Simultaneous Determination of Polyamines and Steroids in Human Serum from Breast Cancer Patients Using Liquid Chromatography–Tandem Mass Spectrometry
Abstract
:1. Introduction
2. Results and Discussion
2.1. Sample Preparation and Optimization
2.2. Liquid Chromatography–Tandem Mass Spectrometry
2.3. Method Validation
2.4. Application of Serum Polyamine and Steroid Profiles to Patients with Breast Cancer and Normal Controls
2.5. Receiver Operating Characteristic Curve
3. Materials and Methods
3.1. Chemicals
3.2. Preparation of Standard Solution
3.3. Sample Information and Ethics Statement
3.4. Sample Preparation
3.5. Liquid Chromatography–Tandem Mass Spectrometry
3.6. Validation
3.7. Statistical Testing and Data Processing
4. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
Sample Availability
References
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Analytes | Calibration Range | Linear Regression Equation | Standard Errors of the Slope | Standard Errors of the Intercept | R2 | LOQ | Matrix Effect (%) | Recovery (%) |
---|---|---|---|---|---|---|---|---|
N-PUT | 1–5000 | y = 0.0008x + 0.0986 | 1.42−5 | 0.03 | 0.998 | 1 | 101.4 | 107.4 |
CAD | 1–5000 | y = 0.0001x + 0.0718 | 6.07−6 | 0.01 | 0.995 | 1 | 83.1 | 87.9 |
N-SPM | 0.1–5000 | y = 0.084x − 0.6771 | 1.76−4 | 0.34 | 0.998 | 0.1 | 86.8 | 101.8 |
PUT | 1–5000 | y = 0.001x + 0.0949 | 7.90−5 | 0.15 | 0.992 | 1 | 115.5 | 98.2 |
SPD | 1–5000 | y = 0.01x − 0.6062 | 1.59−3 | 0.31 | 0.999 | 1 | 126.3 | 90.1 |
DAP | 1–5000 | y = 0.0013x + 0.0449 | 5.89−5 | 0.11 | 0.996 | 1 | 89.7 | 101.7 |
N-SPD | 1–5000 | y = 0.0067x + 0.419 | 3.76−4 | 0.73 | 0.990 | 1 | 118.4 | 123.6 |
N-CAD | 1–5000 | y = 0.0003x + 0.13 | 1.26−5 | 0.02 | 0.992 | 1 | 87.5 | 100.7 |
SPM | 1–5000 | y = 6E-05x + 0.0088 | 1.79−5 | 0.03 | 0.995 | 1 | 86.4 | 107.8 |
T | 1–2000 | y = 0.0032x + 0.1867 | 7.92−5 | 0.15 | 0.996 | 1 | 106.7 | 89.9 |
EpiT | 1–2000 | y = 0.0045x + 0.4609 | 1.59−4 | 0.31 | 0.993 | 1 | 103.4 | 100.4 |
DHT | 1–2000 | y = 0.0019x + 0.0017 | 6.13−5 | 0.12 | 0.994 | 1 | 82.1 | 104.7 |
PREG | 1–2000 | y = 0.0003x − 0.0244 | 1.11−5 | 0.02 | 0.991 | 1 | 79.3 | 97.7 |
17α-OHP | 0.1–2000 | y = 0.0115x + 0.1561 | 3.94−4 | 0.77 | 0.993 | 0.1 | 84.6 | 87.8 |
