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Testosterone Testing: Measurement Technologies and Standardization Hubert W Vesper, PhD and Julianne Cook Botelho, PhD Clinical Chemistry Brach Division of Laboratory Sciences National Center for Environmental Health Division of Laboratory Sciences

Overview • Variability in Testosterone Testing – Current Status • CDC Testosterone Standardization Project • Analytical Measurement Procedures

Testing for testosterone is recommended or suggested in clinical guidelines for many years • ASCO Initial Hormonal Management of Androgen-Sensitive Metastatic, Recurrent, or Progressive Prostate Cancer J Clin Oncol 2007;25:1596-1605 • Endocrine Society Clinical Practice Guideline: Testosterone Therapy in Adult Men with androgen Deficiency Syndromes. J Clin Endocrinol Metab 2006;91:1995-2010 • AACE Medical Guideline for Clinical Practice for the Diagnosis and Treatment of Menopause Endocr Pract. 2006;12:315-337 • The role of testosterone therapy in postmenopausal women: position statement of the North American Menopause society. Menopause 2005;12:497-511. • Guidelines on Testicular Cancer . European Urology 2005;48:885–894 • AACE Position Statement on Metabolic and Cardiovascular Consequences of Polycystic Ovary Syndrome Endocr Pract. 2005;11:126-134 • AACE Medical Guideline for Clinical Practice for the Evaluation and Treatment of Male Sexual Dysfunction: A Couple’s Problem Endocr Pract. 2003;9:77-95 • AACE Medical Guideline for Clinical Practice for Evaluation and Treatment of Hypogonadism in Adult Male Patients Endocr Pract. 2002;8:439-456 • AACE Medical Guidelines for Clinical Practice for the diagnosis and treatment of hyperandrogenic disorders Endocr Pract. 2001;7:120-137

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Testosterone testing is increasingly used in research • 177 clinical trials in recruitment or planning phase (clinicaltrials.gov 06/03/2010) • 3,823 articles within the last 3 years (PubMed search from 06/03/2010 for “testosterone” limited to humans) Testosterone levels in blood have been associated with a number of chronic conditions: • metabolic syndrome • diabetes • cardiovascular disease • fractures • neurodegenerative disorder • higher mortality in men

New research directions and clinical applications pose new challenges on testosterone assays Testosterone tests need to be • highly sensitive and specific for reliable detection at low testosterone concentrations (i.e., testosterone in women and children) • highly precise to allow detection of subtle differences in testosterone levels (i.e., to distinguish androgen deficient men from non-deficient men and women with androgen access from those with normal androgen levels) • highly accurate to enable consistent implementation of cut-off values and reference ranges.

Testosterone testing is highly variable across assays

1800

Highest and lowest value reported on one sample by different assays

1600 1400

1000 800 600 400 200

1991

Steinberger Endocr. Pract. 4(1998)

Steinberger 1998

2004

Wang Wang JCEM 2004 89(2004)

N/A

N/A

Y-04

1995

Steinberger Endocr. Pract. 4(1998)

Steinberger 1998

N/A†

Y-10

Y-09

Y-06

Y-05

Y-02

Y-01

Y-95

Y-94

Y-14

Y-13

Y-10

Y-09

Y-06

Y-05

Y-02

0 Y-01

ng/dL

1200

2007

Rosner JCEM 92(2007)

Rosner 2007

Results from the College of American Pathologists Proficiency Testing Surveys

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Variability in testosterone testing does not seem to improve over time Ratio of highest and lowest reported value Ratio 14 12 10 8 6 4 2 0 Y-01

Y-02

Y-05

Y-06

Y-09

Y-10

Y-13

Y-14

Y-94

Y-95

Y-01

Y-02

Y-05

Y-06

1991

1995

Steinberger Steinberger 1998 Endocr. Pract. 4(1998)

Steinberger Steinberger 1998

Y-09

Y-10

Y-04 2004

N/A†

N/A

N/A

2007

Wang Rosner Wang Rosner 2007 JCEM JCEM 2004 89(2004) 92(2007)

Endocr. Pract. 4(1998)

Results from the College of American Pathologists Proficiency Testing Surveys

Measurement bias and variability increases with decreasing testosterone concentrations

GC/MS vs. different immunoassays

Taieb Clin Chem 2003;49:1381-95

Bias patterns differ among immunoassays

LC/MS/MS vs. different immunoassays Wang Clin Endocrin & Metab 2004;89:534

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MS assays show similar bias patterns than immunoassays

