Cato Brede (Presenter)
Stavanger University Hospital
Bio: 1999: PhD in analytical chemistry from University of Oslo, Norway. Invented a new ion source for element-selective detection in GC separations, introducing microplasma mass spectrometry. 1999-2004: Studied specific migration from food contact materials into food and food simulants, mainly as part of projects for the Norwegian Food Control Authority. Results were presented as scientific papers and reports. 2005-now: Stavanger University Hospital, Norway. Working on LC-MS/MS methods, such as drugs of abuse in urine, total homocysteine, methyl malonic acid and a 1.2 min assay for 25-OH-D3/D2 which includes epimer information. We recently started offering glycated albumin (525K) and hepcidin by LC-MS/MS. Participates in medical research and has developed a new polymer monolithic nanoLC column.
Authorship: Cato Brede (1), Nirosa Nadarajah(2), Øyvind Skadberg(1), Jo Adaway (3)
(1) Stavanger University Hospital (Norway), (2) Oslo and Akershus University College of Applied Sciences (Norway), and (3) University Hospital of South Manchester (UK)
| Short Abstract Measurement of multiple salivary steroid hormones requires LC-MS/MS methods with the ability to detect many of these at the low pg/mL level. Sample preparation for analyte enrichment is required. We have optimized a liquid-liquid extraction procedure for saliva, including the use of tannic acid as a novel emulsion preventing reagent, and applied robot pipetting in the 96-well plate format. We tested 5 different derivatization reagents, in order to achieve the most sensitive detection with low LC complexity. The final validated assay included cortisol, cortisone, testosterone, DHEA, progesterone, and 17-OHP. |
Long Abstract
Introduction
The measurement of late night salivary cortisol has become a routine screening tool for Cushing´s syndrome. However, there are many more steroid hormones in saliva, which could be included in a multiplexed LC-MS/MS assay. With such a tool for salivary steroidomics, easy accessible saliva samples can be used to study biological variation and correlation with other clinical chemistry parameters. Hence, we would be able to discover new relationships and perhaps find clinical utility for the measurement of even more steroid hormones in saliva than cortisol alone. Sample preparation by standard liquid-liquid extraction can be a challenging task because of variation in steroid polarity, but also due to formation of emulsions that are difficult to break. Even after successful analyte enrichment, the liquid chromatography and MS detectability present further challenges. Derivatization is a well-known option for improving the sensitivity.
Methods
We optimized a liquid-liquid extraction (LLE) procedure for saliva, including the use of tannic acid as a novel emulsion preventing reagent, which is suitable for robot pipetting in the 96-well plate format. We tested 5 different derivatization reagents, in order to achieve the most sensitive detection with low LC complexity. The final assay included cortisol, cortisone, testosterone, DHEA, progesterone, and 17-OHP. Validation was done according to the Eurachem guidelines (www.eurachem.org) and we also conducted a method comparison of the new multiplexed method with the LC-MS/MS methods established at University Hospital of South Manchester. Normal reference ranges were established for women and men, both early morning and late night.
Results
10% butanol in MTBE was used in the optimized LLE for the six steroid hormones. Tannic acid was found to be a better protein precipitant than TCA, perchloric acid, and acetonitrile. It was also very efficient in preventing emulsions in the LLE, hence it was included in the final method protocol. Of the four hydrazide and the one hydrazine derivatization reagents tested, 2-hydrazinopyridine (2-HP) was found to produce high signal response for five ketosteroid hydrazones. Furthermore, 2-HP was the only reagent to produce single peaks for each of the five hydrazones that were included in the method optimization. Hence, 2-HP was included in the final method protocol. Except for 17-OHP, we obtained good correlation with the methods established at University Hospital of South Manchester, although some negative bias was observed with the new method. We found similar cut-off for late night salivary cortisol as two other laboratories in Norway. Normal reference ranges were established for women and men, both early morning and late night, for the six steroid hormones.
Conclusions & Discussion
With similar cut-off values for late night cortisol as implemented in other laboratories, the method is fit to be used as screening tool for Cushing’s syndrome. Future research will be aimed at studying the clinical utility of the other steroid hormones.
References & Acknowledgements:
| Description | Y/N | Source |
| Grants | no | |
| Salary | yes | Stavanger University Hospital |
| Board Member | no | |
| Stock | yes | G&T Septech AS |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |