R.J. Singh, Ph.D. Mayo Clinic
55
Impact of Tandem Mass Spectrometry in Clinical Diagnostics R.J. Singh, Ph.D. Mayo Clinic
Transcript of R.J. Singh, Ph.D. Mayo Clinic
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
