Journal of Southern Medical University ›› 2026, Vol. 46 ›› Issue (2): 301-315.doi: 10.12122/j.issn.1673-4254.2026.02.08
Qiqi ZHOU(
), Junhe CHEN, Chengtao NIE, Qian WANG, Fangbo XIA(), Hongwei ZHOU()
Received:2025-10-21
Online:2026-02-20
Published:2026-03-10
Contact:
Fangbo XIA, Hongwei ZHOU
E-mail:zqq19950928@126.com;xiafb08@163.com;hzhou@smu.edu.cn
Supported by:Qiqi ZHOU, Junhe CHEN, Chengtao NIE, Qian WANG, Fangbo XIA, Hongwei ZHOU. Development of an LC-MS/MS method for 32 steroid hormones and exploration of their gestational variations[J]. Journal of Southern Medical University, 2026, 46(2): 301-315.
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URL: https://www.j-smu.com/EN/10.12122/j.issn.1673-4254.2026.02.08
| Elution time (min) | A (0.5 mmol/L NH4F, %) | B (Methanol, %) | Flow (mL/min) |
|---|---|---|---|
| 0.00 | 50.0 | 50.0 | 0.4 |
| 4.80 | 40.0 | 60.0 | 0.4 |
| 7.00 | 25.0 | 75.0 | 0.4 |
| 8.50 | 10.0 | 90.0 | 0.4 |
| 11.00 | 10.0 | 90.0 | 0.4 |
| 11.10 | 50.0 | 50.0 | 0.4 |
| 13.50 | 50.0 | 50.0 | 0.4 |
Tab.1 Liquid phase washing procedure
| Elution time (min) | A (0.5 mmol/L NH4F, %) | B (Methanol, %) | Flow (mL/min) |
|---|---|---|---|
| 0.00 | 50.0 | 50.0 | 0.4 |
| 4.80 | 40.0 | 60.0 | 0.4 |
| 7.00 | 25.0 | 75.0 | 0.4 |
| 8.50 | 10.0 | 90.0 | 0.4 |
| 11.00 | 10.0 | 90.0 | 0.4 |
| 11.10 | 50.0 | 50.0 | 0.4 |
| 13.50 | 50.0 | 50.0 | 0.4 |
| NO. | Analyte | Q1 Mass (m/z) | Q3 Mass (m/z) | DP (V) | CE (V) | Retention time (min) |
|---|---|---|---|---|---|---|
| 1 | Melatonin | 233.00 | 159.10 | 25 | 36 | 1.74 |
| 2 | 18-oxocortisol | 377.00 | 313.10 | 110 | 27 | 2.13 |
| 3 | 18-hydroxycortisol | 379.10 | 267.10 | 40 | 24 | 2.40 |
| 4 | Estriol | 287.10 | 171.00 | -180 | -45 | 2.72 |
| 5 | Aldosterone | 361.10 | 315.20 | 40 | 28 | 2.80 |
| 6 | Cortisone | 361.30 | 163.10 | 40 | 32 | 3.12 |
| 7 | 11β-Hydroxyandrost-4-ene-3,17-dione | 303.10 | 267.20 | 40 | 24 | 3.13 |
| 8 | 18-Hydroxycorticosterone | 363.10 | 269.20 | 60 | 28 | 3.22 |
| 9 | Adrenosterone | 301.10 | 257.30 | 75 | 32 | 3.40 |
| 10 | Cortisol | 363.22 | 121.03 | 40 | 36 | 3.57 |
| 11 | Dehydroepiandrosterone Sulfate | 367.10 | 97.00 | -280 | -60 | 3.79 |
| 12 | 11-Ketotestosterone | 303.10 | 121.20 | 40 | 36 | 3.93 |
| 13 | 21-Deoxycortisol | 347.30 | 311.20 | 40 | 24 | 4.58 |
| 14 | Dexamethasone | 393.30 | 373.40 | 25 | 16 | 4.70 |
| 15 | 11β-Hydroxytestosterone | 305.10 | 269.10 | 40 | 24 | 4.87 |
| 16 | Corticosterone | 347.10 | 329.14 | 80 | 20 | 4.96 |
| 17 | 11-Deoxycortisol | 347.10 | 97.10 | 60 | 52 | 5.21 |
| 18 | Androstenedione | 287.40 | 97.00 | 80 | 28 | 6.38 |
| 19 | Estrone | 269.10 | 145.10 | -100 | -45 | 6.55 |
| 20 | Estradiol | 271.20 | 145.10 | -40 | -45 | 6.75 |
| 21 | 21-Hydroxyprogesterone | 331.20 | 97.03 | 60 | 32 | 6.94 |
| 22 | 11β-Hydroxyprogesterone | 331.20 | 313.10 | 85 | 24 | 7.10 |
| 23 | Testosterone | 289.24 | 97.30 | 25 | 28 | 7.25 |
| 24 | 17-Hydroxyprogesterone | 331.15 | 97.10 | 40 | 28 | 7.56 |
| 25 | Dehydroepiandrosterone | 271.00 | 213.10 | 60 | 16 | 7.82 |
| 26 | 17α-Hydroxypregnenolone | 331.10 | 287.30 | -70 | -30 | 7.87 |
| 27 | Epitestosterone | 289.10 | 109.10 | 65 | 36 | 8.20 |
| 28 | Epiandrosterone | 291.00 | 273.30 | 40 | 15 | 8.32 |
| 29 | Dihydrotestosterone | 291.10 | 255.30 | 25 | 23 | 8.51 |
| 30 | Androsterone | 291.00 | 273.30 | 40 | 15 | 9.20 |
| 31 | Pregnenolone | 299.00 | 159.30 | 40 | 31 | 9.62 |
| 32 | Progesterone | 315.30 | 97.10 | 40 | 32 | 9.73 |
Tab.2 Mass spectrometry parameters of LC-MS/MS method
| NO. | Analyte | Q1 Mass (m/z) | Q3 Mass (m/z) | DP (V) | CE (V) | Retention time (min) |
|---|---|---|---|---|---|---|
| 1 | Melatonin | 233.00 | 159.10 | 25 | 36 | 1.74 |
| 2 | 18-oxocortisol | 377.00 | 313.10 | 110 | 27 | 2.13 |
| 3 | 18-hydroxycortisol | 379.10 | 267.10 | 40 | 24 | 2.40 |
| 4 | Estriol | 287.10 | 171.00 | -180 | -45 | 2.72 |
| 5 | Aldosterone | 361.10 | 315.20 | 40 | 28 | 2.80 |
| 6 | Cortisone | 361.30 | 163.10 | 40 | 32 | 3.12 |
| 7 | 11β-Hydroxyandrost-4-ene-3,17-dione | 303.10 | 267.20 | 40 | 24 | 3.13 |
| 8 | 18-Hydroxycorticosterone | 363.10 | 269.20 | 60 | 28 | 3.22 |
| 9 | Adrenosterone | 301.10 | 257.30 | 75 | 32 | 3.40 |
| 10 | Cortisol | 363.22 | 121.03 | 40 | 36 | 3.57 |
| 11 | Dehydroepiandrosterone Sulfate | 367.10 | 97.00 | -280 | -60 | 3.79 |
