title: A validated LC-MS/MS method for the sensitive quantitation of serum 7alpha hydroxy-, 7beta hydroxy- and 7keto-dehydroepiandrosterone using a novel derivatization reagent.
abstract: 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.
authors: EndoCeutics Laboratory, 1405 Parc Technologique Blvd, Suite 250, Québec, QC G1P 4P5, Canada.
authors: Yuyong
authors: Y
authors: Ke
authors: EndoCeutics Laboratory, 1405 Parc Technologique Blvd, Suite 250, Québec, QC G1P 4P5, Canada.
authors: Renaud
authors: R
authors: Gonthier
authors: EndoCeutics Laboratory, 1405 Parc Technologique Blvd, Suite 250, Québec, QC G1P 4P5, Canada.
authors: Jean-Nicolas
authors: JN
authors: Simard
authors: EndoCeutics Laboratory, 1405 Parc Technologique Blvd, Suite 250, Québec, QC G1P 4P5, Canada. Electronic address: fernand.labrie@endoceutics.com.
authors: Fernand
authors: F
authors: Labrie
mesh_terms: D000328:Adult; D001774:Blood Chemical Analysis / Q000379:methods*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D003687:Dehydroepiandrosterone / Q000097:blood* / Q000737:chemistry* / Q000494:pharmacology; D005260:Female; D006461:Hemolysis / Q000187:drug effects; D006801:Humans; D006949:Hyperlipidemias / Q000139:chemically induced; D007202:Indicators and Reagents / Q000737:chemistry; D057230:Limit of Detection; D016014:Linear Models; D008875:Middle Aged; D017698:Postmenopause / Q000097:blood; D017697:Premenopause / Q000097:blood; D053719:Tandem Mass Spectrometry / Q000379:methods*; D055815:Young Adult
chemical_list: D007202:Indicators and Reagents; D003687:Dehydroepiandrosterone
keywords: 7 oxo-DHEA; 7α OH-DHEA; 7β OH-DHEA; DHEA; LC–MS/MS