evaluation of bench-top quadrupole orbitrap ultra...
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Poster Note 64374
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma
Mindy Gao and Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
2 Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
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121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
Purpose To evaluate a rapid quantitative analysis of Vitamin D in human plasma samples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
Introduction Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™
Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
Instruments LC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
Methods Calibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ). 2.Vortex, centrifuge 3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Column: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all data processing and analysis.
Method Performance Evaluation QC samples were prepared by spiking previously analyzed donor plasma to concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
Results FIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma Mindy Gao, Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase) 0 0.70 20 0.2 0.70 20 0.3 0.70 5 1.5 0.70 5 1.6 0.70 20 2.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
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395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
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NL: 7.56E4m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
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NL: 1.54E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
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NL: 2.38E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL 20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff 3.98 -0.450 9.97 -0.280 26.1 4.50 50.1 0.120 96.1 -3.88
Calculated Amt %Diff 3.96 -1.10 10.2 1.95 26.3 5.08 46.7 -6.58 101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive 25OHD3 383.3308 257.2264 15 Positive D6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
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NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
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NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of each calibration standard (Table 6). TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mL and calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentration ng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.0 25-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
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383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
Purpose To evaluate a rapid quantitative analysis of Vitamin D in human plasma samples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
Introduction Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™
Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
Instruments LC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
Methods Calibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ). 2.Vortex, centrifuge 3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Column: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all data processing and analysis.
Method Performance Evaluation QC samples were prepared by spiking previously analyzed donor plasma to concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
Results FIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma Mindy Gao, Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase) 0 0.70 20 0.2 0.70 20 0.3 0.70 5 1.5 0.70 5 1.6 0.70 20 2.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
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395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
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NL: 1.99E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
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NL: 3.63E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
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NL: 2.38E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL 20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff 3.98 -0.450 9.97 -0.280 26.1 4.50 50.1 0.120 96.1 -3.88
Calculated Amt %Diff 3.96 -1.10 10.2 1.95 26.3 5.08 46.7 -6.58 101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive 25OHD3 383.3308 257.2264 15 Positive D6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
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NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
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NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of each calibration standard (Table 6). TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mL and calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentration ng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.0 25-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
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35
40
45
50
55
60
65
70
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80
85
90
95
100
Re
lativ
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bu
nd
an
ce
383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
Purpose To evaluate a rapid quantitative analysis of Vitamin D in human plasma samples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
Introduction Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™
Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
Instruments LC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
Methods Calibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ). 2.Vortex, centrifuge 3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Column: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all data processing and analysis.
Method Performance Evaluation QC samples were prepared by spiking previously analyzed donor plasma to concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
Results FIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma Mindy Gao, Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase) 0 0.70 20 0.2 0.70 20 0.3 0.70 5 1.5 0.70 5 1.6 0.70 20 2.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
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100
Re
lativ
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bu
nd
an
ce
395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
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1001.17
1.13
1.13
NL: 7.56E4m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
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1001.18
1.12
1.12
NL: 1.54E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
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ativ
e A
bund
ance
0
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90
1001.17
1.12
1.12
NL: 2.38E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL 20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff 3.98 -0.450 9.97 -0.280 26.1 4.50 50.1 0.120 96.1 -3.88
Calculated Amt %Diff 3.96 -1.10 10.2 1.95 26.3 5.08 46.7 -6.58 101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive 25OHD3 383.3308 257.2264 15 Positive D6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
20
40
60
80
1000
20
40
60
80
100
Rela
tive
Abun
danc
e
0
20
40
60
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100 1.18
1.13
1.14
NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
20
40
60
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1000
20
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60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
100 1.18
1.13
1.14
NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of each calibration standard (Table 6). TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mL and calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentration ng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.0 25-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
3Thermo Scientific Poster Note • MASCL • PN64374-EN 0315S
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
Purpose To evaluate a rapid quantitative analysis of Vitamin D in human plasma samples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
Introduction Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™
Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
Instruments LC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
Methods Calibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ). 2.Vortex, centrifuge 3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Column: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all data processing and analysis.
Method Performance Evaluation QC samples were prepared by spiking previously analyzed donor plasma to concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
Results FIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma Mindy Gao, Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase) 0 0.70 20 0.2 0.70 20 0.3 0.70 5 1.5 0.70 5 1.6 0.70 20 2.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
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60
70
80
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1000
10
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40
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60
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lativ
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0
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50
60
70
80
90
1001.17
1.13
1.13
NL: 7.56E4m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
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1001.18
1.12
1.12
NL: 1.54E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
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ativ
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ance
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40
50
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90
1001.17
1.12
1.12
NL: 2.38E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL 20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff 3.98 -0.450 9.97 -0.280 26.1 4.50 50.1 0.120 96.1 -3.88
Calculated Amt %Diff 3.96 -1.10 10.2 1.95 26.3 5.08 46.7 -6.58 101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive 25OHD3 383.3308 257.2264 15 Positive D6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
20
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60
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1000
20
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100
Rela
tive
Abun
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1.13
1.14
NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
20
40
60
80
1000
20
40
60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
100 1.18
1.13
1.14
NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of each calibration standard (Table 6). TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mL and calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentration ng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.0 25-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
Purpose To evaluate a rapid quantitative analysis of Vitamin D in human plasma samples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
Introduction Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™
Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
Instruments LC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
Methods Calibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ). 2.Vortex, centrifuge 3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Column: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all data processing and analysis.
Method Performance Evaluation QC samples were prepared by spiking previously analyzed donor plasma to concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
Results FIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma Mindy Gao, Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase) 0 0.70 20 0.2 0.70 20 0.3 0.70 5 1.5 0.70 5 1.6 0.70 20 2.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
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0
10
20
30
40
50
60
70
80
90
1001.17
1.13
1.13
NL: 7.56E4m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
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lativ
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ce
0
10
20
30
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50
60
70
80
90
1001.18
1.12
1.12
NL: 1.54E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0
10
20
30
40
50
60
70
80
90
1001.17
1.12
1.12
NL: 2.38E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL 20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff 3.98 -0.450 9.97 -0.280 26.1 4.50 50.1 0.120 96.1 -3.88
Calculated Amt %Diff 3.96 -1.10 10.2 1.95 26.3 5.08 46.7 -6.58 101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive 25OHD3 383.3308 257.2264 15 Positive D6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
20
40
60
80
1000
20
40
60
80
100
Rela
tive
Abun
danc
e
0
20
40
60
80
100 1.18
1.13
1.14
NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
20
40
60
80
1000
20
40
60
80
100
Relat
ive A
bund
ance
0
20
40
60
80
100 1.18
1.13
1.14
NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of each calibration standard (Table 6). TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mL and calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentration ng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.0 25-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
Purpose To evaluate a rapid quantitative analysis of Vitamin D in human plasma samples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
Introduction Clinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™
Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
Instruments LC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
Methods Calibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ). 2.Vortex, centrifuge 3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanol Column: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all data processing and analysis.
Method Performance Evaluation QC samples were prepared by spiking previously analyzed donor plasma to concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
Results FIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human Plasma Mindy Gao, Marta Kozak Thermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase) 0 0.70 20 0.2 0.70 20 0.3 0.70 5 1.5 0.70 5 1.6 0.70 20 2.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Re
lativ
e A
bu
nd
an
ce
0
10
20
30
40
50
60
70
80
90
1001.17
1.13
1.13
NL: 7.56E4m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
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30
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50
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lativ
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an
ce
0
10
20
30
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50
60
70
80
90
1001.18
1.12
1.12
NL: 1.54E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
20
30
40
50
60
70
80
90
1000
10
20
30
40
50
60
70
80
90
100
Rel
ativ
e A
bund
ance
0
10
20
30
40
50
60
70
80
90
1001.17
1.12
1.12
NL: 2.38E5m/z= 269.1848-269.1948 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL 20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff 3.98 -0.450 9.97 -0.280 26.1 4.50 50.1 0.120 96.1 -3.88
Calculated Amt %Diff 3.96 -1.10 10.2 1.95 26.3 5.08 46.7 -6.58 101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive 25OHD3 383.3308 257.2264 15 Positive D6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
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NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
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NL: 2.66E4m/z= 269.1885-269.1911 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00] MS 021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F: FTMS + c APCI corona Full ms2 [email protected] [50.00-415.00] MS 021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of each calibration standard (Table 6). TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mL and calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentration ng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.0 25-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
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www.thermofisher.com©2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries This information is presented as an example of the capabilities of Thermo Fish
er Scientific products. It is not intended to encourage use of these products
in any manners that might infringe the intellectual property rights of others. Specifications, terms and pricing are subject to change. Not all products are available in all countries. Please consult your local sales representative for details.
For research use only. Not for use in diagnostic procedures.
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
50 100 150 200 250 300 350 400m/z
0
5
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383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
PurposeTo evaluate a rapid quantitative analysis of Vitamin D in human plasmasamples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
IntroductionClinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
InstrumentsLC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
MethodsCalibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ).2.Vortex, centrifuge3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanolColumn: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all dataprocessing and analysis.
