Mass Spec Testing for Steroid Hormone Profiles: Making an Impact on Patient Care
R.J. Singh, Ph.D. Mayo Clinic
Objectives •Congenital Adrenal Hyperplasia (CAH) •Sex Steroids •Cushing’s
CAH New Born Screening
1 CAH
CholesterolBiosynthesis of Steroids
Pregnenolone 17- OH Pregnenolone DHEA ase ’ ------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione
21 OH'ase 17b SDH ------Aromatase
21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ------Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ------Cortisone --- Aldosterone
STEROID PROFILE BY LC MS/MS
TIC: from 051200-36
9.5e5 8 9.0e5 1. Cortisone 8.5e5 2. Cortisol, Cortisol d-4 8.0e5 3. 21-Deoxycortisol 4. Corticosterone 7.5e5 5. 11-Deoxycortisol 7.0e5 6. Androstendione 6.5e5 7. DOC 8. 17-Hydroxyprogesterone 6.0e5 17-Hydroxypregnenolone 5.5e5 9. Progesterone 5.0e5 10. Pregnenolone
Intensity, cps 4.5e5
4.0e5
3.5e5 3.0e5 6 2.5e5 5 7 10 2.0e5 1.5e5 34 2 1.0e5 1 5.0e4 9
1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 Time, min 6
2 Basics of MS Method
Basics of MS Method
Lack of Standardization
3 RIA vs. LC-MS/MS
14000
12000
10000
8000
6000
4000
Mayo LC/MS/MS ng/dL LC/MS/MS Mayo 2000
0 0 2000 4000 6000 8000 10000 12000 14000 Ext/RIA ng/dL
Correlation Between Two Sites
4 Bland Altman Plot (N=76) 1000 + 2 SD = 801.4
500 + 1 SD = 405.8
Mean difference= 10.1
0 (ng/dL) - 1 SD = 385.6 -500
- 2 SD = 781.4
-1000
0 200 400 600
Difference in 17-OHPG; MS/MS -DifferenceMS/MS17-OHPG; in 2.122*Pantex RIA Average 17-OHPG Values 2.122*RIA and LC
CholesterolBiosynthesis of Steroids
Pregnenolone 17- OH Pregnenolone DHEA ase ’ ------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione
21 OH'ase 17b SDH ------Aromatase
21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ------Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ------Cortisone --- Aldosterone
Monthly Test Volumes of Testosterone
Total Testo + Free Testo
Total Testo
Total Testo + Bioavail. Testo
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5 Androgens in Health and Disease • General well-being • Energy • Mood • Libido and sexual satisfaction • Bone physiology • Muscle mass • Hot flashes
Labrie F, et al. Endocr Rev. 2003;24:152-582.
Dimitrakakis C, et al. Menopause. 2004;11:531-535. 16
Methodologies for Endocrine Analytes:
•RIA •GC-FID •CIA •LC-UV/EC •ELISA •GC-MS •FIA •LC-MS •ICMA •LC-MS/MS
Low Male Testosterone (<60 ng/dL): ACS-Enhanced vs. LC-MS/MS
50
45
y = 0.8306x - 2.5493 40 R2 = 0.8482
35 n = 46 30
25
20
15 Testosterone by LC-MS/MS (ng/dL) LC-MS/MS by Testosterone 10
5
0 0 102030405060 Testosterone by Enhanced ACS:180 (ng/dL)
6 Correlation between Immunoassays and Gold standard GC-MS method
Serum Testosterone nmol/L GC-MS
GC-MS method is labor intensive % difference % difference and less sensitive compared to LC-MS/MS method
Serum Testosterone nmol/L GC-MS z
Quality of The T Lab Test
Instrument No. Mean S.D. C.V. Median Low High Labs Value Value