| 12 | 11-Ketotestosterone | 303.10 | 121.20 | 40 | 36 | 3.93 |
| 13 | 21-Deoxycortisol | 347.30 | 311.20 | 40 | 24 | 4.58 |
| 14 | Dexamethasone | 393.30 | 373.40 | 25 | 16 | 4.70 |
| 15 | 11β-Hydroxytestosterone | 305.10 | 269.10 | 40 | 24 | 4.87 |
| 16 | Corticosterone | 347.10 | 329.14 | 80 | 20 | 4.96 |
| 17 | 11-Deoxycortisol | 347.10 | 97.10 | 60 | 52 | 5.21 |
| 18 | Androstenedione | 287.40 | 97.00 | 80 | 28 | 6.38 |
| 19 | Estrone | 269.10 | 145.10 | -100 | -45 | 6.55 |
| 20 | Estradiol | 271.20 | 145.10 | -40 | -45 | 6.75 |
| 21 | 21-Hydroxyprogesterone | 331.20 | 97.03 | 60 | 32 | 6.94 |
| 22 | 11β-Hydroxyprogesterone | 331.20 | 313.10 | 85 | 24 | 7.10 |
| 23 | Testosterone | 289.24 | 97.30 | 25 | 28 | 7.25 |
| 24 | 17-Hydroxyprogesterone | 331.15 | 97.10 | 40 | 28 | 7.56 |
| 25 | Dehydroepiandrosterone | 271.00 | 213.10 | 60 | 16 | 7.82 |
| 26 | 17α-Hydroxypregnenolone | 331.10 | 287.30 | -70 | -30 | 7.87 |
| 27 | Epitestosterone | 289.10 | 109.10 | 65 | 36 | 8.20 |
| 28 | Epiandrosterone | 291.00 | 273.30 | 40 | 15 | 8.32 |
| 29 | Dihydrotestosterone | 291.10 | 255.30 | 25 | 23 | 8.51 |
| 30 | Androsterone | 291.00 | 273.30 | 40 | 15 | 9.20 |
| 31 | Pregnenolone | 299.00 | 159.30 | 40 | 31 | 9.62 |
| 32 | Progesterone | 315.30 | 97.10 | 40 | 32 | 9.73 |
Fig.1 Chromatogram of 32 steroid hormones in positive ion mode. 1: Melatonin; 2: 18-oxocortisol; 3: 18-hydroxycortisol; 5: Aldosterone; 6: Cortisone; 7: 11β-Hydroxyandrost-4-ene-3,17-dione; 8: 18-Hydroxycorticosterone; 9: Adrenosterone; 10: Cortisol; 12: 11-Ketotestosterone; 13: 21-Deoxycortisol; 14: Dexamethasone; 15: 11β-Hydroxytestosterone; 16: Corticosterone; 17: 11-Deoxycortisol; 18: Androstenedione; 21: 21-Hydroxyprogesterone; 22: 11β-Hydroxyprogesterone; 23: Testosterone; 24: 17-Hydroxyprogesterone; 25: Dehydroepiandrosterone; 27: Epitestosterone; 28: Epiandrosterone; 29: Dihydrotestosterone; 30: Androsterone; 31: Pregnenolone; 32: Progesterone.
Fig.2 Chromatogram of 32 steroid hormones in negative ion mode. 4: Estriol; 11: Dehydroepiandrosterone Sulfate; 19: Estrone; 20: Estradiol; 26: 17α-Hydroxypregnenolone.