Vesper Steroids 2009;74:498-503

Scatter of biases is less pronounced with MS assays than with immunoassays, especially at high testosterone values

Thienpont Clin Chem 2008;54:1290

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CDC Workshop on Improving Steroid Hormone Measurements in Patient Care and Research Translation March 2008 •

Support from the Endocrine Society

•

Attendance: >60 experts, Endocrine Society, AACE, AACC, ASRM, ACS

•

Aim: Identify and discuss problems in steroid hormone testing in research, clinical and public health applications

Problems in Steroid Hormone Testing

Lack of comparability of data

Patient data ↔ Study Data Hospital ↔ Hospital Patient data ↔ Clinical Guideline, Normal Ranges

Lack of adequate assay performance

Ability to detect low concentrations (i.e., androgen deficiency in men Ability to distinguish between normal and elevated concentrations (i.e., PCOS in women)

Lack of consensus on use

Free, bioavailable or total T measurements Biological variability and interfering medications

Lack of reference ranges

Normal ranges for men, women and children

CDC Steroid Hormone Standardization Project Goal: Improve diagnosis, treatment, and prevention of diseases by standardizing clinical laboratory measurements

Objective: Create measurement results that are traceable to one accuracy basis and thus are comparable across methods, time and location

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Step 1: Establishing metrological traceability

Material

Procedure  Value assignment


Calibration

Serum testosterone [nmol/l] Gravimetry Testosterone Calibrator (A-NMI Material# M914b)

ID/GC/MS or ID-LC/MS/MS

Working calibrator NIST SRM 971 or fresh frozen patient samples

Reference measurement procedures

Assay abc master procedure

Product calibrator calibrators

Assay xyz end user’s procedure

Routine measurement procedures

Routine sample Patient xyz *** nmol/l ISO 17511

Step 1: Establishing metrological traceability

Material

Procedure  Value assignment


Calibration

Serum testosterone [nmol/l] Gravimetry Testosterone Calibrator (A-NMI Material# M914b)

ID/GC/MS or ID-LC/MS/MS

Working calibrator NIST SRM 971 or fresh frozen patient samples

Reference measurement procedures

Assay abc master procedure

Product calibrator calibrators

Assay xyz end user’s procedure

Routine measurement procedures

Routine sample Patient xyz *** nmol/l ISO 17511

Step 2: Verify/monitor consistency of calibration and value assignment (traceability) across individual assays

 Interlaboratory comparison studies  Accuracy-based proficiency testing/external quality assessment

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CDC HORMONE STANDARDIZATION PROJECT (CDC-HoSt PROJECT) 40 single donor serum samples with values assigned

Calibration/Calibration Verification

Challenge 1 (1st Quarter)

Phase 1

Phase 2

Challenge 2 10 blinded single donor serum samples per challenge

(2nd Quarter)

Challenge 3 (3rd Quarter)

Challenge 4 (4th Quarter)

CLSI Protocol EP9

Bias Estimation using all 4 challenges

CDC HORMONE STANDARDIZATION PROJECT (CDC-HoSt PROJECT) • Provides patient samples with reference values for testosterone so labs and assay manufacturers can assess accuracy and verify their calibration • Monitors accuracy over time to assure that results stay accurate • Labs and assay manufacturers that are successfully standardized will receive certificate and will be posted on CDC Website. • Provides technical assistance in problem solving • Program is open to manufacturers of assays (incl. immunoassays) and laboratories • Enrollment is quarterly • The standardization effort is endorsed by AACE, Endocrine Society, AEPCOS, ASRM, NAMS, AACC

Analytical Measurement Procedures

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A wide range of different assays and technologies are used for measuring testosterone in patient care and clinical research Immunoassays • majority of clinical testing • mostly commercial assays • ELISA and RIA – homogenous assays and extraction assays • CAP lists 14 different assay platforms (peer groups) from 6 different companies

Mass spectrometry-based assays • increasingly used for clinical testing • in-house developed assays (“home-brew” assays) • GC/MS, GC/MS/MS and LC/MS/MS • >17 different assays described in literature for LC/MS/MS using human serum alone

Immunoassays 70s: Conventional Assay Include Isolation Steps  Advantages: • Inactivation of binding proteins • Isolation of analyte removes interfering compounds • Highly reliable when properly validated