Method Performance EvaluationQC samples were prepared by spiking previously analyzed donor plasmato concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
ResultsFIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human PlasmaMindy Gao, Marta KozakThermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase)0 0.70 200.2 0.70 200.3 0.70 51.5 0.70 51.6 0.70 202.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
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lativ
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bu
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an
ce
395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
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1001.17
1.13
1.13
NL: 7.56E4m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
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1.12
NL: 1.54E5m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
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ance
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1001.17
1.12
1.12
NL: 2.38E5m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff3.98 -0.4509.97 -0.28026.1 4.5050.1 0.12096.1 -3.88
Calculated Amt %Diff3.96 -1.1010.2 1.9526.3 5.0846.7 -6.58101 0.640
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.30 25-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery 25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 106 25-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3 %RSD
25-Hydroxyvitamin D2 10.5 9.01 4.51 25-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery 25-Hydroxyvitamin D2 93.2 105 104 25-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive25OHD3 383.3308 257.2264 15 PositiveD6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100 %RSD
25OHD2 1.60 2.52 4.27 1.65 2.71 25OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery 25OHD2 99.7 101 96.8 105 96.9 25OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5%RSD
25OHD2 3.65 5.36 9.17 5.90 10.625OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.325OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
0
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1000
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100
Rela
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1.14
NL: 2.66E4m/z= 269.1885-269.1911 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
0.8 0.9 1.0 1.1 1.2 1.3 1.4 1.5Time (min)
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ive A
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1.14
NL: 2.66E4m/z= 269.1885-269.1911 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of eachcalibration standard (Table 6).
TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mLand calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentrationng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.025-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
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85
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95
100
Re
lativ
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bu
nd
an
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383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to 100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limited matrix effects we observed during method evaluation.
PurposeTo evaluate a rapid quantitative analysis of Vitamin D in human plasmasamples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
IntroductionClinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
InstrumentsLC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
MethodsCalibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ).2.Vortex, centrifuge3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanolColumn: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all dataprocessing and analysis.
Method Performance EvaluationQC samples were prepared by spiking previously analyzed donor plasmato concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
ResultsFIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human PlasmaMindy Gao, Marta KozakThermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase)0 0.70 200.2 0.70 200.3 0.70 51.5 0.70 51.6 0.70 202.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
50 100 150 200 250 300 350 400m/z
0
5
10
15
20
25
30
35
40
45
50
55
60
65
70
75
80
85
90
95
100
Re
lativ
e A
bu
nd
an
ce
395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
0
10
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100R
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Ab
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nce
0
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90
1001.17
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1.13
NL: 7.56E4m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
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NL: 1.54E5m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
0.0 0.5 1.0 1.5 2.0Time (min)
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1001.17
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NL: 2.38E5m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff3.98 -0.4509.97 -0.28026.1 4.5050.1 0.12096.1 -3.88
Calculated Amt %Diff3.96 -1.1010.2 1.9526.3 5.0846.7 -6.58101 0.640
Analyte QC1 QC2 QC3%RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.3025-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 10625-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3%RSD
25-Hydroxyvitamin D2 10.5 9.01 4.5125-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery25-Hydroxyvitamin D2 93.2 105 10425-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive25OHD3 383.3308 257.2264 15 PositiveD6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100%RSD
25OHD2 1.60 2.52 4.27 1.65 2.7125OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery25OHD2 99.7 101 96.8 105 96.925OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5 %RSD
25OHD2 3.65 5.36 9.17 5.90 10.6 25OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.3 25OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
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NL: 2.66E4m/z= 269.1885-269.1911 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
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NL: 2.66E4m/z= 269.1885-269.1911 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of eachcalibration standard (Table 6).
TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mLand calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentrationng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.025-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *
TABLE 4. Intra-assay Precision and Accuracy *
021715_APCI_4xP_D1_Cal_7 #752 RT: 1.13 AV: 1 NL: 5.82E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-410.00]
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383.3298
365.3195
257.2257
121.1012245.225695.0857 135.1167 159.1164 191.1792 269.2241 309.256669.0704
225.1631
327.2668
Conclusion Method meets clinical research requirements for analysis of Vitamin D
25-hydroxy metabolites in plasma samples.
The calibration range used in method evaluation experiments was 1 to100 ng/mL, but for improved method robustness, recommended concentration range is 4-100 ng/mL.
Deuterated analog of 25OHD2 is recommended to correct for limitedmatrix effects we observed during method evaluation.
PurposeTo evaluate a rapid quantitative analysis of Vitamin D in human plasmasamples using a bench-top quadrupole orbitrap high resolution mass spectrometer for use in clinical research.
