Simultaneous determination of Androstenedione and Testosterone by LC-MS/MS

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Simultaneous determination of Androstenedione and
Testosterone by LC-MS/MS

• Gallagher.L, Owen.L, Keevil.B
• Department of Clinical Biochemistry SMUHT

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Outline

• Androstenedione and Testosterone
• Disease states with androgen excess in women
• Methods for the measurement of these androgens
• Method by LC- MS/MS
• Validation
• Comparison with methods in use
• Conclusions and further work

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Androstenedione

• C19 steroid
• Produced by adrenals (50) and ovaries (50)
• Principle circulating androgen in women

• Weak androgenic activity
• Precursor to testosterone and esterone

Female reference range 1.8 - 12.2nmol/L
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Testosterone

• C19 steroid
• Produced by adrenals (25) ovaries (25) and
  (50) by peripheral conversion of
  Androstenedione
• Androgenic activity greater than Androstenedione
• Precursor for Oestradiol

Female reference range lt0.5 - 2.4nmol/L
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Androgens in excess

• Hair growth
• Acne
• Temporal balding
• Clitoral growth
• Sebum production
• Deeper voice

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Disease States with Hyperandrogenaemia in Women

• Adrenal causes
• CAH (21-OHase, 11 -OHase deficiency)
• Androgen secreting adrenal tumours
• Ovarian causes
• Polycystic Ovarian Syndrome
• Ovarian stromal hyperthecosis
• Androgen secreting ovarian tumours

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Methods

• Androstenedione
• Immunoassay
• GCMS
• Testosterone
• Immunoassay
• GCMS

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Method by LCMS/MS Sample Preparation (1)

• 200?L serum, standard or QC
• 10?L internal standard
• (d7-A), (d2-T)
• 1mL Methyl-tert-butyl-ether
• Vortex mixed for 4min
• Transferred supernatant into a glass tube

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Method by LCMS/MS Sample Preparation (2)

• Supernatant blown down with a dry block (40C)
• Residue reconstituted with (100?L) 5050 mobile
  phase
• Vortex mixed 1min
• Placed in 96 well microtitre plate

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Liquid Chromatography
FA Formic Acid, AA Ammonium Acetate
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Mass Spectrometer

• Waters Quattro micro HPLC tandem mass
  spectrometer, with a Z spray ion source
• ES ionisation mode

Tuning conditions
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Mass Spectra
Androstenedione
Parent
Daughter
d7-Androstenedione
Parent
Daughter
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Mass Spectra
Testosterone
Parent
Daughter
d2-Testosterone
Parent
Daughter
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Chromatogram
Testosterone
Androstenedione
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Ion suppression
d7-androstenedione
Androstenedione
d7-androstenedione
Androstenedione
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Ion suppression
Testosterone
d2-testosterone
Testosterone
d2-testosterone
d2-testosterone
Testosterone
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Linearity
Androstenedione y 1.0016x - 0.0145 R2 0.9998
Testosterone y 0.976x 0.3109 R2 0.999
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Lower Limit of Quantification
Androstenedione
Testosterone
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Accuracy and Precision
Within Batch
Androstenedione
Testosterone
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Accuracy and Precision
Between Batch
Androstenedione
Testosterone
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Recovery

• Androstenedione
• Mean recovery 98.5, range 93 -104 (n 6)
• Testosterone
• Mean recovery 92.7, range 89 - 95
• (n 6)

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Stability
1. Single extract injected over 15hrs
Androstenedione CV 8.4
Testosterone CV 2.7
2. Batch of extracted samples measured over
24hr Androstenedione Bias 5.2, Testosterone
Bias 4.4
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Comparison
Androstenedione RIA (DSL-3800 Active)
vs LC-MS/MS
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RIA vs LC- MS/MS

• Bland Altman
• Bias -1.965, n 92

Passing Bablock y 0.575x - 0.207
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Comparison

• Testosterone
• Roche (ECLIA E170)
• vs
• LC-MS/MS

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Roche vs LCMS/MS

• Bland Altman
• Bias -0.216, n 129

Passing Bablock y 0.984x - 0.051 r2 0.97
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Roche vs LCMS/MS
Bland Altman Bias -0.349, n 87
Passing Bablock y 0.718x 0.093 r2 0.25
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Roche vs LCMS/MS
Bland Altman Bias -0.216, n 85
Passing Bablock y 0.755x 0.078 r2 0.61
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Accuracy and Precision Roche
Measured the low QCs 10 times on the Roche No
matrix effect, No cross reactivity
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Conclusions

• A simple LC-MS/MS method for simultaneous
  determination of Androstenedione and Testosterone
• This method is sensitive, accurate and precise
• Androstenedione is overestimated by RIA
• At testosterone concentrations lt3nmol/L, the
  Roche is less accurate than LC-MS/MS
• Consider using LC-MS/MS method for all female
  Testosterone and Androstenedione measurements

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Further Work

• Establish in-house reference range for LC-MS/MS
  method
• Comparison of LC-MS/MS method with Immunoassay
  used at Hope Hospital
• Method using protein precipitation with LC-MS/MS

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