 Disadvantages: • Mostly manual operation • Requires experienced operator • Limited throughput • Sample volume requirements

Immunoassays 80s – present: Direct Assay (no isolation steps)  Advantages: • Highly automated •

High throughput

• Low sample volume requirements • relatively inexpensive

 Disadvantages: • Cross-reactivity with other hormones present in the sample solution (specificity) • Matrix dependent • Lack of sensitivity at low concentrations

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Mass spectrometry is used for many years for the analysis of steroids Year

Development

1950s Electron impact/MS studies of sterols and steroids 1960

First gas chromatography of sterols and steroids

1964

First GC/MS publication on human sterol metabolism

1966

Full urinary steroid profile shown; steroids of complexity range from androgens to cortisol separated in one run

1974

“Mass fragmentography” (selected-ion-monitoring). Hormone assay with labeled internal standards

1974

GC/MS first used for steroid doping control in sports

1982

Fast Atom Bombardment (FAB). First MS of steroid conjugates

1991

ESMS and LC/ESMS introduced

1994

Isotope ratio MS introduced in sports doping control

1990s Tandem MS developments, APCI, APPI 2002

Fully automated HPLC/MS/MS assays. Commercialization of clinical steroid analysis by tandem MS

Adopted from Shackleton 2009 JSBMB

Selection of MS Assays Described in Literature Sample Prep

Ionization T Ions/Transitions IS Ions/Transitions

Range (ng/dL)

LOD (ng/dL)

Multi Analyte

15-1500

15.0

Y

7-2880

8.6

N

Guo T et al. J Clin Chem 2006 372 76-82 Cawood ML et al. Clin Chem 2005 51:1472-1479

10-2500

2.0

Y

Ceglarek U et al. Clin Chim Acta 2009 401:114-118

0-1000

7.0

N

Singh R Steroids 2008 73:13391344

10-25000

3.0

Y

Rauh, M et al. Steroids 2006 71:450-458

2-2000

0.3

N

deprotonize w/acetonitrile

APPI +

289→109

Zinc sulfate-methanol precipitation Protein Precipitation/on line extraction Protein Precipitation/on line extraction

ESI +

289.1→96.7

d2 291.1→98.7

APCI +

289→97

IS used but not listed d3 292.4→97.3

online extraction

APCI +

d5 294.3→113.0

online extraction

APCI +

ethyl acetate:hexane LLE

ESI +

ethyl acetate:hexane LLE

APPI +

289→109

d3 292→109

10-1600

1.0

Y

ethyl acetate:hexane LLE

ESI +

289.2→109.0

d2 291.2→110.9

1-2000

2.0

Y

ESI +

289.4→97.3 289.4→108.9 289.2→108.8 289.2→97.2 289→109 289→97 289→109 289→97.1

d2 291→99

d5 294→113 294→100 d3 292.2→109.1 292.2→97.1

5-2000

N

Reference

Salameh WA et al. Steroids 2010 75:169-175 Fitzgerald RL et al. Methods Mol Biol 2010 603:489-500 Harwood TD et al. Clin Chim Acta 2009 409:78-84 Shiraishi et al. Clin Chem 2008 54: 1855 Turpeinen U et al. Scan J Clin & Lab Invest. 2008 68:50-57

ether-ethyl acetate LLE

ESI +

289→97

d2 291→99

6-2881

1.4

N

MTBE LLE

ESI +

289→97

d5 294→100

5-1000

5.0

N

MTBE LLE

ESI +

289.3→109

d2 291.3→ 98.9

7-2881

7.0

Y

MTBE LLE/heptane LLE

APCI +

289.2→109.1

d5 294.2→113.2

0–2305

1.6

Y

ESI +

304→124 304→112

d3 307→124 307→112

1-200

1.0

N

Moal V et al. Clin Chem Acta 2007 386:12-19 Gallagher LM et al. Ann Clin Biochem 2007 44:48-56 Chen Y et al. Clin BioChem 2009 42:484-490 Borrey et al. Clin Chem Acta 2007 382:134-137

API +

318→126

d3 321→126

3-760

3.0

N

Bui HN et al. Ann Clin Biochem 2010 47: 248-252

ESI +

304→124 304→112

d3 307→124 307→112

10-4000

0.5

N

Kushnir MM et al. Clin Chem 2007 52:120-128

hydroxylamine deriv/ MTBE LLE methyloxylamine deriv/hexane:diethyl ether LLE MTBE LLE/hydroxylamine deriv/SPE

