Impact of Tandem Mass Spectrometry in Clinical Diagnostics

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

Definition Diagnosis or Di`ag`nos´tics

• Identification of a disease, disorder, or syndrome through a method of consistent and accurate analysis.

Laboratory Automation

Picture of the UVA labhere

Methodologies for Analysis

RIACLIAELISAFIA ICMA

GC-FID LC-UV/ECGC-MSLC-MSLC-MS/MS

11-Deoxycorticosterone Testosterone11-deoxycortisol

Aldosterone

CorticosteroneEstradiol

Cortisol

Cholesterol

Pregnenolone DHEA17- OH Pregnenolone

3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

Biosynthesis of Steroids

18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -

Progesterone 17-OH Progesterone Androstenedione17

OH

’ase

17,2

0 de

smol

ase

17b SDH

Aromatase

Estrone

Aromatase

17b SDH

Cortisone

Cushing’s SyndromeIntroduction

ACTH-Dependent Cushing’s Syndrome

ACTH-Independent Cushing’s Syndrome

Cushing’s DiseaseEctopic ACTH Syndrome

Adrenal adenomaAdrenal carcinoma

Pituitary

AdrenalGland

ACTH+

Cortisol-

Cortisol

Hypothalamus

CRH

Adrenal Gland

Adrenal Cortex(outer)

Adrenal Medulla(center)

Adrenaline

http://www.pathology.vcu.edu/education/endocrine/endocrine/adrenal/micro/adrad1x.gif

1998

2006

Obesity Trends* Among U.S. AdultsBRFSS, 1990, 1998, 2006

(*BMI ≥30, or about 30 lbs. overweight for 5’4” person)

1990

No Data <10% 10%–14% 15%–19% 20%–24% 25%–29% ≥30%

21-Deoxycorticosterone Testosterone11-deoxycortisol

Aldosterone

CorticosteroneEstradiol

Cortisol

Cholesterol

Pregnenolone DHEA

17- OH Pregnenolone

3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

Biosynthesis of Steroids

18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -

Progesterone 17-OH Progesterone Androstenedione17

OH

’ase

17,2

0 de

smol

ase

17b SDH

Aromatase

Estrone

Aromatase

17b SDH

Cortisone

Lab Tests

• 24 Urine Free Cortisol• Plasma Cortisol• Plasma Free Cortisol• Midnight Plasma Cortisol• Midnight Salivary Cortisol

Steroid Hormone

• Total S + S-BG + S-A

• Free S

• Conjugates S-G + S-Smetabolites

HO OH

OH

O

O

H

H

H

Cortisol

HO OH

OH

HO

O

H

H

H

5α-Tetrahydrocortisol

HO OH

OH

O

HO

H

H

H

20β-Dihydrocortisol

HO OH

OH

O

O

H

H

H

6β-Hydroxycortisol

HO OH

O

O

H

H

H

OH

OH

O

O

H

H

H

11-Deoxycortisol

O OH

OH

O

O

H

H

H

Cortisone

OH

H

O OH

OH

O

HO

H

H

H

20β-Dihydrocortisone

21-Deoxycortisol

O OH

OH

HO

O

H

H

H

5β-TetrahydrocortisoneH

O OH

OH

HO

O

H

H

H

5α-TetrahydrocortisoneH

HO OH

OH

HO

O

H

H

H

5β-TetrahydrocortisolH

HO OH

OH

HO

HO

H

H

H

20β-α-CortolH

HO OH

OH

HO

HO

H

H

H

α-CortolH

O OH

OH

HO

HO

H

H

H

20β-α-CortoloneH

O OH

OH

HO

HO

H

H

H

α-CortoloneH

•1935, 17-ketosteroids by Zimmermann

•1952, paper chromatography, and later column chromatography

•in mid-eighties, 50 steroid metabolites ('profile') in urine (GC-FID)

•radioimmuno-assays (RIA) were introduced for plasma steroids

•at low concentrations cross reactions and interferences recognized

•Nineties, isotope dilution/GC-MS produced highly accurate values for steroids and were considered as gold standard method

Clinical Steroidology-Coming of ageTeller W.M. Hormone Research Volume 50, Issue 1 1998

History of Radioimmunoassay

1950s Berson and Yalow

Immunoassay types

http://www.abcam.co.jp/ps/datasheet/images/ab17544_1.jpg

Immunoassays - central to current lab testing

• Immunoassays allow sensitive detection of a wide range of analytes

• Immunoassays can be very specific• Immunoassays come in a wide range of formats

and can be automated• Today’s reference ranges for a wide range of

analytes have been established with Immunoassays

• Immunoassay problems and limitations are well understood

Immunoassay

Cortisol 24-108 µg/24 hrs

Method for Urine Cortisol Test

Aldosterone ND* 11β-hydroxyprogesterone 0.50 Allotetrahydrocortisol 4.60 17α- hydroxyprogesterone 0.50 Androstenedione ND 17β-hydroxypregnanolone ND Corticosterone 2.80 11-keto-androsterone ND Cortisone 7.40 11-beta-etiocholanolone ND α-Cortol ND Prenanetriol ND α-cortolone ND Prenenolone ND β-Cortol ND Progesterone ND β-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-

deoxycortisol 0.20

20α-dihydrocortisol 0.50 20β-dihydrocortisol 0.70 20α-dihydrocortisone 0.40 20β-dihydrocortisone ND 11β-hydroxyandrosterone ND 6β-hydroxycortisol 2.40 11β-hydroxyetiocholanolone

ND

Synthetic Steroids Prednisolone (100 μg/dl) 27.00 6-methyl-prednisolone 20.90 Dexamethasone 0.20 Prednisone 6.60 Canrenone ND

Steroid interference in Immunoassay

Prednisone

http://www.mpbio.com/includes/technical/Corti-cote%20RIA%20-%2006B256440.PDF

Immunoassays for Cortisol

HPLC-UV

Normal Cortisol 5-55 µg/24 hrsCortisone 16-128 µg/24 hrs

Immunoassay

Cortisol 24-108 µg/24 hrs

Methods for Urine Cortisol Test

Cortisol and Cortisone by HPLC-UV

LC-MS

HPLC-UV

Normal Cortisol 5-55 µg/24 hrsCortisone 16-128 µg/24 hrs

Immunoassay

Cortisol 24-108 µg/24 hrs

Methods for Urine Cortisol Test

Inte

nsity

cps

Time minutes

Cortisol and Cortisone by LC-MS

LC-MS

HPLC-UV

Normal Cortisol 5-55 µg/24 hrsCortisone 16-128 µg/24 hrs

Immunoassay

Cortisol 24-108 µg/24 hrs

Methods for Urine Cortisol Test

0.0 0.4 0.8 1.2 1.6 2.0

Cortisone

Cortisol

d4-Cortisol

Inte

nsity

cps

Cortisol and Cortisone BY LC MS/MS

Time minutes

y = 0.3555x + 3.9311R2 = 0.5441

01020

304050

0 20 40 60 80

Comparison of immunoassay vs. MS/MSC

ortis

ol µ

g/24

hr

by T

ande

m M

S

µg/24 hr Cortisol ACS 180

Sample Cortisol Concentration ug/24hr Steroid ConcentratID Immunoassay LC-UV LC-MS/MS Present ug/24hr

2 1 38.1 1.3 <1.0 Prednisone/Prednisolone 130

Adrenal Suppression with Synth. Steroids

Sample Cortisol Concentration ug/24hr Steroid ConcentratID Immunoassay LC-UV LC-MS/MS Present ug/24hr

2 1 38.1 1.3 <1.0 Prednisone/Prednisolone 130

11 2 1554 2237 10.1 Methylprednisolone 18875

SC6 3 1.8 <1.0 3.5 Dexamethasone 1404

SC7 4 2.8 <1.0 <1.0 Fludrocortisone 5

SC19 5 <6.0 <1.0 <1.0 Betamethasone 29

SC21 6 <7.5 <1.0 1.3 Dexamethasone 36

SC22 7 252 <1.0 <1.0 Prednisone/Prednisolone 776

Adrenal Suppression with Synth. Steroids

Cushing’s

Iatrogenic Cushing’s SyndromeLiterature Reports

Kumar, S, Singh, RJ, Reed, AM, Lteif, AN. Pediatrics 113 (2004) 1820-1824

Iatrogenic Cushing’s SyndromeMechanism

Pituitary

AdrenalGland

ACTH+

SyntheticGlucocorticoid

Cortisol

Hypothalamus

CRH

How to confirm a diagnosis of iatrogenic Cushing’s Syndrome?

Measure level of synthetic steroids in plasma and/or urine

Cortisol

Synthetic SteroidsLaboratory Testing

HPLC Chromatogram

Taylor, RL, Grebe, SK, Singh, RJ. Clin. Chem. 50 (2004) 1-8

New Born Screening

Sisters

21-Deoxycorticosterone Testosterone11-deoxycortisol

Aldosterone

CorticosteroneEstradiol

Cortisol

Cholesterol

Pregnenolone DHEA

17- OH Pregnenolone

3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

Biosynthesis of Steroids

18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -

Progesterone 17-OH Progesterone Androstenedione17

OH

’ase

17,2

0 de

smol

ase

17b SDH

Aromatase

Estrone

Aromatase

17b SDH

Cortisone

0

20

40

60

80

100

120

140

1 2 3Cortisol Androstenedione OHPG

21-Hydroxylase Deficiencyng

/ml

0

20

40

60

80

100

120

140

160

1 2 3

21-Hydroxylase Deficiency

Cortisol Androstenedione OHPG

ng/m

l

Vit- D

History of Vit-D Testing in the Lab

• The HPLC assay in 1981– 10-50 samples/day

• Conversion to Diasorin RIA 3/20/2000– 50-150 samples/day

• Conversion to MS/MS 11/2/2004– 400 samples/day

• 3000 samples/day

25-OH Vit D3-d6internal

standard

25-OH Vitamin D3

25-OH Vitamin D2

Time (min)

Peak

Inte

nsity

LC-MS/MS VITAMIN D

Cohesive TLX4 System

High Throughput Front-End Device (Cohesive/Thermo)

“High Density” MS/MSThe parking lot

Load-No Tee...Loop out

Transfer-Tee...Loop in

Wash-RegenerateNo Tee...Loop in

21-Deoxycorticosterone Testosterone11-deoxycortisol

Aldosterone

CorticosteroneEstradiol

Cortisol

Cholesterol

Pregnenolone DHEA

17- OH Pregnenolone

3βSDH- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -

21 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

11 OH'ase- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - -- - - - --

Biosynthesis of Steroids

18 OH'ase- - - - - - - - - - - - - - - - - - - - - -- - -

Progesterone 17-OH Progesterone Androstenedione17

OH

’ase

17,2

0 de

smol

ase

17b SDH

Aromatase

Estrone

Aromatase

17b SDH

Cortisone

Thanks for your attention!Thanks for your attention!

singh.ravinder@mayo.edusingh.ravinder@mayo.edu