Y-01 Abbott Architect 10 717.0 31.9 4.5 726 658 756 Bayer Acs:180 14 718.3 37.1 5.2 717 653 779 Bayer Advia Centaur 354 769.2 77.2 10.0 767 562 996 Beckman Access/2 146 653.3 49.4 7.6 654 212 858 Beckman Unicel DxI 87 620.0 31.1 5.0 622 540 701 Diagnostic Sys Solid 5 784 724 874 DPC Coat-A-Count 31 720.9 62.9 8.7 733 590 855 DPC Immulite 2000 131 986.9 79.5 8.1 986 776 1243 DPC Immulite 2500 12 976.4 79.2 8.1 946 906 1150 DPC Immulite/1000 57 924.6 100.7 10.9 909 636 1189 Roche E170 76 924.8 33.5 3.6 927 843 1007 Roche Elecsys 1010/2010 76 895.4 40.0 4.5 898 776 976 Tosoh AIA-Pack 15 1024.3 210.1 20.5 920 843 1428 Vitros ECi 80 890.3 58.9 6.6 889 765 1030 All Instruments 1144 799.6 145.0 18.1 796 212 1450 Mass Spectrometry 5 586 334 649
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7 22
An Endocrine Society Position Statement: Precision and Accuracy Matters Laboratory proficiency testing should be based on the ability to measure accurately and precisely samples containing known concentrations of testosterone, not only on agreement with others using the same method
J Clin Endocrinol Metab 92:405-413, 2007 23
8 Instrument Parts to TLX4 – MS/MS 4000
TLX4 (Liquid Chromatography-LC) MS/MS (Tandem Mass Spectrometer)
Injector 8 valve VIM
LX Inj. ports
TX Inj. ports LC System 1
LC System 2
LC System 3 Computer
LC System 4
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9 Dynamic range of competitive immunoassays
(conc. limited)
www.aapsj.org/view.asp?art=aapsj0902029 28
Sex Hormone-Binding Globulin • SHBG is the carrier protein for estrogen and testosterone • SHBG-bound fraction is unavailable for biological activity • Production regulated by estrogen- testosterone balance • Estrogen stimulates SHBG production • Testosterone decreases SHBG synthesis
Selby C. Ann Clin Biochem. 1990;27:532-541. 29
Steroids with Mol. Wt. of 288.4
288.42 5, 7-ANDROSTADIEN-3β, 17β-DIOL 288.42 5α-ANDROSTAN-3, 16-DIONE 288.42 5α-ANDROSTAN-3, 17-DIONE 288.42 5β-ANDROSTAN-3, 17-DIONE 288.42 1, (5α)-ANDROSTEN-17β-OL-3-ONE 288.42 2, (5α)-ANDROSTEN-11α-OL-17-ONE 288.42 4-ANDROSTEN-3α-OL-17-ONE 288.42 4-ANDROSTEN-3β-OL-17-ONE 288.42 4-ANDROSTEN-17α-OL-3-ONE 288.42 4-ANDROSTEN-17β-OL-3-ONE 288.42 5-ANDROSTEN-3β-OL-16-ONE 288.42 5-ANDROSTEN-3α-OL-17-ONE 288.42 5-ANDROSTEN-3β-OL-17-ONE 288.42 5-ANDROSTEN-17α-OL-3-ONE 288.42 5-ANDROSTEN-17β-OL-3-ONE 288.42 9(11), (5α)-ANDROSTEN-3β-OL-17-ONE 288.42 9(11), (5β)-ANDROSTEN-3α-OL-17-ONE
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10 Compound Amount Added % Cross-reactivity 5-dihydrotestosterone 100 ng/mL 5.4 Androstenedione 100 ng/mL 0.94 Methyltestosterone 100 ng/mL 0.68 Estradiol-17 100 ng/mL 0.02 Androsterone 1 g/mL < 0.1 Cortisol 1 g/mL < 0.1 Corticosterone 1 g/mL < 0.1 Cyproterone 100 ng/mL < 0.1 Danazol 1 g/mL < 0.1 DHEA-sulfate 1 g/mL < 0.1 11-deoxycortisol 1 g/mL < 0.1 Dexamethasone 1 g/mL < 0.1 Estrone 100 ng/mL < 0.1 Oxymetholone 100 ng/mL < 0.1 Progesterone 1 g/mL < 0.1
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Reference Ranges for Testosterone by LC-MS/MS from Two Different Endocrine Laboratories
N Total, nmol/L Tanner Stages Reference Range (ng/dL) (ng/dL) Females Females
TS1 147 <0.588 (17) TS1 <7-20 TS2 66 0.138-1.35 (4-39) TS2 <7-47 TS3 85 0.346-2.07 (10-60) TS3 17-75 TS4 77 0.277-2.18 (8-63) TS4 20-75 TS5 123 0.346-2.07 (10-60) TS5 12-60 Males Males
