Innleiðing einnar mæliaðferðar á LC-MS/MS fyrir átján sterahormón

Abstract

The adrenal glands along with the gonads and the placenta, produce and secrete steroid hormones. Cholesterol is the precursor for all steroid hormones. The human steroidogenesis can be separated into three major pathways, glucocorticoids, mineralocorticoids and finally sex steroids (adrenal androgens and estrogens). Steroid hormones regulate both physiological and developmental processes and they all have specific roles in metabolism and other bodily functions. Any deficiency, excess or buildup of intermediate steroids can lead to steroid hormone disorders, depending on the steroid hormone, for example Cushing’s syndrome (CS) and congenital adrenal hyperplasia (CAH). Steroid hormones are measured using both immunoassay (IA) and liquid chromatography tandem mass spectrometry (LC- MS/MS). Today LC-MS/MS is considered a gold standard for steroid hormone measurements. LC- MS/MS technique is both quantitative and qualitative and able to quantitate very low concentrations of analytes and determine complex molecule structures. The European Union In Vitro Diagnostics Regulation (EU IVDR) entered into force in 2017. The regulation includes, among other things, the implementation of a CE or IVDR marked measurement method instead of laboratory developed LC-MS methods.The first aim of this project was to implement on LC-MS/MS an EU IVDR certified steroid panel which measures 17 endogenous steroid hormones as well as the synthetic glucocorticoid drug dexamethasone. The second aim was to increase the methods sensitivity and lower detection limits, especially for estradiol and testosterone. Following that, perform a partial validation and compare the method to other measurement methods already in use at the National University Hospital of Iceland’s (LSH) laboratory for steroid hormone measurements, those are mostly IA methods but also a laboratory developed LC-MS/MS method.The Steroid Panel LC-MS was implemented on Waters Acquity I-Class UPLC system connected to TQ absolute MS and sample preparation performed using the Freedom EVO® with integrated Resolvex® A200, positive pressure unit. Method validation was performed according to European Medicines Agency (EMA) guidelines.Majority of accuracy and precision (A/P) results were within the EMA guideline limits, except for lower limit of quantification (LLOQ) results for e.g. testosterone and estradiol. Selectivity results showed average peak area ratio of calibrator (Cal) zero and LLOQ >20% for testosterone, estradiol and other compounds. Linear regression for comparisons of LC-MS/MS methods showed coefficient of determination (R2) of >0,99.Preliminary partial validation results acquired, showed good promise of A/P as well as sensitivity of the Steroid Panel LC-MS. We determined that low accuracy of LLOQ for testosterone and estradiol was caused by detected peaks in Cal zero. Meaning that lowering the detection limits for some compounds was not possible and therefore their LLOQ adjusted to Cal A. Comparisons of LC-MS/MS measurement methods showed minimal bias between instruments (<5%). Only one aim of this thesis was fully completed, the implementation of an EU IVDR certified steroid panel on LC-MS/MS. With the launching of this steroid panel into routine use at LSH, numerous esoteric steroid measurements will become available locally for the first time and will be useful for diagnosing different disorders and diseases.