11β-OHP | 0.1–2000 | y = 0.0049x + 0.1846 | 1.83−4 | 0.36 | 0.992 | 0.1 | 88.6 | 91.8 |
A | 0.1–2000 | y = 0.0017x + 0.038 | 3.38−5 | 0.07 | 0.998 | 0.1 | 78.7 | 101.0 |
P4 | 0.1–2000 | y = 0.0062x − 0.2893 | 3.01−4 | 0.58 | 0.994 | 0.1 | 100.9 | 96.7 |
Analytes | Spiked Concentration (ng/mL) | Intra-Day (n = 3) | Inter-Day (n = 3) | ||
---|---|---|---|---|---|
Accuracy | Precision | Accuracy | Precision | ||
(%Bias) | (%CV) | (%Bias) | (%CV) | ||
N-PUT | 10 | 91.2 | 8.1 | 88.1 | 11.3 |
50 | 96.8 | 5.4 | 96.4 | 13.6 | |
500 | 110.4 | 7.0 | 92.9 | 18.5 | |
1000 | 104.1 | 7.0 | 106.7 | 2.6 | |
CAD | 10 | 86.5 | 13.0 | 93.8 | 13.4 |
50 | 107.4 | 5.9 | 100.0 | 7.9 | |
500 | 95.4 | 11.1 | 103.2 | 5.2 | |
1000 | 92.1 | 9.2 | 102.5 | 9.8 | |
N-SPM | 1 | 104.0 | 16.9 | 104.6 | 19.2 |
50 | 88.2 | 15.3 | 101.3 | 0.3 | |
500 | 113.6 | 6.2 | 110.5 | 3.1 | |
1000 | 112.8 | 4.8 | 118.3 | 2.9 | |
PUT | 10 | 99.0 | 10.5 | 107.3 | 18.3 |
50 | 103.5 | 8.6 | 105.5 | 5.8 | |
500 | 99.1 | 15.3 | 115.1 | 3.2 | |
1000 | 103.4 | 15.4 | 102.0 | 17.0 | |
SPD | 10 | 105.8 | 16.3 | 96.2 | 19.7 |
50 | 116.2 | 21.2 | 109.6 | 17.5 | |
500 | 93.8 | 18.3 | 92.0 | 6.8 | |
1000 | 98.6 | 19.6 | 104.7 | 11.5 | |
DAP | 10 | 108.6 | 17.1 | 94.3 | 16.4 |
50 | 105.4 | 8.7 | 94.7 | 20.2 | |
500 | 101.2 | 1.7 | 106.6 | 10.1 | |
1000 | 103.9 | 6.2 | 93.6 | 6.6 | |
N-SPD | 10 | 108.5 | 1.6 | 106.5 | 18.3 |
50 | 110.6 | 11.5 | 104.8 | 11.5 | |
500 | 104.3 | 6.5 | 105.6 | 13.6 | |
1000 | 114.5 | 2.7 | 106.3 | 16.5 | |
N-CAD | 10 | 95.5 | 13.9 | 115.6 | 4.1 |
50 | 109.6 | 15.1 | 111.0 | 14.3 | |
500 | 110.5 | 9.6 | 90.7 | 12.4 | |
1000 | 97.5 | 13.5 | 90.3 | 9.5 | |
SPM | 10 | 91.5 | 14.9 | 92.4 | 0.7 |
50 | 104.2 | 13.3 | 119.3 | 7.8 | |
500 | 104.3 | 10.6 | 87.8 | 6.9 | |
1000 | 103.5 | 12.2 | 98.4 | 16.4 | |
T | 10 | 104.9 | 12.5 | 106.8 | 17.6 |
50 | 94.8 | 14.0 | 106.1 | 11.2 | |
500 | 104.0 | 2.4 | 94.6 | 2.5 | |
1000 | 100.7 | 9.0 | 100.5 | 8.1 | |
EpiT | 10 | 100.9 | 7.0 | 101.7 | 5.9 |
50 | 106.8 | 9.0 | 102.1 | 6.4 | |
500 | 108.6 | 10.0 | 104.0 | 4.3 | |
1000 | 105.6 | 6.9 | 99.9 | 12.3 | |
DHT | 10 | 106.6 | 8.7 | 97.7 | 9.5 |
50 | 114.3 | 6.5 | 90.2 | 11.9 | |
500 | 111.5 | 3.9 | 97.6 | 16.0 | |
