.0

0.0 100.0 200.0 300.0 400.0

%C

V (

Calc

ula

ted

Co

ncen

trati

on

; p

g/m

L)

[DHVD3] pg/mL


LOQ: 6-7 pg/mL

%CV vs. Analyte Conc. (DHVD3)_QTRAP® 5500 System (in Stripped Human Serum)


Traces_ QTRAP® 5500 _DHVD3 (in Stripped Human Serum)

6.25 pg/mL

LOQ is in between

(6-7 pg/mL)

12.5 pg/mL


0.0

5.0

10.0

15.0

20.0

25.0

30.0

0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0

%C

V (

Ca

lcu

late

d C

on

cen

tra

tio

n;

pg

/mL

)

[DHVD2] pg/mL


LOQ: 10-11 pg/mL

%CV vs. Analyte Conc. (DHVD2)_ QTRAP® 5500 System (in Stripped Human Serum)


6.25 pg/mL

12.5 pg/mL

LOQ is in between

10-11 pg/mL

Traces_ QTRAP® 5500 _DHVD2 (in Stripped Human Serum)


LOQ values were determined by

1. %CV (<15%)

2. Signal to noise values

3. Quality of the signal in general

Note


Real Serum Sample Data


Endo: 6.4 pg/mL IS: 250 pg/mL

Chromatograms of Samples: DHVD3 Channel


















LLOQ case


























R² = 0.5161

0

20

40

60

80

100

120

0 10 20 30 40 50 60 70 80 90 100

1,2

5 D

HV

D3+

1,2

5D

HV

D2_

RIA

(p

g/m

L)

1,25DHVD3+1,25 DHVD2 _LC-MS (pg/mL)

Not included in

correlation

coefficient

calculation

Sample #33

Sample #8

DHVD3-Correlation: LC-MS (Amplifex™ Diene Derivatized) vs. Radio Immuno Assay


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For Research Use Only. Not for use in diagnostic procedures.

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the property of AB Sciex Pte. Ltd. or their respective owners.

AB SCIEX™ is being used under license.

Documents

Analysis of 1,25 DHVD3 and 1,25 DHVD2 Using … · RUO-MKT-11-3442-A For Research Use Only. Not for use on diagnostic procedures. Analysis of 1,25 DHVD 3 and 1,25 DHVD 2 Using Amplifex™