IntroductionClinical researchers commonly use a triple quadrupole mass spectrometer for analysis of 25-hydroxyvitamin D2 (25OHD2) and 25-hydroxyvitamin D3 (25OHD3) in plasma. We evaluated the Thermo Scientific™ Q Exactive™Focus mass spectrometer equipped with Thermo Scientific™ Orbitrap™ technology using a fast, cost-efficient method that collected high resolution MS/MS spectra for improved method specificity. Protein precipitated plasma samples were analyzed with 3 min LC method, and high resolution MS/MS spectra were collected for each analyte. The most abundant fragment, besides water-loss, in each MS2 spectrum was selected for quantitative analysis (Table 3, Figure 1). The linearity range was 4-100 ng/mL (Figure 3, Figure 4); precision and accuracy were within 15%; matrix effects and interferences were not observed.
InstrumentsLC system
Pump: Thermo Scientific TM Dionex TM Ultimate TM 3000 Autosampler: Thermo Scientific TM OAS-3X00TXRS
Q Exactive Focus orbitrap mass spectrometer
MethodsCalibrators
25-hydroxyvitamin D2 (25OHD2), 25-hydroxyvitamin D3 (25OHD3) were purchased from Cerillant Corporation. Since analyte free matrix was not available, calibration standards were prepared in ethanol. NIST controls and spiked plasma recovery data were used to prove calibration curve accuracy.
Sample Preparation
1.100 µL of plasma sample + 300 uL of acetonitrile containing internal standard (25 ng/mL d6-25-hydroxyvitamin D3 ).2.Vortex, centrifuge3.Inject 50 µL into LC-MS system.
LC method
Mobile phase A: 0.1% formic acid in water Mobile phase B: 0.1% formic acid in methanolColumn: Thermo Scientific TM Hypersil GOLDM aQ 5µ 50x2.1mm
TABLE 1. HPLC Gradient Method
Mass Spectrometry Method
APCI ionization source
Data acquisition method: parallel reaction monitoring (PRM) experiment collecting MS2 spectra for each analyte.
Resolution: 17.5K
Data Analysis
Thermo Scientific™ TraceFinder 3.2 software was used for all dataprocessing and analysis.
Method Performance EvaluationQC samples were prepared by spiking previously analyzed donor plasmato concentrations specified in Table 2 and Figure 2.
TABLE 2. QC Samples Concentrations
ResultsFIGURE 1. 25OHD2, 25OHD3 MS/MS Spectrum
FIGURE 2. Chromatogram of QC Plasma Samples
QC1 QC2 QC3
25OHD2
25OHD3
6-25OHD3
(IS)
FIGURE 3. Calibration Curves with Lowest Calibration Standard Peaks
25OHD2
4.0 ng/mL
25OHD3
4.0 ng/mL
d6-25OHD3
(IS), 25 ng/mL
©2015 Thermo Fisher Scientific. All Rights Reserved.
All other trademarks are the property of Thermo Fisher Scientific and its subsidiaries.
This information is not intended to encourage use of these products in any manners that might infringe the intellectual property rights of others.
Evaluation of Bench-top Quadrupole Orbitrap Ultra High Resolution MS for Use in Clinical Research in Rapid Quantitative Analysis of Vitamin D in Human PlasmaMindy Gao, Marta KozakThermo Fisher Scientific, San Jose, CA, USA
For research use only. Not for use in diagnostic procedures.