MS Assays and Conventional IAs Have Similar Sample Preparation Procedures Isolation/Extraction Chromatographic Separation Molecular Mass Fragmentation of Analyte

Identification

Structural Characteristics of Analyte

Calibrators and Internal Standards

Quantitation

External Calibration

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Advantages of MS-based Methodologies Specificity • Isolation based on analyte polarity • Derivatization of the analyte based (mostly used for GC applications) • Chromatographic separation • Mass separation of the steroid • Mass fragmentation • Verification (confirmation ions) Sensitivity • High specificity leads to improved signal/noise ratio Universal • Detection of multiple analytes in one analytical run

Ionization Electrospray Ionization (ESI) Converts ions in solution into gas phase ions

ESI mostly commonly used for testosterone Other ionization techniques are commonly used for assays that measure testosterone with other steroids that are not amenable to ESI.

Multi

Ionization Analyte

ESI +

APPI +

APCI +

Reference

N

Cawood ML et al. Clin Chem 2005 51:1472-1479

N

Singh R Steroids 2008 73:1339-1344

N

Fitzgerald RL et al. Methods Mol Biol 2010 603:489-500

Y

Shiraishi et al. Clin Chem 2008 54: 1855

N

Turpeinen U et al. Scan J Clin & Lab Invest. 2008 68:50-57

N

Moal V et al. Clin Chem Acta 2007 386:12-19

Y

Gallagher LM et al. Ann Clin Biochem 2007 44:48-56

N

Borrey et al. Clin Chem Acta 2007 382:134-137

N

Kushnir MM et al. Clin Chem 2007 52:120-128

N

Bui HN et al. Ann Clin Biochem 2010 47: 248-252

Y

Harwood TD et al. Clin Chim Acta 2009 409:78-84

Y

Guo T et al. J Clin Chem 2006 372 76-82

Y

Ceglarek U et al. Clin Chim Acta 2009 401:114-118

Y

Rauh, M et al. Steroids 2006 71:450-458

N Y

Chen Y et al. Clin BioChem 2009 42:484-490

Salameh WA et al. Steroids 2010 75:169-175

ESI Positive Ion Mode APPI

APCI

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Tandem Mass Spectrometry with a triple quadrupole (QqQ) mass spectrometer Q1 Fixed

q2- collision

T

109

T

Precursor Ion

Q3 Fixed

109

Fragment Ion

Fragmentation

The resulting precursor/fragment pairs are called mass “transitions”.

T Ions Transitions

Reference Guo T et al. J Clin Chem 2006 372 76-82 Harwood TD et al. Clin Chim Acta 2009 409:78-84

289→109

Shiraishi et al. Clin Chem 2008 54: 1855 Gallagher LM et al. Ann Clin Biochem 2007 44:48-56 Chen Y et al. Clin BioChem 2009 42:484-490 Cawood ML et al. Clin Chem 2005 51:1472-1479 Ceglarek U et al. Clin Chim Acta 2009 401:114-118

289→97 Turpeinen U et al. Scan J Clin & Lab Invest. 2008 68:50-57 Moal V et al. Clin Chem Acta 2007 386:12-19 Singh R Steroids 2008 73:1339-1344 Rauh, M et al. Steroids 2006 71:450-458

289→109 289→97

Salameh WA et al. Steroids 2010 75:169-175 Fitzgerald RL et al. Methods Mol Biol 2010 603:489-500 Borrey et al. Clin Chem Acta 2007 382:134-137

304→124 304→112

Kushnir MM et al. Clin Chem 2007 52:120-128

318→126

Bui HN et al. Ann Clin Biochem 2010 47: 248-252

Derivatization Can improve ionization efficiency and decrease the LOD by increasing the volatility and can reduce the ionization energy of polar compounds allowing it to ionize in the source better. Common Derivative for T: hydroxylamine or methoxylamine hydroxylamine reacts with the keto groups to form oxime derivative H H + O Testosterone m/z 289

N OH

-H2O N OH Testosterone Oxime Derivative m/z 304 (hydroxylamine) m/z 317 (methoxylamine)

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Mass Transitions Transitions to Monitor • Avoid non specific fragments i.e. loss of water (many steroid hormones show loss of water), acetate groups, methyl groups • Monitor 2 mass transitions (“MS/MS ions”) for the T and IS Confirmation Ions • 1 transition - used to quantify • 1 transition - used to verify analyte  increases the specificity  increases the confidence for correct identification of a particular analyte. Typical Transitions Monitored for Testosterone: m/z 89 to m/z 97 and m/z 289 to m/z 109