TS1 143 <0.657 (19) TS1 <7-20 TS2 66 0.069-5.16 (2-149) TS2 8-66 TS3 65 0.242-26.37 (7-762) TS3 26-800 TS4 98 5.71-29.54 (165-854) TS4 85-1200 TS5 42 6.71-27.09 (194-783) TS5 300-950
Inter-individual Variability
Harman, S. M. et al. J Clin Endocrinol Metab 2001;86:724-731
Copyright ©2001 The Endocrine Society
11 Testosterone – Representative Sample
Analyte
Internal Standard
Testosterone – Comparison Curve – 1216 Samples
2500 y = 0.9951x - 26.305 2000 R2 = 0.9879 1500 T (ng/ml) 1000
500
0 0 500 1000 1500 2000 2500 T (ng/ml)
Poor Inter-Laboratory Agreement of Testosterone Measurements
AACC-2008
12 CholesterolBiosynthesis of Steroids
Pregnenolone 17- OH Pregnenolone DHEA ase ’ ------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione
21 OH'ase 17b SDH ------Aromatase
21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ------Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ------Cortisone --- Aldosterone
Estrogens Undergo Extensive CYP Enzymatic Modifications
Zhu BT. Lee AJ. NADPH-dependent metabolism of 17beta-estradiol and estrone to polar and nonpolar metabolites by human tissues and cytochrome P450 isoforms. Steroids. 70(4):225-44, 200
Limitations of Steroid Determination by Direct Immunoassay J. Taieb, C. Benattar, A. S. Birr, and A. Lindenbaum Clin. Chem. 2002; 48: 583 - 585
13 E2 by 7 Immuno-assays and GC-MS/MS
Lee, J. S. et al. J Clin Endocrinol Metab 2006;91:3791-3797 40 Copyright ©2006 The Endocrine Society
Testosterone and Estrogen Circulation in the Body
Bioavailable Testosterone Bioavailable Estrogen
~0.5-2% free ~1.8% free ~30% bound ~60% bound to albumin to albumin
~35% bound ~65% bound to SHBG to SHBG
Simon JA. Fert Steril. 2002;77:S77-S82. Demers LM. In: Redmond, G, ed. Androgenic Disorders. Raven Press, New York, NY; 1995:21-34.
Impact of Drugs
Epilepsia, 32(Suppl. 6):S60-S67, I99 I
14 Anti-aromatase Compounds
Exemestane AromasinR Atamestane
Letrozole Anastrozole FemeraR ArimidixR
Chromatographic Separation of Estrogens
1.80E+05
1.60E+05 Estradiol d5 Estradiol d4-E1 d5-E2 1.40E+05 Estrone d4 Estrone 1.20E+05
1.00E+05
8.00E+04 Peak Intensity (cps) Intensity Peak 6.00E+04 E2 E1 4.00E+04
2.00E+04
0.00E+00 01234 Time (minutes)
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Anastrozole vs. Placebo
Mauras N et al. JCEM 2008;93:823-831 ©2008 by Endocrine Society
15 Potential Steroid-Based Molecules of Ion Pair 279.1/149.2
CH3 OH CH3 OH OH OH CH3 CH3
HO HO H H HO H HO H 5a-ESTRAN-3a, 17a-DIOL 5a-ESTRAN-3a, 17ß-DIOL 5a-ESTRAN-3ß, 17a-DIOL 5a-ESTRAN-3ß, 17ß-DIOL
CH3 OH OH CH OH CH OH CH3 3 3
HO H HO HO HO H H H 5ß-ESTRAN-3a, 17a-DIOL 5ß-ESTRAN-3a, 17ß-DIOL 5ß-ESTRAN-3ß, 17a-DIOL 5ß-ESTRAN-3ß, 17ß-DIOL
CholesterolBiosynthesis of Steroids
Pregnenolone 17- OH Pregnenolone DHEA ase ’ ------3SDH 17,20 desmolase 17 OH Progesterone 17-OH Progesterone Androstenedione
21 OH'ase 17b SDH ------Aromatase
21-Deoxycorti- 11-deoxycortisol Testosterone costerone 11 OH'ase Aromatase ------Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ------Cortisone --- Aldosterone
ACTH
Cortisol
Feedback Inhibition
16 Cushing's Syndrome
• "Cushing's Syndrome" refers to the symptoms and signs of hypercortisolism regardless of etiology • "Cushing's Disease" is Cushing's Syndrome due to pituitary hypersecretion of ACTH.