| NO. | Analytes | Linearity | LOQ (ng/mL) | LOD (ng/mL) | ||
|---|---|---|---|---|---|---|
| Regression | Range (ng/mL) | R2 | ||||
| 1 | Melatonin | y=202x-0.0483 | 0.005-12 | 0.994 | 0.005 | 0.002 |
| 2 | 18-Oxocortisol | y=35.6x-0.0212 | 0.01-15 | 0.993 | 0.010 | 0.007 |
| 3 | 18-Hydroxycortisol | y=5x-0.0353 | 0.03-30 | 0.997 | 0.030 | 0.020 |
| 4 | Estriol | y=2.54e+003x-107 | 0.2-300 | 0.999 | 0.200 | 0.070 |
| 5 | Aldosterone | y=41.4x-0.0763 | 0.007-20 | 0.998 | 0.007 | 0.003 |
| 6 | Cortisone | y=2.24x+0.00123 | 0.02-200 | 0.993 | 0.020 | 0.010 |
| 7 | 11β-Hydroxyandrost-4-ene-3,17-dione | y=49.7x-0.0161 | 0.005-10 | 0.996 | 0.005 | 0.003 |
| 8 | 18-Hydroxycorticosterone | y=12x+0.00666 | 0.003-15 | 0.996 | 0.003 | 0.001 |
| 9 | Adrenosterone | y=0.543x+0.00285 | 0.05-400 | 0.998 | 0.050 | 0.020 |
| 10 | Cortisol | y=3.86x+0.00437 | 0.02-400 | 0.996 | 0.020 | 0.006 |
| 11 | Dehydroepiandrosterone sulfate | y=1.49e+003x+108 | 1-7500 | 0.999 | 1.000 | 0.900 |
| 12 | 11-Ketotestosterone | y=11.5x-0.00131 | 0.001-6 | 0.993 | 0.001 | 0.0007 |
| 13 | 21-Deoxycortisol | y=4.8x-0.00272 | 0.003-15 | 0.996 | 0.003 | 0.001 |
| 14 | Dexamethasone | y=0.409x-0.00365 | 0.1-60 | 0.996 | 0.100 | 0.050 |
| 15 | 11β-Hydroxytestosterone | y=1.2x-0.00209 | 0.01-30 | 0.998 | 0.010 | 0.007 |
| 16 | Corticosterone | y=4.65x+0.00826 | 0.01-30 | 0.998 | 0.010 | 0.007 |
| 17 | 11-Deoxycortisol | y=0.358x+0.000234 | 0.01-15 | 0.997 | 0.005 | 0.001 |
| 18 | Androstenedione | y=2.14x-0.00139 | 0.01-30 | 0.997 | 0.007 | 0.003 |
| 19 | Estrone | y=1.55e+005x-676 | 0.1-900 | 0.997 | 0.100 | 0.050 |
| 20 | Estradiol | y=3.52e+004x-344 | 0.1-600 | 0.996 | 0.100 | 0.070 |
| 21 | 21-Hydroxyprogesterone | y 2.24x+ 0.0106 | 0.007-20 | 0.992 | 0.007 | 0.003 |
| 22 | 11β-Hydroxyprogesterone | y=1.21x+0.000691 | 0.02-50 | 0.996 | 0.020 | 0.010 |
| 23 | Testosterone | y=2.68x+0.0107 | 0.01-15 | 0.997 | 0.005 | 0.001 |
| 24 | 17-Hydroxyprogesterone | y=1.21x+0.00351 | 0.01-100 | 0.995 | 0.010 | 0.006 |
| 25 | Dehydroepiandrosterone | y=0.338x+0.000631 | 0.05-100 | 0.993 | 0.050 | 0.020 |
| 26 | 17α-Hydroxypregnenolone | y=5.74e+004x-846 | 0.1-100 | 0.995 | 0.100 | 0.040 |
| 27 | Epitestosterone | y=0.985x-0.00085 | 0.01-5 | 0.997 | 0.010 | 0.004 |
| 28 | Epiandrosterone | y=0.307x-0.0116 | 0.01-5 | 0.997 | 0.010 | 0.004 |
| 29 | Dihydrotestosterone | y=3.77x+0.000735 | 0.005-10 | 0.996 | 0.005 | 0.002 |
| 30 | Androsterone | y=0.938x-0.027 | 0.1-10 | 0.997 | 0.100 | 0.070 |
| 31 | Pregnenolone | y=0.581x+0.0466 | 0.1-100 | 0.995 | 0.100 | 0.040 |
| 32 | Progesterone | y=4.93x+0.00683 | 0.005-30 | 0.997 | 0.005 | 0.003 |