Sample Prep

Reference

deprotonize w/acetonitrile

Guo T et al. J Clin Chem 2006 372 76-82

Zinc sulfate-methanol precipitation

Cawood ML et al. Clin Chem 2005 51:1472-1479

PPT

Ceglarek U et al. Clin Chim Acta 2009 401:114-118

Protein Precipitation/Extraction in line Singh R Steroids 2008 73:1339-1344

SPE

Rauh, M et al. Steroids 2006 71:450-458

Extraction in line Salameh WA et al. Steroids 2010 75:169-175

MTBE LLE/hydroxylamine deriv/SPE

Kushnir MM et al. Clin Chem 2007 52:120-128 Fitzgerald RL et al. Methods Mol Biol 2010 603:489-500

ethyl acetate:hexane LLE

Harwood TD et al. Clin Chim Acta 2009 409:78-84

ether-ethyl acetate LLE

Turpeinen U et al. Scan J Clin & Lab Invest. 2008 68:50-57

Shiraishi et al. Clin Chem 2008 54: 1855

LLE

Moal V et al. Clin Chem Acta 2007 386:12-19

MTBE LLE

Gallagher LM et al. Ann Clin Biochem 2007 44:4856

MTBE LLE/heptane LLE

Chen Y et al. Clin BioChem 2009 42:484-490

hydroxylamine deriv/ MTBE LLE

Borrey et al. Clin Chem Acta 2007 382:134-137

methyloxylamine deriv/hexane:diethyl ether LLE

Bui HN et al. Ann Clin Biochem 2010 47: 248-252

Impact of Sample Preparation Same LC/MS/MS conditions, calibrators, samples, and analysts Different sample preparation procedures Results: Varying time required to LC/MS/MS analysis Assay Performance – increased backpressure/peak broadening/column life Different Imprecision (ion suppression issues- cleaner samples?) Different Population data distributions Not statistically significant difference in accuracy Male Patient Sample Distribution

20

Female Patient Sample Distribution

18

40

800

35

16 14

30 Concentration ng/dL

Concentration ng/dL

700

Precision (%CV)

900

600 500 400

25

12 10 8 6

20

4

15

300

2

10

200

0 Prep 1

100

5 (a)

(b) Sample Preparation Method

(c )

(a)

(b)

(c )

5 days, 3 replicates/day

Prep 2

Prep 3 Sample 1 (502 ng/dL) Sample 2 (224 ng/dL) Sample 3 (14 ng/dL)

Sample Preparation Method

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Compounds used for interference testing Fitzgerald RL Moal V et al. et al. Methods Clin Chem Acta Mol Biol 2010 2007 386:12-19 603:489-500 DHT DHT DHEA DHEA EpiT EpiT delta 4 Androstenedione 21&17αHydroxyprogesterone Cortisol Cortisone Aldosterone Corticosterone 11&21Deoxycortisol

Shiraishi et al. Clin Chem 2008 54 : 1855-1863 DHT

Androstenedione Hydroxyprogersterone Cortisol

Bui HN et al. Ann Clin Biochem 2010 47:248-252 DHT

Androstenedione

Estradiol Dehydroepiandrosterone Cholesterol

Pre- Analytical Considerations: Technical Factors Tube typeFluoride tubes can cause lower T concentrations than actually present (Wang et al. Steroids 2008 73: 1345-1352)

Clot activator can cause interference (Wang et al. Steroids 2008 73: 1345-1352)

No major difference reported for SST glass vs plastic tubes (Raff et al. Steroids 2008 1297-1304)

Storage stabilitytotal T reported stable in -25oC over 40 yrs in serum (Stroud et al. Psyconeuro 2007 32:140-150)

Freeze-thaw stabilityminimal effect (Raff et al. Steroids 2008 1297-1304)

Method Parameters Important for Validation Parameter

Description

Analysis Requirements

Comparability w/ another (reference) laboratory

Minimum 20 patient samples measured in duplicate

Recovery of expected values for reference materials

3-5 runs of reference material in duplicate

Repeatability : within-day

3 concentrations- 10 replicate measurements

Accuracy

Precision Reproducibility: between-day

LOD Linearity Matrix Effect

3 concentrations- measured in duplicate over 20 days

3* SD0 of lowest points on CC S/N >3 Linear regression on replicate calibration curves, R2 and SE Interfering compounds Ion suppression