Time (min) Flow rate (mL/min) %A (mobile phase)0 0.70 200.2 0.70 200.3 0.70 51.5 0.70 51.6 0.70 202.0 0.70 20
021715_APCI_4xP_D1_Cal_7 #787 RT: 1.17 AV: 1 NL: 3.64E6F: FTMS + c APCI corona Full ms2 [email protected] [50.00-420.00]
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395.3300
377.3193
269.1893
83.0860229.1583119.0856
209.1322251.1792135.1168 159.1163 185.1320 307.2419
279.2106
335.2729
Water lost fragment
Quantifier
25OHD2
Quantifier
Water lost fragment
25OHD3
RT: 0.00 - 2.00 SM: 5B
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NL: 7.56E4m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaL_5
NL: 1.99E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaL_5
NL: 2.07E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaL_5
RT: 0.00 - 2.00 SM: 5B
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NL: 1.54E5m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaM_2
NL: 3.63E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaM_2
NL: 2.65E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaM_2
RT: 0.00 - 2.00 SM: 5B
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NL: 2.38E5m/z= 269.1848-269.1948 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_PlasmaH_2
NL: 5.64E5m/z= 257.2214-257.2314 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_PlasmaH_2
NL: 2.62E6m/z= 263.2588-263.2688 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_PlasmaH_2
10.0 ng/mL 30.0 ng/mL20.0 ng/mL
Collision energy (20 eV) was optimized to maximize response of quantifying fragment ion (m/z=269.1893)
Collision energy (15 eV) was optimized to maximize response of quantifying fragment ion (m/z=257.2257)
25.1 ng/mL 39.9 ng/mL 60.8 ng/mL
4 ng/mL 4 ng/mL
Calculated Amt %Diff3.98 -0.4509.97 -0.28026.1 4.5050.1 0.12096.1 -3.88
Calculated Amt %Diff3.96 -1.1010.2 1.9526.3 5.0846.7 -6.58101 0.640
Analyte QC1 QC2 QC3%RSD
25-Hydroxyvitamin D2 6.30 - 12.7 5.33 - 13.0 2.77 - 5.3025-Hydroxyvitamin D3 3.21 - 7.64 2.64 - 5.89 3.70 - 4.70
%Recovery25-Hydroxyvitamin D2 87.0 - 100 99.9 - 109 102 - 10625-Hydroxyvitamin D3 82.9 - 83.5 94.5 - 96.3 91.1 - 101
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Analyte QC1 QC2 QC3%RSD
25-Hydroxyvitamin D2 10.5 9.01 4.5125-Hydroxyvitamin D3 5.56 3.93 6.1
%Recovery25-Hydroxyvitamin D2 93.2 105 10425-Hydroxyvitamin D3 82.9 95.1 97.3
* 5 replicates of QC samples containing 25OHD2 and 25OHD3 were proceed and analyzed in 3 batches
Compound Precursor (m/z)
Quantitation Fragment (m/z)
Collision Energy (V) Polarity
25OHD2 395.3308 269.1898 20 Positive25OHD3 383.3308 257.2264 15 PositiveD6-25OHD3 (IS) 389.3685 263.2638 20 Positive
Analyte Cal-4 Cal-10 Cal-25 Cal-50 Cal-100%RSD
25OHD2 1.60 2.52 4.27 1.65 2.7125OHD3 4.72 1.37 4.77 2.01 3.86
%Recovery25OHD2 99.7 101 96.8 105 96.925OHD3 100 101 97.0 98.8 103
* 5 replicate injections of each calibration standard containing 25OHD2 and 25OHD3 were performed to demonstrate the system reproducibility
Analyte Plasma-1 Plasma-2 Plasma-3 Plasma-4 Plasma-5%RSD
25OHD2 3.65 5.36 9.17 5.90 10.625OHD3 2.70 3.48 1.09 4.31 6.99
%Recovery 25OHD2 77.2** 91.7 79.9** 86.5 85.325OHD3 112 104 106 109 111
* 15 ng/mL of each analyte was spiked in 5 different plasma samples in 5 replicates
** 25OHD2 % recovery will be increased when deuterated analog will be used as internal standard.
FIGURE 4. Chromatogram of Lowest Calibration Standard
RT: 0.80 - 1.50 SM: 5B
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NL: 2.66E4m/z= 269.1885-269.1911 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_Cal_3
RT: 0.80 - 1.50 SM: 5B
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NL: 2.66E4m/z= 269.1885-269.1911 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-420.00] MS021715_APCI_4xP_D1_Cal_3
NL: 4.23E4m/z= 257.2251-257.2277 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-410.00] MS021715_APCI_4xP_D1_Cal_3
NL: 2.24E6m/z= 263.2625-263.2651 F:FTMS + c APCI corona Fullms2 [email protected] [50.00-415.00] MS021715_APCI_4xP_D1_Cal_3
Chromatogram reconstructed with mass accuracy of 5 ppm
System reproducibility was evaluated by analyzing 5 replicates of eachcalibration standard (Table 6).
TABLE 3. PRM transitions of targets and internal standards
Method accuracy was evaluated by obtaining %recovery for NIST controls.
Method precision was evaluated by analyzing 5 replicates of each QC sample in 3 different days (Table 4, Table 5).
Matrix effects were evaluated by spiking previously analyzed plasma from 5 different donors with 25OHD3 and 25OHD2 concentrations of 15 ng/mLand calculating % RSD and % recovery from 5 replicate analysis (Table 7).
Analyte/Concentrationng/mL QC1 QC2 QC3
25-Hydroxyvitamin D2 10.0 20.0 30.025-Hydroxyvitamin D3 25.1 39.9 60.8
TABLE 7. Matrix Effects *
TABLE 6. System Reproducibility *
TABLE 5. Inter-assay Precision and Accuracy *