Tab.3 Linear equations, linear ranges, correlation coefficients, LOQ, and LOD of the method
| NO. | Analytes | Linearity | LOQ (ng/mL) | LOD (ng/mL) | ||
|---|---|---|---|---|---|---|
| Regression | Range (ng/mL) | R2 | ||||
| 1 | Melatonin | y=202x-0.0483 | 0.005-12 | 0.994 | 0.005 | 0.002 |
| 2 | 18-Oxocortisol | y=35.6x-0.0212 | 0.01-15 | 0.993 | 0.010 | 0.007 |
| 3 | 18-Hydroxycortisol | y=5x-0.0353 | 0.03-30 | 0.997 | 0.030 | 0.020 |
| 4 | Estriol | y=2.54e+003x-107 | 0.2-300 | 0.999 | 0.200 | 0.070 |
| 5 | Aldosterone | y=41.4x-0.0763 | 0.007-20 | 0.998 | 0.007 | 0.003 |
| 6 | Cortisone | y=2.24x+0.00123 | 0.02-200 | 0.993 | 0.020 | 0.010 |
| 7 | 11β-Hydroxyandrost-4-ene-3,17-dione | y=49.7x-0.0161 | 0.005-10 | 0.996 | 0.005 | 0.003 |
| 8 | 18-Hydroxycorticosterone | y=12x+0.00666 | 0.003-15 | 0.996 | 0.003 | 0.001 |
| 9 | Adrenosterone | y=0.543x+0.00285 | 0.05-400 | 0.998 | 0.050 | 0.020 |
| 10 | Cortisol | y=3.86x+0.00437 | 0.02-400 | 0.996 | 0.020 | 0.006 |
| 11 | Dehydroepiandrosterone sulfate | y=1.49e+003x+108 | 1-7500 | 0.999 | 1.000 | 0.900 |
| 12 | 11-Ketotestosterone | y=11.5x-0.00131 | 0.001-6 | 0.993 | 0.001 | 0.0007 |
| 13 | 21-Deoxycortisol | y=4.8x-0.00272 | 0.003-15 | 0.996 | 0.003 | 0.001 |
| 14 | Dexamethasone | y=0.409x-0.00365 | 0.1-60 | 0.996 | 0.100 | 0.050 |
| 15 | 11β-Hydroxytestosterone | y=1.2x-0.00209 | 0.01-30 | 0.998 | 0.010 | 0.007 |
| 16 | Corticosterone | y=4.65x+0.00826 | 0.01-30 | 0.998 | 0.010 | 0.007 |
| 17 | 11-Deoxycortisol | y=0.358x+0.000234 | 0.01-15 | 0.997 | 0.005 | 0.001 |
| 18 | Androstenedione | y=2.14x-0.00139 | 0.01-30 | 0.997 | 0.007 | 0.003 |
| 19 | Estrone | y=1.55e+005x-676 | 0.1-900 | 0.997 | 0.100 | 0.050 |
| 20 | Estradiol | y=3.52e+004x-344 | 0.1-600 | 0.996 | 0.100 | 0.070 |
| 21 | 21-Hydroxyprogesterone | y 2.24x+ 0.0106 | 0.007-20 | 0.992 | 0.007 | 0.003 |
| 22 | 11β-Hydroxyprogesterone | y=1.21x+0.000691 | 0.02-50 | 0.996 | 0.020 | 0.010 |
| 23 | Testosterone | y=2.68x+0.0107 | 0.01-15 | 0.997 | 0.005 | 0.001 |
| 24 | 17-Hydroxyprogesterone | y=1.21x+0.00351 | 0.01-100 | 0.995 | 0.010 | 0.006 |
| 25 | Dehydroepiandrosterone | y=0.338x+0.000631 | 0.05-100 | 0.993 | 0.050 | 0.020 |
| 26 | 17α-Hydroxypregnenolone | y=5.74e+004x-846 | 0.1-100 | 0.995 | 0.100 | 0.040 |
| 27 | Epitestosterone | y=0.985x-0.00085 | 0.01-5 | 0.997 | 0.010 | 0.004 |
| 28 | Epiandrosterone | y=0.307x-0.0116 | 0.01-5 | 0.997 | 0.010 | 0.004 |
| 29 | Dihydrotestosterone | y=3.77x+0.000735 | 0.005-10 | 0.996 | 0.005 | 0.002 |
| 30 | Androsterone | y=0.938x-0.027 | 0.1-10 | 0.997 | 0.100 | 0.070 |
| 31 | Pregnenolone | y=0.581x+0.0466 | 0.1-100 | 0.995 | 0.100 | 0.040 |
