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Simultaneous determination of Androstenedione and Testosterone by LC-MS

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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Simultaneous determination of Androstenedione and Testosterone by LC-MS

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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

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