1000 | 100.6 | 6.8 | 95.5 | 18.5 | |
PREG | 10 | 111.1 | 17.8 | 82.0 | 17.7 |
50 | 92.4 | 0.6 | 96.6 | 7.5 | |
500 | 98.9 | 16.0 | 96.1 | 11.4 | |
1000 | 103.7 | 7.4 | 101.7 | 8.6 | |
17α-OHP | 1 | 101.5 | 17.1 | 107.9 | 8.4 |
50 | 102.2 | 15.4 | 106.8 | 7.9 | |
500 | 109.1 | 13.2 | 100.4 | 3.8 | |
1000 | 96.4 | 1.2 | 95.0 | 1.2 | |
11β-OHP | 1 | 98.9 | 3.5 | 99.4 | 13.5 |
50 | 95.0 | 21.8 | 108.0 | 4.7 | |
500 | 109.3 | 6.0 | 92.0 | 7.7 | |
1000 | 109.9 | 7.1 | 106.1 | 7.3 | |
A | 1 | 106.1 | 5.7 | 107.0 | 10.0 |
50 | 96.9 | 6.9 | 94.8 | 10.2 | |
500 | 101.0 | 1.0 | 107.8 | 4.2 | |
1000 | 93.3 | 8.5 | 91.2 | 9.0 | |
P4 | 1 | 99.9 | 19.7 | 101.0 | 1.7 |
50 | 101.0 | 3.5 | 97.1 | 9.4 | |
500 | 91.0 | 13.1 | 98.3 | 8.6 | |
1000 | 110.8 | 5.3 | 103.0 | 18.1 |
Normal Controls (n = 10) | Patients (n = 10) | p Value | |||
---|---|---|---|---|---|
Mean ± SD | Median, Range | Mean ± SD | Median, Range | ||
Polyamines | |||||
N-PUT | 11.12 ± 4.34 | 11.09, 1.6–16.53 | 61.64 ± 58.88 | 46.57, 15.88–183.08 | 0.021 |
CAD | 60.3 ± 27.33 | 66.35, 12.17–96.11 | 208.21 ± 169.71 | 174.62, 31.9–545.38 | 0.027 |
N-SPM | 0.53 ± 0.45 | 0.45, 0.14–1.81 | 1.1 ± 1.13 | 0.82, 0.16–3.6 | 0.12 |
PUT | 54.88 ± 70.74 | 16.3, 2.65–232.98 | 120.94 ± 170.91 | 38.52, 2.38–586.46 | 0.146 |
SPD | 15.15 ± 20.73 | 4.88, 1.3–76.45 | 19.92 ± 22.53 | 13.62, 1.06–77.13 | 0.551 |
DAP | 14.38 ± 6.08 | 14.99, 2.5–22.12 | 39.48 ± 21.64 | 36.79, 12.02–67.47 | 0.003 |
N-SPD | 31.06 ± 15.91 | 31.05, 4.42–58.37 | 57.75 ± 25.66 | 58.35, 15.51–95.85 | 0.016 |
N-CAD | 20.04 ± 16.63 | 16.45, 2.45–50.16 | 76.8 ± 84.72 | 39.11, 15.28–236.6 | 0.109 |
SPM | 63 ± 48.54 | 60, 13.91–172.96 | 179.37 ± 207.84 | 76.83, 7.58–632.22 | 0.121 |
Steroids | |||||
T | 4.86 ± 3.44 | 3.52, 1.3–10.09 | 8.06 ± 6.24 | 6.52, 1.78–18.88 | 0.095 |
EpiT | 12.64 ± 5.36 | 10.79, 7.51–24.55 | 28.74 ± 13.93 | 24.85, 12.39–53.79 | 0.004 |
DHT | 2.35 ± 1.22 | 1.7, 1.6–3.76 | 2.71 ± 1.94 | 2.71, 1.34–4.08 | 0.809 |
PREG | 32.98 ± 11.37 | 31.97, 16.16–47.4 | 44.87 ± 15.27 | 44.47, 23.56–62.74 | 0.106 |
17α-OHP | 1.15 ± 1.21 | 0.73, 0.14–3.76 | 1.15 ± 1.35 | 0.71, 0.12–4.08 | 0.999 |
11β-OHP | 2.12 ± 1.7 | 1.9, 0.62–5.08 | 1.78 ± 1.47 | 1.42, 0.42–4.6 | 0.698 |
