Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays
Abstract
:1. Introduction
2. Results
2.1. Method Development and Validation
2.1.1. Mass Spectrometry
2.1.2. Liquid Chromatography
2.1.3. Sample Preparation
2.2. Validation
2.2.1. Calibration, Regression Model and Linearity
2.2.2. Sensitivity and Limits of Quantification
2.2.3. Imprecision
2.2.4. Accuracy
2.2.5. Selectivity and Specificity
2.2.6. Interferences
2.2.7. Stability and Robustness
2.3. Assay Comparison
3. Discussion
3.1. Method Development
3.2. Assay Validation
3.3. Assay Comparison
4. Materials and Methods
4.1. Substances and Consumables
4.2. Preparation of Reagents and Mobile Phases
4.3. Calibrators, Controls, and Serum Samples
4.4. Sample Preparation
4.5. Liquid Chromatography–Mass Spectrometry
4.6. Optimizing Mass Spectrometer Settings
4.7. Data Recording and Processing
4.8. Method Validation
4.8.1. Linearity and Calibration Model
4.8.2. Sensitivity
4.8.3. Precision and Accuracy
4.8.4. Proficiency Testing
4.8.5. Recovery
4.8.6. Selectivity and Specificity—Postcolumn Infusion Experiments
4.8.7. Matrix Effects and Extraction Recovery
4.8.8. Interferences
4.8.9. Stability
4.9. Assay Comparison
4.9.1. Immunoassays
4.9.2. Single-Patient Samples
4.9.3. Pooled Serum Samples
4.9.4. Data Analysis
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Analyte | Parameter | Unit | QC-I 1:20 | QC-I 1:10 | QC-I 1:5 | QC-I | QC-II | QC-III |
---|---|---|---|---|---|---|---|---|
11-DOC | (nmol/L) | 0.012 | 0.023 | 0.047 | 0.233 | 0.590 | 3.050 | |
IM-IN | (%) | n.a. | 13.5 | 9.0 | 4.7 | 3.3 | 2.7 | |
IM-ALL | (%) | n.a. | 6.3 | 5.7 | 1.9 | 1.2 | 0.9 | |
BI-ALL | (%) | n.a. | 14.5 | 6.9 | 1.4 | −1.8 | −1.3 | |
17P | (nmol/L) | 0.04 | 0.09 | 0.17 | 0.85 | 4.30 | 25.80 | |
IM-IN | (%) | 14.3 | 7.3 | 5.3 | 3.4 | 2.1 | 1.2 | |
IM-ALL | (%) | 7.9 | 6.1 | 3.9 | 1.4 | 0.8 | 0.6 | |
BI-ALL | (%) | 1.9 | 0.3 | 0.6 | −0.5 | −0.3 | −1.6 | |
ANDRO | (nmol/L) | 0.05 | 0.09 | 0.18 | 0.91 | 3.75 | 31.90 | |
IM-IN | (%) | 11.2 | 6.0 | 5.5 | 2.6 | 2.0 | 2.5 | |
IM-ALL | (%) | 6.1 | 4.5 | 3.6 | 1.2 | 0.8 | 0.8 | |
BI-ALL | (%) | 5.1 | −0.8 | 1.5 | −0.4 | 1.4 | −2.4 | |
DHEA | (nmol/L) | 0.3 | 0.7 | 1.4 | 6.8 | 42.6 | 144.0 | |
IM-IN | (%) | n.a. | n.a. | 23.9 | 7.2 | 5.0 | 3.4 | |
IM-ALL | (%) | n.a. | n.a. | 11.5 | 4.3 | 1.9 | 2.0 | |
BI-ALL | (%) | n.a. | n.a. | 11.2 | −1.2 | −0.9 | −2.1 | |
DHT | (nmol/L) | 0.017 | 0.033 | 0.066 | 0.33 | 1.590 | 3.890 | |
IM-IN | (%) | n.a. | 45.5 | 26.2 | 8.3 | 5.8 | 6.0 | |
IM-ALL | (%) | n.a. | 14.2 | 10.0 | 6.3 | 2.8 | 3.5 | |
BI-ALL | (%) | n.a. | 91.3 | 25.4 | −1.1 | 1.4 | −2.3 | |
PROG | (nmol/L) | 0.05 | 0.09 | 0.19 | 0.94 | 9.72 | 47.90 | |
IM-IN | (%) | 21.7 | 8.0 | 6.3 | 2.8 | 2.0 | 1.5 | |
IM-ALL | (%) | 4.5 | 3.8 | 1.8 | 0.8 | 0.4 | 0.3 | |
BI-ALL | (%) | 20.6 | 7.8 | 2.2 | −2.5 | 1.5 | −0.4 | |
TES | (nmol/L) | 0.03 | 0.07 | 0.14 | 0.68 | 5.13 | 26.50 | |
IM-IN | (%) | 18.6 | 11.1 | 5.6 | 3.9 | 2.3 | 2.5 | |
IM-ALL | (%) | 13.8 | 7.6 | 4.1 | 2.0 | 1.1 | 1.4 | |
BI-ALL | (%) | −0.8 | 0.1 | 2.3 | 0.2 | 1.9 | 0.0 | |
21-DF | (nmol/L) | 0.01 | 0.03 | 0.06 | 0.28 | 1.05 | 6.58 | |
IM-IN | (%) | n.a. | n.a. | 18.4 | 10.6 | 9.6 | 10.9 | |
IM-ALL | (%) | n.a. | n.a. | 11.3 | 5.3 | 3.9 | 2.1 | |
BI-ALL | (%) | n.a. | n.a. | 6.6 | 0.3 | 7.3 | 5.2 | |
11-DF | (nmol/L) | 0.04 | 0.08 | 0.17 | 0.82 | 4.05 | 26.5 | |
IM-IN | (%) | 12.3 | 7.9 | 4.5 | 11.0 | 3.1 | 2.6 | |
IM-ALL | (%) | 5.7 | 4.8 | 3.0 | 1.8 | 1.2 | 0.9 | |
BI-ALL | (%) | 26.1 | 7.5 | 0.9 | −3.1 | −6.9 | 2.9 | |
CC | (nmol/L) | 0.12 | 0.25 | 0.49 | 2.47 | 12.00 | 80.40 | |
IM-IN | (%) | 15.6 | 9.6 | 5.1 | 3.5 | 4.4 | 3.5 | |
IM-ALL | (%) | 7.6 | 5.0 | 3.1 | 1.2 | 0.8 | 1.4 | |
BI-ALL | (%) | −3.7 | 1.7 | 3.4 | 2.4 | 5.9 | −0.3 | |
DHEAS | (nmol/L) | 35 | 70 | 140 | 699 | 4230 | 13800 | |
IM-IN | (%) | 5.2 | 3.4 | 2.0 | 2.2 | 2.1 | 1.1 | |
IM-ALL | (%) | 0.6 | 0.6 | 0.6 | 0.5 | 0.4 | 0.3 | |
BI-ALL | (%) | 38.6 | 14.9 | 3.8 | −6.6 | −5.7 | −12.3 | |
E2 | (nmol/L) | 0.015 | 0.030 | 0.059 | 0.297 | 1.510 | 9.260 | |
IM-IN | (%) | 10.2 | 9.9 | 9.2 | 6.1 | 5.3 | 4.7 | |
IM-ALL | (%) | 8.8 | 7.0 | 7.9 | 3.7 | 2.6 | 2.7 | |
BI-ALL | (%) | 2.4 | −2.1 | −0.8 | −0.5 | −1.3 | −2.7 | |
CL | (nmol/L) | 3 | 7 | 14 | 69 | 166 | 487 | |
IM-IN | (%) | 22.7 | 10.7 | 4.0 | 2.6 | 3.2 | 2.2 | |
IM-ALL | (%) | 2.5 | 1.7 | 1.2 | 1.0 | 1.5 | 1.4 | |
BI-ALL | (%) | −0.9 | −0.6 | 1.3 | 0.5 | 1.6 | 0.7 | |
CN | (nmol/L) | 0.29 | 0.57 | 1.15 | 5.74 | 34.2 | 80.8 | |
IM-IN | (%) | 19.4 | 13.4 | 9.0 | 7.1 | 6.6 | 6.8 | |
IM-ALL | (%) | 6.3 | 7.7 | 5.1 | 3.6 | 4.8 | 4.8 | |
BI-ALL | (%) | 34.5 | 13.4 | 4.8 | −0.3 | 1.0 | −1.4 | |
ALDO | (nmol/L) | 0.013 | 0.026 | 0.051 | 0.255 | 0.699 | 2.430 | |
IM-IN | (%) | n.a. | n.a. | 25.4 | 4.2 | 7.2 | 5.5 | |
IM-ALL | (%) | n.a. | n.a. | 9.1 | 3.7 | 3.2 | 2.9 | |
BI-ALL | (%) | n.a. | n.a. | 17.3 | 6.2 | 2.9 | 14.1 |
Analyte | Unit | F1 | F2 | F3 | F4 | F5 | M1 | M2 | M3 | M4 | M5 |
---|---|---|---|---|---|---|---|---|---|---|---|
11-DOC | (nmol/L) | 0.25 | 0.20 | 0.16 | 0.10 | 0.10 | 0.24 | 0.16 | 0.14 | 0.09 | 0.06 |
(%) | 5.4 | 10.8 | 7.4 | 13.5 | 18.9 | 13.4 | 5.7 | 9.0 | 12.0 | 9.2 | |
17P | (nmol/L) | 2.24 | 2.15 | 2.42 | 1.10 | 0.87 | 1.29 | 1.46 | 2.61 | 2.40 | 2.34 |
(%) | 1.8 | 6.2 | 4.6 | 4.5 | 13.0 | 8.7 | 2.5 | 3.6 | 1.3 | 1.2 | |
ANDRO | (nmol/L) | 0.35 | 4.54 | 4.03 | 2.18 | 1.83 | 0.25 | 1.66 | 3.08 | 2.56 | 1.84 |
(%) | 2.6 | 5.9 | 4.5 | 3.0 | 12.9 | 11.2 | 1.8 | 2.6 | 0.8 | 1.0 | |
DHEA | (nmol/L) | 3.68 | 17.59 | 17.89 | 9.87 | 6.21 | 2.68 | 9.52 | 14.58 | 8.09 | 4.51 |
(%) | 45.0 | 7.3 | 7.4 | 5.5 | 12.6 | 22.1 | 6.3 | 7.4 | 11.5 | 23.9 | |
DHT | (nmol/L) | n.a. | 0.231 | 0.283 | 0.138 | 0.120 | 0.140 | 0.569 | 1.245 | 0.960 | 0.940 |
(%) | n.a. | 23.9 | 12.3 | 16.9 | 16.4 | 24.7 | 7.4 | 7.7 | 6.6 | 10.9 | |
PROG | (nmol/L) | 46.12 | 3.98 | 23.57 | 1.70 | 0.13 | 28.89 | 0.14 | 0.25 | 0.20 | 0.19 |
(%) | 0.6 | 5.8 | 4.9 | 3.9 | 7.5 | 9.0 | 5.9 | 4.4 | 5.8 | 4.3 | |
TES | (nmol/L) | 0.07 | 0.97 | 1.06 | 0.68 | 0.76 | 0.49 | 7.60 | 18.49 | 16.01 | 14.18 |
(%) | 10.5 | 7.9 | 5.9 | 5.8 | 13.5 | 11.1 | 1.6 | 3.1 | 2.1 | 2.2 | |
21-DF | (nmol/L) | 0.03 | 0.06 | 0.06 | 0.07 | 0.08 | 0.02 | 0.05 | 0.04 | 0.07 | 0.08 |
(%) | 38.5 | 19.9 | 40.4 | 18.9 | 22.6 | 35.1 | 47.6 | 28.0 | 35.4 | 32.3 | |
11-DF | (nmol/L) | 1.23 | 1.52 | 0.72 | 0.81 | 0.96 | 0.78 | 1.48 | 0.87 | 1.02 | 0.85 |
(%) | 3.4 | 6.9 | 5.4 | 2.5 | 11.6 | 10.4 | 4.9 | 4.4 | 2.4 | 3.4 | |
CC | (nmol/L) | 5.13 | 15.18 | 11.93 | 16.27 | 10.65 | 6.65 | 7.91 | 15.78 | 10.29 | 7.36 |
(%) | 4.6 | 6.8 | 5.7 | 5.2 | 12.1 | 7.1 | 5.2 | 9.6 | 4.6 | 4.3 | |
DHEAS | (nmol/L) | 451 | 4283 | 4307 | 2013 | 1407 | 419 | 4153 | 6115 | 3189 | 1744 |
(%) | 1.3 | 5.1 | 4.4 | 3.2 | 11.7 | 7.6 | 1.5 | 1.9 | 1.0 | 0.7 | |
E2 | (nmol/L) | 0.393 | 0.391 | 2.365 | 0.102 | 0.027 | 0.104 | 0.029 | 0.093 | 0.080 | 0.079 |
(%) | 4.3 | 5.1 | 5.9 | 7.9 | 10.9 | 11.0 | 12.4 | 5.3 | 8.0 | 5.9 | |
