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Ke Y.,EndoCeutics Laboratory | Gonthier R.,EndoCeutics Laboratory | Simard J.-N.,EndoCeutics Laboratory | Labrie F.,EndoCeutics Laboratory
Steroids | Year: 2016

7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone (7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA) are oxidized metabolites of dehydroepiandrosterone (DHEA). Their concentrations are low in the circulation, especially in postmenopausal women, thus resulting in a considerable challenge for their reliable measurement. A sensitive and accurate LC-MS/MS method has been developed using a simple sample preparation procedure and a novel derivatization with 1-amino-4-methyl piperazine (MP). The derivatized metabolites are stable in high water content reagents. A 10 pg/mL (0.2 pg on column) for the low limit of quantitation (LLOQ) has been achieved for all three compounds. A proper choice of multiple reaction monitoring (MRM) transitions provides good specificity. The excess amount of reagent can be removed from the sample during the derivatization process. Within the calibration range of 10-2000 pg/mL, a good linearity was obtained with R > 0.99 where the weighing factor is 1/X while the bias and coefficient of variance (CV) are within 8% for all levels of QCs and calibration curves. This method has been fully validated according to the FDA guidelines, where the results of the matrix effect meet the acceptance criteria while freeze-thaw stability, short and long term stability in matrix and solution as well as post-processed sample stability meet the requirements. With this method, the concentrations of 7α OH-DHEA, 7β OH-DHEA and 7 oxo-DHEA were measured in premenopausal and postmenopausal serum. The average concentration of 7α OH-DHEA is equivalent to that of 7β OH-DHEA in both types of sera. © 2016 Elsevier Inc. All rights reserved.

Ke Y.,EndoCeutics Laboratory | Bertin J.,EndoCeutics Laboratory | Gonthier R.,EndoCeutics Laboratory | Simard J.-N.,EndoCeutics Laboratory | Labrie F.,EndoCeutics Laboratory
Journal of Steroid Biochemistry and Molecular Biology | Year: 2014

Steroids were first analyzed by immunoassay-based methods followed by gas chromatography mass spectrometry (GC-MS or GC-MS/MS) with derivatization techniques since steroids are neutral and do not ionize at a high level using the electrospray ionization technique. We now report a validated liquid chromatography tandem mass spectrometry (LC-MS/MS) method for the simultaneous quantification of seven steroidal compounds, i.e., estradiol (E2), estrone (E1), testosterone (Testo), dihydrotestosterone (DHT), androst-5-ene-3β, 17β-diol (5-diol), dehydroepiandrosterone (DHEA) and androstenedione (4-dione). The system used is a UPLC-MS/MS (Qtrap 6500) system. With this method, the sample preparation is the combination of liquid-liquid extraction and a simple selective derivatization for only E1 and E2. This assay method is simple and practically eliminates potential contamination. Low quantification limits of 1 pg/mL, 4 pg/mL, 50 pg/mL, 10 pg/mL, 100 pg/mL, 500 pg/mL and 100 pg/mL have been found, respectively for the steroids mentioned above. Without derivatization, DHT sensitivity can be as low as 4 pg/mL with S/N ≥ 5. A full validation has been performed for the seven compounds in compliance with GLP and FDA guidelines for bioanalytical method development and validation. Recovery of all seven compounds in unstripped serum is similar to that in stripped serum: 72.1-84.7% for E2, 83.6-94.5% for E1, 88.2-90.3% for Testo, 82.0-90.6% for DHT, 84.9-92.0% for 5-diol, 88.1-93.8% for DHEA and 86.2-90.3% for 4-dione, respectively. A good linearity is obtained with R > 0.99 for each compound within its calibration range. Accuracies of all levels of QC are within the range of 15% for all seven compounds. The between day variation coefficients are 6.1-8.9% for the low limits of quantification of all seven compounds with 0.7-6.1% for higher levels of QCs for all seven compounds. All results of other test parameters similarly meet the acceptance criteria of EndoCeutics SOPs and FDA guidelines. By comparison of GC-MS/MS and LC-MS/MS data for six derivatized and nonderivatized free steroids, the present data show the crucial importance to use validated assays according to the FDA guidelines to increase specificity, precision and reliability of the absolute values associated with MS/MS-based assays. This method has already been applied to series of samples from clinical trials and is ready for wide clinical use. © 2014 Elsevier Ltd. All rights reserved.

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