| 32 | Progesterone | y=4.93x+0.00683 | 0.005-30 | 0.997 | 0.005 | 0.003 |
| NO. | Analytes | Day 1 (n=3) | Day 2 (n=3) | Day 3 (n=3) | |||
|---|---|---|---|---|---|---|---|
| Con (ng/mL) | CV (%) | Con (ng/mL) | CV (%) | Con (ng/mL) | CV (%) | ||
| 1 | Melatonin | 12.58 | 4.83 | 11.33 | -5.58 | 12.96 | 8.0 |
| 2 | 18-Oxocortisol | 15.89 | 5.93 | 14.17 | -5.53 | 16.02 | 6.8 |
| 3 | 18-Hydroxycortisol | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 4 | Estriol | 312.90 | 4.30 | 287.10 | -4.30 | 324.60 | 8.20 |
| 5 | Aldosterone | 20.78 | 3.90 | 18.46 | -7.70 | 21.60 | 8.00 |
| 6 | Cortisone | 96.24 | -3.76 | 103.72 | 3.72 | 108.13 | 8.13 |
| 7 | 11β-Hydroxyandrost-4-ene-3,17-dione | 10.62 | 6.20 | 9.38 | -6.20 | 10.80 | 8.00 |
| 8 | 18-Hydroxycorticosterone | 14.18 | -5.47 | 15.73 | 4.87 | 14.48 | -3.47 |
| 9 | Adrenosterone | 417.2 | 4.30 | 383.6 | -4.10 | 432.40 | 8.10 |
| 10 | Cortisol | 423.64 | 5.91 | 382.88 | -4.28 | 414.48 | 3.62 |
| 11 | Dehydroepiandrosterone sulfate | 7875.00 | 5.00 | 7125 | -5.00 | 8100.00 | 8.00 |
| 12 | 11-Ketotestosterone | 6.36 | 6.00 | 5.58 | -7.00 | 6.54 | 9.00 |
| 13 | 21-Deoxycortisol | 15.89 | 5.93 | 14.17 | -5.53 | 16.02 | 6.80 |
| 14 | Dexamethasone | 63.60 | 6.00 | 56.52 | -5.80 | 64.80 | 80.00 |
| 15 | 11β-Hydroxytestosterone | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 16 | Corticosterone | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 17 | 11-Deoxycortisol | 14.18 | -5.47 | 15.73 | 4.87 | 14.48 | -3.47 |
| 18 | Androstenedione | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 19 | Estrone | 943.74 | 4.86 | 862.38 | -4.18 | 972.54 | 8.06 |
| 20 | Estradiol | 624.60 | 4.10 | 576.42 | -3.93 | 648.54 | 8.09 |
| 21 | 21-Hydroxyprogesterone | 21.83 | 9.15 | 18.38 | -8.1 | 19.14 | -4.30 |
| 22 | 11β-Hydroxyprogesterone | 53.00 | 6.00 | 46.50 | -7.00 | 54.50 | 9.00 |
| 23 | Testosterone | 14.05 | -4.33 | 14.92 | -0.53 | 15.79 | 5.27 |
| 24 | 17-Hydroxyprogesterone | 107.83 | 5.83 | 92.94 | -7.06 | 103.66 | 3.66 |
| 25 | Dehydroepiandrosterone | 94.61 | -5.39 | 105.27 | 5.27 | 107.83 | 7.83 |
| 26 | 17α-Hydroxypregnenolone | 105.38 | 5.38 | 106.13 | 6.13 | 94.11 | -5.89 |
| 27 | Epitestosterone | 5.26 | 5.20 | 4.75 | -5.00 | 5.40 | 8.00 |
| 28 | Epiandrosterone | 5.31 | 6.20 | 4.69 | -6.20 | 4.60 | -8.00 |
| 29 | Dihydrotestosterone | 9.58 | -4.20 | 10.62 | 6.20 | 9.17 | -8.3 |
| 30 | Androsterone | 9.38 | -6.20 | 10.62 | 6.20 | 10.80 | 8.00 |
| 31 | Pregnenolone | 103.72 | 3.72 | 94.18 | -5.82 | 108.13 | 8.13 |
| 32 | Progesterone | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
