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Outline. Androstenedione and TestosteroneDisease states with androgen excess in womenMethods for the measurement of these androgensMethod by LC- MS/MSValidationComparison with methods in useConclusions and further work. Androstenedione. C19 steroidProduced by adrenals (50%) and ovaries (50%)Principle circulating androgen in women.
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1. Simultaneous determination of Androstenedione and Testosterone by LC-MS/MS Gallagher.L, Owen.L, Keevil.B
Department of Clinical Biochemistry SMUHT
3. Androstenedione C19 steroid
Produced by adrenals (50%) and ovaries (50%)
Principle circulating androgen in women
4. Testosterone C19 steroid
Produced by adrenals (25%) ovaries (25%) and (50%) by peripheral conversion of Androstenedione
Androgenic activity greater than Androstenedione
Precursor for Oestradiol
5. Androgens in excess Hair growth
Acne
Temporal balding
Clitoral growth
Sebum production
Deeper voice
6. 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
7. Methods
Androstenedione
Immunoassay
GCMS
Testosterone
Immunoassay
GCMS
8. 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
9. Method by LCMS/MS Sample Preparation (2) Supernatant blown down with a dry block (40C)
Residue reconstituted with (100?L) 50:50 mobile phase
Vortex mixed 1min
Placed in 96 well microtitre plate
10. Liquid Chromatography
11. Mass Spectrometer
12. Mass Spectra
13. Mass Spectra
14. Chromatogram
15. Ion suppression
16. Ion suppression
17. Linearity To evaluate linearity of the calibration curves, three calibration curves were prepared and analysed. The curves were judged linear if the correlation coefficient R2 was better than 0.99 as calculated by weighted linear regression.To evaluate linearity of the calibration curves, three calibration curves were prepared and analysed. The curves were judged linear if the correlation coefficient R2 was better than 0.99 as calculated by weighted linear regression.
18. Lower Limit of Quantification
21. Recovery Androstenedione
Mean recovery: 98.5%, range 93 -104% (n = 6)
Testosterone
Mean recovery: 92.7%, range 89 - 95%
(n = 6)
22. Stability
23. Comparison
24. RIA vs LC- MS/MS Bland Altman
Bias = -1.965, n = 92
25. Comparison Testosterone:
Roche (ECLIA E170)
vs
LC-MS/MS
26. Roche vs LCMS/MS Bland Altman
Bias = -0.216, n= 129
27. Roche vs LCMS/MS
28. Roche vs LCMS/MS
29. Accuracy and Precision Roche
30. 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 <3nmol/L, the Roche is less accurate than LC-MS/MS
Consider using LC-MS/MS method for all female Testosterone and Androstenedione measurements
31. 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