ACE UHPLC and HPLC Columns
Vi1,25-Dihydroxyvitamins D2 and D3 in Serum by LC-MS/MS
XIC of +MRM (3 pairs): 635.400/314.100 Da ID: 1,25-diOH D2 from Sample 21 (Vit D Free 0.5) of 20151027_standards.wiff (Turbo Spray) Max. 1.8e4 cps.
1.8e4
1.7e4
1.6e4
1.5e4
1.5e4
1.5e4
1.4e4
1.3e4
1.2e4
1.1e4
1.0e4
1.0e4
1.0e4
9000.0
8000.0
7000.0
6000.0
5.0e3
5.0e3
5000.0
4000.0
3000.0
2000.0
1000.0
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 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
40 pg/mL 500 pg/mL
1 3 1 3
1.7e4
1.7e4
1.6e4
1.6e4
1.5e4
1.5e4
1.4e4
1.4e4
1.3e4
1.3e4
1.2e4
1.2e4
1.1e4
1.1e4
1.0e4
9500.0
9000.0
8500.0
8000.0
7500.0
7000.0
6500.0
6000.0
5500.0
5000.0
4500.0
4000.0
3500.0
3000.0
2500.0
2000.0
1500.0
1000.0
500.0
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Time - Minutes
Time, min
0.0
Intensity, cps
1.41
0.0
0.0 1.0 2.0 3.0
Application #AN4070
Conditions Analytes
Column: ACE UltraCore 2.5 SuperC18
Dimensions: 50 x 2.1 mm
Part Number: CORE-25A-0502U
Mobile Phase: MeCN/H2O (50:50 v/v) containing 30
μL methylamine per 500 mL
Flow Rate: 0.5 mL/min
Injection: 20 μL
Temperature: 40 °C
Detection: AB Sciex 5500 triple quad MS
ESI in positive ion mode
IonSpray Voltage: 5500 V
Temperature: 550 °C
Sample: 1,25 diOH vitamin D2 and 1,25 diOH vitamin D3 metabolites are extracted from serum
using supported liquid extraction. Sensitivity of LC-MS/MS analysis is maximised through
use of PTAD (9-phenyl-1,2,4-triazole-3,5-dione) derivatisation and complexation with methylamine.
2
1
0.0 1.0
0.0
2.0 3.0
3
1. 1,25-diOH vitamin D3-PTAD-methylamine complex
(m/z 623.4 314.1)
2. d3-1,25-diOH vitamin D3-PTAD-methylamine complex (I.S.)
(m/z 626.4 317.1)
3. 1,25-diOH vitamin D2-PTAD-methylamine complex
(m/z 635.4 314.1)
1,25-dihydroxy vitamin D3
1,25-dihydroxy vitamin D2
High calibration standard
(500 pg/mL) in vitamin D
stripped serum
Low and high calibration standards in charcoal stripped (vitamin D free) serum,
analysed as PTAD-methylamine complexes
Sample Name: "Vit D Free 0.04" Sample ID: "" File: "20151027_standards.wiff"
Peak Name: "1,25-diOH D2" Mass(es): "635.400/314.100 Da"
Comment: "" Annotation: ""
Sample Index: 13
Sample Type: Standard
Concentration: 0.0400 ng/mL
Calculated Conc: 0.0383 ng/mL
Acq. Date: 10/27/2015
Acq. Time: 5:40:08 PM
Modified: Yes
Proc. Algorithm: IntelliQuan - IQA II
Min. Peak Height: 0.00 cps
Min. Peak Width: 0.00 sec
Smoothing Width: 7 points
RT Window: 30.0 sec
Expected RT: 1.47 min
Use Relative RT: No
Int. Type: Manual
Retention Time: 1.41 min
Area: 8.11e+003 counts
Height: 1.56e+003 cps
Start Time: 1.32 min
End Time: 1.59 min
1500
1450
1400
1350
1300
1250
1200
1150
1100
1050
1000
950
900
850
800
750
700
650
600
550
500
450
400
350
300
250
200
150
100
50
1.10e4
1.08e4
1.06e4
1.04e4
1.02e4
1.00e4
9800.00
9600.00
9400.00
9200.00
9000.00
8800.00
8600.00
8400.00
8200.00
8000.00
7800.00
7600.00
7400.00
7200.00
7000.00
6800.00
6600.00
6400.00
6200.00
6000.00