17 Pseudo-Cushings Disease:
• Condition that mimics the biochemical laboratory results of Cushing's Syndrome • Stress • Morbid obesity • Marathon running • Alcoholism • Depression
18 Lab Tests & Cushing’s
• Urine Free Cortisol • Plasma Cortisol • Plasma Free Cortisol • Midnight Plasma Cortisol • Midnight Salivary Cortisol
100000
10000
1000
100 Salivary Cortsiol ng/dL 10
1 Normals Cushing’s n = 186 n = 32
Steroid Hormone
• Total S + S-BG + S-A • Bio-available S+S-A • Free S • Conjugates S-G + S-S metabolites
19 Methods for Urine Cortisol Test:
HPLC-UV LC- MS Immunoassay
Cortisol 24-108 µg/24 hrs Normal Cortisol 5-55 µg/24 hrs Cortisone 16-128 µg/24 hrs
Steroid interference in Immunoassay Aldosterone ND* 11-hydroxyprogesterone 0.50 Allotetrahydrocortisol 4.60 17- hydroxyprogesterone 0.50 Androstenedione ND 17-hydroxypregnanolone ND Corticosterone 2.80 11-keto-androsterone ND Cortisone 7.40 11-beta-etiocholanolone ND -Cortol ND Prenanetriol ND -cortolone ND Prenenolone ND -Cortol ND Progesterone ND -Cortolone ND Spironolactone ND Dehydrocorticosterone 0.50 Testosterone ND 11-deoxycorticosterone 0.40 Tetrahydrocortisol ND 11-deoxycortisol 7.30 Tetrahydrocortisone 0.54 21-deoxycortisol 4.50 Tetrahydro-11- 0.20 deoxycortisol 20-dihydrocortisol 0.50 20-dihydrocortisol 0.70 20-dihydrocortisone 0.40 20-dihydrocortisone ND 11-hydroxyandrosterone ND 6-hydroxycortisol 2.40 11- ND hydroxyetiocholanolone Synthetic Steroids Prednisolone (100 g/dl) 27.00 6-methyl-prednisolone 20.90 Dexamethasone 0.20 Prednisone 6.60 Canrenone ND
Cortisol – Serum Access Protocol Known Interferences
Substance Added Amount (ng/mL) Reactivity % Corticosterone 1000 2.08 Cortisone 1000 7.6 11-Deoxycorticosterone 10000 0.91 11-Deoxycortisol 1000 17.8 17-α-Hydroxyprogesterone 10000 5.33 Progesterone 10000 0.46 Prednisolone 200 7.60 Tetrahydrocortisone 10000 0.1 Prednisone 10000 3.05 Dexamethasone 10000 0.04
20 Cortisol and Cortisone by HPLC-UV
Cortisol and Cortisone by LC-MS Intensity cps Intensity
Time minutes
Cortisol and Cortisone BY LC MS/MS
Cortisone d4-Cortisol Intensity cps Intensity Cortisol
0.0 0.4 0.8 1.2 1.6 2.0 Time minutes
21 Anatomy of the Adrenal Veins
Daunt N Radiographics 2005;25:S143-S158
Cushing’s
Use of Glucocorticoids
22 OH O OH OH O O O H OH OH O O HO O HO O HO H H H O H H OH H H H H H F H H F H O O O O
6a-methyl-prednisolone Prednisone Triamcinolone Acetonide Triamcinolone
O O O OCOCH3 HO HO OH HO O HO OH H H H HO H H H H F H O O O Fludrocortisone Cortisol Cortisone
CH O 3 OH O O OH O HO OH O HO HO HO OH HO H H H HO H H H H F H F H F H O O O O Prednisolone Fluoromethalone Dexamethasone Betamethasone
HPLC Chromatogram
Taylor, RL, Grebe, SK, Singh, RJ. Clin. Chem. 50 (2004) 1-8
Conclusion • Using MS methods sometimes is not a choice but is neccessity. • Doing a thorough method validation can be tedious, but the consequences of not doing it right are wasted time, money, and resources
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23 Thank you [email protected]
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