Tab.5 Validation of the stability of the high-performance liquid chromatography autosampler
| NO. | Analytes | Day 1 (n=3) | Day 2 (n=3) | Day 3 (n=3) | |||
|---|---|---|---|---|---|---|---|
| Con (ng/mL) | CV (%) | Con (ng/mL) | CV (%) | Con (ng/mL) | CV (%) | ||
| 1 | Melatonin | 12.58 | 4.83 | 11.33 | -5.58 | 12.96 | 8.0 |
| 2 | 18-Oxocortisol | 15.89 | 5.93 | 14.17 | -5.53 | 16.02 | 6.8 |
| 3 | 18-Hydroxycortisol | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 4 | Estriol | 312.90 | 4.30 | 287.10 | -4.30 | 324.60 | 8.20 |
| 5 | Aldosterone | 20.78 | 3.90 | 18.46 | -7.70 | 21.60 | 8.00 |
| 6 | Cortisone | 96.24 | -3.76 | 103.72 | 3.72 | 108.13 | 8.13 |
| 7 | 11β-Hydroxyandrost-4-ene-3,17-dione | 10.62 | 6.20 | 9.38 | -6.20 | 10.80 | 8.00 |
| 8 | 18-Hydroxycorticosterone | 14.18 | -5.47 | 15.73 | 4.87 | 14.48 | -3.47 |
| 9 | Adrenosterone | 417.2 | 4.30 | 383.6 | -4.10 | 432.40 | 8.10 |
| 10 | Cortisol | 423.64 | 5.91 | 382.88 | -4.28 | 414.48 | 3.62 |
| 11 | Dehydroepiandrosterone sulfate | 7875.00 | 5.00 | 7125 | -5.00 | 8100.00 | 8.00 |
| 12 | 11-Ketotestosterone | 6.36 | 6.00 | 5.58 | -7.00 | 6.54 | 9.00 |
| 13 | 21-Deoxycortisol | 15.89 | 5.93 | 14.17 | -5.53 | 16.02 | 6.80 |
| 14 | Dexamethasone | 63.60 | 6.00 | 56.52 | -5.80 | 64.80 | 80.00 |
| 15 | 11β-Hydroxytestosterone | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 16 | Corticosterone | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 17 | 11-Deoxycortisol | 14.18 | -5.47 | 15.73 | 4.87 | 14.48 | -3.47 |
| 18 | Androstenedione | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
| 19 | Estrone | 943.74 | 4.86 | 862.38 | -4.18 | 972.54 | 8.06 |
| 20 | Estradiol | 624.60 | 4.10 | 576.42 | -3.93 | 648.54 | 8.09 |
| 21 | 21-Hydroxyprogesterone | 21.83 | 9.15 | 18.38 | -8.1 | 19.14 | -4.30 |
| 22 | 11β-Hydroxyprogesterone | 53.00 | 6.00 | 46.50 | -7.00 | 54.50 | 9.00 |
| 23 | Testosterone | 14.05 | -4.33 | 14.92 | -0.53 | 15.79 | 5.27 |
| 24 | 17-Hydroxyprogesterone | 107.83 | 5.83 | 92.94 | -7.06 | 103.66 | 3.66 |
| 25 | Dehydroepiandrosterone | 94.61 | -5.39 | 105.27 | 5.27 | 107.83 | 7.83 |
| 26 | 17α-Hydroxypregnenolone | 105.38 | 5.38 | 106.13 | 6.13 | 94.11 | -5.89 |
| 27 | Epitestosterone | 5.26 | 5.20 | 4.75 | -5.00 | 5.40 | 8.00 |
| 28 | Epiandrosterone | 5.31 | 6.20 | 4.69 | -6.20 | 4.60 | -8.00 |
| 29 | Dihydrotestosterone | 9.58 | -4.20 | 10.62 | 6.20 | 9.17 | -8.3 |
| 30 | Androsterone | 9.38 | -6.20 | 10.62 | 6.20 | 10.80 | 8.00 |
| 31 | Pregnenolone | 103.72 | 3.72 | 94.18 | -5.82 | 108.13 | 8.13 |
| 32 | Progesterone | 28.65 | -4.50 | 31.23 | 4.10 | 27.84 | -7.20 |
Fig.3 Optimization of washing step conditions in SPE.