A | 1.41 ± 1.07 | 1.01, 0.29–3.29 | 2.73 ± 2.03 | 2.02, 0.68–6.73 | 0.09 |
P4 | 1.3 ± 0.87 | 1.05, 0.18–3.64 | 1.47 ± 0.82 | 1.34, 0.27–3.06 | 0.569 |
Compound | Abbreviation | Precursor Ion (m/z) | Product Ion (m/z) | Normalized Collision Energy (%) | Retention Time (min) |
---|---|---|---|---|---|
1,3-diaminopropane | DAP | 275.0 | 201.1 | 22 | 9.3 |
Putrescine | PUT | 289.0 | 215.0 | 28 | 10 |
Spermidine | SPD | 446.1 | 372.3 | 35 | 14.6 |
Spermine | SPM | 603.0 | 529.2 | 48 | 17.9 |
1,6-diaminohexane | DAH | 317.0 | 243.0 | 27 | 12.45 |
Cadaverine | CAD | 303.0 | 229.0 | 45 | 11.2 |
N-acetyl putrescine | N-PUT | 231.0 | 157.0 | 28 | 4.8 |
N-acetyl spermidine | N-SPD | 388.0 | 314.2 | 24 | 7.6 |
N-acetyl spermine | N-SPM | 545.4 | 471.3 | 37 | 12.5 |
N-acetyl cadaverine | N-CAD | 245.0 | 171.1 | 51 | 5.1 |
Testosterone | T | 289.2 | 271.3 | 56 | 8.7 |
Dihydrotestosterone | DHT | 291.2 | 255.3 | 24 | 11.1 |
Epitestosterone | EpiT | 289.2 | 271.3 | 26 | 9.8 |
Epitestosterone-d3 | EpiT-d3 | 292.2 | 256.4 | 35 | 9.7 |
Androstenedione | A | 287.2 | 269.3 | 48 | 9.7 |
Pregnenolone | PREG | 317.2 | 299.3 | 29 | 14.1 |
Progesterone | P4 | 315.2 | 297.3 | 30 | 13.9 |
17α-Hydroxyprogesterone | 17α-OHP | 331.2 | 313.3 | 22 | 9.8 |
11β-Hydroxyprogesterone | 11β-OHP | 331.2 | 313.3 | 29 | 9.2 |
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Lee, Y.R.; Lee, J.W.; Hong, J.; Chung, B.C. Simultaneous Determination of Polyamines and Steroids in Human Serum from Breast Cancer Patients Using Liquid Chromatography–Tandem Mass Spectrometry. Molecules 2021, 26, 1153. https://doi.org/10.3390/molecules26041153
Lee YR, Lee JW, Hong J, Chung BC. Simultaneous Determination of Polyamines and Steroids in Human Serum from Breast Cancer Patients Using Liquid Chromatography–Tandem Mass Spectrometry. Molecules. 2021; 26(4):1153. https://doi.org/10.3390/molecules26041153
Chicago/Turabian StyleLee, Yu Ra, Ji Won Lee, Jongki Hong, and Bong Chul Chung. 2021. "Simultaneous Determination of Polyamines and Steroids in Human Serum from Breast Cancer Patients Using Liquid Chromatography–Tandem Mass Spectrometry" Molecules 26, no. 4: 1153. https://doi.org/10.3390/molecules26041153