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 lab here Methodologies for Analysis RIA GC-FID CLIA LC-UV/EC ELISA GC-MS FIA LC-MS ICMA LC-MS/MS Biosynthesis of Steroids Cholesterol Pregnenolone 17- OH Pregnenolone DHEA ---------------------3βSDH -------------------------------------------------------- 17,20 desmolase 17 OH’ase Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase ------ 11-Deoxycorticosterone 11-deoxycortisol Testosterone 11 OH'ase Aromatase ---------------------------------------------- ------ Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone Cushing’s Syndrome Introduction Hypothalamus CRH Pituitary Cortisol ACTH - + Adrenal Gland Cortisol ACTH-Dependent Cushing’s Syndrome Cushing’s Disease Ectopic ACTH Syndrome ACTH-Independent Cushing’s Syndrome Adrenal adenoma Adrenal carcinoma Adrenal Gland Adrenaline Adrenal Cortex (outer) Adrenal Medulla (center) http://www.pathology.vcu.edu/education/endocrine/endocrine/adrenal/micro/adrad1x.gif Obesity Trends* Among U.S. Adults BRFSS, 1990, 1998, 2006 (*BMI ≥30, or about 30 lbs. overweight for 5’4” person) 1990 1998 2006 No Data <10% 10%–14% 15%–19% 20%–24% 25%–29% ≥30% Biosynthesis of Steroids Cholesterol Pregnenolone 17- OH Pregnenolone DHE A ---------------------3βSDH -------------------------------------------------------- 17,20 desmolase 17 OH’ase Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase ------ 21-Deoxycorticosterone11-deoxycortisol Testosterone 11 OH'ase Aromatase ---------------------------------------------- ------ Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone 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-S metabolites O O OH HO OH OH H H H H H H O O 21-Deoxycortisol 11-Deoxycortisol O OH O OH OH HO O OH H H H H H H O O Cortisol Cortisone O OH HO OH HO OH HO OH HO OH O OH H H H H H H H H H O O O OH 6β-Hydroxycortisol 20β-Dihydrocortisol 20β-Dihydrocortisone O OH O OH O OH O OH HO OH HO OH O OH O OH H H H H H H H H H H H H HO HO H H HO HO H H 5α-Tetrahydrocortisol 5β-Tetrahydrocortisol 5α-Tetrahydrocortisone 5β-Tetrahydrocortisone HO OH HO OH HO OH HO OH HO OH HO OH O OH O OH H H H H H H H H H H H H HO HO HO HO H H H H 20β-α-Cortol α-Cortol 20β-α-Cortolone α-Cortolone Clinical Steroidology-Coming of age Teller W.M. Hormone Research Volume 50, Issue 1 1998 •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 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 Method for Urine Cortisol Test Immunoassay Cortisol 24-108 µg/24 hrs Steroid interference in Immunoassay 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- 0.20 deoxycortisol 20α-dihydrocortisol 0.50 20β-dihydrocortisol 0.70 20α-dihydrocortisone 0.40 20β-dihydrocortisone ND 11β-hydroxyandrosterone ND 6β-hydroxycortisol 2.40 11β- ND hydroxyetiocholanolone Synthetic Steroids Prednisolone (100 μg/dl) 27.00 6-methyl-prednisolone 20.90 Dexamethasone 0.20 Prednisone 6.60 Canrenone ND Prednisone Immunoassays for Cortisol http://www.mpbio.com/includes/technical/Corti-cote%20RIA%20-%2006B256440.PDF Methods for Urine Cortisol Test HPLC-UV Immunoassay Cortisol 24-108 µg/24 hrs Normal Cortisol 5-55 µg/24 hrs Cortisone 16-128 µg/24 hrs Cortisol and Cortisone by HPLC-UV 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 Cortisol and Cortisone by LC-MS Intensity cps Time minutes 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 Cortisol and Cortisone BY LC MS/MS Cortisone d4-Cortisol Intensity cps Cortisol 0.0 0.4 0.8 1.2 1.6 2.0 Time minutes Comparison of immunoassay vs. MS/MS 50 40 y = 0.3555x + 3.9311 2 R = 0.5441 30 20 10 0 0 20406080 Cortisol µg/24 hr by Tandem MS µg/24 hr Cortisol ACS 180 Adrenal Suppression with Synth. Steroids Sample Cortisol Concentration ug/24hr Steroid Concentrat ID 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 Concentrat ID 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 Cushing’s Iatrogenic Cushing’s Syndrome Literature Reports Kumar, S, Singh, RJ, Reed, AM, Lteif, AN. Pediatrics 113 (2004) 1820-1824 Iatrogenic Cushing’s Syndrome Mechanism Hypothalamus CRH Cortisol Pituitary Synthetic ACTH Glucocorticoid + Adrenal Gland Cortisol How to confirm a diagnosis of iatrogenic Cushing’s Syndrome? Measure level of synthetic steroids in plasma and/or urine Synthetic Steroids Laboratory Testing HPLC Chromatogram Taylor, RL, Grebe, SK, Singh, RJ. Clin. Chem. 50 (2004) 1-8 New Born Screening Sisters Biosynthesis of Steroids Cholesterol Pregnenolone 17- OH Pregnenolone DHE A ---------------------3βSDH -------------------------------------------------------- 17,20 desmolase 17 OH’ase Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase ------ 21-Deoxycorticosterone11-deoxycortisol Testosterone 11 OH'ase Aromatase ---------------------------------------------- ------ Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone 21-Hydroxylase Deficiency 140 120 100 80 ng/ml 60 40 20 0 Cortisol123Androstenedione OHPG 21-Hydroxylase Deficiency 160 140 120 100 ng/ml 80 60 40 20 0 Cortisol123Androstenedione OHPG 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 LC-MS/MS VITAMIN D 25-OH Vitamin D3 25-OH Vit D -d 3 6 25-OH Vitamin D Peak Intensity internal 2 standard Time (min) High Throughput Front-End Device (Cohesive/Thermo) Cohesive TLX4 System “High Density” MS/MS The parking lot Load- Transfer- No Tee...Loop out Tee...Loop in Wash-Regenerate No Tee...Loop in Biosynthesis of Steroids Cholesterol Pregnenolone 17- OH Pregnenolone DHE A ---------------------3βSDH -------------------------------------------------------- 17,20 desmolase 17 OH’ase Progesterone 17-OH Progesterone Androstenedione 21 OH'ase 17b SDH ---------------------------------------------- Aromatase ------ 21-Deoxycorticosterone11-deoxycortisol Testosterone 11 OH'ase Aromatase ---------------------------------------------- ------ Estrone Corticosterone Cortisol Estradiol 17b SDH 18 OH'ase ---------------------- Cortisone --- Aldosterone ThanksThanks forfor youryour attention!attention! 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