Simultaneous Determination of Six Immunosuppressants in Human Whole Blood by HPLC-MS/MS Using a Modified QuEChERS Method
Abstract
:1. Introduction
2. Results
Validation of the Analytical Method
3. Discussion
3.1. Column and Mobile Phase Selection
3.2. Selecting and Optimizing Preprocessing Conditions
3.2.1. Optimizing Extraction Conditions
3.2.2. Optimization of Salinization Conditions
3.2.3. Optimization of Purification Conditions
3.2.4. Comparative Analysis of QuEChERS and Solid-Phase Supported Liquid-Liquid Extraction (SLE)
3.3. Comparisons with Other Methods
4. Materials and Methods
4.1. Quantitative Analysis by UHPLC-MS/MS
4.1.1. Reagents and Chemicals
4.1.2. Preparation of the Standard Stock Solutions and Working Solutions
4.1.3. Sample Preparation
4.1.4. LC-MS/MS Analysis
4.2. Method Validation
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Analyte | Spiked (ng·mL−1) | ME (%) | Precision (RSD %) | Accuracy (%) | |
---|---|---|---|---|---|
Inter-Day | Intra-Day | ||||
MPA | 20 | 101.99 | 3.52 | 1.13 | 107.79 |
500 | 85.78 | 2.94 | 3.44 | 114.95 | |
800 | 103.19 | 6.73 | 2.31 | 110.75 | |
MMF | 0.2 | 112.69 | 6.44 | 2.78 | 109.56 |
5 | 86.25 | 1.29 | 7.71 | 109.60 | |
8 | 78.95 | 3.76 | 1.33 | 106.42 | |
FK-506 | 2 | 91.23 | 9.98 | 3.88 | 95.66 |
50 | 80.51 | 0.55 | 1.36 | 109.09 | |
80 | 78.97 | 1.40 | 1.19 | 101.50 | |
RAPA | 2 | 94.08 | 4.31 | 5.49 | 109.43 |
50 | 92.29 | 5.40 | 0.54 | 113.47 | |
80 | 47.81 | 3.05 | 4.94 | 113.09 | |
EVER | 2 | 106.65 | 14.92 | 4.59 | 92.24 |
50 | 51.42 | 5.02 | 3.20 | 102.22 | |
80 | 50.26 | 2.07 | 1.61 | 102.44 | |
PIM | 2 | 91.17 | 7.02 | 2.98 | 85.07 |
50 | 84.21 | 0.23 | 4.79 | 99.30 | |
80 | 85.20 | 0.86 | 1.01 | 92.07 |
Analyte | Inter-Day (CV, %) | Intra-Day (CV, %) | LOD (ng·mL−1) | LOQ (ng·mL−1) | Recovery (%) |
---|---|---|---|---|---|
MPA | 4.40 ± 2.0 | 2.29 ± 1.2 | 2.30 | 7.60 | 116 ± 3.6 |
MMF | 3.83 ± 2.6 | 3.94 ± 3.3 | 0.02 | 0.07 | 109 ± 1.8 |
FK-506 | 3.98 ± 5.2 | 2.14 ± 1.5 | 0.03 | 0.09 | 102 ± 6.7 |
RAPA | 4.25 ± 1.2 | 3.66 ± 2.7 | 0.05 | 0.20 | 112 ± 2.2 |
EVER | 7.34 ± 6.7 | 3.13 ± 1.5 | 0.05 | 0.20 | 99.0 ± 5.8 |
PIM | 2.70 ± 3.8 | 2.93 ± 1.9 | 0.02 | 0.06 | 92.1 ± 7.1 |
Method | Solvent | Sample | Analytes | Detection System | LOQ (ng·mL−1) | RSD (%) | Recovery (%) | Ref. |
---|---|---|---|---|---|---|---|---|
QuEChERS | - | Whole blood | MPA/MMF/FK-506/ RAPA/EVER/PIM | LC-MS/MS | 7.60/0.07/0.09/0.20/0.20/0.06 | 0.23~14.92 | 85.07~114.95 | this work |
PP | Methanol | Plasma | FK-506 | LC-MS/MS | 0.1 | - | 107% | [32] |
Online SPE | ZnSO4 | Plasma | MPA/ FK-506/ RAPA/ EVER | LC-MS/MS | 7.0/7.5/ 4.6/6.4 | 0.9~14.7 | 89.0~138.0 | [33] |
PP | ACN/ MTBE | Whole blood | FK-506/ RAPA/EVER | LC-MS/MS | 0.5/0.5/ 0.5 | 1.87~11.2 | 78.6~85.9 | [34] |
Online SPE | MeOH- ZnSO4 (66:34) | Whole blood | FK-506/ RAPA/ EVER | LC-MS/MS | 1.4/0.72/1.15 | <5 | 92.8~95.9 | [35] |
PP | ZnSO4/ ACN | Whole blood | FK-506 | LC-MS/MS | 1.0 | - | 94 | [36] |
SPE | - | Brain | EVER | LC-MS | 4.0 | 3~19 | 82~102 | [37] |
Analyte | Precursor Ion (m/z) | Product Ion (m/z) | Fragmentor Voltage (V) | Collision Voltage (eV) | Retention Time (min) |
---|---|---|---|---|---|
MPA | 338.3 | 207.1 * | 140 | 20 | 6.4 |
338.3 | 302.9 | 140 | 10 | ||
MMF | 434.2 | 114.2 * | 145 | 28 | 7.2 |
434.2 | 285.0 | 145 | 26 | ||
FK-506 | 821.5 | 768.2 * | 165 | 18 | 9.8 |
821.5 | 786.2 | 165 | 14 | ||
RAPA | 931.5 | 864.4 * | 155 | 15 | 10.3 |
931.5 | 882.2 | 155 | 6 | ||
EVER | 975.5 | 908.3 * | 165 | 10 | 10.5 |
975.5 | 926.2 | 165 | 6 | ||
PIM | 827.4 | 774.2 * | 170 | 20 | 11.2 |
827.4 | 792.2 | 170 | 18 |
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Zheng, M.; Song, J.; Xue, H.; Li, H.; Lian, K. Simultaneous Determination of Six Immunosuppressants in Human Whole Blood by HPLC-MS/MS Using a Modified QuEChERS Method. Molecules 2022, 27, 4087. https://doi.org/10.3390/molecules27134087
Zheng M, Song J, Xue H, Li H, Lian K. Simultaneous Determination of Six Immunosuppressants in Human Whole Blood by HPLC-MS/MS Using a Modified QuEChERS Method. Molecules. 2022; 27(13):4087. https://doi.org/10.3390/molecules27134087
Chicago/Turabian StyleZheng, Min, Jianshi Song, Hua Xue, Hui Li, and Kaoqi Lian. 2022. "Simultaneous Determination of Six Immunosuppressants in Human Whole Blood by HPLC-MS/MS Using a Modified QuEChERS Method" Molecules 27, no. 13: 4087. https://doi.org/10.3390/molecules27134087