CC | (nmol/L) | 187 | 355 | 358 | 401 | 406 | 223 | 247 | 343 | 361 | 313 |
(%) | 1.8 | 5.2 | 5.3 | 3.5 | 11.1 | 10.2 | 4.0 | 2.6 | 1.9 | 3.2 | |
CL | (nmol/L) | 47.3 | 68.7 | 51.8 | 48.3 | 49.1 | 54.2 | 54.3 | 52.0 | 51.6 | 41.1 |
(%) | 3.9 | 7.5 | 6.6 | 4.6 | 12.1 | 10.3 | 6.5 | 7.4 | 6.1 | 5.7 | |
ALDO | (nmol/L) | 0.271 | 0.302 | 0.239 | 0.214 | 0.228 | 0.274 | 0.243 | 0.176 | 0.146 | 0.189 |
(%) | 7.1 | 6.6 | 8.2 | 8.1 | 11.0 | 8.8 | 5.6 | 10.3 | 5.2 | 5.7 |
Comparator Assay † | Analyte | Intercept | (95% CI) of Intercept | Slope | (95% CI) of Slope | R2 * | (95% CI) of R2 | Linear § | n |
---|---|---|---|---|---|---|---|---|---|
ECLIA | CL | 0.84 | (−13.51 to 10.00) | 1.03 | (1.00 to 1.08) | 0.966 | (0.948 to 0.978) | Yes | 86 |
ECLIA | DHEAS | −103.84 | (−206.60 to 11.90) | 0.84 | (0.81 to 0.86) | 0.974 | (0.963 to 0.983) | Yes | 107 |
ECLIA | E2 | 0.00 | (−0.02 to 0.02) | 1.04 | (0.99 to 1.16) | 0.891 | (0.835 to 0.928) | Yes | 82 |
ECLIA | PROG | −0.57 | (−0.68 to −0.46) | 1.03 | (0.99 to 1.08) | 0.937 | (0.892 to 0.964) | Yes | 51 |
ECLIA | TES | 0.05 | (−0.01 to 0.13) | 1.03 | (1.00 to 1.06) | 0.959 | (0.939 to 0.972) | Yes | 96 |
ECLIA | 17P | 0.02 | (−0.28 to 0.33) | 0.39 | (0.31 to 0.52) | 0.670 | (0.528 to 0.776) | Yes | 79 |
CLIA | ANDRO | −0.03 | (−0.29 to 0.22) | 0.52 | (0.49 to 0.55) | 0.936 | (0.903 to 0.958) | Yes | 84 |
ELISA | DHEA | 1.09 | (−0.34 to 2.30) | 0.72 | (0.62 to 0.82) | 0.897 | (0.844 to 0.933) | Yes | 79 |
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Braun, V.; Stuppner, H.; Risch, L.; Seger, C. Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays. Int. J. Mol. Sci. 2022, 23, 14691. https://doi.org/10.3390/ijms232314691
Braun V, Stuppner H, Risch L, Seger C. Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays. International Journal of Molecular Sciences. 2022; 23(23):14691. https://doi.org/10.3390/ijms232314691
Chicago/Turabian StyleBraun, Valentin, Hermann Stuppner, Lorenz Risch, and Christoph Seger. 2022. "Design and Validation of a Sensitive Multisteroid LC-MS/MS Assay for the Routine Clinical Use: One-Step Sample Preparation with Phospholipid Removal and Comparison to Immunoassays" International Journal of Molecular Sciences 23, no. 23: 14691. https://doi.org/10.3390/ijms232314691