Fig.4 Optimization of elution conditions in SPE.
Fig.5 Optimization of mobile phase additive conditions.
Fig.6 Representative total ion current chromatogram of the target steroid hormones in a second-trimester serum sample. A-D: Mass spectral peak charts with different intensity ranges, showing the chromatographic separation of target compounds in low, medium-low, medium-high, and high response intervals, respectively. 1: Melatonin; 2: 18-oxocortisol; 3: 18-hydroxycortisol; 4: Estriol; 5: Aldosterone; 6: Cortisone; 7: 11β-Hydroxyandrost-4-ene-3,17-dione; 8: 18-Hydroxycorticosterone; 9: Adrenosterone; 10: Cortisol; 11: Dehydroepiandrosterone Sulfate; 12: 11-Ketotestosterone; 13: 21-Deoxycortisol; 14: Dexamethasone; 15: 11β-Hydroxytestosterone; 16: Corticosterone; 17: 11-Deoxycortisol; 18: Androstenedione; 19: Estrone; 20: Estradiol; 21: 21-Hydroxyprogesterone; 22: 11β-Hydroxyprogesterone; 23: Testosterone; 24: 17-Hydroxyprogesterone; 25: Dehydroepiandrosterone; 26: 17α-Hydroxypregnenolone; 27: Epitestosterone; 28: Epiandrosterone; 29: Dihydrotestosterone; 30: Androsterone; 31: Pregnenolone; 32: Progesterone.
Fig.7 Variations of progesterone, 11-deoxycortisol, 11β-hydroxyprogesterone, estriol, 21-hydroxyprogesterone, 11β-hydroxytestosterone, epitestosterone, 18-hydroxycorticosterone, 17-hydroxyprogesterone and dehydroepiandrosterone sulfate throughout pregnancy. A: Progesterone; B: 11-Deoxycortisol; C: 11β-Hydroxyprogesterone; D: Estriol; E: 21-Hydroxyprogesterone; F: 11β-Hydroxytestosterone; G: Epitestosterone; H: 18-Hydroxycorticosterone; I: 17-Hydroxyprogesterone; J: Dehydroepiandrosterone sulfate.T1:First trimester;T2: Second trimester;T3:Third trimester. *P<0.05 vs T1; #P<0.05 vs T2.
Fig.8 Trends of androstenedione, corticosterone, cortisone, estrone, estradiol, 21-deoxycortisol, cortisol and 18-hydro-xycortisol throughout pregnancy. A:Androstenedione. B: Corticosterone. C: Cortisone. D: Estrone. E: Estradiol. F: 21-Deoxycortisol. G: Cortisol. H: 18-Hydroxycortisol. *P<0.05 vs T1.
Fig.9 Trends of 11-ketotestosterone, pregnenolone and aldosterone throughout pregnancy. A: 11-Ketotestosterone. B: Pregnenolone. C: Aldosterone. *P<0.05 vs T1; #P<0.05 vs T2.
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