quantitative analysis of vit d metabolites in human plasma ... quantitation of 25-oh-vit d2 and...

Clinical Research use only, Not for use in Diagnostics Procedure

Quantitative Analysis of Vit D Metabolites in

Human Plasma using Exactive System

Marta Kozak

Clinical Research Applications Group

Thermo Fisher Scientific San Jose CA

2 Clinical Research use only, Not for use in Diagnostics Procedure

Calibrators and Controls

• Six calibrators at 2, 5 10, 25, 50 and100ng/mL in BSA

• Three QCs at 5ng/mL, 40 ng/mL and 80 ng/mL in BSA

• Cals and QC’s prepared by fortifying bovine serum albumin diluent with 200 ng/mL 25-hydryoxyvitamin D2 and D3 standard mix.

• Precipitating reagent was prepared by adding deuterated D6-25-

hydroxyvitamin D3 to acetonitrile for a final concentration of 75ng/mL.

• Pooled donor serum samples were crashed 2 to 1 with Acetonitrile and spiked with analytes for a final concentration of 20 ng/mL for 25-hydroxyvitamin D2


Presentation Overview

• Goals

• Introduction to Exactive

• Sample preparation and LC/MS method for 25 OH-Vit D

• Calibration curves, LOQ and results on QCs

• Blinded sample results comparison

• Appendix

QQQ: Triple Quadrupole MS/MS system


Goal

• Results comparison between Exactive and QQQ method for

quantitation of 25-OH-Vit D2 and 25-OH-Vit D3 in human plasma

• Exactive@Thermo - Off line sample prep and 2 min gradient

• QQQ@Service lab - Protein precipitation followed by Turboflow and 6min gradient

• Evaluate Exactive system for analysis of 1, 25 di hydroxy Vit D3


Exactive System Introduction


Exactive Benchtop LC-MS

• Resolution100,000 at 1 scan per second 10,000 at 10 scans per second

• Mass accuracySub ppm

•Dynamic range>10,000 within a spectrum

• Scan speedUp to 10 scans per second

• Mass rangem/z 50 - 4000

• Polarity switchingOne positive and one negative scan < 1 second (25K Resolution)


Orbitrap – Principle of Operation

{ })/ln(2/2

),(222

mm RrRrzk

zrU ⋅+−⋅=

z

φ

Hyper-logarithmic potential distribution: “ideal Kingdon trap”

r

12

2

−

=

R

Rmzωωϕ 2

2

−

=

R

Rmzr ωω

qm

kz

/=ω

Makarov A. Anal. Chem. 2000, 72, 1156-1162.

� Characteristic frequencies:• Frequency of rotation ωφ• Frequency of radial oscillations ω

r

• Frequency of axial oscillations ωz


Ethinyl-Estradiol at Different Mass Resolutions

Resolution: 10k, 30k, 50k, 100k

279.12 279.14 279.16 279.18 279.20

m/z

0

10

20

30

40

50

60

70

80

90

100

Re

lative A

bundance

Ethinyl-Estradiol, 279.17434

Butyl-Phthalate, 279.15909 (ubiquitous background ion)

Note:Ethinyl Estradiol used just to demonstrate the power of resolution


100 ppb Hormone Sample (500 pg on-column)

100 ppb Hormone sample measured @ different resolution settings

Ethinyl-Estradiol

The isobaric phthalate

background ion

interferes with the

Ethinyl-Estradiol ion.

At resolution of

10,000 the steroid

mass is off because

the isobaric ions are

not resolved.

17 18 19Time (min)

0

50

100

0

50

100

Rela

tive A

bu

nd

ance

16.68 17.81 18.07 18.9417.5919.04

RP = 10,000Phthalate

Estradiol

17 18 19Time (min)

0

50

100

0

50

100

Rela

tive A

bu

nd

ance

18.03 19.3217.22 18.1716.40 18.76

17.73 RP = 100,000Phthalate

Estradiol

Note:Ethnyl Estradiol used just to demonstrate the power of resolution


Exactive method – Sample preparation

Vortex

100 µL plasma + 200 µL IS 50 ng/mL [2H6]-25OH-D3

Centrifuge 10 min

@ 13200 RPM

Supernatant

50 µL inject

Sample preparation

Note: Stock IS in ACN; Standards, QC and samples were processed using identical procedure

10 µL std + 990 uL of

50% Ethanol

Serial dilutions down

to 100 ng/mL

100 µg/mL 25-OH-D3

and 25-OH-D2 mix in Ethanol

Calibration standards

1 to 200 ng/mL

Standards preparation


Data acceptance criteria

15200Cal Standard 7

15100Cal Standard 6

1550Cal Standard 5

1520Cal Standard 4

1510Cal Standard 3

152Cal Standard 2

201Cal Standard 1

15200QC 3

1550QC 2

205 for Vit-D2 and 20 for Vit-D3*QC 1*

Required Precision [%]Concentration [ng/mL]Samples

* QC1 spiked with 5 ng/mL of Vit-D3 and Vit-D2. Only total concentration was analyzed for QC1 sample.

Current generally acceptable LC/MS precision


25OH-Vit D2 and D3 – Exactive mass spectrum

D2

D2 - H2O

D2 - (2 x H2O)

D3 - H2O

D3 – (2 x H2O)


Exactive – 3 Selected M/Z

Analyte Monitored m/z

25-OH-D3 401.3411 (M+H)

383.3306 (M - H2O + H)

365.3200 (M - 2 x H2O) + H)

25-OH-D2 413.3411 (M+H)

395.3305 (M - H2O + H)

377.3199 (M - 2 x H2O) + H

[2H6] 25-OH-D3

(Internal Standard)

407.3787 (M+H)

389.3679 (M - H2O + H)

371.3576 (M - 2 x H2O) + H

m/z with minimum interference


• LC method

• Thermo Hypersil aQGold 50 x 2.1 mm, 5um (1, 25 Vit D3)

• Thermo Hypersil aQGold 50 x 2.1 mm, 5µm (25 OH Vit D)• Mobile phase A: water containing 0.1% formic acid • Mobile phase B: methanol containing 0.1% formic acid

• Column Temp: 50 Deg C

• Exactive MS method

• HESI source

• Full scan MS acquisition @ 50,000 Resolution

• External mass calibration

• Extract Ion Chromatogram for molecule of interest (m/z)@ better than 5ppm mass accuracy

• Confirm the identity using mass accuracy and RT

• Quantitate using peak area in the chromatogram

Exactive – LC/MS Method

Note: Details in appendix


Linearity and Accuracy – 25OH-Vit D3

% Difference

200 ng/mL

100 ng/mL

50 ng/mL

20 ng/mL

10 ng/mL

2 ng/mL

1 ng/mL

Standard

7.67.913.0

-1.7-2.32.9

-7.9-2.4-7.5

7.84.9-0.02

-6.5-9.6-10.5

0.81.52.2

Precision just below set criteria

Validation_3Validation_2Validation_1

Note: Calibration curve using neat standards

Vit D3 - Validation1

Y = 0.00576964+0.0027313*X R^2 = 0.9918 W: 1/X^2

0 20 40 60 80 100 120 140 160 180 200 220

ng/mL

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

Are

a R

atio

Vit D3 - Validation 2

Y = 0.0056991+0.00298291*X R^2 = 0.9954 W: 1/X^2

0 20 40 60 80 100 120 140 160 180 200 220

ng/mL

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

Are

a R

atio

Vit D3 - Validation 3

Y = 0.00571591+0.00317597*X R^2 = 0.9946 W: 1/X^2

0 20 40 60 80 100 120 140 160 180 200 220

0.00

0.05

0.10

0.15

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

0.65

0.70

0.75

Are

a R

atio


% Difference

200 ng/mL

100 ng/mL

50 ng/mL

20 ng/mL

10 ng/mL

2 ng/mL

1 ng/mL

Standard

2.18.29.7

0.3-5.30.8

-1.4-5.9-8.6

2.02.17.5

-2.9-4.4-4.3

-0.79.7-10.6

0.6-4.45.4

Validation_3Validation_2Validation_1

Vit D2 – Validation 1

Y = 0.00432057+0.0119399*X R^2 = 0.9919 W: 1/X^2

0 20 40 60 80 100 120 140 160 180 200 220

ng/mL

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

Are

a R

atio


Y = 0.0010089+0.0121633*X R^2 = 0.9943 W: 1/X^2

0 20 40 60 80 100 120 140 160 180 200 220

ng/mL

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

Are

a R

atio


Y = -0.000622327+0.0121577*X R^2 = 0.9996 W: 1/X^2

0 20 40 60 80 100 120 140 160 180 200 220

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

Are

a R

atio

Note: Calibration curve using neat standards

Linearity and Accuracy – 25OH-Vit D2


Lower points in calibration curve – Good ppecificity

1ng_std - m/z= 365.32-365.32 SM: 5 RT: 0.75 - 1.50 NL: 4.36E4F: {0,0} + p APCI Full ms [ 350.00-420.00]

0.8 0.9 1.0 1.1 1.2 1.3 1.4

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lati

ve

In

ten

sit

y

RT: 1.12

1.20

1.470.970.77 0.87

Note:Extracted Ion Chromatogram (XIC)@accuracy of 5 ppm


0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lati

ve

In

ten

sit

y

RT: 1.13

1.321.441.250.78 0.960.880.83


0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lati

ve

In

ten

sit

y

RT: 1.13

1.25 1.411.310.81 1.480.980.89 1.03


0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lati

ve

In

ten

sit

y

RT: 1.16

1.29

1.000.86 0.92


0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lativ

e In

ten

sit

y

RT: 1.16

1.411.31 1.351.050.91 0.990.83


0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5

Time (min)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100

Re

lati

ve In

ten

sit

y

RT: 1.17

1.370.990.86

D3 -1ng/mL D3 -10ng/mLD3 -2ng/mL

D2 -1ng/mLD2 -10ng/mL

D2 -2ng/mLLOQ

LOQ

%RSD>20%


QC Results - 25OH-Vit D3 inter and intra assay

2.25.86.5%RSD

%Rec

Mean

5

4

3

2

1

Replicate

QC1 (22 ng/mL)

19.323.121.5

20.021.620.7

NDNDND

20.422.622.1

19.920.919.0

20.119.921.3

20.321.519.6

Val 3Val 2Val 1

%Rec

%RSD

Mean

5

4

3

2

1

Replicate

QC2 (50 ng/mL)

49.452.751.9

47.354.749.6

49.153.953.5

47.754.955.8

Val 3Val 2Val 1

98.9105104

6.34.76.4

54.949.753.1

48.250.247.4

%Rsc

%RSD

Mean

5

4

3

2

1

Replicate

QC3 (200 ng/mL)

205215212

207213220

204212200

201212218

Val 3Val 2Val 1

102108106

1.52.33.8

209224215

204215209

Excellent accuracy, precision and recovery


%Rec

SD

Mean

5

4

3

2

1

Replicate

QC1 (5 ng/mL)

4.44.94.9

4.44.64.8

0.280.400.37

87.493.096.9

4.44.85.3

3.95.14.2

4.74.14.8

4.34.45.0

Val 3Val 2Val1

%Rec

%RSD

Mean

5

4

3

2

1

Replicate

QC2 (50 ng/mL)

49.348.450.4

48.054.052.2

49.145.054.2

51.648.947.3

Val 3Val 2Val 1

98.696.8101

3.07.36.0

48.148.551.1

49.745.647.3

%Rec

%RSD

Mean

5

4

3

2

1

Replicate

QC3 (200 ng/mL)

202193188

190187191

199188177

184186191

Val 3Val 2Val 1

10196.793.9

8.95.03.4

209209191

230197189

Excellent accuracy, precision and recovery

QC Results - 25OH-Vit D2 inter and intra assay


Blinded Sample Results Comparison


Blinded sample - Results comparison

• Seven blinded samples from service lab analyzed

• Comparison with service labs 6 min TurboFlowTM QQQ method

• 5 replicate analysis on Exactive to asses precision


Blinded samples – Results summary

Good agreement betweenExactive and service lab QQQ results

Note: External service lab used Access QQQ

Vitamin D2Vitamin D3

2.4

2.0

2.3

<1.9

7.2

2.1

<1.9

Clinical Lab (ng/mL)

0.6

-1.2

-12.4

3.5

13.7

2.3

-15.4

% Diff

ND<134.034.2G

ND<125.825.5F

ND<136.332.3E

ND<120.419.7D

67.44.316.614.6C

ND<134.834.0B

ND<133.539.6A

% DiffExactive(ng/mL)


Exactive(ng/mL)

Sample#

Vitamin D2Vitamin D3

2.4

2.0

2.3

<1.9

7.2

2.1

<1.9


0.6

-1.2

-12.4

3.5

13.7

2.3

-15.4

% Diff

ND<134.034.2G

ND<125.825.5F

ND<136.332.3E

ND<120.419.7D

67.44.316.614.6C

ND<134.834.0B

ND<133.539.6A

% DiffExactive(ng/mL)


Exactive(ng/mL)

Sample#

Service lab

ng/mL

Service lab

ng/mL


Chromatograms - Blinded sample A

c:\xcalibur\...\rawfiles\sample_a5 7/13/2009 4:47:50 PM

RT: 0.00 - 2.02 SM: 5B

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

Time (min)

0

50

100

0

50

100

0

50

100

Re

lati

ve

Ab

un

da

nc

e

0

50

100

0

50

100

RT: 1.14AA: 2580930

RT: 0.09AA: 10925

RT: 0.37AA: 21678 0.93 1.01 1.991.44 1.491.38 1.57 1.680.78 1.83 1.870.21 0.49 0.720.650.61

RT: 1.14MA: 2040088

1.350.93 1.300.90 1.05 1.911.45 1.540.140.08 1.780.64 0.72 1.720.23 0.36 0.50

RT: 1.15MA: 2178738

1.33 1.370.94 1.950.89 1.480.410.14 1.671.590.35 1.75 1.790.820.22 0.630.45 0.58 0.690.03

RT: 1.15MA: 2205493

0.91 1.361.291.03 1.951.911.51 1.681.63 1.730.05 0.490.20 0.760.27 0.330.09 0.43 0.680.59

RT: 1.14MA: 1964455

0.90 1.05 1.33 1.900.86 1.49 1.821.65 1.690.08 0.14 0.19 0.35 0.55 0.770.59 1.600.48

NL: 5.21E5

m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS ICIS Sample_A1

NL: 4.45E5

m/z= 365.3182-365.3218 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS sample_a2

NL: 4.57E5


NL: 4.72E5


NL: 4.33E5


Exactive39.6 ng/mL

c:\xcalibur\...\rawfiles\sample_a_003 7/14/2009 3:31:06 PM

RT: 0.00 - 2.00 SM: 5B

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Time (min)

0

20

40

60

80

100R

ela

tiv

e A

bu

nd

an

ce

0

20

40

60

80

100

Re

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un

da

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0

20

40

60

80

100

Re

lati

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Ab

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e

RT: 0.84MA: 1076719

0.961.050.74

1.19 1.28 1.46 1.741.67 1.860.09 0.24 0.47 0.540.37

RT: 0.84MA: 1093612

0.970.751.07

1.13 1.18 1.45 1.75 1.801.640.17 1.930.11 0.26 0.54 0.630.38

RT: 0.84MA: 844399

0.970.74 1.071.14 1.19 1.46 1.570.21 1.841.770.39 1.960.11 0.47 0.55

NL: 3.87E5

TIC F: + c APCI SRM ms2 383.280 [257.103-257.153, 365.075-365.125] MS Sample_A_001

NL: 3.70E5

TIC F: + c APCI SRM ms2 383.280 [257.103-257.153, 365.075-365.125] MS sample_a_002

NL: 2.84E5


Access33.5 ng/mL

Vitamin D3

Note: 5 injections on Exactive and 3 injections on Access


Chromatograms - Blinded sample A

Vitamin D2

c:\xcalibur\...\rawfiles\sample_a5 7/13/2009 4:47:50 PM

RT: 0.00 - 2.02 SM: 5B

0.0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2.0

Time (min)

0

50

100

0

50

100

0

50

100

Re

lati

ve

Ab

un

da

nc

e

0

50

100

0

50

1001.25

0.90

1.46 1.52 1.671.421.82 1.89

0.03 0.34 0.670.230.15

1.19

1.151.98

1.94

1.631.46 1.841.760.12 0.680.33 0.430.26 0.56

1.17

0.901.290.87

1.48 1.52 1.671.42 1.76 1.890.550.08 0.710.64 0.760.320.28 0.49

1.98

0.930.88 1.161.94

1.131.551.52 1.63 1.891.760.590.13 0.19 0.750.30 0.490.43

1.97

1.920.87

1.63 1.831.570.52 0.71 0.740.24 0.590.280.180.13

NL: 5.32E3

m/z= 395.3285-395.3325 F: FTMS {0,0} + p APCI Full ms [350.00-420.00] MS Sample_A1

NL: 2.14E4


NL: 3.29E4


NL: 3.53E4


NL: 1.74E4


C:\Xcalibur\...\Rawfiles\Sample_A_001 7/14/2009 3:25:28 PM

RT: 0.00 - 2.00 SM: 5B

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0

Time (min)

0

20

40

60

80

100

Re

lati

ve

Ab

un

da

nc

e

0

20

40

60

80

100

Re

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0

20

40

60

80

100

Re

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0.79

0.84

0.951.00

1.18

1.451.24 1.620.03 1.67 1.84 1.940.460.25 0.620.30

0.81

0.76

0.85

0.94

1.011.06

1.15

1.461.26 1.650.20 1.800.570.02 0.39 1.96

0.79

0.84

0.93

1.011.03

1.15

1.25 1.46 1.59 1.66 1.890.07 0.660.560.26 0.420.15

NL: 8.68E4

TIC F: + c APCI SRM ms2 395.290 [269.093-269.143, 377.175-377.225] MS Sample_A_001

NL: 8.68E4


NL: 7.09E4


Exactive<LOQ

Access<LOQ

Note: 5 injections on Exactive and 3 injections on Access

See appendix for other 6 samples


Appendix

25 OH Vit D Analysis


Reagents

• 25-OH-D3 was purchased from Cerilliant, 25-OH-D2 was purchased

from Sigma. Both compounds were stored in -20 deg.C.

• [2H6]-25-OH-D3 (Internal Standard) was purchased from Medical

Isotopes and it was stored in -70 deg.C.


Method validation

Endogenous quantity in

blank plasma measured using IS alone

25-OH-D3 = 16.5 ng/mL25-OH-D2 = 0.8 ng/mL

Total quantity in

QC sample minus =

Intra assay variability - 5 replicates of each QC sampleInter assay variability - 5 replicates of each QC sample in 3

different batches

Confirm method accuracy

Actual spiked

amount in QC sample


25-OH-Vit D - Ionization source parameters

Exactive Mass Spectrometer

Ionization APCI, positive, Ion Max™ source,

Discharge current 5.0

Vaporizer temperature (deg C) 390

Sheath gas 15

Ion Sweep Gas Pressure: 0.0

Aux gas 5

Capillary temperature (deg C) 275


25-OH-Vit D - Exactive Method

Polarity: Positive

Microscans: 1Resolution: Ultra High (100 000)Target: BalancedMax Inject time: 250 Scan range: 350-420

Divert valve: switch events 4

1-0.95 min – to waste0.95-1.4 – to detector1.4-1.9 – to waste1.9-2.0 – to detector


25-OH-Vit D - LC method

• LC Conditions• Surveyor autosampler and pump

• Mobile Phase• A: water containing 0.1% formic acid

• B: methanol containing 0.1% formic acid

• Column• 50 x 2.1 mm id packed with 3 µm,

C18-aQ stationary phase

• Injection volume: 50 µL

• Flow rate: 700 µL/min

• GradientTime (min) % A %B

0 20 80

0.1 20 80

0.2 5 95

1.4 5 95

1.5 20 80

2.0 20 80


25-OH-Vit D - Autosampler method

quantitative analysis of vit d metabolites in human plasma ... quantitation of 25-oh-vit d2 and...

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