Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant
Abstract
:1. Introduction
2. Materials and Methods
2.1. Chemicals and Reagents
2.2. Instrumentation and Chromatographic Conditions
2.3. Stock and Working Solutions
2.4. Human Sample Preparation
2.5. Assay Validation
2.5.1. Selectivity and Sensitivity
2.5.2. Linearity
2.5.3. Intra- and Inter-Day Precision and Accuracy
2.5.4. Recovery
2.5.5. Stability
2.6. Clinical Research Samples
3. Results
3.1. Assay Development and Validation
3.1.1. Chromatographic Conditions
3.1.2. Selectivity and Sensitivity
3.1.3. Linearity
3.1.4. Accuracy and Precision
3.1.5. Recovery
3.1.6. Stability
3.2. Clinical Application
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Artificial Urine | Artificial Urine + NH4 | % Bias | |||
---|---|---|---|---|---|
3-IS (mg/L) | Ratio | Mean | Ratio | Mean | |
1 | 0.336 | 0.337 | 0.343 | 0.341 | 1.25% |
1 | 0.336 | 0.339 | |||
1 | 0.338 | 0.340 | |||
10 | 3.402 | 3.400 | 3.344 | 3.339 | −1.79% |
10 | 3.395 | 3.339 | |||
10 | 3.403 | 3.334 |
Curve Number | Slope | Intercept | r2 | Range (mg/L) |
---|---|---|---|---|
1 | 0.4841 | −0.0014 | 0.997 | 0.10–10.00 |
2 | 0.4645 | 0.0030 | 0.997 | 0.10–10.00 |
3 | 0.4212 | 0.0036 | 0.999 | 0.10–10.00 |
4 | 0.4115 | −0.0012 | 0.997 | 0.10–10.00 |
Intra-Day Precision and Accuracy | Inter-Day Precision and Accuracy | |||||||
---|---|---|---|---|---|---|---|---|
Level | Nominal 3-IS Conc. (mg/L) | Day | Mean Observed Conc. (mg/L) | Precision (%) | Accuracy (%) | Mean Observed Conc. (mg/L) | Precision (%) | Accuracy (%) |
L-LOQ | 0.10 | 1 | 0.105 | 0.47 | 105.3 | 108.4 | ||
2 | 0.113 | 1.37 | 112.6 | 0.108 | 3.10 | |||
3 | 0.107 | 1.47 | 107.3 | |||||
M-QC | 1.00 | 1 | 1.037 | 1.33 | 103.7 | |||
2 | 1.026 | 1.23 | 102.6 | 1.021 | 1.60 | 102.1 | ||
3 | 1.016 | 0.58 | 101.6 | |||||
U-LOQ | 10.00 | 1 | 10.105 | 0.20 | 101.1 | |||
2 | 10.008 | 0.33 | 100.1 | 10.047 | 1.70 | 100.5 | ||
3 | 10.261 | 0.33 | 102.6 |
Sample | 3-IS Concentration before Spiking (mg/L) | %CV | Nominal Spiking Level (mg/L) | 3-IS Urine Concentration after Spiking (mg/L) | %CV | Recovery (%) |
---|---|---|---|---|---|---|
HV 1 | 3.65 | 0.02 | 0.1 | 3.74 | 0.15 | 92.7 |
0.5 | 4.11 | 0.18 | 96.9 | |||
1 | 4.61 | 0.12 | 95.9 | |||
HV 2 | 3.13 | 0.18 | 0.1 | 3.22 | 0.08 | 92.1 |
0.5 | 3.59 | 0.17 | 96.1 | |||
1 | 4.03 | 0.06 | 90.0 |
Condition | Time | Parameter | Sample | ||||
---|---|---|---|---|---|---|---|
L-LOQ | M-QC | U-LOQ | HV 1 | HV 2 | |||
Benchtop | 0 h | 3-IS (mg/L) | 0.101 | 0.995 | 10.205 | 76.268 | 69.485 |
Precision % | 0.35 | 0.04 | 0.29 | 5.15 | 1.48 | ||
1 h | 3-IS (mg/L) | 0.098 | 1.046 | 9.994 | 71.865 | 68.387 | |
Precision % | 0.37 | 0.25 | 1.49 | 1.48 | 0.55 | ||
% vs. t0 | 97.0 | 105.1 | 97.9 | 94.2 | 98.4 | ||
2 h | 3-IS (mg/L) | 0.086 | 0.989 | 9.485 | 70.671 | 67.600 | |
Precision % | 0.62 | 0.21 | 0.29 | 0.16 | 0.14 | ||
% vs. t0 | 85.1 | 99.4 | 92.9 | 92.7 | 97.3 | ||
4 h | 3-IS (mg/L) | 0.100 | 0.968 | 9.434 | 69.984 | 68.912 | |
Precision % | 0.07 | 0.06 | 0.14 | 0.06 | 0.23 | ||
% vs. t0 | 99.0 | 97.3 | 92.4 | 91.8 | 99.2 | ||
6 h | 3-IS (mg/L) | 0.102 | 0.986 | 9.975 | 70.903 | 67.794 | |
Precision % | 0.25 | 1.86 | 0.23 | 0.21 | 0.43 | ||
% vs. t0 | 100.1 | 99.1 | 97.7 | 93.0 | 97.6 | ||
7 days | 3-IS (mg/L) | 0.093 | 0.946 | 8.953 | 81.784 | 70.484 | |
CV% | 0.24 | 0.02 | 0.21 | 0.17 | 0.07 | ||