5800.00
5600.00
5400.00
5200.00
5000.00
4800.00
4600.00
4400.00
4200.00
4000.00
3800.00
3600.00
3400.00
3200.00
3000.00
2800.00
2600.00
2400.00
2200.00
2000.00
1800.00
1600.00
1400.00
1200.00
1000.00
800.00
600.00
400.00
200.00
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 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time, min
0
In te n s ity , cp s
1.41
Sample Name: "Vit D Free 0.5" Sample ID: "" File: "20151027_standards.wiff"
Peak Name: "1,25-diOH D2" Mass(es): "635.400/314.100 Da"
Comment: "" Annotation: ""
Sample Index: 20
Sample Type: Standard
Concentration: 0.500 ng/mL
Calculated Conc: 0.492 ng/mL
Acq. Date: 10/27/2015
Acq. Time: 6:18:14 PM
Modified: No
Proc. Algorithm: IntelliQuan - IQA II
Min. Peak Height: 0.00 cps
Min. Peak Width: 0.00 sec
Smoothing Width: 7 points
RT Window: 30.0 sec
Expected RT: 1.47 min
Use Relative RT: No
Int. Type: Valley
Retention Time: 1.41 min
Area: 8.91e+004 counts
Height: 1.75e+004 cps
Start Time: 1.32 min
End Time: 1.76 min
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 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time, min
0.0
In te n s ity , cp s
1.41
Sample Name: "Vit D Free 0.04" Sample ID: "" File: "20151027_standards.wiff"
Peak Name: "1,25-diOH D3" Mass(es): "623.400/314.100 Da"
Comment: "" Annotation: ""
Sample Index: 12
Sample Type: Standard
Concentration: 0.0400 ng/mL
Calculated Conc: 0.0419 ng/mL
Acq. Date: 10/27/2015
Acq. Time: 5:34:40 PM
Modified: Yes
Proc. Algorithm: IntelliQuan - IQA II
Min. Peak Height: 0.00 cps
Min. Peak Width: 0.00 sec
Smoothing Width: 9 points
RT Window: 30.0 sec
Expected RT: 1.19 min
Use Relative RT: No
Int. Type: Manual
Retention Time: 1.14 min
Area: 3.67e+003 counts
Height: 1.02e+003 cps
Start Time: 1.07 min
End Time: 1.24 min
1000
980
960
940
920
900
880
860
840
820
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760
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60
40
20
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 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time, min
0
In te n s ity , cp s
1.14
0.86
Sample Name: "Vit D Free 0.5" Sample ID: "" File: "20151027_standards.wiff"
Peak Name: "1,25-diOH D3" Mass(es): "623.400/314.100 Da"
Comment: "" Annotation: ""
Sample Index: 20
Sample Type: Standard
Concentration: 0.500 ng/mL
Calculated Conc: 0.501 ng/mL
Acq. Date: 10/27/2015
Acq. Time: 6:18:14 PM
Modified: No
Proc. Algorithm: IntelliQuan - IQA II
Min. Peak Height: 0.00 cps
Min. Peak Width: 0.00 sec
Smoothing Width: 9 points
RT Window: 30.0 sec
Expected RT: 1.19 min
Use Relative RT: No
Int. Type: Valley
Retention Time: 1.14 min
Area: 4.12e+004 counts
Height: 1.13e+004 cps
Start Time: 1.08 min
End Time: 1.33 min
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 3.0 3.2 3.4 3.6 3.8 4.0 4.2 4.4 4.6 4.8 5.0
Time, min
0.00
In te n s ity , cp s
1.14
Reproduced with permission of Biotage GB Ltd, UK. For further details of sample preparation see Biotage App Note AN857