Isidor Minovic (Presenter)
University Medical Center Groningen
Bio: Back in 2008, my interest in chemistry and patient care motivated me to enroll for Pharmacy (at the University of Groningen), of which I successfully completed the Bachelor and Master according to curriculum in 2014. During my Master, I conducted basal research towards clarifying the role of chondrocytes in asthma in a pharmacology laboratory at the University of Melbourne. After Pharmacy, I started a joint PhD in Laboratory Medicine and Nephrology (at the University Medical Center Groningen), that focuses on providing mechanistic insights into the transsulfuration pathway. As part of my PhD, I have been working on developing and validating an improved LC-MS/MS method for the determination of total urinary cortisol and major metabolites (see abstract) and an LC-MS/MS method for quantification of the uremic toxins p-cresol sulfate and indoxyl sulfate.
Authorship: I. Minovic(1,2), M. van Faassen(1), S.J.L. Bakker(2), I.P. Kema(1)
Departments of (1)Laboratory Medicine and (2)Nephrology, University Medical Center Groningen, Groningen, the Netherlands
| Short Abstract Clinical assessment of daily cortisol production is conventionally done by measuring urinary free cortisol, thus ignoring the fact that only 1% of total endogenous cortisol production appears in urine. Additional measurements of cortisol metabolites are likely to provide a more reliable estimation of total daily cortisol production. We therefore developed and validated a sensitive, selective, and rapid LC-MS/MS method for simultaneous assessment of total cortisol, cortisone, tetrahydrocortisol (THF), allo-tetrahydrocortisol (a-THF) and tetrahydrocortisone (THE) in urine. Total run-time of our method was 5.5 minutes and intra- and inter-assay coefficients of variation were <10% for all components. Linearity in the calibration range (r2) was consistently >0.99 for each component and all results were in agreement with those obtained from routine LC-MS/MS and GC-MS/MS analyses. |
Long Abstract
Introduction. Clinical assessment of daily cortisol production is conventionally done by measuring urinary free cortisol, thus ignoring the fact that approximately only 1% of total endogenous cortisol production appears in urine. Additional measurements of cortisol metabolites are likely to provide a more reliable estimation of total daily cortisol production. Current methods for quantification of urinary cortisol metabolites require substantial volumes of urine and lack proper internal validation. In addition, these methods involve cumbersome sample preparation steps and long GC run times, making them unsuitable for high-throughput research. We therefore aimed to develop and validate a rapid LC-MS/MS method for simultaneous assessment of total cortisol, cortisone, tetrahydrocortisol (THF), allo-tetrahydrocortisol (a-THF), and tetrahydrocortisone (THE) in urine.
Methods. Internal standards for each of the five components (all deuterated, except cortisol(13-C)), buffer and enzyme solution were added to 100 µL of urine in 96-well plates. This mixture was incubated for 4 h at 46°C to achieve optimal hydrolytic efficiency. Analytes were extracted using a 96-well Supported Liquid Extraction plate (Phenomenex) and were chromatographically separated with a UPLC phenyl-hexyl column (Waters). Mass spectrometric detection was performed in selective reaction monitoring mode, using a quadrupole tandem mass spectrometer in negative mode (Xevo TQ-S, Waters). As part of the validation procedure, intra and inter-assay coefficients of variation (CV’s) were assessed by analyzing the three quality controls (QC’s) – i.e. low, medium, and high – ten times on one day and on ten different days, respectively. Recoveries and lower limits of quantification (LLOQ’s) were determined by measuring spiked QC’s and diluted QC-low samples, respectively, on six different days. To investigate accuracy, 40 patient samples were analyzed and results were compared with routine LC-MS/MS and GC-MS/MS methods.
Results. Total run-time of our assay was 5.5 minutes and intra- and inter-assay CV’s were <10% for all components. Linearity in the calibration range (r2) was consistently >0.99 for each component. Recoveries all met the predefined requirements of 85-115% and LLOQ’s were 8.45 nM, 9.27 nM, 0.30 µM, 0.52 µM, and 0.29 µM for cortisol, cortisone, THF, a-THF, and THE, respectively. All results were in agreement with those obtained from routine analyses.
Conclusion. We have successfully developed and validated a sensitive, selective and rapid mass spectrometric method, suitable for quantification of total urinary cortisol, cortisone, THF, a-THF, and THE in high-throughput research.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |