Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry
Abstract
:1. Introduction
2. Results
2.1. Sensitivity, Linearity, Imprecision, and Recovery
2.2. Steroid Profiling of Dry Eye and Healthy Control Tear Samples by LC-MS/MS Analysis
3. Discussion
4. Materials and Methods
4.1. Patients
4.2. Samples
4.3. Materials
4.4. Preparation of Standard Solutions, Calibrators and Quality Controls
4.5. Sample Preparation
4.6. LC-MS/MS Analysis
4.7. Quantification
4.8. Assay Performance Characteristics
4.9. Statistical Analysis
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Analyte | Concentration Range (ng/mL) | Calibration Function (y = b1(±SD)x + b0(±SD)) a,b | R2 |
---|---|---|---|
CORT | 0.3–20.08 | y = 0.396(±0.04)x + 0.026(±0.160) | 0.996 |
11-DECOL | 0.05–1.00 | y = 3.041(±0.942)x + 0.077(±0.052) | 0.994 |
ADIONE | 0.05–2.91 | y = 9.492(±0.584)x + 0.204(±0.054) | 0.994 |
TESTO | 0.02–1.00 | y = 9.276 (±2.446)x + 0.171(±0.103) | 0.993 |
17-OHP | 0.05–4.20 | y = 4.372 (±1.218)x − 0.053(±0.045) | 0.993 |
PROG | 0.05–4.20 | y = 11.015 (±1.789)x + 0.279(±0.182) | 0.994 |
Intra-Assay | Inter-Assay | Recovery % | |||||||
---|---|---|---|---|---|---|---|---|---|
Compound | QC Level | Mean Response | SD | %CV | Mean Response | SD | %CV | Mean | SD |
CORT | QC1 | 0.13 | 0.02 | 13.47 | 0.14 | 0.01 | 8.43 | 50.12 | 13.18 |
QC2 | 0.52 | 0.01 | 2.35 | 0.52 | 0.03 | 5.12 | 33.12 | 0.73 | |
QC3 | 2.88 | 0.15 | 5.09 | 2.57 | 0.13 | 5.14 | 44.29 | 1.98 | |
11-DECOL | QC1 | 0.27 | 0.02 | 7.97 | 0.15 | 0.02 | 12.41 | 46.99 | 6.64 |
QC2 | 0.67 | 0.01 | 1.63 | 0.72 | 0.06 | 7.83 | 67.45 | 6.67 | |
QC3 | 2.19 | 0.11 | 4.98 | 2.26 | 0.21 | 9.48 | 58.18 | 2.83 | |
ADIONE | QC1 | 0.83 | 0.04 | 4.99 | 0.99 | 0.03 | 3.15 | 23.26 | 7.16 |
QC2 | 2.14 | 0.10 | 4.77 | 2.30 | 0.15 | 6.72 | 22.01 | 2.26 | |
QC3 | 11.99 | 0.13 | 1.12 | 11.85 | 0.32 | 2.73 | 24.53 | 2.23 | |
TESTO | QC1 | 0.67 | 0.09 | 13.82 | 0.56 | 0.05 | 8.77 | 19.50 | 2.92 |
QC2 | 2.14 | 0.31 | 14.67 | 1.92 | 0.09 | 4.51 | 37.46 | 0.48 | |
QC3 | 6.39 | 0.73 | 11.49 | 5.29 | 0.74 | 13.94 | 44.85 | 5.91 | |
17-OHP | QC1 | 0.21 | 0.03 | 15.24 | 0.35 | 0.04 | 10.84 | 22.78 | 9.92 |
QC2 | 0.78 | 0.07 | 8.86 | 1.04 | 0.06 | 5.99 | 30.61 | 6.49 | |
QC3 | 6.25 | 0.17 | 2.77 | 6.76 | 0.62 | 9.15 | 50.64 | 2.60 | |
PROG | QC1 | 0.83 | 0.03 | 3.20 | 1.01 | 0.07 | 6.90 | 39.56 | 4.31 |
QC2 | 2.26 | 0.21 | 9.54 | 2.95 | 0.19 | 6.70 | 30.60 | 1.92 | |
QC3 | 13.92 | 0.10 | 0.72 | 14.77 | 1.22 | 8.30 | 40.43 | 7.50 |
Analyte | Calibration Levels (ng/mL) | QC Levels (ng/mL) | ||||||||
---|---|---|---|---|---|---|---|---|---|---|
