Serum Vitamin D Metabolites by HPLC-MS/MS Combined with Differential Ion Mobility Spectrometry: Aspects of Sample Preparation without Derivatization
Abstract
:1. Introduction
2. Results
2.1. Principally Different Sample Preparation Approaches
2.2. Vitamin D3 Issue Sample Preparation Selection
2.3. LLE Followed by SPE Procedure
2.3.1. The Fragmentation Patterns
2.3.2. The SelexION Advantage
2.4. LLE Followed by SPE Procedure: Partial Validation, DEQAS Controls
3. Discussion
4. Materials and Methods
4.1. Reagents and Materials
4.2. LC–MS/MS Analysis
Time, Min | Acetonitrile, % | Methanol, % | Formic Acid in Water, % |
---|---|---|---|
0 | 0 | 50 | 50 |
1 | 0 | 50 | 50 |
1.5 | 0 | 60 | 40 |
2 | 0 | 60 | 40 |
3.2 | 0 | 70 | 30 |
4.4 | 3 | 68 | 29 |
4.8 | 5 | 70 | 25 |
5.0 | 5 | 70 | 25 |
6.0 | 5 | 75 | 20 |
6.5 | 0 | 85 | 15 |
8.0 | 0 | 85 | 15 |
8.5 | 0 | 50 | 50 |
12 | 0 | 50 | 50 |
4.3. MALDI Mass Spectrometry
4.4. Sample Preparation Procedures
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Analyte | Concentration, ng/mL 1 | Inter-Day Precision, % | Accuracy, % | IS Normalized MF 2 (% CV) | Analyte Recovery, % (% CV) | IS Recovery, % (% CV) |
---|---|---|---|---|---|---|
1,25(OH)2D3 | 30 | 7.0 | 87.5 | 1.02 (9.7) | 95 (6.5) | 71 (2.3) |
225 | 3.6 | 95.9 | 0.90 (11.1) | 90 (7.7) | 73 (5.0) | |
24,25(OH)2D3 | 0.96 | 2.2 | 92.8 | 0.91 (2.0) | 79 (4.5) | 66 (4.0) |
7.2 | 3.7 | 92.2 | 0.99 (10.5) | 84 (3.8) | 70 (3.9) | |
25(OH)D3 | 7.5 | 3.5 | 95.4 | 0.89 (5.2) | 78 (5.9) | 67 (5.3) |
56.3 | 1.6 | 113.0 | 0.89 (10.2) | 82 (5.7) | 65 (5.2) | |
3-epi-25(OH)D3 | 2.25 | 3.7 | 89.6 | 0.94 (6.9) | 82 (7.2) | 68 (7.5) |
16.9 | 1.4 | 96.8 | 0.90 (10.4) | 76 (7.8) | 61 (8.8) | |
25(OH)D2 | 1.4 | 6.1 | 95.6 | 0.81 (3.8) | 77 (8.8) | 67 (5.3) 3 |
10.8 | 5.9 | 100.8 | 0.79 (10.0) | 81 (10.1) | 65 (5.2) 3 | |
D3 | 37.5 | 5.5 | 90.2 | 0.98 (13.6) | 70 (12.4) | 62 (10.0) |
281 | 4.1 | 91.0 | 1.07 (9.3) | 72 (9.0) | 58 (9.2) |
Analyte | Transition Type | Q1 | Q3 | tR, min | CE, V | DP, V | CXP, V |
---|---|---|---|---|---|---|---|
1,25(OH)2D3 | quantifier | 399.3 | 135.1 | 4.9 | 28 | 89 | 16 |
qualifier | 399.3 | 381.3 | 19 | 89 | 14 | ||
24,25(OH)2D3 | quantifier | 417.3 | 399.3 | 4.7 | 13 | 66 | 15 |
qualifier | 417.3 | 381.3 | 15 | 66 | 14 | ||
25(OH)D3 | quantifier | 401.3 | 383.3 | 6.1 | 13 | 59 | 15 |
qualifier | 401.3 | 365.4 | 17 | 59 | 13 | ||
3-epi-25(OH)D3 | quantifier | 401.29 | 383.2 | 6.3 | 14 | 110 | 9 |
qualifier | 401.29 | 365.3 | 17 | 110 | 9 | ||
25(OH)D2 | quantifier | 413.3 | 355.3 | 6.3 | 15 | 110 | 7 |
qualifier | 413.3 | 395.3 | 13 | 110 | 7 | ||
D3 | quantifier | 385.4 | 259.3 | 7.7 | 20 | 100 | 30 |
qualifier | 385.4 | 159.2 | 32 | 100 | 18 | ||
1,25(OH)2D3-d6 | IS | 405.3 | 135.0 | 4.9 | 30 | 170 | 12 |
24,25(OH)2D3-d6 | IS | 423.3 | 387.5 | 4.7 | 16 | 150 | 7 |
25(OH)D3-d6 | IS | 407.4 | 389.3 | 6.1 | 12 | 120 | 11 |
3-epi-25(OH)D3-d3 | IS | 404.4 | 368.3 | 6.3 | 18 | 150 | 6 |
D3-d7 | IS | 392.4 | 266.3 | 7.7 | 20 | 90 | 30 |
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Usoltseva, L.; Ioutsi, V.; Panov, Y.; Antsupova, M.; Rozhinskaya, L.; Melnichenko, G.; Mokrysheva, N. Serum Vitamin D Metabolites by HPLC-MS/MS Combined with Differential Ion Mobility Spectrometry: Aspects of Sample Preparation without Derivatization. Int. J. Mol. Sci. 2023, 24, 8111. https://doi.org/10.3390/ijms24098111
Usoltseva L, Ioutsi V, Panov Y, Antsupova M, Rozhinskaya L, Melnichenko G, Mokrysheva N. Serum Vitamin D Metabolites by HPLC-MS/MS Combined with Differential Ion Mobility Spectrometry: Aspects of Sample Preparation without Derivatization. International Journal of Molecular Sciences. 2023; 24(9):8111. https://doi.org/10.3390/ijms24098111
Chicago/Turabian StyleUsoltseva, Liliia, Vitaliy Ioutsi, Yuriy Panov, Mariya Antsupova, Liudmila Rozhinskaya, Galina Melnichenko, and Natalia Mokrysheva. 2023. "Serum Vitamin D Metabolites by HPLC-MS/MS Combined with Differential Ion Mobility Spectrometry: Aspects of Sample Preparation without Derivatization" International Journal of Molecular Sciences 24, no. 9: 8111. https://doi.org/10.3390/ijms24098111