Reference CLSI EP-9 CLSI EP-15

CLSI EP-15 CLSI EP-5

CLSI EP-17

5-7 levels in duplicate in similar matrix as patient samples Test unconjugated structure analogs of T or conjugated metabolites under the same assay conditions

CLSI EP-6

CLSI EP-14 CLSI EP-7

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Routine vs Reference Measurement Procedures Routine Measurement Procedure

Reference Measurement Procedure

•

Ensure adequate accuracy and precision for purpose of method

•

•

Same calibration curve (widest range possible) and IS concentration for all samples

•

Use weight instead of volume

•

Adjust calibration (bracketing) and IS concentration for each sample to best match analyte concentration

•

Optimize chromatographic separation for compound of interest for full resolution

•

Typically replicate measurements

•

Generally low throughput

•

Typically single-analyte methods

•

Need to meet JCTLM requirements

•

Balance sample preparation and chromatographic separation with throughput

•

Typically single measurement

•

Generally high throughput

•

Frequently multi-analyte methods

•

Need to meet CLIA and FDA requirements

Optimized for trueness and precision to minimize uncertainty

Routine vs Reference Measurement Procedure

Routine Measurement Procedure

Reference Measurement Procedure

• Mass transitions and confirmation ions • Internal standards • Pure compound calibrators • Isolation of analyte from matrix • Chromatographic separation

 Routine MS methods and reference measurement procedures use the same MS instrumentation and thus have similar detector specificity and sensitivity

Analysis Procedure of the CDC Measurement Reference Method for Testosterone Gravimetric Measurement Serum and IS to obtain 1:1 ratio (based on previously obtained orientation values)

Addition of Sodium Acetate Buffer 1st LLE

Dissociation of T from binding proteins Removal of proteins and lipids

Concentrate of Organic Layer Addition of Ammonium Carbonate Buffer 2nd LLE

Deprotonation of phospholipids Removal of phospholipids

Concentrate Organic Layer LC-MS/MS Analysis

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Analysis Procedure of the CDC Measurement Reference Method for Testosterone MS: Applied Biosystems API 4000

HPLC: Shimadzu LC system Column:

heated to 40oC C18 Hypersil Gold (50 X 3 mm, 3µ)

Buffers:

A- water in 0.1% FA B- acetonitrile in 0.1% FA

Gradient:

Buffer B 10% to 95% in 16 min

ESI in the positive ion mode ESI Voltage: Turbo Gas Temp: 475oC

5000 V

MRMQuantiation Ions m/z 289.5 > 97.1 for testosterone m/z 292.2 > 99.9 for 3C13-testosterone. Confirmation Ions m/z 289.5 > 109.1 for testosterone m/z 292.2 > 112.2 for 3C13-testosterone

Inj Vol.: 100 µL Flow Rate: 700 µL/min

Intensity (cps)

Female Calibrator with a Testosterone concentration of 0.35 ng/g (35 ng/dL)

Accuracy

Precision

0.7% Difference NIST SRM

99.9 m/z

Testosterone 289.5>97.1 m/z

Time (mins)

Time (mins)

Summary •

Problems with testosterone assays are related to assay accuracy, sensitivity and specificity

•

These problems are addressed with the CDC steroid hormone standardization program, especially with its HoSt project

•

Testosterone can be measured by immunoassays and mass spectrometry-based assays

•

Both types of assays can provide useful information for patient care, research and public health activities when used properly

•

Mass spectrometry is a promising new technology for routine testosterone testing

Acknowledgement •

The Endocrine Society

•

Solvay Pharmaceuticals

•

CDC Division of Cancer Prevention

•

CDC Foundation

•

Lisa Sapp, AB/Sciex

•

Linda Thienpont, University Gent

•

Susan Tai, NIST

•

American Association of Clinical Chemists

•

American Association of Clinical Endocrinologists

•

College of American Pathologists

•

Christopher Shacklady Jamie White Brittany Butler Gabriella Gay

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Thank you For further information contact: Hubert Vesper: [email protected], 770-488-4191 Juli Botelho: [email protected], 770-488-7391 Mail: 4770 Buford Hwy Ne MS F25 Atlanta, GA 30341 Website: http://www.cdc.gov/labstandards/hs.html

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