L1 | L2 | L3 | L4 | L5 | L6 | L7 | QC1 | QC2 | QC3 | |
CORT | 0.30 | 0.50 | 1.00 | 1.51 | 3.61 | 8.66 | 20.80 | 0.50 | 1.51 | 8.66 |
CCONE | 0.05 | 0.10 | 0.20 | 0.29 | 0.70 | 1.68 | 4.03 | 0.10 | 0.29 | 1.68 |
11-DECOL | 0.05 | 0.07 | 0.10 | 0.20 | 0.40 | 0.70 | 1.00 | 0.07 | 0.20 | 0.70 |
ADIONE | 0.05 | 0.08 | 0.10 | 0.20 | 0.49 | 1.17 | 2.91 | 0.08 | 0.20 | 1.17 |
TESTO | 0.02 | 0.05 | 0.08 | 0.15 | 0.30 | 0.60 | 1.00 | 0.05 | 0.15 | 0.60 |
17-OHP | 0.05 | 0.08 | 0.12 | 0.29 | 0.70 | 1.69 | 4.20 | 0.08 | 0.29 | 1.69 |
PROG | 0.05 | 0.08 | 0.12 | 0.29 | 0.70 | 1.69 | 4.20 | 0.08 | 0.29 | 1.69 |
MRM Function | Time Window (min) | Analyte | Transitions (m/z) | Cone Volts | Coll Energy (eV) |
---|---|---|---|---|---|
1 | 5.0–8.5 | CORT | 363.2 > 120.8 | 100 | 20 |
CORT | 363.2 > 96.9 | 100 | 20 | ||
2H3-CORT | 366.2 > 120.8 | 100 | 20 | ||
2 | 8.0–10.5 | CCONE | 347.3 > 120.8 | 100 | 18 |
CCONE | 347.3 > 96.8 | 100 | 20 | ||
2H8-CCONE | 355.3 > 124.8 | 100 | 18 | ||
3 | 8.0–10.8 | 11-DECOL | 347.3 > 108.8 | 100 | 22 |
11-DECOL | 347.3 > 96.8 | 100 | 20 | ||
2H5-11-DECOL | 352.3 > 112.8 | 100 | 22 | ||
4 | 9.7–12.0 | ADIONE | 287.2 > 96.9 | 100 | 18 |
ADIONE | 287.2 > 108.8 | 100 | 20 | ||
2H5-ADIONE | 292.2 > 99.9 | 100 | 18 | ||
5 | 10.5–13.0 | TESTO | 289.2 > 96.8 | 100 | 19 |
TESTO | 289.2 > 108.8 | 100 | 18 | ||
2H5-TESTO | 294.2 > 99.8 | 100 | 19 | ||
6 | 11.2–13.5 | 17-OHP | 331.2 > 96.8 | 100 | 21 |
17-OHP | 331.2 > 108.9 | 100 | 23 | ||
2H8-OHP | 339.2 > 99.8 | 100 | 21 | ||
7 | 13.5–16.0 | PROG | 315.2 > 96.9 | 100 | 18 |
PROG | 315.2 > 108.8 | 100 | 19 | ||
2H9-PROG | 324.2 > 99.9 | 100 | 18 |
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Pieragostino, D.; Agnifili, L.; Cicalini, I.; Calienno, R.; Zucchelli, M.; Mastropasqua, L.; Sacchetta, P.; Del Boccio, P.; Rossi, C. Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry. Int. J. Mol. Sci. 2017, 18, 1349. https://doi.org/10.3390/ijms18071349
Pieragostino D, Agnifili L, Cicalini I, Calienno R, Zucchelli M, Mastropasqua L, Sacchetta P, Del Boccio P, Rossi C. Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry. International Journal of Molecular Sciences. 2017; 18(7):1349. https://doi.org/10.3390/ijms18071349
Chicago/Turabian StylePieragostino, Damiana, Luca Agnifili, Ilaria Cicalini, Roberta Calienno, Mirco Zucchelli, Leonardo Mastropasqua, Paolo Sacchetta, Piero Del Boccio, and Claudia Rossi. 2017. "Tear Film Steroid Profiling in Dry Eye Disease by Liquid Chromatography Tandem Mass Spectrometry" International Journal of Molecular Sciences 18, no. 7: 1349. https://doi.org/10.3390/ijms18071349