Endocrine Abstracts

For several years, steroid hormones including testosterone (T), androstenedione (A4) and 17-hydroxyprogesterone (17-OHP) have been utilised in the diagnosis of a range of conditions with clinical features of hyperandrogenaemia including polycystic ovary syndrome (PCOS) and congenital adrenal hyperplasia. In recent years a potential role for the 11-oxygenated androgens has emerged in the diagnosis and characterisation of androgenic disorders. Specifically, the 11-oxygenated androgens including 11-ketotestosterone (11-KT), 11-hydroxyandrostenedione (11-OHA4), 11-ketoandrostenedione (11KA4) and 11-hydroxytestosterone (11-OHT), have been shown to be the major circulating androgens in PCOS and the predominant steroids responsible for hyperandrogenaemia in Cushing Syndrome. The use of saliva for diagnostic tests has advantages over blood sampling. Saliva can be collected non-invasively at home without the presence of a healthcare professional and can then be posted to the laboratory, saving the patient a visit to a phlebotomy clinic for venepuncture. We have developed a multiplex LC-MS/MS assay on a Waters TQXS mass spectrometer for salivary T, A4, 17-OHP, 11-KT, 11-OHA4, 11-KA4 and 11-OHT with a run time injection-to-injection of five minutes. Samples were prepared for analysis by supported liquid extraction; we have found optimal sensitivity and negligible matrix effects using a Waters T3 chromatography column, with mobile phases of ammonium fluoride (mobile phase A) and methanol (mobile phase B) giving a two fold greater sensitivity compared to acidic mobile phases. Herein we describe a full bioanalytical validation based on U.S Food and Drug administration standards including studying accuracy, recovery and imprecision. Additionally, we investigate the stability of the seven analytes included in the assay across different conditions including freezer storage, refrigeration, room temperature and 37^oC in order to determine the requirements for collecting and sending of samples to the laboratory for analysis. The assay is available for clinical studies as well as routine analysis and we aim to assess intraindividual variability.