Mass Spec Testing for Hormone Profiles: Making an Impact on Patient Care

R.J. Singh, Ph.D. Mayo Clinic

Objectives •Congenital Adrenal Hyperplasia (CAH) •Sex •Cushing’s

CAH New Born Screening

1 CAH

CholesterolBiosynthesis of Steroids

Pregnenolone 17- OH DHEA ase ’ ------3SDH 17,20 desmolase 17 OH 17-OH Progesterone

21 OH'ase 17b SDH ------Aromatase

21-Deoxycorti- 11-deoxycortisol costerone 11 OH'ase Aromatase ------ 17b SDH 18 OH'ase ------ ---

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 ’ ------3SDH 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 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

25

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 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- 100 ng/mL 5.4 Androstenedione 100 ng/mL 0.94 Methyltestosterone 100 ng/mL 0.68 Estradiol-17 100 ng/mL 0.02 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 ’ ------3SDH 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

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)

44

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 ’ ------3SDH 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- 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 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

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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