% vs. t0 | 92.1 | 95.1 | 87.7 | 107.2 | 101.4 | ||
14 days | 3-IS (mg/L) | 0.093 | 0.891 | 8.636 | 87.702 | 77.221 | |
Precision % | 0.24 | 0.68 | 0.05 | 0.27 | 0.6 | ||
% vs. t0 | 92.1 | 89.5 | 84.6 | 115.0 | 111.1 | ||
4 °C | 0 h | 3-IS (mg/L) | 0.101 | 0.995 | 10.205 | 76.268 | 69.486 |
Precision % | 0.35 | 0.04 | 0.29 | 5.15 | 1.48 | ||
1 h | 3-IS (mg/L) | 0.112 | 1.086 | 9.892 | 79.635 | 71.544 | |
Precision % | 0.29 | 0.23 | 0.45 | 0.38 | 0.24 | ||
% vs. t0 | 110.9 | 109.1 | 96.9 | 104.4 | 102.03 | ||
2 h | 3-IS (mg/L) | 0.108 | 1.008 | 9.262 | 81.194 | 69.345 | |
Precision % | 0.03 | 0.15 | 0.25 | 0.10 | 0.02 | ||
% vs. t0 | 106.9 | 101.3 | 90.7 | 106.5 | 99.8 | ||
4 h | 3-IS (mg/L) | 0.110 | 0.995 | 9.473 | 80.498 | 70.346 | |
Precision % | 0.04 | 0.08 | 0.98 | 0.39 | 0.47 | ||
% vs. t0 | 108.9 | 100.0 | 92.8 | 105.5 | 101.2 | ||
6 h | 3-IS (mg/L) | 0.112 | 1.039 | 10.099 | 83.783 | 73.008 | |
Precision % | 0.47 | 0.31 | 0.42 | 1.06 | 0.39 | ||
% vs. t0 | 110.9 | 104.4 | 99.0 | 109.9 | 105.1 | ||
7 days | 3-IS (mg/L) | 0.100 | 1.014 | 9.227 | 81.169 | 71.142 | |
Precision % | 0.59 | 1.23 | 0.27 | 0.65 | 0.46 | ||
% vs. t0 | 99.0 | 101.9 | 90.4 | 106.4 | 102.4 | ||
14 days | 3-IS (mg/L) | 0.108 | 0.940 | 9.347 | 80.680 | 70.085 | |
Precision % | 0.75 | 0.45 | 0.77 | 0.49 | 0.63 | ||
% vs. t0 | 106.9 | 94.5 | 91.6 | 105.8 | 100.9 |
Condition | Time | Parameter | Sample | ||||
---|---|---|---|---|---|---|---|
L-LOQ | M-QC | U-LOQ | HV 1 | HV 2 | |||
−20 °C | 1 month | ISu (mg/L) | 0.091 | 0.973 | 10.14 | 85.83 | 70.531 |
Precision% | 1.06 | 0.41 | 0.12 | 0.12 | 0.06 | ||
% vs. t0 | 90.1 | 97.8 | 99.4 | 112.5 | 101.5 | ||
2 months | ISu (mg/L) | 0.101 | 0.962 | 9.939 | 79.157 | 68.649 | |
CV% | 0.9 | 0.7 | 0.26 | 0.12 | 0.2 | ||
% vs. t0 | 100.0 | 96.7 | 97.4 | 103.8 | 98.8 |
Patient ID | Age (Years) | Sex | Time * (Days) | Concomitant Drugs | Underlying Disease | [3-IS] (mg/L) [3-IS] (μmol3-IS/mmolcreat) | |
---|---|---|---|---|---|---|---|
1 | 3 | M | −7 | ACY, CAP, CID, ITRA, PRO, TRI/SULFA, VALGA | Chronic granulomatous disease | 13.6 | 67.9 |
2 | 8 | F | +25 | ACY, AMPT, BU, LOR, MPN, OMP, OND, TRI/SULFA, UDC, VANC | Myelodysplasia | 2.0 | 8.3 |
3 | 6 | M | −14 | DIP, FAM, LVM, TRM/SMX | Acute myeloid leukemia | 31.9 | 22.5 |
4 | 10 | F | −4 | FAM, HYD, TAC, VALGA, VCZ | Medullar aplasia | 8.1 | 3.4 |
5 | 13 | F | −8 | FAM, FLU, LVM, OND, PIP/TAZO, TRI/SULFA | Acute myeloid leukemia | 2.8 | 7.0 |
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Olivetti, C.E.; Fernández, M.F.; Stojanova, J.; Ruvinsky, S.; Mangano, A.; Schaiquevich, P. Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant. Methods Protoc. 2024, 7, 64. https://doi.org/10.3390/mps7040064
Olivetti CE, Fernández MF, Stojanova J, Ruvinsky S, Mangano A, Schaiquevich P. Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant. Methods and Protocols. 2024; 7(4):64. https://doi.org/10.3390/mps7040064
Chicago/Turabian StyleOlivetti, Christian Ezequiel, María Florencia Fernández, Jana Stojanova, Silvina Ruvinsky, Andrea Mangano, and Paula Schaiquevich. 2024. "Full Validation and Application to Clinical Research of a High-Performance Liquid Chromatography Method for the Assessment of Urinary 3-Indoxyl Sulfate in Pediatric Patients with Hematopoietic Stem Cell Transplant" Methods and Protocols 7, no. 4: 64. https://doi.org/10.3390/mps7040064